Mutant KCNJ3 and KCNJ5 Potassium Channels as Novel Molecular Targets in Bradyarrhythmias and Atrial Fibrillation.

BACKGROUND: Bradyarrhythmia is a common clinical manifestation. Although the majority of cases are acquired, genetic analysis of families with bradyarrhythmia has identified a growing number of causative gene mutations. Because the only ultimate treatment for symptomatic bradyarrhythmia has been invasive surgical implantation of a pacemaker, the discovery of novel therapeutic molecular targets is necessary to improve prognosis and quality of life. METHODS: We investigated a family containing 7 individuals with autosomal dominant bradyarrhythmias of sinus node dysfunction, atrial fibrillation with slow ventricular response, and atrioventricular block. To identify the causative mutation, we conducted the family-based whole exome sequencing and genome-wide linkage analysis. We characterized the mutation-related mechanisms based on the pathophysiology in vitro. After generating a transgenic animal model to confirm the human phenotypes of bradyarrhythmia, we also evaluated the efficacy of a newly identified molecular-targeted compound to upregulate heart rate in bradyarrhythmias by using the animal model. RESULTS: We identified one heterozygous mutation, KCNJ3 c.247A>C, p.N83H, as a novel cause of hereditary bradyarrhythmias in this family. KCNJ3 encodes the inwardly rectifying potassium channel Kir3.1, which combines with Kir3.4 (encoded by KCNJ5) to form the acetylcholine-activated potassium channel ( IKACh channel) with specific expression in the atrium. An additional study using a genome cohort of 2185 patients with sporadic atrial fibrillation revealed another 5 rare mutations in KCNJ3 and KCNJ5, suggesting the relevance of both genes to these arrhythmias. Cellular electrophysiological studies revealed that the KCNJ3 p.N83H mutation caused a gain of IKACh channel function by increasing the basal current, even in the absence of m2 muscarinic receptor stimulation. We generated transgenic zebrafish expressing mutant human KCNJ3 in the atrium specifically. It is interesting to note that the selective IKACh channel blocker NIP-151 repressed the increased current and improved bradyarrhythmia phenotypes in the mutant zebrafish. CONCLUSIONS: The IKACh channel is associated with the pathophysiology of bradyarrhythmia and atrial fibrillation, and the mutant IKACh channel ( KCNJ3 p.N83H) can be effectively inhibited by NIP-151, a selective IKACh channel blocker. Thus, the IKACh channel might be considered to be a suitable pharmacological target for patients who have bradyarrhythmia with a gain-of-function mutation in the IKACh channel.

Overexpression of KCNJ2 in induced pluripotent stem cell-derived cardiomyocytes for the assessment of QT-prolonging drugs.

Human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes hold great potentials to predict pro-arrhythmic risks in preclinical cardiac safety screening, although the hiPSC cardiomyocytes exhibit rather immature functional and structural characteristics, including spontaneous activity. Our physiological characterization and mathematical simulation showed that low expression of the inward-rectifier potassium (IK1) channel is a determinant of spontaneous activity. To understand impact of the low IK1 expression on the pharmacological properties, we tested if transduction of hiPSC-derived cardiomyocytes with KCNJ2, which encodes the IK1 channel, alters pharmacological response to cardiac repolarization processes. The transduction of KCNJ2 resulted in quiescent hiPSC-derived cardiomyocytes, which need pacing to elicit action potentials. Significant prolongation of paced action potential duration in KCNJ2-transduced hiPSC-derived cardiomyocytes was stably measured at 0.1 µM E-4031, although the same concentration of E-4031 ablated firing of non-treated hiPSC-derived cardiomyocytes. These results in single cells were confirmed by mathematical simulations. Using the hiPSC-derived cardiac sheets with KCNJ2-transduction, we also investigated effects of a range of drugs on field potential duration recorded at 1 Hz. The KCNJ2 overexpression in hiPSC-derived cardiomyocytes may contribute to evaluate a part of QT-prolonging drugs at toxicological concentrations with high accuracy.

Enhancement of Spontaneous Activity by HCN4 Overexpression in Mouse Embryonic Stem Cell-Derived Cardiomyocytes - A Possible Biological Pacemaker.

