Podcasts about nav1

The world of anesthesia is evolving, and pain management is at the forefront of this transformation. In this episode, we dive into the revolutionary non-opioid analgesic, Suzetrigine, a selective NaV1.8 inhibitor making waves in perioperative and acute pain management. With increasing pressure to reduce opioid dependence while maintaining effective pain control, anesthesia providers must stay ahead of cutting-edge developments. Join us as we break down the pharmacology, safety profile, clinical trial data, and real-world implications of this game-changing drug. Here's some of what we discuss in this episode: We'll talk about dosing in extreme detail because, as anesthesia providers, we know that precision matters. The drug interactions is a critical consideration and we'll run through adjustments that need to be made. We get to the into the physiology and pharmacology implications of Suzetrigine, focusing on what this means for nurse anesthetists. The pharmacological properties of Suzetrigine and how it functions at a molecular level to modulate pain perception. The side effects and contraindications. Scientific trials and comparisons to other analgesics for nurse anesthetists.   Visit us online: https://beyondthemaskpodcast.com/ The 1099 CRNA Institute: https://aana.com/1099 Get the CE Certificate here: https://beyondthemaskpodcast.com/wp-content/uploads/2020/04/Beyond-the-Mask-CE-Cert-FILLABLE.pdf Help us grow by leaving a review: https://podcasts.apple.com/us/podcast/beyond-the-mask-innovation-opportunities-for-crnas/id1440309246 Donate to Our Heart Your Hands here: https://www.ourheartsyourhands.org/donate  Support Team Emma Kate: https://grouprev.com/haloswalk2024-shannon-shannon-brekken

Dr. Amit Goyal, along with episode chair Dr. Dinu Balanescu (Mayo Clinic, Rochester), and FIT leads Dr. Sonu Abraham (University of Kentucky) and Dr. Natasha Vedage (MGH), dive into the fascinating topic of channelopathies with Dr. Michael Ackerman, a genetic cardiologist and professor of medicine, pediatrics, and pharmacology at Mayo Clinic, Rochester, Minnesota. Using a case-based approach, they review the nuances of diagnosis and treatment of channelopathies, including Brugada syndrome, catecholaminergic polymorphic ventricular tachycardia (CPVT), and long QT syndrome. Dr. Sonu Abraham drafted show notes. Audio engineering for this episode was expertly handled by CardioNerds intern, Christiana Dangas. The CardioNerds Beyond the Boards Series was inspired by the Mayo Clinic Cardiovascular Board Review Course and designed in collaboration with the course directors Dr. Amy Pollak, Dr. Jeffrey Geske, and Dr. Michael Cullen. CardioNerds Beyond the Boards SeriesCardioNerds Episode PageCardioNerds AcademyCardionerds Healy Honor Roll CardioNerds Journal ClubSubscribe to The Heartbeat Newsletter!Check out CardioNerds SWAG!Become a CardioNerds Patron! Pearls and Quotes - Channelopathies One cannot equate the presence of type 1 Brugada ECG pattern to the diagnosis of Brugada syndrome. Clinical history, family history, and/or genetic testing results are required to make a definitive diagnosis. The loss-of-function variants in the SCN5A gene, which encodes for the α-subunit of the NaV1.5 sodium channel, is the only Brugada susceptibility gene with sufficient evidence supporting pathogenicity. Exertional syncope is an “alarm” symptom that demands a comprehensive evaluation with 4 diagnostic tests: ECG, echocardiography, exercise treadmill test, and Holter monitor. Think of catecholaminergic polymorphic ventricular tachycardia (CPVT) in a patient with exertional syncope and normal EKG! ICD therapy is never prescribed as monotherapy in patients with CPVT. Medical therapy with a combination of nadolol plus flecainide is the current standard of care. Long QT syndrome is one of the few clinical scenarios where genetic testing clearly guides management, particularly with respect to variability in beta-blocker responsiveness. Notes - Channelopathies 1. What are the diagnostic criteria for Brugada syndrome (BrS)? Three repolarization patterns are associated with Brugada syndrome in the right precordial leads (V1-V2): Type 1: Prominent coved ST-segment elevation displaying J-point amplitude or ST-segment elevation ≥2 mm, followed by a negative T wave. Type 2/3: Saddleback ST-segment configuration with variable levels of ST-segment elevation. It is important to note that only a type 1 pattern is diagnostic for Brugada syndrome, whereas patients with type 2/3 patterns may benefit from further testing. The Shanghai score acknowledges that relying solely on induced type 1 ECG changes has limitations. Therefore, one cannot equate the presence of a type 1 Brugada ECG pattern alone to the diagnosis of Brugada syndrome. The score suggests incorporating additional information—such as clinical history, family history, and/or genetic testing results—to achieve a definitive diagnosis. 2. What is the significance of genetic testing in Brugada syndrome? There are 23 alleged Brugada syndrome susceptibility genes published with varying levels of evidence. However, only one gene mutation, the loss-of-function variants in the SCN5A gene encoding for the α-subunit of the NaV1.5 sodium channel, is considered to have sufficient evidence. The overall yield of BrS genetic testing is 20%. The presence of PR prolongation (>200 ms) along with type I EKG pattern increases the yield to 40%. On the contrary, in the presence of a normal PR interval, the likelihood of SCN5A positivity drops to

Good morning from Pharma and Biotech daily: the podcast that gives you only what's important to hear in Pharma and Biotech world.Biogen is facing additional troubles as the Department of Justice (DOJ) and Securities and Exchange Commission (SEC) have issued subpoenas seeking more information. The DOJ is specifically looking into the company's overseas operations, while the SEC is investigating the launch of Biogen's now-discontinued Alzheimer's disease therapy, Aduhelm.In other news, the Federal Trade Commission (FTC) and Department of Health and Human Services (HHS) have launched a probe into the ongoing generic drug shortage. The investigation will focus on possible involvement of group purchasing organizations and drug wholesalers in exacerbating the shortage.Ipsen's Onivyde has been approved as a first-line treatment for metastatic pancreatic cancer. This marks the first new frontline treatment option in over 10 years for adults living with this type of cancer.Latigo Biotherapeutics has raised $135 million in a Series A funding round to enter the non-opioid pain medicine space. The company plans to develop its own Nav1.8 inhibitor for pain management.Bristol Myers Squibb is leading the future of protein degradation with its targeted protein degradation approach. The company aims to design next-generation degraders with precision, agility, and intention to attack disease targets.In other funding news, European-based venture capital firm Earlybird Health has closed a $186 million fund focused on cancer and neuroscience investments in biopharma. Additionally, ADC-focused ProfoundBio has raised $112 million in an oversubscribed Series B round.Brivant, a subsidiary of Roivant Sciences, has announced plans to shut down its MDS-focused division, Vant, following disappointing Phase I/II trial results.The FDA has pushed back the Prescription Drug User Fee Act (PDUFA) date for Rocket Pharmaceuticals' gene therapy for a rare blood disorder.Patients taking semaglutide have seen benefits after total hip replacement surgery, according to recent studies.In other news, Novo Nordisk has acquired Catalent's gene therapy manufacturing business for $1.2 billion.These are the top stories in the biopharmaceutical industry today.

Today, you'll learn about a possible new treatment for chronic pain, a major obstacle to the world's tree-planting plans, and some bad news for nose pickers. Chronic Pain Treatment “Identification and targeting of a unique Nav1.7 domain driving chronic pain.” by Kimberly Gomez, et al. 2023. https://www.pnas.org/doi/10.1073/pnas.2217800120 “Nav1.7 withholds its pain potential.” by Katie Kingwell. 2023. https://www.nature.com/articles/d41573-019-00065-0 “Chronic Pain Among Adults - United States, 2019 - 2021.” CDC. 2023. https://www.cdc.gov/mmwr/volumes/72/wr/mm7215a1.htm “Prevalence of chronic pain among adults in the United States.” by R Jason Yong & Neil Bhattacharyya. 2022. https://pubmed.ncbi.nlm.nih.gov/33990113/ Tree Planting “Plans to plant billions of trees threatened by massive undersupply of seedlings.” by Joshua Brown. 2023. “A lack of ecological diversity in forest nurseries limits the achievement of tree-planting objectives in response to global change.” by Peter W. Clark, et al. 2023. “Trees Help Fight Climate Change.” Arbor Day Foundation. N.d. “Benefits of Planting Trees.” Tree Advisory Board. N.D. Nose Pickers “Why Not to Pick Your Nose // Association between nose picking and SARS-Cov-2 incident, a cohort study in hospital healthcare workers.” by A.H. Ayesha Lavell, et al. 2023. “Nose picking linked to higher risk of covid, study shows.” by Lindsey Bever. 2023. “The Anatomy and Function of the Nasal Cavity.” by Kristin Hayes, RN. 2023. Follow Curiosity Daily on your favorite podcast app to get smarter with Calli and Nate — for free! Hosted on Acast. See acast.com/privacy for more information.

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.16.549195v1?rss=1 Authors: Loya-Lopez, S. I., Allen, H. N., Duran, P., Calderon-Rivera, A., Gomez, K., Kumar, U., Shields, R., Zeng, R., Dwivedi, A., Saurabh, S., Korczeniewska, O. A., Khanna, R. Abstract: Dysregulation of voltage-gated sodium NaV1.7 channels in sensory neurons contributes to chronic pain conditions, including trigeminal neuropathic pain. We previously reported that chronic pain results in part from increased SUMOylation of collapsin response mediator protein 2 (CRMP2), leading to an increased CRMP2/NaV1.7 interaction and increased functional activity of NaV1.7. Targeting this feed-forward regulation, we developed compound 194, which inhibits CRMP2 SUMOylation mediated by the SUMO-conjugating enzyme Ubc9. We further demonstrated that 194 effectively reduces the functional activity of NaV1.7 channels in dorsal root ganglia neurons and alleviated inflammatory and neuropathic pain. Here, we employed a comprehensive array of investigative approaches, encompassing biochemical, pharmacological, genetic, electrophysiological, and behavioral analyses, to assess the functional implications of NaV1.7 regulation by CRMP2 in trigeminal ganglia (TG) neurons. We confirmed the expression of Scn9a, Dpysl2, and UBE2I within TG neurons. Furthermore, we found an interaction between CRMP2 and NaV1.7, with CRMP2 being SUMOylated in these sensory ganglia. Disrupting CRMP2 SUMOylation with compound 194 uncoupled the CRMP2/NaV1.7 interaction, impeded NaV1.7 diffusion on the plasma membrane, and subsequently diminished NaV1.7 activity. Compound 194 also led to a reduction in TG neuron excitability. Finally, when intranasally administered to rats with chronic constriction injury of the infraorbital nerve (CCI-ION), 194 significantly decreased nociceptive behaviors. Collectively, our findings underscore the critical role of CRMP2 in regulating NaV1.7 within TG neurons, emphasizing the importance of this indirect modulation in trigeminal neuropathic pain. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.06.30.547216v1?rss=1 Authors: Obeidat, A. M., Ishihara, S., Li, J., Lammlin, L., Junginger, L., Maerz, T., Miller, R. J., Miller, R., Malfait, A.-M. Abstract: Objective: Knee joints are densely innervated by nociceptors. Sprouting of nociceptors has been reported in late-stage osteoarthritis (OA), both in human knees and in rodent models. Here, we sought to describe progressive nociceptor remodeling in four mouse models of knee OA, capturing early and late-stage disease. Methods: Sham surgery, destabilization of the medial meniscus (DMM), partial meniscectomy (PMX), or non-invasive anterior cruciate ligament rupture (ACLR) was performed in the right knee of 10-12-week old male C57BL/6 NaV1.8-tdTomato mice. Mice were euthanized (1) 4, 8 or 16 weeks after DMM or sham surgery; (2) 4 or 12 weeks after PMX or sham; (3) 1 or 4 weeks after ACLR injury or sham. Additionally, a cohort of naive male wildtype mice was evaluated at 6 and 24 months. Twenty-m thick mid-joint cryosections were assessed qualitatively and quantitatively for NaV1.8+ and PGP9.5+ innervation. Cartilage damage (using a modified OARSI score), synovitis, and osteophytes were assessed blindly. Results: Progressive OA developed in the medial compartment after DMM, PMX, and ACLR. Synovitis and associated neo-innervation by nociceptors peaked in early-stage OA. In the subchondral bone, channels containing sprouting nociceptors appeared early, and progressed with worsening joint damage. Two-year old mice developed primary OA in both the medial and the lateral compartment, accompanied with neuroplasticity in the synovium and the subchondral bone. All 4 models had an increased nerve signal in osteophytes. Conclusion: Anatomical neuroplasticity of nociceptors was observed in association with joint damage in 4 distinct mouse models, suggesting that it is intrinsic to OA pathology. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.06.30.547260v1?rss=1 Authors: Tian, J., Bavencoffe, A. G., Zhu, M. X., Walters, E. T. Abstract: Nociceptor cell bodies generate spontaneous discharge that can promote ongoing pain in persistent pain conditions. Little is known about the underlying mechanisms. Recordings from nociceptor cell bodies (somata) dissociated from rodent and human dorsal root ganglia (DRGs) have shown that prior pain in vivo is associated with low-frequency discharge controlled by irregular depolarizing spontaneous fluctuations of membrane potential (DSFs), likely produced by transient inward currents across the somal input resistance. Here we show that DSFs are associated with high somal input resistance over a wide range of membrane potentials, including depolarized levels where DSFs approach action potential (AP) threshold. Input resistance and both the amplitude and frequency of DSFs were increased in neurons exhibiting spontaneous activity. Ion substitution experiments indicated that the depolarizing phase of DSFs is generated by spontaneous opening of channels permeable to Na+ and/or Ca2+, and that Ca2+-permeable channels are especially important for larger DSFs. Partial reduction of the amplitude and/or frequency of DSFs by perfusion of pharmacological inhibitors indicated small but significant contributions from Nav1.7, Nav1.8, TRPV1, TRPA1, TRPM4, and N-type Ca2+ channels. Less specific blockers suggested a contribution from NALCN channels, and global knockout suggested a role for Nav1.9. The combination of high somal input resistance plus background activity of diverse ion channels permeable to Na+ and/or Ca2+ produces DSFs that are poised to reach AP threshold if resting membrane potential (RMP) depolarizes, AP threshold decreases, and/or DSFs become enhanced -- all of which have been reported under painful neuropathic and inflammatory conditions. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.04.18.537411v1?rss=1 Authors: Drouillas, B., Brocard, C., zanella, S., Bos, R., Brocard, F. Abstract: Persistent sodium current (INaP) in the spinal locomotor network promotes two distinct nonlinear firing patterns: a self-sustained spiking triggered by a brief excitation in bistable motoneurons and bursting oscillations in interneurons of the central pattern generator (CPG). Here, we identified the NaV channels responsible for INaP and their role in motor behaviors. We report the axonal Nav1.6 as the main molecular player for INaP in lumbar motoneurons. The motoneuronal inhibition of Nav1.6, but not of Nav1.1, impairs INaP, bistability, postural tone and locomotor performance. In interneurons of the CPG region, Nav1.6 with Nav1.1 equally mediate INaP and the inhibition of both channels is required to abolish oscillatory bursting activities and the locomotor rhythm. Overall, Nav1.6 plays a significant role both in posture and locomotion by governing INaP-dependent bistability in motoneurons and working in tandem with Nav1.1 to provide INaP-dependent rhythmogenic properties of the CPG. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.04.18.537407v1?rss=1 Authors: zhou, l. x., Zhao, J. Abstract: The voltage-gated sodium channel Nav1.7 plays a crucial role in the initiation and propagation of pain signals. Our previous study has successfully identified the interacting proteins of mouse Nav1.7 (mNav1.7). In this study, we aimed to further elucidate the protein-protein interactions associated with human Nav1.7 (hNav1.7). Stable epitope (TAP)-tagged HEK293 cells expressing hNaV1.7 were utilized for the identification of hNav1.7-interacting proteins. The hNaV1.7-associated complexes were isolated through tandem affinity purification and further characterized by mass spectrometry. Bioinformatics analysis was carried out using the PANTHER classification system. Electrophysiological recording was performed to assess Nav1.7 current. Tap-tagged hNav1.7 was expressed effectively in HEK293 cells, exhibiting normal functional Nav1.7 currents. A total of 261 proteins were identified as interactors of hNav1.7, mainly located across the cell membrane and cytoplasm, and primarily involved in biological processes related to protein translation and expression. Comparison between human and mouse Nav1.7-interacting proteins revealed shared proteins (such as Eef1a1, Eef2, Tcp1, Cct2, Cct3, Cct5, Cct6a, and Cct7) as well as protein families (such as kinesin and Rab GTPases family). Knockdown of two of the shared interacting proteins, CCT5 and TMED10, resulted in reduced Nav1.7 current density. In conclusion, the protein interactions of hNaV1.7 were successfully mapped in the current work. These novel findings offer essential insights into the regulatory mechanisms that govern Nav1.7 function. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.03.28.534533v1?rss=1 Authors: Lenert, M., Burton, M. D. Abstract: Treatments for reproductive disorders in women primarily consist of hormone replacement therapy, which can have negative health impacts. Bidirectional communication between sensory neurons and innervated organs is an emerging area of interest in tissue physiology with potential relevance for reproductive disorders. Indeed, the metabolic activity of sensory neurons can have profound effects on reproductive phenotypes. To investigate this phenomenon, we utilized a murine model with conditional deletion in sensory neurons of liver kinase B1 (LKB1), a serine/threonine kinase that regulates cellular metabolism. Female mice with this LKB1 deletion (Nav1.8cre;LKB1fl/fl) had significantly more pups per litter compared to wild-type females. Interestingly, the LKB1 genotype of male breeders had no effect on fertility outcomes, thus indicating a female-specific role of sensory neuron metabolism in fertility. LKB1 deletion in sensory neurons resulted in reduced ovarian innervation from dorsal root ganglia neurons and increased follicular turnover compared to littermate controls. In summary, LKB1 expression in peripheral sensory neurons plays an important role in modulating fertility of female mice via ovarian sensory innervation. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.03.21.533690v1?rss=1 Authors: Sanchez, J. P., Middleton, S. J., Pattison, L. A., Hilton, H., Awadelkareem, M. A., Zuberi, S. R., Renke, M. B., Hu, H., Yang, X., Clark, A. J., Smith, E. S. J., Bennett, D. L. Abstract: Hyperexcitability in sensory neurons is known to underlie many of the maladaptive changes associated with persistent pain. Chemogenetics has shown promise as a means to suppress such excitability, yet chemogenetic approaches suitable for human applications are needed. PSAM4-GlyR is a modular system based on the human 7 nicotinic acetylcholine and glycine receptors, which responds to inert chemical ligands and the clinically-approved drug, varenicline. Here, we demonstrated the efficacy of this channel in silencing both mouse and human sensory neurons by the activation of large shunting conductances after agonist administration. Virally-mediated expression of PSAM4-GlyR in mouse sensory neurons produced behavioural hyposensitivity upon agonist administration, which was recovered upon agonist washout. Importantly, stable expression of the channel led to similar reversible behavioural effects even after 10 months of viral delivery. Mechanical and spontaneous pain readouts were also ameliorated by PSAM4-GlyR activation in acute and joint pain inflammation models. Furthermore, suppression of mechanical hypersensitivity generated by a spared nerve injury model of neuropathic pain was also observed upon activation of the channel. Effective silencing of behavioural hypersensitivity was reproduced in a human model of hyperexcitability and clinical pain: PSAM4-GlyR activation decreased the excitability of human induced pluripotent stem-cell-derived sensory neurons and spontaneous activity due to a gain of function NaV1.7 mutation causing inherited erythromelalgia. Our results demonstrate the contribution of sensory neuron hyperexcitability to neuropathic pain and the translational potential of an effective, stable and reversible human-based chemogenetic system for the treatment of pain. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.03.16.532982v1?rss=1 Authors: Yuan, T., Wang, Y., Jin, Y., Xu, S., Zhang, H., Chen, Q., Li, N., Ma, X., Song, H., Peng, C., Yang, H., Geng, Z., Dong, J., Duan, G., Sun, Q., Yang, Y., Yang, F., Huang, Z. Abstract: Quinidine has been used as an anticonvulsant to treat patients with KCNT1-related epilepsy by targeting gain-of-function KCNT1 pathogenic mutant variants. However, the detailed mechanism underlying quinidine's blockade against KCNT1 (Slack) remains elusive. Here, we report a functional and physical coupling of the voltage-gated sodium channel NaV1.6 and Slack. NaV1.6 binds to and highly sensitizes Slack to quinidine blockade. Homozygous knockout of NaV1.6 reduces the sensitivity of native sodium-activated potassium currents to quinidine blockade. NaV1.6-mediated sensitization requires the involvement of NaV1.6's N- and C-termini binding to Slack's C-terminus, and is enhanced by transient sodium influx through NaV1.6. Moreover, disrupting the Slack-NaV1.6 interaction by viral expression of Slack's C-terminus can protect against SlackG269S-induced seizures in mice. These insights about a Slack-NaV1.6 complex challenge the traditional view of "Slack as an isolated target" for anti-epileptic drug discovery efforts, and can guide the development of innovative therapeutic strategies for KCNT1-related epilepsy. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.02.23.529757v1?rss=1 Authors: Thompson, C. H., Potet, F., Abramova, T. V., DeKeyser, J.-M., Ghabra, N., Vanoye, C. G., Millichap, J. J., George, A. L. Abstract: Pathogenic variants in neuronal voltage-gated sodium (NaV) channel genes including SCN2A, which encodes NaV1.2, are frequently discovered in neurodevelopmental disorders with and without epilepsy. SCN2A is also a high confidence risk gene for autism spectrum disorder (ASD) and non-syndromic intellectual disability (ID). Previous work to determine the functional consequences of SCN2A variants yielded a paradigm in which predominantly gain-of-function (GoF) variants cause epilepsy whereas loss-of-function (LoF) variants are associated with ASD and ID. However, this framework is based on a limited number of functional studies conducted under heterogenous experimental conditions whereas most disease-associated SCN2A variants have not been functionally annotated. We determined the functional properties of more than 30 SCN2A variants using automated patch clamp recording to assess the analytical validity of this approach and to examine whether a binary classification of variant dysfunction is evident in a larger cohort studied under uniform conditions. We studied 28 disease-associated variants and 4 common population variants using two distinct alternatively spliced forms of NaV1.2 that were heterologously expressed in HEK293T cells. Multiple biophysical parameters were assessed on 5,858 individual cells. We found that automated patch clamp recording provided a valid high throughput method to ascertain detailed functional properties of NaV1.2 variants with concordant findings for a subset of variants that were previously studied using manual patch clamp. Additionally, many epilepsy-associated variants in our study exhibited complex patterns of gain- and loss-of-function properties that are difficult to classify overall by a simple binary scheme. The higher throughput achievable with automated patch clamp enables study of a larger number of variants, greater standardization of recording conditions, freedom from operator bias, and enhanced experimental rigor valuable for accurate assessment of NaV channel variant dysfunction. Together, this approach will enhance our ability to discern relationships between variant channel dysfunction and neurodevelopmental disorders. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.02.20.529310v1?rss=1 Authors: Berecki, G., Bryson, A., Polster, T., Petrou, S. Abstract: SCN1A gain-of-function variants are associated with early onset developmental and epileptic encephalopathies (DEEs) that possess distinct clinical features compared to Dravet syndrome caused by SCN1A loss-of-function. However, it is unclear how SCN1A gain-of-function may predispose to cortical hyper-excitability and seizures. Here, we first report the clinical features of a patient carrying a de novo SCN1A variant (T162I) associated with neonatal-onset DEE, and then characterize the biophysical properties of T162I and three other SCN1A variants associated with neonatal-onset or early infantile DEE (I236V, P1345S, R1636Q). In voltage clamp experiments, three variants (T162I, P1345S and R1636Q) exhibited changes in activation and inactivation properties that enhanced window current, consistent with gain-of-function. Dynamic action potential clamp experiments utilising model neurons incorporating Nav1.1. channels supported a gain-of-function mechanism for all four variants. Here, the T162I, I236V, P1345S, and R1636Q variants exhibited higher peak firing rates relative to wild type and the T162I and R1636Q variants produced a hyperpolarized threshold and reduced neuronal rheobase. To explore the impact of these variants upon cortical excitability, we used a spiking network model containing an excitatory pyramidal cell (PC) and parvalbumin positive (PV) interneuron population. SCN1A gain-of-function was modeled by enhancing the excitability of PV interneurons and then incorporating three simple forms of homeostatic plasticity that restored pyramidal cell firing rates. We found that homeostatic plasticity mechanisms exerted differential impact upon network function, with changes to PV-to-PC and PC-to-PC synaptic strength predisposing to network instability. Overall, our findings support a role for SCN1A gain-of-function and inhibitory interneuron hyperexcitability in early onset DEE. We propose a mechanism through which homeostatic plasticity pathways can predispose to pathological excitatory activity and contribute to phenotypic variability in SCN1A disorders. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.02.14.528217v1?rss=1 Authors: Mao, M., Mattei, C., Rollo, B., Byars, S. G., Cuddy, C., Berecki, G., Heighway, J., Pachernegg, S., Menheniott, T., Apted, D., Jia, L., Dalby, K., Nemiroff, A., Mullen, S., Reid, C., Maljevic, S., Petrou, S. Abstract: SCN2A encodes Nav1.2, an excitatory neuron voltage-gated sodium channel and major monogenic cause of neurodevelopmental disorders, including developmental and epileptic encephalopathies (DEE) and autism. Clinical presentation and pharmocosensitivity vary with nature of SCN2A variant dysfunction with gain-of-function (GoF) cases presenting with pre- or peri-natal seizures and loss-of-function (LoF) patients typically having infantile spasms after 6 months of age. Here, we established and assessed patient induced pluripotent stem cell (iPSC) - derived neuronal models for two recurrent SCN2A DEE variants with GoF R1882Q and LoF R853Q associated with early- and late-onset DEE, respectively. Patient-derived iPSC lines were differentiated using a Neurogenin-2 overexpression yielding populations of cortical-like glutamatergic neurons. Electrophysiological and transcriptomic profiles were assessed after 2-4 weeks in culture. Increased neuronal activity at both cellular and network level was observed for R1882Q iPSC-derived neurons at three weeks of differentiation. In contrast, R853Q neurons showed only subtle changes in excitability after four weeks in vitro. In alignment with the reported efficacy in some GoF SCN2A patients, phenytoin (sodium channel blocker) reduced excitability of neurons to the control levels in R1882Q neuronal cultures. Transcriptomic alterations in neurons were detected for each variant and convergent pathways pointed at the shared mechanisms underlying SCN2A DEE. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.02.14.528229v1?rss=1 Authors: Wu, J., Quraishi, I. H., Zhang, Y., Bromwich, M., Kaczmarek, L. K. Abstract: KCNT1 encodes the sodium-activated potassium channel Slack (KCNT1, KNa1.1), an important mediator of neuronal membrane excitability. Gain-of-function (GOF) mutations in humans lead cortical network hyperexcitability and seizures, as well as very severe intellectual disability. Using a mouse model of Slack GOF-associated epilepsy, we found that both excitatory and inhibitory neurons of the cerebral cortex have increased Na+-dependent K+ (KNa) currents and voltage-dependent sodium (NaV) currents. The characteristics of the increased KNa currents were, however, different in the two cell types such that the intrinsic excitability of excitatory neurons was enhanced but that of inhibitory neurons was suppressed. We further showed that the expression of NaV channel subunits, particularly that of NaV1.6, is upregulated and that the length of the axon initial segment (AIS) and of axonal NaV immunostaining is increased in both neuron types. We found that the proximity of the AIS to the soma is shorter in excitatory neurons than in inhibitory neurons of the mutant animals, potentially contributing to the different effects on membrane excitability. Our study on the coordinate regulation of KNa currents and the expression of NaV channels may provide a new avenue for understanding and treating epilepsies and other neurological disorders. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Summary: Tarantulas have taught us some pretty cool things! Join Kiersten and a guest co-host as they talk about what tarantulas have taught us about colors and pain killers.   For my hearing impaired listeners, a complete transcript of this podcast follows the show notes on Podbean.   Show Notes:  “Tarantula venom could be used as a potent pain reliever” by Angela Betsaida B. Laguipo, BSN; https://www.news-medical.net/news/20200414/Tarantula-venom-could-be-used-as-a-potent-pain-reliver.aspx “Tarantula Venom Helps Reveal How We Sense Pain” by Ben Taub; https://www.iflscience.com/tarantula-venom-helps-reveal-how-we-sense-pain-36091 “Blue Tarantula Hair Inspires Nonfading Color Pigment” by Kacey Deamer; https://www.livescience.com/58031-tarantula-hair-inspired-nonfading-color.html   Transcript (Piano music plays) Kiersten - This is Ten Things I Like About…a ten minute, ten episode podcast about unknown or misunderstood wildlife. (Piano music stops) Kiersten - Welcome to Ten Things I Like About… I'm Kiersten, your host, and this is a podcast about misunderstood or unknown creatures in nature. Some we'll find right out side our doors and some are continents away but all are fascinating.  This podcast will focus ten, ten minute episodes on different animals and their amazing characteristics. Please join me on this extraordinary journey, you won't regret it. This episode continues tarantulas and the sixth thing I like about tarantulas is what we're learning from them that changes the way we see our world! Today I have a special guest to help me, my husband Georgiy! Thanks for helping me with this episode on biomimicry, Georgiy.   Georgiy - You're welcome. You said bio-what?    Kiersten - Biomimicry.    Georgiy - What is biomimicry?   Kiersten - Biomimicry means that a structure or process built or designed by humans was influenced by biological creatures or processes that happen naturally in the world.    Georgiy - Cool! So this episode is about what we are learning from studying tarantulas?   Kiersten - Yes! Exactly! And we're going to start off with a study done by scientists researching color in tarantulas. Georigy, do you know that tarantulas come in many different colors?   Georgiy - I do because you showed me pictures.  My favorite tarantula is the bright blue Sapphire Tarantula.   Kiersten - Ooo. Good choice! That one is amazing!   Georgiy - What did they find out researching the blue color of these tarantulas?   Kiersten - I'm so glad you asked because it's super cool! They discovered that the blue on the tarantula is a structural color. Many of the colors we see are pigments that produce color when the electrons interact with light. Our clothing and our paint are based on these kinds of pigments. The problem is that they will eventually fade and is often made with chemicals that can harm our environment.    The tarantula's blue color is a structural color, which means there are tiny nanostructures on their exoskeleton that scatter light at a specific wavelength producing the blue color we see. Now structural colors, which are produced when light interacts with nanostructures that are about the same size as a specific color's wavelength, are nothing new. We've known about structural colors for a while, but most of them are iridescent.    Georgiy - Like some bird feathers?    Kiersten - Yes! Just like certain bird's feathers. Have you ever looked at at peacock feather in the sun?    Georgiy - I have. It's very pretty.   Kiersten - What happened when you twisted that feather between your fingers?   Georgiy - It looks like it changed colors.   Kiersten - Exactly! When the light reflects off the nanostructures at different angles the light changes m  aking the color change. It's beautiful but as one of the researchers from the University of Akron in Ohio, Bor-Kai Hsiung (suhng) said in an interview with LiveScience, “It's beautiful out in nature, but not very functional when we're watching television and we move to a new seat.”    Georgiy - So how does the Sapphire Tarantula fit in?     Kiersten - Researchers took a closer look at several different species of blue tarantulas and discovered that their pigments are not iridescent. The nanostructres of their hair are covered with distinct flower-like structures that limit the iridescence.   Georgiy - Wow! What does that mean?   Kiersten - It means that we could use this structure to create more vibrant, longer lasting, and less toxic colors for use in paints, clothing, and digital screens!   Georgiy - Well, that is just cool! What other things are tarantulas teaching us?   Kiersten - The venom of Heteroscodra maculata, or the Togo Starburst Tarantula native to West Africa, is helping us understand how our bodies process pain.   Georgiy - Hmmm. Tell me more.   Kiersten - Certainly. Researchers at the University of California, San Fransisco were interested in isolating the specific pathways that indicate pain to our central nervous system. The impulses that tell our CNS that we are in pain use voltage-gated sodium channels known as Nav channels. We have so many different types of Nav channels that we don't currently know which ones actually indicate pain. So when you use a local anesthetic it blocks all the Nav channels so that patient does not feel pain. But if we could understand which channels actually transmit the pain signals we could better treat certain CNS disorders.    Georgiy - That sounds complicated.   Kiersten - It kind of is, but that's it for the hard part. Next the researchers injected the tarantulas venom into the feet of mice and then mapped the Nav channels that reacted.     Georgiy - What happened?   Kiersten - The mice got some itchy feet and the researchers discovered that the Nav1.1 channels are the ones that reacted to the proteins in the venom. We now understand that these Nav1.1 channels are the ones that react to a mechanical pain but not a thermal pain. They took this information and applied it to a disorder called irritable bowl syndrome, or IBS, in which people often describe pain in their guts. These Nav1.1 channels are found in the gut. So by using this tarantula's venom we now have a new idea of how to better treat the symptoms of IBS.   Georgiy - All of that from tarantula venom?   Kiersten - Yep! And speaking of venom, there's more.   Georgiy - More? What else have we learned from studying tarantula venom?   Kiersten - Researchers at the University of Queensland have discovered that molecules in tarantula venom could be used as pain killers for people that suffer from chronic, or long-term, pain.   Georgiy - That's interesting, but taking pain killers long term can be pretty addictive, right?   Kiersten - Yes, if you're taking an opioid pain killer. These can be extremely addictive and as anyone who listens to the news knows, it has been a big problem that many people are battling as of late. The venom of the Chinese Bird Spider, also known as the Chinese Black Earth Tiger Tarantula, was broken down into its individual molecules, then scientists replicated some of the molecules creating mini-molecules and gave them to mice. It helped reduce pain without any addictive side effects. This could be a non-addictive alternative to opioids for people with long-term pain.   Georgiy - It seems like we are learning a lot from tarantulas.   Kiersten - We really are and as long as we keep their natural habitats from disappearing who know what else we can learn!   That's it for this episode of Ten Things I Like About Tarantulas! Thanks for co-hosting with me this week, Georgiy!   Georgiy - You're welcome!    Kiersten -  I hope you all enjoyed learning about what tarantulas have taught us because it's my sixth favorite thing about these amazing arachnids.   If you're enjoying this podcast please recommend me to friends and family and take a moment to give me a rating on whatever platform your listening. It will help me reach more listeners and give the animals I talk about an even better chance at change.    Join me next week for another thing I like about tarantulas!   (Piano Music plays)  This has been an episode of Ten Things I like About with Kiersten and Company. Original music written and performed by Katherine Camp, piano extraordinaire.

