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Am J Physiol Heart Circ Physiol 293: H3643-H3649, 2007. First published October 5, 2007; doi:10.1152/ajpheart.01357.2006
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Impaired activation of ATP-sensitive K+ channels in endocardial myocytes from left ventricular hypertrophy

Junichi Shimokawa, Hisashi Yokoshiki, and Hiroyuki Tsutsui

Department of Cardiovascular Medicine, Hokkaido University Graduate School of Medicine, Sapporo, Japan

Submitted 13 December 2006 ; accepted in final form 29 September 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
ATP-sensitive K+ (KATP) channels are essential for maintaining the cellular homeostasis against metabolic stress. Myocardial remodeling in various pathologies may alter this adaptive response to such stress. It was reported that transmural electrophysiological heterogeneity exists in ventricular myocardium. Therefore, we hypothesized that the KATP channel properties might be altered in hypertrophied myocytes from endocardium. To test this hypothesis, we determined the KATP channel currents using the perforated patch-clamp technique, open cell-attached patches, and excised inside-out patches in both endocardial and epicardial myocytes isolated from hypertrophied [spontaneous hypertensive rats (SHR)] vs. normal [Wistar-Kyoto rats (WKY)] left ventricle. In endocardial cells, KATP channel currents (IK,ATP), produced by 2 mM CN– and no glucose at 0 mV, were significantly smaller (P < 0.01), and time required to reach peak currents after onset of KATP channel opening (Timeonset to peak) was significantly longer (319 ± 46 vs. 177 ± 37 s, P = 0.01) in the SHR group (n = 9) than the WKY group (n = 13). However, in epicardial cells, there were no differences in IK,ATP and Timeonset to peak between the groups (SHR, n = 12; WKY, n = 12). The concentration-open probability-response curves obtained during the exposure of open cells and excised patches to exogenous ATP revealed the impaired KATP channel activation in endocardial myocytes from SHR. In conclusion, KATP channel activation under metabolic stress was impaired in endocardial cells from rat hypertrophied left ventricle. The deficit of endocardial KATP channels to decreased intracellular ATP might contribute to the maladaptive response of hypertrophied hearts to ischemia.

adenosine 5' -trisphosphate-sensitive potassium ion channel; ischemia; transmural heterogeneity; patch-clamp technique

LEFT VENTRICULAR HYPERTROPHY is a major risk factor for sudden cardiac death. The incidence of sudden death after myocardial infarction is significantly higher in patients with hypertension and left ventricular hypertrophy (6, 12, 26). This increased risk may result, in part, from hypertrophy-induced destabilization of cardiac electrical activity, especially the prolongation of the action potential, the dispersion of refractoriness, and triggered activity (22, 32). On the other hand, the extent of shortening of action potentials caused by simulated ischemia was greater in hypertrophied myocytes (10, 22). Therefore, the electrophysiological responsiveness to metabolic stress has been shown to be altered in hypertrophied cardiac myocytes (4, 43, 47).

Cardiac ATP-sensitive K+ (KATP) channels, first described by Kakei et al. (17) and Noma (30), have been subjected to extensive scrutiny because they are activated and pass current when the intracellular concentration of ATP decreases. Thus the KATP channel provides a linkage of metabolism in cardiac cells to membrane electrical activity and vice versa, especially during the depletion of energy stores, which may occur during myocardial ischemia (4, 31 39). Based on these properties of KATP channel, it is speculated that this channel may play a significant role in the electrophysiological response to myocardial ischemia (28, 33).

On the other hand, a number of studies demonstrated the presence of electrophysiological heterogeneity across the left ventricular wall. The electrophysiological properties of epicardial myocytes are reported to be more susceptible to ischemia than endocardial myocytes (8, 13, 20). Therefore, we hypothesized that the heterogeneity of KATP channel property and their response to ischemia could be altered in hypertrophied myocytes. To test this hypothesis, we determined the activation of KATP channels of myocytes isolated from left ventricular epicardium (EPI) and endocardium (ENDO) from normal and hypertrophied hearts.


   MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
RESULTS
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The animal experiments were approved by the Animal Care and Use Committee at Hokkaido University Graduate School of Medicine. All procedures complied with theGuideline for the Care and Use of Laboratory Animals (NIH Pub. No. 85–23, 1996 revision).

Cell isolation. Hearts were excised from adult male 18- to 22-wk-old Wistar-Kyoto rats (WKY) and spontaneous hypertensive rats (SHR) after anesthesia with inhalation of diethyl ether and peritoneal injection of heparin sodium (400 IU/kg). The heart was mounted on a Langendorff apparatus and was retrogradely perfused with Tyrode solution (37°C) containing (in mM) 143 NaCl, 5.4 KCl, 0.33 NaH2PO4, 5 HEPES, 5.5 glucose, 0.5 MgCl2, and 1.8 CaCl2 (pH 7.4 using NaOH) gassed with 100% O2 until the beating rate became stable (3–5 min). The perfusate was then changed to nominally Ca2+-free Tyrode solution (otherwise identical to above) for 10 min, resulting in the cessation of the heart beating. The quiescent heart was perfused with the nominally Ca2+-free Tyrode solution containing 0.5 mg/ml collagenase (Wako Chemicals, Osaka, Japan) and 0.1 mg/ml protease (Sigma Chemical, St. Louis) for 35–40 min. At the end of the digestion by collagenase, the perfusate was changed to the nominally Ca2+-free Tyrode solution to wash off the collagenase/protease solution. After the atria and the right ventricle were removed, small pieces of the left ventricular tissues were dissected from the EPI and the ENDO surfaces (to a depth not exceeding 30% of the thickness of the ventricular wall) with fine scissors. The tissue pieces in modified "Kraftbrühe" solution containing (in mM) 70 KOH, 50 L-glutamic acid, 40 KCl, 20 taurine, 20 KH2PO4, 10 glucose, 10 HEPES, 1 EGTA, and 3 MgCl2 (pH 7.4 using KOH) were gently stirred and filtered through a stainless-steel mesh. The cell suspension was stored in a refrigerator (4°C). The cells were used for the experiments 2–12 h after isolation.

Electrophysiological recording technique. Whole cell currents and single channel currents were recorded using the patch-clamp method. The electrode was connected to an input of a current-voltage converter with a feedback resistance of 100 M{Omega} for recording whole cell current and 10 G{Omega} for recording single channel current. The isolated cells were placed in a perfusion chamber (1 ml volume) attached to an inverted microscope (model IX70; Olympus) and constantly superfused with Tyrode solution. After the cells settled to the chamber bottom, they were perfused with bath solution by a fine tube located close to cells to ensure fast solution exchange with the use of a pressure-driven perfusion system (models BPS-8 and PR-10; ALA Scientific Instruments). During superfusion with Tyrode solution containing 1.8 mM CaCl2, 30–40% of the cells were Ca2+ tolerant and rod shaped. Single rod-shaped cells having smooth surfaces with clearly demarked striations were selected for the electrical measurements. Pipettes were fabricated from 1.5-mm outer diameter to 0.85-mm inner diameter borosilicate glass capillaries (World Precision Instruments, Sarasota, FL) using a multistage horizontal puller (model P-97; Sutter Instrument, Novato, CA).

Membrane currents were measured with an Axopatch 200 patch-clamp amplifier controlled by a personal computer using a Digidata 1200 acquisition board driven by pCLAMP 6.0/8.0 software (Axon Instruments). All experiments were performed at room temperature (22–25°C).

Whole cell current recordings. The perforated patch-clamp technique was achieved to record whole cell currents using pipettes with tip resistances of 1–2 M{Omega} filled by pipette (intracellular) solution containing (in mM) 100 potassium glutamate (H2O), 40 KCl, 4 NaOH, 5 HEPES, 1 EGTA, 1 MgCl2, and 50–200 µg/ml nystatin, pH 7.2, using KOH (or HCl) when needed. Cell membrane capacitance (Cm) was determined from the amplitude of the current elicited by hyperpolarizing voltage-ramp pulses from a holding potential of 0 to –5 mV (duration 25 ms at 0.2 V/s); this procedure avoided interference by any time-dependent ionic currents.

