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Cardiovascular Research 1998 37(3):691-699; doi:10.1016/S0008-6363(97)00249-6
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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 (KATP) 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 KATP opener, cromakalim also provided protection similar to H2O2 against the Ca2+ PD injury. However, a specific KATP 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; KATP 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 Formula ) 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 (KATP) [13–15]and protein kinase C (PKC) [16–18]. The accumulated experimental findings suggest that KATP 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 KATP 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 KATP. To elucidate the role of KATP in H2O2-induced protective effects, we examined whether a KATP opener, cromakalim, could mimic the effects of H2O2 and a selective inhibitor of KATP, 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 KATP 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).


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  		Table 1 Effect of various interventions on hemodynamic changes in hearts subjected to the calcium paradox

 

Figure 1
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  		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).

 

Figure 2
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  		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).


Figure 3
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  		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).

 

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  		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 2Go).

3.4 Effects of H2O2 and KATP 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 4GoGo). 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).


Figure 4
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  		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 KATP 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 5GoGo). 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.


Figure 5
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  		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 KATP reduced but did not abolish the H2O2-induced effects. However, cromakalim, a specific KATP opener yielded less protection than H2O2 or PMA. These findings suggest that KATP, 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 KATP 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 KATP 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 KATP 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 KATP inhibitor further suggests that H2O2 also activates KATP.

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-{alpha} and PKC-{delta}, 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 KATP by PKC has recently been reported by several investigators [33–35]. Light et al. [35]suggests that PKC can activate KATP in physiological level of tissue ATP concentration as observed in brief (within 5 min) preconditioning ischemia, although KATP 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 KATP 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 KATP 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
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 

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