Ischemic preconditioning (IPC) is one of the most powerful interventions to reduce ischemia-reperfusion injury. The aim of the present study was to investigate the involvement of the phosphatidylinositol-3-kinases (PI3Ks) family in cardioprotection exerted by IPC and the relationship between preservation of mitochondrial morphology and ATP synthesis capacity. In this regard, macroautophagy (autophagy) is considered a dynamic process involved in the replacement of aged or defective organelles under physiological conditions. IPC consisted of four 5-min cycles of ischemia-reperfusion followed by sustained ischemia. Wortmannin (W), a PI3K family inhibitor, was added to the perfusion medium to study the involvement of autophagy in the beneficial effects of IPC. In the present study, LC3-II/I expression was significantly increased in the IPC group when compared with the control group. The hearts subjected to IPC showed greater degradation of p62 than control groups, establishing the existence of an autophagic flow. Electron microscopy showed that IPC preserves the structural integrity of mitochondria after ischemia and at the end of reperfusion. Moreover, hearts subjected to IPC exhibited increased mitochondrial ATP synthesis. The beneficial effects of IPC were abolished by W in all trials of this study, abolishing the differences between the IPC and control groups. These results suggest that IPC could partly reduce injury by ischemia-reperfusion (I/R) by decreasing mitochondrial damage and promoting autophagy. Since W is a nonspecific inhibitor of the PI3Ks family, further research is required to confirm participation of PI3K in the response to IPC.
Although autophagy is a prominent feature of myocardial ischaemia and reperfusion, its functional significance is unclear and controversial. In order to gain a deeper insight into the role of autophagy in myocardial ischaemia-reperfusion, we explored the effects of the pharmacological inhibitor of autophagy 3-methyladenine (3-MA). Isolated rat atria subjected to simulated 75-min ischaemia/75-min reperfusion (Is-Rs) in the presence or absence of 3-MA were used. The LC3-II/LC3-I ratio, an indicator of autophagosome formation, did not increase after ischaemia either in the presence or absence of 3-MA, but there was significant enhancement during reperfusion, which was prevented by the presence of 3-MA. The autophagy inhibitor also increased p62 protein, one of the specific substrates degraded through the autophagy-lysosomal pathway. Electron micrographs showed double membrane autophagosome-like structures during reperfusion, which were absent in atria subjected to Is-Rs in the presence of 3-MA. These findings suggest that this agent inhibited the autophagic flux under the present experimental conditions. Inhibition of autophagy during Is-Rs was accompanied by a high incidence of tachyarrhythmias during reperfusion, and a decrease in the maximal inotropic response to β-adrenergic and to calcium stimulation at the end of Is-Rs. Deterioration of mitochondrial morphology and function, without affecting cell viability, was observed in atria subjected to Is-Rs in the presence of 3-MA. The present results suggest an association between the inhibition of autophagy and functional alterations of the cells that have undergone sublethal stress, and have been able to recover in this experimental model of ischaemia-reperfusion.
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