Cardiovascular Research

Cardiovascular Research 1998 37(3):691-699; doi:10.1016/S0008-6363(97)00249-6
© 1998 by European Society of Cardiology

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Copyright © 1998, European Society of Cardiology

Oxidant stress with hydrogen peroxide attenuates calcium paradox injury: role of protein kinase C and ATP-sensitive potassium channel

Hiroshi Miyawaki, Yigang Wang and Muhammad Ashraf^*

Department of Pathology and Laboratory Medicine University of Cincinnati Medical Center, 231 Bethesda Avenue, Cincinnati, Ohio 45267-0529, USA

^* Corresponding author. Tel. +1-513-558-0145; Fax (+1-513) 558 2289.

Received 24 March 1997; accepted 30 September 1997

	Abstract

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
Objective: We tested the hypotheses that low concentration of H₂O₂ attenuates the Ca²⁺ paradox (Ca²⁺ PD) injury, and that activation of protein kinase C (PKC) and/or ATP-sensitive potassium channel (K_ATP) are involved in the protective effects of H₂O₂. Methods: Langendorff-perfused rat hearts were subjected to the Ca²⁺PD (10 min of Ca²⁺ depletion followed by 10 min of Ca²⁺ repletion). Functional and biochemical effects of H₂O₂ and other interventions on the cell injury induced by the Ca²⁺ PD were assessed. Results: In the Ca²⁺ PD hearts pretreated with 20 µmol/l H₂O₂, left ventricular end-diastolic pressure and coronary flow were significantly preserved. Furthermore, peak lactate dehydrogenase release was significantly decreased and ATP contents were more preserved, compared with non-treated Ca²⁺ PD hearts. H₂O₂-treated hearts also showed remarkable preservation of cell structure. Addition of a specific PKC inhibitor, chelerythrine during H₂O₂ treatment completely abolished the beneficial effects of H₂O₂ on the Ca²⁺ PD. Similarly, an activator of PKC, Phorbol 12-myristate 13 acetate mimicked the protection by H₂O₂. Furthermore, pretreatment with a K_ATP opener, cromakalim also provided protection similar to H₂O₂ against the Ca²⁺ PD injury. However, a specific K_ATP inhibitor, glibenclamide was not able to completely block the effects of H₂O₂. Conclusions: These findings suggest that pretreatment with low concentration of H₂O₂ provides significant protection against the lethal injury of Ca²⁺ PD in rat hearts. PKC-mediated signaling pathways appear to play a crucial role in the protection against the Ca²⁺ PD injury.

KEYWORDS Calcium; K_ATP channel; Myocytes; Protein kinase C; Preconditioning

	1 Introduction

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
It is believed that reactive oxygen species (i.e. H₂O₂, ·O₂^–, ·OH, and ) are implicated in the pathogenesis of ischemia/reperfusion injury [1, 2]. These reactive species are generated during ischemia/reperfusion and cause injury to myocardial cells [3–9]. However, a brief exposure of myocytes to oxygen radicals generated by reaction of xanthine oxidase with xanthine elicits both early (60 min later) and late (24 h later) protection against anoxia/reoxygenation injury [10]. On the other hand, extended perfusion with exogenous H₂O₂ [11]generated through xanthine and xanthine oxidase reaction [12]causes cardiac dysfunction and irreversible pathological changes. It is based on our previous study [10]that a brief exposure of cardiac cells to ·O₂^–/ H₂O₂ provides protection against subsequent prolonged ischemia. Furthermore, H₂O₂ which is produced as a result of ^·O₂^– dismutation, has been reported to cause the activation of ATP-sensitive potassium channels (K_ATP) [13–15]and protein kinase C (PKC) [16–18]. The accumulated experimental findings suggest that K_ATP and /or PKC are actively involved in preconditioning phenomenon [19–23].

We examined the preconditioning effects of H₂O₂ on the Ca²⁺ paradox (Ca²⁺ PD) which is a far lethal experimental model than the sustained ischemia/reperfusion. A disruption of the electrical and mechanical properties of the cells, calcified mitochondria, a loss of the cellular contents, high-energy phosphate depletion and massive accumulation of calcium within the cells are common features of the Ca²⁺ PD [24–26]. Although Ca²⁺ PD induces more severe cellular damage than ischemia/reperfusion injury (I/R), Ca²⁺ PD is a useful model for investigating the pathogenesis of Ca²⁺ overload-associated injury which also occurs during I/R. Nevertheless, a recent evidence suggests that a brief ‘Ca²⁺’ stress attenuates the Ca²⁺ PD as well as ischemic damage [22, 25].

