Expression Of Nav1 Research Articles

Study of the cardiac sodium channel in a mouse model of Scn5a haploinsufficiency towards a novel approach of therapy

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Sorbonne Université doctoral scolarship INSERM Introduction Brugada syndrome (BrS) is an inherited cardiac arrhythmia that greatly increases the risk of sudden death as a result of ventricular fibrillation. Approximately 20% of BrS cases can be attributed to a mutation in SCN5A, the gene encoding the alpha-subunit Nav1.5 of the cardiac sodium-channel, which carries the sodium current INa. These mutations typically lead to loss-of-function of Nav1.5, resulting in a decrease in INa and most of them (»70 %) are responsible for haploinsufficiency. In the Scn5a+/- mouse model, a sodium-channel dysfunction phenotype is observed, characterized by conduction abnormalities (PR and QRS interval prolongation), a 50% reduction in INa, and an increased susceptibility to arrhythmias. Aim The aim of this study was to assess the impact of the overexpression of a Nav1.5-peptide, which has been found to enhance INa, in Scn5a+/- mice, as a novel approach of therapy. Methods We used AAV9-systemic injection to overexpress the Nav1.5-peptide into the heart of Scn5a+/- mice to gauge their susceptibility to arrhythmias induced by intracardiac pacing. We also examined the Nav1.5-peptide effects on endogenous Nav1.5 expression through molecular biology and biochemistry analyses. Results The duration of PR and QRS intervals in the hearts of Scn5a+/- mice was restored to normal by overexpression of the Nav1.5-peptide (PR interval = 46.3 ± 0.9 ms, n=22 in Scn5a+/- mice vs 40.4 ± 0.8 ms, n= 23 in WT mice, P<0.0001 and 42.6 ± 0.9 ms, n=11 in Scn5a+/- +Nav1.5-peptide, P<0.05; QRS interval = 15.2 ± 0.4, n=22 ms in Scn5a+/- mice vs 13.2 ± 0.4 ms, n=23 in WT mice, P<0.01 and 13.6 ± 0.4 ms, n=14 in Scn5a+/- +Nav1.5-peptide, P<0.05). Additionally, overexpression of the Nav1.5-peptide seemed to protect Scn5a+/- mice from induced arrhythmic events caused by programmed-ventricular stimulation (total premature beats = 1.3 ± 0.4 in Scn5a+/- mice, n = 13 vs 0.2 ± 0.2, n = 6 in injected Scn5a+/- mice, NS and 0.2 ± 0.2, n = 12 in WT mice, P< 0.05). There were no significant differences in total Nav1.5 expression between injected and non-injected mice as shown by RT-qPCR and Western-blots, but the expression of Nav1.5 at the plasma membrane appeared to be restored by overexpression of the Nav1.5-peptide (Nav1.5/N-cadherin ratio = 0.50 ± 0.08, n = 11 in Scn5a+/- mice vs 1 ± 0.15, n= 11 in WT mice, P< 0.05 and 0.94 ± 0.13, n = 11 in injected Scn5a+/- mice, P< 0.05). Conclusion Gene therapy with Nav1.5-peptide can attenuate both conduction defects and increased arrhythmia inducibility observed in Scn5a+/- mice. Although additional studies will be needed to confirm and explore further our results, this could be promising to treat patients with « sodium-channel dysfunction » such as BrS.

Molecular and cellular context influences SCN8A variant function.

Pathogenic variants in SCN8A, which encodes the voltage-gated sodium (NaV) channel NaV1.6, associate with neurodevelopmental disorders, including developmental and epileptic encephalopathy. Previous approaches to determine SCN8A variant function may be confounded by use of a neonatally expressed, alternatively spliced isoform of NaV1.6 (NaV1.6N) and engineered mutations rendering the channel tetrodotoxin (TTX) resistant. We investigated the impact of SCN8A alternative splicing on variant function by comparing the functional attributes of 15 variants expressed in 2 developmentally regulated splice isoforms (NaV1.6N, NaV1.6A). We employed automated patch clamp recording to enhance throughput, and developed a neuronal cell line (ND7/LoNav) with low levels of endogenous NaV current to obviate the need for TTX-resistance mutations. Expression of NaV1.6N or NaV1.6A in ND7/LoNav cells generated NaV currents with small, but significant, differences in voltage dependence of activation and inactivation. TTX-resistant versions of both isoforms exhibited significant functional differences compared with the corresponding WT channels. We demonstrated that many of the 15 disease-associated variants studied exhibited isoform-dependent functional effects, and that many of the studied SCN8A variants exhibited functional properties that were not easily classified as either gain- or loss-of-function. Our work illustrates the value of considering molecular and cellular context when investigating SCN8A variants.

