Chelerythrine increases Na-K-ATPase activity and limits ischemic injury in isolated rat hearts

¹ Division of Cardiovascular Medicine, Department of Internal Medicine, School of Medicine, and ² Department of Molecular Biosciences, School of Veterinary Medicine, University of California, Davis, California 95616

	ABSTRACT

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Myocardial ischemia results in an increase in intracellular sodium concentration ([Na]_i), which may lead to cellular injury via cellular swelling and calcium overload. Because protein kinase C (PKC) has been shown to reduce Na-K-ATPase activity, we postulated that pharmacological inhibition of PKC would directly increase Na-K-ATPase activity, reduce [Na]_i during ischemia, and provide protection from ischemic injury. Isolated rat hearts were subjected to 30 min of global ischemia with and without the specific PKC inhibitor chelerythrine. Intracellular pH, ATP, and [Na]_i were assessed using ³¹P and ²³Na NMR spectroscopy, whereas Na-K-ATPase and PKC activity were determined using biochemical assays. Na/H exchanger activity was determined using the ammonium prepulse technique under nonischemic conditions. Chelerythrine increased Na-K-ATPase activity (13.76 ± 0.89 vs. 10.89 ± 0.80 mg ADP · h¹ · mg protein¹; P = 0.01), reduced PKC activity in both the membrane and cytosolic fractions (39% and 28% of control, respectively), and reduced creatine kinase release on reperfusion (48 ± 5 IU/g dry wt vs. 689 ± 63 IU/g dry wt; P = 0.008). The rise in [Na]_i during ischemia was significantly reduced in hearts treated with chelerythrine (peak [Na]_i chelerythrine: 21.5 ± 1.2 mM; control: 31.9 ± 1.2 mM; P < 0.0001), without an effect on either acidosis (nadir pH 6.16 ± 0.05 for chelerythrine vs. 6.08 ± 0.04 for control), the rate of ATP depletion or Na/H exchanger activity. These data support the hypothesis that pharmacological inhibition of PKC before ischemia induces cardioprotection by reducing intracellular sodium overload via an increase in Na-K-ATPase activity.

intracellular sodium; protein kinase C; cardioprotection

	INTRODUCTION

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MYOCARDIAL ISCHEMIA results in an increase in intracellular sodium concentration ([Na]_i), which secondarily increases intracellular calcium via Na/Ca exchange, resulting in cellular injury (29, 43). The rise in [Na]_i can be ameliorated by limiting the stimulus for sodium-proton exchange (e.g., reducing intracellular acidosis) or limiting sodium influx via either the Na/H exchanger (33, 40) or the Na-K-2Cl cotransporter (35). Theoretically, the rise in [Na]_i could also be limited by increasing sodium efflux during ischemia via the sodium-potassium pump (Na-K-ATPase). However, because the Na-K-ATPase is an energy-requiring enzyme, the limitation of [Na]_i secondary to increased Na-K-ATPase activity may be countered by increased ATP hydrolysis, potentially resulting in more rapid ATP depletion and acidification (14). Thus it is unknown whether, on balance, stimulation of the Na-K-ATPase would be beneficial or detrimental to the ischemic heart.

Whereas Na-K-ATPase function is primarily driven by ionic gradients and the availability of ATP (18), there is evidence that activity of the Na-K-ATPase is phosphorylation dependent and, specifically, that phosphorylation by protein kinase C (PKC) independently decreases Na-K-ATPase function (15, 46). From these data, we postulated that sustained PKC inhibition would enhance Na-K-ATPase activity in the ischemic heart and increase sodium efflux during ischemia, resulting in lower [Na]_i during ischemia and limitation of ischemic injury. This hypothesis was tested using an isolated perfused rat heart model of global ischemia with inhibition of PKC using the specific inhibitor chelerythrine. The effects of this intervention were measured using functional parameters, creatine kinase release, and, using nuclear magnetic resonance spectroscopy, intracellular pH, ATP, and [Na]_i.

	METHODS

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All experiments were performed with the approval of the University of California, Davis, Animal Research Committee.

