Jingxiong Wang

Normal function of HERG K+ channels expressed in HEK293 cells requires basal protein kinase B activity

The potential role of protein kinase B (PKB), a serine/threonine protein kinase, in regulating HERG (human ether‐a‐go‐go related gene) K+ channel function was investigated. Wortmannin (a phosphoinositide 3‐kinase (PI3K) inhibitor) caused ∼30% reduction of HERG current (I HERG) stably expressed in HEK293 cells. Transient transfection with the constitutively active PI3K in HERG‐expressing HEK293 cells slightly increased (∼7%) I HERG while a dominant negative PI3K significantly reduced I HERG (∼25%) relative to results in vehicle‐transfected cells. I HERG was ∼35% greater in cells transfected with the constitutively activated PKB (caPKB), whereas it was ∼47% smaller in cells transfected with dominant negative PKB (dnPKB). Basal activation of PKB was detected by immunocytochemistry. PKB activity was significantly enhanced in caPKB‐transfected cells and nearly abolished in dnPKB‐transfected cells. We conclude that normal HERG function in HEK293 cells requires basal activity of PKB. Our data represent the first evidence that PKB phosphorylation regulates K+ channels.

Ionic mechanisms underlying abnormal QT prolongation and the associated arrhythmias in diabetic rabbits: a role of rapid delayed rectifier K+ current.

Abnormal QT prolongation with the associated arrhythmias is considered the major cardiac electrical disorder and a significant predictor of mortality in diabetic patients. The precise ionic mechanisms for diabetic QT prolongation remained unclear. We performed whole-cell patch-clamp studies in a rabbit model of alloxan-induced insulin-dependent diabetes mellitus. We demonstrated that heart rate-corrected QT interval and action potential duration (APD) were prolonged by ñ20% with frequent occurrence of ventricular tachyarrhythmias. Several K+ currents were found decreased in diabetic rabbits including transient outward K+current (Ito) that was reduced by ñ60%, rapid delayed rectifier K+ current (IKr) reduced by ñ70% and slow delayed rectifier K+ current (IKs) reduced by ñ40%. The time-dependent kinetics of these currents remained unaltered. The peak amplitude of L-type Ca% current (ICaL) was reduced by ñ22% and the inactivation kinetics was slowed; the integration of these two effects yielded ñ15% reduction of ICaL. The inward rectifier K+ current (IK1) and fast sodium current (INa) were unaffected. Simulation with LabHEART, a computer model of rabbit ventricular action potentials, revealed that inhibition of Ito or IKs alone fails to alter APD whereas inhibition of IKr alone results in 30% APD prolongation and inhibition of ICaL alone causes 10% APD shortening. Integration of changes of all these currents leads to ñ20% APD lengthening. Protein levels of the pore-forming subunits for these ion channels were decreased to varying extents, as revealed by immunoblotting analysis. Our study represents the first documentation of IKr channelopathy as the major ionic mechanism for diabetic QT prolongation.

HERG K+ Channel Conductance Promotes H2O2-Induced Apoptosis in HEK293 Cells: Cellular Mechanisms

The human ether-a-go-go-related gene (HERG) encodes a delayed rectifier K+ channel, which is expressed in a variety of tissues and cells. Besides its well-recognized function in cellular electrophysiology, HERG channels have also been implicated in neuronal differentiation and cell cycle regulation. We have recently found that HERG regulates apoptosis. To elucidate the signaling pathways, we performed studies in HEK293 cells stably expressing HERG channels. ELISA was used to quantify DNA fragmentation, a biochemical hallmark of apoptosis. In HERG-transfected HEK cells, the degree of DNA fragmentation was found consistently higher (ñ4-times) than in non-transfected cells. Correspondingly, remarkable activation of caspase 3, caspase 9 and cleavage of PARP were seen in HERG-expressing cells, which were otherwise minimal in non-transfected cells. Exposure of cells to H2O2 (10 hrs) at concentrations up to 1 mM, which is known to induce apoptosis in a variety of cells, caused minimal DNA fragmentation in non-transfected cells. HERG expression facilitates DNA fragmentation induced by H2O2 at a concentration-dependent fashion, starting at 200 µM and reaching maximum at 1 mM. Selective HERG channel inhibitors, dofetilide or E-4031 (5 µM) prevented DNA fragmentation. Inhibition of p38 by SB-203580 alleviated DNA-F and PD-98059, which inhibited activation of ERKs, nearly abolished DNA-F. Immunoblotting analysis demonstrated that p38, SAPKs and ERKs MAP kinases were all substantially activated (>10-fold higher) in HERG-expressing cells vs. non-transfected cells. Akt activity was ñ4-fold lower in HERG cells vs. non-transfected cells in the absence of H2O2 and was slightly increased (ñ2-fold) after H2O2 exposure. We conclude that HERG channels facilitate cellular DNA fragmentation in HEK cells via concomitant activation of MAP kinases and inactivation of Akt.