Membrane currents were recorded every 6 s by applying voltage clamp to 0 mV for 500 ms from a holding potential of –40 mV. After control currents for first 30 s were recorded, the cell was exposed to simulated ischemia for 510 s. Ischemia was simulated by perfusing the surface of the cell with glucose-free Tyrode solution containing 2 mM CN–.

Single-channel recordings. Single channel currents were recorded by the open cell-attached method and the excised inside-out patch method. These methods were similar to procedures previously reported in mouse, rat, and guinea pig myocytes (17, 21, 29, 44, 46). The pipette resistances were ~2–5 M{Omega} when filled with pipette (extracellular) solution containing (in mM) 150 KCl, 0.5 MgCl2, 1.8 CaCl2, and 5 HEPES, pH 7.4 with KOH. The cells were perfused with the bath (intracellular) solution containing (in mM) 140 KCl, 10 KOH, 2 MgCl2, 5 HEPES, and 5 EGTA, pH 7.3 with KOH after forming the cell-attached patch. To obtain open cell-attached patch currents, the cell membrane was then ruptured by briefly exposing one end of the cell to 1% digitonin applied by fine tube. Opening of KATP channels can be observed 60–120 s after applying digitonin. To obtain excised inside-out patch currents, the pipette tip was withdrawn from the cell surface after forming of the cell-attached patch without a decrease in seal resistance. Single channel current signals were filtered at 2 kHz, sampled at 4 kHz, and stored on a personal computer using AxoScope 1 software (Axon Instruments). Channel openings were determined by using the 50% threshold criterion. The number of functional channels in the patch was approximated as the maximum number of overlaps of the openings in the absence of ATP. Open state probability (PO) was estimated using PO = I/(Ni), where I is the mean patch current, N is the number of channels in the patch, and i is the unit amplitude of the single channel current.

Statistical analysis. All data are expressed as means ± SE. Simple between-group analyses were conducted by using a Student's unpaired t-test. The incidence of channel activation in whole cell current recordings was analyzed using the chi square test. Between-group comparisons across the time or the intracellular ATP concentration were made by using a repeated-measures ANOVA. Differences with P < 0.05 were considered significant.


   RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
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Cell Cm. Mean Cm was calculated to be 146 ± 13.7 pF in epicardial myocytes from WKY (n = 15); 164 ± 14.9 pF in endocardial myocytes from WKY (n = 15); 278 ± 34.7 pF in epicardial myocytes from SHR (n = 13); and 237 ± 34.5 pF in endocardial myocytes from SHR (n = 16). Cm in SHR was greater than WKY in both groups (P = 0.0005 in EPI group, P = 0.034 in ENDO group).

Activation of whole cell IK,ATP currents by metabolic inhibition. Figure 1 shows the examples of whole cell membrane current characteristics on the voltage step from holding potential of –40 to 0 mV before and after metabolic inhibition by exposure to 2 mM CN– and no glucose in endocardial myocytes from the left ventricle of both WKY (Fig. 1, left) and SHR (Fig. 1, right). After exchange of bath solution with 2 mM CN– and no glucose, the outward whole cell currents at 0 mV increased in almost all of the experimented cells. The electrophysiological changes during such metabolic inhibitions are thought to be mediated through KATP channel openings (36, 45, 46). Thus we defined the difference between the currents at the end of test pulse to 0 mV before and after metabolic inhibition as KATP channel current (IK,ATP). In some of the experimented cells, the increase of outward current was not observed. Therefore, we evaluated the incidence of KATP channel activation after exposure to 2 mM CN– and no glucose. We considered the KATP channel of the experimented cell to be activated when the outward current at 0 mV pulse reached 1 pA/pF within 540 s from the beginning of the experiment (within 510 s after exposure to metabolic inhibition), and the time point was defined to be the onset of KATP channel activation. Table 1 shows the number of cells in which KATP channels were activated in each group. In ENDO myocytes, the KATP channels tended to be less activated in SHR than in WKY [87% in WKY (n = 13/15) vs. 57% in SHR (n = 9/16), P = 0.06]. However, it did not reach statistical significance. In contrast, the incidence of KATP channel openings in EPI myocytes was nearly identical between the groups [80% in WKY (n = 12/15) vs. 90% in SHR (n = 12/13), P = 0.4].