Isolated Heart Model

Experiments were performed using an isovolumic, isolated perfused rat heart preparation, as described previously (34, 36). Briefly, male Sprague-Dawley rats (~350 g) were pretreated with heparin (1,000 U ip), followed by pentobarbital sodium (65 mg/kg ip). After deep anesthesia was achieved in each rat, the heart was rapidly excised and placed into iced saline. The arrested heart was retrograde perfused through the aorta. Left ventricular pressure was determined using a latex balloon placed in the left ventricle with high-pressure tubing connected to a pressure transducer. Perfusion pressure was monitored using high-pressure tubing off the perfusion line. Hemodynamic measurements (perfusion pressure, left ventricular end-diastolic and systolic pressure, heart rate) were recorded on a four-channel Gould Windowgraf recorder (Gould, Valley View, OH). The perfusate was delivered using a Rainin Rabbit/Plus roller pump (Rainin Instrument, Emeryville, CA). The perfusate consisted of (in mM) 118 NaCl, 4.7 KCl, 1.25 CaCl₂, 1.2 MgSO₄, and 25 NaHCO₃, with the substrate being 11 mM glucose. The perfusion apparatus was temperature controlled, with heated baths used for the perfusate and for the water jacketing around the perfusion tubing to maintain heart temperature at 37 ± 0.5°C under all conditions. Oxygen was directly bubbled in the perfusate container and also in the heating chambers immediately proximal to the heart using gas-permeable tubing.

Nuclear Magnetic Resonance Spectroscopy

Phosphorus-31 spectroscopy. All spectroscopy was performed on a GE Omega 300 vertical bore spectrometer using a dual-tuned probe. After standard tuning, matching, and shimming on the water signal was established, ³¹P spectroscopy was performed using 224 acquisitions of a 60° pulse and 1.2-s interpulse delay, with spectra processed using an exponential multiplication of 10 Hz and manual phasing. Each time point was therefore acquired over ~5 min. Peak intensities (areas) were determined using spectrometer software. Intracellular pH was determined from the chemical shift of the inorganic phosphate resonance using a titration curve established in this laboratory. ATP resonances were referenced to their control value determined in duplicate at the start of the experiment.

²³Na spectroscopy. [Na]_i was determined using the shift reagent thulium-DOTP⁵ ([TmDOTP]⁵), supplied by Magnetic Resonance Solutions (Dallas, TX), which has little hemodynamic effect on the heart (27). [TmDOTP]⁵ was added to the perfusate throughout the entire experimental period, and ²³Na spectra were acquired using a broadband probe tuned to 79.9 MHz. Free induction decays (496) were signal averaged over 5 min using a 45° pulse with an 0.8-s interpulse delay. [Na]_i was calculated using the formula where A_Nai and A_Nao are the intracellular and extracellular areas of the sodium resonances, respectively, and f_o and f_i are the fractional visibilities of extra- and intracellular sodium (assumed as 1.0 and 0.4, respectively).

Na-K-ATPase activity. Hearts were rapidly frozen in liquid nitrogen 1) after the 40-min preparation period, 2) after 15 min of ischemia, and 3) after 5 min of reperfusion following 30 min of ischemia. The microsomal fraction of frozen tissue was assayed for overall and ouabain-sensitive ATPase activity using a previously described spectrophotometric assay (38). Heart tissue was homogenized at 4°C in 2 ml of a homogenizing solution (0.2 M sucrose-0.02 M Tris · HCl buffer, pH 7.5), containing 100 µl of a protease inhibitor [57 mM phenylmethylsulfonyl fluoride (PMSF) + 10 µl of 1 mg/ml leupeptin]. The homogenate was then centrifuged at 100 g for 10 min. The microsomes were prepared from the homogenates using previously published procedures (24). The linear rate of oxidation of NADH to NAD⁺ was monitored at 340 nM using a Perkin-Elmer Lambda 12 Spectrophotometer (Perkin-Elmer, Norwalk, CT). ATPase activity was calculated from the linear portion of the curve using the extinction coefficient of NADH, the volume of the reaction mixture, and the amount of heart homogenate. The Na-K-ATPase activity was obtained by subtracting the ATPase activity obtained in the presence of 10 mM ouabain from that obtained without ouabain.

