Cardiac-specific deletion of BRG1 ameliorates ventricular arrhythmia in mice 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 ß-catenin, forming a transcription complex, which suppressed the transcription activity of SCN5A and KCND3, thereby influencing the incidence of VA post-MI.

Synthesis and In Vivo Antiarrhythmic Activity Evaluation of Novel Scutellarein Analogues as Voltage-Gated Nav1.5 and Cav1.2 Channels Blockers.

Malignant cardiac arrhythmias with high morbidity and mortality have posed a significant threat to our human health. Scutellarein, a metabolite of Scutellarin which is isolated from Scutellaria altissima L., presents excellent therapeutic effects on cardiovascular diseases and could further be metabolized into methylated forms. A series of 22 new scutellarein derivatives with hydroxyl-substitution based on the scutellarin metabolite in vivo was designed, synthesized via the conjugation of the scutellarein scaffold with pharmacophores of FDA-approved antiarrhythmic medications and evaluated for their antiarrhythmic activity through the analyzation of the rat number of arrhythmia recovery, corresponding to the recovery time and maintenance time in the rat model of barium chloride-induced arrhythmia, as well as the cumulative dosage of aconitine required to induce VP, VT, VF and CA in the rat model of aconitine-induced arrhythmia. All designed compounds could shorten the time of the arrhythmia continuum induced by barium chloride, indicating that 4'-hydroxy substituents of scutellarein had rapid-onset antiarrhythmic effects. In addition, nearly all of the compounds could normalize the HR, RR, QRS, QT and QTc interval, as well as the P/T waves' amplitude. The most promising compound 10e showed the best antiarrhythmic activity with long-term efficacy and extremely low cytotoxicity, better than the positive control scutellarein. This result was also approved by the computational docking simulation. Most importantly, patch clamp measurements on Nav1.5 and Cav1.2 channels indicated that compound 10e was able to reduce the INa and ICa in a concentration-dependent manner and left-shifted the inactivation curve of Nav1.5. Taken together, all compounds were considered to be antiarrhythmic. Compound 10e even showed no proarrhythmic effect and could be classified as Ib Vaughan Williams antiarrhythmic agents. What is more, compound 10e did not block the hERG potassium channel which highly associated with cardiotoxicity.

Inhibitory Effects of Nobiletin on Voltage-Gated Na+ Channel in Rat Ventricular Myocytes Based on Electrophysiological Analysis and Molecular Docking Method.

Nobiletin (NOB) has attracted much attention owing to its outstanding bioactivities. This study aimed to investigate its anti-arrhythmic effect through electrophysiological and molecular docking studies. We assessed the anti-arrhythmic effects of NOB using aconitine-induced ventricular arrhythmia in a rat model and the electrophysiological effects of NOB on rat cardiomyocytes utilizing whole-cell patch-clamp techniques. Moreover, we investigated the binding characters of NOB with rNav1.5, rNav1.5/QQQ, and hNaV1.5 via docking analysis, comparing them with amiodarone and aconitine. NOB pretreatment delayed susceptibility to ventricular premature and ventricular tachycardia and decreased the incidence of fatal ventricular fibrillation. Whole-cell patch-clamp assays demonstrated that the peak current density of the voltage-gated Na+ channel current was reversibly reduced by NOB in a concentration-dependent manner. The steady-state activation and recovery curves were shifted in the positive direction along the voltage axis, and the steady-state inactivation curve was shifted in the negative direction along the voltage axis, as shown by gating kinetics. The molecular docking study showed NOB formed a π-π stacking interaction with rNav1.5 and rNav1.5/QQQ upon Phe-1762, which is the homolog to Phe-1760 in hNaV1.5 and plays an important role in antiarrhythmic action This study reveals that NOB may act as a class I sodium channel anti-arrhythmia agent.

Disruption of asparagine-linked glycosylation to rescue and alter gating of the NaV1.5-Na+ channel.

