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in chronically hypoxic rat myocardium: involvement in cardioprotection
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ek Novák,2t'ádal,1ek Kolá11Institute of Physiology, Academy of Sciences of the Czech Republic and Centre for Experimental Cardiovascular Research, 2Faculty of Science, Charles University, Prague, Czech Republic
Submitted 14 June 2004 ; accepted in final form 24 November 2004
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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and -
was performed in the cytosol and three particulate fractions of left ventricular myocardium. Infarct size was determined in open-chest animals subjected to 20-min coronary artery occlusion and 3-h reperfusion. The PKC inhibitors chelerythrine (1 or 5 mg/kg) or rottlerin (selective for PKC- isoform; 0.3 mg/kg) were administered intravenously as a single bolus 15 min before ischemia. Chronic hypoxia had no effect on the expression and distribution of PKC-. The relative amount of PKC- increased in the cytosol and nuclear-cytoskeletal, mitochondrial, and microsomal fractions of chronically hypoxic myocardium by 100%, 212%, 237%, and 146%, respectively, compared with corresponding normoxic values. Chronic hypoxia decreased the size of myocardial infarction (normalized to the area at risk) by about one-third on the average (P < 0.05). Both doses of chelerythrine tended to reduce infarction in controls, and only the high dose completely abolished the improvement of ischemic tolerance in hypoxic hearts (P < 0.05). Rottlerin attenuated the infarct size-limiting effect of chronic hypoxia (P < 0.05), and it had no effect in controls. These results suggest that chronic intermittent hypoxia-induced cardioprotection in rats is partially mediated by PKC-; the contribution of other isoforms remains to be determined.
ischemia-reperfusion; infarct size; protein kinase C
Recent studies demonstrated that mitochondrial ATP-sensitive K+ (mitoKATP) channels play a crucial role in the cardioprotective mechanism of long-term chronic hypoxia (3, 7, 32, 49) as well as in short-lived protection induced by various preconditioning stimuli including its early and delayed forms (for a review, see Ref. 11). The opening of myocardial mitoKATP channels is associated with the activation of PKC, which appears to be a key player in signal transduction of preconditioning (28, 38, 46), although the sequence of these events is a matter of debate. Among all PKC isoforms that have been detected in the myocardium, mainly two of the novel isoforms, and , have been proposed as the most likely candidates for mediating protection in preconditioned rat hearts. Western blot analysis has demonstrated that cardiac protection by preconditioning is associated with a distinct pattern of translocation of - and -isoforms from the cytosol to various membrane compartments (8, 19, 33, 41).
Compared with preconditioning, much less is known about the involvement of PKC in the mechanism by which chronic hypoxia protects the heart against acute ischemia-reperfusion injury. As recently reported (36), only PKC- is activated and translocated from the cytosol to the particulate fraction in chronically hypoxic infant human and neonatal rabbit myocardium. Chelerythrine completely abolished both the PKC- translocation and cardioprotection induced by chronic hypoxia but had no effect in normoxic rabbit hearts. It has been proposed that the activation of PKC- mediated the increased cardiac ischemic tolerance in this experimental model (36). To our knowledge, the potential role of PKC in cardioprotection by chronic hypoxia has not been examined in other species and models. The present study was, therefore, designed to characterize the influence of chronic intermittent hypoxia on the expression and distribution of PKC isoforms- and - in the left ventricular (LV) myocardium of adult rats. The potential involvement of PKC in the protection conferred by chronic hypoxia was studied by examining the effects of the PKC inhibitors chelerythrine and rottlerin (-isoform selective) on the size of myocardial infarction induced by coronary artery occlusion in the in vivo open-chest animals.
