Impairment of Human Ether-à-Go-Go-related Gene (HERG) K+ Channel Function by Hypoglycemia and Hyperglycemia

SIMILAR PHENOTYPES BUT DIFFERENT MECHANISMS*

Yiqiang ZhangDagger §, Hong HanDagger , Jingxiong WangDagger §, Huizhen WangDagger , Baofeng Yang||, and Zhiguo WangDagger §**

From the Dagger  Research Center, Montreal Heart Institute, Montreal, Quebec H1T 1C8, the § Department of Medicine, University of Montreal, Montreal, Quebec H3C 3J7, Canada and the || Department of Pharmacology, Harbin Medical University, Harbin, Heilongjiang 150086, Peoples Republic of China

Received for publication, October 29, 2002, and in revised form, December 20, 2002

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Hyperglycemia and hypoglycemia both can cause prolongation of the Q-T interval and ventricular arrhythmias. Here we studied modulation of human ether-à-go-go-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 [Glu]o = 10 or 20 mM) and hypoglycemia ([Glu]o = 2.5, 1, or 0 mM) impaired HERG function by reducing HERG current (IHERG) density, as compared with normoglycemia ([Glu]o = 5 mM). Complete inhibition of glucose metabolism (glycolysis and oxidative phosphorylation) by 2-deoxy-D-glucose mimicked the effects of hypoglycemia, but inhibition of glycolysis or oxidative phosphorylation alone did not cause IHERG depression. Depletion of intracellular ATP mimicked the effects of hypoglycemia, and replacement of ATP by GTP or non-hydrolysable ATP failed to prevent the effects. Inhibition of oxidative phosphorylation by NaCN or application of antioxidants vitamin E or superoxide dismutase mimetic (Mn(III) tetrakis(4-benzoic acid) porphyrin chloride) abrogated and incubation with xanthine/xanthine oxidase mimicked the effects of hyperglycemia. Hyperglycemia or xanthine/xanthine oxidase markedly increased intracellular levels of reactive oxygen species, as measured by 5-(and-6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate (CM-H2DCFDA) fluorescence dye, and this increase was prevented by NaCN, vitamin E, or Mn(III) tetrakis(4-benzoic acid) porphyrin chloride. We conclude that ATP, derived from either glycolysis or oxidative phosphorylation, is critical for normal HERG function; depression of IHERG in hypoglycemia results from underproduction of ATP and in hyperglycemia from overproduction of reactive oxygen species. Impairment of HERG function might contribute to Q-T prolongation caused by hypoglycemia and hyperglycemia.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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+ current in the heart, which is the major repolarizing current in the plateau voltage range of cardiac action potentials. HERG K+ 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+ channels and the potential mechanisms.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Cell Culture-- HEK293 cells stably expressing HERG (a kind gift from Drs. Zhou and January) (14) were grown in Dulbecco's modified Eagle's medium supplemented with 10% heat-inactivated fetal bovine serum, 200 µM G418, 100 units/ml penicillin, and 100 µg/ml streptomycin. The cells subcultured to ~85% confluency were harvested by trypsinization and stored in the Tyrode's solution containing 0.5% bovine serum albumin at 4 °C (12). Electrophysiological recordings were conducted within 10 h of storage.

Whole-cell Patch Clamp Recording-- Patch clamp techniques have been described in detail elsewhere (15-16). Currents were recorded with whole-cell voltage clamp with an Axopatch-200B amplifier (Axon Instruments). Borosilicate glass electrodes had tip resistances of 1-3 megohms when filled with the internal solution containing (mM) 130 KCl, 1 MgCl2, 5 Mg-ATP, 10 EGTA, and 10 HEPES (pH 7.3). The extracellular (Tyrode's) solution contained (mM) 136 NaCl, 5.4 KCl, 1 CaCl2, 1 MgCl2, 10 HEPES (pH 7.4), and glucose at concentrations as specified. Experiments were conducted at 36 ± 1 °C. Junction potentials were zeroed before formation of the membrane-pipette seal. Series resistance and capacitance were compensated, and leak currents were subtracted.

Pharmacological Probes-- D-Glucose (Glu), 2-deoxy-D-glucose (2dG), sodium cyanide (NaCN), pyruvate, ATP, GTP, AMP-PCP (non-hydrolysable analogue of ATP), xanthine (X), xanthine oxidase (XO), and vitamin E (VitE) were all purchased from Sigma. Xanthine was prepared in 2 N NaOH and diluted in the Tyrode's solution 800 times with the pH adjusted to 7.4 with HCl. Xanthine oxidase was added to the xanthine preparation to form the X/XO-reactive oxygen species (ROS) generating system. VitE was dissolved in ethanol and diluted 1000 times to reach the final concentration. Pyruvate in liquid was diluted into the Tyrode's solution, and pH was adjusted to 7.4 with NaOH before use. All other compounds were directly dissolved into the patch clamp recording solutions as specified. Mn(III) tetrakis(4-benzoic acid) porphyrin chloride (MnTBAP) purchased from Calbiochem was dissolved in 1 N NaOH and diluted by 5000 times to reach the desired experimental concentrations. All compounds and reagents were prepared fresh before the experiments.

Intracellular Reactive Oxygen Species (ROS) Measurement-- 5- (and-6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate (CM-H2DCFDA) (Molecular Probes) is a ROS-sensitive probe that can be used to detect oxidative activity in living cells. It passively diffuses into cells, where its acetate groups are cleaved by intracellular esterases, releasing the corresponding dichlorodihydrofluorescein derivative. Its thiol-reactive chloromethyl group reacts with intracellular glutathione and other thiols. Subsequent oxidation yields a fluorescent adduct that is trapped inside the cell. When it is excited at 480 nm, its emissions at 505-530 nm can be captured. CM-H2DCFDA is prepared in dimethyl sulfoxide immediately prior to loading. Glass coverslips were coated with laminin and placed in the wells of a 12-well culture plate before the cells were seeded into the well in a density of 5.0 × 104/well. After overnight incubation, the cells were washed with pre-warmed (37 °C) phosphate-buffered saline once and then incubated in the Tyrode's solution containing glucose of varying concentrations or exogenous superoxide-generating system (xanthine/xanthine oxidase) or the reagents as to be otherwise specified, together with the fluorescence dye CM-H2DCFDA (10 µM). After 30 min of incubation, the coverslips were washed with pre-warmed phosphate-buffered saline twice before being mounted to the glass slides with anti-fading mounting medium and were examined immediately under a laser scanning confocal microscope (Zeiss LSM 510). The percentage of positively stained cells and the fluorescence intensity of staining were determined by densitometric scanning with LSM software (Zeiss).

Data Analysis-- Group data are expressed as mean ± S.E. Comparisons among groups were made by analysis of variance (F-test), and Bonferroni-adjusted t tests were used for multiple group comparisons, and paired or unpaired t test was used, as appropriate, for single comparisons. A two-tailed p < 0.05 was taken to indicate a statistically significant difference. Nonlinear least square curve fitting was performed with CLAMPFIT in pCLAMP 8.0 or GraphPad Prism.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Effects of Glucose on IHERG-- To study the effects of varying concentrations of glucose (Glu, ranging from 0 to 20 mM) on IHERG, our experiments were designed for group comparisons. For each experiment, HERG-expressing HEK293 cells were divided into six groups each superfused with the Tyrode's solution containing a given concentration of Glu for 30 min prior to patch clamp recordings. In addition, recordings were performed immediately after formation of whole-cell configuration and adjustments of capacitance and series resistance compensation, and all recordings were made complete within 3 min. In this manner, there was minimal dialysis through the recording pipette and thereby minimal current run-down (time-dependent current decay), and the data best reflect the effects of Glu on IHERG in cells with intact intracellular contents. In addition, such an experimental design also allowed us to study the effect of Glu on IHERG under conditions devoid of influence from exogenous ATP included in the pipette, which is an important issue to be described later. IHERG was elicited by 2.5-s depolarizing steps from -60 to +40 mV to record the activating current, followed by a repolarizing pulse to -50 mV for another 2.5 s to observe the deactivating tail current, before being returned to a holding potential of -80 mV. The results are illustrated in Fig. 1 with both representative raw data and analyzed mean data. Glucose produced two characteristic alterations of HERG channel functions as follows: changes of IHERG amplitude and density and shifts of I-V relationships and activation curves.