Figure 1
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  		Fig. 1. Effect of metabolic inhibition on whole cell membrane currents in endocardial myocytes from both Wistar-Kyoto rats (WKY, left) and spontaneous hypertensive rats (SHR, right); representative whole cell membrane currents before and after exposure to metabolic inhibition in isolated myocytes recorded in the perforated patch configuration. Membrane was voltage clamped to 0 mV for 500 ms from a holding potential of –40 mV. In control, outward currents were <100 pA in almost all of the cells examined ({circ}). Exposure to metabolic inhibition produced by 2 mM CN– in the absence of glucose increased outward currents ({triangleup}). Horizontal lines indicate zero current level.

 

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  		Table 1. Incidence of KATP channel openings in whole cell experiments

 
Figure 2 shows the time course of IK,ATP activation induced by metabolic inhibition in epicardial and endocardial myocytes of left ventricle from WKY and SHR. Mean IK,ATP of the activated myocytes from ENDO were significantly greater in WKY (n = 13) than SHR (n = 9, P < 0.01), although there was no difference in myocytes from EPI (n = 12 in WKY vs. n = 12 in SHR, P = 0.76).


Figure 2
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  		Fig. 2. ATP-sensitive K+ (KATP) channel current (IK,ATP) density after exposure to metabolic inhibition by 2 mM CN– and no glucose. During the exposure to metabolic inhibition >120 s, IK,ATP of WKY ({circ}, n = 13) was significantly greater than SHR (bullet, n = 9) in endocardium (bottom, P < 0.01, by repeated two-way ANOVA), although there was no difference between WKY (n = 12) and SHR (n = 12) in epicardium (top, P = 0.76).

 
As shown in Fig. 3, time required to reach peak current after the onset of IK,ATP activation (Timeonset to peak) was longer in endocardial myocytes from SHR than WKY (319 ± 46 vs. 177 ± 37 s, P = 0.024). In contrast, it did not differ in epicardial myocytes between the groups (274 ± 46 s in SHR vs. 235 ± 45 s in WKY, P = 0.55). There were no differences in time required to the onset of IK,ATP activation after exposure to 2 mM CN– and no glucose between WKY and SHR in both EPI and ENDO.


Figure 3
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  		Fig. 3. Time required to reach peak IK,ATP after the onset of IK,ATP activation (Timeonset to peak). In the endocardial (ENDO) group, Timeonset to peak was longer in SHR than WKY (318.7 ± 45.6 vs. 176.8 ± 36.8 s, P = 0.01). However, no difference was observed in the epicardium (EPI, 273.5 ± 45.1 s in SHR vs. 234.5 ± 45.5 s in WKY). *P < 0.05 between WKY and SHR.

 
Differences of single KATP channel response to intracellular ATP concentration. To gain further insights into the different responses to metabolic inhibition in SHR, single KATP channel characteristics were compared between WKY and SHR.

Figure 4 shows representative single KATP channel currents in isolated left ventricular myocytes of EPI and ENDO from WKY and SHR recorded by open cell-attached patch configuration at –60 mV. Upon formation of the open cell-attached patch configuration in the ATP-free bath solution, activation of KATP channels appeared immediately. When extracellular solution was switched to that containing 3 mM ATP, KATP channels closed rapidly in all experimented cells. Decreasing the intracellular ATP concentration gradually unmasked the channel activity. Compared with WKY, the KATP channels in myocytes of SHR, especially from ENDO, were less activated.