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MATERIALS AND METHODS |
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Tissue fractionation. The animals designed for PKC determination were killed by decapitation, and their hearts were rapidly excised, washed in cold (0°C) saline, and dissected into the left and right free ventricular walls and the septum on the ice dish. All parts were frozen in liquid nitrogen, weighed separately, and stored at –70°C until use. The LV myocardium was cut by scissors and minced using Turrax (twice for 30 s), followed by Potter-Elvehjem homogenization in 10 vol of ice-cold buffer composed of (in mmol/l) 12.5 Tris·HCl (pH 7.4), 250 sucrose, 2.5 EGTA, 1 EDTA, 100 NaF, 5 DTT, 0.3 phenylmethylsulfonyl fluoride, 0.2 leupeptin, and 0.02 aprotinin. The homogenate was fractionated according to the method described previously (13). Briefly, the homogenate was centrifuged at 100 g for 20 min to remove cellular debris and unbroken cells. The supernatant was then centrifuged at 1,000 g for 10 min to obtain a nuclear-cytoskeletal-enriched fraction in the pellet, followed by centrifugation of the supernatant at 8,000 g for 10 min to obtain a mitochondria-enriched fraction in the pellet. The remaining supernatant was subjected to centrifugation at 100,000 g for 60 min. The resulting pellet represented the microsomal fraction; the last supernatant was the cytosolic fraction. The homogenate and pellets of nuclear-cytoskeletal, mitochondrial, and microsomal fractions were resuspended in homogenization buffer containing 1% Triton X-100 held on ice for 60 min and then centrifuged at 100,000 g for a further 60 min. The resulting detergent-treated supernatants were used for Western blot analyses. Triton X-100 was added to the cytosolic fraction to reach the final concentration of 1%. Protein content was determined according to the Lowry assay modified by Peterson (35). All of the chemicals were purchased from Sigma unless otherwise indicated.
Western blot analysis of PKC isoforms.
Detergent-treated extracts of subcellular fractions were subjected to SDS-PAGE electrophoresis on a 8% bis-acrylamide polyacrylamide gel at 20 mA/gel for 90 min on a Mini-Protean II apparatus (Bio-Rad). After electrophoresis, the resolved proteins were transferred to a nitrocellulose membrane (Amersham). Membranes were incubated in 5% dry low-fat milk in Tris-buffered saline with Tween 20 (TTBS) for 60 min at room temperature to block nonspecific protein binding. After being washed in TTBS buffer (3 times quickly, 3x 5 min each), the membranes were probed with PKC- and PKC- isoform-specific primary rabbit antisera (Sigma; 1:8,000 in TTBS) for 90 min at room temperature. It has been proved that these PKC- and PKC- antibodies are specific and do not cross-react with other PKC isoforms (2). The membranes were washed again and incubated with the secondary swine anti-rabbit IgG antibody labeled with horseradish peroxidase (Sevapharma; 1:4,000 in TTBS) for 60 min at room temperature. Before enhanced chemiluminescence (ECL), the membranes were washed as described above and stored in TTBS for at least 2 h. For ECL, substrates A (Luminol solution) and B (H2O2 solution) were prepared, mixed 1:1 just before use, and poured on membranes. The specific signal was detected on the autoradiographic film (Amersham). Scanning (Epson Perfection 1240U Scanner) and ImageQuant software were used for quantification of the relative abundance of individual PKC isoforms. The amount of protein applied to the gel varied for each isoform and fraction to achieve linearity with the intensity x area (volume) of the band on the Western blot. To ensure the specificity of PKC-- and PKC--immunoreactive proteins, prestained molecular weight protein standards (Fluka), recombinant human PKC- and PKC- standards (Sigma), rat brain extract, and the respective blocking immunizing peptides (Sigma) were used.
Infarct size determination. Animals were anesthetized with pentobarbital sodium (60 mg/kg ip, Sanofi). Heparinized cannulas were placed in the left carotid artery for blood pressure monitoring with a pressure transducer (Gould P23Gb) and in the right jugular vein for drug administration. Tracheotomy was performed, and the rats were intubated with a cannula connected to a rodent ventilator (Columbus Instruments) and ventilated with room air at 68 strokes/min (tidal volume of 1.2 ml/100 g body wt). Blood pressure signal was stored in a computer and subsequently analyzed by our custom-designed software. The heart rate (HR) was derived from the blood pressure curve. The rectal temperature was maintained between 36.5 and 37.5°C by a heated table throughout the experiment.