View larger version (36K):
[in this window]
[in a new window]
 
Fig. 1.   Effects of glucose on HERG K+ current (IHERG) stably expressed in HEK293 cells. A, typical examples of IHERG traces recorded in the Tyrode's solution containing 0, 5, 10, or 20 mM D-glucose (Glu) with the voltage protocol shown in the inset and presented as current density (pA/pF) for better group comparisons. The same voltage protocol is applied for the IHERG recordings shown in the subsequent figures, except as otherwise indicated. B, current density-voltage relationships of IHERG. The steady-state step IHERG measured at the end of 2.5-s pulses was normalized to the capacitance of the respective cells and plotted as a function of test potentials. Shown are data averaged from 22, 9, 11, 25, 24, and 15 cells for 0, 1, 2.5, 5, 10, and 20 mM Glu, respectively. *, p < 0.05 versus 5 mM [Glu]o. C, ratio of the step IHERG recorded with varying [Glu]o over the IHERG with 5 mM [Glu]o as a function of test potentials. D, ratio of the step IHERG recorded with varying [Glu]o over the IHERG with 5 mM [Glu]o as a function of glucose concentrations. Shown are the data obtained at test potentials of -40, -10, and +20 mV. E, normalized I-V relationships obtained by dividing the current amplitude at various potentials by the maximum current for each concentration of glucose. For clarity, only the data with 0, 5, and 20 mM [Glu]o are shown. Note the negative shifts of the curves with low and high [Glu]o, relative to 5 mM [Glu]o, along the voltage axis. F, steady-state voltage-dependent activation of IHERG. The activation curves were constructed by plotting the conductance G as a function of potentials. G was calculated by normalizing the tail currents at -50 mV by dividing the amplitude of the tail currents evoked at various antecedent step potentials by that of the tail current at +40 mV. The symbols are mean of experimental data, and the lines represent the Boltzmann fit: G/Gmax = 1/{1 + exp((V1/2 - V)/k)}, where Gmax represents the maximal conductance at +40 mV, V1/2 is a half-maximal activation voltage, and k is a slope factor. The numbers in the legends represent the extracellular glucose concentrations ([Glu]o) in mM.

Comparison of IHERG recorded at varying extracellular concentrations of Glu ([Glu]o = 0, 1, 2.5, 5, 10, or 20 mM) consistently showed that IHERG density was maximal at a physiological [Glu]o (5 mM), and it was depressed at [Glu]o below or above 5 mM (Fig. 1, A-C). In other words, under normoglycemia ([Glu]o = 5 mM) the HERG K+ channel operates at its maximum function level, whereas under hypoglycemia ([Glu]o = 0, 1, or 2.5 mM) or hyperglycemia ([Glu]o = 10 or 20 mM), the HERG channel function is impaired, and the degree of functional impairment is proportional to the degrees of hypoglycemia or hyperglycemia (Fig. 1D). For example, with 5 mM [Glu]o, the IHERG current density was 88.5 ± 7.4 pA/pF (n = 25) at a test potential of 0 mV, whereas with 0 and 20 mM, the values were 35.4 ± 3.0 pA/pF (n = 22, p < 0.05 versus 5 mM [Glu]o) and 35.8 ± 4.9 (n = 15, p < 0.05 versus 5 mM [Glu]o), respectively, approximately a 2-fold difference. The HERG tail current density at -10 mV was also significantly depressed by hypoglycemia (0, 1, and 2.5 mM [Glu]o) or hyperglycemia (10 and 20 mM [Glu]o) (data not shown).

Also noticeable is the hypolarization shifts of IHERG I-V relationships (Fig. 1E) and the activation curve by hypoglycemia (Fig. 1F). For example, the half-activation voltage (V1/2) was -25.4 ± 2.7 mV (n = 19) for 5 mM [Glu]o and -34.3 ± 3.8 mV (n = 22) for 0 mM [Glu]o (p < 0.05, unpaired t test), which accounted for around 10 mV shift of the steady-state voltage-dependent activation of IHERG. This negative shift resulted in a crossover of the I-V curves between normoglycemia and hypoglycemia. Only slight (~4 mV) hyperpolarization shift of activation was seen with hyperglycemia (20 mM [Glu]o).

Several potential mechanisms could explain the observed effects of [Glu]o on IHERG. First, there was a possibility that the effects were a consequence of alterations of extracellular osmolarity with varying [Glu]o. Second, Glu might act directly on HERG proteins to modify the channel function. Finally, Glu metabolism, which generates ATP as well as other metabolic intermediates, also has the potential to modulate IHERG. The following experiments were performed to clarify these issues.

To test the first possibility, we performed experiments in which cells were first superfused with a given concentration of Glu (1, 5, or 20 mM) for >30 min, followed by IHERG recording within 3 min bathing in the Tyrode's solution containing 10 mM Glu. Under such conditions, IHERG demonstrated the same pattern of changes as described above; cells pre-exposed to 1 or 20 mM Glu had markedly smaller IHERG density than those pre-exposed to 5 mM Glu (Fig. 2A), despite that all recordings were made under isotonic conditions with 10 mM [Glu]o. For instance, at 0 mV, the IHERG density was 56.1 ± 6.9 pA/pF (n = 8) with 5 mM [Glu]o, whereas with 1 and 20 mM [Glu]o, the values were 32.4 ± 3.2 pA/pF (n = 7, p < 0.05 versus 5 mM [Glu]o) and 35.8 ± 2.2 (n = 7, p < 0.05 versus 5 mM [Glu]o), respectively. Consistently, the activation curve of IHERG was also shifted to more negative potentials by hypoglycemia (Fig. 2B). A similar difference of IHERG between 5 and 20 mM [Glu]o was also consistently seen when 15 mM cellobiose was added to the Tyrode's solution containing 5 mM Glu (Fig. 2D).


View larger version (34K):
[in this window]
[in a new window]
 
Fig. 2.   Effects of glucose on IHERG under conditions with corrected osmolarity. A and B, mean current density (pA/pF)-voltage relationships and activation conductance (G) curves, respectively (n = 7, 9, and 7 for 1, 5, and 20 mM [Glu]o, respectively). The cells of different groups were superfused with solutions containing a given concentration of glucose (1, 5, or 20 mM) for >30 min and then switched to the same solution with an identical glucose concentration (10 mM). The IHERG values recorded within 3 min at 10 mM [Glu]o were used for analysis. *, p < 0.05 versus 5 mM [Glu]o, unpaired t tests. C and D, depression of IHERG and negative shift of IHERG activation, respectively, in high glucose (20 mM [Glu]o, n = 5), as compared with those in normal glucose (5 mM [Glu]o) + 15 mM cellobiose (n = 5) to balance the extracellular osmolarity. *, p < 0.05 versus Glu-5 + cellobiose 15, unpaired t-tests.

The above data indicate that changes of osmolarity is unlikely the mechanism by which glucose modulates IHERG. Apart from that, the fact that differences of IHERG among the cells pretreated with three different concentrations of Glu (1, 5, and 20 mM) persisted even though IHERG was recorded 10 min after superfusion with the normal Tyrode's solution containing 10 mM [Glu]o suggests that the effects of glucose on IHERG are mediated by some intracellular events, and direct interactions between glucose and HERG channels do not likely play a major role.