Figure 4
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  		Fig. 4. Representative tracings of single KATP channel currents from open cell-attached patches during exposure of intracellular membrane to the solution containing no ATP, 3 mM ATP, 1 mM ATP, 300 µM ATP, 100 µM ATP, or 30 µM ATP in epicardial myocytes (A) and endocardial myocytes (B) from WKY (A and B, top) and SHR (A and B, bottom). Membrane potential was held at –60 mV. Solid lines indicate the zero current level; inward currents are shown as downward deflections. The currents were displayed through a low-pass filter of 2 kHz. As shown in A and B, the KATP channels in SHR were less activated, especially in endocardial myocytes.

 
Figure 5 shows the concentration-response relationship for ATP inhibition of KATP channels in myocytes of EPI and ENDO from WKY and SHR in the open cell-attached patch configuration. The data points were fitted to the Hill equation: relative Po = 1/[1+([ATP]/IC50)n], where [ATP] is the ATP concentration, IC50 is the concentration required for half-maximal inhibition, and n is the Hill coefficient. In EPI, IC50 obtained from the fitted curve was 50.7 µM in WKY (n = 8) and 40.6 µM in SHR (n = 8). Hill's coefficient obtained from the fitted curve was 1.64 in WKY and 2.76 in SHR. In ENDO, IC50 obtained from the fitted curve was 75.1 µM in WKY (n = 10) and 41.3 µM in SHR (n = 9). Hill's coefficient obtained from the fitted curve was 1.27 in WKY and 2.63 in SHR. The relative Po at 100 µM ATP was smaller in SHR than WKY in ENDO (0.12 ± 0.05 vs. 0.42 ± 0.09, P = 0.01), although there was no difference in EPI (0.09 ± 0.04 vs. 0.25 ± 0.12, P = 0.11).


Figure 5
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  		Fig. 5. Concentration-dependent inhibition of KATP channels by ATP in open cell-attached patch recordings. Relationship between concentration of ATP in intracellular solution and relative open-state probability (Po) in WKY ({circ}) and SHR (bullet) are given. Left: in epicardial myocytes, half-maximal channel inhibition (IC50) of the fitted curve was 50.7 µM in WKY (n = 8) and 40.6 µM in SHR (n = 8). Hill's coefficient of the fitted curve was 1.64 in WKY and 2.76 in SHR. Right: in endocardial myocytes, IC50 of the fitted curve was 75.1 µM in WKY (n = 10) and 41.3 µM in SHR (n = 9). Hill's coefficient of the fitted curve was 1.27 in WKY and 2.63 in SHR. The relative Po at 100 µM ATP was smaller in SHR than WKY from endocardial myocytes (P = 0.01), although there was no difference in myocytes from epicardial myocytes (P = 0.11).

 
Figure 6 shows representative single KATP channel currents in isolated left ventricular myocytes of EPI and ENDO from WKY and SHR recorded by the excised inside-out membrane patch configuration at –60 mV. The concentration-response relationship for ATP inhibition of KATP channels in myocytes of EPI and ENDO from WKY and SHR in the excised inside-out patch configuration was shown in Fig. 7. In EPI, IC50 obtained from the fitted curve was 111.5 µM in WKY (n = 9) and 86.4 µM in SHR (n = 8). Hill's coefficient obtained from the fitted curve was 1.55 in WKY and 1.89 in SHR. In ENDO, IC50 obtained from the fitted curve was 155.2 µM in WKY (n = 8) and 41.1 µM in SHR (n = 8). Hill's coefficient obtained from the fitted curve was 2.55 in WKY and 1.30 in SHR. The relative Po was smaller in SHR than WKY in ENDO (P = 0.015, repeated-measures ANOVA ), although there was no difference in EPI (P = 0.2). The relative Po at 100 µM ATP was smaller in SHR than WKY in ENDO (0.25 ± 0.08 vs. 0.76 ± 0.15, P = 0.008), although there was no difference in EPI (0.43 ± 0.08 vs. 0.53 ± 0.07, P = 0.4).


