CGRP sensory neurons promote tissue healing via neutrophils and macrophages.

The immune system has a critical role in orchestrating tissue healing. As a result, regenerative strategies that control immune components have proved effective1,2. This is particularly relevant when immune dysregulation that results from conditions such as diabetes or advanced age impairs tissue healing following injury2,3. Nociceptive sensory neurons have a crucial role as immunoregulators and exert both protective and harmful effects depending on the context4-12. However, how neuro-immune interactions affect tissue repair and regeneration following acute injury is unclear. Here we show that ablation of the NaV1.8 nociceptor impairs skin wound repair and muscle regeneration after acute tissue injury. Nociceptor endings grow into injured skin and muscle tissues and signal to immune cells through the neuropeptide calcitonin gene-related peptide (CGRP) during the healing process. CGRP acts via receptor activity-modifying protein 1 (RAMP1) on neutrophils, monocytes and macrophages to inhibit recruitment, accelerate death, enhance efferocytosis and polarize macrophages towards a pro-repair phenotype. The effects of CGRP on neutrophils and macrophages are mediated via thrombospondin-1 release and its subsequent autocrine and/or paracrine effects. In mice without nociceptors and diabetic mice with peripheral neuropathies, delivery of an engineered version of CGRP accelerated wound healing and promoted muscle regeneration. Harnessing neuro-immune interactions has potential to treat non-healing tissues in which dysregulated neuro-immune interactions impair tissue healing.

Bacteria hijack a meningeal neuroimmune axis to facilitate brain invasion.

The meninges are densely innervated by nociceptive sensory neurons that mediate pain and headache1,2. Bacterial meningitis causes life-threatening infections of the meninges and central nervous system, affecting more than 2.5 million people a year3-5. How pain and neuroimmune interactions impact meningeal antibacterial host defences are unclear. Here we show that Nav1.8+ nociceptors signal to immune cells in the meninges through the neuropeptide calcitonin gene-related peptide (CGRP) during infection. This neuroimmune axis inhibits host defences and exacerbates bacterial meningitis. Nociceptor neuron ablation reduced meningeal and brain invasion by two bacterial pathogens: Streptococcus pneumoniae and Streptococcus agalactiae. S. pneumoniae activated nociceptors through its pore-forming toxin pneumolysin to release CGRP from nerve terminals. CGRP acted through receptor activity modifying protein 1 (RAMP1) on meningeal macrophages to polarize their transcriptional responses, suppressing macrophage chemokine expression, neutrophil recruitment and dural antimicrobial defences. Macrophage-specific RAMP1 deficiency or pharmacological blockade of RAMP1 enhanced immune responses and bacterial clearance in the meninges and brain. Therefore, bacteria hijack CGRP-RAMP1 signalling in meningeal macrophages to facilitate brain invasion. Targeting this neuroimmune axis in the meninges can enhance host defences and potentially produce treatments for bacterial meningitis.

Identification and targeting of a unique NaV1.7 domain driving chronic pain.

Small molecules directly targeting the voltage-gated sodium channel (VGSC) NaV1.7 have not been clinically successful. We reported that preventing the addition of a small ubiquitin-like modifier onto the NaV1.7-interacting cytosolic collapsin response mediator protein 2 (CRMP2) blocked NaV1.7 function and was antinociceptive in rodent models of neuropathic pain. Here, we discovered a CRMP2 regulatory sequence (CRS) unique to NaV1.7 that is essential for this regulatory coupling. CRMP2 preferentially bound to the NaV1.7 CRS over other NaV isoforms. Substitution of the NaV1.7 CRS with the homologous domains from the other eight VGSC isoforms decreased NaV1.7 currents. A cell-penetrant decoy peptide corresponding to the NaV1.7-CRS reduced NaV1.7 currents and trafficking, decreased presynaptic NaV1.7 expression, reduced spinal CGRP release, and reversed nerve injury-induced mechanical allodynia. Importantly, the NaV1.7-CRS peptide did not produce motor impairment, nor did it alter physiological pain sensation, which is essential for survival. As a proof-of-concept for a NaV1.7 -targeted gene therapy, we packaged a plasmid encoding the NaV1.7-CRS in an AAV virus. Treatment with this virus reduced NaV1.7 function in both rodent and rhesus macaque sensory neurons. This gene therapy reversed and prevented mechanical allodynia in a model of nerve injury and reversed mechanical and cold allodynia in a model of chemotherapy-induced peripheral neuropathy. These findings support the conclusion that the CRS domain is a targetable region for the treatment of chronic neuropathic pain.