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.02.04.527110v1?rss=1 Authors: Shiers, S., Funk, G., Cervantes, A., Horton, P., Dussor, G., Hennen, S., Price, T. J. Abstract: NaV1.7, a membrane-bound voltage-gated sodium channel, is preferentially expressed along primary sensory neurons, including their peripheral & central nerve endings, axons, and soma within the dorsal root ganglia and plays an integral role in amplifying membrane depolarization and pain neurotransmission. Loss- and gain-of-function mutations in the gene encoding NaV1.7, SCN9A, are associated with a complete loss of pain sensation or exacerbated pain in humans, respectively. As an enticing pain target supported by human genetic validation, many compounds have been developed to inhibit NaV1.7 but have disappointed in clinical trials. The underlying reasons are still unclear, but recent reports suggest that inhibiting NaV1.7 in central terminals of nociceptor afferents is critical for achieving pain relief by pharmacological inhibition of NaV1.7. We report for the first time that NaV1.7 mRNA is expressed in putative projection neurons (NK1R+) in the human spinal dorsal horn, predominantly in lamina 1 and 2, as well as in deep dorsal horn neurons and motor neurons in the ventral horn. NaV1.7 protein was found in the central axons of sensory neurons terminating in lamina 1-2, but also was detected in the axon initial segment of resident spinal dorsal horn neurons and in axons entering the anterior commissure. Given that projection neurons are critical for conveying nociceptive information from the dorsal horn to the brain, these data support that dorsal horn NaV1.7 expression may play an unappreciated role in pain phenotypes observed in humans with genetic SCN9A mutations, and in achieving analgesic efficacy in clinical trials. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.02.01.526678v1?rss=1 Authors: Mavashov, A., Brusel, M., Liu, J., Woytowicz, V., Bae, H., Chen, Y.-H., Dani, V. S., Cardenal, E., Spinosa, V., Aibar, J. A., Rubinstein, M. Abstract: Dravet syndrome (Dravet) is a severe congenital developmental genetic epilepsy caused by de novo mutations in the SCN1A gene. Nonsense mutations are found in ~20% of the patients, and the R613X mutation was identified in multiple patients. Here we characterized the epileptic and non-epileptic comorbidities of a novel preclinical Dravet mouse model harboring this nonsense Scn1a mutation. Heterozygous Scn1a R613X mutation on a mixed C57BL/6J:129S1/SvImJ background exhibited spontaneous seizures, susceptibility to heat-induced seizures, and premature mortality, recapitulating the core epileptic phenotypes of Dravet. In addition, these mice, available as an open-access model, demonstrated increased locomotor activity in the open-field test, mimicking some non-epileptic Dravet-associated comorbidities. Conversely, Scn1aWT/R613X mice on the pure 129S1/SvImJ background had a normal life span and were easy to breed. Homozygous Scn1aR613X/R613X mice died before P16. Our molecular analyses of hippocampal and cortical expression demonstrated that the premature stop codon induced by the R613X mutation reduced Scn1a mRNA and NaV1.1 protein levels to ~50% in heterozygous Scn1aWT/R613X mice, with marginal expression in homozygous Scn1aR613X/R613X mice. Together, we introduce a novel Dravet model carrying the R613X Scn1a nonsense mutation that can. be used to study the molecular and neuronal basis of Dravet, as well as the development of new therapies associated with SCN1A nonsense mutations in Dravet. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.01.11.523598v1?rss=1 Authors: van Hugte, E. J. H., Lewerissa, E., Wu, K. M., Parodi, G., van Voorst, T., Kogo, N., Keller, J., Schubert, D., Schelhaas, H. J., Verhoeven, J., Majoie, M., van Bokhoven, H., Nadif Kasri, N. Abstract: Dravet syndrome is a severe epileptic encephalopathy, characterized by (febrile) seizures, behavioral problems and developmental delay. 80% of Dravet syndrome patients have a mutation in SCN1A, encoding NaV1.1. Milder clinical phenotypes, such as GEFS+ (generalized epilepsy with febrile seizures plus), can also arise from SCN1A mutations. Predicting the clinical phenotypic outcome based on the type of mutation remains challenging, even when the same mutation is inherited within one family. Both this clinical and genetic heterogeneity add to the difficulties of predicting disease progression and tailored prescription of anti-seizure medication. A better understanding of the neuropathology of different SCN1A mutations, might give insight in differentiating the expected clinical phenotype and best fit treatment choice. Initially it was recognized that loss of Na+ -current in inhibitory neurons specifically resulted in disinhibition and consequently seizure generation. However, the extent to which excitatory neurons contribute to the pathophysiology is currently debated, and might depend on the patient clinical phenotype or the specific mutation in SCN1A. To examine the genotype-phenotype correlations of SCN1A mutations in relation to excitatory neurons, we investigated a panel of patient-derived excitatory neuronal networks differentiated on multi-electrode arrays. We included patients with different clinical phenotypes, harboring different mutations in SCN1A, plus a family where the same mutation leads to both GEFS+ and Dravet syndrome. We hitherto describe a previously unidentified functional excitatory neuronal network phenotype in the context of epilepsy, which corresponded to seizurogenic network prediction patterns elicited by proconvulsive compounds. We find that excitatory neuronal networks were differently affected, dependent on the type of SCN1A mutation, but not on clinical severity. Specifically, pore domain mutations could be distinguished from voltage sensing domain mutations. Furthermore, all patients showed aggravated neuronal network responses upon febrile temperatures. While the basal neuronal network phenotypes could not be distinguished based on patient clinical severity, retrospective drug screening revealed that anti-seizure medication only affected GEFS+ patient-, but not Dravet patient-derived neuronal networks in a patient specific and clinically relevant manner. In conclusion, our results indicate a mutation-specific excitatory neuronal network phenotype, which recapitulates the foremost clinically relevant features, providing future opportunities for precision therapies. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Editor's note: The North American Pain School (NAPS) took place 19-24 June 2022, in Montebello, Québec City, Canada. NAPS – an educational initiative of the International Association for the Study of Pain (IASP) and Analgesic, Anesthetic, and Addiction Clinical Trial Translations, Innovations, Opportunities, and Networks (ACTTION), and presented by the Quebec Pain Research Network (QPRN) – brings together leading experts in pain research and management to provide trainees with scientific education, professional development, and networking experiences. This year's theme was, “Controversies in Pain Research.” Five of the trainees were also selected to serve as PRF-NAPS Correspondents, who provided firsthand reporting from the event, including interviews with NAPS' Visiting Faculty members and Patient Partners, summaries of scientific sessions, and coverage on social media.  In the podcast below, PRF-NAPS Correspondent Joseph Lesnak, a PhD candidate at the University of Iowa, US, spoke with NAPS Visiting Faculty member Rajesh Khanna. Rajesh is a Professor of Molecular Pathobiology and the Director of New York University's Pain Research Center in the US. His research focuses on the functions of voltage-gated ion channels and the discovery of novel biologics and small molecules targeting pain and neurodegenerative diseases (see related PRF news article). Joseph and Rajesh discussed targeting Nav1.7 for pain relief, the challenge of moving a pharmacologic through the regulatory process, and a serendipitous finding that arose during the COVID-19 pandemic. This podcast is also available on Apple Podcasts here and Spotify here.

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.10.04.510784v1?rss=1 Authors: Xie, Y.-F., Yang, J., Ratte, S., Prescott, S. A. Abstract: Nociceptive sensory neurons convey pain signals to the CNS. Nociceptor hyperexcitability amplifies those signals, causing pain hypersensitivity. Reducing nociceptor excitability should mitigate that hypersensitivity, consistent with the effects of loss-of-function mutations in voltage-gated sodium (NaV) channels like NaV1.7. Yet efforts to phenocopy such mutations pharmacologically have failed in clinical trials. This failure may stem from the degenerate nature of nociceptor excitability. Here, we show that nociceptors can achieve equivalent excitability using different combinations of NaV1.3, NaV1.7, and NaV1.8. If NaV1.3 and/or NaV1.8 levels are high enough to maintain nociceptor excitability, selectively blocking NaV1.7 (e.g. with PF-05089771) becomes inconsequential. We demonstrate shifts in drug efficacy by comparing neurons tested after different numbers of days in vitro (DIV): Nociceptor excitability relies on NaV1.8 at DIV0 but that responsibility shifts to NaV1.7 and NaV1.3 on DIV4-7. A similar shift in NaV-dependence occurs in vivo, following inflammation, and impacts the ability of PF-05089771 to modulate pain sensitivity. These results demonstrate that nociceptors are surprisingly flexible in using different NaV isoforms to regulate excitability. This flexibility poses a serious problem for subtype-selective drugs whose efficacy hinges on such vagaries. Degeneracy at the cellular level must be considered when choosing drug targets at the molecular level. Copy rights belong to original authors. Visit the link for more info Podcast created by PaperPlayer

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.09.15.508178v1?rss=1 Authors: Mitchell, M., Cook, L. F., Shiers, S., Tavares-Ferreira, D., Akopian, A. N., Dussor, G., Price, T. J. Abstract: Fragile X Mental Retardation Protein (FMRP) regulates activity-dependent RNA localization and local translation to modulate synaptic plasticity throughout the CNS. Mutations in the FMR1 gene that hinder or ablate FMRP function cause Fragile X Syndrome (FXS), a disorder associated with sensory processing dysfunction. FXS pre-mutations are associated with increased FMRP expression and neurological impairments including sex dimorphic presentations of chronic pain. In mice, FMRP ablation causes dysregulated DRG neuron excitability and synaptic vesicle exocytosis, spinal circuit activity, and decreased translation-dependent nociceptive sensitization. Activity-dependent, local translation is a key mechanism for enhancing primary nociceptor excitability which promotes pain in animals and humans. These works indicate that FMRP likely regulates nociception and pain at the level of the primary nociceptor or spinal cord. Therefore, we sought to better understand FMRP expression in the human dorsal root ganglion (DRG) and spinal cord using immunostaining in organ donor tissues. We find that FMRP is highly expressed in DRG and spinal neuron subsets with substantia gelatinosa exhibiting the most abundant immunoreactivity in spinal synaptic fields. Here, it is expressed in nociceptor axons. FMRP puncta colocalized with Nav1.7 and TRPV1 receptor signals suggesting a pool of axoplasmic FMRP localizes to plasma membrane-associated loci in these branches. Interestingly, FMRP puncta exhibited notable colocalization with calcitonin gene-related peptide (CGRP) immunoreactivity selectively in female spinal cord. Our results support a regulatory role for FMRP in human nociceptor axons of the dorsal horn and implicate it in the sex dimorphic actions of CGRP signaling in nociceptive sensitization and chronic pain. Copy rights belong to original authors. Visit the link for more info Podcast created by PaperPlayer

Barry S. Ticho, MD, PhD, Chief Medical Officer at Stoke Therapeutics, gives a detailed overview of Dravet syndrome.As Dr. Ticho explains, Dravet syndrome is a rare neurological condition that usually appears during the first year of life as frequent febrile seizures. As the condition progresses, other types of seizures typically occur, including myoclonus and status epilepticus. Moderate to severe cognitive impairment is also common. Most cases of Dravet syndrome occur due to a mutation of the SCN1A gene. The SCN1A gene codes for the protein NaV1.1. With only one functional SCN1A gene, people with Dravet syndrome produce less of the NaV1.1 protein. NaV1.1 is an important protein for the nerves in the brain to work properly. Low levels of NaV1.1 in the brain can lead to seizures and other symptoms of Dravet syndrome.

In this week's episode of the Spine & Nerve podcast Drs. Nicolas Karvelas and Brian Joves take a look back at basic physiology to try to look into the future. An area of research that has really piqued the interest of Dr. Karvelas in recent years has been the discussion/possibility of selective voltage gated sodium channel (NaV) modulators. NaV are transmembrane proteins that are an integral part of the initiation and propagation of action potentials in neurons and other electrically excitable cells. We have seen that small changes in NaV function are biologically relevant because there are several human diseases that are the result of mutations in these channels. This has led to research into selective NaV modulators as a potential target as we continue to search for treatment options with significant analgesic potential and decreased risk of side effects / adverse effects. The medical / research community continues to work to optimize medication options to treat painful disease processes. From an analgesic medication perspective, although there are a variety of different medications available including: topical medications, acetaminophen, non-steroidal anti-inflammatory drugs, gabapentin, pregabalin, serotonin norepinephrine reuptake inhibitors, tricyclic anti-depressant medications, non-selective sodium channel blockers, NMDA receptor modulations (Memantine, Ketamine), alpha-2 agonists, glial cell modulators (Low Dose Naltrexone), Buprenorphine, full mu opioids. These Medications are not without their limitations for multiple reasons including but not limited to side effects, risks, and contraindications depending on patient's age and/or comorbidities. To the best of our knowledge there are 10 different NaV subtypes; and specifically NaV 1.3, 1.7, 1.8, 1.9 have been demonstrated to play a critical role in pain signaling. NaV 1.8 is a sensory neuron specific channel with preferential expression in the dorsal root ganglion and trigeminal ganglion neurons, and it is highly expressed on nociceptors. Similar to the other NaV subtypes that have been identified to play essential roles in pain, mutations in NaV 1.8 have been demonstrated to lead to significant alterations in the nervous system / pain pathways; specifically gain of function NaV 1.8 mutations clinically manifest as painful small fiber peripheral polyneuropathy. NaV 1.8 modulation is being aggressively researched with the goal of positive impact on painful diseases. VX-150 is a oral pro-drug that is a highly selective inhibitor of NaV1.8, and a recent study by Dr. Hijma and colleagues was published evaluating the analgesic potential and safety of VX-150. Listen as the doctors discuss this exciting and important area of research. The discussion includes a detailed review of the fore-mentioned recent research article. This podcast is for information and educational purposes only, it is not meant to be medical or career advice. If anything discussed may pertain to you, please seek council with your healthcare provider. The views expressed are those of the individuals expressing them, they may not represent the views of Spine & Nerve. References: 1. Hijma HJ, Siebenga PS, de Kam ML, Groeneveld GJ. A Phase 1, Randomized, Double-Blind, Placebo-Controlled, Crossover Study to Evaluate the Pharmacodynamic Effects of VX-150, a Highly Selective NaV1.8 Inhibitor, in Healthy Male Adults. Pain Med. 2021 Aug 6;22(8):1814-1826.

