Role Of PKC In Cardioprotective Mechanisms Of Exercise Preconditioning During Exhaustive Exercise In Rats

Objective: Ischemic preconditioning (IP) is a phenomenon that repeated episodes of brief and moderate vascular occlusion and reperfusion can render the ischemic heart more tolerant to a subsequent ischemic insult. Similar with IP, repeated short terms of high intensity exercise can also enhance the tolerance of the heart to a subsequent injury, termed“exercise preconditioning (EP)”. Although the powerful cardioprotective effect of EP has been proved, the underlied mechanisms still need further investigation. Based on the research of IP, this work examined the cardioprotection of EP to the exhausitive exercise, focused on the role of protein kinase C (PKC) in the transmembrane and intracellular signal transduction pathway of EP, and illustrated the alteration of PKC delta isoform (?PKC) and effector HSP70 in early and late protective phase of EP, and the relationship between them, then afforded new evidences to the cardioprotective effect of EP and its cellular and molecular mechanisms.Methods: 250 male healthy SD rats were randomly assigned to 10 groups. C group was control. EE group run to exhaustion on the treadmill, as a myocardial injury model. EEP group were subjected to a high-intensity interval exercise protocol on the treadmill, as EP model, and 0.5 h time point after EP was selected to observe the early cardioprotection of EP. CHE+ EEP group were injected PKC inhibitor chelerythrine ( CHE )intraperitoneally before EP. EEP+EE group run to exhaustion 0.5 h after EP. CHE+EEP+EE group were injected PKC inhibitor chelerythrine(CHE)intraperitoneally before EP, 0.5 h after EP, run to exhaustion. LEP group were subjected to the same EP protocol, and 24 h time point after EP was selected to observe the late cardioprotection of EP. CHE+LEP group were injected CHE intraperitoneally before EP. LEP+EE group run to exhaustion 24 h after EP. CHE+LEP+EE group were injected CHE intraperitoneally before EP, 24 h after EP, run to exhaustion. Myocardial injury was evaluated quantitatively in terms of the serum cardiac troponin I (cTnI) and N-terminal pro-brain natriuretic peptide (NT-proBNP) levels, the myocardial ischemia/hypoxia area, and the integral optical density (IOD) of haematoxylin–basic fuchsin–picric acid (HBFP) staining, and qualitatively in terms of the myocardial ultrastructure. The cardiac protein content of ?PKC?p-?PKC^Thr507and HSP70 were evaluated quantitatively in terms of westernblot, the cardiac gene expression of?PKC and HSP70 were evaluated by RT-PCR, and immunohistochemistry was performed to detect the distribution of?PKC?p-?PKC^Thr507and HSP70 in cardiomyocytes,?PKC mRNA expression in cardiomyocytes was examined by in situ hybridization.Results: (1) Compared with C group, exhaustive exercise induced significant high levels of serum cTnI, NT-proBNP, myocardial ischemia/hypoxia area and IOD value of HBFP staining, with the severe ultrastructural changes. During the early protective phase of EP, the serum cTnI and NT-proBNP levels, myocardial ischemia/hypoxia area and IOD value of HBFP staining after running to exhaustion decreased significantly, and the ultrastructural damage was attenuated obviously. During the late protective phase of EP, exercise tolerance of rats increased significantly, however, myocardial injury after running to exhaustion was aggravated, including worse ultrastructure, and significant high levels of myocardial ischemia/hypoxia area and IOD value, while the serum cTnI level was low, NT-proBNP level had no changes. During the early protective phase of EP, with PKC inhibitor chelerythrine (CHE)injection before EP, the serum cTnI levels, myocardial ischemia/hypoxia area and IOD value of HBFP stain, and myocardial ultrastructure after running to exhaustion had no obvious changes, except the lower serum NT-proBNP levels. During the late protective phase of EP, with CHE injection before EP, the serum NT-proBNP levels, myocardial ischemia/hypoxia area and IOD value of HBFP staining decreased significantly, and myocardial ultrastructure damage was attenuated, while the serum cTnI levels had no obvious changes. (2) Compared with C group, after running