Accordingly, the aim of the present study was to determine whether oxidative stress by H₂O₂ reduces the lethal injury induced by Ca²⁺ PD and to examine the roles of K_ATP and PKC in the H₂O₂-mediated effects on the Ca²⁺ PD injury.

	2 Methods

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
2.1 Materials
H₂O₂, cromakalim, and phorbol 12-myristate 13-acetate (PMA) were purchased from Sigma Chemical; glibenclamide and chelerythrine chloride were obtained from Research Biochemical.

2.2 Heart preparation
Male Sprague-Dawley rats weighing 250 to 300 g were anesthetized by intraperitoneal injection of 30 mg/kg pentobarbital. After intraperitoneal injection of 500 U/kg heparin sodium, hearts were removed and retrogradely perfused through the aorta in a noncirculating Langendorff apparatus with Krebs–Henseleit (KH) buffer consisted of (mmol/l) NaCl 118, KCl 4.7, MgSO₄ 1.2, KH₂PO₄ 1.2, CaCl₂ 1.8, NaHCO₃ 25, and glucose 11. The buffer was saturated with 95% O₂– 5% CO₂ (pH 7.4, 37°C) for 50 min. Hearts were perfused at a constant pressure of 80 mmHg. A water-filled latex balloon-tipped catheter was inserted into the left ventricle through the left atrium and was adjusted to a left ventricular end-diastolic pressure of 5 mmHg during the initial equilibration. The distal end of the catheter was connected to a pressure transducer (Gould P23Db). Cardiac function was determined using the double product of heart rate multiplied by left ventricular developed pressure (LVDP) divided by 1000 [27]. LVDP was calculated from the difference between LV peak systolic pressure and end-diastolic pressure (EDP). Heart rate, left ventricular pressure, were monitored on a Grass 7D polygraph (model 7P20, Grass Instrument, Quincy, Mass.). After perfusion with the oxygenated KH buffer for an equilibration period of 20 min, a three-way stopcock above the aortic root was used to perfuse Ca²⁺ free KH buffer for 10 min, followed by KH buffer containing Ca²⁺ for 10 min in order to induce a typical Ca²⁺ PD [26]. The coronary effluent was collected in a beaker, and coronary flow (CF) was determined volumetrically at the end of equilibration, during treatment period, the Ca²⁺ depletion period, and at 1.5, 3, 5, and 10 min during Ca²⁺ repletion. Glibenclamide and chelerythrine were directly administered through the aortic cannula at a rate of 0.2 ml/min by a Harvard infusion pump. The present study conforms with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1985).

2.3 Experimental groups
After equilibration, hearts were randomly divided into following experimental groups:

Group 1: normal control. Hearts (n=6) were perfused for 40 min with KH buffer as a normal control for different experimental groups.

Group 2: calcium paradox (Ca²⁺ PD). After 20 min perfusion with normal KH buffer, hearts (n=6) were subjected to Ca²⁺-free KH buffer for 10 min followed by Ca²⁺ containing KH buffer for 10 min.

Group 3: Ca²⁺ PD+H₂O₂. Hearts (n=6) were perfused with KH buffer containing 20 µmol/l H₂O₂ for 10 min. After 10 min wash-out period, hearts were subjected to Ca²⁺ PD as in group 2. Cytotoxity of H₂O₂ had been systemically investigated in our laboratory [11, 12, 28]. Onodera et al. [11]examined the dose- and time-dependent effects of exogenous H₂O₂ and showed that perfusion of 100 µmol/l H₂O₂ for 15 min caused no significant changes in cardiac function and cell morphology. We chose 20 µmol/l H₂O₂ for this study as the appropriate concentration for causing minimal and reversible injury.

Group 4: H₂O₂ and activation of PKC. To determine whether the beneficial effects of H₂O₂ were mediated by PKC activation, we used a PKC activator, PMA, in order to mimic the beneficial effects by H₂O₂ and a specific PKC inhibitor, chelerythrine to reverse effects of PMA during H₂O₂ treatment. Hearts were perfused with KH buffer containing both PMA and chelerythrine.

Group 4A: Ca²⁺ PD+PMA. Hearts (n=6) were perfused with KH buffer containing a PKC activator, PMA (1 nmol/l) for 10 min, prior to Ca²⁺ PD, and continued during Ca²⁺ PD.

