Inhibition of the voltage-gated potassium channel Kv1.5 by hydrogen sulfide attenuates remodeling through S-nitrosylation-mediated signaling.

The voltage-gated K+ channel plays a key role in atrial excitability, conducting the ultra-rapid rectifier K+ current (IKur) and contributing to the repolarization of the atrial action potential. In this study, we examine its regulation by hydrogen sulfide (H2S) in HL-1 cardiomyocytes and in HEK293 cells expressing human Kv1.5. Pacing induced remodeling resulted in shorting action potential duration, enhanced both Kv1.5 channel and H2S producing enzymes protein expression in HL-1 cardiomyocytes. H2S supplementation reduced these remodeling changes and restored action potential duration through inhibition of Kv1.5 channel. H2S also inhibited recombinant hKv1.5, lead to nitric oxide (NO) mediated S-nitrosylation and activated endothelial nitric oxide synthase (eNOS) by increased phosphorylation of Ser1177, prevention of NO formation precluded these effects. Regulation of Ikur by H2S has important cardiovascular implications and represents a novel and potential therapeutic target.

8-hydroxypinoresinol-4-O-ß-D-glucoside from Valeriana officinalis L. Is a Novel Kv1.5 Channel Blocker.

ETHNOPHARMACOLOGY RELEVANCE: In folkloric medicine of many cultures, one of the medical uses of Valeriana officinalis Linn is to treat heart-related disease. Recently, it was shown that the ethanol extracts from V. officinalis could effectively prevent auricular fibrillation, and 8-hydroxypinoresinol-4-O-ß-D-glucoside (HPG) from the extracts is one of the two active compounds showing antiarrhythmia activities. AIM OF THE STUDY: The human Kv1.5 channel (hKv1.5) has potential antiarrhythmia activities, and this study arms at investigating the current blocking effects of HPG on hKv1.5 channel. MATERIAL AND METHODS: HPG was obtained from V. officinalis extracts, and hKv1.5 channels were expressed in HEK 293 cells. HPG was perfused while recording the current through hKv1.5 channels. Patch-clamp recording techniques were used to study the effects of HPG at various concentrations (10 µM, 30 µM, and 50 µM) on hKv1.5 channels. RESULTS: The present study demonstrated that HPG inhibited hKv1.5 channel current in a concentration-dependent manner; the higher the concentration, the greater is the inhibition at each depolarization potential. During washout, the channels did not full recover indicating that the un-coupling between HPG and hKv1.5 channels is a slow process. CONCLUSION: HPG may be an effective and safe active ingredient for AF having translational potential.

Evaluation of Batch Variations in Induced Pluripotent Stem Cell-Derived Human Cardiomyocytes from 2 Major Suppliers.

Drug-induced proarrhythmia is a major safety issue in drug development. Developing sensitive in vitro assays that can predict drug-induced cardiotoxicity in humans has been a challenge of toxicology research for decades. Recently, induced pluripotent stem cell-derived human cardiomyocytes (iPSC-hCMs) have become a promising model because they largely replicate the electrophysiological behavior of human ventricular cardiomyocytes. Patient-specific iPSC-hCMs have been proposed for personalized cardiac drug selection and adverse drug response prediction; however, many procedures are involved in cardiomyocytes differentiation and purification process, which may result in large line-to-line and batch-to-batch variations. Here, we examined the purity, cardiac ion channel gene expression profile, and electrophysiological response of 3 batches of iPSC-hCMs from each of 2 major cell suppliers. We found that iPSC-hCMs from both vendors had similar purities. Most of the cardiac ion channel genes were expressed uniformly among different batches of iCells, while larger variations were found in Cor.4U cells, particularly in the expression of CACNA1C, KCND2, and KCNA5 genes, which could underlie the differences in baseline beating rate (BR) and field potential duration (FPD) measurements. Although, in general, the electrophysiological responses of different batches of cells to Na+, Ca2+, Ikr, and Iks channel blockers were similar, with Ikr blocker-induced proarrhythmia, the sensitivities were depended on baseline BR and FPD values: cells that beat slower had longer FPD and greater sensitivity to drug-induced proarrhythmia. Careful evaluation of the performance of iPSC-hCMs and methods of data analysis is warranted for shaping regulatory standards in qualifying iPSC-hCMs for drug safety testing.

