Cardiac Isoform NaV1 Research Articles

New aryl and acylsulfonamides as state-dependent inhibitors of Nav1.3 voltage-gated sodium channel

Voltage-gated sodium channels (Navs) play an essential role in neurotransmission, and their dysfunction is often a cause of various neurological disorders. The Nav1.3 isoform is found in the CNS and upregulated after injury in the periphery, but its role in human physiology has not yet been fully elucidated. Reports suggest that selective Nav1.3 inhibitors could be used as novel therapeutics to treat pain or neurodevelopmental disorders. Few selective inhibitors of this channel are known in the literature. In this work, we report the discovery of a new series of aryl and acylsulfonamides as state-dependent inhibitors of Nav1.3 channels. Using a ligand-based 3D similarity search and subsequent hit optimization, we identified and prepared a series of 47 novel compounds and tested them on Nav1.3, Nav1.5, and a selected subset also on Nav1.7 channels in a QPatch patch-clamp electrophysiology assay. Eight compounds had an IC50 value of less than 1 μM against the Nav1.3 channel inactivated state, with one compound displaying an IC50 value of 20 nM, whereas activity against the inactivated state of the Nav1.5 channel and Nav1.7 channel was approximately 20-fold weaker. None of the compounds showed use-dependent inhibition of the cardiac isoform Nav1.5 at a concentration of 30 μM. Further selectivity testing of the most promising hits was measured using the two-electrode voltage-clamp method against the closed state of the Nav1.1-Nav1.8 channels, and compound 15b displayed small, yet selective, effects against the Nav1.3 channel, with no activity against the other isoforms. Additional selectivity testing of promising hits against the inactivated state of the Nav1.3, Nav1.7, and Nav1.8 channels revealed several compounds with robust and selective activity against the inactivated state of the Nav1.3 channel among the three isoforms tested. Moreover, the compounds were not cytotoxic at a concentration of 50 μM, as demonstrated by the assay in human HepG2 cells (hepatocellular carcinoma cells). The novel state-dependent inhibitors of Nav1.3 discovered in this work provide a valuable tool to better evaluate this channel as a potential drug target.

Molecular Pathology of Sodium Channel Beta-Subunit Variants

The voltage-gated Na+ channel regulates the initiation and propagation of the action potential in excitable cells. The major cardiac isoform NaV1.5, encoded by SCN5A, comprises a monomer with four homologous repeats (I-IV) that each contain a voltage sensing domain (VSD) and pore domain. In native myocytes, NaV1.5 forms a macromolecular complex with NaVβ subunits and other regulatory proteins within the myocyte membrane to maintain normal cardiac function. Disturbance of the NaV complex may manifest as deadly cardiac arrhythmias. Although SCN5A has long been identified as a gene associated with familial atrial fibrillation (AF) and Brugada Syndrome (BrS), other genetic contributors remain poorly understood. Emerging evidence suggests that mutations in the non-covalently interacting NaVβ1 and NaVβ3 are linked to both AF and BrS. Here, we investigated the molecular pathologies of 8 variants in NaVβ1 and NaVβ3. Our results reveal that NaVβ1 and NaVβ3 variants contribute to AF and BrS disease phenotypes by modulating both NaV1.5 expression and gating properties. Most AF-linked variants in the NaVβ1 subunit do not alter the gating kinetics of the sodium channel, but rather modify the channel expression. In contrast, AF-related NaVβ3 variants directly affect channel gating, altering voltage-dependent activation and the time course of recovery from inactivation via the modulation of VSD activation.

Small G-protein RhoA is a potential inhibitor of cardiac fast sodium current.

Small G-proteins of Rho family modulate the activity of several classes of ion channels, including K+ channels Kv1.2, Kir2.1, and ERG; Ca2+ channels; and epithelial Na+ channels. The present study was aimed to check the RhoA potential regulatory effects on Na+ current (INa) transferred by Na+ channel cardiac isoform NaV1.5 in heterologous expression system and in native rat cardiomyocytes. Whole-cell patch-clamp experiments showed that coexpression of NaV1.5 with the wild-type RhoA in CHO-K1 cell line caused 2.7-fold decrease of INa density with minimal influence on steady-state activation and inactivation. This effect was reproduced by the coexpression with a constitutively active RhoA, but not with a dominant negative RhoA. In isolated ventricular rat cardiomyocytes, a 5-h incubation with the RhoA activator narciclasine (5 × 10-6M) reduced the maximal INa density by 38.8%. The RhoA-selective inhibitor rhosin (10-5M) increased the maximal INa density by 25.3%. Experiments with sharp microelectrode recordings in isolated right ventricular wall preparations showed that 5 × 10-6M narciclasine induced a significant reduction of action potential upstroke velocity after 2h of incubation. Thus, RhoA might be considered as a potential negative regulator of sodium channels cardiac isoform NaV1.5.