PKC Assay

PKC was measured by biochemical assay of the membrane and cytosolic fractions according to the method of Inoguchi et al. (21), with minor changes. Briefly, myocardial tissue was obtained after 40 min of normoxic perfusion in both control and chelerythrine-treated hearts (n = 8 in each group) and after 30 min of global ischemia (n = 5 in each group) and then rapidly freeze clamped. The tissue was homogenized using Polytron and Dounce homogenizers in iced buffer containing 20 mM Tris · HCl, 2 mM EDTA, 0.5 mM EGTA, 2 mM dithiothreitol, 1 mg/ml leupeptin, 0.1 mg/ml aprotinin, and 1 mM PMSF. This solution was centrifuged at low speed (1,000 g) for 10 min. The resulting supernatant was ultracentrifuged at 100,000 g for 30 min, yielding the cytosolic fraction. The remaining pellet was resuspended in buffer containing 1% Triton X-100 and spun at 100,000 g for 30 min, with the resultant supernatant retained as the membrane fraction. Both membrane and cytosolic fractions were passed through 0.5-ml Mono-Q columns; washed with 2 ml of buffer containing Tris · HCl, EDTA, EGTA, and dithiothreitol (in previously described concentrations); and eluted with 0.5 ml of this same buffer containing 400 mM NaCl. PKC activity was measured by its ability to transfer ³²P from [-³²P]ATP (NEN) into a specific substrate octapeptide (RKRTLRRL) in the presence of Ca²⁺ (1 mM), phosphatidylserine (1 mg/ml), and diacylglycerol (0.02 mg/ml). Hence, PKC activity is expressed as specific activity (pmol ATP · min¹ · mg protein¹) for each cellular fraction. Protein determination was performed according to the method of Bradford (7).

Na/H Exchanger Activity

Because the accumulation of intracellular sodium during ischemia is modulated, in part, by the Na/H exchanger (39), the effect of chelerythrine on this exchanger was assessed using the ammonium chloride prepulse technique. Briefly, hearts (either control or chelerythrine treated as in the other protocols, n = 6 in each group) were exposed to 20 mM NH₄Cl for 10 min using buffer modified by elimination of K⁺ and addition of ouabain 100 µM (to inhibit the Na-K-2Cl cotransporter and the Na-K-ATPase), followed by washout with NH₄Cl-free buffer. In this procedure, NH₄Cl acidifies the cell by initially dissociating extracellularly into NH₃ and H⁺; the NH₃ freely diffuses into the cell, where it establishes a new NH⁺₄-NH₃ equilibrium with available H⁺. On NH₄Cl washout, this new equilibrium is rapidly destroyed as NH₃ quickly diffuses out of the cell, resulting in a sudden increase in free H⁺ and a fall in intracellular pH (6, 16). The increase in pH is largely modulated by the Na/H exchanger, and the slope of pH recovery has been widely used to assess Na/H exchanger activity (16). In the current experiments, intracellular pH was measured using ³¹P spectroscopy as described above; however, in contrast to the ischemia protocols, pH was measured with 1-min time resolution to accurately define pH recovery. The slope of pH recovery in the first 5 min after the pH nadir was used to determine Na/H exchanger activity.

Statistical Methods

Data were analyzed using INSTAT (GraphPad, San Diego, CA) software operating on an IBM-compatible personal computer. Differences between the same measurements at different time points were assessed using ANOVA for repeated measures, with subsequent Student-Newman-Keuls post hoc tests. Differences between two groups were assessed using unpaired Student's t-tests. A P value of <0.05 was used to reject the null hypothesis. All data are expressed as means ± SE.

Ischemia Protocols

Each group of hearts (control and chelerythrine treated) was subjected to two ischemic protocols. Protocol I used ³¹P NMR and measured functional recovery, creatine kinase release, and intracellular pH and ATP under baseline conditions, during 30 min of total global ischemia, and during reperfusion. Protocol II measured the changes in [Na]_i using ²³Na NMR under the same conditions. PKC in the cytosol and membrane fractions, as well as Na-K-ATPase activity, were measured in the absence of ischemia in separate groups of hearts treated as described above.

Protocol I: Functional Recovery, Creatine Kinase Release, Intracellular pH, and High-Energy Phosphates

Control hearts (n = 7) received 40 min of perfusion with normal buffer before 30 min of global ischemia and 60 min of reperfusion. Chelerythrine hearts (n = 8) received 40 min of buffer containing chelerythrine (1 µM) before the 30 min of ischemia and 60 min of reperfusion. The concentration of chelerythrine was chosen on the basis of the reported IC₅₀ of this drug for PKC (<0.7 µM) (20).