Developmental expression of Kv1 voltage-gated potassium channels in the avian hypothalamus.

Specialized hypothalamic neurons integrate the homeostatic balance between food intake and energy expenditure, processes that may become dysregulated during the development of diabetes, obesity, and other metabolic disorders. Shaker family voltage-gated potassium channels (Kv1) contribute to the maintenance of resting membrane potential, action potential characteristics, and neurotransmitter release in many populations of neurons, although hypothalamic Kv1 channel expression has been largely unexplored. Whole-cell patch clamp recordings from avian hypothalamic brain slices demonstrate a developmental shift in the electrophysiological properties of avian arcuate nucleus neurons, identifying an increase in outward ionic current that corresponds with action potential maturation. Additionally, RT-PCR experiments identified the early expression of Kv1.2, Kv1.3, and Kv1.5 mRNA in the embryonic avian hypothalamus, suggesting that these channels may underlie the electrophysiological changes observed in these neurons. Real-time quantitative PCR analysis on intact microdissections of embryonic hypothalamic tissue revealed a concomitant increase in Kv1.2 and Kv1.5 gene expression at key electrophysiological time points during development. This study is the first to demonstrate hypothalamic mRNA expression of Kv1 channels in developing avian embryos and may suggest a role for voltage-gated ion channel regulation in the physiological patterning of embryonic hypothalamic circuits governing energy homeostasis.

Functional Characterization of Rare Variants Implicated in Susceptibility to Lone Atrial Fibrillation.

BACKGROUND: Few rare variants in atrial fibrillation (AF)-associated genes have been functionally characterized to identify a causal relationship between these variants and development of AF. We here sought to determine the clinical effect of rare variants in AF-associated genes in patients with lone AF and characterized these variants electrophysiologically and bioinformatically. METHODS AND RESULTS: We screened all coding regions in 12 AF-associated genes in 90 patients with lone AF, with an onset of 47±11 years (66 men; mean age, 56±13 years) by high-resolution melting curve analysis and DNA sequencing. The potassium and sodium currents were analyzed using whole-cell patch clamping. In addition to using 4 individual in silico prediction tools, we extended those predictions to an integrated tool (Combined Annotation Dependent Depletion). We identified 7 rare variants in KCNA5, KCNQ1, KCNH2, SCN5A, and SCN1B genes in 8 patients: 2 of 8 probands had a family history of AF. Electrophysiological studies revealed that 2 variants showed a loss-of-function, and 4 variants showed a gain-of-function. Five of 6 variants with electrophysiological abnormalities were predicted as pathogenic by Combined Annotation Dependent Depletion scores. CONCLUSIONS: In our cohort of patients with lone AF, 7 rare variants in cardiac ion channels were identified in 8 probands. A combination of electrophysiological studies and in silico predictions showed that these variants could contribute to the development of lone AF, although further in vivo study is necessary to confirm these results. More than half of AF-associated rare variants showed gain-of-function behavior, which may be targeted using genotype-specific pharmacological therapy.

Role for myosin-V motor proteins in the selective delivery of Kv channel isoforms to the membrane surface of cardiac myocytes.

