Kobayashi Satoru, Zhao Fengyi, Zhang Ziying, Kobayashi Tamayo, Huang Yuan, Shi Bingyin, Wu Weihua, Liang Qiangrong
Department of Biomedical Sciences, New York Institute of Technology, College of Osteopathic Medicine, Old Westbury, NY, United States.
Department of Endocrinology, The First affiliated Hospital of Xi'an Jiaotong University, Xi'an, China.
Front Physiol. 2020 Dec 10;11:604069. doi: 10.3389/fphys.2020.604069. eCollection 2020.
Hyperglycemia-induced mitochondrial dysfunction plays a key role in the pathogenesis of diabetic cardiomyopathy. Injured mitochondrial segments are separated by mitochondrial fission and eliminated by autophagic sequestration and subsequent degradation in the lysosome, a process termed mitophagy. However, it remains poorly understood how high glucose affects the activities of, and the relationship between, mitochondrial fission and mitophagy in cardiomyocytes. In this study, we determined the functional roles of mitochondrial fission and mitophagy in hyperglycemia-induced cardiomyocyte injury. High glucose (30 mM, HG) reduced mitochondrial connectivity and particle size and increased mitochondrial number in neonatal rat ventricular cardiomyocytes, suggesting an enhanced mitochondrial fragmentation. SiRNA knockdown of the pro-fission factor dynamin-related protein 1 (DRP1) restored mitochondrial size but did not affect HG toxicity, and Mdivi-1, a DRP1 inhibitor, even increased HG-induced cardiomyocyte injury, as shown by superoxide production, mitochondrial membrane potential and cell death. However, DRP1 overexpression triggered mitochondrial fragmentation and mitigated HG-induced cardiomyocyte injury, suggesting that the increased mitochondrial fission is beneficial, rather than detrimental, to cardiomyocytes cultured under HG conditions. This is in contrast to the prevailing hypothesis that mitochondrial fragmentation mediates or contributes to HG cardiotoxicity. Meanwhile, HG reduced mitophagy flux as determined by the difference in the levels of mitochondria-associated LC3-II or the numbers of mitophagy foci indicated by the novel dual fluorescent reporter mt-Rosella in the absence and presence of the lysosomal inhibitors. The ability of HG to induce mitochondrial fragmentation and inhibit mitophagy was reproduced in adult mouse cardiomyocytes. Overexpression of Parkin, a positive regulator of mitophagy, or treatment with CCCP, a mitochondrial uncoupler, induced mitophagy and attenuated HG-induced cardiomyocyte death, while Parkin knockdown had opposite effects, suggesting an essential role of mitophagy in cardiomyocyte survival under HG conditions. Strikingly, Parkin overexpression increased mitochondrial fragmentation, while DRP1 overexpression accelerated mitophagy flux, demonstrating a reciprocal activation loop that controls mitochondrial fission and mitophagy. Thus, strategies that promote the mutual positive interaction between mitochondrial fission and mitophagy while simultaneously maintain their levels within the physiological range would be expected to improve mitochondrial health, alleviating hyperglycemic cardiotoxicity.
高血糖诱导的线粒体功能障碍在糖尿病性心肌病的发病机制中起关键作用。受损的线粒体片段通过线粒体分裂被分离，并通过自噬隔离以及随后在溶酶体中的降解而被清除，这一过程称为线粒体自噬。然而，高糖如何影响心肌细胞中线粒体分裂和线粒体自噬的活性以及它们之间的关系，目前仍知之甚少。在本研究中，我们确定了线粒体分裂和线粒体自噬在高血糖诱导的心肌细胞损伤中的功能作用。高糖（30 mM，HG）降低了新生大鼠心室心肌细胞的线粒体连通性和颗粒大小，并增加了线粒体数量，提示线粒体碎片化增强。促分裂因子动力相关蛋白1（DRP1）的小干扰RNA敲低恢复了线粒体大小，但不影响HG毒性，而DRP1抑制剂Mdivi-1甚至增加了HG诱导的心肌细胞损伤，超氧化物生成、线粒体膜电位和细胞死亡情况均表明了这一点。然而，DRP1过表达引发线粒体碎片化并减轻了HG诱导的心肌细胞损伤，这表明在HG条件下培养的心肌细胞中，增加的线粒体分裂是有益而非有害的。这与线粒体碎片化介导或促成HG心脏毒性的主流假说相反。同时，如通过线粒体相关的LC3-II水平差异或在有无溶酶体抑制剂情况下新型双荧光报告基因mt-Rosella所示的线粒体自噬灶数量所确定的，HG降低了线粒体自噬通量。HG诱导线粒体碎片化和抑制线粒体自噬的能力在成年小鼠心肌细胞中也得到了重现。线粒体自噬的正向调节因子Parkin的过表达或线粒体解偶联剂CCCP的处理诱导了线粒体自噬并减轻了HG诱导的心肌细胞死亡，而Parkin敲低则产生相反的效果，这表明线粒体自噬在HG条件下心肌细胞存活中起重要作用。引人注目的是，Parkin过表达增加了线粒体碎片化，而DRP1过表达加速了线粒体自噬通量，证明了一个控制线粒体分裂和线粒体自噬的相互激活环。因此，促进线粒体分裂和线粒体自噬之间相互正向作用同时将它们的水平维持在生理范围内的策略有望改善线粒体健康，减轻高血糖心脏毒性。