Protocol II: Intracellular Sodium

Control (n = 4) and chelerythrine (n = 4) hearts were studied using ²³Na NMR. Hearts in each group were perfused for 40 min with their respective buffers, followed by 30 min of ischemia and 30 min of reperfusion. All buffers in this protocol were enriched with 4 mM [TmDOTP]⁵ that was added at the start of perfusion without recirculation of the buffer. In both protocols, hearts were reperfused with buffer not containing chelerythrine.

	RESULTS

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Na-K-ATPase Activity

Ouabain-sensitive Na-K-ATPase activity was measured by biochemical assay before the onset of ischemia, after 15 min of global ischemia, and after 5 min of reperfusion. Hearts receiving chelerythrine had significantly higher Na-K-ATPase activity values than control hearts under baseline conditions (Table 1).

View this table: [in this window] [in a new window]   Table 1.   Na-K-ATPase activity under baseline conditions (after 40 min of perfusion), after 15 min of global ischemia, and after 5 min of reperfusion

PKC Activity

PKC activity in both the cytosolic and membrane fractions was significantly reduced after exposure to chelerythrine under normoxic conditions (Table 2). PKC activity after 30 min of ischemia was markedly lower in both groups, with no difference in either cytosolic or membrane activities between the control and chelerythrine-treated hearts.

View this table: [in this window] [in a new window]   Table 2.   Protein kinase C activity in control and chelerythrine-treated hearts immediately before global ischemia and after 30 min of ischemia without reperfusion

Na/H Exchanger

As shown in Fig. 1, there was no difference in the pH recovery after acidification using the ammonium prepulse technique, indicating that chelerythrine had no effect on the Na/H exchanger under nonischemic conditions. It is interesting to note, however, that the initial acidification on exposure to NH₄Cl was significantly blunted in the chelerythrine hearts.

View larger version (20K): [in this window] [in a new window]   Fig. 1.   Intracelluar pH in control (Ctl) hearts and hearts exposed to chelerythrine (Chel) for 40 min. After exposure to 20 mM NH4Cl and washout (solid line, time = 0 min), hearts acidified to a pH nadir (time = 2 min). Recovery of pH was identical in the 2 groups as evidenced by the slope of pH recovery (J) in the 5 min after pH nadir. These data indicate that Na/H exchanger was unaffected by Chel. Differences in pH were found during NH4Cl exposure (time = 10 to 0 min), perhaps indicating differences in NH3 transport.

Ischemic Injury and Functional Recovery

Creatine kinase release, a measure of myocardial injury, was significantly reduced after 30 min of ischemia in hearts receiving chelerythrine before ischemia (48 ± 5 IU/g dry wt) compared with control hearts (689 ± 63 IU/g dry wt, P < 0.0001).

Left ventricular developed pressure was similar in the control and chelerythrine hearts at the preischemic time point (67 ± 4 and 58 ± 5 mmHg, respectively) and rapidly fell to zero at the onset of ischemia. On reperfusion, recovery of developed pressure was limited in the control hearts (developed pressure of 17% of preischemic baseline), whereas chelerythrine-treated hearts had recovery of left ventricular developed pressure to 60% of preischemic baseline (Fig. 2). These differences in functional recovery paralleled the effect of chelerythrine on creatine kinase release.

View larger version (12K): [in this window] [in a new window]   Fig. 2.   Left ventricular developed pressure (LVDP) immediately before 30 min of ischemia (time = 0 min), after 5 min of reperfusion (time = 35 min), and after 60 min of reperfusion (time = 90 min) in Ctl and Chel hearts. Hearts receiving Chel buffer recovered significantly more LVDP than did Ctl hearts over same time period. There was no recovery of developed pressure at 35 min in Ctl hearts. * P < 0.05 Chel vs. Ctl hearts.

End-diastolic pressure (EDP) was set to 10 mmHg in both groups before ischemia and rose similarly during ischemia in both groups (end-ischemic EDP: control 54 ± 5, chelerythrine 46 ± 6 mmHg) and slowly fell on reperfusion. The EDP, although similar in the two groups at the onset of reperfusion, was significantly lower in the chelerythrine-treated hearts after 40 min of reperfusion (control, 44 ± 1; chelerythrine, 30 ± 4 mmHg, P < 0.05) and remained lower for the subsequent 20 min. Experiments (n = 4) using only the diluent for chelerythrine (DMSO 1 µM) did not show any reduction in creatine kinase release (1,036 ± 185 IU/g dry wt) or greater functional recovery compared with control hearts.