Role of Glycolysis and Oxidative Phosphorylation on Glucose-induced IHERG Enhancement-- Glucose, once being taken up into the cell, is metabolized via glycolysis to generate 2 molecules of ATP and 2 molecules of pyruvate (a substrate for oxidative phosphorylation), which is further metabolized via oxidative phosphorylation to produce more ATP molecules. To investigate whether the effects of Glu on IHERG are associated with glucose metabolism, we studied the effects of the glycolysis inhibitor 2-deoxy-D-glucose (2dG). Glucose was replaced by the non-hydrolysable analogue of glucose 2dG to eliminate the glucose metabolism (both glycolysis and subsequent oxidative phosphorylation). Cells were superfused with the Glu-free Tyrode's solution containing 5 mM 2dG for 30 min before patch clamp recordings. IHERG recorded under such a condition was compared with IHERG recorded with the Tyrode's solution containing 5 mM Glu. As shown in Fig. 3, 2dG substitution for Glu reproduced the two characteristic changes of IHERG as observed under hypoglycemia. First, marked depression of IHERG was seen in the presence of 2dG, with IHERG density only ~38% that in the presence of 5 mM [Glu]o at 0 mV. Second, similar to the results with 0 mM [Glu]o, the I-V relationship and activation curve were shifted by 10 mV toward hyperpolarizing potentials by 2dG (-28.2 ± 3.4 mV for 5 mM [Glu]o and -37.6 ± 4.5 mV for 5 mM 2dG, p < 0.05, n = 8 for both groups) (Fig. 3B). Because 2dG is a competitive inhibitor of glycolysis, its effects on IHERG in the presence of 5 mM Glu were also investigated. Cells were superfused with the Glu-containing Tyrode's solution with or without 5 mM 2dG for 30 min before patch clamp recordings. The IHERG density was ~45% smaller in cells treated with 2dG than in untreated cells (Fig. 3C). For example, the IHERG density at 0 mV was 40.2 ± 4.1 pA/pF for cells treated with 2dG at 5 mM [Glu]o (n = 14) and was 75.6 ± 6.6 pA/pF for cells without 2dG treatment (n = 14, p < 0.05). These results indicate the importance of glucose metabolism in maintaining the normal HERG function. Intriguingly, when 2dG was added to the hyperglycemic solution containing 20 mM [Glu]o, IHERG was increased as compared with that measured in the hyperglycemic solution without 2dG (Fig. 3E). In other words, 2dG partly reversed the depressed IHERG caused by hyperglycemia toward the normal HERG function seen under normoglycemia.


View larger version (32K):
[in this window]
[in a new window]
 
Fig. 3.   Effects of complete inhibition of glucose metabolism or inhibition of glycolysis on IHERG. A and B, mean current density (pA/pF)-voltage relationships and activation conductance (G) curves of IHERG. Complete inhibition of glucose metabolism was achieved by the addition of 5 mM 2dG to the Glu-free solution, and inhibition of glycolysis alone was achieved by the addition of pyruvate to the 2dG-containing Glu-free solution. The numbers in the legends represent the concentrations of Glu, 2dG, or pyruvate in mM. The number of cells was 8 for the Glu 5 group, 7 for Glu 0 + 2dG 5 group, 7 for Glu 0 + 2dG 5 + pyruvate 5 group, and 8 for Glu 0 + 2dG 5 + pyruvate 20 group. *, p < 0.05 versus 5 mM [Glu]o. C and D, comparison between the effects of 2dG (5 mM) on IHERG under 0 mM [Glu]o and 5 mM [Glu]o. *, p < 0.05 versus 5 mM [Glu]o. E and F, effects of 2dG (5 mM) on IHERG under 20 mM [Glu]o. The number of cells was 10 for Glu 5 group, 8 for Glu 20 group, and 11 for Glu 20 + 2dG 5 group. *, p < 0.05 versus 20 mM [Glu]o, unpaired t tests.

To dissect further which of the two, glycolysis or oxidative phosphorylation, is truly responsible for HERG modulation, the following experiments were carried out. In the first set of experiments, pyruvate was supplied to the 2dG-containing Glu-free Tyrode's solution. Under such a condition, the glycolysis was inhibited, but the oxidative phosphorylation was maintained. As displayed in Fig. 3A, addition of pyruvate at a concentration of 5 mM restored the depressed IHERG induced by glycolysis inhibition. However, the negative shifts of I-V relationship and activation curve, as seen with hypoglycemia or 2dG substitution for Glu, were still consistently observed with pyruvate (Fig. 3B). In contrast, elevation of pyruvate to 20 mM weakened the ability to restore the suppressed IHERG caused by glycolysis inhibition with 2dG substitution for Glu. Thus, the IHERG density with 20 mM pyruvate in the 2dG-containing Glu-free Tyrode's solution was considerably smaller than that with normal [Glu]o (5 mM). However, this high concentration of pyruvate still failed to prevent the negative shifts of IHERG I-V relationship and activation curve produced by glycolysis inhibition (Fig. 3B).

In the second set of experiments, the oxidative phosphorylation was inhibited by inclusion of NaCN (2 mM), an uncoupler of oxidative phosphorylation, in the bathing solution, and the glycolysis was kept intact with 5 or 20 mM [Glu]o. Under normoglycemia (5 mM [Glu]o), inhibition of oxidative phosphorylation by NaCN produced a slight non-significant decrease in IHERG (Fig. 4A). Under hyperglycemia (20 mM [Glu]o), however, IHERG was markedly diminished, and NaCN restored the depressed IHERG toward the IHERG amplitude seen under normoglycemia (5 mM). For instance, the step and tail IHERG densities in 20 mM glucose were 50.4 ± 5.9 and 56.4 ± 5.3 pA/pF (n = 14) at -10 mV and were restored by NaCN to 73.9 ± 7.9 and 81.6 ± 6.4 pA/pF (n = 13, p < 0.05 versus 20 mM [Glu]o), respectively. Also important is that the oxidative phosphorylation inhibition did not produce any significant voltage shifts of I-V relationships and activation curves, regardless of different [Glu]o values (5 or 20 mM) (Fig. 4B). It appears from the above data that either glycolysis or oxidative phosphorylation was sufficient to sustain the normal function of HERG channels, and significant negative shifts of IHERG I-V relationships and activation curves occurred when glycolysis was inhibited, regardless of whether oxidative phosphorylation was maintained or not.


View larger version (21K):
[in this window]
[in a new window]
 
Fig. 4.   Effects of inhibition of oxidative phosphorylation on IHERG. Mean current density (pA/pF)-voltage relationships (A) and activation conductance (G) curves (B). Inhibition of oxidative phosphorylation with intact glycolysis was achieved by the addition of NaCN (2 mM) to the Glu (5 or 20 mM)-containing solution. The numbers in the legends represent the concentrations of Glu or NaCN in mM. n = 23 for Glu 5 group, 15 for Glu 5 + NaCN 2 group, 14 for Glu 20 group, and 13 for Glu 20 + NaCN 2 group. *, p < 0.05 versus Glu 5 mM; +, p < 0.05 versus Glu 20 mM, unpaired t tests.

Role of Intracellular ATP in Maintaining HERG Function-- The above experiments indicate that glucose metabolism (glycolysis and oxidative phosphorylation) is critical for IHERG modulation by Glu. Yet it was unclear whether the IHERG modulation by glucose metabolism is associated with the generation of high energy phosphates (i.e. ATP), and if so whether the ATP generated from the glucose metabolism is glycolysis-derived or oxidative phosphorylation-derived. To clarify this issue, we first assessed the influence of intracellular ATP depletion on HERG function in the presence of 5 mM Glu, by omitting ATP from the pipette (internal) solution. IHERG recorded immediately after membrane rupture and capacitance/resistance compensation was taken as base-line control data, and the same measurement was repeated every 5 min up to 15 min. Under our experimental conditions, 10 min is sufficient to allow complete dialysis, thereby the equilibrium between pipette solution and cytoplasm. As illustrated in Fig. 5, the IHERG recorded with the normal ATP-containing pipette showed only slight run-down over a 15-min period, whereas the IHERG recorded with ATP-free pipette was found significantly reduced with time. There was an ~46% decrease in IHERG at 10 min after dialysis at -10 mV (Fig. 5C), being similar to the reduction of IHERG under hypoglycemia or with the inhibition of glucose metabolism by 2dG. Also consistent with the hypoglycemia and metabolic inhibition was the negative shifts of the I-V relationship (Fig. 5D) and voltage-dependent activation (Fig. 5E) of IHERG with ATP depletion; the V1/2 was changed by ~8 mV from -31.2 ± 3.6 mV before to -38.9 ± 8.1 mV (p < 0.05, n = 7) after [ATP]i depletion.


