Search

BackgroundGenetic variants in calsequestrin-2 (CASQ2) cause an autosomal recessive form of catecholaminergic polymorphic ventricular tachycardia (CPVT), although isolated reports have identified arrhythmic phenotypes among heterozygotes. Improved insight into the inheritance patterns, arrhythmic risks, and molecular mechanisms of CASQ2-CPVT was sought through an international multicenter collaboration.MethodsGenotype-phenotype segregation in CASQ2-CPVT families was assessed, and the impact of genotype on arrhythmic risk was evaluated using Cox regression models. Putative dominant CASQ2 missense variants and the established recessive CASQ2-p.R33Q variant were evaluated using oligomerization assays and their locations mapped to a recent CASQ2 filament structure.ResultsA total of 112 individuals, including 36 CPVT probands (24 homozygotes/compound heterozygotes and 12 heterozygotes) and 76 family members possessing at least 1 presumed pathogenic CASQ2 variant, were identified. Among CASQ2 homozygotes and compound heterozygotes, clinical penetrance was 97.1% and 26 of 34 (76.5%) individuals had experienced a potentially fatal arrhythmic event with a median age of onset of 7 years (95% CI, 6-11). Fifty-one of 66 CASQ2 heterozygous family members had undergone clinical evaluation, and 17 of 51 (33.3%) met diagnostic criteria for CPVT. Relative to CASQ2 heterozygotes, CASQ2 homozygote/compound heterozygote genotype status in probands was associated with a 3.2-fold (95% CI, 1.3-8.0; P=0.013) increased hazard of a composite of cardiac syncope, aborted cardiac arrest, and sudden cardiac death, but a 38.8-fold (95% CI, 5.6-269.1; P

Long-QT syndrome mutations can cause syncope and sudden death by prolonging the cardiac action potential (AP). Ion channels affected by mutations are various, and the influences of cellular calcium cycling on LQTS cardiac events are unknown. To better understand LQTS arrhythmias, we performed current-clamp and intracellular calcium ([Ca(2+)]i) measurements on cardiomyocytes differentiated from patient-derived induced pluripotent stem cells (iPS-CM). In myocytes carrying an LQT2 mutation (HERG-A422T), APs and [Ca(2+)]i transients were prolonged in parallel. APs were abbreviated by nifedipine exposure and further lengthened upon releasing intracellularly stored Ca(2+). Validating this model, control iPS-CM treated with HERG-blocking drugs recapitulated the LQT2 phenotype. In LQT3 iPS-CM, expressing NaV1.5-N406K, APs and [Ca(2+)]i transients were markedly prolonged. AP prolongation was sensitive to tetrodotoxin and to inhibiting Na(+)-Ca(2+) exchange. These results suggest that LQTS mutations act partly on cytosolic Ca(2+) cycling, potentially providing a basis for functionally targeted interventions regardless of the specific mutation site.

Mutations in human ether-a-go-go-related gene 1 (hERG) are linked to long QT syndrome type 2 (LQT2). hERG encodes the pore-forming [alpha]-subunits that coassemble to form rapidly activating delayed rectifier [K.sup.+] current in the heart. LQT2-1inked missense mutations have been extensively studied in noncardiac heterologous expression systems, where biogenic (protein trafficking) and biophysical (gating and permeation) abnormalities have been postulated to underlie the loss-of-function phenotype associated with LQT2 channels. Little is known about the properties of LQT2-1inked hERG channel proteins in native cardiomyocyte systems. In this study, we expressed wild-type (WT) hERG and three LQT2-1inked mutations in neonatal mouse cardiomyocytes and studied their electrophysiological and biochemical properties. Compared with WT hERG channels, the LQT2 missense mutations G601S and N470D hERG exhibited altered protein trafficking and underwent pharmacological correction, and N470D hERG channels gated at more negative voltages. The [DELTA]Y475 hERG deletion mutation trafficked similar to WT hERG channels, gated at more negative voltages, and had rapid deactivation kinetics, and these properties were confirmed in both neonatal mouse cardiomyocyte and human embryonic kidney (HEK)-293 cell expression systems. Differences between the cardiomyocytes and HEK-293 cell expression systems were that hERG current densities were reduced 10-fold and deactivation kinetics were accelerated 1.5- to 2-fold in neonatal mouse cardiomyocytes. An important finding of this work is that pharmacological correction of trafficking-deficient LQT2 mutations, as a potential innovative approach to therapy, is possible in native cardiac tissue. human ether-a-go-go-related gene I; long QT syndrome 2; protein trafficking; E4031 doi: 10.1152/ajpheart.01236.2009.