SCN5A gene encodes the voltage-gated sodium channel NaV1.5 which is composed of a pore-forming α subunit of the channel. Asparagine (N)-linked glycosylation is one of the common post-translational modifications in proteins. The aim of this study was to investigate impact of N-linked glycosylation disruption on the Na+ channel, and the mechanism by which glycosylation regulates the current density and gating properties of the Na+ channel. The NaV1.5-Na+ channel isoform (α submit) derived from human was stably expressed in human embryonic kidney (HEK)-293 cells (Nav1.5-HEK cell). We applied the whole-cell patch-clamp technique to study the impact of N-linked glycosylation disruption in Nav1.5-HEK cell. Inhibition of the N-glycosylation with tunicamycin caused a significant increase of NaV1.5 channel current (INa) when applied for 24 h. Tunicamycin shifted the steady-state inactivation curve to the hyperpolarization direction, whereas the activation curve was unaffected. Recovery from inactivation was prolonged, while the fast phase (τfast) and the slow phase (τslow) of the current decay was unaffected by tunicamycin. INa was unaffected by tunicamycin in the present of a proteasome inhibitor MG132 [N-[(phenylmethoxy)carbonyl]-L-leucy-N-[(1S)-1-formyl-3-methylbutyl]-L-leucinamide], while it was significantly increased by tunicamycin in the presence of a lysosome inhibitor butyl methacrylate (BMA). These findings suggest that N-glycosylation disruption rescues the NaV1.5 channel possibly through the alteration of ubiquitin-proteasome activity, and changes gating properties of the NaV1.5 channel by modulating glycan milieu of the channel protein.

A Mathematical Model of the Human Cardiac Na+ Channel.

Sodium ion channel is a membrane protein that plays an important role in excitable cells, as it is responsible for the initiation of action potentials. Understanding the electrical characteristics of sodium channels is essential in predicting their behavior under different physiological conditions. We investigated several Markov models for the human cardiac sodium channel NaV1.5 to derive a minimal mathematical model that describes the reported experimental data obtained using major voltage clamp protocols. We obtained simulation results for peak current-voltage relationships, the voltage dependence of normalized ion channel conductance, steady-state inactivation, activation and deactivation kinetics, fast and slow inactivation kinetics, and recovery from inactivation kinetics. Good agreement with the experimental data provides us with the mechanisms of the fast and slow inactivation of the human sodium channel and the coupling of its inactivation states to the closed and open states in the activation pathway.

Epidermal Growth Factor Potentiates Migration of MDA-MB 231 Breast Cancer Cells by Increasing NaV1.5 Channel Expression.

INTRODUCTION: Breast cancer is one of the leading causes of death worldwide and is the result of dysregulation of various signaling pathways in mammary epithelial cells. The mortality rate in patients suffering from breast cancer is high because the tumor cells have a prominent invasive capacity towards the surrounding tissues. Previous studies carried out in tumor cell models show that voltage-gated ion channels may be important molecular actors that contribute to the migratory and invasive capacity of the tumor cells. METHODS: In this study, by using an experimental strategy that combines cell and molecular biology assays with electrophysiological recording, we sought to determine whether the voltage-dependent sodium channel NaV1.5 regulates the migratory capacity of the human breast cancer cell line MDA-MB 231, when cells are maintained in the presence of epidermal growth factor (EGF), as an inductor of the epithelial-mesenchymal transition. RESULTS: Our data show that EGF stimulates the migratory capacity of MDA-MB 231 cells, by regulating the functional expression of NaV1.5 channels. Consistent with this, the stimulatory actions of the growth factor were prevented by the use of tetrodotoxin, an Na+ channel selective blocker, as well as by resveratrol, an antioxidant that can also affect Na+ channel activity. DISCUSSION: The understanding of molecular mechanisms, such as the EGF pathway in the progression of breast cancer is fundamental for the design of more effective therapeutic strategies for the disease.

Cardiac Nav 1.5 is modulated by ubiquitin protein ligase E3 component n-recognin UBR3 and 6.

