Impairment of HERG K+ Channel Function by Tumor Necrosis Factor-α

Congestive heart failure (CHF) is associated with susceptibility to lethal arrhythmias and typically increases levels of tumor necrosis factor-α (TNF-α) and its receptor, TNFR1. CHF down-regulates rapid delayed-rectifier K+ current (IKr) and delays cardiac repolarization. We studied the effects of TNF-α on cloned HERG K+ channel (human ether-a-go-go-related gene) in HEK293 cells and native IKr in canine cardiomyocytes with whole-cell patch clamp techniques. TNF-α consistently and reversibly decreased HERG current (IHERG). Effects of TNF-α were concentration-dependent, increased with longer incubation period, and occurred at clinically relevant concentrations. TNF-α had similar inhibitory effects on IKr 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-α slightly increased HERG protein level in CHF but not in healthy hearts. In cells pretreated with the inhibitory anti-TNFR1 antibody, TNF-α lost its ability to suppress IHERG, 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-α-induced IHERG depression. TNF-α 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-α/TNFR1 system impairs HERG/IKr 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-α and may be a novel mechanism for electrophysiological abnormalities and sudden death in CHF. Congestive heart failure (CHF) is associated with susceptibility to lethal arrhythmias and typically increases levels of tumor necrosis factor-α (TNF-α) and its receptor, TNFR1. CHF down-regulates rapid delayed-rectifier K+ current (IKr) and delays cardiac repolarization. We studied the effects of TNF-α on cloned HERG K+ channel (human ether-a-go-go-related gene) in HEK293 cells and native IKr in canine cardiomyocytes with whole-cell patch clamp techniques. TNF-α consistently and reversibly decreased HERG current (IHERG). Effects of TNF-α were concentration-dependent, increased with longer incubation period, and occurred at clinically relevant concentrations. TNF-α had similar inhibitory effects on IKr 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-α slightly increased HERG protein level in CHF but not in healthy hearts. In cells pretreated with the inhibitory anti-TNFR1 antibody, TNF-α lost its ability to suppress IHERG, 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-α-induced IHERG depression. TNF-α 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-α/TNFR1 system impairs HERG/IKr 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-α and may be a novel mechanism for electrophysiological abnormalities and sudden death in CHF. TNF-α 1The abbreviations used are: TNF-α, tumor necrosis factor-α; CHF, congestive heart failure; APD, action potential duration; EAD, early afterdepolarization; ROS, reactive oxygen species; CM-H2DFDA, 5-(and-6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate; VitE, vitamin E; MnTBAP, Mn(III) tetrakis(4-benzoic acid) porphyrin chloride; X/XO, xanthine/xanthine oxidase. is a potent inducible cytokine with pleiotropic biological effects (1McTiernan C.F. Feldman A.M. Curr. Cardiol. Rep. 2000; 2: 189-197Google Scholar). Up-regulation of TNF-α is a consistent finding in clinical (2Aukrust P. Ueland T. Lien E. Bendtzen K. Muller F. Andreassen A.K. Nordoy I. Aass H. Espevik T. Simonsen S. Froland S.S. Gullestad L. Am. J. Cardiol. 1999; 83: 376-382Google Scholar) and experimental CHF (3Irwin M.W. Mak S. Mann D.L. Qu R. Penninger J.M. Yan A. Dawood F. Wen W.H. Shou Z. Liu P. Circulation. 1999; 99: 1492-1498Google Scholar). Circulating concentrations of TNF-α and soluble TNF receptors are independent predictors of mortality in CHF (4Deswal A. Petersen N.J. Feldman A.M. Young J.B. White B.G. Mann D.L. Circulation. 