Regulation of NaV1.5 Sodium Channels by Small G-Proteins of the Rho Family in a Heterologous Expression System.

The effect of small G-proteins of the Rho family on sodium current conducted by cardiac isoform NaV1.5 of voltage-gated sodium channels was studied in heterologous expression system, CHO-K1 cell line transfected with a plasmid containing the NaV1.5 gene. The influence of cotransfection with genes of wild-type, constitutively-active, and dominant-negative small G-proteins RhoA, Rac1, and Cdc2 on the parameters of sodium current and its noninactivating component (INa,late) was estimated. Among three studied small G-proteins, only RhoA (wild-type and constitutively-active type) strongly affected sodium current reducing its peak amplitude, but not the value of INa,late. Cotransfection with wild-type Rac1 resulted in a minor decrease in sodium current. Thus, small G-protein RhoA has potential capability for suppression of sodium current, although physiological relevance of this property has to be verified.

Abstract 493: An Age-associated Reduction in Na V 1.5 Protein Expression Correlates With an Increase in Na V 1.8 Protein Expression in the Right Atrial Appendage of the Human Heart

Background: Na v 1.5 is the predominantly expressed voltage-gated sodium channel (VGSC) isoform in the heart, responsible for phase 0 of the action potential. Our rodent studies have shown expression of neuronal isoform Na v 1.8 protein in the adult heart. Others have associated Na v 1.8 expression with aberrant sodium currents leading to increased frequency of arrhythmogenic episodes. Aim: To investigate age-associated changes in the protein expression of VGSCs Na v 1.5 and Na v 1.8 in the right atrial appendage of the human heart. Methods: Tissues from the right atrial appendage were obtained with NHS ethical approval (IRAS 197493) from patients undergoing routine cardiac surgery, coronary artery bypass or valve repair. The protein expression of VGSC isoforms Na v 1.5 and Na v 1.8 were quantified using specific protein-directed primary antibodies (Alomone, Israel) coupled with a secondary antibody conjugated to HRP for western blot (Abcam, UK), or Alexa Fluor 488 (ThermoFisher, UK) for immunohistochemistry with confocal microscopy (LSM 710 Zeiss, UK). Densities were quantified using Image J software. Results: Derived from 26 patients, the protein expression of Na v 1.5 arranged in chronological age order (43-87 years of age) demonstrated a negative correlation between increasing age and Na v 1.5 protein expression (Pearson correlation coefficient of -0.25). The contrary was shown for Na v 1.8, which had a positive correlation with increasing age (Pearson correlation coefficient of 0.26), as older patients show increased Na v 1.8 protein expression. This novel finding was reflected when we compared confocal images from elderly patients with their younger counterparts, as the elderly showed a higher density of labelled Na v 1.8 protein and a lower density of labelled Na v 1.5 protein, particularly located at the t-tubules and intercalated disks. Conclusion: With increasing chronological age, the human heart reduces expression of the predominant cardiac isoform Na v 1.5 whilst increasing the expression of Na v 1.8: This finding has the potential to increase the risk of an elderly person’s heart to an increased susceptibility of arrhythmias.

The role of HIF-1α, CBP and p300 in the regulation of Nav1.5 expression in breast cancer cells

Up-regulation of voltage-gated sodium channels (VGSCs) in breast cancer, especially the cardiac isoform Nav1.5 is correlated with lymph-node metastasis and poor prognosis. In this study, the role of the transcription factor hypoxia inducible factor-1α (HIF-1α) and its co-activators CBP and p300 in regulating Nav1.5 expression in breast cancer cells was investigated. Modulation of HIF-1α expression in MDA-MB-231 (aggressive type) and MCF-7 cells (less aggressive type) was achieved using siRNA targeting HIF-1α and the hypoxia-mimetic agent cobalt chloride (CoCl2), respectively, whereas C646 was used to inhibit CBP and p300 in MDA-MB-231 cells. HIF-1α protein and mRNA of CBP, p300 and Nav1.5 were all expressed at higher levels in MDA-MB-231 cells than in MCF-7 cells. In siHIF-1α–treated MDA-MB-231 cells, Nav1.5, CBP mRNA expression, cell viability and cell migration were all significantly reduced. In CoCl2-treated MCF-7 cells, HIF-1α protein expression, CA9, p300 and Nav1.5 mRNA expressions were all increased. Although CoCl2 caused growth suppression, motility and migration of MCF-7 cells remained intact. Treatment with C646 also successfully inhibited CBP, p300, Nav1.5 mRNA expression followed by suppression of motility and migration in MDA-MB-231 cells. Overall, HIF-1α, CBP and p300 are important transcriptional regulators of Nav1.5 expression in breast cancer.