BACKGROUND: Establishment of a biological pacemaker is expected to solve the persisting problems of a mechanical pacemaker including the problems of battery life and electromagnetic interference. Enhancement of the funny current (If) flowing through hyperpolarization-activated cyclic nucleotide-gated (HCN) channels and attenuation of the inward rectifier K+ current (IK1) flowing through inward rectifier potassium (Kir) channels are essential for generation of a biological pacemaker. Therefore, we generated HCN4-overexpressing mouse embryonic stem cells (mESCs) and induced cardiomyocytes that originally show poor IK1 currents, and we investigated whether the HCN4-overexpressing mESC-derived cardiomyocytes (mESC-CMs) function as a biological pacemaker in vitro. METHODS AND RESULTS: The rabbit Hcn4 gene was transfected into mESCs, and stable clones were selected. mESC-CMs were generated via embryoid bodies and purified under serum/glucose-free and lactate-supplemented conditions. Approximately 90% of the purified cells were troponin I-positive by immunostaining. In mESC-CMs, expression level of the Kcnj2 gene encoding Kir2.1, which is essential for generation of IK1 currents that are responsible for stabilizing the resting membrane potential, was lower than that in an adult mouse ventricle. HCN4-overexpressing mESC-CMs expressed about a 3-times higher level of the Hcn4 gene than did non-overexpressing mESC-CMs. Expression of the Cacna1h gene, which encodes T-type calcium channel and generates diastolic depolarization in the sinoatrial node, was also confirmed. Additionally, genes required for impulse conduction including Connexin40, Connexin43, and Connexin45 genes, which encode connexins forming gap junctions, and the Scn5a gene, which encodes sodium channels, are expressed in the cells. HCN4-overexpressing mESC-CMs showed significantly larger If currents and more rapid spontaneous beating than did non-overexpressing mESC-CMs. The beating rate of HCN4-overexpressing mESC-CMs responded to ivabradine, an If inhibitor, and to isoproterenol, a beta-adrenergic receptor agonist. Co-culture of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) with aggregates composed of mESC-CMs resulted in synchronized contraction of the cells. The beating rate of hiPSC-CMs co-cultured with aggregates of HCN4-overexpressing mESC-CMs was significantly higher than that of non-treated hiPSC-CMs and that of hiPSC-CMs co-cultured with aggregates of non-overexpressing mESC-CMs. CONCLUSIONS: We generated HCN4-overexpresssing mESC-CMs expressing genes required for impulse conduction, showing rapid spontaneous beating, responding to an If inhibitor and beta-adrenergic receptor agonist, and having pacing ability in an in vitro co-culture system with other excitable cells. The results indicated that these cells could be applied to a biological pacemaker.

Image-based evaluation of contraction-relaxation kinetics of human-induced pluripotent stem cell-derived cardiomyocytes: Correlation and complementarity with extracellular electrophysiology.

In this study, we used high-speed video microscopy with motion vector analysis to investigate the contractile characteristics of hiPS-CM monolayer, in addition to further characterizing the motion with extracellular field potential (FP), traction force and the Ca(2+) transient. Results of our traction force microscopy demonstrated that the force development of hiPS-CMs correlated well with the cellular deformation detected by the video microscopy with motion vector analysis. In the presence of verapamil and isoproterenol, contractile motion of hiPS-CMs showed alteration in accordance with the changes in fluorescence peak of the Ca(2+) transient, i.e., upstroke, decay, amplitude and full-width at half-maximum. Simultaneously recorded hiPS-CM motion and FP showed that there was a linear correlation between changes in the motion and field potential duration in response to verapamil (30-150nM), isoproterenol (0.1-10µM) and E-4031 (10-50nM). In addition, tetrodotoxin (3-30µM)-induced delay of sodium current was corresponded with the delay of the contraction onset of hiPS-CMs. These results indicate that the electrophysiological and functional behaviors of hiPS-CMs are quantitatively reflected in the contractile motion detected by this image-based technique. In the presence of 100nM E-4031, the occurrence of early after-depolarization-like negative deflection in FP was also detected in the hiPS-CM motion as a characteristic two-step relaxation pattern. These findings offer insights into the interpretation of the motion kinetics of the hiPS-CMs, and are relevant for understanding electrical and mechanical relationship in hiPS-CMs.

Ginsenoside Re, a main phytosterol of Panax ginseng, activates cardiac potassium channels via a nongenomic pathway of sex hormones.