Intracellular pH and ATP Measurements

Intracellular pH was measured using ³¹P NMR spectroscopy. Chelerythrine had no effect on pH during normoxic perfusion (pH in each group = 7.05). Control and chelerythrine-treated hearts had similar degrees of acidification during the 30-min ischemic period, with nadir pH values (6.08 ± 0.04 for control; 6.16 ± 0.05 for chelerythrine) attained 20 min after the onset of ischemia for both groups. The rapidity and extent of pH recovery on reperfusion did not differ between them.

Neither control perfusion nor exposure to chelerythrine under normoxic conditions reduced ATP levels below initial measurements (104 ± 2 and 96 ± 4% of baseline, respectively). ATP depletion rates during ischemia (ATP/min) did not significantly differ between the groups, with slopes of 3.58 ± 0.06 for control and 3.64 ± 0.18 for hearts receiving chelerythrine. Both groups of hearts experienced complete ATP loss 20 min after the onset of ischemia concurrent with the absence of further acidification. ATP recovery was similar between the groups at the onset of reperfusion, averaging 7.9 ± 8.6% of baseline in control hearts and 8.2 ± 5.8% in chelerythrine-treated hearts. However, chelerythrine-treated hearts maintained ATP levels throughout reperfusion (11.9 ± 6.2%), whereas ATP was completely depleted in control hearts.

[Na]_i Measurements

[Na]_i, as measured by ²³Na NMR spectroscopy, was readily distinguished from extracellular sodium by adding the shift reagent [TmDOTP]⁵ to all buffers in this protocol. As shown in Fig. 3, the increase in [Na]_i was greater in control hearts during ischemia, with an end-ischemic value of 31.9 ± 1.2 mM compared with 21.5 ± 1.2 mM in the chelerythrine-treated hearts (P < 0.001). After 30 min of reperfusion, [Na]_i of control hearts was 74% higher than baseline, whereas that of chelerythrine-treated hearts had returned to preischemic values.

View larger version (19K): [in this window] [in a new window]   Fig. 3.   Changes in intracellular sodium concentration ([Na]i, in mM) during 30 min of global ischemia in Ctl and Chel hearts. Chel hearts had significantly lower levels of [Na]i during ischemia compared with Ctl hearts after 15 min of ischemia and during the entire reperfusion period. * P < 0.05 Chel vs. Ctl hearts.

	DISCUSSION

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This study has demonstrated that PKC inhibition with chelerythrine protected the ischemic heart and that this cardioprotection was associated with activation of the Na-K-ATPase at the beginning of ischemia and significant limitation in the rise in [Na]_i during ischemia. These data are consistent with the paradigm that PKC inhibition results in increased Na-K-ATPase activity before ischemia and greater sodium efflux during ischemia, thereby limiting the rise in [Na]_i during ischemia. As a consequence of this lower [Na]_i, it is likely that calcium accumulation during ischemia and reperfusion is also limited.

[Na]_i

[Na]_i normally rises at the onset of ischemia in the heart (34), and interventions that limit the rise in [Na]_i (such as Na/H exchange inhibition) have been associated with reductions in ischemic injury (37, 40), presumably by limiting the stimulus for increased intracellular Ca concentration via Na/Ca exchange (1, 19). Although not previously examined, one would postulate that increasing sodium efflux during ischemia would also be protective by a similar mechanism. Because the Na-K-ATPase is the primary mechanism for sodium efflux, stimulation of this enzyme would be expected to limit the rise in [Na]_i during ischemia. The data from this study support such a mechanism, with a lower level of [Na]_i during ischemia and lower cellular injury (as assessed by functional recovery and creatine kinase release) coupled with higher Na-K-ATPase activity under baseline and ischemic conditions. A concern that stimulation of the Na-K-ATPase would be detrimental by increasing ATP utilization was not supported, because the rates of ATP depletion were identical in the two groups.

These findings of a beneficial role of increased Na-K-ATPase activity are supported by other experiments in which maintenance of Na-K-ATPase activity was observed during global ischemia in ischemically preconditioned hearts (31). Other investigators have also shown that increased Na-K-ATPase activity during low-flow ischemia limited the rise in sodium and improved functional recovery (12). Finally, increased activity of the Na-K-ATPase using acidic reperfusion after regional no-flow ischemia limited reperfusion arrhythmias (3), suggesting another possible mechanism for the protective effect of increased Na-K-ATPase activity.