View larger version (29K):
[in this window]
[in a new window]
 
Fig. 5.   Effects of depletion of intracellular ATP on IHERG. A, raw IHERG recorded with the ATP-free pipette solution right after membrane rupture as base-line control data (left) and 10 min after membrane rupture with complete dialysis (right). B, mean I-V relationships (n = 5 cells) showing the depression of IHERG caused by depletion of intracellular ATP. C, time-dependent changes of IHERG at -10 mV showing the current run down with the ATP-free pipette solution, which is otherwise minimal with the ATP-containing pipette solution. D, normalized I-V relationships showing the negative shift of voltage dependence of IHERG caused by the depletion of intracellular ATP. E, activation conductance (G) curves before and after ATP depletion. *, p < 0.05 versus control (Ctl), paired t tests; +, p < 0.05, F test indicating the statistical significance of the time dependence.

To investigate whether the requirement of intracellular ATP for HERG function relies on hydrolysis of ATP or is simply because of nucleotide interaction with the nucleotide binding domain of HERG channels (17), we carried out the following series of experiments. We first used the pipette containing the non-hydrolysable AMP-PCP to replace ATP. With 5 mM AMP-PCP in the pipette, the IHERG demonstrated a rapid run-down as observed with the ATP-free internal solution (Fig. 6C). For instance, at -10 mV the IHERG recorded 10 min after dialysis was 32.2 ± 2.1% smaller than the basal current recorded right after membrane rupture. We then went on to test if substitution of GTP for ATP could prevent IHERG run-down. With 5 mM GTP in the ATP-free pipette solution, the IHERG developed a similar degree of rapid run-down to what was seen with the intracellular ATP depletion alone (Fig. 6F). For example, at -10 mV, there were 34.4 ± 5.1% decreases in the IHERG amplitude 10 min after dialysis. Moreover, the negative shifts of I-V relationships and activation curves were also seen with AMP-PCP or GTP (Fig. 6, B and E). For instance, V1/2 was changed from -33.4 ± 0.8 mV for base line to -37.3 ± 1.0 mV for 10 min of dialysis with AMP-PCP, and similarly, V1/2 was shifted from -31.4 ± 1.5 mV to -38.9 ± 2.2 mV by GTP (p < 0.05).


View larger version (26K):
[in this window]
[in a new window]
 
Fig. 6.   Effects of non-hydrolysable ATP (AMP-PCP) and GTP on IHERG. A and D, I-V relationships. IHERG was recorded right after membrane rupture and 10 min after dialysis with the ATP-free pipette solution containing AMP-PCP (n = 10) (A) or GTP (n = 7) (D). B and E, activation conductance (G) curves before and after AMP-PCP or GTP in the ATP-free internal solution. The numbers in the legends represent AMP-PCP or GTP concentrations in mM. C and F, time-dependent changes of IHERG at -10 mV recorded with the pipette containing ATP (control (Ctl)) or AMP-PCP (C) or GTP (F). *, p < 0.05 versus control, paired t tests; +, p < 0.05, F test indicating the significance of time dependence.

The same effects of ATP depletion on IHERG were consistently reproduced when glyburide (10 µM) was included in the superfusate to inhibit ATP-sensitive K+ current (KATP), if any (data not shown).

Role of ROS on Hyperglycemia-induced IHERG Depression-- Collectively from the above experiments, glucose metabolism is necessary for maintaining the HERG channel function, and the ATP produced by either glycolysis or oxidative phosphorylation seems to be a key factor for the regulation; on the other hand, the fact that NaCN restores the depressed IHERG induced by 20 mM [Glu]o or by 20 mM pyruvate suggests that oxidative phosphorylation also produces negative (suppressive) regulation on HERG function. This would imply that the IHERG suppression by high glucose via oxidative phosphorylation is ATP-independent or is the balance between the enhancement by ATP and the suppression by other factors associated with oxidative phosphorylation. It has been well established that mitochondria produce most of the endogenous reactive oxygen species (ROS) through oxidative phosphorylation (18-23), and hyperglycemia stimulates massive ROS production (19, 24-29). It is therefore rational to propose that the endogenously produced ROS via oxidative phosphorylation stimulated by hyperglycemia could impair HERG channel function to suppress IHERG. To test this hypothesis, we performed the following experiments. We first evaluated the effects of an antioxidant vitamin E (VitE) on 20 mM [Glu]o-induced IHERG depression. Cells were divided into three groups as follows: 5 mM [Glu]o, 20 mM [Glu]o, and 20 mM [Glu]o + 0.1 mM VitE. As illustrated in Fig. 7, A and B, pretreatment of cell with VitE effectively prevented the IHERG suppression by hyperglycemia; the IHERG density in VitE group was virtually identical to that in the normoglycemia group. There was no significant shift of the activation curve along the voltage axis. These data suggest a participation of ROS in the HERG regulation by hyperglycemia. Next, we studied the effects of another antioxidant, superoxide dismutase (SOD) mimetic MnTBAP, on the IHERG depression induced by 20 mM [Glu]o. Because the compound does not readily penetrate the cells, it was intracellularly applied through dialysis of the pipette solution at a concentration of 5 µM. Cells were superfused with 20 mM Glu for 30 min prior to formation of the whole-cell membrane patch. To correct for the potential current run-down, the IHERG recorded at various time points after membrane rupture was normalized to the IHERG recorded with the normal Tyrode's solution without MnTBAP at the corresponding time points. As shown in Fig. 7C, MnTBAP caused a time-dependent increase in the IHERG amplitude, indicating a restoration of the depressed HERG function. By comparison, no alterations of IHERG, or virtually a slight decrease (presumably representing run-down of the current), were found with catalase in the pipette (data not shown). These results indicate that the ROS involved in the IHERG suppression by hyperglycemia was of mainly superoxide anion (O<UP><SUB>2</SUB><SUP>−</SUP></UP>).


View larger version (43K):
[in this window]
[in a new window]
 
Fig. 7.   Effects of antioxidants on IHERG depression induced by hyperglycemia (20 mM [Glu]o). A and B, mean current density (pA/pF)-voltage relationships and activation conductance (G) curves, respectively, showing the effects of VitE (100 µM) on IHERG under 20 mM [Glu]o. The cells were superfused with VitE for 40 min or with the normal Tyrode's solution for control before patch clamp recordings. Group comparison was made between the untreated control cells (Ctl, n = 9) and the VitE-treated (n = 8) cells. *, p < 0.05 versus control, unpaired t tests. C and D, mean current density (pA/pF)-voltage relationships and activation conductance (G) curves, respectively, showing the effects of MnTBAP (5 µM), a superoxide dismutase mimetic, on IHERG under 20 mM [Glu]o. MnTBAP was applied intracellularly through the pipette. IHERG recorded immediately after whole-cell formation and series resistance compensation was taken as base-line control data (Ctl) and that recorded 10 min after dialysis was used for analysis to reflect the effects of MnTBAP. To correct for potential run-down of the current, the IHERG recorded with MnTBAP was normalized to that recorded with the normal internal solution. *, p < 0.05 versus control, paired t tests.

To obtain further evidence for this notion, we assess the effects of exogenously produced O<UP><SUB>2</SUB><SUP>−</SUP></UP> by the ROS-generating system xanthine/xanthine oxidase (X/XO) on IHERG. Cells were incubated with or without X/XO (500 µM/5 milliunits/ml) in Tyrode's solution containing 5 mM [Glu]o for 40 min before IHERG was recorded under 5 mM [Glu]o. The IHERG density was consistently smaller in X/XO-treated cells than in X/XO non-treated cells (Fig. 8, A and B).