Exploring the mechanism of Nav1.3 in the ION-CCI rat model based on the TLR4/TRAF6/NF-κB pathway

BackgroundTrigeminal neuralgia (TN) is a common and difficult-to-treat neuropathic pain disorder in clinical practice. Previous studies have shown that Toll-like receptor 4 (TLR4) modulates the activation of the NF-κB pathway to affect neuropathic pain in rats. Voltage-gated sodium channels (VGSCs) are known to play an important role in neuropathic pain electrical activity. ObjectiveTo investigate whether TLR4 can regulate Nav1.3 through the TRAF6/NF-κB p65 pathway after infraorbital nerve chronic constriction injury (ION-CCI).Study design: ION-CCI modeling was performed on SD (Sprague Dawley) rats. To verify the success of the modeling, we need to detect the mechanical pain threshold and ATF3. Then, detecting the expression of TLR4, TRAF6, NF-κB p65, p-p65, and Nav1.3 in rat TG. Subsequently, investigate the role of TLR4/TRAF6/NF-κB pathway in ION-CCI model by intrathecal injections of LPS-rs (TLR4 antagonist), C25-140 (TRAF6 inhibitor), and PDTC (NF-κB p65 inhibitor). ResultsION-CCI surgery decreased the mechanical pain threshold of rats and increased the expression of ATF3, TLR4, TRAF6, NF-κB p-p65 and Nav1.3, but there was no difference in NF-κB p65 expression. After inject antagonist or inhibitor of the TLR4/TRAF6/NF-κB pathway, the expression of Nav1.3 was decreased and mechanical pain threshold was increased. ConclusionIn the rat model of ION-CCI, TLR4 in the rat trigeminal ganglion regulates Nav1.3 through the TRAF6/NF-κB p65 pathway, and TLR4 antagonist alleviates neuropathic pain in ION-CCI rats.

Development and application of a multiple reaction monitoring method for the simultaneous quantification of sodium channels Nav1.1, Nav1.2, and Nav1.6 in solubilized membrane proteins from stable HEK293 cell lines, rodents, and human brain tissues

RationaleNav1.1, 1.2, and 1.6 are transmembrane proteins acting as voltage‐gated sodium channels implicated in various forms of epilepsy. There is a need for knowing their actual concentration in target tissues during drug development.MethodsUnique peptides for Nav1.1, Nav1.2, and Nav1.6 were selected as quantotropic peptides for each protein and used for their quantification in membranes from stably transfected HEK293 cells and rodent and human brain samples using ultra‐high‐performance liquid chromatography–electrospray ionization tandem mass spectrometry.ResultsNav 1.1, 1.2, and 1.6 protein expressions in three stably individually transfected HEK293 cell lines were found to be 2.1 ± 0.2, 6.4 ± 1.2, and 4.0 ± 0.6 fmol/μg membrane protein, respectively. In brains, Nav1.2 showed the highest expression, with approximately three times higher (P < 0.003) in rodents than in humans at 3.05 ± 0.57, with 3.35 ± 0.56 in mouse and rat brains and 1.09 ± 0.27 fmol/μg in human brain. Both Nav1.1 and 1.6 expressions were much lower in the brains, with approximately 40% less expression in human Nav1.1 than rodent Nav1.1 at 0.49 ± 0.1 (mouse), 0.43 ± 0.3 (rat), and 0.28 ± 0.04 (humans); whereas Nav1.6 had approximately 60% less expression in humans than rodents at 0.27 ± 0.09 (mouse), 0.26 ± 0.06 (rat), and 0.11 ± 0.02 (humans) fmol/μg membrane proteins.ConclusionsMultiple reaction monitoring was used to quantify sodium channels Nav1.1, 1.2, and 1.6 expressed in stably transfected HEK293 cells and brain tissues from mice, rats, and humans. We found significant differences in the expression of these channels in mouse, rat, and human brains. Nav expression ranking among the three species was Nav1.2 ≫ Nav1.1 > Nav1.6, with the human brain expressing much lower concentrations overall compared to rodent brain.

Voluntary running wheel exercise induces cognitive improvement post traumatic brain injury in mouse model through redressing aberrant excitation regulated by voltage-gated sodium channels 1.1, 1.3, and 1.6