This week's episode features author Kieran Docherty and Associate Editor Torbjørn Omland as they discuss the article "The Effect of Neprilysin Inhibition on Left Ventricular Remodeling in Patients with Asymptomatic Left Ventricular Systolic Dysfunction Late After Myocardial Infarction." Dr. Carolyn Lam: Welcome to Circulation on the Run, your weekly podcast, summary, and backstage pass to the journal and its editors. We're your co-hosts: I'm Dr. Carolyn Lam, associate editor from the National Heart Center and Duke National University of Singapore. Dr. Greg Hundley: And I'm Dr. Greg Hundley, associate, editor, director of the Pauley Heart Center at VCU Health in Richmond, Virginia. Well, Carolyn, we've got an exciting feature this week involving Neprilysin license inhibition and left ventricular remodeling in patients with asymptomatic left ventricular systolic function after they've sustained myocardial infarction. But before we get to that feature discussion, how about we grab a cup of coffee and jump in on some of the other articles in the issue? Would you like to go first? Dr. Carolyn Lam: I'd love to, and I want to talk about transcatheter aortic valve replacement, or TAVR, that we all know is really transforming our management of aortic stenosis. Despite rapid improvements, however, serious complications remain relatively common and are not well-described by single outcome measures. So the purpose of this paper was to determine if there was site-level variation in TAVR outcomes in the United States using a novel 30-day composite measure. And this is from Dr. Desai and colleagues from Hospital of University of Pennsylvania. So they performed a retrospective cohort study using data from the STS/ACC TVT registry to develop a novel-ranked competent performance measure that incorporates mortality and serious complications. Based on the associations with one-year risk adjusted mortality and health status, they identified for peri-procedural complications to include in the composite risk model, in addition to mortality. And ranked empirically, according to severity, these were: stroke, major life-threatening or disabling bleeding, stage three acute kidney injury, and moderate or severe perivalvular regurgitation. Dr. Carolyn Lam: Now, based on these ranked outcomes, they found that there was significant site-level variation in quality of care in TAVR in the United States. Overall, better-than-expected site performance was observed in 8% of sites, whereas performance as-expected was observed in 80%, and worse-than-expected performance was observed in 11% of sites. Dr. Greg Hundley: Carolyn, really interesting comprehensive data. So how do we put this all together? And what's the take-home message for us, clinically? Dr. Carolyn Lam: Well, there are substantial variations in the quality of TAVR care received in the United States, and 11% of sites were identified as providing care below the average level of performance. Further study is necessary to determine the structural, process-related, and technical factors associated with high- and low-performing sites. And all this is discussed in a beautifully, beautiful accompanying editorial by Drs. Dharam Kumbhani and Eric Peterson.   Dr. Greg Hundley: Oh, fantastic. You know, Carolyn, those editorials are so helpful in helping us put these new data in perspective. Well, my next paper comes to us from the world of preclinical science, and it's from Professor Vincent Christoffels from Amsterdam in UMC. So genetic variants of SCN10A, encoding the neural voltage-gated sodium channel NaV1.8 are strongly associated with atrial fibrillation, Brugada syndrome, cardiac conduction velocities, and heart rate. And these investigators studied the cardiac expression of SCN10A and the function of a variant-sensitive intronic enhancer previously linked to the regulation of SCN5A, and coding the major essential cardiac sodium channel NaV1.5. Dr. Carolyn Lam: Wow, great. So what did they find, Greg? Sounds like a first-of-its-kind study. Dr. Greg Hundley: Right, Carolyn. So genetic variants in and around SCN10A modulate enhancer function and expression of the cardiac-specific NCN10A short transcript, and the authors propose that non-encoding genetic variation modulates transcriptional regulation of a functional C-terminal portion of NaV .8 and cardiomyocytes that impacts NaV1.5 function, cardiac conduction velocities, and arrhythmia susceptibility. Dr. Carolyn Lam: Wow, that was a lot. So what are the implications, Greg? Could you simplify it for us? Dr. Greg Hundley: Yes. Right, Carolyn. So three things. First, the authors uncovered a novel alternative mechanism for how the SCN10A locus regulates cardiac conduction. Second, their data implicate that genetic variation-sensitive regulation of expression of NCN10A short modulates conductivity of the heart, and can predispose to arrhythmia in the human population. And then finally, Carolyn, in deciphering the underlying mechanism of the increased NaV1.5 mediated current density by NaV1.8 short, the authors believe their findings could ultimately lead to the development of novel therapeutic strategies for particular conduction disorder. Dr. Carolyn Lam: Thanks, Greg. Well, this next paper is really interesting. It's the first validation of the enhanced potency of human-induced pluripotent stem cells-derived cardiomyocytes over-expressing Cyclin D2, or CCND2, under the control of myosin heavy chain promoter, and differentiated into cardiomyocytes. Now, that was a mouthful, but so interesting, because Dr. Zhang and colleagues from University of Alabama in Birmingham used infarcted pig hearts, and transplanted these amazing cardiomyocytes, and found that they were associated with proliferation of recipient heart cardiomyocytes, epithelial cells, and smooth muscle cells, all, at least partly, by paracrine activity. Dr. Greg Hundley: Well, Carolyn. Really an involved clinical design. So, what are the clinical implications of all this research?   Dr. Carolyn Lam: Well, first, I think the paper validated a novel therapeutic strategy aimed at upregulating proliferation of recipient cardiac cells using human-induced pluripotent stem cells-derived cell or cell products. Furthermore, targeting the myocyte cell cycle regulators, such as Cyclin D2, holds a strategic potential for re-muscularization of an infarcted region. Dr. Greg Hundley: Very good, Carolyn. Well, how about we see what else is in this issue? So I'll start first. There's a Research Letter by Professor Marston, entitled 'Combining High-Sensitivity Troponin with the American Heart Association/American College of Cardiology Cholesterol Guidelines to Guide of Avelumab Therapy'. Next, there's an ECG challenge from Dr. Feliciano, entitled 'An Ominous EKG'. And then finally, there's a very nice exchange of letters from Drs. Lang and Sattar regarding a prior publication: volume status is the key in heart failure. Dr. Carolyn Lam: And finally, a very important perspective piece by Dr. Catapano on omega-3 for cardiovascular disease: where do we stand after reduce it in strength? Wow, that was great, Greg. But let's move on now to our feature discussion. Dr. Greg Hundley: You bet. Dr. Greg Hundley: Well, listeners, we are on to our feature discussion today, on this July 20 issue. And we're very excited to have with us Dr. Kieran Docherty from University of Glasgow in Glasgow, Scotland, and our own associate editor, Dr. Torbjørn Omland from University of Oslo in Oslo, Norway. Welcome, gentlemen. And Kieran, let's start with you. Could you describe some of the background related to your study, and what was the hypothesis that you wanted to address? Dr. Kieran Docherty: Well, firstly, thank you very much for the invitation to discuss our trial today on the podcast. The background of our trial was that we are all aware that the development of left ventricular systolic dysfunction following acute myocardial infarction places patients at a subsequent increased risk of the development of heart failure, and the process of progressive dilatation of the left ventricle and a reduction in stroke volume, known as adverse left ventricular remodeling, is the process which underlies this elevated risk of heart failure. And many of the treatments that have been shown to be beneficial following myocardial infarction, such as [inaudible 00:09:24] , and angiotensin receptor blockers and beta blockers, the benefit of these medications, in part, is due to their ability to attenuate this process of adverse remodeling. Now, our hypothesis was that it would be possible to further attenuate, prevent, or delay the process of adverse remodeling in patients at risk of heart failure following myocardial infarction, by the addition of a Neprilysin inhibitor to current standard of care. Dr. Kieran Docherty: Now, as we all know, a Neprilysin inhibitor in the form of sacubitril valsartan when combined with an angiotensin receptor blocker, has been shown to improve outcomes in patients with symptomatic heart failure, with reduced ejection fraction in the PARADIGM-HF trial, and Neprilysin inhibitors increase endogenous levels of natriuretic peptides, amongst a range of other substrates for Neprilysin, including adrenomedullan, GLP-1, and bradykinin. And our hypothesis was that adding in a Neprilysin inhibitor, thereby increasing endogenous levels of these peptides with potentially beneficial effects, such as reducing fibrosis, reducing hypertrophy, [inaudible 00:10:34] and diuresis, may have an additive beneficial effect on left ventricular remodeling in these high-risk patients with left ventricular systolic dysfunction following myocardial infarction. Dr. Greg Hundley: Very nice hypothesis. So, how did you set up, Kieran, your study design, and what study population, how many patients and whatnot, did you include in your study? Dr. Kieran Docherty: Well, the first consideration when designing the study was broadly, what group of patients should we involve? And the main limitation was the indication for the use of sacubitril valsartan in patients with symptomatic heart failure, so we did not feel that we could include these patients. Therefore, our study population included patients who had asymptomatic left ventricular systolic dysfunction following previous myocardial infarction. And specifically, we wanted patients who were at least three months following my cardiac infarction. And the reason for that was to try and exclude patients who had transient systolic dysfunction or left ventricular stunning. And we performed a screening transthoracic echo at this time point. And if patients had an ejection fraction of 40% or less on echo, and if they were tolerant of a minimum dose of an ACE inhibitor, 2.5 milligrams of ramipril BD or equivalent, and they were taking a beta blocker, unless contraindicated or not tolerated, then they were suitable for randomization. Dr. Greg Hundley: Very good. And what did you find? Dr. Kieran Docherty: So we find that in comparison with the ARB Valsartan, sacubitril valsartan did not have any beneficial effects on cardiac MRI-based measures of left ventricular remodeling. And the primary end point of our study was left ventricular end systolic volume index. There was also no improvements in biomarkers of myocardial stress, i.e. NT-proBNP, or my cardio injury, i.e. high sensitivity to Troponin I. Dr. Greg Hundley: Very nice. And any pertinent issues relevant to, perhaps, some subgroups, regardless of age or perhaps gender? Dr. Kieran Docherty: So in a post-hoc analysis, we performed an analysis of the primary endpoint in patients who were below or at or above the median NT-proBNP level, which is 238 p-grams per mil. And we found, very interestingly, the suggestion of a benefit, in terms of left ventricular remodeling with a reduction and systolic volume index in patients who had higher levels of NT-proBNP compared to those who had lower levels. Dr. Greg Hundley: Very good. Well, listeners, let's turn now to our associate editor, Dr. Torbjørn Omland, who... Torbjørn, you see many papers come across your desk. What attracted you to this manuscript? And then how do you put the results of this study in the context with other studies that have been published, particularly recently, in patients with heart failure that have received sacubitril valsartan? Dr. Torbjørn Omland: So, Greg, there were many aspects of this trial that made it very attractive for circulation. I think the hypothesis was very interesting, and also it is a very well-conducted study using the reference methods for assessing left ventricular function, using that for assessing the primary endpoint. And they also have a broad array of secondary end points that also sort of provide insight in potential pathways or mechanisms that can explain the effect sacubitril Valsartan. So, that's also a very sort of hot topic within the cardiology research currently, and we know that the ACC, actually a much larger study, PARADISE-MI, was presented. And we knew that this study was also very interesting, because we knew when we received this manuscript, that another, bigger trial that's sort of related would be presented at the ACC at the late-breaking clinical trial sessions there the PARADISE-MI study. But this sort of provided insight that nicely complimented the results of that study. Dr. Torbjørn Omland: And I think as Kieran alluded to, we already have the very impressive results from PARADIGM-HF is showing a very substantial benefit in patients with chronic heart failure and reduced ejection fraction. And then we have sort of the borderline results from the Paragon trial, in those with preserved ejection fraction, where it actually was a gradient from those with mildly elevated, where there seemed to be a beneficial effect to those with more normal EF, where there was no effect. So, this study sort of provided new information, very relevant to the whole field, I think. Dr. Greg Hundley: Very nice. Well, gentlemen, I want to turn back to you and ask each of you, first Kieran, and then Torbjørn. Kieran, what do you think is the next study that needs to be performed in, really, this sphere of research? Dr. Kieran Docherty: As Torbjørn has already alluded to, PARADISE-MI kind of... It fills the gap across the spectrum of heart failure. So in patients who are at high risk of heart failure immediately following myocardial infarction, that that group of patients were studied in PARADISE-MI. And there is an echocardiographic sub-study of PARADISE-MI, which we await the results for. And I think that will be very interesting, because our patient population was distinct from the group studied in PARADISE-MI, namely the fact that the median time from MI was 3.6 years. So, these patients were not in the throes of the neural humoral activation at the time of acute myocardial infarction and prior to the development of established my cardio scar and fibrosis. And so, it may be that the addition of a Neprilysin inhibitor to patients immediately following myocardial infarction may have some benefits, in terms of attenuating or preventing ventricular dilatation reduction and injection fraction that is observed. So I think we await the echocardiographic results of PARADISE-MI with great interest. Dr. Greg Hundley: Very good. And Torbjørn , do you have anything to add? Dr. Kieran Docherty: Yes. I found observations that actually, in terminal proBNP measurements, could potentially identify a higher-risk group that actually could benefit from the intervention. It was very interesting. So I think we always speak about precision medicine and cardiology, and I think this is sort of one avenue that we should pursue and see whether we use biomarkers like NT-proBNP to identify those patients who will benefit most from interventions like sacubitril Valsartan Dr. Greg Hundley: Excellent. Well, listeners, we've heard a really interesting discussion today. Another study investigating Neprilysin inhibition in combination with angiotensin receptor blockers, and basically highlighting that in patients with asymptomatic left ventricular dysfunction following several years after myocardial infarction, that treatment with sacubitril Valsartan did not have a significant reverse remodeling effect just compared with valsartan alone. Well, on behalf of Carolyn and myself, we want to thank Dr. Kieran Docherty and his submission here to circulation, and also our own associate editor, Dr. Torbjørn Omland. Dr. Greg Hundley: And for all of you, we wish you a great week, and we hope to catch you next week on The Run. Dr. Greg Hundley: This program is copyright of the American Heart Association, 2021. The opinions expressed by speakers in this podcast are their own, and not necessarily those of the editors or of the American Heart Association. For more visit ahajournals.org.  

In this week’s podcast, articles “The Cardiac Late Sodium Channel Current is a Molecular Target for the Sodium-Glucose Co-Transporter 2 Inhibitor Empagliflozin” by Light et al (www.ahajournals.org/doi/10.1161/CIRCULATIONAHA.121.053350) and “Metabolic effects of empagliflozin in heart failure: A randomized, double-blind, and placebo-controlled trial (Empire HF Metabolic) by Jensen et al (www.ahajournals.org/doi/10.1161/CIRCULATIONAHA.120.053463) are discussed. Dr. Carolyn Lam: Welcome to Circulation on the Run, your weekly podcast, summary and backstage pass to the journal and its editors. We're your co-hosts, I'm Dr. Carolyn Lam, associate editor from the National Heart Center and Duke National University of Singapore. Dr. Greg Hundley: And I'm Dr. Greg Hundley, associate editor, director of the Pauley Heart Center at VCU Health in Richmond, Virginia, Dr. Carolyn Lam: Greg, it's double feature day. And guess what? Both papers that we're going to talk about are regarding the SGLT2 inhibitors, and really look at the mechanism of action of these amazing compounds, from both a pre-clinical and clinic point of view. That's all I want to say, because we've got to tune in, a very interesting discussion coming right up. Dr. Carolyn Lam: But first I'd like to ask you a question. What do you think is the association between health-related quality of life and mortality in heart failure around the world? Dr. Greg Hundley: Well, Carolyn, I would think that, actually, they might be linked. Dr. Carolyn Lam: That's a really clever answer. Thanks Greg. Well, the authors are actually going to tell you with this next paper. It's from Dr. Salim Yusuf from Population Health Research Institute and McMaster University in Hamilton, Canada, and colleagues, who looked at the global congestive heart failure, or GCHF study, which is the largest study that has systematically examined health-related quality of life, measured by the Kansas City Cardiomyopathy Questionnaire, which is the largest study that has systematically examined health-related quality of life and its association with outcomes in heart failure, across eight major geographic regions, spanning five continents. Dr. Greg Hundley: Wow, Carolyn. So what did they find here? Dr. Carolyn Lam: Health-related quality of life, as measured by the Kansas City Cardiomyopathy Questionnaire, or KCCQ, really differs considerably between geographic regions, with markedly lower quality of life related to heart failure in Africa compared to elsewhere. Health-related quality of life was also a strong predictor of death and heart failure hospitalization in all regions, irrespective of symptoms class, and with both preserved and reduced ejection fraction. Dr. Carolyn Lam: Indeed, this paper really highlighted a great need to address disparities that impact health-related quality of life in patients with heart failure in different regions of the world. Dr. Greg Hundley: Fantastic, Carolyn. Well, I have two studies to discuss, Carolyn, and they're kind of similar, so we're going to do them back to back. The first study reports the results of the Sort Out X Trial, a large scale randomized multi-center, single-blind, two-arm, non-inferiority trial, with registry based follow-up designed to evaluate the Dual Therapy Sirolimus-Eluting, and CD34 positive antibody coated combo stent or DTS versus the Sirolimus-Eluting Orsiro Stent or SES. Dr. Greg Hundley: And the study comes to us from Dr. Lars Jakobson, from Arhus University Hospital. The primary endpoint target lesion failure, or TLF was a composite of cardiac death, myocardial infarction, or target lesion revascularization within 12 months, all analyzed using intention to treat. Dr. Carolyn Lam: All right, Greg. So the DTS compared to the SES, what did they find? Dr. Greg Hundley: Thanks, Carolyn. So the DTS did not confirm non-inferiority to the SES stent for target lesion failure at 12 months. The SES was superior to the DTS, mainly because the DTS was associated with an increased risk of target lesion revascularization. However, rates of death, cardiac death, and myocardial infarction at 12 months did not differ significantly between the two stent groups. Dr. Greg Hundley: Now Carolyn, in this same issue, we have another study evaluating endothelial function and implantation of intercoronary stents. And this second study comes to us from Professor Alexandra Lansky, from the Yale University School of Medicine and Yale Cardiovascular Research Group. And Carolyn, the study evaluated whether implantation of an intercoronary stent that facilitated endothelialization after the four to six weeks smooth muscle anti-proliferative effects post-stent implantation would be non-inferior to traditional drug-eluting stents. Dr. Carolyn Lam: Okay, another interesting study. And so, how did they do that? What did they find? Dr. Greg Hundley: Yeah, so Carolyn, a total of 1,629 patients were randomly assigned in a two to one fashion to the supreme DES stent, so 1,086 patients, or the DPDES stent, which was 543 patients. And there were no significant differences in rates of device success, clinically driven, target lesion revascularization, or stent thrombosis at 12 months. Dr. Greg Hundley: And the safety composite of cardiovascular death and target vessel revascularization or myocardial infarction was 3.5 versus 4.6% with the supreme DES stent compared to the DPDES stent. But target revascularization for this new stent was two and a half fold higher. Dr. Greg Hundley: So Carolyn looking at these two papers, what have we learned? So first, the Jakobsen, et al, tested whether the stainless steel COMBO Sirolimus-Eluting Stent coated luminally with CD34 positive antibody could theoretically capture endothelial progenitor cells and regrow endothelium. Dr. Greg Hundley: And the investigators observed that this stent had higher, not lower or equivalent, target lesion revascularization relative to the current generation Cobalt-Chrome Stent that only eluted sirolimus. Dr. Greg Hundley: In the second study, Lansky and associates examined an approach which was touted as enhancing endothelial recovery, where the early erosion of material and release of drug was thought to allow earlier endothelial recovery enhancing vascular response. Non-inferiority of the rapid release was demonstrated, but rather than hints of superiority, there were signs of inferiority. Hereto, target lesion revascularization was problematic and was two and a half fold higher. Dr. Greg Hundley: And so, Carolyn, there's a wonderful editorial from Professor Elazer Edelman from the Massachusetts Institute of Technology entitled, “Karnovsky's Dictum that Endothelium is Good Looking and Smart,” where Dr. Edelman emphasizes that while some endothelial cells may have been present after deployment of these devices, perhaps a fully constituted functioning endothelium may not have been achieved. Dr. Greg Hundley: And as we know, it is a fully functioning endothelium with nitric oxide release, buried platelet adhesion that is most protective. It is a really provocative read that reflects on previous thoughts from Morris Karnovsky, who suggests preservation of endothelial function is optimized by minimizing injury to it. And so, Carolyn, these combined articles really highlight the current state of new developments within interventional cardiology to thwart re-stenosis and highly recommend them to our readers. Dr. Carolyn Lam: Wow, thank you, Greg. That was amazing. But you know what, so's this next paper, because it really provides novel insights into that enigma of the role that the epicardium plays in the pathogenesis of arrhythmogenic cardiomyopathy. Dr. Carolyn Lam: Now, to delineate the contributions of the epicardium to the pathogenesis of arrhythmogenic cardiomyopathy, doctors Marian from University of Texas Health Science Center at Houston, Texas and colleagues performed a series of elegant mouse experiments using conditional deletion of the gene encoding desmoplakin in the epicardial cells of mice. Mutations in genes and coding desmosome proteins, including desmoplakin are known to be major causes of arrhythmogenic cardiomyopathy. Dr. Greg Hundley: Wow, Carolyn, very interesting. So what did they find here? Dr. Carolyn Lam: Epicardial derived cardiac fibroblasts and epithelial cells expressed paracrine factors, including TGF-β1 and fibroblasts growth factors, which mediated epithelial mesenchymal transition and contributed to the pathogenesis of myocardial fibrosis, apoptosis, arrhythmias, and cardiac dysfunction in a mouse model of arrhythmogenic cardiomyopathy. These findings really uncover contributions of the epicardial derived cells to the pathogenesis of arrhythmogenic cardiomyopathy. Dr. Carolyn Lam: Greg, there's a whole lot of other interesting stuff in today's series, as well. There's an exchange of letters among doctors Mehmood, doctors Moayedi and Dr. Birks regarding the article “Prospective Multicenter Study of Myocardial Recovery Using Left Ventricular Assist Device.” There's an ECG challenge by Dr. Ezekowitz on a silent arrhythmia. How would you treat this patient? Go quiz yourself. Dr. Carolyn Lam: There is an AHA Update by Dr. Churchwell on how federal policy changes can advance the AHAs mission to achieve health equity. And finally, a Perspective by Dr. Talbert on rheumatic fever and the American Heart Association, The Nearly 100 hundred-Year War. Well, that wraps it up for the summaries. Let's go to the double feature, shall we? Dr. Greg Hundley: You bet. Dr. Carolyn Lam: Wow, today's feature discussion is really all about SGLT2 inhibitors, and that question that we're still asking, how do they work? And today, we are discussing two papers, very interestingly, looking at it from different aspects, one from a preclinical lens, finding a very novel target for SGLT2 inhibitors, and the other from a clinical lens, and really looking at the metabolic effects of the SGLT2 inhibitors in a way you've not seen before. Dr. Carolyn Lam: I'm very pleased to have with us the authors of these very exciting papers. We have Dr. Jesper Jensen from Herlev and Gentofte University Hospital in Denmark. We have Dr. Peter Light from University of Alberta, in Canada, and we have our associate editors, Dr. Thomas Eschenhagen from University Medical Center, Hamburg, and Dr. Justin Ezekowitz from University Alberta. Dr. Carolyn Lam: So, welcome gentlemen, thank you so much for joining us today. I suggest, let's start from the mice before we go to the men, and Peter, if you don't mind by starting us in, please tell us about this novel target you found, why you looked at it, how you found it, what it means. Dr. Peter Light: Hi, Carolyn, yeah, happy to discuss that. So, we all know that through numerous clinical trials, there's a very unexpected and exciting cardioprotective effect against heart failure with the SGLT2 inhibitors. And we decided to investigate some of the molecular mechanisms, which may underlie that protection. And in looking at the literature previously, and from my own lab's work, we're very interested in control of electrical excitability and ionic homeostasis in cells. Dr. Peter Light: So we investigated a known target or a known iron channel, which is involved in the etiology of heart failure as well as cardiac arrhythmias. And that would be the cardiac sodium channel. So, we investigated the effects specifically on a component of the cardiac sodium channel called the late sodium current, which is only induced in disease states, and they could be that during heart failure or ischemia, or can actually be in congenital conditions such as Long QT Syndrome Three, which involves certain mutations in this sodium channel. Dr. Peter Light: So we basically investigate the effects of empagliflozin, dapagliflozin and canagliflozin, in several different models of a sodium channel dysfunction, including mice with heart failure. And really what we've found is that this class of drug, and this is a class effect, it's not specific to just one of these SGLT2 inhibitors, what we found, they are very good inhibitors of this late current of the sodium channel. And in fact, they don't even affect the peak current at all. Dr. Peter Light: And when we did this and we analyzed the data, we found the IC 50s were in the low micromolar or even sub micromolar range for these drugs, which is exciting. And we extended those studies into cardiac myocytes and looked at calcium handling in those cardiac myocytes and saw that we get a very nice reduction in abnormal calcium handling in cardiac myocytes. Dr. Peter Light: We also used in silico molecular docking of these drugs to the cryo-EM structure of the NaV1.5, which is the cardiac sodium channel and identified that these drugs bind to a known region of that channel, which also binds the local anesthetics or anti-arrhythmic drug, Lidocaine, as well as the anti-anginal drug, Ranolazine. Dr. Peter Light: And finally, we showed that these drugs also reduce inflammation through the NLRP3 inflammasome in an isolated beating heart model. So collectively, we provide evidence that the late component of the sodium channel is a really important, or maybe a really important target for the molecular actions of this drug, and may underlie those observations received from the clinical trials relating both to heart failure, as well as sudden cardiac death. Dr. Carolyn Lam: Thomas, could you put this in context for us? Dr. Thomas Eschenhagen: Thanks, Carolyn. I mean, we immediately liked the story because as you said, and Peter as well, these drugs have amazing effects and every clinical paper and indeed some new ones, but it's really unclear how they do that. And what is, besides the established target, the SGL2 in the kidney, what could be the reason for all of this or some of this?   Dr. Thomas Eschenhagen: And then, of course, other examples proposed, like the sodium hydrogen exchanger, but this story didn't go so far. So we saw now this data from Peter showing that, and this is, of course, for a pharmacologist, just like me, very important, it's very potent binding. It's not a binding which happens in a millimolar or high micromolar, but as Peter said in low micromolar range. So that makes it a very realistic effect, for example, much more potent than ranolazine. Dr. Thomas Eschenhagen: And, of course, now the question is, to which extent could this, now I would say, establish the effect on the late sodium current, explain some of the findings which came out of the clinical studies, and actually, a question I would have to Peter, now that I think most of you know, the late sodium current is a reason for the increased sodium for LQT3 syndrome, very rare. Dr. Thomas Eschenhagen: But, of course it would be tempting to say, okay, maybe that would be a very good drug, particularly for people with LQT3. Did you think about that, Peter? Is it something on your list, mexiletine has been tried. Dr. Peter Light: Yeah, so I think that it's a certainly intriguing possibility. In fact, in our study, we did test out several different Long QT3 mutations and saw a reduction in the late component as also sodium channel. It's tempting to speculate that, indeed, these could actually be a rather effective anti-arrhythmic drug in patients with these LQT3 mutations or specific ones. I would love to be able to test that in at least some of the genetic mouse models of Long QT3 and to see whether this concept holds water or not. Dr. Carolyn Lam: Wow, this is incredible. SGLT2 inhibitors from anti-diabetic to now anti failure, and now anti-arrhythmic drugs? That's just amazing. Thank you, Peter. We should move on to this next paper, and this one all the way on the other spectrum, a clinical paper called Empire Heart Failure, Empire Heart Failure Metabolic, actually. Jesper, could you tell us about your trial and what you found? Dr. Jesper Jensen: Sure, thanks for the invitation to take part in the podcast, first of all. I'll tell you a little bit, we designed this study to try to get behind mechanisms, so the clinical benefits of the SGLT2 inhibitors in order to try to make a clinical outcome trial. But as you know, the DAPA-HF and the EMPEROR-Reduced were competed very fast, demonstrating the clinical benefits in HFrEF patients. Dr. Jesper Jensen: So, the data of our study provides some detailed mechanistic insights to these findings. And from the literature, we know that SGLT2 inhibitors improve glucose metabolism in patients with diabetes, and these changes might not be surprising in the diabetes population, but moreover, alterations in glucose metabolism may not be the main mechanism for the early occurring clinical benefits. Dr. Jesper Jensen: However, we know that many of our heart failure patients without diabetes are insulin resistant as a metabolic feature of the heart failure, and the insulin resistance is associated with an increased risk of developing future diabetes, which in turn reduces the long-term survival and quality of life. So, the targeting insulin resistance in HFrEF patients is, therefore, of clinical relevance to our patients. Dr. Jesper Jensen: So, the population of the Empire HF Metabolic consisted of patients with chronic HFrEF, with or without type two diabetes, who are on a stable guideline directed heart failure therapy, and have also indicated on anti-diabetic therapy. And we randomized patients to receive empagliflozin 10 milligrams once daily, or matching placebo as an-add on for 12 weeks. Dr. Jesper Jensen: And this was a modest sized randomized control trial, including 120 patients. A very large proportion of patients received guideline directed heart failure therapy, and they generally consisted of the best one third of atypical HFrEF population, and only 10% had concomitant history of type two diabetes. Dr. Jesper Jensen: We then, at baseline and after 12 weeks, we formed an oral glucose tolerance test to assess the hepatic and a peripheral insulin sensitivity and performed a whole body DXA scan to investigate alterations in body composition. We know that patients lose weight when they get an SGLT2 inhibitor with and without diabetes, but we don't know what it consists of in a HFrEF population. Dr. Carolyn Lam: Tell us what you found after 12 weeks. Dr. Jesper Jensen: Yeah, so a large proportion, actually half of the patients without a history of diabetes, had a new onset diabetes, or impact glucose tolerance at baseline. So even though few have no diabetes, this population were at very high risk of developing future diabetes. And the main finding was that empagliflozin improved insulin sensitivity. So the hepatic insulin sensitivity was improved by 13% and the peripheral insulin resistance was improved by 20% compared to placebo. Dr. Jesper Jensen: And moreover, both fasting and postprandial glucose were significantly reduced. And regarding the body composition, patients in a mean lost at 1.2 kilos, or 2.6 pounds, which mainly consisted of a loss in lean mass and no significant changes were observed in fatness, and this is from the DXA scan. Dr. Carolyn Lam: Hmm. Justin, could you shed some light on what the editors thought about this, and there's lots of questions still, huh? Dr. Justin Ezekowitz: Yeah, absolutely, Carolyn, and thanks Jesper for sharing this paper with Circulation. Thanks for summarizing it so well. I think the questions that come up and the reason why we liked it so much was we're all trying to understand mechanism of how these medications work so profoundly for our patients. Dr. Justin Ezekowitz: Now, in this predominantly non-diabetic population, the fact that the liver and the peripheral insulin sensitivity improves, how does that bear out for the fact that there is no fat loss in the early stages, yet that's all been linked to later improved exercise capacity and increased fat loss later on in life. Dr. Justin Ezekowitz: So, do you think those two are going to be linked if you went to say from 12 weeks beyond the 52 or two years down the road? Dr. Jesper Jensen: Yes, that is what we've seen from diabetes populations, at least. So you could imagine that the same would be the case also in the HFrEF primarily non-diabetic population, but again, we don't know. But early loss is the mass loss. Dr. Justin Ezekowitz: So Jesper, when you think about the peripheral insulin sensitivity improvement, is that largely indicating mostly muscle based insulin sensitivity improvement, and that would indicate that the muscles, perhaps, are functioning better in the short term with just a simple change in therapy. Dr. Jesper Jensen: Yeah, that could be a way to put it. I would agree upon that. Dr. Justin Ezekowitz: So thanks, Jesper, I think that may indicate the quality of life improvement that we may be seeing in the functional status there, Carolyn. Dr. Carolyn Lam: Yeah, but as you said, Justin, there just seems so many other questions. To Jesper, I want to know, what further might you want to do to find out what's happening with this? The loss of lean mass surprised me, frankly. I thought it would have been fat mass. So, what are you doing to look at that? And then to Peter, I want to go the other direction. What are you planning next that might bring this closer to humans and a clinical study? So maybe I'll ask Jesper to go first. Dr. Jesper Jensen: So, I definitely agree with you, Carolyn. We would also have to put our money on the fat from the beginning, before the study. So with respect to the weight loss, then a loss in lean mass is not preferable if it represents muscle. So however, the weight loss works to mediate the observed change in insulin resistance. And additionally, a significant loss in muscle would result in reduced insulin sensitivity. And we observed the opposite. Therefore, the observed loss in lean mass may be speculated to represent water and pointing towards the early diuretic effect SGLT2 inhibitors. So, the DXA scan is good at looking at body composition, but it has difficulties in separating lean mass from whether it's muscle or water, but combined with the findings on the insulin resistance, we speculate that the lean mass loss is more. Dr. Carolyn Lam: Thank you. And Peter, could you very quickly tell us what are next steps, in your view? Dr. Peter Light: Yeah, obviously we were studying mouse model of heart failure. We'd like to make a more of a translational step in the next experiments we do by studying human tissues. So getting access to ventricular tissue from ex-planted hearts, human hearts, too, and then measure electrical activity as well as some calcium imaging. Dr. Peter Light: Looking at some of these Long QT3 animal models would be another thing that we're going to do. And also start looking at to see whether we can get access to any electrophysiological data from electronic medical records to start looking for DCGs and measuring QT interval, for example, would be another nice step to that. Dr. Peter Light: And then, more of a drug development side of things, we are actively synthesizing new derivatives of these drugs and seeing whether we can enhance the cardio-protective effects on the late sodium current, but actually remove the ability to inhibit SGLT2. So we would no longer have a glucose-lowering drug, but we'd have a cardioprotective drug. So, it's all very exciting work going on right now. Dr. Carolyn Lam: You've been listening to Circulation on the Run. From Greg and I, don't forget to tune in again next week. Dr. Greg Hundley: This program is copyright of the American Heart Association, 2021. The opinions expressed by speakers in this podcast are their own and not necessarily those of the editors or of the American Heart Association. For more, visit ahajournals.org.  