View larger version (29K):
[in this window]
[in a new window]
 
Fig. 8.   Effects of the oxidant generating system X/XO on IHERG under normoglycemia (5 mM [Glu]o). A, analogue data of IHERG with and without treatment with X/XO. The cells were superfused with X/XO (500 µM/5 milliunits/ml) for >30 min before patch clamp recordings. B and C, mean current density (pA/pF)-voltage relationships and activation conductance (G) curves, respectively. Group comparison was made between the untreated control cells (Ctl) and the X/XO-treated cells. *, p < 0.05 versus control, unpaired t tests, n = 8, for control and n = 7 for X/XO.

To confirm that ROS production was indeed increased by 20 mM [Glu]o and the hyperglycemia-induced ROS was mainly of O<UP><SUB>2</SUB><SUP>−</SUP></UP>, we proceeded to measure the intracellular ROS levels using CM-H2DCFDA fluorescence dye. The ROS level was measured in cells preincubated with the Tyrode's solution containing 5 or 20 mM glucose for 30 min. The staining of the cells demonstrated two distinct patterns as follows: one localized to the defined rod-shaped structures, and the other one diffused evenly throughout the cytoplasm. The former presumably represents the physiological production of ROS as a by-product of oxidative phosphorylation in mitochondria, and the latter indicates overproduction of ROS as a result of metabolic stress and damage to mitochondria. The cells with diffused staining and with fluorescence intensity >= 5 times the background were defined as positive staining, and the number of cells with positive staining was pooled from 5 fields. The intensity of staining by the fluorescent probe for ROS was analyzed by densitometric scanning using the LSM program, and cells with either localized or diffused staining were taken for analysis, and the data were normalized to the control (5 mM [Glu]o) values. Under normoglycemia, a majority of cells that was stained by CM-H2DCFDA demonstrated the localized pattern, and the diffused staining was sparse. Yet in the cells treated with 20 mM Glu, the number of the cells with positive staining as well as the intensity of staining was consistently higher, as compared with the cells treated with 5 mM Glu (Fig. 9). This high level of ROS production was markedly suppressed in the cells pretreated with NaCN (2 mM, Fig. 9), an uncoupler of oxidative phosphorylation, indicating that the mitochondrion is most likely where the ROS was massively produced. Because we have demonstrated that pyruvate at high concentrations decreased IHERG (Fig. 3A), the ROS level in the cells pretreated with 20 mM pyruvate was also measured. As shown in Fig. 9, like 20 mM Glu, 20 mM pyruvate also significantly increased the ROS production although to a less extent. Moreover, the glycolysis inhibitor 2dG (5 mM) also reduced the ROS level in high glucose (20 mM), which is indicated by fewer positively stained cells and lower intensity of staining (Fig. 9). The results explain why 2dG partly restored the depressed IHERG in 20 mM Glu (Fig. 3E).


View larger version (89K):
[in this window]
[in a new window]
 
Fig. 9.   Oxidative phosphorylation and intracellular levels of ROS measured by CM-H2DCFDA fluorescence dye. A, laser scanning confocal microscopic images of CM-H2DCFDA staining reflecting the intracellular ROS levels. The numbers in the labels indicate the concentrations in mM. Note the focused staining on the rod-shaped structures in the cells under normoglycemia and the diffused staining throughout the cytoplasm in the cells treated with high glucose or pyruvate. B, percentage of positively stained cells (mean ± S.E.), obtained from 5 fields of 3 experiments by counting the cells with staining intensity >= 5 times the background. C, averaged intensity of CM-H2DCFDA fluorescence measured from the positively stained cells. Shown are the data normalized to 5 mM [Glu]o. *, p < 0.05 versus Glu 5 mM and ×, p < 0.05 versus Glu 20 mM, unpaired t tests.

We have shown that VitE prevented, and MnTBAP partly reversed, the IHERG depression in hyperglycemia (Fig. 7). To see whether this is indeed attributable to their antioxidant actions, effects of VitE and MnTBAP on hyperglycemia-induced ROS production were also studied. As shown in Fig. 10, A and B, the ROS level was significantly lower, as indicated by the smaller number of cells with positive staining and the weaker intensity of staining in individual cells, in the cells pretreated with VitE than in non-treated cells, at 20 mM [Glu]o. Consistently, MnTBAP abolished the hyperglycemia-induced ROS generation; the number of stained cells and the intensity of staining in the presence of MnTBAP were nearly the same as those under normoglycemia.


View larger version (57K):
[in this window]
[in a new window]
 
Fig. 10.   Effects of antioxidants vitamin E (100 µM) and SOD mimetic (MnTBAP, 10 µM) on intracellular ROS levels under hyperglycemia ([Glu]o = 20 mM). A, confocal microscopic images of CM-H2DCFDA staining showing decreases in the intracellular ROS levels in the cells treated with VitE or MnTBAP. B, percentage of the positively stained cells (mean ± S.E.), obtained from 5 fields of 3 experiments by counting the cells with staining intensity >= 5 times the background. C, averaged intensity of CM-H2DCFDA fluorescence measured from the positively stained cells. Shown are the data normalized to the control (Ctl) cells without antioxidant treatment. ×, p < 0.05 versus Glu 20 mM, unpaired t tests.

It has been well documented that the X/XO ROS-generating system stimulates mainly the generation of O<UP><SUB>2</SUB><SUP>−</SUP></UP>. To test whether this is also true in our conditions, the ROS that was exogenously generated by X/XO and penetrated cells was measured, and the effect of MnTBAP was studied at 5 mM [Glu]o. As displayed in Fig. 11, the ROS level was significantly higher in the cells treated with X/XO alone, and this increase in ROS level was prevented in the cells pretreated with MnTBAP.


View larger version (58K):
[in this window]
[in a new window]
 
Fig. 11.   Effects of the superoxide generating system X/XO on the intracellular ROS levels under normoglycemia ([Glu]o = 5 mM). A, confocal microscopic images of CM-H2DCFDA staining showing the increases in the intracellular ROS levels by X/XO (0.5 mM/5 milliunits/ml) and the abrogation of ROS increase by the SOD mimetic MnTBAP (10 µM). B, percentage of the positively stained cells (mean ± S.E.), obtained from 5 fields of 3 experiments by counting the cells with intensity of staining >= 5 times the background. C, averaged intensity of staining measured from the positively stained cells. Shown are the data normalized to the control (Ctl) cells without X/XO treatment. *, p < 0.05 versus Glu 5 mM and × p < 0.05 versus Glu 5 mM + X/XO, unpaired t tests.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The work described here documents a previously unreported role of glucose in regulating the function of HERG K+ channels. Our data revealed that glucose produces two characteristic effects on the HERG channel function: changes of HERG current (IHERG) amplitude/density and activation voltage. The maximum HERG function operates under physiological [Glu]o (5 mM, normoglycemia), and depressed HERG function occurs with [Glu]o <5 mM (hypoglycemia) or with [Glu]o >5 mM (hyperglycemia); hypoglycemia but not hyperglycemia causes shifts of HERG activation toward hyperpolarizing voltages. The modulation of HERG by glucose is critically mediated by glucose metabolism (glycolysis and oxidative phosphorylation), and ATP and ROS are crucial in defining the HERG function with changing extracellular glucose levels.

Depression of HERG Function in Hypoglycemia Likely Results from Underproduction of ATP-- In our study, lower glucose levels and inhibition of glucose metabolism both produced similar suppression of HERG function as reflected by the substantial diminishment of HERG current (IHERG), pointing to a requirement of glucose metabolism for HERG modulation by glucose. Our data allowed us to reach the following conclusions.

Either Glycolysis- or Oxidative Phosphorylation-derived ATP Is Sufficient for Maintaining the Normal HERG Function-- The end point of glucose metabolism is the generation of high energy phosphates for maintaining cellular functions, and depletion of ATP could impair cellular processes dependent on high energy phosphates. One of the major findings of this study is that the normal HERG function critically relies on the level of intracellular ATP; depletion of intracellular ATP impairs HERG function to an extent similar to what severe hypoglycemia (0 mM [Glu]o) does (see Figs. 1, 2, and 5). Complete inhibition of glucose metabolism by 2dG substitution for glucose reproduces the effects of hypoglycemia or ATP depletion on IHERG. Yet, neither inhibition of glycolysis alone by 2dG substitution of Glu with a supply of pyruvate to sustain oxidative phosphorylation nor inhibition of oxidative phosphorylation alone by NaCN in the presence of 5 mM Glu to maintain glycolysis is able to cause depression of HERG function. The results imply that the ATP generated by glucose metabolism plays an important role in maintaining HERG function, and either the glycolytic or oxidative ATP is adequate for the regulation.