Nav1.8 in small dorsal root ganglion neurons contributes to vincristine-induced mechanical allodynia.

Vincristine-induced peripheral neuropathy is a common side effect of vincristine treatment, which is accompanied by pain and can be dose-limiting. The molecular mechanisms that underlie vincristine-induced pain are not well understood. We have established an animal model to investigate pathophysiological mechanisms of vincristine-induced pain. Our previous studies have shown that the tetrodotoxin-sensitive voltage-gated sodium channel Nav1.6 in medium-diameter dorsal root ganglion (DRG) neurons contributes to the maintenance of vincristine-induced allodynia. In this study, we investigated the effects of vincristine administration on excitability in small-diameter DRG neurons and whether the tetrodotoxin-resistant (TTX-R) Nav1.8 channels contribute to mechanical allodynia. Current-clamp recordings demonstrated that small DRG neurons become hyper-excitable following vincristine treatment, with both reduced current threshold and increased firing frequency. Using voltage-clamp recordings in small DRG neurons, we now show an increase in TTX-R current density and a -7.3 mV hyperpolarizing shift in the half-maximal potential (V1/2) of activation of Nav1.8 channels in vincristine-treated animals, which likely contributes to the hyperexcitability that we observed in these neurons. Notably, vincristine treatment did not enhance excitability of small DRG neurons from Nav1.8 knockout mice, and the development of mechanical allodynia was delayed but not abrogated in these mice. Together, our data suggest that sodium channel Nav1.8 in small DRG neurons contributes to the development of vincristine-induced mechanical allodynia.

Unique electrophysiological property of a novel Nav1.7, Nav1.8, and Nav1.9 sodium channel blocker, ANP-230.

Voltage-gated sodium channel subtypes, Nav1.7, Nav1.8, and Nav1.9 are predominantly expressed in peripheral sensory neurons. Recent genetic studies have revealed that they are involved in pathological pain processing and that the blockade of Nav1.7, Nav1.8, or Nav1.9 will become a promising pharmacotherapy especially for neuropathic pain. A growing number of drug discovery programs have targeted either of the subtypes to obtain a selective inhibitor which can provide pain relief without affecting the cardiovascular and central nervous systems, though none of them has been approved yet. Here we describe the in vitro characteristics of ANP-230, a novel sodium channel blocker under clinical development. Surprisingly, ANP-230 was shown to block three pain-related subtypes, human Nav1.7, Nav1.8, and Nav1.9 with similar potency, but had only low inhibitory activity to human cardiac Nav1.5 channel and rat central Nav channels. The voltage clamp experiments using different step pulse protocols revealed that ANP-230 had a "tonic block" mode of action without state- and use-dependency. In addition, ANP-230 caused a depolarizing shift of the activation curve and decelerated gating kinetics in human Nav1.7-stably expressing cells. The depolarizing shift of activation curve was commonly observed in human Nav1.8-stably expressing cells as well as rat dorsal root ganglion neurons. These data suggested a quite unique mechanism of Nav channel inhibition by ANP-230. Finally, ANP-230 reduced excitability of rat dorsal root ganglion neurons in a concentration dependent manner. Collectively, these promising results indicate that ANP-230 could be a potent drug for neuropathic pain.

Comparison of Nocifensive Behavior in NaV1.7-, NaV1.8-, and NaV1.9-Channelrhodopsin-2 Mice by Selective Optogenetic Activation of Targeted Sodium Channel Subtype-Expressing Afferents.