Traumatic brain injury (TBI) leads to disturbed brain discharge rhythm, elevated excitability, anxiety-like behaviors, and decreased learning and memory capabilities. Cognitive dysfunctions severely affect the quality of life and prognosis of TBI patients, requiring effective rehabilitation treatment. Evidence indicates that moderate exercise after brain injury decreases TBI-induced cognitive decline. However, the underlying mechanism remains unelucidated. Our results demonstrate that TBI causes cognitive impairment behavior abnormalities and overexpression of Nav1.1, Nav1.3 and Nav1.6 proteins inside the hippocampus of mice models. Three weeks of voluntary running wheel (RW) exercise treatments before or/and post-injury effectively redressed the aberrant changes caused by TBI. Additionally, a 10% exercise-conditioned medium helped recover cell viability, neuronal sodium current and expressions of Nav1.1, Nav1.3 and Nav1.6 proteins across cultured neurons after injury. Therefore, the results validate the neuroprotection induced by voluntary RW exercise treatment before or/and post-TBI. The RW exercise-induced improvement in cognitive behaviors and neuronal excitability could be associated with correcting the Nav1.1, Nav1.3, and Nav1.6 expression levels. The current study proves that voluntary exercise is an effective treatment strategy against TBI. The study also highlights novel potential targets for rehabilitating TBI, including the Navs proteins.

Characterization of Na+ currents regulating intrinsic excitability of optic tectal neurons.

Developing neurons adapt their intrinsic excitability to maintain stable output despite changing synaptic input. The mechanisms behind this process remain unclear. In this study, we examined Xenopus optic tectal neurons and found that the expressions of Nav1.1 and Nav1.6 voltage-gated Na+ channels are regulated during changes in intrinsic excitability, both during development and becsuse of changes in visual experience. Using whole-cell electrophysiology, we demonstrate the existence of distinct, fast, persistent, and resurgent Na+ currents in the tectum, and show that these Na+ currents are co-regulated with changes in Nav channel expression. Using antisense RNA to suppress the expression of specific Nav subunits, we found that up-regulation of Nav1.6 expression, but not Nav1.1, was necessary for experience-dependent increases in Na+ currents and intrinsic excitability. Furthermore, this regulation was also necessary for normal development of sensory guided behaviors. These data suggest that the regulation of Na+ currents through the modulation of Nav1.6 expression, and to a lesser extent Nav1.1, plays a crucial role in controlling the intrinsic excitability of tectal neurons and guiding normal development of the tectal circuitry.

Cardiac-specific deletion of BRG1 ameliorates ventricular arrhythmia inmice with myocardial infarction.

Malignant ventricular arrhythmia (VA) after myocardial infarction (MI) is mainly caused by myocardial electrophysiological remodeling. Brahma-related gene 1 (BRG1) is an ATPase catalytic subunit that belongs to a family of chromatin remodeling complexes called Switch/Sucrose Non-Fermentable Chromatin (SWI/SNF). BRG1 has been reported as a molecular chaperone, interacting with various transcription factors or proteins to regulate transcription in cardiac diseases. In this study, we investigated the potential role of BRG1 in ion channel remodeling and VA after ischemic infarction. Myocardial infarction (MI) mice were established by ligating the left anterior descending (LAD) coronary artery, and electrocardiogram (ECG) was monitored. Epicardial conduction of MI mouse heart was characterized in Langendorff-perfused hearts using epicardial optical voltage mapping. Patch-clamping analysis was conducted in single ventricular cardiomyocytes isolated from the mice. We showed that BRG1 expression in the border zone was progressively increased in the first week following MI. Cardiac-specific deletion of BRG1 by tail vein injection of AAV9-BRG1-shRNA significantly ameliorated susceptibility to electrical-induced VA and shortened QTc intervals in MI mice. BRG1 knockdown significantly enhanced conduction velocity (CV) and reversed the prolonged action potential duration in MI mouse heart. Moreover, BRG1 knockdown improved the decreased densities of Na+ current (INa) and transient outward potassium current (Ito), as well as the expression of Nav1.5 and Kv4.3 in the border zone of MI mouse hearts and in hypoxia-treated neonatal mouse ventricular cardiomyocytes. We revealed that MI increased the binding among BRG1, T-cell factor 4 (TCF4) and β-catenin, forming a transcription complex, which suppressed the transcription activity of SCN5A and KCND3, thereby influencing the incidence of VA post-MI.

Reduction of SIRT1-Mediated Epigenetic Upregulation of Nav1.7 Contributes to Oxaliplatin-Induced Neuropathic Pain