ATP synthesis and utilization are subcellularly compartmentalized; glycolysis-derived ATP primarily regulates membrane proteins because the glycolytic pathway is associated with sarcolemma (31), whereas oxidative phosphorylation-derived ATP preferentially supports cytosolic processes because oxidative ATP is generated within the mitochondria and subsequently transported to the cytoplasm (32). Regulation of cardiac ATP-sensitive K+ channel (KATP) (34) and L-type Ca2+ channel (ICa) (33) by intracellular ATP has been well documented by some previous studies. It was found that both KATP and ICa were preferentially regulated by glycolytic ATP (33, 34).

Glycolysis-derived ATP May Be Responsible for Maintaining the Normal Voltage-dependent Activation of IHERG-- Although as mentioned above, either glycolytic or oxidative ATP is sufficient for maintaining the normal HERG current amplitude/density, only glycolysis-derived ATP seems to affect the steady-state voltage-dependent activation property of HERG channels. This notion is supported by several lines of evidence from our experiments. 1) Hypoglycemia (0 or 1 mM [Glu]o), a situation with inadequate ATP production but not hyperglycemia (10 or 20 mM [Glu]o), causes negative shifts of I-V relationships and voltage-dependent activation curves. 2) Inhibition of glycolysis (Fig. 3), but not oxidative phosphorylation (Fig. 4), abolishes the negative shifts of the HERG activation. 3) When glycolysis is inhibited by 2dG, preservation of oxidative phosphorylation by addition of pyruvate fails to prevent the negative shift caused by hypoglycemia (see Fig. 3). 4) Depletion of intracellular ATP reproduces negative shifts similar to those seen with hypoglycemia. Similar dependence of glycolytic ATP regulation of KATP and ICa has been documented (33-34).

Role of ATP in Maintaining HERG Function Is Most Likely Due to the Phosphorylation-dependent Mechanisms-- Two alternative mechanisms could account for intracellular ATP regulation of ion channels: ATP acts as a substrate for phosphorylation of channel proteins by protein kinase which requires ATP hydrolysis, and ATP interacts with the nucleotide binding domains of channel proteins to produce allosteric regulation not requiring ATP hydrolysis. The latter mechanism has been shown to operate for KATP and ICa regulation (33, 35). In our case, neither the non-hydrolysable analogue of ATP AMP-PCP nor GTP prevented the IHERG run-down caused by ATP depletion (Fig. 6); instead substitution of AMP-PCP or GTP for ATP in the internal solution produced nearly identical effects as seen with ATP depletion alone. The results suggest that the HERG regulation by ATP under our experimental conditions is phosphorylation-dependent requiring ATP hydrolysis. In other words, ATP serves as a substrate for phosphorylation of HERG channels by protein kinases. Indeed, we have recently found that the normal HERG function requires basal activity of protein kinase B and inhibition of protein kinase B markedly suppresses IHERG and shifts HERG activation along the voltage axis toward more negative potentials (36). These results are in good agreement with the HERG regulation by ATP. Studies are currently undertaken to clarify the link between ATP and protein kinase B modulation of HERG channels.

Depression of HERG Function in Hyperglycemia Results from Overproduction of ROS-- The consequence of the physiological role in oxidative phosphorylation is the generation of ROS as by-products of the consumption of molecular oxygen in the electron transport chain (23). Physiologically, these ROS are mostly trapped within mitochondria and rapidly scavenged by endogenous antioxidants like SOD, catalase, glutathione, etc. Yet under metabolic stress, ROS can be overproduced and can cause damages to mitochondria. Consequently, the ROS may diffuse throughout the cytoplasm and cause further deleterious effects on other cellular processes. Abnormally high concentrations of glucose can enhance ROS damage at least in three different ways. First, it has been known that high glucose (25 mM) evoked ROS generation, which was blocked by antioxidants, inhibitors of mitochondrial electron transport chain complex, inhibitors of glycolysis-derived pyruvate transport into mitochondria, uncouplers of oxidative phosphorylation, SOD mimetics, catalase, etc. (19). Superoxide anion (O<UP><SUB>2</SUB><SUP>−</SUP></UP>) is found to be the major ROS produced under hyperglycemia (37-42), and increases in ROS can be prevented by SOD. Second, glucose itself can auto-oxidize to form ROS including O<UP><SUB>2</SUB><SUP>−</SUP></UP>, OH-, and H2O2 (43). Finally, acute elevations in glucose also depress natural antioxidant defenses. It has been found that incubation of purified bovine CuZn-SOD with 10 to 100 mM glucose reduces the enzyme activity by 60% (44).

Elevated glucose or pyruvate level is expected to enhance oxidative phosphorylation and produce more ATP molecules to support HERG function or increase IHERG. However, our observations are contrary to this expectation. Our results showed that hyperglycemia or excessive pyruvate markedly depressed the HERG function. A reasonable explanation for this is that the ROS produced under hyperglycemia counteract the effects of ATP, and the net outcome is a balance between enhancing effects of ATP and suppressing effects of ROS. Evidently, under our experimental conditions, the effects of increased ROS overwrite the effects of increased ATP, resulting in suppression of IHERG. This notion is supported by the following evidence. First, the depression of IHERG induced by hyperglycemia was prevented or reversed by the antioxidants vitamin E and MnTBAP (SOD mimetic). Second, inhibition of the glycolysis and thereby the subsequent oxidative phosphorylation by 2dG partially reversed the depressed IHERG under hyperglycemia (Fig. 3). Weakened oxidative phosphorylation due to inhibition of the glycolysis would reduce both ATP and ROS productions, but the net result was an increase in IHERG, indicating again that in our experimental conditions ROS overweighs ATP in terms of their effects on IHERG. This is in agreement with the notion that the suppressing effects of ROS overproduction overwhelm the effects of ATP increase. Moreover, 2dG also can compete with glucose for access to glucose transporters and thus decreases glucose uptake which in turn can result in reduction of ROS production in the cells. Finally, our data indeed demonstrated the ability of high glucose to stimulate an overproduction of ROS (see Fig. 9). The fact that a high concentration of pyruvate mimicked, whereas VitE or NaCN abrogated, the ROS overproduction suggests that the ROS were mainly produced via the oxidative phosphorylation in mitochondria in our cells.

It has been reported that the ROS, which generate highly reactive hydroxyl group (OH-), such as H2O2 or FeSO4/ascorbic acid (an oxidative stimulus analogous to H2O2), increased IHERG at negative potentials by shifting the HERG activation to more negative voltages (45-46). These results are opposite to our observations. One explanation is that the ROS generated under our experimental conditions may be different from the OH--generating system (H2O2 or FeSO4/ascorbic acid). As already mentioned, previous studies have confirmed that the ROS induced by hyperglycemia is mainly of O<UP><SUB>2</SUB><SUP>−</SUP></UP>. Here, we also showed that the SOD mimetic MnTBAP reduced the hyperglycemia-induced ROS overproduction (Fig. 10), and the O<UP><SUB>2</SUB><SUP>−</SUP></UP>-generating system X/XO produced ROS which were also abolished by MnTBAP, evidence for O<UP><SUB>2</SUB><SUP>−</SUP></UP> as a major ROS generated in our cells. Consistently, depressive effects of hyperglycemia or X/XO on IHERG were significantly weakened by MnTBAP. Indeed, it has been reported that the O<UP><SUB>2</SUB><SUP>−</SUP></UP> generated by high glucose (23 mM) or by X/XO in rat small coronary arteries impairs voltage-gated K+ (Kv) current (39, 47); reducing the current density by around 60%, which was partially restored by SOD and catalase. All together, we believe that different ROS might have different effects on IHERG; OH- enhances, whereas O<UP><SUB>2</SUB><SUP>−</SUP></UP> depresses, IHERG.