Voltage-gated sodium channels, including NaV1.7, NaV1.8, and NaV1.9, play important roles in pain transmission and chronic pain development. However, the specific mechanisms of their action remain unclear, highlighting the need for in vivo stimulation studies of these channels. Optogenetics, a novel technique for targeting the activation or inhibition of specific neural circuits using light, offers a promising solution. In our previous study, we used optogenetics to selectively excite NaV1.7-expressing neurons in the dorsal root ganglion of mice to induce nocifensive behavior. Here, we further characterize the impact of nocifensive behavior by activation of NaV1.7, NaV1.8, or NaV1.9-expressing neurons. Using CRISPR/Cas9-mediated homologous recombination, NaV1.7-iCre, NaV1.8-iCre, or NaV1.9-iCre mice expressing iCre recombinase under the control of the endogenous NaV1.7, NaV1.8, or NaV1.9 gene promoter were produced. These mice were then bred with channelrhodopsin-2 (ChR2) Cre-reporter Ai32 mice to obtain NaV1.7-ChR2, NaV1.8-ChR2, or NaV1.9-ChR2 mice. Blue light exposure triggered paw withdrawal in all mice, with the strongest response in NaV1.8-ChR2 mice. These light sensitivity differences observed across NaV1.x-ChR2 mice may be dependent on ChR2 expression or reflect the inherent disparities in their pain transmission roles. In conclusion, we have generated noninvasive pain models, with optically activated peripheral nociceptors. We believe that studies using optogenetics will further elucidate the role of sodium channel subtypes in pain transmission.

Evaluation of Nav1.8 as a therapeutic target for Pitt Hopkins Syndrome.

Pitt Hopkins Syndrome (PTHS) is a rare syndromic form of autism spectrum disorder (ASD) caused by autosomal dominant mutations in the Transcription Factor 4 (TCF4) gene. TCF4 is a basic helix-loop-helix transcription factor that is critical for neurodevelopment and brain function through its binding to cis-regulatory elements of target genes. One potential therapeutic strategy for PTHS is to identify dysregulated target genes and normalize their dysfunction. Here, we propose that SCN10A is an important target gene of TCF4 that is an applicable therapeutic approach for PTHS. Scn10a encodes the voltage-gated sodium channel Nav1.8 and is consistently shown to be upregulated in PTHS mouse models. In this perspective, we review prior literature and present novel data that suggests inhibiting Nav1.8 in PTHS mouse models is effective at normalizing neuron function, brain circuit activity and behavioral abnormalities and posit this therapeutic approach as a treatment for PTHS.

Complex alterations in inflammatory pain and analgesic sensitivity in young and ageing female rats: involvement of ASIC3 and Nav1.8 in primary sensory neurons.

OBJECTIVE AND DESIGN: Our aim was to determine an age-dependent role of Nav1.8 and ASIC3 in dorsal root ganglion (DRG) neurons in a rat pre-clinical model of long-term inflammatory pain. METHODS: We compared 6 and 24 months-old female Wistar rats after cutaneous inflammation. We used behavioral pain assessments over time, qPCR, quantitative immunohistochemistry, selective pharmacological manipulation, ELISA and in vitro treatment with cytokines. RESULTS: Older rats exhibited delayed recovery from mechanical allodynia and earlier onset of spontaneous pain than younger rats after inflammation. Moreover, the expression patterns of Nav1.8 and ASIC3 were time and age-dependent and ASIC3 levels remained elevated only in aged rats. In vivo, selective blockade of Nav1.8 with A803467 or of ASIC3 with APETx2 alleviated mechanical and cold allodynia and also spontaneous pain in both age groups with slightly different potency. Furthermore, in vitro IL-1ß up-regulated Nav1.8 expression in DRG neurons cultured from young but not old rats. We also found that while TNF-α up-regulated ASIC3 expression in both age groups, IL-6 and IL-1ß had this effect only on young and aged neurons, respectively. CONCLUSION: Inflammation-associated mechanical allodynia and spontaneous pain in the elderly can be more effectively treated by inhibiting ASIC3 than Nav1.8.

Identification of N-(((1S,3R,5S)-adamantan-1-yl)methyl)-3-((4-chlorophenyl)sulfonyl)benzenesulfonamide as novel Nav1.8 inhibitor with analgesic profile.