to exhaustion, myocardial?PKC and p-?PKC^Thr507 content increased significantly, while HSP70 content had no changes. The granular?PKC immunoreactive stain distributed homogeneously in the cytoplasm; HSP70 immunoreactive stain, fine sand, dispersed homogeneously in the cytoplasm; p-?PKC^Thr507 immunoreactive stain, pocked shape, scattered in the cytoplasm. The expression and distribution of?PKC mRNA in cardiomyocytes had no changes after exhaustive exercise, while HSP70 mRNA levels increased significantly. (3) Compared with C group, in the early protective phase of EP, myocardial?PKC and p-?PKC^Thr507 content increased significantly, while HSP70 content had no changes,?PKC and HSP70 distribution had no obviously changes, while p-?PKC^Thr507 translocated to intercalated disc, gap junction and nucleus from cytoplasm.?PKC mRNA and HSP70 mRNA expression changes had not been observed. After running to exhaustion, compared to the direct exhaustive exercise, p-?PKCThr507 maintained on intercalated disc, gap junction and nucleus myocardial?PKC and p-?PKCThr507 content decreased significantly,?PKC mRNA expression and distribution had no changes, while HSP70 mRNA expression decreased significantly. Although the myocardial HSP70 content had no alterations, the distribution of HSP70 in some cardiomyocytes was lost. (4) Compared with C group, in the late protective phase of EP, myocardial?PKC and HSP70 content were still significantly high, while p-?PKCThr507 content was normal, and still on the intercalated disc. Myocardial?PKC mRNA levels increase significantly, the signal of in situ hybridization enhanced in the cytoplasm, while HSP70 mRNA levels had no changes. After running to exhaustion, compared to the direct exhaustive exercise, p-?PKCThr507 content decreased significantly, and immunoreactive stain diminished. Myocardial?PKC content and distribution,?PKC mRNA expression and distribution had no alterations. HSP70 mRNA levels decreased significantly, HSP70 content had no changes, but the lost of HSP70 immunoreactive stain in cardiomyocytes was more obviously. (5) CHE has no influence on the expression and distribution of?PKC mRNA, but promotes?PKC protein expression. With injection of CHE, myocardial?PKC content increased significantly after running to exhaustion during the early protective phase of EP, during the late protective phase of EP, and after running to exhaustion during the late protective phase of EP, p-?PKCThr507 content increased significantly after running to exhaustion during the late protective phase of EP. After CHE injection, HSP70 content had a decreased tendency, but increased significantly in the late protective phase of EP, no distribution alteration was observed. With injection of CHE, HSP70 mRNA levels decreased significantly in the early protective phase of EP, and after exhaustive exercise in the late protective phase of EP.Conclusion: (1) Running to exhaustion on treadmill causes acute myocardial injury in rats. EP induces an early phase cardioprotection and enhances exercise tolerance in the late phase, however, had no significant cardioprotective effect to exhaustive exercise in the late phase. PKC inhibitor CHE has no influence on the early phase protective effect of EP, while attenuates the myocardial damage caused by running to exhaustion in the late protective phase. (2) p-?PKCThr507 translocating to intercalated disc, gap junction and nucleus from cytoplasm is an essential step of cellular signal transduction in cardioprotection of EP. CHE has no effect on?PKC mRNA expression and distribution, but promotes?PKC protein expression, especially in the late protective phase, which needs further investigations. (3) EP induces myocardial adaptation to stress with no alteration of HSP70 content in early phase, and attenuates the stress response during exhaustive exercise, leading to a less extent myocardial injury. CHE regulates the expression of HSP70, especially the gene expression, myocardial?PKC and HSP70 protein level increased together in the late protective phase, suggesting a relationship between?PKC and HSP70 regulation. (4) The high levels of myocardial?PKC and HSP70 content in the late protective phase of EP is important for the protective effect.