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.

Adrenergic regulation of the rapid component of delayed rectifier K+ current: implications for arrhythmogenesis in LQT2 patients.

BACKGROUND: KCNH2 gene mutations disrupting rapid component of I(K) (I(Kr)) underlie type 2 congenital long QT syndrome (LQT2). Startled auditory stimuli are specific symptomatic triggers in LQT2, thus suggesting fast arrhythmogenic mechanism. OBJECTIVE: We investigated acute alpha(1A)- and cyclic adenosine monophosphate (cAMP)-related beta-adrenergic modulation of I(Kr) in HL-1 cardiomyocytes, wild type (WT)- and 2 LQT2-associated mutant Kv11.1 channels (Y43D- and K595E-Kv11.1) reconstituted in Chinese hamster ovary (CHO) cells. METHODS: I(Kr) and Kv11.1 currents were recorded using the whole-cell patch-clamp technique and confocal microscopy of HL-1 cardiomyocytes transfected with green fluorescent protein (GFP)-tagged pleckstrin homology domain of phospholipase C-delta(1) visualized fluctuations of membrane phosphatidylinositol 4,5-bisphosphate (PIP(2)) content. RESULTS: In HL-1 cardiomyocytes expressing human alpha(1A)-adrenoceptor, superfusion with phenylephrine significantly reduced I(Kr) amplitude, shifted current activation to more positive potentials, and accelerated kinetics of deactivation. Confocal images showed a decline of membrane PIP(2) content during phenylephrine exposure. Simultaneous application of adenylyl cyclase activator forskolin and phosphodiesterase inhibitor 3-isobutyl-1-methylxantine (IBMX) shifted I(Kr) activation to more negative potentials and decreased tail current amplitudes after depolarizations between +10 and +50 mV. In CHO cells, alpha(1A)-adrenoceptor activation downregulated WT-Kv11.1 channels and forskolin/IBMX produced a dual effect. Expressed alone, the Y43D-Kv11.1 or K595E-Kv11.1 channel had no measurable function. However, co-expression of WT-Kv11.1 and each mutant protein evoked currents with loss-of-function alterations but identical to WT-Kv11.1 alpha(1A)- and forskolin/IBMX-induced regulation. CONCLUSION: Acute adrenergic regulation of at least 2 Kv11.1 mutant channels is preserved as in WT-Kv11.1 and native I(Kr). Suppression of alpha(1A)-adrenoceptor-related transduction might have therapeutic implications in some cases of LQT2.

N- and C-terminal KCNE1 mutations cause distinct phenotypes of long QT syndrome.

BACKGROUND: Long QT syndromes (LQTS) are inherited diseases involving mutations to genes encoding a number of cardiac ion channels and a membrane adaptor protein. The MinK protein is a cardiac K-channel accessory subunit encoded by the KCNE1 gene, mutations of which are associated with the LQT5 form of LQTS. OBJECTIVE: The purpose of this study was to search for the KCNE1 mutations and clarify the function of those mutations. METHODS: We conducted a genetic screen of KCNE1 mutations in 151 Japanese LQTS patients using the denaturing high-performance liquid chromatography-WAVE system and direct sequencing. In two LQTS patients, we identified two KCNE1 missense mutations, located in the MinK N- and C-terminal domains. The functional effects of these mutations were examined by heterologous coexpression with KCNQ1 and KCNH2. RESULTS: One mutation, which was identified in a 67-year-old woman, A8V, was novel. Her electrocardiogram (ECG) revealed marked bradycardia and QT interval prolongation. Another mutation, R98W, was identified in a 19-year-old woman. She experienced syncope followed by palpitation in exercise. At rest, her ECG showed bradycardia with mild QT prolongation, which became more prominent during exercise. In electrophysiological analyses, R98W produced reduced I(Ks) currents with a positive shift in the half activation voltages. In addition, when the A8V mutation was coexpressed with KCNH2, this reduced current magnitude, which is suggestive of a modifier effect by the A8V KCNE1 mutation on I(Kr). CONCLUSION: KCNE1 mutations may be associated with mild LQTS phenotypes, and KCNE1 gene screening is of clinical importance for asymptomatic and mild LQTS patients.

Blockade of angiotensin II type 1 receptor improves the arrhythmia morbidity in mice with left ventricular hypertrophy.

BACKGROUND: Stimulation of angiotensin II type 1 (AT(1)) receptors has been shown to generate the arrhythmogenic substrate in ventricular hypertrophy. We examined whether candesartan, an AT1 receptor blocker, has antiarrhythmic effects on mouse model of left ventricular hypertrophy created by transverse aorta constriction (TAC). METHODS AND RESULTS: Forty-eight male mice were divided into 3 groups: TAC, candesartan (TAC plus candesartan) and control groups. Echocardiographic examination was performed before the operation and 2 and 4 weeks after the operation. Four weeks after the operation, electrophysiological studies were conducted by inserting a 1.7 F octapolar electrode catheter through the right external jugular vein into the right ventricle. The effective refractory period of the atrioventricular node (AVNERP) in TAC group was significantly prolonged, and short episodes of ventricular tachycardia (VT) and atrial fibrillation (AF) could be induced in 12 of 16 mice (75%) and 8 of 16 (50%), respectively. In contrast, in candesartan group, the incidence of VT was significantly reduced (12.5%) and no AF was induced. Moreover, the drug produced a significant left ventricular hypertrophy regression and restored the AVNERP to normal. CONCLUSIONS: Candesartan reduced both ventricular and atrial arrhythmias in the TAC mice, presumably by preventing the electrical remodeling by inhibiting the AT(1) receptor.

Open-state unblock characterizes acute inhibition of I potassium current by amiodarone in guinea pig ventricular myocytes.

INTRODUCTION: The aim of the present study was to investigate the acute action of amiodarone on the slow component of delayed rectifier K+ current (IKs) under basal conditions and during beta-adrenoceptor stimulation in guinea pig ventricular myocytes. METHODS AND RESULTS: Using the whole-cell patch-clamp method, IKs was evoked by depolarizing voltage-clamp steps, during superfusion with the Na+-, K+-, and Ca2+-free solution supplemented with 0.4 microM nisoldipine and 5 microM E-4031. The acute effect of amiodarone was evaluated, within approximately 10 minutes after starting the bath application, by the amplitude of deactivating tail currents at -50 mV. Amiodarone concentration dependently blocked I(Ks) and exerted a more potent effect on IKs when activated by shorter pulse durations; the degree of block by 30 microM amiodarone on IKs activated by 200 ms, 500 ms, and 2000 ms depolarizing pulses to +30 mV was 55.9 +/- 5.8%, 38.6 +/- 6.0%, and 27.1 +/- 4.0% (n = 5 each), respectively. An envelope of tails test conducted at +10, +30, and +60 mV demonstrated that the degree of IKs block by amiodarone was gradually attenuated during membrane depolarization, which can be described by a monoexponential function, thus supporting the presence of open channel unblock. Amiodarone also blocked IKs maximally stimulated by 1 microM isoprenaline, to an extent similar to control, when IKs was activated by pulse durations of < or =2000 ms. CONCLUSION: We propose that amiodarone acutely blocks native IKs with characteristics associated with open channel unblock, and that the protein kinase A-mediated phosphorylation of channel proteins only minimally affects the amiodarone block.