The voltage-gated Na(+) channel Nav 1.5 is essential for action potential (AP) formation and electrophysiological homoeostasis in the heart. The ubiquitin-proteasome system (UPS) is a major degradative system for intracellular proteins including ion channels. The ubiquitin protein ligase E3 component N-recognin (UBR) family is a part of the UPS; however, their roles in regulating cardiac Nav 1.5 channels remain elusive. Here, we found that all of the UBR members were expressed in cardiomyocytes. Individual knockdown of UBR3 or UBR6, but not of other UBR members, significantly increased Nav 1.5 protein levels in neonatal rat ventricular myocytes, and this effect was verified in HEK293T cells expressing Nav 1.5 channels. The UBR3/6-dependent regulation of Nav 1.5 channels was not transcriptionally mediated, and pharmacological inhibition of protein biosynthesis failed to counteract the increase in Nav 1.5 protein caused by UBR3/6 reduction, suggesting a degradative modulation of UBR3/6 on Nav 1.5. Furthermore, the effects of UBR3/6 knockdown on Nav 1.5 proteins were abolished under the inhibition of proteasome activity, and UBR3/6 knockdown reduced Nav 1.5 ubiquitylation. The double UBR3-UBR6 knockdown resulted in comparable increases in Nav 1.5 proteins to that observed for single knockdown of either UBR3 or UBR6. Electrophysiological recordings showed that UBR3/6 reduction-mediated increase in Nav 1.5 protein enhanced the opening of Nav 1.5 channels and thereby the amplitude of the AP. Thus, our findings indicate that UBR3/6 regulate cardiomyocyte Nav 1.5 channel protein levels via the ubiquitin-proteasome pathway. It is likely that UBR3/6 have the potential to be a therapeutic target for cardiac arrhythmias.

Diagnostic yield and variant reassessment in the genes encoding Nav1.5 channel in Russian patients with Brugada syndrome.

Brugada syndrome (BrS) is an inherited cardiac arrhythmia characterized by ST-elevation, negative T-wave, and a high risk of sudden cardiac death (SCD) due to ventricular tachycardia. It is associated with mutations in over 20 genes but only SCN5A is recommended for routine genetic screening. This study was performed to estimate diagnostic yield and pathogenicity assessment of rare genetic variants in the genes encoding Nav1.5 channel in Russian patients with Brugada syndrome (BrS). Targeted genes panel sequencing of the five genes were screened using IonTorrent PGM with following Sanger confirmation. Detailed clinical evaluation of 75 unrelated BrS probands with a deep phenotyping of SCN5A (+) probands was performed. Twelve rare genetic variants (six missense, six truncating) were initially identified and classified as disease-causing. Reassessment of the clinical significance in the light of the current guidelines revealed: 2 Pathogenic (P) variants; 8 Likely Pathogenic (LP); two missense variants (p.G274S and p. S1778H) were re-classified later as a variant of uncertain significance (VUS). Unique VUS (p.Arg100Ser) was detected in the SCN4B gene. Lone Brugada-pattern was observed in 46% probands; 54% patients had concomitant arrhythmias. PR interval, the only electrocardiography parameter correlating with SCN5A-mutation, was longer (207 ± 24 ms) than normal in SCN5A (+) probands. SCD cases were registered in 31 families. Depression was the only recurring extra-cardiac complaint in SCN5A (+) probands; it was self-reported in five SCN5A (+) probands, and co-segregated with Brugada pattern in 2 families. After variants reassessment, the ratio of SCN5A (+) probands with Brugada syndrome accounts for 13% in Russian cohort.

Alterations of Nedd4-2-binding capacity in PY-motif of NaV 1.5 channel underlie long QT syndrome and Brugada syndrome.