2001; 103: 2055-2059Google Scholar). Patients with CHF are at increased risk of sudden death due to cardiac arrhythmias. CHF increases action potential duration (APD) (5Beuckelmann D.J. Nabauer M. Erdmann E. Circ. Res. 2002; 73: 379-385Google Scholar), leading to early afterdepolarizations (EADs) and lethal ventricular tachyarrhythmias (6Marban E. Nature. 2002; 415: 213-218Google Scholar). Polymorphic ventricular tachycardias, likely related to arrhythmogenic afterdepolarizations, are common in CHF (6Marban E. Nature. 2002; 415: 213-218Google Scholar, 7Nuss H.B. Kaab S. Kass D.A. Tomaselli G.F. Marban E. Am. J. Physiol. 1999; 277: H80-H91Google Scholar). The molecular mechanisms underlying APD prolongation in CHF remain incompletely understood. The rapid delayed rectifier K+ current (IKr) is crucial in cardiac repolarization. The human ether-a-go-go-related gene (HERG) encodes the pore-forming α-subunit of IKr and congenital or drug-induced abnormalities in HERG protein function are a common cause of the long QT syndrome. Simulations of cellular electrophysiology predict IKr inhibition to cause EADs in failing, but not nonfailing, myocytes (8Priebe L. Beuckelmann D.J. Circ. Res. 1998; 82: 1206-1223Google Scholar). A recent study demonstrated that transgenic mice overexpressing TNF-α with heart failure had significantly prolonged APD (9London B. Baker L.C. Lee J.S. Shusterman V. Choi B-R. Kubota T. McTiernan C.F. Feldman A.M. Slama G. Am. J. Physiol. 2003; 284: H431-H441Google Scholar). It is unknown whether TNF-α affects cardiac K+ channels. We therefore examined the hypothesis that TNF-α might affect HERG/IKr, thereby potentially contributing to CHF-related repolarization abnormalities. Cell Disposition—HEK293 cells stably expressing HERG were a kind gift from Drs. Zhou and January. Cell culture and handling procedures have been described previously (10Zhou Z. Gong Q. Ye B. Fan Z. Makielski J.C. Robertson G.A. January C.T. Biophys. J. 1998; 74: 230-241Google Scholar). Cardiomyocytes were isolated from healthy adult mongrel dogs as described in detail previously (11Shi H. Wang H. Wang Z. Mol. Pharmacol. 1999; 55: 497-507Google Scholar, 12Yue L. Feng J. Li G.R. Nattel S. Am. J. Physiol. 1996; 270: H2157-H2168Google Scholar). The procedures for animal use were in accordance with institutional guidelines. Whole-cell Patch Clamp Recording—Patch clamp techniques have been described in detail elsewhere (13Wang J. Wang H. Han H. Zhang Y. Yang B. Nattel S. Wang Z. Circulation. 2001; 104: 2645-2648Google Scholar, 14Zhang Y. Wang H. Wang J. Han H. Nattel S. Wang Z. FEBS Lett. 2003; 534: 125-132Google Scholar, 15Wang H. Zhang Y. Cao L. Han H. Wang J. Yang B. Nattel S. Wang Z. Cancer Res. 2003; 62: 4843-4848Google Scholar, 16Zhang Y. Han H. Wang J. Wang H. Yang B. Wang Z. J. Biol. Chem. 2003; 278: 10417-10426Google Scholar). Experiments were conducted at 36 ± 1 °C. For current recordings in canine myocyte studies, the following were included in the bath to block contaminating currents: CdCl2 (200-μmol/liter, L-type Ca2+ current), 4-aminopyridine (1 mmol/liter, transient outward K+ currents), glyburide (10 μmol/liter, ATP-sensitive K+ current), and 293B (10 μmol/liter, slow delayed-rectifier K+ current). Action potentials were recorded in the current clamp mode with Tyrode solution free of ion channel blockers. TNF-α was either added to the extracellular solution 10 min after formation of whole-cell configuration (acute studies), or cells were incubated with TNF-α in the medium for 10 h before patch clamp recording (long term exposure). Western Blot—The procedures were similar to those described previously (15Wang H. Zhang Y. Cao L. Han