RATIONALE: Kv1.5 (KCNA5) mediates the ultra-rapid delayed rectifier current that controls atrial action potential duration. Given its atrial-specific expression and alterations in human atrial fibrillation, Kv1.5 has emerged as a promising target for the treatment of atrial fibrillation. A necessary step in the development of novel agents that selectively modulate trafficking pathways is the identification of the cellular machinery controlling Kv1.5 surface density, of which little is yet known. OBJECTIVE: To investigate the role of the unconventional myosin-V (MYO5A and MYO5B) motors in determining the cell surface density of Kv1.5. METHODS AND RESULTS: Western blot analysis showed MYO5A and MYO5B expression in the heart, whereas disruption of endogenous motors selectively reduced IKur current in adult rat cardiomyocytes. Dominant negative constructs and short hairpin RNA silencing demonstrated a role for MYO5A and MYO5B in the surface trafficking of Kv1.5 and connexin-43 but not potassium voltage-gated channel, subfamily H (eag-related), member 2 (KCNH2). Live-cell imaging of Kv1.5-GFP and retrospective labeling of phalloidin demonstrated motility of Kv1.5 vesicles on actin tracts. MYO5A participated in anterograde trafficking, whereas MYO5B regulated postendocytic recycling. Overexpression of mutant motors revealed a selective role for Rab11 in coupling MYO5B to Kv1.5 recycling. CONCLUSIONS: MYO5A and MYO5B control functionally distinct steps in the surface trafficking of Kv1.5. These isoform-specific trafficking pathways determine Kv1.5-encoded IKur in myocytes to regulate repolarizing current and, consequently, cardiac excitability. Therapeutic strategies that manipulate Kv1.5 selective trafficking pathways may prove useful in the treatment of arrhythmias.

Intermittent hypoxia-induced increases in reactive oxygen species activate NFATc3 increasing endothelin-1 vasoconstrictor reactivity.

Sleep apnea (SA), defined as intermittent respiratory arrest during sleep, is associated with increased incidence of hypertension, peripheral vascular disease, stroke, and sudden cardiac death. We have shown that intermittent hypoxia with CO2 supplementation (IH), a model for SA, increases blood pressure and circulating ET-1 levels, upregulates lung pre-pro ET-1 mRNA, increases vasoconstrictor reactivity to ET-1 in rat small mesenteric arteries (MA) and increases vascular reactive oxygen species (ROS). NFAT activity is increased in the aorta (AO) and MA of mice exposed to IH in an ET-1-dependent manner, and the genetic ablation of the isoform NFATc3 prevents IH-induced hypertension. We hypothesized that IH causes an increase in arterial ROS generation, which activates NFATc3 to increase vasoconstrictor reactivity to ET-1. In support of our hypothesis, we found that IH increases ROS in AO and MA. In vivo administration of the SOD mimetic tempol during IH exposure prevents IH-induced increases in NFAT activity in mouse MA and AO. We found that IH causes an NFATc3-dependent increase in vasoconstrictor reactivity to ET-1, accompanied by an increase in vessel wall [Ca²âº]. Our results indicate that IH exposure causes an increase in arterial ROS to activate NFATc3, which then increases vasoconstrictor reactivity and Ca²âº response to ET-1. These studies highlight a novel regulatory pathway, and demonstrate the potential clinical relevance of NFAT inhibition to prevent hypertension in SA patients.

Torilin from Torilis japonica (Houtt.) DC. blocks hKv1.5 channel current.

Torilin was purified from Torilis japonica (Houtt.) DC., and its effects on a rapidly activating delayed rectifier K+ channel (hKv1.5), cloned from human heart and stably expressed in Ltk- cells, as well as the corresponding K+ current (the ultrarapid delayed rectifier, I(KUR)) were assessed in human atrial myocytes. Using the whole cell configuration of the patch-clamp technique, torilin was found to inhibit the hKv1.5 current in time and voltage-dependent manners, with an IC50 value of 2.51+/-0.34 microM at +60 mV. Torilin accelerated the inactivation kinetics of the hKv1.5 channel, and slowed the deactivation kinetics of the hKv1.5 current, resulting in a tail crossover phenomenon. Additionally, torilin inhibited the hKv1.5 current in a use-dependent manner. These results strongly suggest that torilin is a type of open-channel blocker of the hKv1.5 channel.