Estradiol promotes sudden cardiac death in transgenic long QT type 2 rabbits while progesterone is protective.

BACKGROUND: Postpubertal women with inherited long QT syndrome type 2 (LQT2) are at increased risk for polymorphic ventricular tachycardia (pVT) and sudden cardiac death (SCD), particularly during the postpartum period. OBJECTIVE: To investigate whether sex hormones directly modulate the arrhythmogenic risk in LQTS. METHODS: Prepubertal ovariectomized transgenic LQT2 rabbits were treated with estradiol (EST), progesterone (PROG), dihydrotestosterone (DHT), or placebo (OVX). RESULTS: During 8 weeks of treatment, major cardiac events-spontaneous pVT or SCD-occurred in 5 of the 7 EST rabbits and in 2 of the 9 OVX rabbits (P <.05); in contrast, no events occurred in 9 PROG rabbits and 6 DHT rabbits (P <.01 vs PROG; P <.05 vs DHT). Moreover, EST increased the incidence of pVT (P <.05 vs OVX), while PROG reduced premature ventricular contractions, bigeminy, couplets, triplets, and pVT (P <.01 vs OVX; P <.001 vs EST). In vivo electrocardiographic monitoring, in vivo electrophysiological studies, and ex vivo optical mapping studies revealed that EST promoted SCD by steepening the QT/RR slope (P <.05), by prolonging cardiac refractoriness (P <.05), and by altering the spatial pattern of action potential duration dispersion. Isoproterenol-induced Ca(2+) oscillations resulted in early afterdepolarizations in EST-treated hearts (4 of 4), while PROG prevented SCD by eliminating this early afterdepolarization formation in 4 of the 7 hearts (P = .058 vs EST; P <.05 vs OVX). Analyses of ion currents demonstrated that EST increased the density of I(Ca,L) as compared with OVX (P <.05) while PROG decreased it (P <.05). CONCLUSION: This study reveals the proarrhythmic effect of EST and the antiarrhythmic effect of PROG in LQT2 in vivo, outlining a new potential antiarrhythmic therapy for LQTS.

Differential conditions for early after-depolarizations and triggered activity in cardiomyocytes derived from transgenic LQT1 and LQT2 rabbits.

Early after-depolarization (EAD), or abnormal depolarization during the plateau phase of action potentials, is a hallmark of long-QT syndrome (LQTS). More than 13 genes have been identified as responsible for LQTS, and elevated risks for EADs may depend on genotypes, such as exercise in LQT1 vs. sudden arousal in LQT2 patients. We investigated mechanisms underlying different high-risk conditions that trigger EADs using transgenic rabbit models of LQT1 and LQT2, which lack I(Ks) and I(Kr) (slow and fast components of delayed rectifying K(+) current), respectively. Single-cell patch-clamp studies show that prolongation of action potential duration (APD) can be further enhanced by lowering extracellular potassium concentration ([K(+)](o)) from 5.4 to 3.6 mm. However, only LQT2 myocytes developed spontaneous EADs following perfusion with lower [K(+)](o), while there was no EAD formation in littermate control (LMC) or LQT1 myocytes, although APDs were also prolonged in LMC myocytes and LQT1 myocytes. Isoprenaline (ISO) prolonged APDs and triggered EADs in LQT1 myocytes in the presence of lower [K(+)](o). In contrast, continuous ISO perfusion diminished APD prolongation and reduced the incidence of EADs in LQT2 myocytes. These different effects of ISO on LQT1 and LQT2 were verified by optical mapping of the whole heart, suggesting that ISO-induced EADs are genotype specific. Further voltage-clamp studies revealed that ISO increases L-type calcium current (I(Ca)) faster than I(Ks) (time constant 9.2 s for I(Ca) and 43.6 s for I(Ks)), and computer simulation demonstrated a high-risk window of EADs in LQT2 during ISO perfusion owing to mismatch in the time courses of I(Ca) and I(Ks), which may explain why a sympathetic surge rather than high sympathetic tone can be an effective trigger of EADs in LQT2 perfused hearts. In summary, EAD formation is genotype specific, such that EADs can be elicited in LQT2 myocytes simply by lowering [K(+)](o), while LQT1 myocytes require sympathetic stimulation. Slower activation of I(Ks) than of I(Ca) by ISO may explain why different sympathetic modes, i.e. sympathetic surge vs. high sympathetic tone, are associated with polymorphic ventricular tachycardia in LQTS patients.