H. Wang J. Yang B. Nattel S. Wang Z. Cancer Res. 2003; 62: 4843-4848Google Scholar). Polyclonal anti-HERG raised in rabbit against highly purified peptide (CY)EEL PAGAPELPQD GPT, corresponding to residues 1118–1133 of human HERG was purchased from Alomone Laboratories (Jerusalem, Israel). Intracellular Reactive Oxygen Species (ROS) Measurement—5-(and-6)-Chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate (CM-H2DFDA) from Molecular Probes was used to detect oxidative activity in living cells as detailed previously (16Zhang Y. Han H. Wang J. Wang H. Yang B. Wang Z. J. Biol. Chem. 2003; 278: 10417-10426Google Scholar). Data Analysis—Group data are mean ± S.E. Paired t tests were used for single comparisons. Kinetics were analyzed with CLAMPFIT (pCLAMP 8.0) or Graphpad Prism. IHERG was elicited by 2-s depolarizations followed by 2-s repolarizing steps (Fig. 1, inset). Currents were recorded immediately after formation of whole-cell configuration and series resistance compensation. Comparisons were made between control cells (without TNF-α) and cells incubated for 10 h with various TNF-α concentrations from 0.01 to 10 ng/ml, which are within the pathophysiological range of TNF-α levels (∼0.1 ng/ml) (17Anker S.D. Volterrani M. Egerer K.R. Felton C.V. Kox W.J. Poole-Wilson P.A. Coats A.J. Q. J. Med. 1998; 91: 199-203Google Scholar, 18Ferrari R. Bachetti T. Confortini R. Opasich C. Febo O. Corti A. Cassani G. Visioli O. Circulation. 1995; 92: 1479-1486Google Scholar, 19Liu L. Zhao S.P. Int. J. Cardiol. 1999; 69: 77-82Google Scholar). IHERG density was reduced by TNF-α, with effects that were concentration- and voltage-dependent, being larger at more negative potentials (Fig. 1A). IHERG kinetics were unaltered by TNF-α. Exposure to TNF-α for 15 min concentration-dependently decreased IHERG. IHERG amplitude was decreased by 9, 16, and 35% by TNF-α at 1, 10, and 100 ng/ml, respectively. Results at 100 ng/ml are shown in Fig. 1B. Depression of IKr by TNF-α was reproduced in both dog atrial and ventricular myocytes (Fig. 1C). APD50 and APD90, duration at 50 and 90% repolarization, respectively, were both significantly longer in single ventricular cells preincubated with TNF-α at 10 ng/ml in Tyrode solution for 10 h relative to control cells (Fig. 1D). Western blot analysis of HERG protein levels in the membrane preparations extracted from HERG-expressing HEK293 cells and from the ventricular myocytes of healthy dogs or dogs with tachypacing induced CHF was performed. A band of around 135 kDa was identified by anti-HERG antibody, and the band was abolished after the antibody had been neutralized by its antigenic peptide. TNF-α treatment neither significantly alter HERG protein level in HEK293 cells nor in healthy dogs. HERG protein level was slightly higher in CHF than in healthy dogs and was slightly increased by TNF-α in CHF dogs (Fig. 2A). To clarify whether TNF-α acts on IHERG via activation of TNF receptor I (TNFR1), we incubated HEK293 cells with H389 (an inhibitory anti-TNFR1 antibody) for 1 h before patch clamp recording upon acute exposure to 100 ng/ml TNF-α or beginning 1 h before prolonged (10 h) exposure to 1 ng/ml TNF-α. H389 prevented suppression of IHERG by subsequent acute or prolonged application of TNF-α. Data from prolonged exposure experiments are shown in Fig. 2B. Activation of TNFR1 can stimulate overproduction of intracellular ROS (20Suematsu N. Tsutsui H. Wen J. Kang D. Ikeuchi M. Ide T. Hayashidani S. Shiomi T. Kubota T. Hamasaki N. Takeshita A. Circulation. 