Hyperglycemia and hypoglycemia both can cause prolongation of the Q-T interval and ventricular arrhythmias. Here we studied modulation of human ether-à -gogo-related gene (HERG) K ؉ channel, the major molecular component of delayed rectifier K ؉ current responsible for cardiac repolarization, by glucose in HEK293 cells using whole-cell patch clamp techniques. We found that both hyperglycemia (extracellular glucose concentration Glucose, the primary end product of the digestion of glycogen, is essential for maintaining life activities in organisms. As a major source of metabolic fuel, degradation of glucose via glycolysis and subsequent oxidative phosphorylation generates high energy phosphates to power the biological processes in the cell. Yet, through an exquisitely complex network of control mechanisms, the rate of glucose metabolism is only as great as needed by the organisms. Moreover, glucose also has other regulatory effects on many cellular functions. Either inadequate or excessive glucose can be harmful to the living system. Therefore, the blood glucose level is dynamically controlled. However, under pathological conditions like diabetes, glucose cannot be efficiently utilized, and the blood glucose level rises. When the blood level of glucose is maintained higher than 7 mM, it is considered as hyperglycemia. Diabetes therapy, on the other hand, can lead to an overly low level of blood glucose, which is referred to as hypoglycemia when the level falls below 3 mM.Either hypoglycemia or hyperglycemia can have deleterious effects on the cells. One common feature of electrophysiological alterations caused by both hypoglycemia and hyperglycemia in the heart is prolongation of Q-T interval and the associated ventricular arrhythmias that are presumably responsible for sudden cardiac death in diabetic patients (1-10). However, the ionic mechanisms by which hyperglycemia and hypoglycemia prolong Q-T interval remained unclear, which is at least a part of the reasons why diabetic patients die of mainly cardiac complications.The human either-à -go-go-related gene (HERG) 1 encodes the rapid component of delayed rectifier K ϩ current in the heart, which is the major repolarizing current in the plateau voltage range of cardiac action potentials. HERG K ϩ channels are susceptible to genetic defects and environmental cues, with the consequence being depression of HERG function in most situations (9). Indeed, most of the cases of long Q-T syndrome are ascribed to dysfunction of HERG channels, particularly that induced by therapeutic drugs (13). It is conceivable that HERG alteration might also be involved in the Q-T prolongation induced by hyperglycemia and hypoglycemia. This thought prompted us to carry out a series of experiments to study the effects of glucose on HERG K ϩ channels and the potential mechanisms.

Congestive heart failure (CHF) is associated with susceptibility to lethal arrhythmias and typically increases levels of tumor necrosis factor-␣ (TNF-␣) and its receptor, TNFR1. CHF down-regulates rapid delayed-rectifier K ؉ current (I Kr ) and delays cardiac repolarization. We studied the effects of TNF-␣ on cloned HERG K ؉ channel (human ether-a-go-go-related gene) in HEK293 cells and native I Kr in canine cardiomyocytes with whole-cell patch clamp techniques. TNF-␣ consistently and reversibly decreased HERG current (I HERG ). Effects of TNF-␣ were concentration-dependent, increased with longer incubation period, and occurred at clinically relevant concentrations. TNF-␣ had similar inhibitory effects on I Kr and markedly prolonged action potential duration (APD) in canine cardiomyocytes. Immunoblotting analysis demonstrated that HERG protein level was slightly higher in canine hearts with tachypacing-induced CHF than in healthy hearts, and TNF-␣ slightly increased HERG protein level in CHF but not in healthy hearts. In cells pretreated with the inhibitory anti-TNFR1 antibody, TNF-␣ lost its ability to suppress I HERG , indicating a requirement of TNFR1 activation for HERG suppression. Vitamin E or MnTBAP (Mn(III) tetrakis(4-benzoic acid) porphyrin chloride), a superoxide dismutase mimic) prevented, whereas the superoxide anion generating system xanthine/xanthine oxidase mimicked, TNF-␣-induced I HERG depression. TNF-␣ caused robust increases in intracellular reactive oxygen species, and vitamin E and MnTBAP abolished the increases, in both HEK293 cells and canine ventricular myocytes. We conclude that the TNF-␣/TNFR1 system impairs HERG/I Kr function mainly by stimulating reactive oxygen species, particularly superoxide anion, but not by altering HERG expression; the effect may contribute to APD prolongation by TNF-␣ and may be a novel mechanism for electrophysiological abnormalities and sudden death in CHF.