Chronic pain is a common and challenging clinical problem that significantly impacts patients' quality of life. The sodium channel Nav1.8 plays a crucial role in the occurrence and development of chronic pain, making it one of the key targets for treating chronic pain. In this article, we combined virtual screening with cell membrane chromatography techniques to establish a novel method for rapid high-throughput screening of selective Nav1.8 inhibitors. Using this approach, we identified a small molecule compound 6, which not only demonstrated high affinity and inhibitory activity against Nav1.8 but also exhibited significant inhibitory effects on CFA-induced chronic inflammatory pain. Compared to the positive drug VX-150, compound 6 showed a more prolonged analgesic effect, making it a promising candidate as a Nav1.8 inhibitor with potential clinical applications. This discovery provides a new therapeutic option for the treatment of chronic pain.

Frontiers in Acute Pain Management: Emerging Concepts in Pain Pathways and the Role of VX-548 as a Novel NaV1.8 Inhibitor: A Narrative Review.

PURPOSE OF REVIEW: Despite ongoing research into alternative postsurgical pain treatments, opioids remain widely used analgesics regardless of associated adverse effects, including dependence and overdose, as demonstrated throughout the current opioid crisis. This is likely related to a failure in proving the efficacy of alternative analgesics in clinical trials, despite strong evidence supporting the potential for effective analgesia through in vitro studies. While NaV1.7 and NaV1.8 channels have shown to be key components of pain perception, studies regarding pharmacological agents utilizing these channels as targets have largely failed to demonstrate the efficacy of these proposed analgesics when compared to current multimodal pain treatment regimens. RECENT FINDINGS: However, the novel NaV1.8 channel inhibitor, VX-548 has surpassed previously studied NaV1.8 inhibitors in clinical trials and continues to hold promise of a novel efficacious analgesic to potentially be utilized in multimodal pain treatment on postsurgical patients. Additionally, NaV1.8 is encoded by the SCN10A, which has been shown to be minimally expressed in the brain, suggesting a lower likelihood of adverse effects in the CNS, including dependence and abuse. Novel pharmacologic analgesics that are efficacious without the significant side effects associated with opioids have lacked meaningful development. However, recent clinical trials have shown promising results in the safety and efficacy of the pharmacological agent VX-548. Still, more clinical trials directly comparing the efficacy of VX-548 to standard of care post-surgical drugs, including opioids like morphine and hydromorphone are needed to demonstrate the long-term viability of the agent replacing current opioids with an unfavorable side effect profile.

Genetic Analysis of SCN11A, SCN10A, and SCN9A in Familial Episodic Pain Syndrome (FEPS) in Japan and Proposal of Clinical Diagnostic Criteria.

Familial episodic pain syndrome (FEPS) is an early childhood onset disorder of severe episodic limb pain caused mainly by pathogenic variants of SCN11A, SCN10A, and SCN9A, which encode three voltage-gated sodium channels (VGSCs) expressed as key determinants of nociceptor excitability in primary sensory neurons. There may still be many undiagnosed patients with FEPS. A better understanding of the associated pathogenesis, epidemiology, and clinical characteristics is needed to provide appropriate diagnosis and care. For this study, nationwide recruitment of Japanese patients was conducted using provisional clinical diagnostic criteria, followed by genetic testing for SCN11A, SCN10A, and SCN9A. In the cohort of 212 recruited patients, genetic testing revealed that 64 patients (30.2%) harbored pathogenic or likely pathogenic variants of these genes, consisting of 42 (19.8%), 14 (6.60%), and 8 (3.77%) patients with variants of SCN11A, SCN10A, and SCN9A, respectively. Meanwhile, the proportions of patients meeting the tentative clinical criteria were 89.1%, 52.0%, and 54.5% among patients with pathogenic or likely pathogenic variants of each of the three genes, suggesting the validity of these clinical criteria, especially for patients with SCN11A variants. These clinical diagnostic criteria of FEPS will accelerate the recruitment of patients with underlying pathogenic variants who are unexpectedly prevalent in Japan.

NaV1.8 as Proarrhythmic Target in a Ventricular Cardiac Stem Cell Model.