John Mulcahy PhD, President and CEO, SiteOne Therapeutics talks about broadening their pipeline from acute pain to hypersensitivity disorders like chronic cough and itching. Approach is to inhibit voltage-gated subtype sodium channel Nav1.7, a genetically validated target for pain. Stopping the pain signal in the peripheral nervous system before it gets to the spinal cord avoids some of the liabilities with existing pain medication like opioids. Lead candidate is now an intravenous analgesic showing proof of concept for the treatment of acute pain and itch. #pain #hypersensitivity #sodiumchannels #opioids SiteOneTherapeutics.com Download the transcript here  

John Mulcahy PhD, President and CEO, SiteOne Therapeutics talks about broadening their pipeline from acute pain to hypersensitivity disorders like chronic cough and itching. Approach is to inhibit voltage-gated subtype sodium channel Nav1.7, a genetically validated target for pain. Stopping the pain signal in the peripheral nervous system before it gets to the spinal cord avoids some of the liabilities with existing pain medication like opioids. Lead candidate is now an intravenous analgesic showing proof of concept for the treatment of acute pain and itch. #pain #hypersensitivity #sodiumchannels #opioids SiteOneTherapeutics.com Listen to the podcast here  

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.11.05.370429v1?rss=1 Authors: DeKeyser, J.-M., Thompson, C. H., George, A. L. Abstract: Mutations in genes encoding the human brain-expressed voltage-gated sodium (NaV) channels NaV1.1, NaV1.2, and NaV1.6 are associated with a variety of human diseases including epilepsy, autism spectrum disorder, familial migraine, and other neurodevelopmental disorders. A major obstacle hindering investigations of the functional consequences of brain NaV channel mutations is an unexplained instability of the corresponding recombinant complementary DNA (cDNA) when propagated in commonly used bacterial strains manifested by high spontaneous rates of mutation. Here we investigated the cause for instability of human NaV1.1 cDNA. We identified cryptic prokaryotic promoter-like elements that are presumed to drive transcription of translationally toxic mRNAs in bacteria as the cause of the instability, and demonstrated that mutations in these elements can mitigate the instability. Extending these observations, we generated full-length human NaV1.1, NaV1.2, and NaV1.6 plasmids using one or two introns that interrupt the cryptic reading frames along with a minimum number of silent nucleotide changes that achieved stable propagation in bacteria. Expression of the stabilized sequences in cultured mammalian cells resulted in functional NaV channels with properties that matched their parental constructs. Our findings explain a widely observed instability of recombinant neuronal human NaV channels, and we describe re-engineered plasmids that attenuate this problem. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.10.31.363416v1?rss=1 Authors: Martinez-Espinosa, P. L., Yang, C., Xia, X., Lingle, C. J. Abstract: Adrenal chromaffin cells (CCs) in rodents express a rapidly inactivating, TTX-sensitive sodium current. The current has generally been attributed to Nav1.7, although a possible role for Nav1.3 has also been suggested. Nav channels in rat CCs rapidly inactivate into two separable pathways, which differ in their time course of recovery from inactivation. One population recovers with time constants similar to traditional fast inactivation and the other about 10-fold slower. Inactivation properties suggest that the two pathways result from a single homogeneous population of channels. Here we probe the properties and molecular components of the Nav current present in mouse CCs. We first confirm that functional properties of Nav current in rat and mouse cells are generally similar in terms of activation range, steady-state inactivation, and dual pathway fast inactivation. The results then show that all inward Nav current is absent in CCs from Nav1.3 KO mice. Subsequently, in a mouse with KO of fibroblast growth factor homology factor 14 (FGF14), we find that the slow component of recovery from fast inactivation is completely absent in most CCs, with no change in the time constant of fast recovery. Experiments probing the use-dependence of Nav current diminution between WT and FGF14 KO mice directly demonstrate a role of slow recovery from inactivation in determination of Nav current availability. Overall, the results indicate that the FGF14-mediated inactivation is the major determinant in defining use-dependent changes in Nav availability in CCs. We also consider the potential impact that inactivating FGFs with different recovery kinetics can exert on differential use-dependent changes in Nav availability. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.10.14.339382v1?rss=1 Authors: Shiers, S., Sankaranarayanan, I., Jeevakumar, V., Cervantes, A., Reese, J. C., Price, T. J. Abstract: Peripheral sensory neurons are characterized by their size, molecular profiles, and physiological responses to specific stimuli. In mouse, the peptidergic and non-peptidergic subsets of nociceptors are distinct and innervate different lamina of the spinal dorsal horn. The unique molecular signature and neuroanatomical organization of these neurons supports a labeled line theory for certain types of nociceptive stimuli. However, long standing evidence supports the polymodal nature of nociceptors in many species. We have recently shown that the peptidergic marker, CGRP, and the non-peptidergic marker, P2X3R, show largely overlapping expression at the mRNA level in human dorsal root ganglion (DRG). Herein, our aim was to assess the protein distribution of nociceptor markers, including their central projections, in the human DRG and spinal cord. Using DRGs obtained from organ donors, we observed that CGRP and P2X3R were co-expressed by approximately 33% of human DRG neurons and TrpV1 was expressed in ~60% of human DRG neurons. In the dorsal spinal cord, CGRP, P2X3R, TrpV1 and Nav1.7 protein stained the entirety of lamina II, with only P2XR3 showing a gradient of expression. This was confirmed by measuring the size of the substantia gelatinosa using Hematoxylin and Eosin staining of adjacent sections. Our findings are consistent with the known polymodal nature of most primate nociceptors and indicate that the central projection patterns of nociceptors are different between mice and humans. Elucidating how human nociceptors connect to subsets of dorsal horn neurons will be important for understanding the physiological consequences of these species differences. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.10.01.308460v1?rss=1 Authors: Speigel, I. A., Hemmings, H. C. Abstract: General anesthesia is critical to modern medicine and animal research, but the cellular and molecular actions of general anesthetics on the central nervous system remain poorly understood. Volatile anesthetics such as isoflurane disrupt synaptic transmission and inhibit synaptic vesicle release in a neurotransmitter-selective manner. For example, GABA release from interneurons is less sensitive to isoflurane inhibition than are glutamate or dopamine release. Hippocampal and cortical interneuron subpopulations have diverse neurophysiological and synaptic properties, and their individual subtype-specific responses to isoflurane are unknown. We used live-cell optical imaging of exocytosis using fluorescent biosensors expressed in transgenic mouse hippocampal neuron cultures to delineate interneuron subtypespecific effects of isoflurane on synaptic vesicle exocytosis. We found that a clinically relevant concentration of isoflurane (0.5 mM) differentially modulated action potential-mediated exocytosis from GABAergic interneurons: parvalbumin-expressing interneurons were inhibited to 83.1plus-or-minus sign11.7% of control, whereas somatostatin-expressing and interneurons glutamatergic neurons were inhibited to 58.6plus-or-minus sign13.3% and 64.5plus-or-minus sign8.5% of control, respectively. The role of presynaptic voltage-gated sodium channel (Nav) subtype expression in determining isoflurane sensitivity was probed by overexpression or knockdown of specific Nav subtypes, which have distinct sensitivities to isoflurane and are differentially expressed between glutamatergic and GABAergic neurons. We found that the sensitivity of exocytosis to isoflurane was determined by the relative expression of Nav1.1 (associated with lower sensitivity) and Nav1.6 (associated with higher sensitivity). Thus the selective effects of isoflurane on synaptic vesicle exocytosis from hippocampal interneuron subtypes is determined by synaptic diversity in the relative expression of Nav1.1 and Nav1.6. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.09.30.320788v1?rss=1 Authors: Lignani, G., Liavas, A., Kullmann, D. M., Schorge, S. Abstract: Neuronal excitability is tightly regulated, requiring rapidly activating and inactivating voltage-gated sodium channels to allow accurate temporal encoding of information. Alternative splicing greatly broadens the repertoire of channels, but the adaptive significance of this phenomenon is incompletely understood. An alternative splicing event that is conserved across vertebrates affects part of the first domain of sodium channels and modulates their availability after inactivation. Here we use this conserved splicing event to ask whether this modulation has consistent effects in different neuronal backgrounds, or whether a conserved splicing event can be exploited to produce distinct effects in different cell types. We show that the consequences of alternate splicing of human Nav1.1 and Nav1.2 for neuronal activity depend on whether they are expressed in the cell types where they normally predominate (interneurons or excitatory neurons, respectively). Splicing in the adult isoform in both channels is sufficient to slow action potential rise times in all neurons. However, changes to both action potential half width and maximal firing rate are specific to cell type and channel, with each channel appearing tuned to mediate effects in its predominant neuronal background. Finally, we use dynamic clamp to demonstrate that alternative splicing in Nav1.1 changes how interneurons fire during epileptiform events. Our data show that, for sodium channels, despite conserved amino acid changes and similar effects on channel gating, alternative splicing has distinct impacts on neuronal properties, thus highlighting how closely sodium channels are tuned to distinct cellular backgrounds. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.09.29.318774v1?rss=1 Authors: Gomez, K., Ran, D., Madura, C. L., Moutal, A., Khanna, R. Abstract: Voltage-gated sodium channels are key players in neuronal excitability and pain signaling. Functional expression of the voltage-gated sodium channel NaV1.7 is under the control of SUMOylated collapsin response mediator protein 2 (CRMP2). If not SUMOylated, CRMP2 forms a complex with the endocytic proteins Numb, the epidermal growth factor receptor pathway substrate 15 (Eps15), and the E3 ubiquitin ligase Nedd4-2 to promote clathrin-mediated endocytosis of NaV1.7. We recently reported that CRMP2 SUMO-null knock-in (CRMP2K374A/K374A) female mice have reduced NaV1.7 membrane localization and currents in their sensory neurons. Preventing CRMP2 SUMOylation was sufficient to reverse mechanical allodynia in CRMP2K374A/K374A female mice with neuropathic pain. Here we report that inhibiting clathrin assembly in nerve-injured male and female CRMP2K374A/K374A mice, increased pain sensitivity in allodynia-resistant animals. Furthermore, Numb, Nedd4-2 and Eps15 expression was not modified in basal conditions in the dorsal root ganglia (DRG) of male and female CRMP2K374A/K374A mice. Finally, silencing these proteins in DRG neurons from female CRMP2K374A/K374A mice, restored the loss of sodium currents. Our study shows that the endocytic complex composed of Numb, Nedd4-2 and Eps15, is necessary for non SUMOylated CRMP2-mediated internalization of sodium channels in vivo. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.09.01.275099v1?rss=1 Authors: de Clauser, L., Luiz, A. P., Santana-Varela, S., Wood, J. N., Sikandar, S. Abstract: Cancer-induced bone pain (CIBP) is a complex condition comprising components of inflammatory and neuropathic processes, but changes in the physiological response profiles of bone-innervating afferents remain poorly understood. We used a combination of retrograde labelling and in vivo calcium imaging of bone marrow-innervating DRG neurons to determine the contribution of these cells in the establishment and maintenance of CIBP. We found a majority of femoral bone afferent cell bodies in L3 DRG that also express the sodium channel subtype Nav1.8 - a marker of nociceptive neurons - and lack expression of parvalbumin - a marker for proprioceptive primary afferents. Surprisingly, the response properties of bone marrow afferents to both increased intraosseous pressure and acid were unchanged by the presence of cancer. On the other hand, we found increased excitability and polymodality of cutaneous afferents innervating the ipsilateral paw in cancer bearing animals, as well as a behavioral phenotype that suggests changes at the level of the DRG contribute to secondary hypersensitivity. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.08.04.235861v1?rss=1 Authors: Capurro, A., Thornton, J., Cessac, B., Armstrong, L., Sernagor, E. Abstract: Chronic pain is a global healthcare problem with a huge societal impact. Its management remains unsatisfactory, with no single treatment clinically approved in most cases. In this study we use an in vitro experimental model of erythromelalgia consisting of sensory neurons derived from human induced pluripotent stem cells obtained from a patient (carrying the mutation F1449V) and a control subject. We combine neurophysiology and computational modelling to focus on the Nav1.7 voltage gated sodium channel, which acts as an amplifier of the receptor potential in nociceptive neurons and plays a critical role in erythromelalgia due to gain of function mutations causing the channel to open with smaller depolarisations. Using multi-electrode array (extracellular) recordings, we found that the scorpion toxin OD1 increases the excitability of sensory neurons in cultures obtained from the control donor, evidenced by increased spontaneous spike rate and amplitude. In erythromelalgia cultures, the application of the Nav1.7 blocker PF-05089771 effectively stopped spontaneous firing. These results, which are in accordance with current clamp and voltage clamp recordings reported in the literature, are explained with a conductance-based computational model of a single human nociceptive neuron. The disease was simulated through a decrease of the Nav1.7 half activation voltage, which decreased the rheobase and increased the response to supra threshold depolarizing currents. This enhanced response could be successfully supressed by blocking the Nav1.7 channels. The painful effects of OD1 were simulated through a slower establishment and a quicker removal of Nav1.7 inactivation, reproducing the effects of the toxin on the spike frequency and amplitude. Our model simulations suggest that the increase in extracellular spike amplitude observed in the MEA after OD1 treatment can be due mainly to a slope increase in the ascending phase of the intracellular spike caused by impaired inactivation gating. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.07.31.231753v1?rss=1 Authors: Tavares-Ferreira, D., Ray, P., Sankaranarayanan, I., Mejia, G. L., Wangzhou, A., Shiers, S., Uttarkar, R. S., Megat, S., Barragan-Iglesias, P., Dussor, G., Akopian, A. N., Price, T. J. Abstract: Background: There are clinically relevant sex differences in acute and chronic pain mechanisms, but we are only beginning to understand their mechanistic basis. Transcriptome analyses of rodent whole dorsal root ganglion (DRG) have revealed sex differences, mostly in immune cells. We examined the transcriptome and translatome of the mouse DRG with the goal of identifying sex differences. Methods: We used Translating Ribosome Affinity Purification (TRAP) sequencing and behavioral pharmacology to test the hypothesis that nociceptor (Nav1.8 expressing neurons) translatomes would differ by sex. Results: We found 66 genes whose mRNA were sex-differentially bound to nociceptor ribosomes. Many of these genes have known neuronal functions but have not been explored in sex differences in pain. We focused on Ptgds, which was increased in female mice. The mRNA encodes the prostaglandin D2 (PGD2) synthesizing enzyme. We observed increased Ptgds protein and PGD2 in female mouse DRG. The Ptgds inhibitor AT-56 caused intense pain behaviors in male mice but was only effective at high doses in females. Conversely, female mice responded more robustly to another major prostaglandin, PGE2, than did male mice. Ptgds protein expression was also higher in female cortical neurons, suggesting DRG findings may be generalizable to other nervous system structures. Conclusions: Nociceptor TRAP sequencing (TRAP-seq) reveals unexpected sex differences in one of the oldest known nociceptive signaling molecule families, the prostaglandins. Our results demonstrate that translatome analysis reveals physiologically relevant sex differences important for fundamental protective behaviors driven by nociceptors. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.07.09.190777v1?rss=1 Authors: Dimitri Ryczko, Maroua Hanini-Daoud, Steven Condamine, Benjamin J. B. Bréant, Maxime Fougère, Roberto Araya, Arlette Kolta Abstract: AbstractThe most complex cerebral functions are performed by the cortex which most important output is carried out by its layer 5 pyramidal neurons. Their firing reflects integration of sensory and contextual information that they receive. There is evidence that astrocytes influence cortical neurons firing through the release of gliotransmitters such as ATP, glutamate or GABA. These effects were described at the network and at the synaptic levels, but it is still unclear how astrocytes influence neurons input-output transfer function at the cellular level. Here, we used optogenetic tools coupled with electrophysiological, imaging and anatomical approaches to test whether and how astrocytic activation affected processing and integration of distal inputs to layer 5 pyramidal neurons (L5PN). We show that optogenetic activation of astrocytes near L5PN cell body prolonged firing induced by distal inputs to L5PN and potentiated their ability to trigger spikes. The observed astrocytic effects on L5PN firing involved glutamatergic transmission to some extent but relied on release of S100β, an astrocytic Ca2+-binding protein that decreases extracellular Ca2+ once released. This astrocyte-evoked decrease of extracellular Ca2+ elicited firing mediated by activation of Nav1.6 channels. Our findings suggest that astrocytes contribute to the cortical fundamental computational operations by controlling the extracellular ionic environment.Key Points SummaryIntegration of inputs along the dendritic tree of layer 5 pyramidal neurons is an essential operation as these cells represent the most important output carrier of the cerebral cortex. However, the contribution of astrocytes, a type of glial cell to these operations is poorly documented.Here we found that optogenetic activation of astrocytes in the vicinity of layer 5 in the mouse primary visual cortex induce spiking in local pyramidal neurons through Nav1.6 ion channels and prolongs the responses elicited in these neurons by stimulation of their distal inputs in cortical layer 1.This effect partially involved glutamatergic signalling but relied mostly on the astrocytic calcium-binding protein S100β, which regulates the concentration of calcium in the extracellular space around neurons.These findings show that astrocytes contribute to the fundamental computational operations of the cortex by acting on the ionic environment of neurons.Competing Interest StatementThe authors have declared no competing interest.View Full Text Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.06.25.096552v1?rss=1 Authors: Jorgensen, H. S., Jensen, D. B., Dimintiyanova, K. P., Bonnevie, V. S., Hedegaard, A., Lehnhoff, J., Moldovan, M., Grondahl, L. K., Meehan, C. F. Abstract: Amyotrophic lateral sclerosis is a neurodegenerative disease preferentially affecting motoneurones. Transgenic mouse models have been used to investigate the role of abnormal motoneurone excitability in this disease. Whilst an increased excitability has repeatedly been demonstrated in vitro in neonatal and embryonic preparations from SOD1 mouse models, the results from the only studies to record in vivo from spinal motoneurones in adult SOD1 models have produced conflicting findings. Deficits in repetitive firing have been reported in G93A SOD1 mice but not in presymptomatic G127X SOD1 mice despite shorter motoneurone axon initial segments (AISs) in these mice. These discrepancies may be due to the earlier disease onset and prolonged disease progression in G93A SOD1 mice with recordings potentially performed at a later sub-clinical stage of the disease in this mouse. To test this, and to explore how the evolution of excitability changes with symptom onset we performed in vivo intracellular recording and AIS labelling in G127X SOD1 mice immediately after symptom onset. No reductions in repetitive firing were observed showing that this is not a common feature across all ALS models. Immunohistochemistry for the Na+ channel Nav1.6 showed that motoneurone AISs increase in length in G127X SOD1 mice at symptom onset. Consistent with this, the rate of rise of AIS components of antidromic action potentials were significantly faster confirming that this increase in length represents an increase in AIS Na+ channels occurring at symptom onset in this model. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.06.23.150367v1?rss=1 Authors: Eaton, M., Zhang, J., Ma, Z., Park, A. C., Lietzke, E. E., Romero, C. M., Liu, Y., Coleman, E. R., Chen, X., Xiao, T., Huang, Z., Skarnes, W. C., Koss, W. A., Yang, Y. Abstract: Recent large-scale genomic studies have revealed SCN2A as one of the most frequently mutated gene in patients with neurodevelopmental disorders including autism spectrum disorder and intellectual disability. SCN2A encodes for voltage-gated sodium channel isoform 1.2 (Nav1.2), which is mainly expressed in the central nervous system and responsible for the propagation of neuronal action potentials. Homozygous knockout (null) of Scn2a is perinatal lethal, whereas heterozygous knockout of Scn2a results in mild behavior abnormalities. To achieve a more substantial, but not complete, reduction of Scn2a expression, we characterized a Scn2a deficient mouse model using a targeted gene trap knockout (gtKO) strategy to recapitulate loss-of-function SCN2A disorders. This model produces viable homozygous mice (Scn2agtKO/gtKO) that can survive to adulthood, with markedly low but detectable Nav1.2 expression. Although Scn2agtKO/gtKO adult mice possess normal olfactory, taste, hearing, and mechanical sensitivity, they have decreased thermal and cold tolerance. Innate behaviors are profoundly impaired including impaired nesting, marble burying, and mating. These mice also have increased food and water intake with subsequent increases in fecal excretion of more but smaller fecal boli. This novel Scn2a gene trap knockout mouse thus provides a unique model to study pathophysiology associated with Scn2a deficiency. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.06.01.127183v1?rss=1 Authors: macdonald, d., sikandar, s., weiss, j., pyrski, m., luiz, a., millet, q., emery, e., mancini, f., Iannetti, G. D., alles, s., zhao, j., cox, j., brownstone, R., Zufall, f., wood, j. Abstract: Deletion of SCN9A encoding the voltage-gated sodium channel NaV1.7 in humans leads to profound pain insensitivity and anosmia. Conditional deletion of NaV1.7 in sensory neurons of mice also abolishes pain suggesting the locus of analgesia is the nociceptor. Here we demonstrate that NaV1.7 knockout mice have essentially normal nociceptor activity using in vivo calcium imaging and extracellular recording. However, glutamate and substance P release from nociceptor central terminals in the spinal cord is greatly reduced by an opioid-dependent mechanism. Analgesia is also substantially reversed by central but not peripheral application of opioid antagonists. In contrast, the lack of neurotransmitter release from olfactory sensory neurons is opioid-independent. Male and female humans with NaV1.7 null mutations show naloxone reversible analgesia. Thus opioid-dependent inhibition of neurotransmitter release is the principal mechanism of NaV1.7 null analgesia in mice and humans. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.05.29.124693v1?rss=1 Authors: Patel, R. R., Ping, X., Patel, S., McDermott, J. S., Krajewski, J. L., Chi, X. X., Nisenbaum, E. S., Jin, X., Cummins, T. R. Abstract: Brain isoforms of voltage-gated sodium channels (VGSCs) have distinct cellular and subcellular expression patterns as well as functional roles that are critical for normal physiology as aberrations in their expression or activity lead to pathophysiological conditions. In this study, we asked how inhibition of select isoforms of VGSCs alters epileptiform activity to further parse out the roles of VGSCs in the brain. We first determined the relative selectivity of recently discovered small molecule, aryl sulfonamide, inhibitors (ICA-121431 and Compound 801) against Nav1.1, Nav1.2, and Nav1.6 activity using whole-cell patch clamp recordings obtained from HEK293 cells. To test the effects of these inhibitors on epileptiform activity, we obtained multielectrode array (MEA) recordings from mouse cortical slices in the presence of 4-aminopyridine (4-AP) to induce epileptiform activity. We found that the ICA-121431 and Compound 801 compounds are relatively selective for Nav1.1 and Nav1.6, respectively. From the MEA recordings, we found that inhibition of Nav1.6 and Nav1.2 with 500nM of the Compound 801 compound completely abolishes ictal local field potentials induced by 4-AP, whereas inhibition of Nav1.1 with 500nM of the ICA-121431 compound had minimal effect on epileptiform activity induced by 4-AP. Due to the prominent expression of Nav1.1 in inhibitory neurons, we asked whether inhibition of Nav1.1 alone alters activity. We found that, indeed, inhibition of Nav1.1 with the ICA-121431 compound increased basal activity in the absence of 4-AP. These findings expand our current understanding of the roles of VGSC isoforms in the brain and suggest that selective targeting of Nav1.6 may be a more efficacious treatment strategy for epileptic syndromes. Copy rights belong to original authors. Visit the link for more info