Potential mechanisms for the enhancement of HERG K+ channel function by phospholipid metabolites

Phospholipid metabolites lysophospholipids cause extracellular K+ accumulation and action potential shortening with increased risk of arrhythmias during myocardial ischemia. Here we studied effects of several lysophospholipids with different lengths of hydrocarbon chains and charged headgroups on HERG K+ currents (IHERG) expressed in HEK293 cells and the potential mechanisms using whole‐cell patch‐clamp techniques. Only the lipids with 16 hydrocarbons such as 1‐palmitoyl‐lysophosphatidylcholine (LPC‐16) and 1‐palmitoyl‐lysophosphatidylglycerol (LPG‐16) were found to produce significant enhancement of IHERG and negative shifts of HERG activation, although the voltage dependence of the effects was different between LPC‐16 and LPG‐16 which have differently charged headgroups. The lipid with 18 hydrocarbons modestly increased IHERG. The lipids with 6 or 24 hydrocarbons had no effect or slightly decreased IHERG. Inhibition or activation of protein kinase C did not alter the effects of LPC‐16 and LPG‐16. Participation of phosphatidylinositol‐4,5‐bisphosphate in IHERG enhancement by LPC‐16/LPG‐16 was also excluded. Vitamin E augmented the effects of LPC‐16/LPG‐16 whereas xanthine/xanthine oxidase reduced IHERG: indicating that LPC‐16/LPG‐16 produced dual effects on IHERG: direct enhancement of IHERG and indirect suppression via production of superoxide anion. We conclude that enhancement of HERG function by lysophospholipids is specific to the lipids with 16‐hydrocarbon chain structure and the pattern of voltage dependence is determined by the polar headgroups. The increase in IHERG is best described by direct interactions between lipid molecules and HERG proteins, which is consistent with lack of effects via membrane destabilization or modulation by intracellular signaling pathways.

Sphingolipid Metabolite Ceramide Causes Metabolic Perturbation Contributing to HERG K+ Channel Dysfunction

Ceramide, a sphingolipid metabolite, has emerged as a key second messenger molecule that mediates multiple cellular functions. Its de nova synthesis and accumulation in ischemic myocardium, congestive heart failure and diabetic cardiomyopathy is associated with the abnormalities such as abnormal QT prolongation and increased risk of arrhythmias. To investigate how ceramide is involved in modulating cardiac repolarization, we performed whole-cell patch-clamp studies on HERG current (IHERG), a critical determinant of cardiac repolarization, expressed in HEK293 cells. Acute application (superfusion for 25min) of membrane permeable ceramide (C2, 5 µM) did not alter IHERG. Prolonged incubation with C2 for 10hrs caused pronounced IHERG inhibition in a concentration-dependent and voltage-independent fashion and positive shift of voltage-dependent HERG activation. The IC50 for IHERG suppression was 19.5 µM. C2 did not affect the inactivation property and time-dependent kinetics of IHERG. Similar effects were observed with production of endogenous ceramide catalyzed by sphingomyelinase. Tyrosine kinase inhibitors failed to reverse C2-induced suppression of HERG function, and PKA and PKC inhibitors only slightly reversed the IHERG depression. Western blotting and immunocytochemical analyses indicate that C2 does not alter HERG protein expression on the cytoplasmic membrane. The inhibitory effect of C2 on IHERG was reversed by antioxidants vitamin E or MnTBAP. C2 caused considerable production of intracellular reactive oxygen species (ROS), which was prevented by vitamin E or MnTBAP. We conclude that ceramide depresses IHERG mainly via ROS overproduction and ceramide-induced IHERG impairment may contribute to QT prolongation in prolonged myocardial ischemia, heart failure and diabetic cardiomyopathy.

