In the current study, we have demonstrated that IPostC or HPostC can attenuate postischemic injury reflected by reduced infarct size, cell viability, plasma CK-MB, ROS generation and cellular apoptosis in nondiabetic mice or in H9c2 cells exposed to normal glucose. Moreover, we found that the cardioprotective effect induced by IPostC or HPostC is associated with the attenuation of PHLPP1 expression to mediate not only through the activation of Akt but also through the inactivation of Mst1. Meanwhile, we showed that inhibition of PHLPP1 or Mst1 can decrease the cellular damage in in vitro, but the effect is blocked by LY294002. Importantly, we showed that impaired IPostC cardioprotection in the diabetic heart is associated with excessive expression of PHLPP1. PHLPP1 knockdown preserves the protective effect induced by HPostC in H9c2 cells exposed to high glucose not only through the activation of Akt but also through the inactivation of Mst1.
IPostC has been proposed as a strategy against myocardial IR injury, and the cardioprotective effect has been confirmed in animal studies . Our previous study also confirmed that remote limb ischemic postconditioning could protect against myocardial IR injury in mice . In this study, a cardiac IR injury mouse model was established. AAR/LV ratio did not differ among all groups, indicating that the model in our study is reliable. Our data demonstrated that IPostC obviously attenuated the myocardial infarct size, plasma CK-MB release, cardiomyocytes apoptosis and oxidative stress after 45 min of ischemia and 2 h of reperfusion in mice. Meanwhile, HPostC significantly decreased the cellular damage, cleaved Caspase3 expression, oxidative stress and mPTP opening level reflected by JC-1 ratio (green/red) in H9c2 cells exposed to normal glucose after 4 h of hypoxia and 4 h of reoxygenation. Taken together, IPostC has a potent protective effect against cardiac IR injury.
Akt is an important protective kinase in the heart injury. It is well known that activation of Akt kinase protects the heart against IR injury . PHLPP1 is an endogenous negative regulator of Akt signaling in the heart. PHLPP1 knockdown elevates the Akt activity in cardiomyocytes and, in turn, provides protection against IR injury . Mst1 is the second important PHLPP1 target . Mst1 is a vital kinase of the Hippo signaling pathway that regulates the development and homeostasis of heart by mediating cell proliferation, apoptosis and cell fate decisions . The data demonstrated that HR stimulation gradually elevated PHLPP1 expression from 2 to 6 h, with the peak increase in PHLPP1 expression occurring after 4 h of reoxygenation. This result suggests that PHLPP1 is involved in the pathological process of myocardial HR injury. Meanwhile, to investigate the role of PHLPP1 in cardiomyocyte death, we examined whether PHLPP1 knockdown alone is sufficient to attenuate cellular apoptosis. The data indicated that siRNA-mediated knockdown of PHLPP1 gene could mitigate the cardiomyocyte damage induced by HR under normal glucose condition, accompanied with increased Akt activity and decreased Mst1 activity. However, the protective effect of PHLPP1 knockdown was blocked by PI3K/Akt inhibitor LY294002. Meanwhile, we found that Mst1 inhibitor XMU-MP-1 could also reduce HR-mediated cardiomyocyte injury. After myocardial IR or HR injury, the levels of PHLPP1 expression as well as Akt and Mst1 phosphorylation were elevated in the cardiomyocytes. Moreover, IPostC or HPostC could greatly attenuate the PHLPP1 expression in the cardiomyocyte, concomitant with the significant increment of Akt phosphorylation and reduction of Mst1 phosphorylation. Therefore, the data indicated that downregulation of PHLPP1 expression plays an important role in the effect of IPostC cardioprotection not only through the activation of Akt but also through the inactivation of Mst1.
Ischemic postconditioning results in a significant reduction in infarct size in young, healthy animals. However, major cardiovascular comorbidities such as hyperlipidemia, diabetes, and aging interfere with these cardioprotective mechanisms, thereby limiting the protective effects of myocardial ischemic postconditioning . It is tempting to see the effect of comorbidities on the failure of ischemic postconditioning in clinical translation. Therefore, such studies would be necessary to identify important pathways of cardioprotection that could be inactive in the presence of major cardiovascular comorbidities. Our data indicated that the myocardial infarct size, apoptosis and cellular injury were markedly increased in diabetic mice or H9c2 cells exposed to high glucose, and the result was consistent with our previous studies [9, 10, 20]. In addition, we observed that hyperglycemia eliminated the cardiac protective effects of IPostC, which fits well with previously reported studies that hyperglycemia could abolish the benefits of ischemic postconditioning [26, 27]. Previous studies have shown that increased cardiac PTEN and decreased cardiac TOPK or DJ-1 or STAT3 are responsible for the loss of diabetic heart sensitivity to ischemic or pharmacological postconditioning [10, 26–28], but the underlying molecular mechanism is completely unclear. Meanwhile, some studies indicated that several agents such hydrogen sulfide, deferoxamine and N-acetylcysteine were utilized to restore the myocardial protective effect of sevoflurane or ischemic postconditioning in diabetes [29–31], but most of them alone could protect heart against IR injury [32–34]. Therefore, the efficient and effective method has yet to be developed. have myocardial protection. In our study, our data indicated that either in STZ-induced diabetic mice or in H9c2 cells exposed to high glucose, the cardiac protective effects of IPostC were blocked, accompanied by increased PHLPP1 expression and Mst1 phosphorylation as well as decreased Akt phosphorylation. We have mentioned above that IPostC protected heart against IR injury via inhibition of PHLPP1 expression to elevate Akt phosphorylation and reduce Mst1 phosphorylation. Therefore, these data suggest that the underlying mechanism attributable to the limiting cardioprotection induced by IPostC is likely the abnormality of PHLPP1/Akt/Mst1 signaling pathway in diabetes. Then, we further confirmed these observations by inhibiting PHLPP1 expression in H9c2 cells exposed to high glucose. Our data showed that PHLPP1 knockdown by adenoviral vectors encoding siRNA decreased the cellular damage and oxidative stress in H9c2 cells exposed to high glucose, accompanied by increased Akt phosphorylation and decreased Mst1 phosphorylation. Moreover, the protective effects induced by PHLPP1 knockdown were blocked by LY294002. IPostC was added in the PHLPP1 knockdown H9c2 cells exposed to high glucose to partly decrease the cellular injury and oxidative stress, but it was not statistically significant. Taken together, these findings indicated that hyperglycemia-induced overexpression of PHLPP1 played a key role in the lost effect of IPostC cardioprotection.