Acute pain signaling has a key protective role and is highly evolutionarily conserved. Chronic pain, however, is maladaptive, occurring as a consequence of injury and disease, and is associated with sensitization of the somatosensory nervous system. Primary sensory neurons are involved in both of these processes, and the recent advances in understanding sensory transduction and human genetics are the focus of this review. Voltage-gated sodium channels (VGSCs) are important determinants of sensory neuron excitability: they are essential for the initial transduction of sensory stimuli, the electrogenesis of the action potential, and neurotransmitter release from sensory neuron terminals. Nav1.1, Nav1.6, Nav1.7, Nav1.8, and Nav1.9 are all expressed by adult sensory neurons. The biophysical characteristics of these channels, as well as their unique expression patterns within subtypes of sensory neurons, define their functional role in pain signaling. Changes in the expression of VGSCs, as well as posttranslational modifications, contribute to the sensitization of sensory neurons in chronic pain states. .

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.05.02.073999v1?rss=1 Authors: macdonald, d. I., Luiz, a. P., Millet, Q., Emery, E. C., wood, j. n. Abstract: Neuropathic pain patients often experience innocuous cooling as excruciating pain. The cell and molecular basis of this cold allodynia is little understood. We used in vivo calcium imaging of sensory ganglia to investigate the activity of peripheral cold-sensing neurons in three mouse models of neuropathic pain: oxaliplatin-induced neuropathy, partial sciatic nerve ligation and ciguatera poisoning. In control mice, cold-sensing neurons were few in number and small in size. In neuropathic animals with cold allodynia, a set of normally silent large-diameter neurons became sensitive to cooling. Many silent cold-sensing neurons expressed the nociceptor markers NaV1.8 and CGRP. Ablating these neurons diminished cold allodynia. Blocking KV1 voltage-gated potassium channels was sufficient to trigger de novo cold sensitivity in silent cold-sensing neurons. Thus silent cold-sensing neurons are unmasked in diverse neuropathic pain states and cold allodynia results from peripheral sensitization caused by altered nociceptor excitability. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.04.25.061663v1?rss=1 Authors: Paige, C., Barba-Escobedo, P. A., Mecklenburg, J., Patil, M., Goffin, V., Grattan, D., Dussor, G., Akopian, A. N., Price, T. J. Abstract: Many clinical and preclinical studies report higher prevalence and severity of chronic pain in females. We used hyperalgesic priming with interleukin 6 (IL6) priming and PGE2 as a second stimulus as a model for pain chronicity. Intraplantar IL6 induced hypersensitivity was similar in magnitude and duration in both males and females, while both paw and intrathecal PGE2 hypersensitivity was more persistent in females. This difference in PGE2 response was dependent on both circulating estrogen and translation regulation signaling in the spinal cord. In males, the duration of hypersensitivity was regulated by testosterone. Since the prolactin receptor (Prlr) is regulated by reproductive hormones and is female-selectively activated in sensory neurons, we evaluated whether Prlr signaling contributes to hyperalgesic priming. Using a competitive Prlr antagonist, and a mouse line with ablated Prlr in the Nav1.8 sensory neuronal population, we show that Prlr in sensory neurons is necessary for the development of hyperalgesic priming in female but not male mice. Overall, sex-specific mechanisms in the initiation and maintenance of chronic pain are regulated by the neuroendocrine system and, specifically, sensory neuronal Prlr signaling. SIGNIFICANCE STATEMENT: Females are more likely to experience chronic pain than males, but the mechanisms that underlie this sexual dimorphism are not completely understood. Here, we demonstrate that the duration of mechanical hypersensitivity is dependent on circulating sex hormones in mice, where estrogen caused an extension of sensitivity and testosterone was responsible for a decrease in the duration of the hyperalgesic priming model of chronic pain. Additionally, we demonstrated that Prolactin receptor expression in Nav1.8+ neurons was necessary for hyperalgesic priming in female, but not male mice. Our work demonstrates a female-specific mechanism for the promotion of chronic pain involving the neuroendrocrine system and mediated by sensory neuronal prolactin receptor. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.04.23.057851v1?rss=1 Authors: Verma, P., Eaton, M., Kienle, A., Flockerzi, D., Yang, Y., Ramkrishna, D. Abstract: Chemotherapy-induced peripheral neuropathy (CIPN) is a prevalent, painful side effect which arises due to a number of chemotherapy agents. CIPN can have a prolonged effect on quality of life. Chemotherapy treatment is often reduced or stopped altogether because of the severe pain. Currently, there are no FDA-approved treatments for CIPN partially due to its complex pathogenesis in multiple pathways involving a variety of channels, specifically, voltage-gated ion channels. A surrogate of neuropathic pain in an in vitro setting is hyperexcitability in dorsal root ganglia (DRG) peripheral sensory neurons. Our study employs bifurcation theory to investigate the role of voltage-gated ion channels in inducing hyperexcitability as a consequence of spontaneous firing, due to the common chemotherapy agent paclitaxel. Our mathematical investigation suggests that the sodium channel Nav1.8 and the delayed rectifier potassium channel conductances are the most critical for hyperexcitability in normal firing small DRG neurons. Introducing paclitaxel into the model, our bifurcation analysis predicts that hyperexcitability is extreme for a medium dose of paclitaxel, which is validated by multi-electrode array recordings. Our findings using multi-electrode array experiments reveal that the Nav1.8 blocker A-803467 and the delayed rectifier potassium enhancer L-alpha-phosphatidyl-D-myo-inositol 4,5-diphosphate, dioctanoyl (PIP2) have a protective effect on the firing rate of DRG when administered separately together with paclitaxel as suggested by our bifurcation analysis. Copy rights belong to original authors. Visit the link for more info

Jane Ferguson:                Hello. Welcome to episode 33 of Getting Personal: Omics Of The Heart, your podcast from Circulation: Genomic and Precision Medicine. I'm Jane Ferguson. This episode is from October 2019. Let's get started.                                            First up is a paper from Sébastien Thériault, Yohan Bossé, Jean-Jacques Schott and colleagues from Laval University, Quebec and INSERM in Mont. They published on genetic association analyses, highlight IL6, ALPL and NAV1 as three new susceptibility genes underlying Calcific Aortic Valve Stenosis.                                            In this paper, they were interested in finding out whether they could identify novel susceptibility genes for Calcific Aortic Valve Stenosis, or CAVS, which is a severe and often fatal condition with limited treatment options other than surgical aortic valve replacement. They conducted a GWAS meta-analysis across four European ancestry cohorts comprising over 5,000 cases and over 354,000 controls. They identified four loci at genome-wide significance, including two known loci in LPA and PALMD as well as two novel loci, IL6 which encodes the interleukin six cytokine, and ALPL, which encodes an alkaline phosphatase. They then integrated transcriptomic data from 233 human aortic valves to conduct the transcriptome wide association study and find an additional risk locus associated with higher expression of NAV1 encoding neuron navigator one. Through fine mapping, integrating conservation scores, and methylation peaks, they narrowed down the putative causal variants at each locus identifying one snip in each of PALMD and IL6 as likely causal in addition to two candidates snips at ALPL and three plausible candidate snips in NAV1.                                            Phenome-Wide Association Analysis, or PheWAS of the top candidate functional snips found that the IL6 risk variant associated with higher eosinophil count, pulse pressure and systolic blood pressure. Overall, this study was able to identify novel loci associated with CAVS potentially implicating inflammation and hypertension in CAVS etiology. Additional functional studies are required to further explore these potential mechanisms.                                            Next up is a paper from Elisavet Fotiou, Bernard Keavney and colleagues from the University of Manchester. Their paper entitled Integration of Large-Scale Genomic Data Sources With Evolutionary History Reveals Novel Genetic Loci for Congenital Heart Disease explored the genetic etiology of sporadic non syndromic congenital heart disease using an evolution informed approach. Ohnologs are related genes that have been retained following ancestral whole genome duplication events which occurred around 500 million years ago. The authors hypothesized that ohnologs which were retained versus duplicated genes that were lost were likely to have been under greater evolutionary pressure due to the need to maintain consistent gene dosage. For example, as could occur when the resulting proteins form complexes that require stochiometric balance.                                            Thus, ohnologs may be enriched for genes that are sensitive to dosage. The group analyzed copy number variant data from over 4,600 non syndromic coronary heart disease patients as well as whole exome sequence data from 829 cases of Tetralogy of Fallot. Compared to control data obtained from public databases, there was evidence for significant enrichment in CHD associated variants in ohnologs but not in other duplicated genes arising from small scale duplications. Through this and various other filtering steps to prioritize likely variants, the group was able to identify 54 novel candidate genes for congenital CHD highlighting the utility of considering the evolutionary origin of genes in the search for disease relevant biology.                                            Next, we have a clinical letter entitled Pathological Overlap of Arrhythmogenic Right Ventricular Cardiomyopathy and Cardiac Sarcoidosis from Ashwini Kerkar, Victoria Parikh and colleagues at Stanford University. They describe a case of a 50 year old woman previously healthy and a long distance runner who presented with tachycardia. She was found to have normal left ventricular size but severe right ventricular enlargement and systolic dysfunction. Genetic testing using an Arrhythmogenic Right Ventricular Cardiomyopathy or ARVC panel identified a variant in DSG2. through cascade testing it was found that two of the patient's three children also carried this variant. The patient experienced worsening RV failure and subsequently underwent heart transplantation at age 55. Pathology of the heart showed evidence of cardiac sarcoidosis. There have been some previous reports of overlap in ARVC and cardiac sarcoid pathology but not in cases with a high confidence genetic diagnosis such as this one.                                            This case raises the possibility of shared disease mechanisms underlying ARVC and cardiac sarcoidosis and suggests that therapies aimed at immune modulation may also have utility in ARVC. However, further work is required to test this hypothesis. Our next paper is a perspective piece from Babken Asatryan and Helga Servatius from Bern University Hospital. In Revisiting the Approach to Diagnosis of Arrhythmogenic Cardiomyopathy: Stick to the Arrhythmia Criterion!, they outline the challenges in defining diagnostic criteria for a Arrhythmogenic Right Ventricular Cardiomyopathy or ARVC, given the variable presentation of the disease. Given recent advances in knowledge, particularly in recognizing disease overlap with Arrhythmogenic Left Ventricular Cardiomyopathy or ALVC and Biventricular Arrhythmogenic Cardiomyopathy, a new clinical perspective was warranted. The Heart Rhythm Society updated their recommendations this year to introduce a new umbrella term that better encompasses the spectrum of disease, Arrhythmogenic Cardiomyopathy or ACM. This recommends the arrhythmia criterion Should be used as a first line screening criteria for ACM.                                            This is a broad criteria and a definitive diagnosis of ACM requires exclusion of systemic disorders such as sarcoidosis, amyloidosis, mild carditis, Chagas disease, and other cardiomyopathies. Implementation of this new approach to diagnosis may require more extensive investigation of arrhythmias including the use of ambulatory ECG monitors or cardiac loop recorders. These changes may also affect who's referred for genetic testing, potentially shifting diagnoses towards genotype rather than phenotype based disease classifications. Despite challenges and adopting new approaches, it is hoped that these changes will ultimately serve to improve risk stratification and allow for improved disease management and intervention to prevent sudden cardiac death.                                            We end with a scientific statement chaired by Sharon Cresci and co-chaired by Naveen Pereira with a writing group representing the AHA Councils on Genomic and Precision Medicine, Cardiovascular and Stroke Nursing and Quality of Care and Outcomes Research entitled Heart Failure in the Era of Precision Medicine: A Scientific Statement From the American Heart Association. This paper provides a comprehensive overview of the current state of omics technologies as they relate to the development and progression of heart failure and considers the current and potential future applications of these high throughput data for precision medicine with respect to prevention, diagnosis and therapy of heart failure. They discuss advances in genomics, pharmacogenomics, epigenomics, proteomics, metabolomics, and the microbiome, and integrate the findings from this rapidly developing field as they pertain to new methods to diagnose, treat, and prevent heart failure.                                            And that's it for October. I hope to see many of you at AHA Scientific Sessions in Philadelphia in November and look forward to bringing you more of the best new science next month. Thanks for listening. This podcast was brought to you by Circulation: Genomic and Precision Medicine and the American Heart Association Council on Genomic and Precision Medicine. This program is copyright American Heart Association 2019.  

Jane Ferguson:                 Hi, everyone. Welcome to Getting Personal: Omics of the Heart. This is podcast episode 16 from May 2018. I'm Jane Ferguson from Vanderbilt University Medical Center and this podcast is brought to you by Circulation Genomic and Precision Medicine and the AHA Council on Genomic and Precision Medicine. Jane Ferguson:                 This month we talked to Dr. Caitrin McDonough from the University of Florida. We briefly mentioned her paper in last month's episode Genetic Variants Influencing Plasma Renin Activity in Hypertensive Patients From the PEAR Study, but we wanted to go into it in more depth this month. Caitrin shared with us that this manuscript actually resulted from student course work and was a collaborative effort between students and instructors. The manuscript highlights has successful as approach can be both in increasing student engagement and as an effective way to do high quality research. You can hear her talk more about her innovative approach to student learning and the study findings later in this episode. Jane Ferguson:                 Of course, we have a great lineup of papers in Circulation Genomic and Precision Medicine this month. First up, a paper entitled, "SCN5A Variant Functional Perturbation and Clinical Presentation Variants of a Certain Significance" by Brett Kroncke, Andrew Glazer, and Dan M. Roden and colleagues from Vanderbilt University Medical Center. They were interested in investigating the functional significance of variants in the cardiac sodium channel in particular to see if they could explain why some variant carriers present with cardiac arrhythmias while others remain asymptomatic. Through a comprehensive literature search, they identified 1712 SCN5A variants and characterized the carriers by disease presentation. Variants associated with disease were more likely to fall in transmembrane domains consistent with the importance of these domains for channel function. Jane Ferguson:                 Using American College of Medical Genetics Criteria for variant classification, they found that variants classified as more pathogenic were also more penetrant. Penetrance was also associated with electrophysiological parameters. This approach highlights how modeling the penetrance of different variants can help define disease risk for individuals who carry potentially pathogenic variants. Jane Ferguson:                 Next we have a paper from Vincenzo Macri, Jennifer Brody, Patrick Ellinor, Nona Sotoodehnia and colleagues from the University of Washington and Massachusets General Hospital. This is also related to sodium channels and the paper is entitled, "Common Coding Variants in SCN10A Are Associated With the Nav1.8 Late Current and Cardiac Conduction". They were interested in SCN10A and sequenced this gene in over 3600 individuals from the CHARGE consortium to identify variants associated with cardiac conduction. They were able to replicate associations between variants and PR and the QRS intervals in a sample of almost 21,000 individuals from the CHARGE Exome sample. They identified several missense variants have clustered into distinct haplotypes and they showed that these haplotypes were associated with late sodium current. Jane Ferguson:                 Continuing the cardiac conduction theme, Honghuang Lin,  Aaron Isaacs and colleagues published a manuscript entitled, "Common and Rare Coding Genetic Variation Underlying the Electrocardiographic PR Interval". They conducted a meta-analyses of PR interval in over 93000 individuals which included over 9000 individuals of African ancestry. They identified 31 loci, 11 of which have not been reported before. We see SCN5A come up again as a gene of interest in this study but their analyses also implicated a novel locus, MYH6. Jane Ferguson:                 Next up moving from the heart to the vasculature, Janne Pott, Markus Scholz and colleagues from the University of Leipzig published a manuscript entitled, "Genetic Regulation of PCSK9 Plasma Levels and Its Impact on Atherosclerotic Vascular Disease Phenotypes". They were interested in whether circulating PCSK9 can be used as a diagnostic or predictive biomarker. To address this, they conducted a GWAS of plasma PCSK9 in over 3000 individuals from the LIFE-Heart study. They found that several independent variants within the PCSK9 gene were associated with plasma PCSK9 as well as some suggestive variants in another gene locus FBXL18. They used Mendelian randomization to probe causality and the data suggest that PCSK9 variants have a causal role in the presence and severity of atherosclerosis. Jane Ferguson:                 Moving on to another biomarker, Lisanne Blauw, Ko Willems van Dijk and colleagues from the  Einthoven Laboratory for Experimental Vascular Medicine report on CETP in their manuscript Cholesteryl Ester Transfer Protein Concentration A Genome-Wide Association Study Followed by Mendelian Randomization on Coronary Artery Disease. They aimed to assess potential causal effects of circulating CDP on cardiovascular disease through GWAS and Mendelian randomization. Jane Ferguson:                 In a study of over 4000 individuals from the Netherlands Epidemiology of Obesity Study, they identified three variants in CTP that associated with plasma levels of CETP and explained over 16% in the total variation in CDP levels. Genetically predicted in CETP was associated with reduced HDL and LDL cholesterol suggesting that CETP may be causally associated with coronary disease. Jane Ferguson:                 Rounding out the table of contents we also have a clinical case perspective from Nosheen Reza, Anjali The Importance of Timely Genetic Evaluation in family members in cases of cardiac disfunction and cardiomyopathy. We have a report from Adrianna Vlachos, Jeffrey Lipton and colleagues on the Diamond Blackfan Anemia Registry and we have a clinical case from Yukihiro Saito, Hiroshi Ito and colleagues on TRP and poor mutations in patients with ventricular non-compaction and cardiac conduction disease. Jane Ferguson:                 To read all of these papers and the accompanying commentaries, log on to circgenetics.aha.journals.org and if you're a visual learner or you need a work related excuse to spend time on YouTube, you can also access video summaries of all our articles from the CircGen website or directly from our YouTube channel Circulation Journal. Lastly, follow us on Twitter at circ_gen or on Facebook to get new content directly in your feed. Jane Ferguson:                 I'm joined today by Caitrin McDonough from the University of Florida and Caitrin is an Assistant Professor in the Department of Pharmacotherapy and Translational research in the College of Pharmacy and she's the first author on a recently published manuscript entitled, "Genetic Variants Influencing Plasma Renin Activity in Hypertensive Patients From the PEAR Study". This was published in the April 2018 issue of Circulation Genomic and Precision Medicine. Welcome, Caitrin. Caitrin M.:                           Thank you. Jane Ferguson:                 For listeners who haven't had a chance to read the paper yet, I wonder could you give us a brief overview of what prompted you to do this study? Caitrin M.:                           Sure so this looks at plasma renin activity and just initially a GWAS but it was done in a hypertensive population from the pharmogenomic evaluation of antihypertensive responses study. Particularly, since our group here at the University of Florida is more interested in pharmacogenomics we wanted to address plasma renin since it can influence blood pressure response to antihypertensive medication particularly if you use it as something to predict but also to correlate it with that as there have been also prior data from our group that shows if you have different levels of plasma renin that would predict if you would respond better to certain types of antihypertensive medications such as a beta-blocker or a diuretic. Caitrin M.:                           We used both a GWAS approach as well as a prioritization through blood pressure response to focus in on signals and then furthered by using prioritization using data from RNA seq and looking at eQTLs and then finally looking at more of just a traditional net replication of the original plasma renin activity signal. Caitrin M.:                           Overall, one of the interesting things and why we were initially doing this study was really in connection with a graduate course that myself and another faculty member here who's also an author on the paper, Yan Gong [inaudible 00:09:12]. We often have the students analyze data from the PEAR study as we have a lot of data from that study and it helped us analyze additional papers but we didn't necessarily know if this was going to be an interesting phenotype but through that course work which turned out that it really did have some interesting signals so we wanted to follow up more on. Jane Ferguson:                 Yeah, I love that approach so I think that's a really smart way to do it. To actually get your students to analyze your data and get them really involved in the process. How much then did the students ... how much were then they able to get involved when it started transpiring that their results would actually be something that could be put together for a manuscript? Caitrin M.:                           Overall, they are fairly involved. During the course work, what we usually do is give them just directly types data since a lot of them have not done this type of genetic analysis before and we split it up where each student gets about four to five chromosomes of data and then different phenotypes in the different race groups as we have both whites and African Americans. They get a certain race group, certain number of chromosomes and so they're able to conduct the analysis just using the Uplink software which is fairly user-friendly and straightforward. Then they get experience making Manhattan plots and using LocusZoom. Caitrin M.:                           After they have the basic techniques, then we teach them how to start following up top signals and determine what is a good signal. They're looking at the LD or SNP function or possibly gene function or looking at their genotype, phenotype relationships and making sure that it's not just one person who's driving the whole signal. Then selecting what top reasons and top SNPs may need a follow up. That part they all do there in the class and learn more of the basics. Caitrin M.:                           After the class, the students who want to continue to participate we get together and redistribute data where they would then move on to working on the imputed data sets and we teach them how to do that. Then we give them ... operate it somewhat similar to a consortia level meta-analysis type thing. I write up an analysis plan, each student does some part of the analysis. They have to bring it all back to me. I sort through it. We meet and go through it. Then we set our next steps to follow up. Then different students get different SNPs to investigate the function of or different subanalyses to do. Caitrin M.:                           One of our graduate students who is on this particular project, her dissertation project was very focused on our RNA seq data so that was how we were able to bring in the eQTL analysis using the RNA seq data as she had done a lot of the groundwork with that already. In one of our discussions that was one of the ways that we were able to incorporate the prioritization since she was intimately familiar with that data set. Jane Ferguson:                 Yeah and I think that's great. I can imagine that, that's a much more compelling way for students to learn about how to analyze data when they see the natural follow through. Do you find that some of the students maybe get more excited about research or are more likely to pursue future research opportunities by having had this hands on experience with the publication process and completing a project really did to this very end? Caitrin M.:                           They do, yeah. I see some of the students that end up sticking with it more are the students who I work more closely with and see more closely some of the students who are from other departments still stay involved but sometimes don't stay quite as involved. But, all of them really do continue to follow up and ask if they can still help or if there's anything they need to do until we get it to publication which is really nice. Jane Ferguson:                 Yeah, right. I think that's fantastic and I'm sure every study has its challenges. I'm interested what were the challenges you encountered in doing this study and which one of them may be unique to the way you have a lot of different people analyzing different aspects of the data versus the regular challenges that would come up in a study like this. Caitrin M.:                           Yeah so some of it I think is just keeping everyone on track and keeping it organized, making sure I think some of our challenges with this study was just me making I think on a lot of other studies, while I had certainly hands on the data it was more of an oversight rule for some pieces of it and just making sure everything looked the way that I thought it did, double checking. Some of it I think the teaching aspect of it just making sure everything was also done correctly and then keeping everything organized made the study a little bit more challenging. Caitrin M.:                           I think part of it too was with the PEAR study, it is a very rich data set. Determining what we wanted for our prioritization scheme and how to work through the different types of data sets that we had and put it all together as initially we just assign each student a different piece and we had a vague plan but it was a little bit more tricky as to work through how it was all coming together then when everyone came back together since a lot of people were doing as opposed to just one person doing it. Jane Ferguson:                 Right, so yeah and I think you're touching on the part that all of us have when we're writing papers that you sometimes end up with a lot of data at the beginning, you're trying to sift through it and then sometimes at a certain point you see something and you're like, "Okay, yes. This is interesting." Then you start following it up. Jane Ferguson:                 I wonder at what point did that happen? I suppose you probably ... You ran the GWAS for plasma renin activity and then find a number of suggested SNPs that were significant you associated but then ... Describe your strategy and you did so the second screening stuff to look at the pharmacological aspect defining [crosstalk 00:15:12]? Caitrin M.:                           Yeah, our initial plan going in was the first two steps, to do the GWAS for plasma renin activity and then to do the prioritization through blood pressure responses. I was very familiar with what our lab was familiar with but then after we got there, I think we were then troubled with what we did next and where to go. When we decided to bring in the RNA seq data, I think that was when it really started coming together as our top signal, the SNN-TXNDC11 gene region really stuck out then and it showed up. That seemed like a much stronger signal and it gave us a little bit more focus and also brought it much more of a functional aspect where we would maybe start to believe that signal more. That I think was really when we did that more of a turning point for the study and helped us focus more on where then to go with the results. Jane Ferguson:                 As far as the data you had I think over 700 people for your GWAS. Then you had a pretty large number of ... Was it the same subjects or different subjects where you also had the RNA seq data to do the QTL analysis? Caitrin M.:                           The same subject so not everyone has RNA seq. We have RNA seq data on 50 individuals and they were selected from whites and at the extremes of the blood pressure response so that it has a slightly interesting selection process. It's the main data analysis there was a best responder, worse responder to thiazide diuretics. Caitrin M.:                           When we do the eQTL analysis, we aren't always sure what we're going to get since we're missing the middle of blood pressure response. But, when we're just looking strictly at eQTL analysis sometimes we get lucky and sometimes it looks weird. Jane Ferguson:                 In your case as well you had the added issue of subjects were randomized to drug treatment so it was some where responders were ... I guess some people got the drug that worked for them and some people did not get the drug that worked for them. Caitrin M.:                           Yeah. Jane Ferguson:                 Did you I guess were incorporating both groups so good responders to either and some of that was because of their gene. They got the right drug for their genotype. Caitrin M.:                           Exactly, yeah. Jane Ferguson:                 It's good and then you were able to replicate this. After you were able to prioritize your gene region based on the GWAS and the drug response and the eQTL data, you actually ended up being able to go to a second sample to replicate the association right? Caitrin M.:                           Yes, it was in a lot of the same investigators, we have a second study PEAR-2, which has a very similar design to the PEAR study but used different drugs but also collected baseline plasma renin activity. We were able to use that phenotype again. We did have slight differences in GWAS to imputation panels at that point in time for when we were conducting this study so we ended up using a proxy to replicate but we did see the same signal in the second population which was very nice to see. Jane Ferguson:                 What is known about this gene region or either of these two genes? Caitrin M.:                           Overall, that was I think one of our harder points when we started trying to make the connections back to our phenotype. This was one of the areas where we did also have help from our students and the students as that was part of their initial training where they really looked to see what function was of various different genes and how to follow them up. That was one area where they came in, was to help look up some of the function of these ... there have been some connections with the various genes, the other phenotypes and with SNN and to atherosclerosis and other inflammatory cytokines such as TNF-alpha. Then there have been data also from [inaudible 00:19:37] that really show that there is an eQTL in this region that which supports what we saw in our own data. However, there really wasn't any direct connections with renin and the renin angiotensin aldosterone system and blood pressure regulation that we could find in the literature. Caitrin M.:                           We're not exactly sure how it connects but based off of our functional data and levels of evidence and then we saw some of that in publicly available data, we're still very interested in the region. Jane Ferguson:                 I think the data is compelling enough that it looks like you've identified the new region that probably is the mechanistically related that will require a whole bunch of basic mechanistic research to figure out what exactly the genes in this region are doing and how this ultimately connect back to blood pressure and response to drug therapy. Caitrin M.:                           Exactly. Jane Ferguson:                 I could see this ... I mean obviously there's a whole lot of potential functional work there and then probably also the clinical work, I wonder what you think about how this would affect any pharmacogenetic therapeutic ... You know at present I think you can look at plasma renin activity and use that as a predictor of drug response to help guide therapies. Would you think that a genotype guided therapy may end up being more effective than the plasma renin activity measurement in this case? Caitrin M.:                           In this case since this is so connected with a phenotype that you could use with plasma renin, I think if you're able to draw a plasma renin you may just want to do that. I think our overall goal would be if someone had preexisting genetic data and you weren't wanting to do an additional test or if you're contemplating response to a lot of different drugs that perhaps you could use a genetic data. One of the issues that was brought up on review and that are a lot of group considers quite a lot is that we have a lot of signals and that our group has certainly published a lot in this area and there's a lot of signals that we have to a lot of different drugs and how do you incorporate all of them together, is there overlap between them or where do they all fall? Caitrin M.:                           That is certainly something that we're still working on as more I think ultimate goal would be more to delve more of a SNP score or gene score and some type of risk score that would help you determine what drug you would best respond to. We've done that a little bit in some of our prior publications but we haven't yet taken all of our data together and help to build something that would if you had a lot of data on an individual and various different alleles at various different genes, how that would respond. Caitrin M.:                           Overall, when we look at blood pressure response as a pharmacogenetic signal, certainly we see larger affect sizes than you would in disease genetics but we're not seeing affect sizes like you would with more of an adverse drug event. We're in between there and we're often times it's not necessarily just going to be one SNP or one gene that would tell your whole story but a combination of quite a few of them. Jane Ferguson:                 I wonder are there more similar stories like this from the same data set? You know you've been through this process from start to finish and building in the functional work and do you think that next year's class will be able to do this again with the same data set? That maybe pick one of the next priority candidate down the list and maybe find another interesting story like this? Caitrin M.:                           Yeah, so we actually just finished our class this year and they looked at potassium. We just got done grading final papers and submitting grades so we will over the summer be working with them a little bit more. I think some of our new graduate students too are starting to work on trying to make more connections between a lot of our different phenotypes and as you start to layer those together what it exactly means for a patient or implementation perspective. Jane Ferguson:                 Yeah, interesting. We'll have to look for that story whenever you guys get done with it. Otherwise, are you planning on following up this specific SNP region in any other way or any other studies? Where's next for you guys overall? Caitrin M.:                           I think one of the things we would like to do is look at this more in PEAR-2. We really just brought the PEAR-2 data set in here as replication of the top region in that last stage but we have that data set and we can certainly look at that data set. Caitrin M.:                           The other thing that I would like to do is as we started this project in conjunction with the class that was a couple of years ago at this point in time, we used [TAP/MAP 3 00:24:35] imputed data since that was what we had in the lab and what we were using at that point in time. At this point in time, we have now imputed both PEAR and PEAR-2 2000 genomes phase three data. It'd be interesting to see if we are able to see any additional signals or if these regions become stronger or exactly what would happen using a more imputation panel that has more coverage and where we would have the same panel between both PEAR and PEAR-2. Jane Ferguson:                 Right because you may or may not have identified the causal SNPs in the previous access but- Caitrin M.:                           Yeah. Jane Ferguson:                 -yeah so it'd be nice to see if you can actually get that out. That potentially could end being a drugable target maybe suitable for something more specific but who knows. Is there anything else that we haven't covered yet that you'd like to mention? Caitrin M.:                           Overall, I think that just this type of model of utilizing more of a real world analysis and data in a class project really certainly engages our students a lot and I think they all enjoy actually being able to work with data that came out of this study and have a lot more hands-on experience and really project-based analysis experience. We've been very happy with this model and have used it multiple times. We have an HDL paper, the renin paper, our glucose response paper and now we're working on the potassium project. It's been a good model for us here with our pharmacogenomics class. Jane Ferguson:                 Yeah, I mean I think it's a really smart and intuitive way to think about education. It's mutually beneficial it sounds like, so it's helping you guys get your data analyzed. It's really helping the students learn so I think it's a win-win situation. I think it's a model that a lot of other people would really be interested in adopting. Caitrin M.:                           Yeah. Jane Ferguson:                 Okay well thanks so much for talking to me and talking about your model and your research. It's been great. Caitrin M.:                           Yes, thank you very much for having me. Jane Ferguson:                 That's it from us for May. Thank you for listening and come back for more next month.  