BACKGROUND: Clinically, neuropathic pain is a severe side effect of oxaliplatin chemotherapy, which usually leads to dose reduction or cessation of treatment. Due to the unawareness of detailed mechanisms of oxaliplatin-induced neuropathic pain, it is difficult to develop an effective therapy and limits its clinical use. OBJECTIVES: The aim of the present study was to identify the role of sirtuin 1 (SIRT1) reduction in epigenetic regulation of the expression of voltage-gated sodium channels 1.7 (Nav1.7) in the dorsal root ganglion (DRG) during oxaliplatin-induced neuropathic pain. STUDY DESIGN: Controlled animal study. SETTING: University laboratory. METHODS: The von Frey test was performed to evaluate pain behavior in rats. Real-time quantitative polymerase chain reaction, western blotting, electrophysiological recording, chromatin immunoprecipitation, and small interfering RNA (siRNA) were used to illustrate the mechanisms. RESULTS: In the present study, we found that both the activity and expression of SIRT1 were significantly decreased in rat DRG following oxaliplatin treatment. The activator of SIRT1, resveratrol, not only increased the activity and expression of SIRT1, but also attenuated the mechanical allodynia following oxaliplatin treatment. In addition, local knockdown of SIRT1 by intrathecal injection of SIRT1 siRNA caused mechanical allodynia in naive rats. Besides, oxaliplatin treatment enhanced the action potential firing frequency of DRG neurons and the expression of Nav1.7 in DRG and activation of SIRT1 by resveratrol reversed this effect. Furthermore, blocking Nav1.7 by ProTx II (a selective Nav1.7 channel blocker) reversed oxaliplatin-induced mechanical allodynia. In addition, histone H3 hyperacetylation at the Nav1.7 promoter in DRG of rats following oxaliplatin treatment was significantly suppressed by activation of SIRT1 with resveratrol. Moreover, both the expression of Nav1.7 and histone H3 acetylation at the Nav1.7 promoter were upregulated in the DRG by local knockdown of SIRT1 with SIRT1 siRNA in naive rats. LIMITATIONS: More underlying mechanism(s) of SIRT1 reduction after oxaliplatin treatment needs to be explored in future research. CONCLUSIONS: These findings suggest that reduction of SIRT1-mediated epigenetic upregulation of Nav1.7 in the DRG contributes to the development of oxaliplatin-induced neuropathic pain in rats. The intrathecal drug delivery treatment of activating SIRT1 might be a novel therapeutic option for oxaliplatin-induced neuropathic pain. KEY WORDS: SIRT1, neuropathic pain, dorsal root ganglion, NAV1.7 voltage-gated sodium channel, oxaliplatin

Functional Epicardial Conduction Disturbances Due to a SCN5A Variant Associated With Brugada Syndrome

BackgroundBrugada syndrome is a significant cause of sudden cardiac death (SCD), but the underlying mechanisms remain hypothetical. ObjectivesThis study aimed to elucidate this knowledge gap through detailed ex vivo human heart studies. MethodsA heart was obtained from a 15-year-old adolescent boy with normal electrocardiogram who experienced SCD. Postmortem genotyping was performed, and clinical examinations were done on first-degree relatives. The right ventricle was optically mapped, followed by high-field magnetic resonance imaging and histology. Connexin-43 and NaV1.5 were localized by immunofluorescence, and RNA and protein expression levels were studied. HEK-293 cell surface biotinylation assays were performed to examine NaV1.5 trafficking. ResultsA Brugada-related SCD diagnosis was established for the donor because of a SCN5A Brugada-related variant (p.D356N) inherited from his mother, together with a concomitant NKX2.5 variant of unknown significance. Optical mapping demonstrated a localized epicardial region of impaired conduction near the outflow tract, in the absence of repolarization alterations and microstructural defects, leading to conduction blocks and figure-of-8 patterns. NaV1.5 and connexin-43 localizations were normal in this region, consistent with the finding that the p.D356N variant does not affect the trafficking, nor the expression of NaV1.5. Trends of decreased NaV1.5, connexin-43, and desmoglein-2 protein levels were noted; however, the RT-qPCR results suggested that the NKX2-5 variant was unlikely to be involved. ConclusionsThis study demonstrates for the first time that SCD associated with a Brugada-SCN5A variant can be caused by localized functionally, not structurally, impaired conduction.

Age-dependent and modality-specific changes in the phenotypic markers Nav1.8, ASIC3, P2X3 and TRPM8 in male rat primary sensory neurons during healthy aging

The effects during healthy aging of the tetrodotoxin-resistant voltage-gated sodium channel 1.8 (Nav1.8), the acid-sensing ion channel-3 (ASIC3), the purinergic-receptor 2X3 (P2X3) and transient receptor potential of melastatin-8 (TRPM8) on responses to non-noxious stimuli are poorly understood. These effects will influence the transferability to geriatric subjects of findings obtained using young animals. To evaluate the involvement of these functional markers in mechanical and cold sensitivity to non-noxious stimuli and their underlying mechanisms, we used a combination of immunohistochemistry and quantitation of immunostaining in sub-populations of neurons of the dorsal root ganglia (DRG), behavioral tests, pharmacological interventions and Western-blot in healthy male Wistar rats from 3 to 24months of age. We found significantly decreased sensitivity to mechanical and cold stimuli in geriatric rats. These behavioural alterations occurred simultaneously with differing changes in the expression of Nav1.8, ASIC3, P2X3 and TRPM8 in the DRG at different ages. Using pharmacological blockade in vivo we demonstrated the involvement of ASIC3 and P2X3 in normal mechanosensation and of Nav1.8 and ASIC3 in cold sensitivity. Geriatric rats also exhibited reductions in the number of A-like large neurons and in the proportion of peptidergic to non-peptidergic neurons. The changes in normal sensory physiology in geriatric rats we report here strongly support the inclusion of aged rodents as an important group in the design of pre-clinical studies evaluating pain treatments.