Differential regulation of sodium channels as a novel proarrhythmic mechanism in the human failing heart.

In heart failure (HF), enhanced persistent Na+ current (INaL) exerts detrimental effects on cellular electrophysiology and can induce arrhythmias. However, the underlying regulatory mechanisms remain unclear. Our aim was to potentially investigate the regulation and electrophysiological contribution of neuronal sodium channel NaV1.8 in failing human heart and eventually to reveal a novel anti-arrhythmic therapy. By western blot, we found that NaV1.8 protein expression is significantly up-regulated, while of the predominant cardiac isoform NaV1.5 is inversely reduced in human HF. Furthermore, to investigate the relation of NaV1.8 regulation with the cellular proarrhythmic events, we performed comprehensive electrophysiology recordings and explore the effect of NaV1.8 on INaL, action potential duration (APD), Ca2+ spark frequency, and arrhythmia induction in human failing cardiomyocytes. NaV1.8 inhibition with the specific blockers A-803467 and PF-01247324 decreased INaL, abbreviated APD and reduced cellular-spontaneous Ca2+-release and proarrhythmic events in human failing cardiomyocytes. Consistently, in mouse cardiomyocytes stressed with isoproterenol, pharmacologic inhibition and genetically knockout of NaV1.8 (SCN10A-/-), were associated with reduced INaL and abbreviated APD. We provide first evidence of differential regulation of NaV1.8 and NaV1.5 in the failing human myocardium and their contribution to arrhythmogenesis due to generation of INaL. We propose inhibition of NaV1.8 thus constitutes a promising novel approach for selective anti-arrhythmic therapy in HF.

Design of Conformationally Constrained Acyl Sulfonamide Isosteres: Identification of N-([1,2,4]Triazolo[4,3- a]pyridin-3-yl)methane-sulfonamides as Potent and Selective hNaV1.7 Inhibitors for the Treatment of Pain.

The sodium channel NaV1.7 has emerged as a promising target for the treatment of pain based on strong genetic validation of its role in nociception. In recent years, a number of aryl and acyl sulfonamides have been reported as potent inhibitors of NaV1.7, with high selectivity over the cardiac isoform NaV1.5. Herein, we report on the discovery of a novel series of N-([1,2,4]triazolo[4,3- a]pyridin-3-yl)methanesulfonamides as selective NaV1.7 inhibitors. Starting with the crystal structure of an acyl sulfonamide, we rationalized that cyclization to form a fused heterocycle would improve physicochemical properties, in particular lipophilicity. Our design strategy focused on optimization of potency for block of NaV1.7 and human metabolic stability. Lead compounds 10, 13 (GNE-131), and 25 showed excellent potency, good in vitro metabolic stability, and low in vivo clearance in mouse, rat, and dog. Compound 13 also displayed excellent efficacy in a transgenic mouse model of induced pain.

The role of REST and HDAC2 in epigenetic dysregulation of Nav1.5 and nNav1.5 expression in breast cancer