PKC Inhibition and Na-K-ATPase Activity

PKC has been shown in several models to inhibit the activity of the Na-K-ATPase (5, 15). PKC appears to affect both the serine and threonine residues of the -subunit of the Na-K-ATPase, resulting in a conformational change in the protein that subsequently reduces its overall activity (11). Whereas there is little experimental evidence in myocardial tissue, studies on cultured rat vascular smooth muscle have shown that Na-K-ATPase activity is reduced by PKC (46). These data suggest that inhibition of PKC would remove this inhibition and therefore increase Na-K-ATPase activity. The present study clearly supports this hypothesis, because Na-K-ATPase activity was significantly increased following 40 min of PKC inhibition with chelerythrine.

PKC Inhibition and Na/H Exchanger

Because the Na/H exchanger has profound effects on [Na]_i accumulation during myocardial ischemia (40), we investigated whether PKC inhibition with chelerythrine had an inhibitory effect on the exchanger as a potential mechanism of reduced sodium accumulation. This effect would be parallel to that of pharmacological inhibition of the exchanger with compounds such as ethylisopropyl amiloride (40) or HOE-642 (44). The prepulse experiments, however, demonstrated that pH recovery was identical in the two experimental groups. Therefore, in this model, the effect of chelerythrine on sodium accumulation was likely not modulated by the Na/H exchanger. Unexplained at this time is the mechanism of lower acidification during NH₄Cl exposure in the chelerythrine hearts. Because NH₃ may enter the cell via the Na-K-2Cl cotransporter (as well as by diffusion), these data suggest that PKC inhibition may have an effect on this transport protein.

PKC and Myocardial Ischemia

Whereas the current study has shown a beneficial effect of PKC inhibition, activation and translocation of PKC has also been shown to frequently, but not universally, be an important mechanism of ischemic preconditioning (2, 25, 28, 42, 47). In these studies, hearts preconditioned in the presence of a PKC inhibitor exhibit larger infarct size and reduced functional recovery compared with hearts preconditioned with standard buffer (8, 28). Notably, studies in preconditioned rat hearts have not shown a significant reduction in [Na]_i during ischemia (34). The current findings, while consistent with the previously observed effects of PKC activation and translocation inhibiting Na-K-ATPase activity (4, 41), demonstrate an opposite mechanism of protection. Further support for differences between preconditioned and PKC-inhibited hearts is the 36% reduction in end-ischemic [Na]_i in the current experiments in contrast with the minimal changes in [Na]_i in preconditioned hearts (26, 34).

PKC inhibition has been utilized in other studies with similar positive results. Lasley et al. (23) showed that inhibition of PKC using either chelerythrine or bisindolylmaleimide before 45 min of coronary artery occlusion in the rabbit reduced myocardial infarct size up to 50% and was associated with a reduction in adenine nucleotide catabolism (23). A similar protective effect was seen in perfused hearts (23). However, these authors did not explore possible mechanisms of protection, and they did not measure whether bisindolymaleimide or chelerythrine lowered PKC activity. Vogt et al. (45), in an in situ pig model, also demonstrated a protective effect of bisindolymaleimide infusion preceding 60 min of coronary occlusion in the pig. Thus the efficacy of PKC inhibition before regional and global ischemia has been shown, although its mechanism of action has not been previously elucidated.

There are, of course, several studies in which exposure to PKC inhibitors before ischemia had no salutory effect. For example, exposure of isolated rat hearts to 2 µM chelerythrine chloride for 15 min before global ischemia had no effect on functional recovery, intracellular pH, or ATP depletion (10). In another study, exposure of hearts for 30 min to 50 µM polymyxin B (a nonspecific inhibitor of protein kinases) before global ischemia did not affect hemodynamic recovery (9), although infarct size was not measured. In neither of these earlier studies was PKC activity, Na-K-ATPase, or [Na]_i measured, thus limiting comparison with the present study. Lasley et al. (23) speculated that, based on the absence of a protective effect of a higher concentration of chelerythrine, the protective effect was only present at a lower concentration of the inhibitor. Whereas the concentration used in the present study (1 µM) is consistent with this hypothesis, other studies (such as Ref. 10) also used low concentrations without any protective effect. Thus at this time an explanation for the differences in effect is absent.