Figure 6
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  		Fig. 6. Representative tracings of single KATP channel currents from excised inside-out patches during exposure of intracellular membrane to the solution containing no ATP, 3 mM ATP, 1 mM ATP, 300 µM ATP, 100 µM ATP, 30 µM ATP in epicardial myocytes (A) and endocardial myocytes (B) from WKY (A and B, top) and SHR (A and B, bottom). Membrane potential was held at –60 mV. Solid lines indicate the zero current level; inward currents are shown as downward deflections. The currents were displayed through a low-pass filter of 2 kHz. As shown in B, the KATP channels in SHR were less activated in the presence of 30, 100, and 300 µM ATP in endocardial myocytes.

 

Figure 7
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  		Fig. 7. Concentration-dependent inhibition of KATP channels by ATP in excised inside-out patch recordings. Relationship between concentration of ATP in intracellular solution and relative Po in WKY ({circ}) and SHR (bullet) are given. Left: in epicardial myocytes, IC50 of the fitted curve was 111.5 µM in WKY (n = 9) and 86.4 µM in SHR (n = 8). Hill's coefficient of the fitted curve was 1.55 in WKY and 1.89 in SHR. Right: In endocardial myocytes, IC50 of the fitted curve was 155.2 µM in WKY (n = 8) and 41.1 µM in SHR (n = 8). Hill's coefficient of the fitted curve was 2.55 in WKY and 1.30 in SHR. The relative Po was smaller in SHR than WKY from endocardial myocytes (P = 0.015), although there was no difference in myocytes from epicardial myocytes (P = 0.2). This difference was statistically determined by repeated two-way ANOVA.

 
The number of functional KATP channels included in each patch and the Po of KATP channels during exposure to ATP-free bath solution were not different between WKY and SHR in either open-cell attached patch experiments or excised inside-out patch experiments (Table 2).


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  		Table 2. Number and open probability of functional KATP channels included in experimented patches during exposure to ATP-free bath solution

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The major findings in this study are as follows: 1) in perforated whole cell current recordings, the increase of IK,ATP by metabolic inhibition was smaller in the endocardial myocytes from SHR than WKY; 2) the time required to reach peak IK,ATP currents after the onset of KATP channel activation was longer in the endocardial myocytes from SHR than WKY; 3) in single KATP channel recordings using the open cell-attached patch method, the relative PO at 100 µM of intracellular ATP was smaller in the endocardial myocytes from SHR than WKY; and 4) in single KATP channel recordings using the excised inside-out patch method, the relative PO was smaller in endocardial myocytes from SHR than WKY.

To our knowledge, this is the first study to compare the change of whole cell IK,ATP induced by metabolic inhibition between normal and hypertrophied hearts. In addition, the present study evaluated, for the first time, the transmural difference of single KATP channel currents between normal and hypertrophied hearts. These results might suggest that the adaptive response against the reduction of intracellular ATP induced by ischemia is diminished in the hypertrophied left ventricle, especially in the ENDO.

In contrast to our findings, it has been reported that using, excised inside-out or open cell-attached patches, the IC50 value for intracellular ATP was increased in hypertrophied feline left ventricular myocytes (3, 47). These discrepancies might be in part explained by species differences and experimental conditions, which include the age of experimented rats or temperature of experimental condition. In the present study, we employed both whole cell and single channel configurations and clearly demonstrated the impairment of KATP channel activation to simulated ischemia in rat hypertrophied left ventricular cells.

Kohya et al. (22) reported that the occurrence of ventricular tachyarrhythmias induced by left coronary artery occlusion was increased in hypertrophied rats. A recent study, using transgenic mice expressing dominant-negative Kir6 subunits, has proposed that cardiac KATP channels are essential for protecting the heart from lethal arrhythmias and adaptation to stress situations (37). Our results support this view because the deficit and transmural abnormality of the KATP channel activation, which plays a protective role toward the ischemia, of hypertrophied heart well explain the susceptibility of ventricular arrhythmias.

