Treatment options for myocarditis are currently limited. Inhibition of calpains has been shown to prevent Coxsackievirus B3 (CVB3)-induced cardiac injuries, but the underlying mechanism of action of calpains has not been elucidated. We investigated whether NOD-, LRR-, and pyrin domain-containing 3 (NLRP3) inflammasome participated in CVB3-induced myocarditis, and investigated the effects of calpain-1 on CVB3-induced cardiac injury. NLRP3 inflammasome was activated in CVB3-infected hearts, evidenced by elevated protein levels of NLRP3, N-terminal domain of Gasdermin D, and cleaved caspase-1, and the increased co-localization of NLRP3 and apoptosis-associated speck-like protein. The intraperitoneal administration of MCC950, a selective inhibitor of the NLRP3 inflammasome, led to decreased levels of serum creatine kinase-MB, cardiac troponin I, lactate dehydrogenase, interleukin-18, interleukin-1β, prevention of the infiltration of inflammatory cells, and improvement of cardiac function under CVB3 infection. Transgenic mice overexpressing the endogenous calpain inhibitor calpastatin (Tg-CAST mice) exhibited not only decreased apoptosis, inflammation, fibrosis, and enhanced cardiac function but also inhibition of NLRP3 inflammasome and pyroptosis. The selective inhibition of calpain-1 using PD151746 protected cardiomyocytes in vitro from CVB3 infection by downregulating NLRP3 inflammasome and, thus, preserved cell viability. Mechanistically, we showed that mitochondrial dysfunction preceded inflammatory response after CVB3 treatment and elimination of mitochondrial reactive oxygen species (ROS) using mitochondria-targeted antioxidants (mito-TEMPO) recapitalized the phenotype observed in Tg-CAST mice. Furthermore, the promotion or inhibition of calpain-1 activation in vitro regulated the mitochondrial respiration chain. Mito-TEMPO reversed calpain-1-mediated NLRP3 inflammation activation and cell death. We also found that mitochondrial calpain-1, which was increased after CVB3 stimulation, activated the NLRP3 inflammasome and resulted in cell death. Furthermore, ATP synthase-α (ATP5A1) was revealed to be the cleaving target of calpain-1 after CVB3 treatment. Downregulating ATP5A1 using ATP5A1-small interfering RNA impaired mitochondrial function, decreased cell viability, and induced NLRP3 inflammasome activation. In conclusion, CVB3 infection induced calpain-1 accumulation in mitochondria, and led to subsequent ATP5A1 cleavage, mitochondrial ROS overproduction, and impaired mitochondrial function, eventually causing NLRP3 inflammasome activation and inducing pyroptosis. Therefore, our findings established the role of calpain in viral myocarditis and unveiled its underlying mechanism of its action. Calpain appears as a promising target for the treatment of viral myocarditis.

目前，心肌炎的治疗选择有限。钙蛋白酶的抑制已被证明可以预防柯萨奇病毒 B3 (CVB3) 引起的心脏损伤，但钙蛋白酶的潜在作用机制尚未阐明。我们研究了含有 NOD-、LRR- 和 pyrin 结构域的 3 (NLRP3) 炎性体是否参与了 ​​CVB3 诱导的心肌炎，并研究了钙蛋白酶 1 对 CVB3 诱导的心脏损伤的影响。NLRP3 炎性体在 CVB3 感染的心脏中被激活，NLRP3 蛋白水平升高、Gasdermin D 的 N 末端结构域和裂解的 caspase-1，以及 NLRP3 和凋亡相关斑点样蛋白的共定位增加证明了这一点。MCC950（NLRP3 炎性体的选择性抑制剂）的腹膜内给药导致血清肌酸激酶-MB、心肌肌钙蛋白 I、乳酸脱氢酶、IL-18、IL-1β，预防炎症细胞浸润，改善CVB3感染下的心功能。过表达内源性钙蛋白酶抑制剂 calpastatin 的转基因小鼠（Tg-CAST 小鼠）不仅表现出细胞凋亡减少、炎症、纤维化和心脏功能增强，而且还表现出对 NLRP3 炎性体和细胞焦亡的抑制。使用 PD151746 选择性抑制钙蛋白酶 1 通过下调 NLRP3 炎性体在体外保护心肌细胞免受 CVB3 感染，从而保持细胞活力。从机械上讲，我们表明，在 CVB3 处理后线粒体功能障碍先于炎症反应，并且使用线粒体靶向抗氧化剂 (mito-TEMPO) 消除线粒体活性氧 (ROS) 重塑了在 Tg-CAST 小鼠中观察到的表型。此外，体外促进或抑制 calpain-1 激活可调节线粒体呼吸链。Mito-TEMPO 逆转了钙蛋白酶 1 介导的 NLRP3 炎症激活和细胞死亡。我们还发现在 CVB3 刺激后增加的线粒体钙蛋白酶 1 激活 NLRP3 炎性体并导致细胞死亡。此外，ATP 合成酶-α (ATP5A1) 被发现是 CVB3 处理后钙蛋白酶-1 的切割靶标。使用 ATP5A1-小干扰 RNA 下调 ATP5A1 会损害线粒体功能，降低细胞活力，并诱导 NLRP3 炎性体激活。总之，CVB3 感染诱导线粒体中的钙蛋白酶 1 积累，并导致随后的 ATP5A1 切割、线粒体 ROS 过量产生和线粒体功能受损，最终导致 NLRP3 炎性体激活并诱导细胞焦亡。因此，我们的研究结果确立了钙蛋白酶在病毒性心肌炎中的作用，并揭示了其潜在的作用机制。钙蛋白酶似乎是治疗病毒性心肌炎的有前途的靶点。我们的研究结果确立了钙蛋白酶在病毒性心肌炎中的作用，并揭示了其潜在的作用机制。钙蛋白酶似乎是治疗病毒性心肌炎的有前途的靶点。我们的研究结果确立了钙蛋白酶在病毒性心肌炎中的作用，并揭示了其潜在的作用机制。钙蛋白酶似乎是治疗病毒性心肌炎的有前途的靶点。