Jane Ferguson:                Hi, everyone. Welcome to Episode Four of Getting Personal: -Omics of the Heart." I'm Jane Ferguson, an assistant professor at Vanderbilt University Medical Center. This month, we have a special feature from early career member, Andrew Landstrom, who went to the Heart Rhythm Scientific Sessions in Chicago earlier this month and talked to some of the scientists who presented their research. So listen on for interviews Andrews conducted with Anneline te Riele, discussing the challenges and opportunities related to incidental findings in genetic testing, with Ernesto Fernandez, describing his research into whole exome sequencing and Long QT syndrome, and with David Tester, discussing novel variance and pathway analysis in Sudden Infant Death Syndrome. Andrew :                           My name is Andrew Landstrom and I am from the Baylor College of Medicine Department of Pediatrics' section on Cardiovascular Disease. I'm here at the 2017 Heart Rhythm Society Scientific Sessions. Anneline, will you tell us a little bit more about yourself, and what brought you to HRS? Anneline:                          Sure. So my name is Anneline Te Riele, I am a physician from The Netherlands. I finished my medical training in 2012 basically, in The Netherlands, and I started doing a PhD on ARVC in a combined project of our Netherlands patient as well as a group at Hopkins. So what brought me to HRS? I think of course the science. There's a lot of very good science. Actually, I think it's the best meeting for my purposes. Andrew :                           Absolutely. So will you just start by telling us a little bit about the spectrum of genetic testing in the clinic and about both the opportunities and the challenges that it brings? Anneline:                          Sure. So what we do in clinic, and I think this is really the challenge that we're facing currently, is we have moved from just testing on gene or one small panel of genes to bigger panels and then to whole exome or even whole genome sequencing. And I think the good part of that is that in certain cases, certain well-selected cases, you'll get a higher change of actually finding that gene that is responsible for disease.                                            On the contrary, it also leads to a lot of incidental findings. So findings that you were not expecting based on the phenotype of the patient and then you need to deal with those abnormalities that you've found and that brings on a lot of challenges as well for the family but also for us as physicians. Do we then need to screen those families, what do we do with this patient, do we treat them with medical therapies or drugs or do we give them ICDs? That kinds of question. So that I think is a virtually important part of what we're currently dealing with in clinical practice. Andrew :                           It does seem to be a very widespread problem. And here in the US of course we have the American College of Medical Genetics guidelines about reporting a variance. How do you think that that plays into the increased genetic uncertainty here in the US at least? Anneline:                          So that's a great questions. In 2013, the ACMG produced a guideline on which genes to report if you find these incidental findings. So 24 of these genes, and that's actually a big number, 24 of these genes are cardiovascular genes and that's mainly because changes in cardiovascular genes may detrimental effects down the line and really cause death or certain morbidities that are really important for the patient so we do need to deal with that.                                            And the problem with the ACMG guidelines and especially the pathogenicity guidelines is that they require two aspects. They basically require first that the variant was seen before in other cardiomyopathies or in this case other patients with disease. And that's really difficult for cardiomyopathy genes because these are large genes, they have a lot of novel or private mutations in there, so it's really hard to fulfill that requirement of having been seen before.                                            And the second thing is that the ACMG guidelines require functional studies as another proof of evidence of pathogenicity and of course, I think we would all like to do that in all of our patients, but it's just not feasible for financial purposes and all that. So that's a problem that we're facing. There are options and solutions but I think we'll talk about that later, but yeah, I think that's a problem that we're facing. Andrew :                           So on the one hand you have the ability to make a diagnostic decision based on a clear finding, but oftentimes the threshold to calling it a clearly pathologic variant is very high and oftentimes it never rises to that so it becomes more genetic uncertainty. Anneline:                          Yeah. I think that's basically right. And of course in an ideal world, we'll have certainty and say this is likely or this is definitely pathogenic, and this is likely or definitely benign, but in the real world, really, I think maybe even 80, 90% of the cases were in that gray zone in between and we need to deal with that. Andrew :                           Yeah, yeah. And you had some great resources that both scientists and clinicians alike can apply to these unknown, uncertain variants that might clarify things at least a little bit, and what are these tools? Anneline:                          So of course, from a traditional perspective, we have always looked at in silico predictive programs, we'll look at segregation data, and I think they're all very important, but they all have limitations, so for example, in silico predictive programs, they likely overcall mutations deleterious and segregation data is nothing more than evidence of pathogenicity of a locus to a disorder, not necessarily that variant, so the new things that are on the horizon, and a thing that could be the future of [inaudible 00:06:04] interpretation is collaborative project so really we should be collaborating, we should not be having our own little islands. The collaboration is the key here.                                            And collaborative efforts in the US have been for example, ClinVar and NHLBI funded effort, as well as ClinGen and ClinGen, or Clinical Genome, is perhaps the, at least it claims to be, the authoritative central resource to go back to that curates variants as being pathogenic yes or no. And I think these databases, ClinVar finally has a database entry, so the variants will be in ClinVar, but ClinGen provides an expert panel of individuals who will curate these variants as being pathogenic yes or no. I think that is a central resource that we should all be aware of. I know these are not the only ones, there are other collaborative efforts out there.                                            I mean, there are ways to connect clinicians, so for example, Match Maker Exchange is a website that you could use to enter your variant and the phenotype of the patient and you submit your own information and then you'll get matches in other databases, but not only your own match shows up. So if, say, two years later, another physician comes up and looks for the same variant, you'll get a pop up, which will actually be very nice for these clinicians to get in touch. So that's, I think, the feature ... future of variant interpretation is collaboration. That's basically my, I think my main important message here. Andrew :                           I think that's absolutely right. I think this has become sort of a big data question that requires many perspectives, and a lot of resources to be able to curate accurately. What are some of the limitations of these tools that you've seen that kind of, you have to keep in mind in terms of trying to determine whether a variant is truly pathologic or not with a patient that you have sitting in front of you? Anneline:                          So that is, I mean, of course, there's many limitations in the things that we currently do because there's so much that we don't know. But for example, to give you an example, ClinVar I think, is one central resource that we should all be aware of and if you go to ClinVar, there is actually data from two years ago, and I'm sure the numbers are high if we would look now, but if we look in ClinVar two years ago, we already saw that of the, say 120,000 variants that were in the database, 21% of these variants were called VUSes but if you look at these variants, 17% of the cases, the labs or the individual submitters of ClinVar didn't agree on the actual classification of that variant.                                            So the limitations that we all should be aware of is that there is not one single solution and you should look for evidence and really research your variants. So look at Popmap, look at what is out there, look the patient of course, look at the clinical phenotype, does it match what you think the gene should be doing or not, or is it completely unrelated? And then of course search these databases but be aware of the fact that there may be errors there.                                            Another thing I want to highlight too is that we typically go to population databases, so Exome Variant Server, ExAC, I think these are very popular databases that we use to look at the frequency of variants in a selected population. But really these databases may have sub-clinical disease patients, so I know ExAC has three NYBPC-3 mutations that are known to cause HCM, so this is something to keep in mind. There's not a gold standard truth if you open these databases, but you should have multiple pieces of information when interpreting your variant. Andrew :                           And that's a good point. I think with a lot of these cardiomyopathies and channelopathies, particularly some of the more frequent ones, when you have a database of 60,000 people, at least a couple of them are going to have disease. Anneline:                          Yeah. I think that is part of the problem. I mean HCM is pretty prevalent, I mean one in 500 individuals likely, I mean these are recent numbers, has the disease. So I think the cutoff of a minor allele frequency of five percent, which is in the ACMG guidelines, I think is way too high for this disease. So this is what the cardiovascular expert panel of ClinGen has done, so they ... This is, ClinGen, as you might know, Clinical Genome, is a one-on-one team of curators that know the framework of ClinGen and then there is disease experts that are very well accustomed with the disease and the genes associated with it. So they provide teams and these teams work together, and the cardiovascular expert group has recently published a modified, or customized, ACMG guidelines on how to deal with the intricacies of the cardiomyopathies and for example, NYH-7 which is the first genotype deposed in ClinGen or in ClinVar finally.                                            So they modify that cutoff, the minor allele frequency of five percent, which is the BA-1 ACMG guideline cutoff, they changed that to 0.1% and I think that's exactly what you were saying, that is important to keep in mind, some of the cardiomyopathies are way more prevalent so you should not consider that if you see it in a population database that you think that it's, then it's normal, it's not necessarily the case because this is a prevalent disease. Andrew :                           Yeah, and particularly when commercial genetic testing companies all can't agree that a variant is bad, and we all can't agree that a healthy variant may or may not be good, there is definitely a lot of genetic uncertainty there. Anneline:                          Exactly, exactly. Andrew :                           Now, whole-exome sequencing certainly has its role clinically, even with that genetic uncertainty that we spoke about, but it has a clear role in genetic discovery as well. Anneline:                          Sure. Andrew :                           And you were part of a very recent paper, and you led a very long list of authors, speaking more about your collaborative approach to genetics research that evaluated a novel substrate for ARVC, is that correct? Anneline:                          Yes. So this is something I'm actually pretty proud of. As you said, it's a collaborative effort, so it literally take a village to do these kind of studies and we're lucky enough to collaborate with a lot of people who are interested in the same topic. So what we did ... and I metnioned to you in the beginning, I come from the ARVC field ... So what we did is we had one ARVC patient that was discovered by whole-exome sequencing to carry an SCN5A variant and we, in and of itself, found that that was very interesting, because SCN5A, as you know, has been associated with Brugada syndrome predominantly but many other cardiomyopathies as well, so DCM, even ACM. There's been a lot of controversy about SCN5A in that matter.                                            So the computational data, the population data, it all pointed to the fact that this variant may be pathogenic, but we weren't really able to connect those dots just yet. So we then collaborated with the group in NYU with Mario Delmar, who did, first of all, functional studies on the sodium channel, but what was nice is that he was able to use his novel method of super-resolution microscopy which is a way in which we can look at the nano-scale structure of the cardiomyocytes, or really the small, small levels of molecules that you see in these cells. And what we did is we found that not only NAV1.5 which is the gene product of SCN5A but also [inaudible 00:13:53] which is an adherence structure molecule, which links the cells together was actually less present in our ARVC patient compared to the control. And this was in the IPS so cardiomyocyte molecule, which we corrected using CRISPR-Cas9 technology so I think at least in current practice, on of the best pieces of evidence that we can get.                                            So I think this shows that our SCN5A variant, I mean, in this case, probably really was pathogenic, but also in a pathophysiological standpoint, explains to us how SCN5A mutations, which are typically thought to be only affecting the sodium channel, can also lead to cardiomyopathy phenotype which has implications beyond the ARVC world, but also in DCM I think this is a nice finding of collaboration that I think ... I hope more people will look into this. Andrew :                           Absolutely I think the trouble with SCN5A is exactly like you were saying, it's been implicated in Long QT, Brugada Syndrome, SIDS, [inaudible 00:14:57], now ARVC, and even nodal disease, like sinus syndrome and things like that. So the ability to show sort of mechanistically, that while you have a change in your sodium channel gating that you also have a change in the way that the cells can connect with each other and form contractile force is, I guess, key to your study. Anneline:                          Yeah, yeah. I think this really, I mean, I'm hoping at least, it was also finally published in a journal that looks more into functional studies, so not necessarily only genetics, and I think we need to work closely not only on the genetic side, but look closely at the pathophysiological standpoint for gene discovery purposes because this will really explain to us why one gene is implicated in one disease, and also it points to possible directions to perhaps stop the disease process and treat these patients, which I think is vital in our clinical practice. Andrew :                           So are SCN5A mutations in ARVC a common finding or are they rare? Anneline:                          So they are pretty rare. I mean, we do find them every now and then and maybe they're modifiers. So what we did to follow up on that one individual, we check 281 ARVD patients who were screened just by regular screening, not by whole-exome but we did a targeted screening of SCN5A and we found five variants in these 281 patients, so that's two percent. I mean, it's still rare, but it is as rare as any other minor gene causing ARVC, but it is a rare feature, so I mean, I think it could be a player. And interestingly, the phenotype didn't change much. It wasn't really different from the ARVC patients without an SCN5A mutation which is reassuring.                                            What we also saw is that the prevalence of mutations in those with desmosomal mutations. So ARVC is, as you know, typically associated with diseases or mutations in the desmosome. It was more often seen in those without a desmosomal mutation. That was almost double as frequent as in those with a desmosomal mutation. So it does give us some direction to the fact that this may be a player out there. I mean of course it's not Plakophilin-2 which is the major player, I think, in ARVC, but I think it may cause a, at least a certain form of cardiomyopathy of arrhythmogenic cardiomyopathy that we need to be aware of. Andrew :                           And how do you think your new discovery of SCN5A being associated with ARVC, how do you think that plays into the bigger discussion we were having about expansive genetic testing and what that may mean for a patient as far as diagnostic utility but also limitations of variant interpretation? Anneline:                          That's a great question. So I think we should be cautious of saying this gene causes only this disease, and I think this is a common feature not only in ARVC but in a lot of cardiomyopathies and even in channelopathies. I think the concept of one gene causes one disease is outdated. We know that multiple genes have multiple effects and this SCN5A, of course the gene product is NAV1.5 which is the major alpha subunit of the sodium channels so it is really not the canonical function of SCN5A or NAV1.5 that causes cardiomyopathy here but it's a non-canonical function so I think we should be aware of the fact that gene products have different functions and that there can be overlap of the cardiomyopathies. So of course I think we should be screwing SCN5A in our ARVC patients and I'm hoping a lot of labs and a lot of physicians are already doing that, but it's really not the only thing that is associated with ARVC. So that's important to keep in mind. Andrew :                           What do you think the next steps are for sort of broadening the implication of your finding? Anneline:                          So what we are doing currently, and is a little bit of a sneak peek, because this data is not really out there yet, but we have, in this cohort, we found these five variants in 281 individuals, and we're currently working on one of these individuals to get another IPSO cardiomyocyte cell line and look into the functional components to that. And interestingly, this variant, that exact variant in that ARVC patient was also found in a Brugada Syndrome patient. So wouldn't it be nice to actually set them side by side and see what the differences are?                                            Of course this is a little bit of a future music, if you know what I'm saying, like this is something that we don't have just yet, but I think what we need to figure out is how epigenetic or environmental factors play into this field and to explain how one gene or one variant, even, can cause opposite functional effects in different phenotypes. Andrew :                           What do you think is needed to help clarify some of the genetic uncertainty you see clinically? Anneline:                          I think a lot of collaboration, a lot of money, quite frankly. I think we need to ... I mean, the functional data is really helping us not only for understanding that single variant, but also for gene discovery, and as I said, for treatment down the line, that is necessary, and I think the variant of uncertain significance, I mean, if we all live on our little islands and only do our little practices, then we're not going to go a lot further. So we need to work together to understand what your patient has in this variant, my patient had in that variant, and this is our phenotype, so we need to connect those dots to be able to make certain conclusions. Andrew :                           Well, I'm all for collaboration, as well as additional money, that's good. Anneline:                          Good. Andrew :                           Well, thank you so much for spending time with us. Anneline:                          Sure. Andrew :                           And again, congratulations on a wonderful presentation. Anneline:                          Thank you very much. Andrew :                           I'm joined by Dr. Ernesto Fernandez from the Baylor College of Medicine to talk about his research project. Ernesto, I'm wondering if we can just start by introducing yourself and what your project is. Ernesto:                            I am a second-year pediatric resident, I'm applying to a cardiology fellowship right now and I'm interested in, obviously, all aspects of pediatric cardiology. We're trying to figure out whether testing for Long QT genes or Long QT syndrome is actually warranted in otherwise healthy individuals. We're trying to see what the yield is on these testings, specifically whole-exome sequencing. Andrew :                           And I think this project really hits on an important point, whereby, because we've been able to interrogate the genome more comprehensively with clinical testing, that we've run into more incidentally identified variants. And these variants can pop up in genes, like the genes responsible for Long QT syndrome. Talk a little bit more about these variants, what the implication is of finding these variants incidentally, and what your project hoped to target as far as the diagnostic value of these variants. Ernesto:                            Yeah. So I guess the answer to your first question is that we are coming up with these marvelous new techniques of analyzing the genome and now we're using whole-exome sequence testing to look up is someone has any exome that's abnormal and this has caused a huge problem whereby we're now finding all these variants that we don't really know what they mean. We call them variants of undetermined significance.                                            Our study is basically premised by the fact that if you have no underlying suspicion for any arrhythmic disease, there's really no need or no indication to be referred for whole-exome sequencing testing, given that the most likely result is a variant that we don't really know what it means. And it's probably going to be benign. Andrew :                           So on the one hand, you have a well-established gene panel that's being used for diagnostic purposes with you index of suspicion being high for Long QT syndrome versus something like a whole-exome gene screen where somebody may not be thinking about Long QT syndrome as a diagnosis and have low pre-test suspicion but then comes back with a variant found in these genes sort of incidentally. Is that sort of the dichotomy you're drawing? Ernesto:                            Yeah. I think the best way of explaining it is through Bay's Theorem whereby if you have someone with a high index of suspicion when you start off to have sudden cardiac death, a family history of an arrhythmic disease, and you get a test for it, such as a gene panel for Long QT syndrome, and they come up with a positive test result, then you're going to say, "Oh. I should probably evaluate this further," whereas if you have someone who has some dysmorphism, they have delay, they might have seizures, but there's no family history of sudden cardiac death, no personal history of syncope, then there's really no need to send off this big gun, the whole-exome sequence, because you're likely to either get a normal variant or you're likely to get a variant that we don't know what to make of. Andrew :                           So I think, Ernesto, that nicely summarizes the clinical question that you had in mind. What was your hypothesis going into the study, and how did you seek to approach that hypothesis, sort of experimentally? Ernesto:                            So we came up with the hypothesis that if you have an incidentally identified variant within the whole-exome sequencing tests without any other clinical suspicion, it's likely to represent a benign finding. We went about by analyzing the data from the Baylor Miraca labs on the whole-exome sequencing data that they achieved, and we looked specifically at individuals who had gotten these tests and found to have a variant of undetermined significance, or had a pathologic variant for either one or all 17 of the genes for Long QT syndrome. We compared them to individuals who had known Long QT syndrome that had undergone genotype testing, and we [inaudible 00:25:21] these individuals from the literature. And we wanted to compare the whole-exome sequencing cohort to individuals who were otherwise healthy and had obtained a whole-exome sequence. So these are patients or individuals from the well-established ExAC database that are believed to be ostensibly healthy individuals. Andrew :                           So if I understand you correctly, you're comparing this unknown cohort, that being the rare variants found in whole-exome sequencing, against a positive control cohort of pathologic cases versus a negative control cohort of healthy individuals derived from the ExAC database to look for whether those west variants are more similar to the cases or the controls. With regards to the west cohort, what was the prevalence of individuals with these incidentally identified variants, how many did you find? Ernesto:                            So we actually found just about 49% of individuals had some variant in Long QT syndrome gene, and noted that about 12% of them had a mutation in the major causes of Long QT syndrome, and just over a third, or 36% had a mutation in the more rare causes of long QT syndrome. Andrew :                           That's a pretty surprising finding. So you're saying that one in two individuals who get whole-exome sequencing sent for whatever reason, have a variant in a Long QT-associated gene? Ernesto:                            That's what the data suggests. Andrew :                           And where did you go from here? Ernesto:                            So from there, we went onto compare the variant frequency between the case's cohort, those individuals with known Long QT syndrome, those individuals in our west cohort from the Baylor Miraca labs, and those individuals from the ExAC database who are otherwise healthy. So we noted that in our west cohort, there was about 13% of individuals who had a positive variant in the Long QT syndrome one through three genes, the major causes of Long QT syndrome. When we compare that to the ostensibly healthy individuals from the ExAC database, it was 12% in that study that had some variant in Long QT syndrome genes that are major causes of Long QT syndrome itself.                                            This was statistically similar, it was indistinguishable. And then when we compared it to the pathologic cases, it was actually about 50% of those cases who had a positive variant in a Long QT syndrome gene one through three. Andrew :                           So there was a relatively low frequency of individuals who had variants in one of the big three Long QT genes in both controls and the west cohort, and was obviously much higher among individuals with a diagnosis of Long QT syndrome. Ernesto:                            Yep. That's exactly what we found. Andrew :                           And where did you go from here? Ernesto:                            And then from there, we had a good idea that there was probably a big difference between cases and west, but we wanted to make sure, gene by gene, that there was no difference between our west cases and the ExAC database, the control cases. So we mapped each variant frequency by gene for the major causes of Long QT syndrome. There was no statistically significant difference between the west and the controls. Andrew :                           So the gene frequencies between the controls and the west were indistinguishable and very much different, both of them, it would seem, to the pathologic cases. Ernesto:                            Correct. Andrew :                           And you then looked at the position of these variants, the actual amino acid residues, correct? Ernesto:                            Yeah. So we looked at, for KCNQ1, KCNH2, and SCM5A, the three major causes of Long QT syndrome, one, two, three respectively, and we mapped out the amino acid positions where there was actually a mutation for each individuals. So the cases, controls, and pathologic cohorts. We determined the percent overlap between the west cohort and the controls and the percent overlap between the west cohort and the cases and noticed that for all three, there is a huge preference for west and control versus west and cases. Andrew :                           So if you're a west variant you're more likely to reside in the residue also occupied by a healthy individual variant as opposed to a pathologic variant? Ernesto:                            Yeah. Exactly. Andrew :                           And so what did you do next? You retrospectively looked at some of the charts of the patients who were seen at Texas Children's Hospital, correct? Ernesto:                            Mm-hmm (affirmative). So then we had 223 total individuals that had an incidentally identified variant within one of the major three genes, the Long QT syndrome genes. We looked at the reasons for their referrals and noticed that the vast majority of individuals were referred for some developmental delay, for some dysmorphism, for a non-cardiac cause, and then it was only about 23% of these individuals that actually had a reason for referral that was cardiac in nature. And less than on percent of individuals were referred for a solely cardiovascular reason. And we concluded that it's unlikely that these individuals were referred for a cardiac reason, as the data suggests, and that as a result, the index of suspicion for an arrhythmia is likely lower in these individuals. Andrew :                           And what did you find when you looked at the charts of those individuals? Ernesto:                            We had EKG data for a good number of them, and we excluded individuals who obviously had no EKG data, and we excluded individuals who had some congenital abnormality and then anyone with any other arrhythmia that would make the QTC interpretation more difficult, such as interventricular conduction defects.                                            We ended up with 62 individuals and 61 of them had a normal QTC, so there was no evidence of QT prolongation at all. There was one individual who was left who had borderline elevated QTC of 460, which was our cutoff for borderline elevation and this individual had actually been seen by pediatric cardiology at Texas Children's Hospital and found to have ... a history of syncope and it was found to be non-cardiogenic in nature. Andrew :                           So matching the variant data which suggested that you had likely found background variation in the west, you found no evidence of Long QT syndrome in these individuals who had variants in Long QT genes. Ernesto:                            That's correct. So, the overall percent was very similar between the healthy individuals and the west individuals. The variant frequencies were almost indistinguishable, and then the variant co-mapping for all, for both the west and the controls, was preferential to the western cases. So that kind of matched what we found in our study, that there was no clinical suspicion or clinical diagnosis of Long QT syndrome in these individuals who had been found incidentally. Andrew :                           Well that sounds to me to be a pretty big finding. Ernesto:                            Yeah. I think it's pretty important to get this information out there. Andrew :                           So what do you think the take home message for your study is? Ernesto:                            I think the take home message is if you don't have a suspicion of Long QT syndrome or of an arrhythmia, there's low likelihood that such a big gun test as the whole-exome sequence is likely going to change your mind. Andrew :                           So Ernesto, what would you advise a cardiologist who maybe gets a patient in clinic with a chief complaint of a VUS in a Long QT associated gene picked up on west, what would you advise based on your study findings? Ernesto:                            They're going to have to determine their own pre-test suspicion. They're going to have to get a good history and physical, probably get a baseline EKG to determine what the QTC intervals are, and if there's really no other clinical suspicion for Long QT syndrome, they're likely to be able to provide reassurance at that point in time. Andrew :                           Ernesto, what do you think the next steps are for this project, and what do you think still needs to be done in the field to reinforce your conclusions? Ernesto:                            I think my study is one of the early studies of this field, so getting more studies like this and other channelopathies, getting not just looking at Long QT one through three but looking at all of them, and in patients who've been evaluated at Texas Children's or any other institution would be helpful. And then moving forward to give more credence to the idea that if you have history that's reassuring and physical exam that's reassuring, then you probably don't need to have further testing. Andrew :                           What do you recommend if your index of suspicion is high for Long QT syndrome, so maybe a QTC in the low 480s, maybe a family history of syncope or seizures, do you think whole-exome sequencing is the way to go? Ernesto:                            Right now, that's probably not the best test, given all these incidental findings that we don't really know what to do with. There's other tests that are more high-tailored for those specific diseases, like Long QT syndrome panel among others, that are probably more likely to give you a positive post-test probability. Andrew :                           So testing for the disease you're suspicious for as opposed to testing indiscriminately? Ernesto:                            Yeah. Andrew :                           So Ernesto, thank you so much for taking the time our of your day to speak with us. Ernesto:                            Thank you, Andrew. Andrew :                           I'm here with David Tester, senior research technologist working with Mike Ackerman at Mayo Clinic, and he just gave a wonderful talk on whole-exome sequencing and next-generation sequencing as an unbiased look to determine underlying causes of Sudden Infant Death Syndrome, or SIDS. So David, I'm wondering if you can introduce yourself and talk a little bit about your project. Dave:                                 Sure. I'm Dave Tester and I'm at the Mayo Clinic, again with Mike Ackerman. Dr. Ackerman and I have been together for about 18 years now, with a real focus on genetics of sudden cardiac death disorders. So this latest study was looking at whole-exome sequencing in a population of SIDS cases in collaboration with Dr. Elijah Behr at St. George's University in London.                                            And really the approach, what we were aiming for is really kind of two-fold. First we were looking to determine what is the yield of ultra-rare variance within genes that have been implicated in cardiovascular disorders? These would be the cardiac channelopathies and some of the cardiomyopathies such as ACM or ARVC, for example.                                            And the second thing that we were wanting to look at was can we use this to search for sort of novel candidate genes for Sudden Infant Death Syndrome susceptibility? And so we took that aim and really the main result was to show that about 14% of our SIDS cases had what we term potentially informative variants. And those are going to be variants that were within sort of the major channelopathy genes that are implicated in Long QT syndrome or CPVT as well as loss of function variants within the 90 ICC genes that we had examined.                                            Using the ACMG guidelines for determining the pathogenicity of variants, about 4.3% of our SIDS cases hosted an ACMG guideline predicated likely pathogenic to pathogenic variant. And most of those variants represent either a frame shift or splice site error variance really in minor cardiomyopathy genes and channelopathy genes. So there's still a lot of work that needs to be done in terms of looking at specifically missense variance within channel genes and that sort of thing, and really kind of functionally characterizing those to determine whether or not they truly are pathogenic or if they should remain variants of uncertain significance. Andrew :                           And so you took a very complex disease like SIDS with probably a number of differens ideologies and found a pretty good percentage have suspicious variants, that 14% or so, and then 4% had variants that were so suspicious they would meet American College of Medical Genetics guidelines for being a possible or likely pathologic variant. Where do you think this study lies in sort of the continuum of identifying the genetic ideology of SIDS, and what do you think these findings sort of add to that overall picture? Dave:                                 Well I think these findings in general really just kind of show the complexity of SIDS. Whether or not SIDS is really truly genetic or not, or perhaps it just, if it's not monogenic, perhaps it's polygenic, and so those are some things that we should be considering and looking at. Now some of those questions might be able to be answer through our whole-exome sequencing data set that we have, and I think those are really going to be kind of the next phases.                                            We can also take and do some pathway analyses of the exome sequencing data, for example, and see our variance kind of lining up on certain pathways that may contribute to certain pathologies that could contribute to SIDS. Andrew :                           And in your study, you had a few genes where the number of variants that were found in SIDS cases were higher than in your controls. Can you speak some more about what those genes may tell you in the context of pathway analysis for SIDS? Dave:                                 Yes. So there was ... There were not genes that came out with sort of a genome-wide significance level. But there were at least 400 genes that had a p-value of 0.05 over representation in SIDS versus our ethnic match controls and 17 of those genes have a p-value of 0.005 and we're really kind of focused on some of those that have a little bit higher p-value for us to assess. A few of those genes may represent biologically plausible candidate genes for SIDS and we were kind of actually going through and considering which ones we'd like to follow up on in terms of function. Some of these genes do play a role in, say, cardiorespiratory system and function of the heart as well as in the brain. Andrew :                           So then given all these findings, and the fact that you may have some candidate genes and candidate pathways that might be interesting to look at further, what are the next steps that you think would help this project move forward, and what do you think the field of Sudden Infant Death Syndrome and Sudden Unexplained Death Syndrome needs to kind of move forward? Dave:                                 Well I think from a genetic standpoint, the study that we just complete was really on a large set of unrelated infants that had died suddenly. We did not have access to parental DNA and so moving forward in terms of the genetics, I think incorporating sort of a trio analysis I think would get at the question of sort of [inaudible 00:42:01] variance for example. The other things, in terms of genetic standpoint is perhaps looking at different genetic mechanisms. Whether these are copy number variance that may be missed by exome sequencing, perhaps some of the SIDS could be due to epigenetic abnormalities or even small chromosomal abnormalities that perhaps may not be detected on certain arrays on there being used. So I think going forward, kind of taking those approaches to look for sort of unique genetic variation. Andrew :                           Well Dave, thank you so much for taking the time to speak with me and congratulations on a great project. Dave:                                 All right, great, thank you. Jane Ferguson:  Thanks to Andrew for highlighting the interesting precision medicine research presented at HRS and thanks to you all for listening. We'll be back with more next month.