A left thoracotomy was performed, and the pericardium was removed to reveal the location of the left anterior descending (LAD) coronary artery. A polyester ligature 6/0 (Ethibond, Ethicon) was then placed around the LAD coronary artery about 1–2 mm distal to the origin, and an occlusive snare was placed around it. The ends of the suture were threaded through a polyethylene tube. After the surgical preparation, the rats were allowed to stabilize for 10 min before the ischemic interventions. Regional myocardial ischemia was induced by tightening the ligature placed around the coronary artery. A transient decrease in blood pressure verified the coronary artery occlusion. After the occlusion, the snare was released; reperfusion of previously ischemic tissue was indicated by transient decrease of blood pressure and appearance of reperfusion arrhythmias.
In the first experimental series, the effect of rottlerin on myocardial infarct size (IS) was examined in chronically hypoxic and normoxic rats. Rottlerin (BioMol) was dissolved in DMSO and then diluted with saline and administered into the jugular vein in a dose of 0.3 mg/kg as a single bolus (1 ml/kg) 15 min before ischemia; the final dose of DMSO was 6 µl/kg. Control rats were treated in the corresponding way with saline containing the same dose of DMSO. In subsequent experiments, other groups of hypoxic and normoxic animals were treated with chelerythrine (Sigma) dissolved in saline, sonicated, and administered in the same way as rottlerin in a dose of 1 or 5 mg/kg. Corresponding controls were treated with saline (1 ml/kg). All animals were subjected to 20-min coronary artery occlusion followed by 3-h reperfusion.
After reperfusion, the hearts were excised and washed with 20 ml saline through the cannulated aorta. The area at risk (AAR) and IS were determined as described earlier (31) by staining with potassium permanganate and 2,3,5-triphenyltetrazolium chloride. The hearts were cut perpendicularly to the long axis of the ventricle into slices 1 mm thick and stored overnight in 10% neutral formaldehyde solution. The next day after the infarct size staining, the right ventricular (RV) free wall was separated, and both sides of slices were photographed by a digital camera (Olympus). The IS, the size of the AAR, and the size of the LV were determined by a computerized planimetric method. The IS was normalized to the AAR and LV (IS/AAR) and the size of the AAR was normalized to the LV (AAR/LV).
Statistical analysis. The results are expressed as means ± SE. One-way ANOVA and subsequent Student-Newman-Keuls test were used for comparison of differences in normally distributed variables between groups. Differences were taken to be statistically significant when P < 0.05.
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RESULTS |
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and PKC- immunoreactivities were detected on Western blots as single bands that were confirmed by respective blocking peptides, recombinant human PKC standards, and a positive control from rat brain homogenate (Fig. 1, A and B).
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in all fractions compared with the respective normoxic control values (Fig. 2, A and B). This upregulation was more pronounced in particulate fractions (nuclear by 212%, mitochondrial by 237%, microsomal by 146%) than in the cytosol (by 100%). Figure 2C shows the distribution of PKC- between the cytosol and particulate fractions from control normoxic and chronically hypoxic hearts. In normoxic tissue, PKC- was localized predominantly in particulate fractions: it was most abundant in microsomal and nuclear-cytoskeletal fractions and to a lesser extent in the mitochondrial one. Chronic hypoxia led to a significant redistribution of PKC- from the cytosol to nuclear-cytoskeletal and mitochondrial fractions but not to the microsomal fraction; no appreciable redistribution of this isoform among particulate fractions occurred.
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was not significantly affected by chronic hypoxia in any subcellular fraction (Fig. 3, A and B). In normoxic tissue, this isoform was evenly distributed between cytosolic and nuclear fractions and doubled in the microsomal fraction, whereas a lower expression was observed in the mitochondrial fraction. Chronic hypoxia did not affect the distribution of PKC- among the fractions (Fig. 3C).