Radiocaine: An Imaging Marker of Neuropathic Injury.

Voltage-gated sodium channels (Navs) play a crucial electrical signaling role in neurons. Nav-isoforms present in peripheral sensory neurons and dorsal root ganglia of the spinal cord are critically involved in pain perception and transmission. While these isoforms, particularly Nav1.7, are implicated in neuropathic pain disorders, changes in the functional state and expression levels of these channels have not been extensively studied in vivo. Radiocaine, a fluorine-18 radiotracer based on the local anesthetic lidocaine, a non-selective Nav blocker, has previously been used for cardiac Nav1.5 imaging using positron-emission tomography (PET). In the present study, we used Radiocaine to visualize changes in neuronal Nav expression after neuropathic injury. In rats that underwent unilateral spinal nerve ligation, PET/MR imaging demonstrated significantly higher uptake of Radiocaine into the injured sciatic nerve, as compared to the uninjured sciatic nerve, for up to 32 days post-surgery. Radiocaine, due to its high translational potential, may serve as a novel diagnostic tool for neuropathic pain conditions using PET imaging.

IN SILICO MOLECULAR DOCKING STUDIES OF OVARIAN EXTRACT SINGKARAK LAKE PUFFERFISH (TETRAODON LEIURUS) AGAINST BREAST CANCER

Targets the development of breast cancer chemoprevention by blocking the expression of NaV 1.5 channels and NHE, decreasing the expression of Bcl-2 and Bcl-XL, and increasing the expression of Bax. The ovarian extract Singkarak Lake Pufferfish (Tetraodon leiurus) has the potential as chemoprevention in breast cancer cell lines (MCF-7). This study aimed to analyze the interaction of STX, neoSTX, and dcSTX ligands against NaV1.5, NHE, Bcl-2, Bcl-XL, and Bax receptors. This study used the molecular docking method using STX, neoSTX, and dcSTX ligands against NaV1.5, NHE, Bcl-2, Bcl-XL, and Bax receptors. The result showed that the STX ligand has a more stable interaction with NaV1.5, Bcl-2, Bcl-XL, and Bax receptors with ΔG values of -8.72, -7.32 -6.86, and -6.31 kcal/mol compared to neoSTX and dcSTX ligands. Furthermore, the neoSTX ligand has a more stable interaction with the NHE receptor with an ΔG value of -7.5 kcal/mol compared to the STX and dcSTX ligands. This study shows that the ovarian extract of Singkarak Lake Pufferfish (Tetraodon leiurus) has the potential to be developed as cancer chemoprevention.

Investigation on Circulating Neonatal Nav1.5 (nNav1.5) and Its Antibodies in the Blood and Serum of 4T1 Orthotopic Mice Model and Breast Cancer Patients from Hospital Universiti Sains Malaysia (HUSM)

Investigation on Circulating Neonatal Nav1.5 (nNav1.5) and Its Antibodies in the Blood and Serum of 4T1 Orthotopic Mice Model and Breast Cancer Patients from Hospital Universiti Sains Malaysia (HUSM)