Moreover, it has been shown that excessive ROS inhibits glycolysis and the subsequent glycolytic ATP production and even depletes intracellular ATP levels in isolated perfused hearts (48-50). This fact together with our data suggests that besides the potential direct modulation of IHERG by ROS, ATP reduction potentially caused by ROS may also contribute to the IHERG depression under hyperglycemia. This provides an alternative explanation for the depressive effects of the ROS overproduction overcoming the enhancing effects of the expected ATP increase.

In our study, the effects of pyruvate and X/XO were smaller than those of hyperglycemia. This may be because the O<UP><SUB>2</SUB><SUP>−</SUP></UP> generated by hyperglycemia occurs inside the cell but pyruvate does not readily penetrate cells, and the effect of pyruvate observed in the present study may underestimate the true role of oxidative phosphorylation in IHERG modulation. Likewise, the O<UP><SUB>2</SUB><SUP>−</SUP></UP> generated by X/XO was primarily extracellular with subsequent entry into the cell which could also underestimate the effects of O<UP><SUB>2</SUB><SUP>−</SUP></UP>.

It was shown that within minutes of exposure to dihydroxyfumaric acid or xanthine plus xanthine oxidase, both of which produce the superoxide anion, action potential duration was prolonged in canine myocytes, and this effect was followed by the appearance of early after depolarization (51). X/XO caused a 30% increase in action potential duration in superfused papillary muscle or small strips of right ventricular walls of guinea pig hearts (52). However, whether the action potential duration prolongation was associated with inhibition of delayed rectifier K+ current (IKr) is unknown. Our study provides a potential explanation for these observations.

Impairment of HERG Function Might Contribute to Q-T Prolongation Caused by Hypoglycemia and Hyperglycemia-- Heart disease is a leading cause of death in diabetic patients. In patients with diabetes a prolongation of the Q-T interval has been associated with an increased risk of sudden cardiac death (2) due to the occurrence of lethal ventricular arrhythmias, particularly Torsade de pointes following bradycardia (1). Several cardiovascular pathological consequences of diabetes such as hypertension and arteriosclerosis affect the heart to varying degrees. Hyperglycemia, as a consequence of diabetes and an independent risk factor, also can directly cause cardiac damage. On the other hand, insulin therapy increases the risk of hypoglycemia in type 2 diabetic patients; according to the United Kingdom Prospective Diabetes Study, approximately one-third of the insulin-treated patients reported one or more hypoglycemic episodes per year during the first 3 years (3). Hypoglycemia is presumed to be the cause of death in about 3% of insulin-treated diabetic patients (11). Intriguingly, it is well recognized that both hyperglycemia and hypoglycemia can cause prolongation of Q-T interval. In type 2 diabetes, the prevalence of Q-T prolongation is as high as 26%, and Q-T prolongation during experimentally induced and spontaneously occurring hypoglycemia or diabetic hyperglycemia has also been shown to occur in healthy subjects and in diabetic patients with increased risk of malignant ventricular arrhythmias (4-11). Yet the potential ionic mechanisms by which hypoglycemia and hyperglycemia cause Q-T prolongation remained poorly understood. Studies on glucose modulation of cardiac ion channels are sparse and have been mostly limited to ATP-sensitive K+ current (KATP). On the contrary to IHERG, KATP is closed with increased intracellular ATP levels (34). One study reported by Xu et al. (53) demonstrated that hyperglycemia (18 mM [Glu]o) decreased the density of transient outward K+ current but did not alter the inward rectifier K+ current in rat ventricular myocytes that do not express delayed rectifier K+ current (IKr), the physiological counterpart of IHERG. In arterial smooth muscles, high glucose diminished shaker-type delayed rectifier K+ current (39). In rat myelinated nerve fibers, 30 mM glucose increased Ca2+-activated K+ current (30). Whereas none of the data from these studies could fully account for the Q-T prolongation, particularly the Q-T prolongation induced by hypoglycemia, our study provides a plausible, or at least an alternative, explanation; HERG K+ channel may be a mechanistic link for the Q-T prolongation induced by both hyperglycemia and hypoglycemia. Yet one should keep it in mind that HERG may be only one of the multiple factors contributing to the Q-T prolongation in hypoglycemia and hyperglycemia.

    ACKNOWLEDGEMENTS

We thank XiaoFan Yang for excellent technical support and Louis R. Villeneuve for assistance with the confocal microscopic examinations.

    FOOTNOTES

* This work was supported in part by the Canadian Institute of Health Research, the Heart and Stroke Foundation of Quebec, and the Fonds de la Recherche de l'Institut de Cardiologie de Montreal (to Z. W.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Research fellow of the Canadian Institute of Health Research.

** Research scholar of the Fonds de Recherche en Sante de Quebec. To whom correspondence should be addressed: Research Center, Montreal Heart Institute, 5000 Belanger East, Montreal, Quebec H1T 1C8, Canada. Tel.: 514-376-3330; Fax: 514-376-4452; E-mail: wangz@icm.umontreal.ca.

Published, JBC Papers in Press, January 16, 2003, DOI 10.1074/jbc.M211044200

    ABBREVIATIONS

The abbreviations used are: HERG, human ether-à-go-go related gene; ROS, reactive oxygen species; X, xanthine; XO, xanthine oxidase; 2dG, deoxy-D-glucose; MnTBAP, Mn(III) tetrakis(4-benzoic acid) porphyrin chloride; AMP-PCP, beta ,gamma -methyleneadenosine 5'-triphosphate; VitE, vitamin E; SOD, superoxide dismutase; CM-H2DCFDA, 5-(and-6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