BackgroundIncreased expression of voltage-gated sodium channels (VGSCs) have been implicated with strong metastatic potential of human breast cancer in vitro and in vivo where the main culprits are cardiac isoform Nav1.5 and its ‘neonatal’ splice variant, nNav1.5. Several factors have been associated with Nav1.5 and nNav1.5 gain of expression in breast cancer mainly hormones, and growth factors.AimThis study aimed to investigate the role of epigenetics via transcription repressor, repressor element silencing transcription factor (REST) and histone deacetylases (HDACs) in enhancing Nav1.5 and nNav1.5 expression in human breast cancer by assessing the effect of HDAC inhibitor, trichostatin A (TSA).MethodsThe less aggressive human breast cancer cell line, MCF-7 cells which lack Nav1.5 and nNav1.5 expression was treated with TSA at a concentration range 10–10,000 ng/ml for 24 h whilst the aggressive MDA-MB-231 cells was used as control. The effect of TSA on Nav1.5, nNav1.5, REST, HDAC1, HDAC2, HDAC3, MMP2 and N-cadherin gene expression level was analysed by real-time PCR. Cell growth (MTT assay) and metastatic behaviors (lateral motility and migration assays) were also measured.ResultsmRNA expression level of Nav1.5 and nNav1.5 were initially very low in MCF-7 compared to MDA-MB-231 cells. Inversely, mRNA expression level of REST, HDAC1, HDAC2, and HDAC3 were all greater in MCF-7 compared to MDA-MB-231 cells. Treatment with TSA significantly increased the mRNA expression level of Nav1.5 and nNav1.5 in MCF-7 cells. On the contrary, TSA significantly reduced the mRNA expression level of REST and HDAC2 in this cell line. Remarkably, despite cell growth inhibition by TSA, motility and migration of MCF-7 cells were enhanced after TSA treatment, confirmed with the up-regulation of metastatic markers, MMP2 and N-cadherin.ConclusionsThis study identified epigenetics as another factor that regulate the expression level of Nav1.5 and nNav1.5 in breast cancer where REST and HDAC2 play important role as epigenetic regulators that when lacking enhances the expression of Nav1.5 and nNav1.5 thus promotes motility and migration of breast cancer. Elucidation of the regulatory mechanisms for gain of Nav1.5 and nNav1.5 expression may be helpful for seeking effective strategies for the management of metastatic diseases.

Nav1.5 channels can reach the plasma membrane through distinct N-glycosylation states

BackgroundLike many voltage-gated sodium channels, the cardiac isoform Nav1.5 is well known as a glycoprotein which necessarily undergoes N-glycosylation processing during its transit to the plasma membrane. In some cardiac disorders, especially the Brugada syndrome (BrS), mutations in Nav1.5 encoding gene lead to intracellular retention and consequently trafficking defect of these proteins. We used two BrS mutants as tools to clarify both Nav1.5 glycosylation states and associated secretory behaviors. MethodsPatch-clamp recordings and surface biotinylation assays of HEK293T cells expressing wild-type (WT) and/or mutant Nav1.5 proteins were performed to assess the impact of mutant co-expression on the membrane activity and localization of WT channels. Enzymatic deglycosylation assays and brefeldin A (BFA) treatments were also employed to further characterize recombinant and native Nav1.5 maturation. ResultsThe present data demonstrate that Nav1.5 channels mainly exist as two differentially glycosylated forms. We reveal that dominant negative effects induced by BrS mutants upon WT channel current result from the abnormal surface expression of the fully-glycosylated forms exclusively. Furthermore, we show that core-glycosylated channels can be found at the surface membrane of BFA-treated or untreated cells, but obviously without generating any sodium current. ConclusionsOur findings provide evidence that native and recombinant Nav1.5 subunits are expressed as two distinct matured forms. Fully-glycosylated state of Nav1.5 seems to determine its functionality whereas core-glycosylated forms might be transported to the plasma membrane through an unconventional Golgi-independent secretory route. General significanceThis work highlights that N-linked glycosylation processing would be critical for Nav1.5 membrane trafficking and function.

Substituted 4-phenyl-2-aminoimidazoles and 4-phenyl-4,5-dihydro-2-aminoimidazoles as voltage-gated sodium channel modulators

Voltage-gated sodium channels play an integral part in neurotransmission and their dysfunction is frequently a cause of various neurological disorders. On the basis of the structure of marine alkaloid clathrodin, twenty eight new analogs were designed, synthesized and tested for their ability to block human NaV1.3, NaV1.4 and NaV1.7 channels, as well as for their selectivity against human cardiac isoform NaV1.5, using automated patch clamp electrophysiological assay. Several compounds exhibited promising activities on different NaV channel isoforms in the medium micromolar range and some of the compounds showed also moderate isoform selectivities. The most promising results were obtained for the NaV1.3 channel, for which four compounds were found to possess IC50 values lower than 15 μM. All of the active compounds bind to the open-inactivated states of the channels and therefore act as state-dependent modulators. The obtained results validate the approach of using natural products driven chemistry for drug discovery starting points and represent a good foundation for future design of selective NaV modulators.