Phospholipid Lysophosphatidylcholine as a Metabolic Trigger and HERG as an Ionic Pathway for Extracellular K + Accumulation and "Short QT Syndrome" in Acute Myocardial Ischemia

The most profound abnormalities during acute myocardial ischemia are extracellular K⁺ accumulation ([K⁺]_o–?) and shortening of action potential duration or QT interval (APD–? or QT–?), which are pivotal in the genesis of ischemic arrhythmias and sudden cardiac death. The ionic mechanisms however remained obscured. We performed studies in a rabbit model of acute global myocardial ischemia in order to explore ionic and metabolic mechanisms for ischemic [K⁺]_o–? and QT–?. Exogenous 1-palmitoyl-lysophosphatidylcholine (LPC-16) mimicked the low-perfusion ischemia to produce significant [K⁺]_o–? and QT–?. The [K⁺]_o–? and QT–? induced by either LPC-16 or ischemia were prevented by dofetilide, a blocker of rapid delayed rectifier K⁺ current (I_Kr), but not by blockers for other K⁺ channels. Consistently, dofetilide efficiently abolished the ventricular tachy-arrhythmias induced by ischemia or LPC-16. LPC-16 remarkably shortened APD and enhanced the function of I_Kr and HERG (the pore-forming subunit of I_Kr). The effects of LPC-16 manifested with shorter APD (faster repolarization rate) and at more negative potential (membrane repolarization). Dofetilide abolished the I_Kr/HERG enhancing and APD shortening effects of LPC-16. Our results suggest that LPC-16 accumulation/HERG enhancement may be a link between metabolic trigger and ionic pathway for ischemic [K⁺]_o– ? and QTc– ?. This represents the first documentation of I_Kr/HERG as the ionic mechanism in ischemic [K⁺]_o–? and QTc–?. Inhibition of LPC-16 production and accumulation and/or of I_Kr/HERG may be a promising therapeutic strategy to attenuate the incidence of lethal arrhythmias associated with ischemic heart disease.

Potential Therapeutic Value of Antioxidants for Abnormal Prolongation of QT Interval and the Associated Arrhythmias in a Rabbit Model of Diabetes

Abnormal QT prolongation is the major cardiac electrical disorder and a predictor of mortality in diabetic patients. Our previous studies suggest that dysfunction of delayed rectifier K+ current (IKr) is the main cause for the problem. Here we report the potential therapeutic role and mechanisms of vitamin E in the rabbit model of diabetes. The QT interval and action potential duration were considerably prolonged with frequent occurrence of ventricular tachyarrhythmias in diabetic rabbits. Administration of vitamin E corrected the abnormal QT prolongation and abolished the arrhythmic incidence. IKr was found markedly reduced resulting in slowing of cardiac repolarization thereby QT prolongation in diabetic hearts. The diabetic depression of IKr is primarily ascribed to oxidative damages to the cardiac membrane and proteins, as indicated by the overproduction of reactive oxygen species leading to severe lipid peroxidation and protein oxidation. Moreover, IKr depression is most likely due to the dysfunction of HERG K+ channel, the major subunit underlying native IKr, in response to oxidative stress, for peroxide anion-generating system produced similar depression of HERG channels. Vitamin E restored the depressed IKr and HERG by its antioxidant actions which likely underlie its beneficial effects on diabetic QT prolongation and the associated arrhythmias. The data indicate that an antioxidant is sufficient for reversing the IKr/IHERG dysfunction and the consequent electrical disorders in diabetic hearts. Our study also conceptually simplifies the complex nature of diabetic electrical disorders to primarily oxidative stress, and should stimulate interest in antioxidants as a therapeutic strategy for diabetic QT prolongation.