The sodium channel NaV1.8, encoded by the SCN10A gene, has recently emerged as a potential regulator of cardiac electrophysiology. We have previously shown that NaV1.8 contributes to arrhythmogenesis by inducing a persistent Na+ current (late Na+ current, INaL) in human atrial and ventricular cardiomyocytes (CM). We now aim to further investigate the contribution of NaV1.8 to human ventricular arrhythmogenesis at the CM-specific level using pharmacological inhibition as well as a genetic knockout (KO) of SCN10A in induced pluripotent stem cell CM (iPSC-CM). In functional voltage-clamp experiments, we demonstrate that INaL was significantly reduced in ventricular SCN10A-KO iPSC-CM and in control CM after a specific pharmacological inhibition of NaV1.8. In contrast, we did not find any effects on ventricular APD90. The frequency of spontaneous sarcoplasmic reticulum Ca2+ sparks and waves were reduced in SCN10A-KO iPSC-CM and control cells following the pharmacological inhibition of NaV1.8. We further analyzed potential triggers of arrhythmias and found reduced delayed afterdepolarizations (DAD) in SCN10A-KO iPSC-CM and after the specific inhibition of NaV1.8 in control cells. In conclusion, we show that NaV1.8-induced INaL primarily impacts arrhythmogenesis at a subcellular level, with minimal effects on systolic cellular Ca2+ release. The inhibition or knockout of NaV1.8 diminishes proarrhythmic triggers in ventricular CM. In conjunction with our previously published results, this work confirms NaV1.8 as a proarrhythmic target that may be useful in an anti-arrhythmic therapeutic strategy.

Use-Dependent Relief of Inhibition of Nav1.8 Channels by A-887826.

Sodium channel inhibitors used as local anesthetics, antiarrhythmics, or antiepileptics typically have the property of use-dependent inhibition, whereby inhibition is enhanced by repetitive channel activation. For targeting pain, Nav1.8 channels are an attractive target because they are prominent in primary pain-sensing neurons, with little or no expression in most other kinds of neurons, and a number of Nav1.8-targeted compounds have been developed. We examined the characteristics of Nav1.8 inhibition by one of the most potent Nav1.8 inhibitors so far described, A-887826, and found that when studied with physiologic resting potentials and physiologic temperatures, inhibition had strong "reverse use dependence", whereby inhibition was relieved by repetitive short depolarizations. This effect was much stronger with A-887826 than with A-803467, another Nav1.8 inhibitor. The use-dependent relief from inhibition was seen in both human Nav1.8 channels studied in a cell line and in native Nav1.8 channels in mouse dorsal root ganglion (DRG) neurons. In native Nav1.8 channels, substantial relief of inhibition occurred during repetitive stimulation by action potential waveforms at 5 Hz, suggesting that the phenomenon is likely important under physiologic conditions. SIGNIFICANCE STATEMENT: Nav1.8 sodium channels are expressed in primary pain-sensing neurons and are a prime current target for new drugs for pain. This work shows that one of the most potent Nav1.8 inhibitors, A-887826, has the unusual property that inhibition is relieved by repeated short depolarizations. This "reverse use dependence" may reduce inhibition during physiological firing and should be selected against in drug development.

Polysorbate 80 blocked a peripheral sodium channel, Nav1.7, and reduced neuronal excitability.

Polysorbate 80 is a non-ionic detergent derived from polyethoxylated sorbitan and oleic acid. It is widely used in pharmaceuticals, foods, and cosmetics as an emulsifier. Nav1.7 is a peripheral sodium channel that is highly expressed in sympathetic and sensory neurons, and it plays a critical role in determining the threshold of action potentials (APs). We found that 10 µg/mL polysorbate 80 either abolished APs or increased the threshold of the APs of dorsal root ganglions. We thus investigated whether polysorbate 80 inhibits Nav1.7 sodium current using a whole-cell patch-clamp recording technique. Polysorbate 80 decreased the Nav1.7 current in a concentration-dependent manner with a half-maximal inhibitory concentration (IC50) of 250.4 µg/mL at a holding potential of -120 mV. However, the IC50 was 1.1 µg/mL at a holding potential of -90 mV and was estimated to be 0.9 µg/mL at the resting potentials of neurons, where most channels are inactivated. The activation rate and the voltage dependency of activation of Nav1.7 were not changed by polysorbate 80. However, polysorbate 80 caused hyperpolarizing shifts in the voltage dependency of the steady-state fast inactivation curve. The blocking of Nav1.7 currents by polysorbate 80 was not reversible at a holding potential of -90 mV but was completely reversible at -120 mV, where the channels were mostly in the closed state. Polysorbate 80 also slowed recovery from inactivation and induced robust use-dependent inhibition, indicating that it is likely to bind to and stabilize the inactivated state. Our results indicate that polysorbate 80 inhibits Nav1.7 current in concentration-, state-, and use-dependent manners when used even below commercial concentrations. This suggests that polysorbate 80 may be helpful in pain medicine as an excipient. In addition, in vitro experiments using polysorbate 80 with neurons should be conducted with caution.