Ginseng root is one of the most popular herbs throughout the world and is believed to be a panacea and to promote longevity. It has been used as a medicine to protect against cardiac ischemia, a major cause of death in the West. We have previously demonstrated that ginsenoside Re, a main phytosterol of Panax ginseng, inhibits Ca(2+) accumulation in mitochondria during cardiac ischemia/reperfusion, which is attributable to nitric oxide (NO)-induced Ca(2+) channel inhibition and K(+) channel activation in cardiac myocytes. In this study, we provide compelling evidence that ginsenoside Re activates endothelial NO synthase (eNOS) to release NO, resulting in activation of the slowly activating delayed rectifier K(+) current. The eNOS activation occurs via a nongenomic pathway of each of androgen receptor, estrogen receptor-alpha, and progesterone receptor, in which c-Src, phosphoinositide 3-kinase, Akt, and eNOS are sequentially activated. However, ginsenoside Re does not stimulate proliferation of androgen-responsive LNCaP cells and estrogen-responsive MCF-7 cells, implying that ginsenoside Re does not activate a genomic pathway of sex hormone receptors. Fluorescence resonance energy transfer experiments with a probe, SCCoR (single cell coactivator recruitment), indicate that the lack of genomic action is attributable to failure of coactivator recruitment. Thus, ginsenoside Re acts as a specific agonist for the nongenomic pathway of sex steroid receptors, and NO released from activated eNOS underlies cardiac K(+) channel activation and protection against ischemia-reperfusion injury.

Nitric oxide-dependent modulation of the delayed rectifier K+ current and the L-type Ca2+ current by ginsenoside Re, an ingredient of Panax ginseng, in guinea-pig cardiomyocytes.

1 Ginsenoside Re, a major ingredient of Panax ginseng, protects the heart against ischemia-reperfusion injury by shortening action potential duration (APD) and thereby prohibiting influx of excessive Ca2+. Ginsenoside Re enhances the slowly activating component of the delayed rectifier K+ current (IKs) and suppresses the L-type Ca2+ current (I(Ca,L)), which may account for APD shortening. 2 We used perforated configuration of patch-clamp technique to define the mechanism of enhancement of IKs and suppression of I(Ca,L) by ginsenoside Re in guinea-pig ventricular myocytes. 3 S-Methylisothiourea (SMT, 1 microm), an inhibitor of nitric oxide (NO) synthase (NOS), and N-acetyl-L-cystein (LNAC, 1 mm), an NO scavenger, inhibited IKs enhancement. Application of an NO donor, sodium nitroprusside (SNP, 1 mm), enhanced IKs with a magnitude similar to that by a maximum dose (20 microm) of ginseonside Re, and subsequent application of ginsenoside Re failed to enhance IKs. Conversely, after IKs had been enhanced by ginsenoside Re (20 microm), subsequently applied SNP failed to further enhance IKs. 4 An inhibitor of guanylate cyclase, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, 10 microm), barely suppressed IKs enhancement, while a thiol-alkylating reagent, N-ethylmaleimide (NEM, 0.5 mm), clearly suppressed it. A reducing reagent, di-thiothreitol (DTT, 5 mm), reversed both ginsenoside Re- and SNP-induced IKs enhancement. 5 I(Ca,L) suppression by ginsenoside Re (3 microm) was abolished by SMT (1 microm) or LNAC (1 mm). NEM (0.5 mm) did not suppress I(Ca,L) inhibition and DTT (5 mm) did not reverse I(Ca,L) inhibition, whereas in the presence of ODQ (10 microm), ginsenoside Re (3 microm) failed to suppress I(Ca,L). 6 These results indicate that ginsenoside Re-induced IKs enhancement and I(Ca,L) suppression involve NO actions. Direct S-nitrosylation of channel protein appears to be the main mechanism for IKs enhancement, while a cGMP-dependent pathway is responsible for I(Ca,L) inhibition.

Electrophysiological effects of ginseng and ginsenoside Re in guinea pig ventricular myocytes.

Panax ginseng is a folk medicine with various cardiovascular actions; however, its underlying mechanisms of action are not well known. In the present study, we examined the effects of ginseng and its main component, ginsenoside Re, on action potentials and membrane currents recorded from isolated guinea pig ventricular myocytes with the whole-cell patch clamp technique. Ginseng (1 mg/ml) shortened the action potential duration in a rate-dependent manner. Ginseng depressed the L-type Ca2+ current (I(Ca-L)) in a mode of both tonic block and use-dependent block, and enhanced the slowly activating component of the delayed rectifier K+ current (I(Ks)). Ginsenoside Re 3 microM exhibited similar electrophysiological effects to those of 1 mg/ml ginseng, but of slightly smaller magnitude. Inhibition of I(Ca,L) and enhancement of I(Ks) by ginsenoside Re appear to be one of the main electrophysiological actions of ginseng in the heart, although contributions from other ingredients should be considered.