Empagliflozin and dapagliflozin increase Na current in human cardiomyocytes

Abstract Background Empagliflozin (EMPA) and dapagliflozin (DAPA) are sodium-glucose cotransporter 2 inhibitors (SGLT2i) used for the treatment of type 2 Diabetes Mellitus (T2DM). Both drugs reduce morbidity and mortality in heart failure (HF) patients with reduced or preserved ejection fraction, even in the absence of T2DM. Moreover, these drugs decrease ventricular arrhythmias and sudden cardiac death in HF patients. The sodium current (INa), carried by Nav1.5 channels, is responsible for cardiac action potential (AP) depolarization and determines excitability and conduction velocity. In HF patients, the expression of Nav1.5 channels is reduced, leading to a decrease of ventricular excitability that enhances the arrhythmic risk. Purpose We aimed to determine the effects of EMPA and DAPA on human cardiac INa and AP characteristics. Methods Peak INa and ventricular-like APs were recorded in cardiomyocytes derived from human induced pluripotent stem cells (hiPSC-CM) using patch-clamp techniques. INa was also recorded in CHO cells transiently transfected with human Nav1.5+Navβ1 channels. In all cases, EMPA or DAPA (1 μM) were added to culture media and incubated for 24-h. Results APs recorded in hiPSC-CMs exhibited automatic activity and incubation with EMPA or DAPA did not modify spontaneous beating frequency (0.39±0.04 Hz; P>0.05, n≥16). In cells driven at 1 Hz, none of the drugs modified resting membrane potential (−76.7±1.4 mV; P>0.05, n≥11), but significantly increased AP amplitude from 98.6±3.6 to 105±2.2 (DAPA) and 107±2.3 mV (EMPA) (P<0.05). Interestingly, only EMPA lengthened AP duration measured at 20%, 50%, and 90% (from 605.6±31.3 to 760.5±59.0 ms, P<0.05) of repolarization. In hiPSC-CMs EMPA increased maximum INa density by 64% (from −156.0±28.0 to −256.4±28.1 pA/pF, P<0.05, n≥7) and shifted the midpoint (Vh) of the inactivation curve to more hyperpolarized potentials (from −97.3±4.5 to −108.6±4.4 mV, P<0.05, n≥7). In turn, DAPA increased maximum INa density by 24% (to −193.8±26.6 pA/pF) and shifted the Vh of the activation curve to more negative potentials (from −47.2±1.6 mV to −55.5±2.8 mV, P<0.05), an effect that would increase the INa at negative potentials coinciding with channel opening. None of the drugs modified the time course of current activation or inactivation. In CHO cells, EMPA and DAPA effects on INa were identical to those observed on hiPSC-CM. These results suggest that both SGLT2i increase INa by enhancing Nav1.5 expression into the cell membrane, by a direct gating effect on the channel, or by a combination of both. Conclusions In human cardiomyocytes, EMPA and DAPA increase INa and the AP amplitude. Moreover, EMPA, but not DAPA, prolonged AP duration. We propose that EMPA and DAPA exhibit a unique mechanism that increases cardiac excitability and conduction velocity and could contribute to the prevention of arrhythmic events in HF patients. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Ministerio de Ciencia e innovaciόnInstituto de de Salud Carlos III

BmK DKK13, A Scorpion Toxin, Alleviates Pain Behavior in a Rat Model of Trigeminal Neuralgia by Modulating Voltage-Gated Sodium Channels and MAPKs/CREB Pathway.

BmK DKK13 (DKK13) is a mutated recombinant peptide, which has a significant antinociception in a rat model of the inflammatory pain. The purpose of this study was to evaluate the antinociceptive effect of DKK13 on trigeminal neuralgia (TN) in rats. Male Sprague-Dawley (SD) rats were treated with the chronic constriction injury of the infraorbital nerve (IoN-CCI) model to induce stable symptoms of TN. DKK13 (1.0 mg/kg, 2.0 mg/kg and 4.0 mg/kg, i.v.) or morphine (4.0 mg/kg, i.v.) was administered by tail vein once on day 14 after IoN-CCI injury. Behavioral tests, electrophysiology and western blotting were performed to investigate the role and underlying mechanisms of DKK13 on IoN-CCI model. Behavioral test results showed that DKK13 could significantly increase the mechanical pain and thermal radiation pain thresholds of IoN-CCI rats and inhibit the asymmetric spontaneous pain scratching behavior. Electrophysiological results showed that DKK13 could significantly reduce the current density of Nav1.8 in the ipsilateral side of trigeminal ganglion (TG) neurons in IoN-CCI rats, and the steady-state activation and inactivation curves of Nav1.8 shifted, respectively, to the direction of hyperpolarization and depolarization. Western blotting results showed that DKK13 significantly reduced the expression of Nav1.8 and the phosphorylation levels of key proteins of MAPKs/CREB pathway in TG tissues of IoN-CCI rats. In brief, DKK13 has a significant antinociceptive effect on IoN-CCI rats, which may be achieved by changing the dynamic characteristics of Nav1.8 channel and regulating the protein phosphorylation in MAPKs/CREB pathway.