重庆小面 这一碗里，装满了岁月的故事。——Mr.Nav1山环水绕，雾气氤氲。在这座山城的中心有一座不高的丘陵，这是渝中区龙脊。从李子坝到佛图关，从三层马路到鹅岭之巅，这些弯曲的山路上镶嵌着无数家喻户晓的餐馆。拾级而上就是在翻越重庆的美食地图一般。就像武侠小说里的英雄，最后的归宿总是大隐隐于市。在桂花园路的深处就隐藏着一家非著名小面馆。似乎没人能说得清楚这家店开于何年何月，又在这里多久了。只是，这一带的老住户每天早上都会端起这家店的一碗小面，吃个痛快，一天不吃仿佛少了点什么似的。也不知道是面条更劲道，还是海椒更鲜香，亦或是配料更丰富？这家几个平米的店面的门口总是坐满了、站满了、蹲满了人。要不是因为桂花园路是一条背街小巷，恐怕排队吃面的人都会把机动车道截断。西装革履的上班族挨着提笼架鸟的老年人坐，亿万身家的大老板挨在山城棒棒军的旁边，这幅奇怪又和谐的画面就出现在这家店的门口。抬头一看，瓦片下面挂着一块久经风霜的牌匾，看起来既不庄重也不美观，甚至上面的字似乎都是手写的：兄弟面庄。2听这一带的老人儿讲，这家面馆是由兄弟俩经营的。说是兄弟俩，可是这俩人长得那是一点血缘关系也没有的那般迥异。大哥一脸沧桑，似乎五千年的历史都写在脸上，而二弟却永远神采焕发看不到岁月的痕迹。有好事者猜测也许他们是异姓兄弟，也许其中一个是他们父母捡来的孩子。不过这些说法也得不到佐证，只是捕风捉影罢了。兄弟俩听到了都只是默默一笑，性格温和纯良这一点，两个老男人还真是非常像兄弟俩了。邻里邻居的都知道陈大哥对陈老幺特别好，脏活累活都不让弟弟上手。夏天热的让人发狂的日子里，陈大哥从来不让弟弟进厨房，只让他在外面招呼客人。至于打佐料这类的繁琐的活计也是由大哥包办。这样亲密的关系，让人觉得虽然哥俩长得真的不像，但是的确是手足情深。3清晨5点13分，吃面的客人还没蜂拥而至的时候。旁边猪肉铺的王屠户端着一只空碗进来了，把碗撂在桌子上就转身走了。一边走还留一句话飘在半空中：“今天的面钱还是记账，把碗特意给你们还回来是怕你们碗不够用……”最后几个字还余音袅袅的，似乎带着点猪油的黏腻劲儿。陈老大撇撇嘴笑，对弟弟说：“这个老王，一个月吃面的钱差不多能抵这个月我们买他家的猪肉钱了。”弟弟在门外温文尔雅的说：“老王家的猪肉质量的确好，咱家的生意也多亏老主顾帮衬。”他说话的嗓音里面透着一丝年轻的感觉，中气十足，完全不似大哥那烟熏火燎的嗓子。杂货铺的赵婆婆慢慢的走进来了，挑了一张离送面窗口最近的桌子缓慢的坐下来。她坐在这里是为了同时能看见在厨房忙碌的陈老大和门口招呼客人的老二。她一直一个人生活，承蒙兄弟俩照顾着，所以在感情上她更觉得这哥俩是自己的儿子而不是街坊。陈姓兄弟俩在面馆里养的那只肥嘟嘟的橘猫走过来，在赵婆婆的腿上来回的蹭着，蹭了一会就乖乖的趴在了她的脚边。“老大，我怎么这几天没有看见你家娃儿啊？”婆婆问道。“啊……嬢嬢，他前几天不是高考完了嘛，出了成绩之后跟同学出去旅游啦。”老大的腔调里充满了自豪。隔壁桌的客人问：“老板你家孩子考了多少分啊？”陈老幺抢着回答：“630多分哟！我大侄子可争气咯！”赵婆婆又问：“630分能考哪个学校呀？我这老婆子也不懂这些。”隔壁桌的客人听见分数,立刻肃然起敬的说：“嬢嬢，630分差不多可以上清华北大哟！我孩子也考大学，这瓜娃子不争气，上个专科就不错了。”陈老大听见了立即更正说：“上不了清华北大，他报的人民大学不知道能不能考得上呢。”陈老大不好意思的笑笑，觉得嘴角甜甜的。“都是我嫂子教子有方，我大哥天天在面馆哪有空看孩子的功课哟。”众人一直都沉浸在老陈学霸儿子的话题之中，在这个安详而宁静的早晨。4叶时雨穿着一件丝质中式对襟上衣，脚蹬一双黑色布鞋走在街上。他的这身打扮略显老气，可是看皮肤完全没有他刻意包装出来的那种年代感。他今天要去与房屋中介谈一套房屋出售的事宜。一般情况下这样的事情他都不会亲自去谈的，只是今天陈老大儿子在家里招待同学，脱不开身，所以他只好躬亲了。“陈先生，您看一下这是您这套房产目前的总价341万，如果资料确认无误就请您在这里签字。”房屋中介小姐一脸谄媚的看着叶时雨，觉得这个男人应该怎么看都是个成功的企业家，穿着打扮都和某宝的马大大一样的style。“陈先生，在我看来目前房价上涨那么快，您该长期持有不该出手的……”叶时雨也不理她的话问道：“这样就完成手续了么？”“是的陈先生。您提出过，不想接触买家。所以都有我们全权处理了。”“那很好。”他简略的回答“就这样。”售楼小姐再见俩字都没落地，他已经走出去老远了。5兄弟面庄过了中午就不营业了，这在一般的餐馆看来是不可想象的。有人说他们只做早场，是因为他们家的汤头要花时间熬制，所以下午开始必须关门准备食材。有人说他们家女人都不出来干活，老爷们儿都是懒散惯了不愿意太过劳累。还有人说这兄弟俩经营面馆多年有很厚的积蓄，是不差这点钱的，毕竟晚上吃小面的人要少一些。陈老幺清楚这些说的都没错，但是还有一点没说对，那就是他和哥哥有一个天大的秘密。报摊的小王看见陈老幺走过来打招呼：“诶哟，陈哥这才下午三点多您就关板了？这日子过的也太潇洒了！怪不得您保养的这么好，这多年硬是不见你变老。”陈老二打着哈哈就搪塞了过去，但是心里的阴影满满弥漫上来，他想：“最近说我年轻的人也来越多了，难道又到了该搬家的时候了？”6成都，水井街。这条街住着一个漂亮的遗孀，她的美貌闻名遐迩。尤其是那一头黑发中的一缕白发，美的惊心动魄。她的丈夫战死疆场，她一个人带着三胞胎儿子生活。她特别会做泡菜，蓝色的坛子、金黄色的银杏叶、紫色的卤水，她的泡菜色香味都是一流的。他还是少年的时候就吃过她做的泡菜，他觉得这真的是天上才有的美味啊。几块酸萝卜，就可以送下一大碗饭。狼吞虎咽吃完之后，她总是用精致的手绢给他揩干净嘴角的饭粒，然后温柔的看着少年。少年心里想：怪不得蓉姐姐出嫁的时候，有一个叫骡子的轿夫不辞千山万水，也要护送她安全抵达夫家。他是深深的眷恋蓉姐姐的吧，如果我长大了之后也能娶到这样的女子就好了。蓉，比他想象的更耐老一些。他也是。7他的父母早亡，很小就要一个人出来谋生，幸好家里给他留下了一点点遗产——做面条的手艺。裤带面，铺盖面，圆的细面，扁的粗面，韭菜叶面……一个少年竟然能做出这么多花样。那个时候他挑着一个扁担走街串巷卖着担担面，味道非常鲜美深受大家喜爱。慢慢的他发现了自己身上的秘密，他长得比别人慢。孩提时代的玩伴，长着长着就变成了他的哥哥姐姐！他的时间坐标系好像和正常人都不一样。渐渐的流言四起，有人说他是一个妖怪，没有人敢接近他，只有蓉姐姐敢和他说话。在极度的自卑和恐惧中他决定远走他乡，到一个陌生的地方重新开始。他一路向东沿着官道走，又翻过来几座大山，就看到了这座被山环水绕的城，重庆。他的味蕾在少年时就被泡菜激活，又经历过麻婆豆腐的洗礼，他又借鉴了担担面的做法，发明了一种风味面条。一碗里面只有不多的一小把面条，蔬菜煮熟铺底，加上猪油、油辣子、海椒、味精、糖、醋、酱油、花椒面、花生碎、榨菜粒、芝麻酱等等不同的佐料，以非常微妙的配比混合。有的面浇上高汤，有的面直接干拌，有的面配有肥肠和牛肉、有的面配有豌豆和杂酱，就形成了“不可言说之小的重庆小面。”他的第一家面馆：川渝面馆，开门迎客。一个地方住久了，总有细心的人发现，他不会变老。每当这个时候，他就不得不放弃原有的居住地，搬家。所以他这貌似只有43岁的年纪却走过了300多年的岁月。这期间也在身后留下了无数的面馆：三鲜面、梯坎面、一碗面馆、周记面庄……他也有过无数的姓氏和化名。这些年唯一不变的就是他做的小面的品质。随着岁月的积累他的技艺更加炉火纯青。8公元纪年法的56年前，他在山中隐居的时候，路过一座废弃的农舍，却听见里面隐隐传来婴儿的哭声。他走进去查看，发现了一个襁褓中的婴儿，由于很久没进食哭声细若游丝。他立即动了恻隐之心打算带回去先救活了再说。抱起孩子的时候，看见包袱的里面塞着一张纸条：陈六娃。这可能是婴儿的名字吧，除此之外再无他物。他没有喂养婴儿的经验，真是手足无措。往往这个时候就显得家里缺一个女人。他想起来蓉姐姐，美丽而贤惠。他少年时是暗恋过蓉的，但是她也许在200多年前就去世了吧！他知道自己不会变老，担心没有任何女人敢和自己这样一个怪物生活在一起，于是他总是不近女色。即使是这样，他也有过很多风流韵事，只不过都非常短暂。就真的没有一个女人让他彻底心动，不管自己的怪异，不管世俗爱一次么？当然有。他陪她一起度过了漫长的一生，目睹她老去、死亡并亲手把她埋葬。他注视着水面上倒映着的自己这张不老的脸，他想，自己是个正常人该多好啊！往事历历在目，却不能不管这个濒死的婴孩。他用米汤把他养大，并且尊重了孩子生身父母的愿望，就叫他陈六娃，而他自己也改名叫陈昌福。六娃发现，开始他是爸爸，后来变成哥哥，再后来变成弟弟！陈六娃知道叶时雨所有的秘密，并帮他保守一切。连六娃后来娶的老婆陈嫂对这一切都搞不大清楚。兄弟俩的日子过的讳莫如深，他们本不用如此辛劳的卖面条，因为叶时雨积累的财富是惊人的。但是他觉得做小面是他生命很重要的一部分。就好像这一头是他自己，另一头牵着他所有的过去。9进入新千年，重庆搞了一个什么“重庆小面一百强”评选活动。兄弟面庄当然是榜上有名，且位列探花。他们的面馆也越来越出名，可是叶时雨不想这样。他只想过简单的生活，和他的养子亦或者是“大哥”安安静静的隐居在桂花园这条小巷子里。一个早春的清晨他迎来了一个特别的客人。据说这个年轻人可以用舌头感知食材生前的故事或者是厨师的心情。叶时雨像是故意想看看这个年轻人到底有几分功力，于是就让六娃退到一旁，自己亲自操刀做了一碗干熘碗杂面。拿云吃了第一口，就惊讶的望着叶时雨说：“老板！你！？……”他没等说什么，叶时雨用眼色止住了他说：“我只是一个普通的师傅，老板是我的哥哥陈老大。”神秘的笑了。后来，拿云十年如一日的来到这家面馆，和叶时雨混的非常熟识。后来又来了一个漂亮的少女跟在拿云的后面像个小尾巴。再后来他看见女孩子对拿云表白，漫天的银杏叶像一场金色的梦。他抬头望着秋风里的天色，自言自语道：“又该搬家咯。”  作者：安业2017年7月12日110984489@qq.com  