Abnormal QT prolongation (QT-P) in diabetic patients has become a nonnegligible clinical problem and has attracted increasing attention from basic scientists, because it increases the risk of lethal ventricular arrhythmias. Correction of QT-P may be an important measure in minimizing sudden cardiac death in diabetic patients. Here we report the efficacy of insulin in preventing QT-P and the associated arrhythmias and the mechanisms underlying the effects in a rabbit model of type 1 insulin-dependent diabetes mellitus (IDDM). The heart rate-corrected QT (QTc) interval and action potential duration were considerably prolonged, with frequent ventricular tachycardias. The rapid delayed rectifier K+ current (IKr) was markedly reduced in IDDM hearts, and hyperglycemia depressed the function of the human ether-a-go-go-related gene (HERG), which conducts IKr. The impairment was primarily ascribed to the enhanced oxidative damage to the myocardium, as indicated by the increased intracellular level of reactive oxygen species and simultaneously decreased endogenous antioxidant reserve and by the increased lipid peroxidation and protein oxidation. Moreover, IDDM or hyperglycemia resulted in downregulation of HERG protein level. Insulin restored the depressed IKr/HERG and prevented QTc/action potential duration prolongation and the associated arrhythmias, and the beneficial actions of insulin are partially due to its antioxidant ability. Our study represents the first documentation of oxidative stress as the major metabolic mechanism for HERG K+ dysfunction, which causes diabetic QT-P, and suggests IKr/HERG as a potential therapeutic target for treatment of the disorder.

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 (I_to) that was reduced by ñ60%, rapid delayed rectifier K⁺ current (I_Kr) reduced by ñ70% and slow delayed rectifier K⁺ current (I_Ks) reduced by ñ40%. The time-dependent kinetics of these currents remained unaltered. The peak amplitude of L-type Ca% current (I_CaL) was reduced by ñ22% and the inactivation kinetics was slowed; the integration of these two effects yielded ñ15% reduction of I_CaL. The inward rectifier K⁺ current (I_K1) and fast sodium current (I_Na) were unaffected. Simulation with LabHEART, a computer model of rabbit ventricular action potentials, revealed that inhibition of I_to or I_Ks alone fails to alter APD whereas inhibition of I_Kr alone results in 30% APD prolongation and inhibition of I_CaL 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 I_Kr channelopathy as the major ionic mechanism for diabetic QT prolongation.

The potential role of protein kinase B (PKB), a serine/threonine protein kinase, in regulating HERG (human ethera-go-go related gene) K + channel function was investigated. Wortmannin (a phosphoinositide 3-kinase (PI3K) inhibitor) caused V V30% 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 (V V7%) I HERG while a dominant negative PI3K signi¢cantly reduced I HERG (V V25%) relative to results in vehicle-transfected cells. I HERG was V V35% greater in cells transfected with the constitutively activated PKB (caPKB), whereas it was V V47% smaller in cells transfected with dominant negative PKB (dnPKB). Basal activation of PKB was detected by immunocytochemistry. PKB activity was signi¢cantly 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 ¢rst evidence that PKB phosphorylation regulates K + channels. ß 2002 Published by Elsevier Science B.V. on behalf of the Federation of European Biochemical Societies.

Many pathophysiological processes are associated with oxidative stress and progressive cell death. Oxidative stress is an apoptotic inducer that is known to cause rapid cell death. Here we show that a brief oxidative insult (5-min exposure to 400 microM H(2)O(2)), although it did not kill H9c2 rat ventricular cells during the exposure, triggered an intracellular death cascade leading to delayed time-dependent cell death starting from 1 h after the insult had been withdrawn, and this post-H(2)O(2) cell death cumulated gradually, reaching a maximum level 8 h after H(2)O(2) withdrawal. By comparison, sustained exposure to H(2)O(2) caused complete cell death within a narrow time frame (2 h). The time-dependent post-H(2)O(2) cell death was typical of apoptosis, both morphologically (cell shrinkage and nuclear condensation) and biochemically (DNA fragmentation, extracellular exposure of phosphatidylserines, and caspase-3 activation). A dichlorofluorescein fluorescent signal showed a time-dependent endogenous increase of reactive oxygen species (ROS) production, which was almost abolished by inhibition of the mitochondrial electron transport chain. Application of antioxidants (vitamin E or DTT) before H(2)O(2) addition or after H(2)O(2) withdrawal prevented the H(2)O(2)-triggered progressive ROS production and apoptosis. Sequential appearance of events associated with activation of the mitochondrial death pathway was found, including progressive dissipation of mitochondrial membrane potential, cytochrome c release, and late activation of caspase-3. In conclusion, transient oxidative stress triggers an intrinsic program leading to self-sustained apoptosis in H9c2 cells via cumulative production of mitochondrial ROS and subsequent activation of the mitochondrial death pathway. This pattern of apoptosis may contribute to the progressive and long-lasting cell loss in some degenerative diseases.