AIMS: Pathogenic variants of the SCN5A gene can cause Brugada syndrome (BrS) and long QT syndrome (LQTS), which predispose individuals to potentially fatal ventricular arrhythmias and sudden cardiac death. SCN5A encodes the NaV 1.5 protein, the pore forming α-subunit of the voltage-dependent cardiac Na+ channel. Using a WW domain, the E3 ubiquitin ligase Nedd4-2 binds to the PY-motif ([L/P]PxY) within the C-terminus of NaV 1.5, which results in decreased protein expression and current through NaV 1.5 ubiquitination. Here, we investigate the role of E3 ubiquitin ligase Nedd4-2-mediated NaV 1.5 degradation in the pathological mechanisms of the BrS-associated variant SCN5A-p.L1239P and LQTS-associated variant SCN5A-p.Y1977N. METHODS AND RESULTS: Using a combination of molecular biology, biochemical and electrophysiological approaches, we examined the expression, function and Nedd4-2 interactions of SCN5A-p.L1239P and SCN5A-p.Y1977N. SCN5A-p.L1239P is characterized as a loss-of-function, whereas SCN5A-p.Y1977N is a gain-of-function variant of the NaV 1.5 channel. Sequence alignment shows that BrS-associated SCN5A-p.L1239P has a new Nedd4-2-binding site (from LLxY to LPxY). This new Nedd4-2-binding site increases the interaction between NaV 1.5 and Nedd4-2, enhancing ubiquitination and degradation of the NaV 1.5 channel. Disruption of the new Nedd4-2-binding site of SCN5A-p.L1239P restores NaV 1.5 expression and function. However, the LQTS-associated SCN5A-p.Y1977N disrupts the usual Nedd4-2-binding site (from PPxY to PPxN). This decreases NaV 1.5-Nedd4-2 interaction, preventing ubiquitination and degradation of NaV 1.5 channels. CONCLUSIONS: Our data suggest that the PY-motif plays an essential role in modifying the expression/function of NaV 1.5 channels through Nedd4-2-mediated ubiquitination. Alterations of NaV 1.5-Nedd4-2 interaction represent a novel pathological mechanism for NaV 1.5 channel diseases caused by SCN5A variants.

DPP10 is a new regulator of Nav1.5 channels in human heart.

BACKGROUND: Cardiac accessory ß-subunits are part of macromolecular Nav1.5 channel complexes modulating biophysical properties and contributing to arrhythmias. Recent studies demonstrated the structural interaction between ß-subunits of Na+ (Nav1.5) and K+ (Kv4.3) channels. Here, we identified the dipeptidyl peptidase-like protein-10 (DPP10), which is known to modulate Kv4.3-current kinetics, as a new regulator of Nav1.5 channels. METHODS: We assessed DPP10 expression in the healthy and diseased human heart and we studied the functional effects of DPP10 on the Na+ current in isolated rat cardiomyocytes expressing DPP10 after adenoviral gene-transfer (DPP10ad). RESULTS: DPP10 mRNA and proteins were detected in human ventricle, with higher levels in patients with heart failure. In rat cardiomyocytes, DPP10ad significantly reduced upstroke velocity of action potentials indicating reduction in Na+-current density. DPP10 significantly shifted the voltage-dependent Na+ channel activation and inactivation curve to more positive potentials, resulting in greater availability of Na+ channels for activation, along with increasing window Na+ current. In addition, time-to-peak Na+ current was reduced, whereas time course of recovery from inactivation was significantly accelerated by DPP10ad. DPP10 co-immunoprecipitated with Nav1.5 channels in human ventricles, confirming their physical interaction. CONCLUSION: We provide first evidence that DPP10 interacts with Nav1.5 channels, linking Na+- and K+-channel complexes in the heart. Our data suggest that increased ventricular DPP10 expression in heart failure might promote arrhythmias by decreasing peak Na+ current, while increasing window Na+ current and channel re-openings due to accelerated recovery from inactivation.

Basal late sodium current is a significant contributor to the duration of action potential of guinea pig ventricular myocytes.

In cardiac myocytes, an enhancement of late sodium current (INaL) under pathological conditions is known to cause prolongation of action potential duration (APD). This study investigated the contribution of INaL under basal, physiological conditions to the APD Whole-cell INaL and the APD of ventricular myocytes isolated from healthy adult guinea pigs were measured at 36°C. The INaL inhibitor GS967 or TTX was applied to block INaL The amplitude of basal INaL and the APD at 50% repolarization in myocytes stimulated at a frequency of 0.17 Hz were -0.24 ± 0.02 pA/pF and 229 ± 6 msec, respectively. GS967 (0.01-1 µmol/L) concentration dependently reduced the basal INaL by 18 ± 3-82 ± 4%. At the same concentrations, GS967 shortened the APD by 9 ± 2 to 25 ± 1%. Similarly, TTX at 0.1-10 µmol/L decreased the basal INaL by 13 ± 1-94 ± 1% and APD by 8 ± 1-31 ± 2%. There was a close correlation (R2 = 0.958) between the percentage inhibition of INaL and the percentage shortening of APD caused by either GS967 or TTX MTSEA (methanethiosulfonate ethylammonium, 2 mmol/L), a NaV1.5 channel blocker, reduced the INaL by 90 ± 5%, suggesting that the NaV1.5 channel isoform is the major contributor to the basal INaL KN-93 (10 µmol/L) and AIP (2 µmol/L), blockers of CaMKII, moderately reduced the basal INaL Thus, this study provides strong evidence that basal endogenous INaL is a significant contributor to the APD of cardiac myocytes. In addition, the basal INaL of guinea pig ventricular myocytes is mainly generated from NaV1.5 channel isoform and is regulated by CaMKII.