PO-615-04 SODIUM CHANNEL REMODELING UNDERLIES CARDIAC ARRHYTHMIAS IN A MURINE MODEL OF SUDDEN DEATH IN EPILEPSY

Dravet Syndrome (DS) is an inherited epileptic condition mostly associated with loss of SCN1A (NaV1.1) function. DS patients have a higher risk of sudden unexpected death in epilepsy (SUDEP) linked to arrhythmia. But mechanisms of arrhythmia in DS remain unknown. Intriguingly, DS cardiac myocytes exhibit a paradoxical increase in late Na+ currents (INa). Furthermore, reducing Nav1.6 decreases the incidence of seizures, suggesting a possible link between Nav1.6 and arrhythmia in DS. Here we hypothesize that NaV1.6 remodeling, particularly near ryanodine receptor 2 (RyR2) and Na+/Ca2+ exchange (NCX), underlies Ca2+ mishandling and arrhythmias in DS. To investigate dysregulation of cardiac NaVs and its arrhythmogenic impact in DS. Western blot, proximity ligation assay (PLA), stochastic optical reconstruction microscopy (STORM), patch clamp, Ca2+ imaging, in vivo ECG. A mouse model of DS (NaV1.1+/-) exhibited similar NaV1.5 expression levels relative to wild type (WT); however, expression of NaV1.6 was increased in DS hearts. To investigate the structural context of increased NaV1.6 expression we used PLA (<40 nm) and STORM (< 20 nm lateral resolution) to assess the density of NaV1.6 near RyR2 and NCX. PLA demonstrated higher frequency of co-localization between NaV1.6 and RyR2 and NCX in DS relative to WT. This correlated with increase in fraction of NaV1.6 clusters near (<100 nm) RyR2 in DS comparing to WT as revealed by STORM. To asses functional consequences of NaV1.6 cluster remodeling, we used whole-cell INa patch clamp recording and confocal Ca2+ imaging. These studies revealed that DS cardiomyocytes evidenced enhanced late INa coupled with increased frequency of Ca2+ waves. Global suppression of NaV1.6 (DS × NaV1.6+/-) mitigated late INa as well as aberrant Ca2+ handling. Using in vivo ECG we found that during bradycardia challenge (carbachol 0.5 mg/kg intraperitoneal) DS mice exhibited higher incidence of VT relative to WT and DS × NaV1.6+/-. Importantly, VT incidence correlated with increased mortality of DS mice. Remodeling within NaV1.6-rich nanodomains contributes to Ca2+ mishandling and arrhythmia in DS that may underlie SUDEP. NaV1.6 may serve as a druggable target for arrhythmia and SUDEP prevention.

Nerve growth factor-mediated Na+ channel plasticity of bladder afferent neurons in mice with spinal cord injury

AimTo investigate the effect of nerve growth factor (NGF) neutralization on Na+ channel plasticity of bladder afferent neurons in mice with spinal cord injury (SCI). Main methodsFemale C57/BL6 mice were randomly divided into spinal intact (SI) group, SCI group and SCI + NGF-Ab group. SCI was induced by spinal cord transection at the Th8/9 level. In SCI + NGF-Ab group, anti-NGF antibodies (10 μg·kg-1 per hour) were continuously administered for 2 weeks using osmotic pumps. Bladder afferent neurons were labelled with Fluoro‑gold (FG) injected into the bladder wall. L6-S1 dorsal root ganglion (DRG) neurons were dissociated and whole-cell patch clamp recordings were performed on FG-labelled neurons. Expression of Nav1.7 and Nav1.8 was examined by immunofluorescent staining. Key findingsWhole-cell patch clamp recordings showed that TTX only partially inhibited action potentials (AP) and Na+ currents of bladder afferent neurons in SI mice, but it almost completely inhibited them in SCI mice. Total and TTX-sensitive Na+ currents were increased and TTX-resistant currents were decreased in bladder afferent neurons from SCI mice vs. SI mice. These changes in SCI mice were significantly reversed by NGF-antibody treatment. Immunostaining results showed the increased and decreased levels of Nav1.7 and Nav1.8, respectively, in FG-labelled bladder afferent neurons in SCI mice vs. SI mice, which was significantly reversed in SCI + NGF-Ab mice. SignificanceNGF mediates the Na+ channel plasticity with a shift from TTX-resistant Nav1.8 to TTX-sensitive Nav1.7 in bladder afferent neurons, which could be a possible underlying mechanism of bladder afferent hyperexcitability and detrusor overactivity after SCI.

Cilostazol Ameliorates Peripheral Neuropathic Pain in Streptozotocin-Induced Type I Diabetic Rats.

Background: Cilostazol is an antiplatelet agent with vasodilating, endothelial function restoration, and anti-inflammatory effects. This study aims to investigate the efficacy of oral cilostazol for preventing the development of diabetic peripheral neuropathy (DPN). Materials and Methods: Ninety adult male Sprague-Dawley rats were divided into five groups: 1) naïve (control); 2) diabetic (DM); 3) DM receiving 10 mg/kg cilostazol (cilo-10); 4) DM receiving 30 mg/kg cilostazol (cilo-30); and 5) DM receiving 100 mg/kg cilostazol (cilo-100). Hindpaw responses to thermal and mechanical stimuli were measured. Activation of microglia and astrocytes in the spinal dorsal horn (SDH) and expression of NaVs in the dorsal root ganglia (DRG) were examined with Western blots and immunofluorescence. Results: DM rats displayed decreased withdrawal thresholds to mechanical stimuli (mechanical allodynia) and blunted responses to thermal stimuli. In addition, the expression of microglia increased, but astrocytes were reduced in the SDH. Upregulation of Nav −1.1, 1.2, −1.3, −1.6, and −1.7 and downregulation of Nav-1.8 were observed in the DRG. The DM rats receiving cilostazol all returned DM-induced decrease in withdrawal threshold to mechanical stimuli and attenuated neuropathic pain. Additionally, all cilostazol treatments suppressed the level of activated microglial cells and ameliorated the DM-induced decline in astrocyte expression levels in the SDH. However, only the rats treated with cilo-100 demonstrated significant improvements to the aberrant NaV expression in the DRG. Conclusion: Oral cilostazol can blunt the responses of mechanical allodynia and has the potential to treat diabetic neuropathy by attenuating NaV and glial cell dysregulation.