1. Abo, K., Ishida, Y., Yoshida, R., Hozumi, T., Ueno, H., Shiotani, H., Matsunaga, K., and Kazumi, T. (1996) Diabetes Care 19, 1010[Medline] [Order article via Infotrieve]
2. Kahn, J. K., Sisson, J. C., and Vinik, A. I. (1987) J. Clin. Endocrinol. Metab. 64, 751-754[Abstract]
3. United Kingdom Prospective Diabetes Study. (1995) Br. Med. J. 310, 83-88[Abstract/Free Full Text]
4. Marfella, R., Rossi, F., and Giugliano, D. (2001) Diabetes Nutr. Metab. 14, 63-65[Medline] [Order article via Infotrieve]
5. Chelliah, Y. R. (2000) Anaesth. Intensive Care 28, 698-700[Medline] [Order article via Infotrieve]
6. Veglio, M., Borra, M., Stevens, L. K., Fuller, J. H., and Perin, P. C. (1999) Diabetologia 42, 68-75[CrossRef][Medline] [Order article via Infotrieve]
7. Marques, J. L., George, E., Peacey, S. R., Harris, N. D., Macdonald, I. A., Cochrane, T., and Heller, S. R. (1997) Diabet. Med. 14, 648-654[CrossRef][Medline] [Order article via Infotrieve]
8. Landstedt-Hallin, L., Englund, A., Adamson, U., and Lins, P.-E. (1999) J. Intern. Med. 246, 299-307[CrossRef][Medline] [Order article via Infotrieve]
9. Lindstrom, T., Jorfeldt, L., Tegler, L., and Arnqvist, H. J. (1992) Diabet. Med. 9, 536-541[Medline] [Order article via Infotrieve]
10. Shimada, R., Nakashima, T., Nunoi, K., Kohno, Y., Takeshita, A., and Omae, T. (1984) Arch. Intern. Med. 144, 1068-1069[Abstract]
11. Eckert, B., and Agardh, C.-D. (1998) Clin. Physiol. (Oxf.) 18, 570-575
12. Sanguinetti, M. C. (1999) Ann. N. Y. Acad. Sci. 868, 406-413[Abstract/Free Full Text]
13. Taglialatela, M., Castaldo, P., and Pannaccione, A. (1998) Biochem. Pharmacol. 55, 1741-1746[CrossRef][Medline] [Order article via Infotrieve]
14. Zhou, Z., Gong, Q., Ye, B., Fan, Z., Makielski, J. C., Robertson, G. A., and January, C. T. (1998) Biophys. J. 74, 230-241[Abstract/Free Full Text]
15. Wang, J., Wang, H., Han, H., Zhang, Y., Yang, B., Nattel, S., and Wang, Z. (2001) Circulation 104, 2645-2648[Abstract/Free Full Text]
16. Wang, H., Yang, B., Zhang, Y., Han, H., Wang, J., Shi, H., and Wang, Z. (2001) J. Biol. Chem. 276, 40811-40816[Abstract/Free Full Text]
17. Splawski, I., Shen, J., Katherine, W., Timothy, G., Vincent, M., Lehmann, M. H., and Keating, M. T. (1998) Genomics 51, 86-97[CrossRef][Medline] [Order article via Infotrieve]
18. Ludwig, B., Bender, E., Arnold, S., Huttemann, M., Lee, I., and Kadenbach, B. (2001) Chembiochem. 2, 392-403[CrossRef][Medline] [Order article via Infotrieve]
19. Hsieh, T. J., Zhang, S. L., Filep, J. G., Tang, S. S., Ingelfinger, J. R., and Chan, J. S. (2002) Endocrinology 143, 2975-2985[Abstract/Free Full Text]
20. Wallace, D. C. (2002) Methods Mol. Biol. 197, 3-54[Medline] [Order article via Infotrieve]
21. Siraki, A. G., Pourahmad, J., Chan, T. S., Khan, S., and O'Brien, P. J. (2002) Free Radic. Biol. Med. 32, 2-10[CrossRef][Medline] [Order article via Infotrieve]
22. Kirkinezos, I. G., and Moraes, C. T. (2001) Semin. Cell Dev. Biol. 12, 449-457[CrossRef][Medline] [Order article via Infotrieve]
23. Wallace, D. C. (2001) Novartis. Found. Symp. 235, 247-263[Medline] [Order article via Infotrieve]
24. Koo, J. R., Ni, Z., Oviesi, F., and Vaziri, N. D. (2002) Clin. Exp. Hypertens. 24, 333-344[CrossRef][Medline] [Order article via Infotrieve]
25. Marfella, R., Quagliaro, L., Nappo, F., Ceriello, A., and Giugliano, D. (2001) J. Clin. Invest. 108, 635-636[Free Full Text]
26. Srivastava, A. K. (2002) Int. J. Mol. Med. 9, 85-89[Medline] [Order article via Infotrieve]
27. Li, P. A., Liu, G. J., He, Q. P., Floyd, R. A., and Siesjo, B. K. (1999) Free Radic. Biol. Med. 27, 1033-1040[CrossRef][Medline] [Order article via Infotrieve]
28. van Dam, P. S., van Asbeck, B. S., Bravenboer, B., van Oirschot, J. F., Gispen, W. H., and Marx, J. J. (1998) Free Radic. Biol. Med. 24, 18-26[CrossRef][Medline] [Order article via Infotrieve]
29. Giardino, I., Edelstein, D., and Brownlee, M. (1996) J. Clin. Invest. 97, 1422-1428[Abstract/Free Full Text]
30. Takigawa, T., Yasuda, H., Terada, M., Maeda, K., Haneda, M., Kashiwagi, A., Kitasato, H., and Kikkawa, R. (2000) Neuroreport 11, 2547-2551[Medline] [Order article via Infotrieve]
31. Hazen, S. L., Wolf, M. J., Ford, D. A., and Gross, R. W. (1994) FEBS Lett. 339, 213-216[CrossRef][Medline] [Order article via Infotrieve]
32. Weiss, J. N., and Hiltbrand, B. (1985) J. Clin. Invest. 75, 436-447[Medline] [Order article via Infotrieve]
33. Losito, V. A., Tsushima, R. G., Diaz, R. J., Wilson, G. J., and Backx, P. H. (1998) J. Physiol. (Lond.) 511, 67-78[Abstract/Free Full Text]
34. Weiss, J. N., and Lamo, S. T. (1989) J. Gen. Physiol. 94, 911-935[Abstract]
35. Yazawa, K., Kameyama, A., Yasui, K., Li, J. M., and Kameyama, M. (1997) Pfluegers Arch. 433, 557-562[CrossRef][Medline] [Order article via Infotrieve]
36. Zhang, Y., Wang, H., Wang, J., Han, H., Nattel, S., and Wang, Z. (2003) FEBS Lett. 534, 125-132[CrossRef][Medline] [Order article via Infotrieve]
37. Nishikawa, T., Edelstein, D., Du, X. L., Yamagishi, S., Matsumura, T., Kaneda, Y., Yorek, M. A., Beebe, D., Oates, P. J., Hammes, H. P., Giardino, I., and Brownlee, M. (2000) Nature 404, 787-790[CrossRef][Medline] [Order article via Infotrieve]
38. Du, X. L., Edelstein, D., Rossetti, L., Fantus, I. G., Goldberg, H., Ziyadeh, F., Wu, J., and Brownlee, M. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 12222-12226[Abstract/Free Full Text]
39. Liu, Y., Terata, K., Rusch, N. J., and Gutterman, D. D. (2001) Circ. Res. 89, 146-152[Abstract/Free Full Text]
40. Graier, W. F., Posch, K., Wascher, T. C., and Kostner, G. M. (1997) Horm. Metab. Res. 29, 622-629[Medline] [Order article via Infotrieve]
41. Graier, W. F., Posch, K., Fleischhacker, E., Wascher, T. C., and Kostner, G. M. (1999) Diabetes Res. Clin. Pract. 45, 153-160[CrossRef][Medline] [Order article via Infotrieve]
42. Esposito, K., Marfella, R., and Giugliano, D. (2002) J. Endocrinol. Invest. 25, 485-488[Medline] [Order article via Infotrieve]
43. Wolff, S. P., and Dean, R. T. (1987) Biochem. J. 245, 243-250[Medline] [Order article via Infotrieve]
44. Oda, A., Bannai, C., Yamaoka, T., Katori, T., Matsushima, T., and Yamashita, K. (1994) Horm. Metab. Res. 26, 1-4[Medline] [Order article via Infotrieve]
45. Bérubé, J., Caouette, D., and Daleau, P. (2001) J. Pharmacol. Exp. Ther. 297, 96-102[Abstract/Free Full Text]
46. Taglialatela, M., Castaldo, P., Iossa, S., Pannaccione, A., Fresi, A., Ficker, E., and Annunziato, L. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 11698-11703[Abstract/Free Full Text]
47. Liu, Y., and Gutterman, D. D. (2002) Clin. Exp. Pharmacol. Physiol. 29, 305-311[CrossRef][Medline] [Order article via Infotrieve]
48. Ytrehus, K., Myklebust, R., and Mjøs, O. D. (1986) Cardiovasc. Res. 20, 597-603[Medline] [Order article via Infotrieve]
49. Miki, S., Ashraf, M., Salka, S., and Sperelakis, N. (1988) J. Mol. Cell. Cardiol. 20, 1009-1024[Medline] [Order article via Infotrieve]
50. Patane, G., Anello, M., Piro, S., Vigneri, R., Purrello, F., and Rabuazzo, A. M. (2002) Diabetes 51, 2749-2756[Abstract/Free Full Text]
51. Barrington, P. L., Meier, C. F., Jr., and Weglicki, W. B. (1988) J. Mol. Cell. Cardiol. 20, 1163-1178[Medline] [Order article via Infotrieve]
52. Aiello, E. A., Jabr, R. I., and Cole, W. C. (1995) Circ. Res. 77, 153-162[Abstract/Free Full Text]
53. Xu, Z., Patel, K. P., and Rozanski, G. J. (1998) Am. J. Physiol. 271, H2190-H2196


Copyright © 2003 by The American Society for Biochemistry and Molecular Biology, Inc.