Melatonin attenuated chronic visceral pain by reducing Nav1.8 expression and nociceptive neuronal sensitization.

BACKGROUND: Irritable bowel syndrome (IBS) is a common functional gastrointestinal disorder, and its specific pathogenesis is still unclear. We have previously reported that TTX-resistant (TTX-R) sodium channels in colon-specific dorsal root ganglion (DRG) neurons were sensitized in a rat model of visceral hypersensitivity induced by neonatal colonic inflammation (NCI). However, the detailed molecular mechanism for activation of sodium channels remains unknown. This study was designed to examine roles for melatonin (MT) in sensitization of sodium channels in NCI rats. METHODS: Colorectal distention (CRD) in adult male rats as a measure of visceral hypersensitivity. Colon-specific dorsal root ganglion (DRG) neurons were labeled with DiI and acutely dissociated for measuring excitability and sodium channel current under whole-cell patch clamp configurations. Western blot and Immunofluorescence were employed to detect changes in expression of Nav1.8 and MT2. RESULTS: The results showed that rats exhibited visceral hypersensitivity after NCI treatment. Intrathecal application of melatonin significantly increased the threshold of CRD in NCI rats with a dose-dependent manner, but has no role in the control group. Whole-cell patch clamp recording showed that melatonin remarkably decreased the excitability and the density of TTX-R sodium channel in DRG neurons from NCI rats. The expression of MT2 receptor at the protein level was markedly lower in NCI rats. 8MP, an agonist of MT2 receptor, enhanced the distention threshold in NCI rats. Application of 8MP reversed the enhanced hypersensitivity of DRG neurons from NCI rats. 8MP also reduced TTX-R sodium current density and modulated dynamics of TTX-R sodium current activation. CONCLUSIONS: These data suggest that sensitization of sodium channels of colon DRG neurons in NCI rats is most likely mediated by MT2 receptor, thus identifying a potential target for treatment for chronic visceral pain in patients with IBS.

Common variants in SCN10A gene associated with Brugada syndrome.

Genome-wide association studies indicate that SCN10A plays an important role in cardiac electrophysiology. Common and rare SCN10A variants are suggested to contribute to Brugada Syndrome (BrS), an inherited channelopathy resulting from genetic-determined loss-of-function in cardiac sodium channel. This study sought to characterize the role of SCN10A common variants in BrS. Clinical and genetic analyses were performed in 197 patients diagnosed with BrS. Baseline ECG parameters were evaluated in patients carrying each of four common variants associated with BrS. Cellular electrophysiological study was performed in SCN5A-SCN10A co-transfected TSA201 cells to investigate the possible electrophysiological characteristics of the allele of rs6795970, which displayed the most significant association with BrS. Four SCN10A common variants (rs7630989, rs57326399, rs6795970, rs12632942) displayed significant association with BrS susceptibility. There were no evident associations between baseline ECG parameters in BrS patients and the different genotypes of the four variants. Rs6795970 (V1073) was strongly associated with a risk for BrS, which suggests the different electrophysiological characters between these two alleles. Functional study showed a positive shift in steady-state activation (V1/2: -62.2 ± 2.6 vs. -53.5 ± 1.6 for A1073 and V1073 group, respectively; P < 0.05) and slower recovery from inactivation in mutant SCN5A-SCN10A co-transfected cells with, which contribute to the slow conduction in BrS patients with rs6795970. In conclusion, SCN10A common variants are associated with increased susceptibility to BrS. An allele rs6795970 (V1073) increases the risk for BrS. The electrophysiological changes in a positive shift in steady-state activation and slower recovery from inactivation by SCN10A-V1073 contribute to this variant associated BrS.