Pore mutants of HERG and KvLQT1 downregulate the reciprocal currents in stable cell lines.

We previously reported a transgenic rabbit model of long QT syndrome based on overexpression of pore mutants of repolarizing K(+) channels KvLQT1 (LQT1) and HERG (LQT2).The transgenes in these rabbits eliminated the slow and fast components of the delayed rectifier K(+) current (I(Ks) and I(Kr), respectively), as expected. Interestingly, the expressed pore mutants of HERG and KvLQT1 downregulated the remaining reciprocal repolarizing currents, I(Ks) and I(Kr), without affecting the steady-state levels of the native polypeptides. Here, we sought to further explore the functional interactions between HERG and KvLQT1 in heterologous expression systems. Stable Chinese hamster ovary (CHO) cell lines expressing KvLQT1-minK or HERG were transiently transfected with expression vectors coding for mutant or wild-type HERG or KvLQT1. Transiently expressed pore mutant or wild-type KvLQT1 downregulated I(Kr) in HERG stable CHO cell lines by 70% and 44%, respectively. Immunostaining revealed a severalfold lower surface expression of HERG, which could account for the reduction in I(Kr) upon KvLQT1 expression. Deletion of the KvLQT1 NH(2)-terminus did not abolish the downregulation, suggesting that the interactions between the two channels are mediated through their COOH-termini. Similarly, transiently expressed HERG reduced I(Ks) in KvLQT1-minK stable cells. Coimmunoprecipitations indicated a direct interaction between HERG and KvLQT1, and surface plasmon resonance analysis demonstrated a specific, physical association between the COOH-termini of KvLQT1 and HERG. Here, we present an in vitro model system consistent with the in vivo reciprocal downregulation of repolarizing currents seen in transgenic rabbit models, illustrating the importance of the transfection method when studying heterologous ion channel expression and trafficking. Moreover, our data suggest that interactions between KvLQT1 and HERG are mediated through COOH-termini.

Mechanism of C-type natriuretic peptide-induced endothelial cell hyperpolarization.

C-type natriuretic peptide (CNP) has a demonstrated hyperpolarizing effect on vascular smooth muscle cells. However, its autocrine function, including its electrophysiological effect on endothelial cells, is not known. Here, we report the effect of CNP on the membrane potential (E(m)) of pulmonary microvascular endothelial cells and describe its target receptors, second messengers, and ion channels. We measured changes in E(m) using fluorescence imaging and perforated patch-clamping techniques. In imaging experiments, samples were preincubated in the potentiometric dye DiBAC(4)(3), and subsequently exposed to CNP in the presence of selective inhibitors of ion channels or second messengers. CNP exposure induced a dose-dependent decrease in fluorescence, indicating that CNP induces endothelial cell hyperpolarization. CNP-induced hyperpolarization was inhibited by the K(+) channel blockers, tetraethylammonium or iberiotoxin, the nonspecific cation channel blocker, La(3+), or by depletion or repletion of extracellular Ca(2+) or K(+), respectively. CNP-induced hyperpolarization was also blocked by pharmacological inhibition of PKG or by small interfering RNA (siRNA)-mediated knockdown of natriuretic peptide receptor-B (NPR-B). CNP-induced hyperpolarization was mimicked by the PKG agonist, 8-bromo-cGMP, and attenuated by both the endothelial nitric oxide synthase (eNOS) inhibitor, N(omega)-nitro-l-arginine methyl ester (l-NAME), and the soluble guanylyl cyclase (sGC) inhibitor, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one. Presence of iberiotoxin-sensitive, CNP-induced outward current was confirmed by perforated patch-clamping experiments. We conclude that CNP hyperpolarizes pulmonary microvascular endothelial cells by activating large-conductance calcium-activated potassium channels mediated by the activation of NPR-B, PKG, eNOS, and sGC.

Mechanisms of cardiac arrhythmias and sudden death in transgenic rabbits with long QT syndrome.