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DISCUSSION |
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in the LV myocardium, whereas PKC- remained unchanged. The upregulation of PKC- was associated with its redistribution from the cytosol to nuclear-cytoskeletal and mitochondrial fractions. This process may act in targeting of the activated enzyme to specific substrates located in these subcellular compartments. Previous reports dealing with chronic myocardial hypoxia reached rather diverse results. Rouet-Benzineb et al. (37) found an increase in total PKC content and a transient stimulation of the enzyme activity in hypertrophied RVs of rats adapted to normobaric hypoxia (10% O2) for 3 wk. However, no change occurred in the nonhypertrophied LVs. In line with these data, PKC- and PKC- were upregulated only in the RVs of rats exposed for 2 wk to hypobaric hypoxia of 5,500 m (29). In contrast, the activation and translocation of PKC- from the cytosolic to the particulate fraction were observed in the LVs of chronically hypoxic neonatal rabbits and in the right atrial myocardium of children undergoing surgery for congenital cyanotic heart defects (36). The explanation of these discrepancies is not clear, but obviously species and/or ontogenetic differences may play a role. In addition, the presence or absence of LV hypertrophy may be responsible for the diverse results of the rat studies. Unlike in the above-mentioned reports (29, 37), exposure to more severe hypoxia (7,000 m) of intermittent nature led not only to RV hypertrophy due to pulmonary hypertension but also to moderate hypertrophy of the LV in the present study. This response, which is likely the consequence of stress, is in agreement with our previous reports using the same experimental model (16, 34). It is well known that PKC is involved in hypertrophic signal transduction, and it appears that various isoforms may have different roles in this process (5, 6a, 39). Concerning the -isoform, it has been shown to be involved in both the induction and progression of pressure overload myocardial hypertrophy (5). Thus it seems that the increased abundance of PKC- in the chronically hypoxic LVs in our study is related to the process of hypertrophy rather than to chronic tissue hypoxia itself. This view is supported by the observation that cultured neonatal rat ventricular myocytes, subjected to 24-h hypoxia, exhibited translocation of PKC- from the cytosolic to the particulate compartment, whereas PKC- shifted in the opposite way, i.e., to the cytosol (12). We confirmed previous observations that hearts of chronically hypoxic rats are more tolerant to lethal myocardial injury induced by coronary artery occlusion. The degree of protection was somewhat lower in the chelerythrine series of experiments than in the rottlerin series. The cause of this difference is not clear: besides different solvents used, seasonal variation cannot be excluded as these two experimental series were performed with a 3-mo interval between them.
The novel finding is that the PKC- isoform-selective inhibitor rottlerin significantly attenuated this protective effect. It suggests that the activation of PKC- during the acute ischemia-reperfusion insult is involved in the infarct size-limiting effect of chronic hypoxia. The detailed molecular mechanism by which chronic hypoxia improves cardiac ischemic tolerance is unknown, but adaptive changes of mitochondrial functions appear to be of essential importance. In particular, the activation of mitoKATP channels has been proposed to play a crucial role in this form of protection (32). Our observation of the most prominent upregulation and redistribution of PKC- to the mitochondrial fraction is in line with the view that this enzyme is linked with mitoKATP channel opening (41). Similarly, various preconditioning stimuli that involve mitoKATP channel activation are also associated with the translocation of PKC- to mitochondria. This translocation has been demonstrated in classic ischemic preconditioning (8, 41) as well as in various forms of pharmacological and anesthetic preconditioning (1, 10, 25, 41, 42).
To examine whether other PKC isoforms could also participate in the protective mechanism of chronic hypoxia, we measured the infarct size in rats treated with chelerythrine, a general PKC inhibitor. Chelerythrine had no effect on cardioprotection in chronically hypoxic rats at the low dose of 1 mg/kg, which has been shown to be sufficient to abolish the IS-lowering effects of both ischemic and pharmacological preconditioning (9, 47). Only at the high dose of 5 mg/kg, which is commonly used for in vivo rat experiments, did chelerythrine completely abolish the protection induced by chronic hypoxia. This suggests that, apart from PKC-, other PKC isoform(s) play a role in mediating this protective phenomenon. However, the absence of any appreciable effect of chronic hypoxia on PKC- abundance and distribution seems to exclude this particular isoform as a potential candidate.