2003; 107: 1418-1423Google Scholar). To investigate whether ROS mediates TNF-α-induced HERG depression, we assessed the effects of TNF-α on IHERG in cells pretreated with the antioxidant vitamin E (VitE). Pretreatment with VitE for 2 h prevented IHERG reduction by TNF-α (Fig. 2C). Another antioxidant MnTBAP (Mn(III) tetrakis(4-benzoic acid) porphyrin chloride), a superoxide dismutase mimetic, produced similar preventive effects on TNF-α-induced HERG impairment (Fig. 2D). By contrast, preincubation of cells with superoxide anion generating system xanthine/xanthine oxidase (X/XO) mimicked the inhibitory effect of TNF-α on IHERG (Fig. 2E). To confirm that intracellular ROS production was indeed stimulated by TNF-α, we detected ROS level using CM-H2DFDA fluorescence dye to stain the cells. The cells stained with fluorescence intensity ≥5 times the background were defined as positive staining, and the number of cells with positive staining was pooled from five fields. The intensity of staining was analyzed by densitometric scanning using the LSM program, and the data were normalized to the control values without TNF-α (0.1 and 10 ng/ml) treatment (16Zhang Y. Han H. Wang J. Wang H. Yang B. Wang Z. J. Biol. Chem. 2003; 278: 10417-10426Google Scholar). Under control conditions, cells stained by CM-H2DCFDA were sparse, and the staining was weak. Yet with TNF-α treatment, the number of the cells with positive staining was considerably higher and the cells were stained evenly throughout the cytoplasm. Pretreatment with VitE or MnTBAP drastically diminished the number and the intensity of staining (Fig. 2F). Similar results were obtained with isolated canine ventricular myocytes; TNF-α (0.1 ng/ml) markedly increased ROS level and co-application with ViTE (100 μm) or MnTBAP (5 μm) prevented the effects of TNF-α (Fig. 2G). Heart failure is associated with APD and QT interval prolongation, believed to contribute to the occurrence of sudden cardiac death (6Marban E. Nature. 2002; 415: 213-218Google Scholar, 7Nuss H.B. Kaab S. Kass D.A. Tomaselli G.F. Marban E. Am. J. Physiol. 1999; 277: H80-H91Google Scholar). We show here that TNF-α suppresses IHERG in HEK293 cells and IKr in dog cardiomyocytes and prolonged APD. Depression of IHERG/IKr, as produced by TNF-α in this study, may contribute to delayed repolarization and associated malignant ventricular tachyarrhythmias with increased TNF-α level in patients with CHF. Ionic remodeling in CHF has been studied (21Tomaselli G.F. Marban E. Cardiovasc. Res. 1999; 42: 270-283Google Scholar). L-type Ca2+ current density appears to be unaltered (20Suematsu N. Tsutsui H. Wen J. Kang D. Ikeuchi M. Ide T. Hayashidani S. Shiomi T. Kubota T. Hamasaki N. Takeshita A. Circulation. 2003; 107: 1418-1423Google Scholar). The inward-rectifier K+ current is consistently reduced (5Beuckelmann D.J. Nabauer M. Erdmann E. Circ. Res. 2002; 73: 379-385Google Scholar). The transient outward K+ current (Ito) is also reduced, potentially causing APD prolongation (5Beuckelmann D.J. Nabauer M. Erdmann E. Circ. Res. 2002; 73: 379-385Google Scholar, 22Nabauer M. Beuckelmann D.J. Erdmann E. Circ. Res. 1993; 73: 386-394Google Scholar). However, inhibition of Ito reduces APD in human atrial cells (23Escande D. Coulombe A. Faivre J.F. Deroubaix E. Coraboeuf E. Am. J. Physiol. 1987; 252: H142-H148Google Scholar), canine atrial cells (12Yue L. Feng J. Li G.R. Nattel S. Am. J. Physiol. 1996; 270: H2157-H2168Google Scholar), and dog Purkinje fibers (24Lee J.H. Rosen M.R. J. Cardiovasc. Electrophysiol. 1994; 5: 232-240Google Scholar). The effect of Ito on the AP depends largely on the magnitude of IK (25Nygren A. Fiset C. Firek L. Clark J.W. Lindblad D.S. Clark R.B. Giles W.R. Circ. Res. 1998; 82: 63-81Google Scholar). Tsuji et al. (26Tsuji Y. Opthof T. Kamiya K. Yasui K. Liu W. Lu Z. Kodama I. Cardiovasc. Res. 2000; 48: 300-309Google Scholar) showed IKr, measured as E-4031-sensitive tail current, to be ∼36% smaller in rabbits with ventricular tachypacing-induced CHF than in healthy rabbits. Lodge and Normandin (27Lodge N.J. Normandin D.E. J. Mol. Cell. Cardiol. 