Long QT syndrome (LQTS) is a heritable disease associated with ECG QT interval prolongation, ventricular tachycardia, and sudden cardiac death in young patients. Among genotyped individuals, mutations in genes encoding repolarizing K+ channels (LQT1:KCNQ1; LQT2:KCNH2) are present in approximately 90% of affected individuals. Expression of pore mutants of the human genes KCNQ1 (KvLQT1-Y315S) and KCNH2 (HERG-G628S) in the rabbit heart produced transgenic rabbits with a long QT phenotype. Prolongations of QT intervals and action potential durations were due to the elimination of IKs and IKr currents in cardiomyocytes. LQT2 rabbits showed a high incidence of spontaneous sudden cardiac death (>50% at 1 year) due to polymorphic ventricular tachycardia. Optical mapping revealed increased spatial dispersion of repolarization underlying the arrhythmias. Both transgenes caused downregulation of the remaining complementary IKr and IKs without affecting the steady state levels of the native polypeptides. Thus, the elimination of 1 repolarizing current was associated with downregulation of the reciprocal repolarizing current rather than with the compensatory upregulation observed previously in LQTS mouse models. This suggests that mutant KvLQT1 and HERG interacted with the reciprocal wild-type alpha subunits of rabbit ERG and KvLQT1, respectively. These results have implications for understanding the nature and heterogeneity of cardiac arrhythmias and sudden cardiac death.

Posttranslational modification of voltage-dependent potassium channel Kv1.5: COOH-terminal palmitoylation modulates its biological properties.

The physiological function of ion channels is affected by protein-protein and protein-membrane interactions that modulate their activity and/or localization. Palmitoylation modulates protein function by facilitating targeted membrane association, interaction with other proteins, and determining subcellular localization. In this study, we demonstrate that the voltage-dependent potassium (Kv) channel Kv1.5 is palmitoylated and that the mutation of COOH-terminal cysteines is sufficient to abolish the palmitoylation of the Kv1.5 polypeptide in Chinese hamster ovary (CHO) cells. The labeling represented the thioester linkage of the labeled palmitic acid to cysteine rather than amide and oxygen ester linkages as judged by the release of the palmitic acid upon the treatment of the gel with hydroxylamine at a neutral pH. Site-directed mutagenesis and radiolabeling studies revealed that C593 was the sole site of palmitoylation. The elucidation of the biological function of palmitoylation revealed that the expression of the FLAG-Kv1.5 palmitoylation-deficient mutant (FL-Kv1.5(Palm-)) in stable CHO cells increased membrane expression as determined by the biotinylation of surface proteins and quantitative immunofluorescence analyses of these cells, in turn enhancing the outward potassium current. This enhanced surface expression and the currents were consequential to the slower rate of internalization, causing an increased localization of FL-Kv1.5(Palm-) in the plasma membrane compared with the wild-type FL-Kv1.5 channels. We conclude that the Kv1.5 channel is palmitoylated and that its palmitoylation modulates its biological functions and, therefore, might provide a physiological link between the metabolic state and the expression of Kv1.5 on the plasma membrane.

Modulation of human cardiovascular outward rectifying chloride channel by intra- and extracellular ATP.

The macroscopic volume-regulated anion current (VRAC) is regulated by both intracellular and extracellular ATP, which has important implications in signaling and regulation of cellular excitability. The outwardly rectifying Cl(-) channel (ORCC) is a major contributor to the VRAC. This study investigated the effects of intracellular and extracellular ATP on the ORCCs expressed in the human cardiovascular system. With inside-out single-channel patch-clamp techniques, ORCCs were recorded from myocytes isolated from human atrium and septal ventricle and from primary cells originating from human coronary artery endothelium and human coronary artery smooth muscle. ORCCs from all of these tissues had similar biophysical properties, i.e., they were outwardly rectifying in symmetrical Cl(-) solutions, exhibited a slope conductance of approximately 90-100 pS at positive potentials and approximately 22 pS at negative potentials, and had a high open probability that was independent of voltage or time. The presence of ATP at the cytosolic face of the membrane increased the number of patches that contained functional ORCC but had no effect on gating. In contrast, "extracellular" ATP (in pipette solution) had no effect on the proportion of patches in which ORCC was detected but strongly reduced the open probability by increasing the closed dwell time. The potency order for nucleotides to affect gating was ATPgammaS > ATP = UTP > ADP > AMP, which suggests that a negatively charged phosphate group is involved in ORCC block. Our findings are consistent with a role of ORCC in the human cardiovasculature (atrium, ventricle, and coronary arteries). Regulation of ORCC by extracellular ATP suggests that this channel may have an important role in maintaining electrical activity and membrane potential under conditions in which extracellular ATP levels are elevated, such as with ATP release from nerve endings or during pathophysiological conditions.