Fast voltage-dependent sodium (NaV ) currents are functionally expressed in mouse corpus cavernosum smooth muscle cells.

Corpus cavernosum smooth muscle (CCSM) exhibits phasic contractions that are coordinated by ion channels. Mouse models are commonly used to study erectile dysfunction, but there are few published electrophysiological studies of mouse CCSM. We describe the voltage-dependent sodium (NaV ) currents in mouse CCSM and investigate their function. We used electrophysiological, pharmacological and immunocytochemical methods to study the NaV currents in isolated CCSM cells from C57BL/6 mice. Tension measurements were carried out using crural sections of the corpus cavernosum in whole tissue. Fast, voltage-dependent, sodium currents in mouse CCSM were induced by depolarising steps. Steady-state activation and inactivation curves revealed a window current between -60 and -30 mV. Two populations of NaV currents, 'TTX-sensitive' and 'TTX-insensitive', were identified. TTX-sensitive currents showed 48% block with the NaV channel subtype-specific blockers ICA-121431 (NaV 1.1-1.3), PF-05089771 (NaV 1.7) and 4,9-anhydro-TTX (NaV 1.6). TTX-insensitive currents were resistant to blockade by A803467, specific for NaV 1.8 channels. Immunocytochemistry confirmed expression of NaV 1.5 and NaV 1.4 in freshly dispersed CCSM cells. Veratridine, a NaV channel activator, reduced time-dependent inactivation of NaV currents and increased duration of evoked action potentials. Veratridine induced phasic contractions in CCSM strips, reversible with TTX and nifedipine but not KB-R7943. There are fast, voltage-dependent, sodium currents in mouse CCSM. Stimulation of these currents increased contractility of CCSM in vitro, suggesting an involvement in detumescence and potentially providing a clinically relevant target in erectile dysfunction. Further work will be necessary to define its role.

Prostaglandin E2 sensitizes the cough reflex centrally via EP3 receptor-dependent activation of NaV 1.8 channels

BackgroundCough hypersensitivity is a major characteristic feature associated with several types of cough, including chronic cough, but its underlying mechanisms remain to be fully understood. Inflammatory mediators, such as prostaglandin E2 (PGE2), have been implicated in both peripheral induction and sensitization of the cough reflex. In this study, using a conscious guinea pig model of cough, we investigated whether PGE2 can sensitize the cough reflex via central actions and, if so, via which mechanisms.MethodsAll drugs were administered by intracerebroventricular (i.c.v.) route and whole-body plethysmograph set-up was used for both induction, using aerosolized citric acid (0.2 M), and recording of cough. Immunohistochemistry was performed to confirm the expression of NaV 1.8 channels in the nucleus tractus solitarius (nTS).ResultsWe show that both PGE2 and the non-selective EP1/EP3 agonist, sulprostone, dose-dependently enhanced the citric acid-induced cough (P ≤ 0.001, P ≤ 0.01, respectively). Pretreatment with the EP1 antagonist, ONO-8130, did not affect the sulprostone-induced cough sensitization, whilst the EP3 antagonist, L-798,106, dose-dependently inhibited this effect (P ≤ 0.05). Furthermore, treatment with either the EP2 agonist, butaprost or the EP4 agonist, L-902,688, had no effect on cough sensitization. Additionally, pretreatment with either the TRPV1 antagonist, JNJ-17203212 or the TRPA1 antagonist, HC-030031, alone or in combination, nor with the NaV 1.1, 1.2, 1.3, 1.4, 1.6 and 1.7 channel blocker, tetrodotoxin, had any effect on the cough. In contrast, pretreatment with the NaV 1.8 antagonist, A-803467, dose-dependently inhibited this effect (P ≤ 0.05). Furthermore, NaV 1.8 channels were shown to be expressed in the nTS.ConclusionCollectively, our findings show that PGE2 sensitizes the cough reflex centrally via EP3 receptor-dependent activation of NaV 1.8 but independently of TRPV1,TRPA1 and TTX-sensitive sodium channel activation. These results indicate that PGE2 plays an important role in central sensitization of the cough reflex and suggest that central EP3 receptors and/or NaVv 1.8 channels may represent novel antitussive molecular targets.Graphical