Multiple subtypes (␣ 1A , ␣ 1B , and ␣ 1D ) of ␣ 1 -adrenoreceptors (␣ 1 ARs) co-exist in the heart and mediate a variety of cellular functions. We studied ␣ 1 AR modulation of inward rectifier (I K1 ) and transient outward (I to ) K ؉ currents in canine ventricular myocytes. Phenylephrine at 10 M depressed only I to without affecting I K1 and at 100 M inhibited both I to and I K1 . The effect of phenylephrine on I to was abolished by (؉)niguldipine (10 nM Over the past decade, evidence from pharmacological studies and molecular cloning has been accumulating indicating that ␣ 1 -adrenoreceptors (␣ 1 ARs) 1 are actually a heterogeneous group of distinct but related protein subsets. Many cellular responses to ␣ 1 ARs are mediated by multiple subtypes (␣ 1A , ␣ 1B , and ␣ 1D ) (1-4). In the heart, whereas the ␣ 1A and ␣ 1B subtypes have been well characterized, the presence of ␣ 1D AdR was indicated only recently (5-7). Moreover, although the pathophysiological roles of ␣ 1A and ␣ 1B receptors have been well appreciated, those of ␣ 1D subtype in the heart remain to be determined. Enhanced ␣ 1 AR activity has been implicated in various types of arrhythmias, particularly those in the pathogenesis of myocardial ischemia, ischemia-reperfusion and preconditioning, cardiac hypertrophy, etc. (1, 3). Drug intervention with ␣ 1 ARs has thus become an attractive issue for developing new compounds for potential therapy. A significant mechanism underlying ␣ 1 AR-induced alteration of cardiac electrical activity is attributable to the ability of ␣ 1 ARs to modulate ion channels. To date, no less than seven cardiac ionic currents are on the list of ␣ 1 AR modulation, including inward rectifier K ϩ current (I K1 ), transient outward K ϩ current (I to ), delayed rectifier K ϩ current (I K ), ultrarapid delayed rectifier K ϩ current (I Kur ), acetylcholine-induced K ϩ current (I KACh ), calcium current (I Ca ), and chloride current (1, 3, 8 -11). However, it is not known whether the effects are the results from participation of all three different subtypes of ␣ 1 ARs or of a particular individual subtype, although evidence is accumulating that different subtypes may have different roles in regulating cardiac contraction and electrical activities (12-16). Moreover, recent studies also demonstrated subtype differences in the signal transduction (17-19). In light of these studies, we speculated that different subtypes of ␣ 1 ARs may have distinct effects on ion channels. Understanding subtype specificity of ␣ 1 ARs in ion channel regulation is of theoretical and practical importance. K ϩ currents play critical roles in determining cardiac electrical activities. Besides stabilizing resting potential, I K1 in cardiac cells also plays an important role in modulating cellular excitability and regulating membrane repolarization, therefore an important determinant of action potential initiation. Another important cardiac K ϩ current is transient outward K ϩ current (I to ), which is known to be critical for initiating cardiac repolarization...

Background-Lysophosphatidylcholine (LPC), a naturally occurring phospholipid metabolite, accumulates in the ischemic heart and causes extracellular K ϩ accumulation and action potential shortening. LPC has been incriminated as a biochemical trigger of lethal cardiac arrhythmias, but the underlying mechanisms remain poorly understood. Methods and Results-We studied the effect of 1-palmitoyl-LPC (Pal-LPC) on currents resulting from human ether-a-go-go-related gene (HERG) expression in human embryonic kidney (HEK) cells using whole-cell patch-clamp techniques. Bath application of Pal-LPC consistently and reversibly increased HERG current (I HERG ). The effects of Pal-LPC were apparent as early as 3 minutes after application of the drug, reached maximum within 10 minutes, and were reversible on washout. Pal-LPC increased I HERG at voltages between Ϫ20 and ϩ30 mV, with greater effects at stronger depolarization. However, Pal-LPC did not affect the voltage-dependence of I HERG activation. In contrast, Pal-LPC significantly shifted the inactivation curve toward more positive potentials, causing a mean 20.0Ϯ2.2 mV shift in half-inactivation voltage relative to control. Conclusions-Our results indicate that apart from being a well-recognized target for drug inhibition, I HERG can also be enhanced by natural substances. An increase in I HERG by Pal-LPC may contribute to K ϩ loss, abnormal electrophysiology, and arrhythmia occurrence in the ischemic heart. (Circulation. 2001;104:2645-2648.)