重庆小面 这一碗里，装满了岁月的故事。——Mr.Nav1山环水绕，雾气氤氲。在这座山城的中心有一座不高的丘陵，这是渝中区龙脊。从李子坝到佛图关，从三层马路到鹅岭之巅，这些弯曲的山路上镶嵌着无数家喻户晓的餐馆。拾级而上就是在翻越重庆的美食地图一般。就像武侠小说里的英雄，最后的归宿总是大隐隐于市。在桂花园路的深处就隐藏着一家非著名小面馆。似乎没人能说得清楚这家店开于何年何月，又在这里多久了。只是，这一带的老住户每天早上都会端起这家店的一碗小面，吃个痛快，一天不吃仿佛少了点什么似的。也不知道是面条更劲道，还是海椒更鲜香，亦或是配料更丰富？这家几个平米的店面的门口总是坐满了、站满了、蹲满了人。要不是因为桂花园路是一条背街小巷，恐怕排队吃面的人都会把机动车道截断。西装革履的上班族挨着提笼架鸟的老年人坐，亿万身家的大老板挨在山城棒棒军的旁边，这幅奇怪又和谐的画面就出现在这家店的门口。抬头一看，瓦片下面挂着一块久经风霜的牌匾，看起来既不庄重也不美观，甚至上面的字似乎都是手写的：兄弟面庄。2听这一带的老人儿讲，这家面馆是由兄弟俩经营的。说是兄弟俩，可是这俩人长得那是一点血缘关系也没有的那般迥异。大哥一脸沧桑，似乎五千年的历史都写在脸上，而二弟却永远神采焕发看不到岁月的痕迹。有好事者猜测也许他们是异姓兄弟，也许其中一个是他们父母捡来的孩子。不过这些说法也得不到佐证，只是捕风捉影罢了。兄弟俩听到了都只是默默一笑，性格温和纯良这一点，两个老男人还真是非常像兄弟俩了。邻里邻居的都知道陈大哥对陈老幺特别好，脏活累活都不让弟弟上手。夏天热的让人发狂的日子里，陈大哥从来不让弟弟进厨房，只让他在外面招呼客人。至于打佐料这类的繁琐的活计也是由大哥包办。这样亲密的关系，让人觉得虽然哥俩长得真的不像，但是的确是手足情深。3清晨5点13分，吃面的客人还没蜂拥而至的时候。旁边猪肉铺的王屠户端着一只空碗进来了，把碗撂在桌子上就转身走了。一边走还留一句话飘在半空中：“今天的面钱还是记账，把碗特意给你们还回来是怕你们碗不够用……”最后几个字还余音袅袅的，似乎带着点猪油的黏腻劲儿。陈老大撇撇嘴笑，对弟弟说：“这个老王，一个月吃面的钱差不多能抵这个月我们买他家的猪肉钱了。”弟弟在门外温文尔雅的说：“老王家的猪肉质量的确好，咱家的生意也多亏老主顾帮衬。”他说话的嗓音里面透着一丝年轻的感觉，中气十足，完全不似大哥那烟熏火燎的嗓子。杂货铺的赵婆婆慢慢的走进来了，挑了一张离送面窗口最近的桌子缓慢的坐下来。她坐在这里是为了同时能看见在厨房忙碌的陈老大和门口招呼客人的老二。她一直一个人生活，承蒙兄弟俩照顾着，所以在感情上她更觉得这哥俩是自己的儿子而不是街坊。陈姓兄弟俩在面馆里养的那只肥嘟嘟的橘猫走过来，在赵婆婆的腿上来回的蹭着，蹭了一会就乖乖的趴在了她的脚边。“老大，我怎么这几天没有看见你家娃儿啊？”婆婆问道。“啊……嬢嬢，他前几天不是高考完了嘛，出了成绩之后跟同学出去旅游啦。”老大的腔调里充满了自豪。隔壁桌的客人问：“老板你家孩子考了多少分啊？”陈老幺抢着回答：“630多分哟！我大侄子可争气咯！”赵婆婆又问：“630分能考哪个学校呀？我这老婆子也不懂这些。”隔壁桌的客人听见分数,立刻肃然起敬的说：“嬢嬢，630分差不多可以上清华北大哟！我孩子也考大学，这瓜娃子不争气，上个专科就不错了。”陈老大听见了立即更正说：“上不了清华北大，他报的人民大学不知道能不能考得上呢。”陈老大不好意思的笑笑，觉得嘴角甜甜的。“都是我嫂子教子有方，我大哥天天在面馆哪有空看孩子的功课哟。”众人一直都沉浸在老陈学霸儿子的话题之中，在这个安详而宁静的早晨。4叶时雨穿着一件丝质中式对襟上衣，脚蹬一双黑色布鞋走在街上。他的这身打扮略显老气，可是看皮肤完全没有他刻意包装出来的那种年代感。他今天要去与房屋中介谈一套房屋出售的事宜。一般情况下这样的事情他都不会亲自去谈的，只是今天陈老大儿子在家里招待同学，脱不开身，所以他只好躬亲了。“陈先生，您看一下这是您这套房产目前的总价341万，如果资料确认无误就请您在这里签字。”房屋中介小姐一脸谄媚的看着叶时雨，觉得这个男人应该怎么看都是个成功的企业家，穿着打扮都和某宝的马大大一样的style。“陈先生，在我看来目前房价上涨那么快，您该长期持有不该出手的……”叶时雨也不理她的话问道：“这样就完成手续了么？”“是的陈先生。您提出过，不想接触买家。所以都有我们全权处理了。”“那很好。”他简略的回答“就这样。”售楼小姐再见俩字都没落地，他已经走出去老远了。5兄弟面庄过了中午就不营业了，这在一般的餐馆看来是不可想象的。有人说他们只做早场，是因为他们家的汤头要花时间熬制，所以下午开始必须关门准备食材。有人说他们家女人都不出来干活，老爷们儿都是懒散惯了不愿意太过劳累。还有人说这兄弟俩经营面馆多年有很厚的积蓄，是不差这点钱的，毕竟晚上吃小面的人要少一些。陈老幺清楚这些说的都没错，但是还有一点没说对，那就是他和哥哥有一个天大的秘密。报摊的小王看见陈老幺走过来打招呼：“诶哟，陈哥这才下午三点多您就关板了？这日子过的也太潇洒了！怪不得您保养的这么好，这多年硬是不见你变老。”陈老二打着哈哈就搪塞了过去，但是心里的阴影满满弥漫上来，他想：“最近说我年轻的人也来越多了，难道又到了该搬家的时候了？”6成都，水井街。这条街住着一个漂亮的遗孀，她的美貌闻名遐迩。尤其是那一头黑发中的一缕白发，美的惊心动魄。她的丈夫战死疆场，她一个人带着三胞胎儿子生活。她特别会做泡菜，蓝色的坛子、金黄色的银杏叶、紫色的卤水，她的泡菜色香味都是一流的。他还是少年的时候就吃过她做的泡菜，他觉得这真的是天上才有的美味啊。几块酸萝卜，就可以送下一大碗饭。狼吞虎咽吃完之后，她总是用精致的手绢给他揩干净嘴角的饭粒，然后温柔的看着少年。少年心里想：怪不得蓉姐姐出嫁的时候，有一个叫骡子的轿夫不辞千山万水，也要护送她安全抵达夫家。他是深深的眷恋蓉姐姐的吧，如果我长大了之后也能娶到这样的女子就好了。蓉，比他想象的更耐老一些。他也是。7他的父母早亡，很小就要一个人出来谋生，幸好家里给他留下了一点点遗产——做面条的手艺。裤带面，铺盖面，圆的细面，扁的粗面，韭菜叶面……一个少年竟然能做出这么多花样。那个时候他挑着一个扁担走街串巷卖着担担面，味道非常鲜美深受大家喜爱。慢慢的他发现了自己身上的秘密，他长得比别人慢。孩提时代的玩伴，长着长着就变成了他的哥哥姐姐！他的时间坐标系好像和正常人都不一样。渐渐的流言四起，有人说他是一个妖怪，没有人敢接近他，只有蓉姐姐敢和他说话。在极度的自卑和恐惧中他决定远走他乡，到一个陌生的地方重新开始。他一路向东沿着官道走，又翻过来几座大山，就看到了这座被山环水绕的城，重庆。他的味蕾在少年时就被泡菜激活，又经历过麻婆豆腐的洗礼，他又借鉴了担担面的做法，发明了一种风味面条。一碗里面只有不多的一小把面条，蔬菜煮熟铺底，加上猪油、油辣子、海椒、味精、糖、醋、酱油、花椒面、花生碎、榨菜粒、芝麻酱等等不同的佐料，以非常微妙的配比混合。有的面浇上高汤，有的面直接干拌，有的面配有肥肠和牛肉、有的面配有豌豆和杂酱，就形成了“不可言说之小的重庆小面。”他的第一家面馆：川渝面馆，开门迎客。一个地方住久了，总有细心的人发现，他不会变老。每当这个时候，他就不得不放弃原有的居住地，搬家。所以他这貌似只有43岁的年纪却走过了300多年的岁月。这期间也在身后留下了无数的面馆：三鲜面、梯坎面、一碗面馆、周记面庄……他也有过无数的姓氏和化名。这些年唯一不变的就是他做的小面的品质。随着岁月的积累他的技艺更加炉火纯青。8公元纪年法的56年前，他在山中隐居的时候，路过一座废弃的农舍，却听见里面隐隐传来婴儿的哭声。他走进去查看，发现了一个襁褓中的婴儿，由于很久没进食哭声细若游丝。他立即动了恻隐之心打算带回去先救活了再说。抱起孩子的时候，看见包袱的里面塞着一张纸条：陈六娃。这可能是婴儿的名字吧，除此之外再无他物。他没有喂养婴儿的经验，真是手足无措。往往这个时候就显得家里缺一个女人。他想起来蓉姐姐，美丽而贤惠。他少年时是暗恋过蓉的，但是她也许在200多年前就去世了吧！他知道自己不会变老，担心没有任何女人敢和自己这样一个怪物生活在一起，于是他总是不近女色。即使是这样，他也有过很多风流韵事，只不过都非常短暂。就真的没有一个女人让他彻底心动，不管自己的怪异，不管世俗爱一次么？当然有。他陪她一起度过了漫长的一生，目睹她老去、死亡并亲手把她埋葬。他注视着水面上倒映着的自己这张不老的脸，他想，自己是个正常人该多好啊！往事历历在目，却不能不管这个濒死的婴孩。他用米汤把他养大，并且尊重了孩子生身父母的愿望，就叫他陈六娃，而他自己也改名叫陈昌福。六娃发现，开始他是爸爸，后来变成哥哥，再后来变成弟弟！陈六娃知道叶时雨所有的秘密，并帮他保守一切。连六娃后来娶的老婆陈嫂对这一切都搞不大清楚。兄弟俩的日子过的讳莫如深，他们本不用如此辛劳的卖面条，因为叶时雨积累的财富是惊人的。但是他觉得做小面是他生命很重要的一部分。就好像这一头是他自己，另一头牵着他所有的过去。9进入新千年，重庆搞了一个什么“重庆小面一百强”评选活动。兄弟面庄当然是榜上有名，且位列探花。他们的面馆也越来越出名，可是叶时雨不想这样。他只想过简单的生活，和他的养子亦或者是“大哥”安安静静的隐居在桂花园这条小巷子里。一个早春的清晨他迎来了一个特别的客人。据说这个年轻人可以用舌头感知食材生前的故事或者是厨师的心情。叶时雨像是故意想看看这个年轻人到底有几分功力，于是就让六娃退到一旁，自己亲自操刀做了一碗干熘碗杂面。拿云吃了第一口，就惊讶的望着叶时雨说：“老板！你！？……”他没等说什么，叶时雨用眼色止住了他说：“我只是一个普通的师傅，老板是我的哥哥陈老大。”神秘的笑了。后来，拿云十年如一日的来到这家面馆，和叶时雨混的非常熟识。后来又来了一个漂亮的少女跟在拿云的后面像个小尾巴。再后来他看见女孩子对拿云表白，漫天的银杏叶像一场金色的梦。他抬头望着秋风里的天色，自言自语道：“又该搬家咯。”  作者：安业2017年7月12日110984489@qq.com  

In what’s being called a genetic ‘Rosetta Stone', researchers at the University of California, San Francisco have discovered molecules that can lead to infantile epilepsy or autism. Specifically, a neuronal protein called NaV1.2 and the gene that produces it, called SCN2A. Both play a crucial role in early brain development, according to the neuroscientist Kevin Bender. “One of the central functions of the brain cell is to fire something that is called an action potential, which is a way of transmitting information from one cell to the next. And SCN2A and NaV1.2 are at the heart of that process.” Genetic defects that increase neuronal activity as they transmit signals lead to epilepsy, and when this process abnormally slows down, it may cause autism. “We now have a roadmap for understanding how different mutations in SCN2A could lead to different diseases and if we understand that, it might uncover new areas for intervention in the future.”

Guest: Simon Tate, Chief Scientific Officer, Convergence Pharmaceuticals Bio and Contact Info Listen (6:09) Sodium channel blockers and the Nav1.7 protein

ATX-II wurde ursprünglich aus dem Gift der Seeanemone Anemonia sulcata isoliert und ist als potenter Aktivator spannungsgesteuerter Natriumionenkanäle (Navs) bekannt, da es einen persistierenden Na+-Strom induziert. Vor Kurzem wurden bestimmte Nav Subtypen mit menschlichem Schmerz in Verbindung gebracht. Somit liegt es nahe, dass detaillierte Kenntnisse über die Regulierung von Navs für die Entwicklung neuer Pharmaka im Bereich der Schmerztherapie von Vorteil sind. Auch der durch einen alternativen Inaktivierungsmechanismus entstehende resurgent current spielt nach Erkenntnissen der jüngsten Zeit eine Rolle im Schmerzgeschehen. Er kann einen Angriffspunkt für neue Analgetika darstellen, so man seine Entstehung und mögliche Modifikationen entschlüsseln kann. Das Ziel dieser Arbeit war es, zu untersuchen, ob ATX-II resurgent currents von Navs in großen und kleinen murinen DRGs verändern bzw. induzieren kann. Zudem sollte beleuchtet werden, ob und wenn ja, welche Wirkung das Toxin auf periphere A- und C Schmerzfasern hat. Die Auswirkungen von ATX-II auf Navs wurden in großen und kleinen DRGs der Maus sowie in Zelllinien mit whole-cell voltage-clamp Messungen untersucht. Gesunde humane Probanden bewerteten Empfindungsänderungen (Schmerz und Juckreiz) nach intrakutaner Injektion von ATX-II. Mit Hilfe des "Laser Doppler Imagers" wurde das mögliche Auftreten eines Axon Reflex Erythems untersucht. Die mittels des FACS Zellsortierers nach Zellgröße sortierten DRGs wurden mit RT-qPCR auf ihren Gehalt an Nav1.6, Nav1.7 und 4 mRNA untersucht. ATX-II verstärkte resurgent currents in großen DRGs, zeigte in kleinen jedoch keinen Effekt. Im heterologen Expressionssystem trat ein resurgent current erst auf, wenn 4 Peptid über die Intrazellulärflüssigkeit zur Verfügung stand. Korrelierend zu den Befunden auf Zellebene rief ATX-II bei intrakutaner Injektion im Menschen nur solange stechenden Schmerz und veränderte mechanische Empfindung hervor, wie die A-Fasern für die Schmerzleitung verfügbar waren. Nachdem ein Nervenkompressionsblock angelegt und die Leitung via A-Fasern blockiert war, waren die ATX-II vermittelten Effekte verschwunden. Zudem konnte kein Axon Reflex Erythem gefunden werden, das ein Zeichen für eine C-Schmerzfaser-Aktivierung wäre. Die Ergebnisse dieser Arbeit legen nahe, dass geringe Konzentrationen von ATX-II eine selektive Wirkung auf große DRGs haben. Erst durch Hinzufügen von 4-Peptid ist es möglich mit dem Toxin resurgent current in kleinen DRGs sowie Zelllinien mit heterolog exprimierten Nav1.6 und Nav1.7 zu induzieren. Dies lässt darauf schließen, dass es kleinen DRGs an 4 in ausreichender Menge mangelt und deshalb kein resurgent current sichtbar ist. Ebenso übt ATX-II einen Effekt auf A Fasern aus, die mit großen DRGs verbunden sind, wohingegen C-Fasern weitgehend unbeeinflusst bleiben. Die intrakutane Injektion von ATX-II führt zu stechendem Schmerz und einer Juckreiz ähnlichen Empfindung, die sich durch A-Faser-Block verhindern lässt. Daraus kann man schließen, dass ATX-II in niedrigen Konzentrationen als spezifischer A-Faser Aktivator fungiert.

Pax6 encodes a specific DNA-binding transcription factor that regulates the development of multiple organs, including the eye, brain and pancreas. Previous studies have shown that Pax6 regulates the entire process of ocular lens development. In the developing forebrain, Pax6 is expressed in ventricular zone precursor cells and in specific populations of neurons; absence of Pax6 results in disrupted cell proliferation and cell fate specification in telencephalon. In the pancreas, Pax6 is essential for the differentiation of α-, β- and δ-islet cells. To elucidate molecular roles of Pax6, chromatin immunoprecipitation experiments combined with high-density oligonucleotide array hybridizations (ChIP-chip) were performed using three distinct sources of chromatin (lens, forebrain and β-cells). ChIP-chip studies, performed as biological triplicates, identified a total of 5,260 promoters occupied by Pax6. 1,001 (133) of these promoter regions were shared between at least two (three) distinct chromatin sources, respectively. In lens chromatin, 2,335 promoters were bound by Pax6. RNA expression profiling from Pax6⁺/⁻ lenses combined with in vivo Pax6-binding data yielded 76 putative Pax6-direct targets, including the Gaa, Isl1, Kif1b, Mtmr2, Pcsk1n, and Snca genes. RNA and ChIP data were validated for all these genes. In lens cells, reporter assays established Kib1b and Snca as Pax6 activated and repressed genes, respectively. In situ hybridization revealed reduced expression of these genes in E14 cerebral cortex. Moreover, we examined differentially expressed transcripts between E9.5 wild type and Pax6⁻/⁻ lens placodes that suggested Efnb2, Fat4, Has2, Nav1, and Trpm3 as novel Pax6-direct targets. Collectively, the present studies, through the identification of Pax6-direct target genes, provide novel insights into the molecular mechanisms of Pax6 gene control during mouse embryonic development. In addition, the present data demonstrate that Pax6 interacts preferentially with promoter regions in a tissue-specific fashion. Nevertheless, nearly 20% of the regions identified are accessible to Pax6 in multiple tissues.

1) Small fiber neuropathy 2) Topic of the month: Diagnosis of MCI and AD. This podcast for the Neurology Journal begins and closes with Dr. Robert Gross, Editor-in-Chief, briefly discussing highlighted articles from the print issue of Neurology. In the second segment Dr. Ted Burns interviews Dr. Steve Waxman about his paper on small fiber neuropathy. Dr. Jennifer Fugate is reading our e-Pearl of the week about diffusion-weighted imaging. In the next part of the podcast Dr. Jeff Burns interviews Dr. John Morris about other biomarkers for mild cognitive impairment and Alzheimer disease. Next week they will discuss preclinical Alzheimer disease. All participants have disclosures.Dr. Burns serves as Podcast Editor for Neurology®; performs EMG studies in his neuromuscular practice (30% effort); and has received research support from the Myasthenia Gravis Foundation of America and Knopp Neurosciences Inc..Dr. Waxman serves on the editorial boards of Clinical Neuroscience, The Neurologist, Neurobiology of Disease, Clinical Neurology and Neurosurgery, SYNAPSE, Molecular Neurobiology, Clinical Neuroscience Research, Neuron-Glia Biology, Neurotherapeutics, Trends in Molecular Medicine, Molecular Pain and Channels; served on the editorial board Journal of Neurotrauma; serves on the scientific advisory board for Brain and Nature Reviews Neurology; Editor-in-Chief of The Neuroscientist and Neuroscience Letters; Editor of The Journal of Physiology; Section Head Multiple Sclerosis, Faculty of 1000 Medicine; is or has been a consultant for Cardiome Pharmaceutical, Bristol-Myers Squibb, Vertex Pharmaceutical, ChromaCell and DaiNippon Sumitomo Pharmaceuticals; receives research support from Pfizer Inc., The Erythromelalgia Association and Department of Veterans Affairs; holds stock options in SITE ONE Research for serving on its Advisory Board and is listed as an inventor for a patent held by Yale University on the Invention of sodium channel NaV1.9.Dr. Fugate serves on the editorial team for the Neurology® Resident and Fellow Section. Dr. Jeff Burns serves on the editorial board of the Journal of Alzheimer's Disease; has served on a scientific advisory board for the American Academy of Physician Education; has received publishing royalties for Early Diagnosis and Treatment of Mild Cognitive Impairment (Wiley Press, 2008) and Dementia: An Atlas of Investigation and Diagnosis (Clinical Publishing, 2007); serves on the speakers' bureau for Novartis; has served as a consultant for Medacorp Consulting, Johnson County Clinical Trials, and PRA International; receives research support from Elan Corporation, Janssen, Wyeth, Pfizer Inc, Danone, and the Dana Foundation; and has served as an expert witness in legal proceedings regarding competency.Dr. Morris serves on scientific advisory boards for Eisai Inc., Esteve, Janssen Alzheimer Immunotherapy Program, GlaxoSmithKline, Novartis, Otsuka Pharmaceuticals Co., Ltd., and Pfizer Inc; serves on the editorial advisory board of Annals of Neurology; receives royalties from the publications of the books Mild Cognitive Impairment and Early Alzheimer's Disease, Dementia, Handbook of Dementing Illnesses, 2nd edition and for an editorial in Lancet Neurology; and receives research support from Janssen Alzheimer Immunotherapy Program and Pfizer Inc.

Background: Gain-of-function mutations of the nociceptive voltage-gated sodium channel Nav1.7 lead to inherited pain syndromes, such as paroxysmal extreme pain disorder (PEPD). One characteristic of these mutations is slowed fast-inactivation kinetics, which may give rise to resurgent sodium currents. It is long known that toxins from Anemonia sulcata, such as ATX-II, slow fast inactivation and skin contact for example during diving leads to various symptoms such as pain and itch. Here, we investigated if ATX-II induces resurgent currents in sensory neurons of the dorsal root ganglion (DRGs) and how this may translate into human sensations. Results: In large A-fiber related DRGs ATX-II (5 nM) enhances persistent and resurgent sodium currents, but failed to do so in small C-fiber linked DRGs when investigated using the whole-cell patch-clamp technique. Resurgent currents are thought to depend on the presence of the sodium channel beta 4-subunit. Using RT-qPCR experiments, we show that small DRGs express significantly less beta 4 mRNA than large sensory neurons. With the beta 4-C-terminus peptide in the pipette solution, it was possible to evoke resurgent currents in small DRGs and in Nav1.7 or Nav1.6 expressing HEK293/N1E115 cells, which were enhanced by the presence of extracellular ATX-II. When injected into the skin of healthy volunteers, ATX-II induces painful and itch-like sensations which were abolished by mechanical nerve block. Increase in superficial blood flow of the skin, measured by Laser doppler imaging is limited to the injection site, so no axon reflex erythema as a correlate for C-fiber activation was detected. Conclusion: ATX-II enhances persistent and resurgent sodium currents in large diameter DRGs, whereas small DRGs depend on the addition of beta 4-peptide to the pipette recording solution for ATX-II to affect resurgent currents. Mechanical A-fiber blockade abolishes all ATX-II effects in human skin (e.g. painful and itch-like paraesthesias), suggesting that it mediates its effects mainly via activation of A-fibers.

An Interview with Stephen Waxman, MD, author of Han C, Rush, AM, Sulayman D, et al. Sporadic onset of erythermalgia: A gain-of-function mutation in Nav1.7. Annals Neurol 2006;59:553-558. And Waxman SG, Sulayman D. Erythromelalgia: A hereditary pain syndrome enters the molecular era. Annals Neurol 2005;57:785-788. Interviewed by Ted Burns, MD and Erik Ensrud, MD

An Interview with Stephen Waxman, MD, author of Han C, Rush, AM, Sulayman D, et al. Sporadic onset of erythermalgia: A gain-of-function mutation in Nav1.7. Annals Neurol 2006;59:553-558. And Waxman SG, Sulayman D. Erythromelalgia: A hereditary pain syndrome enters the molecular era. Annals Neurol 2005;57:785-788. Interviewed by Ted Burns, MD and Erik Ensrud, MD

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.04.20.049106v1?rss=1 Authors: Moutal, A., Cai, S., Yu, J., Stratton, H. J., Chefdeville, A., Gomez, K., Ran, D., Madura, C. L., Boinon, L., Soto, M., Zhou, Y., Shan, Z., Chew, L. A., Rodgers, K. A., Khanna, R. Abstract: The sodium channel NaV1.7 is a master regulator of nociceptive neuronal firing. Mutations in this channel can result in painful conditions as well as produce insensitivity to pain. Despite being recognized as a 'poster child' for nociceptive signaling and human pain, targeting NaV1.7 has not yet produced a clinical drug. Recent work has illuminated the NaV1.7 interactome, offering insights into the regulation of these channels and identifying potentially new druggable targets. Amongst the regulators of NaV1.7 is the cytosolic collapsin response mediator protein 2 (CRMP2). CRMP2, modified at Lysine 374 (K374) by addition of a small ubiquitin-like modifier (SUMO), bound NaV1.7 to regulate its membrane localization and function. Corollary to this, preventing CRMP2 SUMOylation was sufficient to reverse mechanical allodynia in rats with neuropathic pain. Notably, loss of CRMP2 SUMOylation did not compromise other innate functions of CRMP2. To further elucidate the in vivo role of CRMP2 SUMOylation in pain, we generated CRMP2 K374A knock-in (CRMP2K374A/K374A) mice in which Lys374 was replaced with Ala. CRMP2K374A/K374A mice had reduced NaV1.7 membrane localization and function in female, but not male, sensory neurons. Behavioral appraisal of CRMP2K374A/K374A mice demonstrated no changes in depressive or repetitive, compulsive-like behaviors, and a decrease in noxious thermal sensitivity. No changes were observed in CRMP2K374A/K374A mice to inflammatory, acute, or visceral pain. In contrast, in a neuropathic model, CRMP2K374A/K374A mice failed to develop persistent mechanical allodynia. Our study suggests that CRMP2 SUMOylation-dependent control of peripheral NaV1.7 is a hallmark of chronic, but not physiological, neuropathic pain. Copy rights belong to original authors. Visit the link for more info