Although the present study was performed using isolated single myocytes, the differences in intracellular ATP regulation of hypertrophied hearts were reported in the whole heart experiments. For example, not only glycolysis from exogenous glucose and cardiac glycogen but also the rates of ATP utilization and lactate accumulation during ischemia were reported to be higher in hypertrophied hearts (2, 35, 38). However, it is not clear that impaired activation of endocardial KATP channels in hypertrophy results from the differences in energy metabolisms of hypertrophied hearts.

Recently, Hodgson et al. (16) reported, in a mouse model of heart failure induced by transgenic expression of the cytokine tumor necrosis factor-{alpha}, that KATP channel sensitivity to intracellular ATP was altered using open cell-attached patches despite no differences using excised inside-out patches. These results might suggest that heart failure itself did not alter the biophysical channel characteristics and that the difference of sensitivity to intracellular ATP might be caused by the distinct sensing pathway from the cell membrane to the mitochondria, the major producing site of ATP (21).

In our data from single KATP channel current recordings using the open cell-attached patch method, activation of the IK,ATP obtained from endocardial myocytes of SHR was reduced only in physiological submillimolar level of intracellular ATP concentration. This reduction was further confirmed through the micromolar to submillimolar level of intracellular ATP concentration when using the excised inside-out patch method. Therefore, the impaired KATP channel activations in SHR in this study would be attributable to alteration of mechanisms that regulate the KATP channels other than intracellular ATP metabolism and transport.

Membrane phosphatidylinositol phosphates (PIPs) such as PIP2 and PIP were reported to activate KATP channels (1, 25, 40). In hypertrophied rat hearts, the activity of phospholipase C (PLC), which hydrolyzes membrane-bound PIP2, and in turn may potentially decrease KATP channel activity, was reported to be increased (5). Similarly, in isolated myocytes from stroke-prone SHR heart, the increased PLC activity was indicated (19). These alteration of PLC activity in hypertrophied hearts might impair the activation of KATP channels by reducing membrane-bound PIP2. It was also reported that KATP channels were regulated by actin cytoskeleton (11, 44). In hypertrophied rat ventricular myocytes, transient outward K+ current was decreased by cytochalasin D, a disrupter of the actin microfilaments that had no effect in normal myocytes. Therefore, the cytoskeletal regulation of KATP channels could be altered especially in hypertrophied hearts.

In the present study of whole cell currents recordings, we employed the treatment with 2 mM CN– and no glucose to simulate "ischemia" in single isolated myocytes. Even though the CN– treatment has previously been used as a standard method to inhibit oxidative phosphorylation on single cells by several investigators (8, 9, 23, 24, 46), an extrapolation of the present data into in vivo conditions should be done with caution because the in vivo ischemic condition is obviously more complex. For example, in addition to ATP generation and utilization, there are some energy transfer mechanisms such as the creatine kinase system and adenylate kinase system (7, 18, 34).

In conclusion, the activation of endocardial IK,ATP by exposure to CN– and no glucose in cardiac hypertrophy was attenuated. The transmural alterations in KATP channels might contribute to the maladaptive response to ischemia in hypertrophied hearts.


   GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This study was conducted in part with grants from the Hokkaido Heart Association for Research and the Akiyama Foundation.


   ACKNOWLEDGMENTS
 
We thank Dr. Tetsuro Kohya, Department of Cardiovascular Medicine in NTT Sapporo Hospital, for helpful discussion and constant encouragement of this study and Dr. Haruaki Nakaya, chairman of the Department of Pharmacology in Chiba University Graduate School of Medicine, for technical advice of the perforated patch-clamp technique.


   FOOTNOTES
 

Address for reprint requests and other correspondence: H. Yokoshiki, Dept. of Cardiovascular Medicine, Hokkaido Univ. Graduate School of Medicine, Kita-15, Nishi-7, Kita-ku, Sapporo, 060-8638 Japan (e-mail: yokoshh{at}med.hokudai.ac.jp)

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.


   REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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