Molecular and Functional Relevance of NaV1.8-Induced Atrial Arrhythmogenic Triggers in a Human SCN10A Knock-Out Stem Cell Model.

In heart failure and atrial fibrillation, a persistent Na+ current (INaL) exerts detrimental effects on cellular electrophysiology and can induce arrhythmias. We have recently shown that NaV1.8 contributes to arrhythmogenesis by inducing a INaL. Genome-wide association studies indicate that mutations in the SCN10A gene (NaV1.8) are associated with increased risk for arrhythmias, Brugada syndrome, and sudden cardiac death. However, the mediation of these NaV1.8-related effects, whether through cardiac ganglia or cardiomyocytes, is still a subject of controversial discussion. We used CRISPR/Cas9 technology to generate homozygous atrial SCN10A-KO-iPSC-CMs. Ruptured-patch whole-cell patch-clamp was used to measure the INaL and action potential duration. Ca2+ measurements (Fluo 4-AM) were performed to analyze proarrhythmogenic diastolic SR Ca2+ leak. The INaL was significantly reduced in atrial SCN10A KO CMs as well as after specific pharmacological inhibition of NaV1.8. No effects on atrial APD90 were detected in any groups. Both SCN10A KO and specific blockers of NaV1.8 led to decreased Ca2+ spark frequency and a significant reduction of arrhythmogenic Ca2+ waves. Our experiments demonstrate that NaV1.8 contributes to INaL formation in human atrial CMs and that NaV1.8 inhibition modulates proarrhythmogenic triggers in human atrial CMs and therefore NaV1.8 could be a new target for antiarrhythmic strategies.

Cannabidiol Inhibition of Murine Primary Nociceptors: Tight Binding to Slow Inactivated States of Nav1.8 Channels.

The nonpsychoactive phytocannabinoid cannabidiol (CBD) has been shown to have analgesic effects in animal studies but little is known about its mechanism of action. We examined the effects of CBD on intrinsic excitability of primary pain-sensing neurons. Studying acutely dissociated capsaicin-sensitive mouse DRG neurons at 37°C, we found that CBD effectively inhibited repetitive action potential firing, from 15-20 action potentials evoked by 1 s current injections in control to 1-3 action potentials with 2 µm CBD. Reduction of repetitive firing was accompanied by a reduction of action potential height, widening of action potentials, reduction of the afterhyperpolarization, and increased propensity to enter depolarization block. Voltage-clamp experiments showed that CBD inhibited both TTX-sensitive and TTX-resistant (TTX-R) sodium currents in a use-dependent manner. CBD showed strong state-dependent inhibition of TTX-R channels, with fast binding to inactivated channels during depolarizations and slow unbinding on repolarization. CBD alteration of channel availability at various voltages suggested that CBD binds especially tightly [Kd (dissociation constant), ∼150 nm] to the slow inactivated state of TTX-R channels, which can be substantially occupied at voltages as negative as -40 mV. Remarkably, CBD was more potent in inhibiting TTX-R channels and inhibiting action potential firing than the local anesthetic bupivacaine. We conclude that CBD might produce some of its analgesic effects by direct effects on neuronal excitability, with tight binding to the slow inactivated state of Nav1.8 channels contributing to effective inhibition of repetitive firing by modest depolarizations.SIGNIFICANCE STATEMENT Cannabidiol (CBD) has been shown to inhibit pain in various rodent models, but the mechanism of this effect is unknown. We describe the ability of CBD to inhibit repetitive action potential firing in primary nociceptive neurons from mouse dorsal root ganglia and analyze the effects on voltage-dependent sodium channels. We find that CBD interacts with TTX-resistant sodium channels in a state-dependent manner suggesting particularly tight binding to slow inactivated states of Nav1.8 channels, which dominate the overall inactivation of Nav1.8 channels for small maintained depolarizations from the resting potential. The results suggest that CBD can exert analgesic effects in part by directly inhibiting repetitive firing of primary nociceptors and suggest a strategy of identifying compounds that bind selectively to slow inactivated states of Nav1.8 channels for developing effective analgesics.

