Department of Cardiology, Shanghai Institute of Cardiovascular Diseases, Zhongshan Hospital, Shanghai Medical College of Fudan University, Shanghai, 200030, China.
Department of General Practice, Zhongshan Hospital, Shanghai Medical College of Fudan University, Shanghai, 200030, China.
Cell Biol Toxicol. 2022 Jun;38(3):487-504. doi: 10.1007/s10565-021-09634-9. Epub 2021 Aug 8.
Cardiomyocyte apoptosis is critical for the development of viral myocarditis (VMC), which is one of the leading causes of cardiac sudden death in young adults. Our previous studies have demonstrated that elevated calpain activity is involved in the pathogenesis of VMC. This study aimed to further explore the underlying mechanisms. Neonatal rat cardiomyocytes (NRCMs) and transgenic mice overexpressing calpastatin were infected with coxsackievirus B3 (CVB3) to establish a VMC model. Apoptosis was detected with flow cytometry, TUNEL staining, and western blotting. Cardiac function was measured using echocardiography. Mitochondrial function was measured using ATP assays, JC-1, and MitoSOX. Mitochondrial morphology was observed using MitoTracker staining and transmission electron microscopy. Colocalization of dynamin-related protein 1 (Drp-1) in mitochondria was examined using immunofluorescence. Phosphorylation levels of Drp-1 at Ser637 site were determined using western blotting analysis. We found that CVB3 infection impaired mitochondrial function as evidenced by increased mitochondrial ROS production, decreased ATP production and mitochondrial membrane potential, induced myocardial apoptosis and damage, and decreased myocardial function. These effects of CVB3 infection were attenuated by inhibition of calpain both by PD150606 treatment and calpastatin overexpression. Furthermore, CVB3-induced mitochondrial dysfunction was associated with the accumulation of Drp-1 in the outer membrane of mitochondria and subsequent increase in mitochondrial fission. Mechanistically, calpain cleaved and activated calcineurin A, which dephosphorylated Drp-1 at Ser637 site and promoted its accumulation in the mitochondria, leading to mitochondrial fission and dysfunction. In summary, calpain inhibition attenuated CVB3-induced myocarditis by reducing mitochondrial fission, thereby inhibiting cardiomyocyte apoptosis. Calpain is activated by CVB3 infection. Activated calpain cleaves calcineurin A and converts it to active form which could dephosphorylate Drp-1 at Ser637 site. Then, the active Drp-1 translocates from the cytoplasm to mitochondria and triggers excessive mitochondrial fission. Eventually, the balance of mitochondrial dynamics is broken, and apoptosis occurs.
心肌细胞凋亡对于病毒性心肌炎（VMC）的发展至关重要，VMC 是导致年轻人心脏性猝死的主要原因之一。我们之前的研究表明，钙蛋白酶活性升高与 VMC 的发病机制有关。本研究旨在进一步探讨其潜在机制。用柯萨奇病毒 B3（CVB3）感染乳鼠心肌细胞（NRCMs）和过表达钙蛋白酶抑制剂的转基因小鼠，建立 VMC 模型。用流式细胞术、TUNEL 染色和 Western blot 检测细胞凋亡。用超声心动图测量心功能。用 ATP 测定、JC-1 和 MitoSOX 检测线粒体功能。用 MitoTracker 染色和透射电镜观察线粒体形态。用免疫荧光检测动力相关蛋白 1（Drp-1）在线粒体中的共定位。用 Western blot 分析测定 Drp-1 丝氨酸 637 位点的磷酸化水平。我们发现，CVB3 感染损害了线粒体功能，表现为线粒体 ROS 生成增加、ATP 生成和线粒体膜电位降低、诱导心肌细胞凋亡和损伤以及心功能降低。钙蛋白酶的抑制作用（通过 PD150606 处理和钙蛋白酶抑制剂过表达）减轻了 CVB3 感染的这些影响。此外，CVB3 诱导的线粒体功能障碍与 Drp-1 在线粒体外膜的积累以及随后线粒体裂变的增加有关。在机制上，钙蛋白酶切割并激活钙调神经磷酸酶 A，使 Drp-1 丝氨酸 637 位点去磷酸化，促进其在线粒体中的积累，导致线粒体裂变和功能障碍。总之，钙蛋白酶抑制通过减少线粒体裂变来减轻 CVB3 诱导的心肌炎，从而抑制心肌细胞凋亡。钙蛋白酶被 CVB3 感染激活。激活的钙蛋白酶切割钙调神经磷酸酶 A，并将其转化为活性形式，使 Drp-1 丝氨酸 637 位点去磷酸化。然后，活性 Drp-1 从细胞质转移到线粒体并引发过度的线粒体裂变。最终，线粒体动力学的平衡被打破，发生凋亡。
