Objective The purpose of this study was to investigate the potential cardioprotection roles of autophagy induced by ischemia/reperfusion (I/R) against apopotosis in cardiac myocytes, and tries to clarify the effects of PI3K in this process.
Methods We employed simulated I/R of neonatal rat ventricular myocytes as an in vitro model of I/R injury to the heart. Cardiac myocytes were exposed to 3h hypoxia followed by 14h of reoxygenation, and during the reoxygenation cardiac myocytes were treated with 10mM 3-Methyladenine (3-MA) to inhibit autophagy or 50uM rapamycin to enhance autophagy. Then, we used real-time quantitative PCR (qPCR) to investigate the expression levels of Beclin 1, Bim, and caspase-3. Western blot analysis was used to examine variation in the expression of LC3-II/I (a marker of autophagy), Bim, caspase-3 and PI3K.
Results Our results demonstrated that the mRNA expression of Beclin 1 and the ratio of LC3II/I were significantly increased when myocytes followed sI/R (simulated I/R). Moreover, autophagy enhanced by rapamycin during sI/R, significantly reduced the mRNA expression of Bim and caspase-3, and downregulation of the protein expression level of Bim, caspase-3 and PI3K. In contrast, the mRNA expression of Bim and caspase-3 was increased significantly, and the expression of Bim, caspase-3 and PI3K elevated when 3-MA inhibited the activation of autophagy.
Conclusions Our results demonstrate that autophagy induced by sI/R constitutes a powerful and previously uncharacterized protective mechanism against apopotosis in cardiac myocytes via downregulating the pro-apopototic protein Bim and caspase-3, which is downregulated by PI3K. Rapamycin, the autophagic induction, can be used to reduced damage of fatal I/R injury via protecting against apoptosis, which may provide novel strategies for clinical treatment of I/R injury.
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