Group 4B: Ca²⁺ PD+H₂O₂+chelerythrine. The protocol was similar to that of group 3, except chelerythrine chloride (0.02 µmol/min) was infused for 20 min prior to Ca²⁺ depletion and repletion (n=7).

Group 4C: Ca²⁺ PD+PMA+chelerythrine. The protocol was similar to that of group 5A, except that chelerythrine chloride (0.02 µmol/min) was intraaortically infused during treatment with PMA (n=6).

Group 5: H₂O₂ and activation of K_ATP. To elucidate the role of K_ATP in H₂O₂-induced protective effects, we examined whether a K_ATP opener, cromakalim, could mimic the effects of H₂O₂ and a selective inhibitor of K_ATP, glibenclamide, could abolish the salutary effects of H₂O₂. We also tested whether the use of an optimal dose of glibenclamide plus cromakalim could reverse its salutary effects.

Group 5A: Ca²⁺ PD+cromakalim. Hearts (n=7) were perfused with KH buffer containing K_ATP opener, cromakalim (20 µmol/min) for 10 min, and then the hearts were subjected to Ca²⁺ PD.

Group 5B: Ca²⁺ PD+cromakalim+glibenclamide. The protocol was similar to group 4A, except glibenclamide (0.01 µmol/min), was intraaortically infused (n=6). A preliminary study was done to determine the appropriate concentration of cromakalim (20 µmol/min) and glibenclamide (0.01 µmol/min) with reference to LDH release and ATP preservation.

Group 5C: Ca²⁺ PD+H₂O₂+glibenclamide. The protocol was similar to that for group 3, except glibenclamide (0.01 µmol/min), was intraaortically infused for 15 min followed 5 min wash out (n=7).

2.4 Measurement of LDH
LDH, an indicator of myocardial tissue injury, was determined in coronary effluent [25]. This was assayed by a coupled-enzyme spectrometric technique using a Sigma assay kit. Measurement of enzyme activity was based on oxidation of lactate and the rate of increase in absorbance at 340 nm.

2.5 Measurement of tissue ATP
The heart was immediately frozen in liquid nitrogen after Ca²⁺ depletion and repletion or as specified, and was freeze-dried for 24 h. 50 to 100 mg of freeze-dried tissue was crushed in precooled glass tube, and ATP was extracted with 5 ml of cold 6% trichloroacetic acid. The extract was analyzed for ATP by spectrophotometric method [25].

2.6 Morphological examination
Heart tissue from the mid ventricular wall was taken at the end of Ca²⁺ repletion, and was cut into 1.0 mm pieces which were fixed with 2.5% buffered glutaraldehyde. A semi-quantitative estimate of cell damage was carried out on 1 µm-thick sections. Three randomly chosen blocks from each heart were examined for quantification of cell damage without prior knowledge of the treatment. Approximately 500 cells were analyzed in each heart, and one of three degrees of cell damage was assigned to each cell. Cell morphology was assessed according to the following classification [22, 23]: (1) normal (compact myofibers with uniform staining of nucleoplasm, well-defined rows of mitochondria between the myofibrils, and no separation of opposing intercalated discs); (2) mild damage (same as above, except some vacuoles were present adjacent to the mitochondria); (3) severe damage (reduced staining of cytoplasmic organelles, clumped chromatin material, wavy myofibers, and granularity of cytoplasm; the cells with contraction band necrosis were added in this category).

2.7 Statistical analysis
All values are expressed as means±SEM. Group comparisons were done by ANOVA with multiple comparisons or t-test when appropriate. A difference of P<0.05 was considered significant.

	3 Results

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
3.1 Ca²⁺ paradox
During Ca²⁺ repletion of Ca²⁺-depleted hearts, the reddish color of the heart was lost, indicating a release of intracellular contents; LVEDP was significantly increased and coronary flow was significantly reduced, and no beating of hearts was observed (Table 1). At the end of the Ca²⁺-free period, LDH release was not different from the control values. Immediately upon Ca²⁺ repletion, LDH release increased multifold and peaked at 3 min (Fig. 1). Tissue ATP contents in Ca²⁺ PD hearts were obviously depleted as compared with the control hearts (Fig. 2).

View this table: [in this window] [in a new window]   Table 1 Effect of various interventions on hemodynamic changes in hearts subjected to the calcium paradox

 

View larger version (28K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 1 Effect of various interventions on LDH release in hearts subjected to Ca2+ PD. H2O2 (20 µmol/l), PMA (1 nmol/l), and cromakalim (20 µmol/l) reduced LDH release. Beneficial effect of H2O2 was completely abolished by PKC inhibition with chelerythrine (CH, 0.02 µmol/min) and not completely by the KATP channel blocker, glibenclamide (GB, 0.01 µmol/min).