Novel state-dependent voltage-gated sodium channel modulators, based on marine alkaloids from Agelas sponges

Clathrodin, alkaloid isolated from Agelas sponges, was reported in 1995 as a voltage-gated sodium channel modulator. Here we describe the design and synthesis of conformationally restricted clathrodin analogues incorporating the 4,5,6,7-tetrahydrobenzo[d]thiazol-2-amine moiety and evaluation of their modulatory activities on human voltage-gated sodium channel isoforms Nav1.3, Nav1.4 and Nav1.7, as well as their selectivity against cardiac isoform Nav1.5. Compounds were shown to act as state-dependent modulators of Nav1.3, Nav1.4 and Nav1.7 with IC50 values in the lower micromolar range for the open-inactivated state of the channels. Preliminary structure–activity relationship studies have revealed the importance of hydrophobic interactions for binding to all three tested isoforms. Compound 4e with IC50 value of 8 μM against Nav1.4 represents a novel selective state-dependent Nav1.4 channel modulator.

Isoform-selective Effects of Isoflurane on Voltage-gated Na+ Channels

Voltage-gated Na channels modulate membrane excitability in excitable tissues. Inhibition of Na channels has been implicated in the effects of volatile anesthetics on both nervous and peripheral excitable tissues. The authors investigated isoform-selective effects of isoflurane on the major Na channel isoforms expressed in excitable tissues. Rat Nav1.2, Nav1.4, or Nav1.5 alpha subunits heterologously expressed in Chinese hamster ovary cells were analyzed by whole cell voltage clamp recording. The effects of isoflurane on Na current activation, inactivation, and recovery from inactivation were analyzed. The cardiac isoform Nav1.5 activated at more negative potentials (peak INa at -30 mV) than the neuronal Nav1.2 (0 mV) or skeletal muscle Nav1.4 (-10 mV) isoforms. Isoflurane reversibly inhibited all three isoforms in a concentration- and voltage-dependent manner at clinical concentrations (IC50 = 0.70, 0.61, and 0.45 mm, respectively, for Nav1.2, Nav1.4, and Nav1.5 from a physiologic holding potential of -70 mV). Inhibition was greater from a holding potential of -70 mV than from -100 mV, especially for Nav1.4 and Nav1.5. Isoflurane enhanced inactivation of all three isoforms due to a hyperpolarizing shift in the voltage dependence of steady state fast inactivation. Inhibition of Nav1.4 and Nav1.5 by isoflurane was attributed primarily to enhanced inactivation, whereas inhibition of Nav1.2, which had a more positive V1/2 of inactivation, was due primarily to tonic block. Two principal mechanisms contribute to Na channel inhibition by isoflurane: enhanced inactivation due to a hyperpolarizing shift in the voltage dependence of steady state fast inactivation (Nav1.5 approximately Nav1.4 > Nav1.2) and tonic block (Nav1.2 > Nav1.4 approximately Nav1.5). These novel mechanistic differences observed between isoforms suggest a potential pharmacologic basis for discrimination between Na channel isoforms to enhance anesthetic specificity.

Expression of skeletal muscle Na V1.4 Na channel isoform in canine cardiac Purkinje myocytes

Background and Aim The action potential plateau of Purkinje fibers is particularly sensitive to tetrodotoxin (TTX) and this could be due to a TXX-sensitive Na + current. The expression of TTX-sensitive neuronal Na V1.1 and Na V1.2 isoforms has been reported in canine Purkinje myocytes. Our aim was to investigate by means of biochemical and functional techniques whether the TTX-sensitive skeletal Na V1.4 isoform is also expressed in canine cardiac Purkinje myocytes. Methods and Results Using Na V1.4 specific primers, a PCR product corresponding to Na V1.4 was amplified from canine Purkinje fibers RNA and confirmed by sequencing and megablast of the gene bank. Confocal indirect immunostaining using anti-Na V1.4 antibody demonstrates distinct sarcolemmal staining pattern compared to that of the cardiac isoform Na V1.5. Expression of Na V1.4 in tsA201 cells yielded a TTX-sensitive Na + current with an IC 50 of 10 nM. Conclusions These results demonstrate the expression of the TTX-sensitive Na V1.4 channel in canine cardiac Purkinje myocytes. This novel finding suggests a role of Na V1.4 channel in Purkinje myocytes and thus has important clinical implications for the mechanisms and management of ventricular arrhythmias originating in the Purkinje network.