Centre of Clinical Laboratory, the First Affiliated Hospital of Soochow University, Suzhou 215006, China.
Institutes of Biology and Medical Sciences, Soochow University, Suzhou 215123, China.
Acta Pharmacol Sin. 2021 Jun;42(6):909-920. doi: 10.1038/s41401-020-00526-y. Epub 2020 Sep 23.
The protein levels and activities of calpain-1 and calpain-2 are increased in cardiac mitochondria under pathological conditions including ischemia, diabetes, and sepsis, and transgenic overexpression of mitochondrial-targeted calpain-1 induces dilated heart failure, which underscores an important role of increased calpain in mitochondria in mediating myocardial injury. However, it remains to be determined whether selective inhibition of calpain in mitochondria protects the heart under pathological conditions. In this study, we generated transgenic mice overexpressing mitochondrial-targeted calpastatin in cardiomyocytes. Their hearts were isolated and subjected to global ischemia/reperfusion. Hyperglycemia was induced in the transgenic mice by injections of STZ. We showed that transgenic calpastatin was expressed exclusively in mitochondria isolated from their hearts but not from other organs including skeletal muscle and lung tissues. Transgenic overexpression of mitochondrial-targeted calpastatin significantly attenuated mitochondrial oxidative stress and cell death induced by global ischemia/reperfusion in isolated hearts, and ameliorated mitochondrial oxidative stress, cell death, myocardial remodeling and dysfunction in STZ-treated transgenic mice. The protective effects of mitochondrial-targeted calpastatin were correlated with increased ATP5A1 protein expression and ATP synthase activity in isolated hearts subjected to global ischemia/reperfusion and hearts of STZ-treated transgenic mice. In cultured rat myoblast H9c2 cells, overexpression of mitochondrial-targeted calpastatin maintained the protein levels of ATP5A1 and ATP synthase activity, prevented mitochondrial ROS production and decreased cell death following hypoxia/reoxygenation, whereas upregulation of ATP5A1 or scavenging of mitochondrial ROS by mito-TEMPO abrogated mitochondrial ROS production and decreased cell death. These results confirm the role of calpain in myocardial injury, suggesting that selective inhibition of calpain in myocardial mitochondria by mitochondrial-targeted calpastatin is an effective strategy for alleviating myocardial injury and dysfunction in cardiac pathologies.
在包括缺血、糖尿病和败血症在内的病理条件下，钙蛋白酶-1 和钙蛋白酶-2 的蛋白水平和活性在心脏线粒体中增加，并且线粒体靶向钙蛋白酶-1 的转基因过表达诱导扩张性心力衰竭，这突显了增加的钙蛋白酶在介导心肌损伤中的重要作用。然而，仍然需要确定在病理条件下选择性抑制线粒体中的钙蛋白酶是否能保护心脏。在这项研究中，我们生成了在心肌细胞中过表达线粒体靶向钙蛋白酶抑制剂的转基因小鼠。分离它们的心脏并进行整体缺血/再灌注。通过注射 STZ 诱导转基因小鼠发生高血糖症。我们表明，转基因钙蛋白酶抑制剂仅在从其心脏分离的线粒体中表达，而不在其他器官（包括骨骼肌和肺组织）中表达。转基因过表达线粒体靶向钙蛋白酶抑制剂显著减轻了整体缺血/再灌注分离心脏中诱导的线粒体氧化应激和细胞死亡，并改善了 STZ 处理的转基因小鼠中的线粒体氧化应激、细胞死亡、心肌重构和功能障碍。线粒体靶向钙蛋白酶抑制剂的保护作用与在整体缺血/再灌注分离心脏和 STZ 处理的转基因小鼠心脏中增加的 ATP5A1 蛋白表达和 ATP 合酶活性相关。在培养的大鼠成肌细胞 H9c2 细胞中，过表达线粒体靶向钙蛋白酶抑制剂维持 ATP5A1 的蛋白水平和 ATP 合酶活性，防止缺氧/复氧后线粒体 ROS 的产生并减少细胞死亡，而过表达 ATP5A1 或通过 mito-TEMPO 清除线粒体 ROS 则可消除线粒体 ROS 的产生并减少细胞死亡。这些结果证实了钙蛋白酶在心肌损伤中的作用，表明通过线粒体靶向钙蛋白酶抑制剂选择性抑制心肌线粒体中的钙蛋白酶是缓解心脏病理中心肌损伤和功能障碍的有效策略。