 

View larger version (26K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 2 Effects of various interventions on maximum LDH release (upper panel) and tissue ATP contents (lower panel) in hearts subjected to Ca2+ PD. Hearts subjected to Ca2+ PD showed a drastic loss of LDH and ATP. The KATP channel blocker, glibenclamide (GB, 0.01 µmol/min) partially prevented the beneficial effects of H2O2, and chelerythrine (CH), a specific PKC inhibitor, completely blocked the H2O2-induced protection. PMA (1 nmol/l) almost mimicked the effects of H2O2. The KATP channel opener, cromakalim (20 µmol/l) was less effective in reducing cell injury than H2O2 and PMA. Reversal by chelerythrine of H2O2's salutary effect on the Ca2+ PD injury was almost similar to that of hearts treated with PMA plus chelerythrine.

 
In Ca²⁺ PD hearts, most of the cells were hypercontracted, and the cell membranes were ruptured, resulting in the extrusion of intracellular contents (Fig. 3, Table 2).

View larger version (125K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 3 Effect of different interventions on the cell morphology. A: Normal heart. Nuclei showed uniform distribution of chromatin material (arrow). Compact and well-preserved myofibers were observed. (x320) B: Calcium paradox heart. All cells were so hyper-contracted that the membranes were ruptured. As a result, mitochondria were extruded from the cells (arrow). (x320) C: H2O2-treated heart. Myocytes were significantly preserved compared with nontreated calcium paradox heart (B). A few severely damaged cells were also observed (arrow). (x320) D: Heart treated with H2O2 and glibenclamide. Most of myocytes were severely damaged. However, the degree of hypercontraction was less than in nontreated calcium paradox heart. (x320) E: Heart treated with H2O2 and cherlerythrine. All cells were damaged similar to Ca2+ paradox. (x320) F: Cromakalim-treated heart. Approximately 20% cells was normal (arrowhead) and others were severely damaged (arrow). (x320) G: PMA-treated heart. Most of myocytes were well preserved with the exception of some damaged cells (arrow).

 

View this table: [in this window] [in a new window]   Table 2 Semiquantitative estimate of morphological damage in hearts subjected to Ca2+ paradox after various interventions

 
3.2 Effects of H₂O₂ on Ca²⁺ paradox
At the end of Ca²⁺ repletion, a significant improvement in CF and LVEDP was observed in H₂O₂-treated hearts compared to hearts subjected to Ca²⁺ PD alone. Six in 10 hearts treated with H₂O₂ resumed contraction. There were no significant differences in LDH release and tissue ATP levels between beating hearts and non-beating hearts after Ca²⁺ repletion (data not shown). In H₂O₂-treated hearts, maximum LDH release was reduced significantly and ATP contents were also preserved better than control Ca²⁺ PD hearts without H₂O₂ treatment (Fig. 1, Fig. 2). The cellular structure was also markedly preserved in the H₂O₂-treated hearts; 45.2±7.5% of cells were normal; 14.2±2.9% and 40.6±4.9% were mildly and severely damaged respectively; and the damage was significantly lesser than that in the Ca²⁺ PD hearts without pretreatment (Table 2, Fig. 3).

3.3 Effects of H₂O₂ and activation of PKC on Ca₂₊ paradox
Pre-administration of chelerythrine to the hearts treated with H₂O₂ caused cessation of the heart beats during Ca²⁺ repletion. Chelerythrine exacerbated the maximum LDH release and caused depletion of tissue ATP contents in H₂O₂-treated hearts (Fig. 1, Fig. 2). Furthermore, the degree of cellular damage in chelerythrine-treated hearts was almost the same as in non-treated Ca²⁺ PD hearts (Table 2, Fig. 3). Thus, PKC inhibitor abolished the beneficial effects of H₂O₂ on the Ca²⁺ PD injury.