Variant Intronic Enhancer Controls SCN10A-short Expression and Heart Conduction.

BACKGROUND: Genetic variants in SCN10A, encoding the neuronal voltage-gated sodium channel NaV1.8, are strongly associated with atrial fibrillation, Brugada syndrome, cardiac conduction velocities, and heart rate. The cardiac function of SCN10A has not been resolved, however, and diverging mechanisms have been proposed. Here, we investigated the cardiac expression of SCN10A and the function of a variant-sensitive intronic enhancer previously linked to the regulation of SCN5A, encoding the major essential cardiac sodium channel NaV1.5. METHODS: The expression of SCN10A was investigated in mouse and human hearts. With the use of CRISPR/Cas9 genome editing, the mouse intronic enhancer was disrupted, and mutant mice were characterized by transcriptomic and electrophysiological analyses. The association of genetic variants at SCN5A-SCN10A enhancer regions and gene expression were evaluated by genome-wide association studies single-nucleotide polymorphism mapping and expression quantitative trait loci analysis. RESULTS: We found that cardiomyocytes of the atria, sinoatrial node, and ventricular conduction system express a short transcript comprising the last 7 exons of the gene (Scn10a-short). Transcription occurs from an intronic enhancer-promoter complex, whereas full-length Scn10a transcript was undetectable in the human and mouse heart. Expression quantitative trait loci analysis revealed that the genetic variants in linkage disequilibrium with genetic variant rs6801957 in the intronic enhancer associate with SCN10A transcript levels in the heart. Genetic modification of the enhancer in the mouse genome led to reduced cardiac Scn10a-short expression in atria and ventricles, reduced cardiac sodium current in atrial cardiomyocytes, atrial conduction slowing and arrhythmia, whereas the expression of Scn5a, the presumed enhancer target gene, remained unaffected. In patch-clamp transfection experiments, expression of Scn10a-short-encoded NaV1.8-short increased NaV1.5-mediated sodium current. We propose that noncoding genetic variation modulates transcriptional regulation of Scn10a-short in cardiomyocytes that impacts NaV1.5-mediated sodium current and heart rhythm. CONCLUSIONS: Genetic variants in and around SCN10A modulate enhancer function and expression of a cardiac-specific SCN10A-short transcript. We propose that noncoding genetic variation modulates transcriptional regulation of a functional C-terminal portion of NaV1.8 in cardiomyocytes that impacts on NaV1.5 function, cardiac conduction velocities, and arrhythmia susceptibility.

Family-based exome sequencing identifies rare coding variants in age-related macular degeneration.

Genome-wide association studies (GWAS) have identified 52 independent variants at 34 genetic loci that are associated with age-related macular degeneration (AMD), the most common cause of incurable vision loss in the elderly worldwide. However, causal genes at the majority of these loci remain unknown. In this study, we performed whole exome sequencing of 264 individuals from 63 multiplex families with AMD and analyzed the data for rare protein-altering variants in candidate target genes at AMD-associated loci. Rare coding variants were identified in the CFH, PUS7, RXFP2, PHF12 and TACC2 genes in three or more families. In addition, we detected rare coding variants in the C9, SPEF2 and BCAR1 genes, which were previously suggested as likely causative genes at respective AMD susceptibility loci. Identification of rare variants in the CFH and C9 genes in our study validated previous reports of rare variants in complement pathway genes in AMD. We then extended our exome-wide analysis and identified rare protein-altering variants in 13 genes outside the AMD-GWAS loci in three or more families. Two of these genes, SCN10A and KIR2DL4, are of interest because variants in these genes also showed association with AMD in case-control cohorts, albeit not at the level of genome-wide significance. Our study presents the first large-scale, exome-wide analysis of rare variants in AMD. Further independent replications and molecular investigation of candidate target genes, reported here, would assist in gaining novel insights into mechanisms underlying AMD pathogenesis.