PKC Inhibition and Changes in pH and ATP

It has been suggested that attenuating pH changes during ischemia is beneficial in terms of reducing infarct size, potentially by reducing the stimulus for sodium accumulation via Na/H exchange (13, 22, 30). However, some recent studies indicate that pH may be unrelated to myocardial damage, especially under conditions in which ion regulation is altered (33, 36). In the present experiments, pH during ischemia varied little between the groups, with chelerythrine-treated hearts experiencing slightly less acidosis. Given that these groups exhibited large differences in creatine kinase release, it appears that myocardial injury is independent of the degree of ischemic acidosis when sodium accumulation is limited.

The ATP depletion rate was also relatively unchanged between the groups, with both control and chelerythrine-treated hearts experiencing complete loss of ATP within 20 min of ischemia. A reduction in ATP is a potential determinant of myocardial ischemic injury, and ATP preservation during ischemia has been implicated in the cardioprotection provided by preconditioning (22, 30). Whereas slower ATP loss may be important during preconditioning, it does not appear to be linked to the benefits observed with prolonged PKC inhibition. However, the modest preservation of ATP on reperfusion in the chelerythrine-treated hearts may have contributed to improved functional recovery by maintaining ATPase function for ionic homeostasis and contractile function (18).

Limitations

The results from the current study strongly support the hypothesis that prolonged exposure of an isolated rat heart to the PKC inhibitor chelerythrine before ischemia significantly reduces infarct size and improves functional recovery by increasing sodium efflux. However, limitations inherent in the experimental design must be addressed. First, the use of a shift reagent has potential effects on ion transport and hemodynamic function. However, [TmDOTP]⁵ has been shown to provide an accurate means to separate intra- and extracellular sodium, without adversely affecting myocellular function (27, 33), and there are no data suggesting a differential effect or interaction of [TmDOTP]⁵ under conditions of PKC inhibition. Second, other effects of chelerythrine chloride cannot be excluded. This PKC inhibitor was chosen because it is a potent, selective PKC antagonist, interacting with the catalytic domain of the PKC molecule (20). It is considered to bind PKC more specifically than staurosporine, polymyxin B, and others (20). In addition, the dose used (1 µM) is relatively low compared with that used in other studies but is sufficient to inhibit PKC (by its IC₅₀ and the current PKC measurements). Thus it is unlikely that non-PKC effects of chelerythrine were significant in these experiments. Third, the measurements of PKC showed that chelerythrine reduced the activity of PKC in both the membrane and cytosolic fractions. It is unknown whether specific isoforms of PKC were differentially inhibited or translocated by chelerythrine (17). Finally, whereas the findings of increased Na-K-ATPase activity and lower [Na]_i during ischemia are consistent with the hypothesis, a causal relationship of these events has not been established. Indeed, it is possible that PKC inhibition could have resulted in the observed benefits on function and creatine kinase release through reduced phosphorylation of other proteins such as the Na-K-2Cl cotransporter (32).

In conclusion, exposure to the PKC inhibitor chelerythrine significantly reduced creatine kinase release and improved functional recovery after 30 min of ischemia in this isolated heart model. PKC inhibition concurrently increased activity of the Na-K-ATPase before and during ischemia and limited the rise in [Na]_i normally seen during ischemia. These findings are consistent with removal of PKC inhibition from the Na-K-ATPase, resulting in increased sodium efflux limiting sodium and, likely, calcium overload during ischemia and reperfusion. This intervention has potential for providing pharmacological cardioprotection under conditions of severe ischemia.

	ACKNOWLEDGEMENTS

This study was supported by grants from the American Heart Association-California Affiliate (to S. Schaefer) and a National Institutes of Health Training Grant in Cardiovascular and Neurophysiology (to J. L. Lundmark). The spectrometer was funded by an award from the National Science Foundation.

	FOOTNOTES

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. §1734 solely to indicate this fact.

Address for reprint requests: S. Schaefer, Division of Cardiovascular Medicine, TB 172, Univ. of California, Davis, CA 95616
(E-mail: sschaefer{at}ucdavis.edu).

Received 11 August 1998; accepted in final form 31 March 1999.

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