Inhibitory effects of neferine on Nav1.5 channels expressed in HEK293 cells.

Neferine, a bisbenzylisoquinoline alkaloid in Lotus Plumule, was proved to have a wide range of biological activities. In the present study, using whole-cell patch-clamp technique, we investigated the effects of neferine on Nav1.5 channels that are stably expressed in HEK 293 cells. We found that neferine potently and reversibly inhibited Nav1.5 currents in a concentration dependent manner with a half-maximal inhibition (IC50) being 26.15 µmol/L. The inhibitory effects of neferine on Nav1.5 currents were weaker than those of quinidine at the same concentration. The steady-state inactivation curve was significantly shifted towards hyperpolarizing direction in the presence of 30 µmol/L neferine, while the voltage-dependent activation was unaltered. Neferine prolonged the time to peak of activation, increased the inactivation time constants of Nav1.5 currents and markedly slowed the recovery from inactivation. The inhibitory effect of neferine could be potentiated in a frequency-dependent manner. These results suggested that neferine can block Nav1.5 channels under the open state and inactivating state and it is an open channel blocker of Nav1.5 channels.

Jingzhaotoxin-35, a novel gating-modifier toxin targeting both Nav1.5 and Kv2.1 channels.

Jingzhaotoxin-35 (JZTX-35), a 36-residue polypeptide, was purified from the venom of the Chinese tarantula Chilobrachys jingzhao. JZTX-35 inhibited Nav1.5 and Kv2.1 currents with the IC50 value of 1.07 µM and 3.62 µM, respectively, but showed no significant effect on either Na(+) currents or Ca(2+) currents evoked in hippocampal neurons. It shifted the activation of the Nav1.5 and Kv2.1 channels to more depolarized voltages, and markedly shifted the steady-state inactivation of Nav1.5 currents toward more hyperpolarized potentials. Moreover, JZTX-35 can bind to a close state of Nav1.5 and Kv2.1 channels. These results indicate that JZTX-35 is a new gating modifier toxin. JZTX-35 shares high sequence similarity with Jingzhaotoxins (JZTXs) targeting Nav1.5 or Kv2.1 channels, but they showed different ion channel selectivity. Structure-function analysis in this study would provide important clues for the exploration of ion channel selectivity of JZTXs.

Is there a role for voltage-gated Na+ channels in the aggressiveness of breast cancer?

Breast cancer is the most common cancer among women and its metastatic potential is responsible for numerous deaths. Thus, the need to find new targets for improving treatment, and even finding the cure, becomes increasingly greater. Ion channels are known to participate in several physiological functions, such as muscle contraction, cell volume regulation, immune response and cell proliferation. In breast cancer, different types of ion channels have been associated with tumorigenesis. Recently, voltage-gated Na+ channels (VGSC) have been implicated in the processes that lead to increased tumor aggressiveness. To explain this relationship, different theories, associated with pH changes, gene expression and intracellular Ca2+, have been proposed in an attempt to better understand the role of these ion channels in breast cancer. However, these theories are having difficulty being accepted because most of the findings are contrary to the present scientific knowledge. Several studies have shown that VGSC are related to different types of cancer, making them a promising pharmacological target against this debilitating disease. Molecular biology and cell electrophysiology have been used to look for new forms of treatment aiming to reduce aggressiveness and the disease progress.