In a separate group of experiments, we determined whether activation of PKC mimics the beneficial effects of H₂O₂ pretreatment on the Ca²⁺ PD injury. With PKC activation, the effects of LV functional recovery in PMA (1 nmol/l)-treated hearts were almost the same as H₂O₂-treated hearts. After Ca²⁺ repletion, 6 in 11 hearts treated with PMA resumed beating (Table 1). The maximum LDH release and tissue ATP levels in PMA-treated hearts were similar to H₂O₂-treated hearts (Fig. 1). LVEDP, coronary flow and tissue ATP level were also preserved in PMA-treated hearts as compared with Ca²⁺ PD hearts (Table 1, Fig. 2). Thus, PMA almost mimicked the salutary effects of H₂O₂ on Ca²⁺ PD. Morphological examination of PMA-treated hearts also supported the biochemical findings (Table 2, Fig. 3). However, these beneficial effects by PMA were abolished by infusing chelerythrine. Chelerythrine reversed the salutary effects of H₂O₂ on the Ca²⁺ PD injury which was almost similar to those in hearts treated with PMA plus chelerythrine (Table 2, Figs. 1 and 2).

3.4 Effects of H₂O₂ and K_ATP activation on Ca²⁺ paradox injury
A lower concentration of glibenclamide (0.001 µmol/min) did not affect the H₂O₂-induced protection (Fig. 4). The maximum inhibitory effect of glibenclamide on H₂O₂-treatment was observed at 0.01 µmol/min, however, this concentration of glibenclamide could not completely reverse the H₂O₂-induced beneficial effects (Figs. 1 and 2 and 4). A significant reduction in maximum LDH release and increased tissue ATP preservation were also observed in glibenclamide-treated hearts compared to control Ca²⁺ PD. However, ATP values were significantly less compared to H₂O₂-treated hearts (Fig. 2). The morphological findings are also in agreement with the above observations (Table 2, Fig. 3).

View larger version (31K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 4 Dose-response effects of glibenclamide on LDH release and ATP contents in H2O2-treated hearts subjected to the Ca2+ PD. Maximum blocking effect of glibenclamide (GB) on H2O2-induced protection was achieved with 0.01 µmol/min. However, this concentration of glibenclamide attenuated but did not completely block the protective effects of H2O2.

 
In separate series of experiments, we determined the effect of K_ATP opener, cromakalim on the Ca²⁺ PD damage. The maximum effects of cromakalim were observed at 20 µmol/min (Fig. 5). This concentration of cromakalim significantly reduced LVEDP, maximum LDH release, preserved tissue ATP levels, and increased CF as compared with non-treated Ca²⁺ PD hearts. However, the degree of protection was less than H₂O₂-treated hearts (Figs. 1 and 2 and 5). Morphological damage was also greater in hearts treated with cromakalim than H₂O₂ (Table 2, Fig. 3). After glibenclamide (0.01 µmol/min) was infused in cromakalim-treated hearts, the salutary effects by cromakalim were completely reversed.

View larger version (26K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 5 Dose-response effects of cromakalim on LDH release and ATP contents in hearts subjected to the Ca2+ paradox in the absence of H2O2. Maximum protection was achieved with 20 µmol/l cromakalim.

 

	4 Discussion

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
The present study demonstrates that low concentration of H₂O₂ provides cardioprotection to rat hearts subjected to Ca²⁺ PD. The effects of H₂O₂ are mimicked by PMA and are completely blocked by PKC inhibitor, chelerythrine thus indicating the potential role of PKC activation in the H₂O₂-induced protection. Glibenclamide, a specific inhibitor of K_ATP reduced but did not abolish the H₂O₂-induced effects. However, cromakalim, a specific K_ATP opener yielded less protection than H₂O₂ or PMA. These findings suggest that K_ATP, at least partly, acts as an end effect of H₂O₂-induced protection.

A number of studies have indicated that the lethal cellular damage induced by Ca²⁺ PD is mainly due to massive Ca²⁺ influx during Ca²⁺ repletion phase (see review [24]). Therefore, H₂O₂-induced protection appears to be due to attenuation of prominent increase in [Ca²⁺]_i during Ca²⁺ PD. In the Langendorff-perfused rat hearts, Nayler et al. [29]studied the mechanism of the Ca²⁺ entry into the myocardium during Ca²⁺ PD and suggested that Ca²⁺ entry consisted of at least two components, the transient initial component through voltage-dependent Ca²⁺ channel and the second component through Na⁺–Ca²⁺ exchanger. Furthermore, they suggested that the major increase of Ca²⁺ influx occurs via the Na⁺–Ca²⁺ exchanger. Using isolated myocytes, Lambert et al. [30]also reached the same conclusions as Nayler and colleagues.