Unlike in the chronically hypoxic rats, chelerythrine tended to decrease the IS in normoxic animals. This observation is not exceptional, although the majority of studies did not show any protective effect in the controls. For example, Lasley et al. (23) demonstrated a reduction of myocardial infarction by chelerythrine in rabbits that was significant at a low dose (0.1 mg/kg) but not at a higher dose (3.8 mg/kg); a similar protective effect occurred in isolated perfused hearts. A tendency to IS reduction by chelerythrine was seen in open-chest rat studies (9, 22). It has been suggested that the cardioprotective effect of PKC inhibition was mediated by an increase in Na+-K+-ATPase activity and subsequent limitation of intracellular Na+ overload (26). Moreover, in view of the observation that chelerythrine at a dose of 5 mg/kg may exert rapid proapoptotic actions (45), its influence on cardiac ischemic tolerance and cardioprotection should be interpreted with caution with respect to the role of PKC.
Our results are in partial disagreement with a still single study that examined the involvement of PKC in the cardioprotective effect of chronic hypoxia. In isolated perfused hearts of neonatal rabbits raised at hypoxic atmosphere, chelerythrine completely abolished the improved postischemic recovery of contractile function and prevented the activation and translocation of PKC- (36). These authors proposed that adaptation to hypoxia maintained PKC- in a chronically active state with activation shuttling the enzyme between the cytosol and particulate fractions, resulting in the activation of downstream protective pathways. We cannot confirm this view in adult chronically hypoxic rats, where the -isoform rather than the -isoform seems to play a role. Again, both species and ontogenetic differences should be taken into account as well as differences in experimental models and protocols. Interestingly, PKC- does not seem to mediate the IS-limiting effect of chronic low-dose ethanol drinking in adult rats (14).
A similar controversy exists about the role of PKC in short-lived preconditioning: experimental studies are not consistent in determining which PKC isoform is crucial for cardioprotection. The -isoform, which is strongly upregulated in chronically hypoxic hearts in the present study, appears to play a role in various forms of pharmacological preconditioning, particularly in rats and mice. It has been shown, for example, that rottlerin abolished 1-opioid receptor-mediated protection in open-chest rats (10) and anesthetic preconditioning in isolated perfused rat hearts or superfused trabeculae (6, 41) as well as diazoxide- or adenosine-induced delayed protection in isolated mouse hearts (21, 43, 48). PKC- inhibition also abolished ischemic preconditioning in isolated rat hearts (41). In contrast, rottlerin had no effect on the IS-limiting effect of ischemic preconditioning in open-chest rats in the study of Fryer et al. (8). Recent experiments suggest that PKC- may play even an opposite, detrimental role in cardiac ischemic tolerance during the reperfusion phase: the inhibition of this isoform with a selective antagonist peptide V1–1 only at reperfusion resulted in cardioprotection (18, 30). Further studies with isoform-specific PKC inhibitors are needed to resolve the complex involvement of this enzyme in the mechanism of increased ischemic tolerance of preconditioned or chronically hypoxic hearts.
In conclusion, we have shown that PKC-, but not PKC-, is markedly upregulated in various subcellular compartments of the chronically hypoxic rat myocardium. The protective effect of chronic hypoxia on the size of myocardial infarction induced by coronary artery occlusion was completely abolished by the nonselective PKC inhibitor chelerythrine and significantly attenuated by the selective PKC- inhibitor rottlerin. It suggests that the activity of PKC, in particular of the PKC- isoform, during the acute ischemia-reperfusion insult plays a role in the cardioprotective mechanism afforded by chronic hypoxia.
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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.
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P < 0.05 vs. corresponding controls.