1997; 29: 3211-3221Google Scholar) demonstrated earlier that IKr, measured as dofetilide-sensitive tail current, reduced by ∼45% in the BIO TO-2 strain of cardiomyopathic hamster of 10 months old, derived from the BIO 53.58 animals and providing a model of dilated low output heart failure, compared with the 10-month-old control (BIO F1B) hamsters. A recent study by London et al. (9London B. Baker L.C. Lee J.S. Shusterman V. Choi B-R. Kubota T. McTiernan C.F. Feldman A.M. Slama G. Am. J. Physiol. 2003; 284: H431-H441Google Scholar) showed significant APD prolongation in transgenic mice which overexpressed TNF-α and developed heart failure. Our study suggests that TNF-α may be an important mediator of CHF-induced IKr reduction and is the first to demonstrate that TNF-α can modulate cardiac K+ channels. We further demonstrated that pretreatment with VitE or MnTBAP prevented, whereas X/XO mimicked, TNF-α-induced IHERG depression. The effects of VitE and MnTBAP are likely due to their antioxidant actions because TNF-α increased the intracellular ROS level in a concentration-dependent manner in both HEK293 cells and canine ventricular myocytes, more specifically O2·¯ level because VitE or MnTBAP effectively prevented the increase. In line with our finding, a recent study published during the course of this study clearly demonstrated the ability of TNF-α to stimulate mitochondrial production of ROS in cardiomyocytes (20Suematsu N. Tsutsui H. Wen J. Kang D. Ikeuchi M. Ide T. Hayashidani S. Shiomi T. Kubota T. Hamasaki N. Takeshita A. Circulation. 2003; 107: 1418-1423Google Scholar). It has also been shown that ROS is one of the key deleterious factors in failing heart (28Choudhary G. Dudley Jr., S.C. Congest. Heart Fail. 2002; 8: 148-155Google Scholar, 29Byrne J.A. Grieve D.J. Cave A.C. Shah A.M. Arch. Mal. Coeur. Vaiss. 2003; 96: 214-223Google Scholar). Our data therefore indicate that TNF-α-induced HERG depression occurs at the functional level, but not at the expression levels (TNF-α did not alter HERG protein content), and the functional impairment of HERG channels by TNF-α is mediated by ROS, particularly O2·¯. Circulating TNF-α levels predict mortality in CHF, and therapies directed against TNF-α may limit the pathophysiologic consequences (1McTiernan C.F. Feldman A.M. Curr. Cardiol. Rep. 2000; 2: 189-197Google Scholar). In healthy human subjects, the TNF-α level is below 0.01 ng/ml, but in patients with heart failure, it can increase to over 0.1 ng/ml (17Anker S.D. Volterrani M. Egerer K.R. Felton C.V. Kox W.J. Poole-Wilson P.A. Coats A.J. Q. J. Med. 1998; 91: 199-203Google Scholar, 18Ferrari R. Bachetti T. Confortini R. Opasich C. Febo O. Corti A. Cassani G. Visioli O. Circulation. 1995; 92: 1479-1486Google Scholar, 19Liu L. Zhao S.P. Int. J. Cardiol. 1999; 69: 77-82Google Scholar). TNF-α significantly inhibited IHERG over this concentration range (e.g. by ∼35% at plateau voltages from –10 to +10 mV in cells exposed to 0.1 ng/ml TNF-α for 10 h). Our study might have underestimated the effects of TNF-α on APD because the myocytes were incubated with TNF-α at 4 °C to maintain good quality of the cells. Our observations provide new insights into the potential molecular mechanisms underlying electrophysiological abnormalities and sudden arrhythmic death in patients with CHF. We thank XiaoFan Yang for excellent technical support and Louis R. Villeneuve for his assistance for confocal microscopic examinations.