It has been proposed that the activation of K_ATP leads to the shortening of action potential duration subsequent to increased K⁺ efflux and decreases Ca²⁺ influx via voltage-gated Ca²⁺ channels and consequently cellular damage is reduced [19]. Recently, Behring et al. [31]have demonstrated that K_ATP openers depressed the increase in cytosolic Ca²⁺ during sustained ischemia and reperfusion as determined by NMR and this effect was correlated with attenuation of ischemic contracture. We showed that the effects of cromakalim on Ca²⁺ were less pronounced than H₂O₂. This result indicates that Ca²⁺ extrusion by mechanisms associated with K_ATP during Ca²⁺ PD is not sufficient enough to yield the same protective effects as H₂O₂ or PMA on the Ca²⁺ PD. In addition, the partial attenuation of H₂O₂-mediated effect by K_ATP inhibitor further suggests that H₂O₂ also activates K_ATP.

PKC [32]is believed to be an important regulatory enzyme of the myocardium under both normal and abnormal conditions. The abolition of H₂O₂-mediated protection by PKC inhibition strongly points out the pivotal role of PKC in signal transduction pathways leading to protection against the Ca²⁺ PD. This is further strengthened by the fact that the protection can be duplicated by PMA before induction of the Ca²⁺ PD. The pathway by which H₂O₂ activates PKC is not clear. It is known that H₂O₂ activates PKC [16–18], although the mechanism by which H₂O₂ activates PKC is not clear. Gopalakrishna et al. [16]demonstrated that treatment with low concentration of H₂O₂ for a brief period increases PKC activity, which declined after longer periods of H₂O₂-exposure. H₂O₂ is known to increase [Ca²⁺]_I through various mechanisms [33]. We have recently demonstrated that a transient rise in Ca²⁺ as a result of Ca²⁺ preconditioning [22, 23]mediates the translocation of PKC- and PKC-, which seem to participate in different signal transduction pathways leading to cardiac protection against lethal injury associated with the Ca²⁺-overload [25]and ischemia [22, 23]. The role of PKC in preconditioning has been fairly established by several investigators [20–22, 33, 35]. There is likelihood that PKC stimulation influences various membrane ion channels which are important in regulating the ions movement.

Activation of K_ATP by PKC has recently been reported by several investigators [33–35]. Light et al. [35]suggests that PKC can activate K_ATP in physiological level of tissue ATP concentration as observed in brief (within 5 min) preconditioning ischemia, although K_ATP usually opens subsequent to decrease in tissue ATP level [36]. Treatment with low concentration of H₂O₂ as well as brief ischemia hardly influences tissue ATP contents [11]. Thus, the mechanism of protection by H₂O₂ may be due to its direct activation of K_ATP or indirectly mediated by PKC. However, H₂O₂-induced signaling pathway appears to be more complicated, since our results show that the protection was blocked totally by chelerythrine but only partial loss of protection was observed with glibenclamide. Thus it appears that PKC activation by H₂O₂ contributes to the cardioprotection through the opening of K_ATP as well as other mechanisms. For example, the activation of PKC results in decrease in [Ca²⁺]_I through the modification of L-type Ca²⁺ channels [37]and SR Ca²⁺-ATPase [38]. Furthermore, activated PKC might indirectly reduce Ca²⁺ influx through Na⁺–Ca²⁺ exchanger. PKC enhances Na⁺–K⁺ ATPase activity [39], which is usually depressed by severe myocardial injury, leading to decrease in Na⁺ accumulation [40]. The latter mechanism may reduce the Ca²⁺ influx through Na⁺–Ca²⁺ exchanger in myocardium subjected to the Ca²⁺ PD.

It is generally believed that the massive production of oxygen radicals after sustained ischemia and reperfusion is involved with the pathogenesis of ischemia/reperfusion injury. However, exposure of hearts to lower concentration of reactive oxygen species before prolonged ischemia may prime hearts with alterations of ion channels through PKC activation, resulting in protection. A recent report from Tritto et al. [41]in which ischemic preconditioning was induced by low concentration of oxygen radicals and was blocked with oxygen radical scavengers supports our hypothesis that low dose of oxygen radicals plays a crucial role in cardioprotection by preconditioning phenomenon.

In summary, we have demonstrated that H₂O₂ triggers the protective responses and that PKC plays a crucial role in the signal transduction pathways leading to protection against the lethal cellular injury induced by the Ca²⁺ PD.

Time for primary review 16 days.

	Acknowledgements

 
This study was supported in part by NIH research grants HL23597 and HL55678 from the National Heart, Lung and Blood Institute. The authors thank Atif Ashraf for technical assistance.

	References

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