Reduction of repolarization reserve increases the risk of arrhythmia. We hypothesized that inhibition of [K.sup.+] current ([I.sub.K]) to decrease repolarization reserve would unmask the proarrhythmic role of endogenous, physiological late [Na.sup.+] current (late [I.sub.Na]). Monophasic action potentials (MAP) and 12-1ead electrocardiogram were recorded from female rabbit isolated hearts. To block [I.sub.K] and reduce repolarization reserve, E-4031, 4-aminopyridine, and Ba[Cl.sub.2] were used; to block endogenous late [I.sub.Na], tetrodotoxin (TTX) and ranolazine were used. E-4031 (1-60 nM) concentrationdependently prolonged MAP duration ([MAPD.sub.90]) and increased duration of the T wave from [T.sub.peak] to [T.sub.end] ([T.sub.peak] -[T.sub.end]), transmural dispersion of repolarization (TDR), and beat-to-beat variability (BVR) of [MAPD.sub.90]. E-4031 caused spontaneous and pause-triggered polymorphic ventricular tachycardia [torsade de pointes (TdP)]. In the presence of 60 nM E-4031, TTX (0.6-3 [micro]M) and ranolazine (5-10 [micro]M) shortened [MAPD.sub.90], decreased TDR, BVR, and [T.sub.peak]-[T.sub.end] (n = 9-20, P < 0.01), and abolished episodes of TdP. In hearts treated with Ba[Cl.sub.2] or 4-aminopyridine plus E-4031, TTX (0.6-3 [micro]M) shortened [MAPD.sub.90] and decreased [T.sub.peak]-[T.sub.end]. Ranolazine could not reverse the effect of E-4031 to inhibit human ether-a-go-go-related gene (HERG) [K.sup.+] current; thus, the reversal by ranolazine of effects of E-4031 was likely due to inhibition of late [I.sub.Na] and not to antagonism of the HERG-blocking action of E-4031. We conclude that endogenous, physiological late [I.sub.Na] contributes to arrhythmogenesis in hearts with reduced repolarization reserve. Inhibition of this current partially reverses MAPD prolongation and abolishes arrhythmic activity caused by [I.sub.K] inhibitors. late sodium current; repolarization reserve; ventricular tachycardia; monophasic action potential; ion currents

KCNH2 (hERG1) encodes the [alpha]-subunit proteins for the rapidly activating delayed rectifier [K.sup.+] current ([I.sub.Kr]), a major [K.sup.+] current for cardiac myocyte repolarization. In isolated myocytes [I.sub.Kr] frequently is small in amplitude or absent, yet KCNH2 channels and [I.sub.Kr] are targets for drug block or mutations to cause long QT syndrome. We hypothesized that KCNH2 channels and [I.sub.Kr] are uniquely sensitive to enzymatic damage. To test this hypothesis, we studied heterologously expressed [K.sup.+], [Na.sup.+], and L-type [Ca.sup.2+] channels, and in ventricular myoctyes [I.sub.Kr], slowly activating delayed rectifier [K.sup.+] current ([I.sub.Ks]), and inward rectifier [K.sup.+] current ([I.sub.1]), by using electrophysiological and biochemical methods. 1) Specific exogenous serine proteases (protease XIV, XXIV, or proteinase K) selectively degraded KCNH2 current ([I.sub.KCNH2]) and its mature channel protein without damaging cell integrity and with minimal effects on the other channel currents; 2) immature KCNH2 channel protein remained intact; 3) smaller molecular mass KCNH2 degradation products appeared; 4) protease XXIV selectively abolished [I.sub.Kr]; and 5) reculturing HEK-293 cells after protease exposure resulted in the gradual recovery of [I.sub.KCNH2] and its mature channel protein over several hours. Thus the channel protein for [I.sub.KCNH2] and [I.sub.Kr] is uniquely sensitive to proteolysis. Analysis of the degradation products suggests selective proteolysis within the S5-pore extracellular linker, which is structurally unique among Kv channels. These data provide 1) a new mechanism to account for low [I.sub.Kr] density in some isolated myocytes, 2) evidence that most complexly glycosylated KCNH2 channel protein is in the plasma membrane, and 3) new insight into the rate of biogenesis of KCNH2 channel protein within cells. rapidly activating delayed rectifier potassium current; enzymes; myocyte isolation

Mutations in the human ether-a-go-go-related gene (HERG) cause long QT syndrome. We previously showed that the HERG N470D mutation expressed as homotetrameric channels causes a protein trafficking defect, and this can be corrected by the HERG channel blocking drug E-4031. The N470D mutant also has been reported to cause dominant negative suppression of HERG current when coexpressed with wild-type channel subunits. The aims of this study were 1) to investigate the molecular mechanism responsible for the dominant negative effect of the N470D mutant coexpressed with wild-type subunits and 2) to test whether the trafficking defective heteromeric channels could be pharmacologically rescued by E-4031. Using a combination of immunoprecipitation and Western blot methods, we showed that N470D mutant and wild-type HERG subunits were physically associated in the endoplasmic reticulum as heteromeric channels. The coassembly resulted in the retention of both wild-type and N470D subunits in the endoplasmic reticulum. Culturing cells in E-4031 increased the cell surface expression of these channels, although with an altered electrophysiological phenotype. These results suggest that the dominant negative effect of the N470D wild-type coassembled channels is caused by retention of heteromeric channels in the endoplasmic reticulum and that the trafficking defect of these channels can be corrected by specific pharmacological strategies. ion channels; arrhythmia; protein trafficking; patch clamp

Molecular and functional characterization of common polymorphisms in HERG (KCNH2) potassium channels. Am J Physiol Heart Circ Physiol 286: H2434-H2441. 2004. First published February 19, 2004; 10.1152/ajpheart.00891.2003.--Long QT syndrome (LQTS) is a cardiac repolarization disorder that can lead to arrhythmias and sudden death. Chromosome 7-linked inherited LQTS (LQT2) is caused by mutations in human ether-a-go-go-related gene (HERG; KCNH2), whereas drug-induced LQTS is caused primarily by HERG channel block. Many common polymorphisms are functionally silent and have been traditionally regarded as benign and without physiological consequence. However, the identification of common nonsynonymous single nucleotide polymorphisms (nSNPs; i.e., amino-acid coding variants) with functional phenotypes in the SCN5A N[a.sup.+] channel and MiRP1 [K.sup.+] channel [beta]-subunit have challenged this viewpoint. In this report, we test the hypothesis that common missense HERG polymorphisms alter channel physiology. Comprehensive mutational analysis of HERG was performed on genomic DNA derived from a population-based cohort of sudden infant death syndrome and two reference allele cohorts derived from 100 African American and 100 Caucasian individuals. Amino acid-encoding variants were considered common polymorphisms if they were present in at least two of the three study cohorts with an allelic frequency >0.5%. Four nSNPs were identified: K897T, P967L, R1047L, and Q1068R. Wild-type (WT) and polymorphic channels were heterologously expressed in human embryonic kidney cells, and biochemical and voltage-clamp techniques were used to characterize their functional properties. All channel types were processed similarly, but several electrophysiological differences were identified: 1) K897T current density was lower than the other polymorphic channels; 2) K897T channels activated at more negative potentials than WT and R1047L; 3) K897T and Q1068R channels inactivated and recovered from inactivation faster than WT, P967L, and R1047L channels; and 4) K897T channels showed subtle differences compared with WT channels when stimulated with an action potential waveform. In contrast to K897T and Q1068R channels, P967L and R 1047L channels were electrophysiologically indistinguishable from WT channels. All HERG channels had similar sensitivity to block by cisapride. Therefore, some HERG polymorphic channels are electrophysiologically different from WT channels. ion channels; genetics; long QT syndrome; arrhythmia; sudden death

Role of glycosylation in cell surface expression and stability of HERG potassium channels. Am J Physiol Heart Circ Physiol 283: H77-H84, 2002. First published March 7, 2002; 10.1152/ajpheart.00008. 2002.--The human ether-a-go-go-related gene (HERG) encodes the pore-forming subunit of the rapidly activating delayed rectifier potassium channel in the heart. We previously showed that HERG channel protein is modified by N-linked glycosylation. HERG protein sequence contains two extracellular consensus sites for N-linked glycosylation (N598, N629). In this study, we used the approaches of site-directed mutagenesis and biochemical modification to inhibit N-linked glycosylation and studied the role of glycosylation in the cell surface expression and turnover of HERG channels. Our results show that N598 is the only site for N-linked glycosylation and that glycosylation is not required for the cell surface expression of functional HERG channels. In contrast, N629 is not used for glycosylation, but mutation of this site (N629Q) causes a protein trafficking defect, which results in its intracellular retention. Pulse-chase experiments show that the turnover rate of nonglycosylated HERG channel is faster than that of the glycosylated form, suggesting that N-linked glycosylation plays an important role in HERG channel stability. heart; arrhythmia; ion channels; mutations; patch clamp

A study was conducted to characterize human Na+ channel kinetics near physiological temperature and analyze complex gating changes in a mutant channel that may influence the electrophysiological and clinical phenotype of congenital long Q-T syndrome. Results indicated a number of similarities between the temperature dependence of the gating kinetics of the human heart and nonhuman cardiac channels. Findings also identified a mechanism that may influence the shortening of the prolonged Q-T interval with increases in heart rate.

Human ether-a-go-go-related gene (HERG) stably transfected HEK293 cells were utilized to examine whether cisparide was a potent blocker of HERG channel blocker. Results have shown that cisparide in low concentrations can suppress HERG, and may be the underlying mechanism for the Q-T prolongation and ventricular arrhythmias. Cisparide can block HERG-encoded channels expressed in HEK293 cells.