心肌炎的治疗选择目前是有限的。抑制钙蛋白酶已被证明可以预防柯萨奇病毒B3(CVB3)引起的心脏损伤,但是钙蛋白酶的潜在作用机制尚未阐明。我们调查了NOD-,LRR-,含pyrin结构域3(NLRP3)炎性体参与CVB3诱导的心肌炎,并研究了钙蛋白酶-1对CVB3引起的心脏损伤的影响。NLRP3炎性体在CVB3感染的心脏中被激活,NLRP3,GasderminD的N末端结构域的蛋白质水平升高,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。NLRP3炎性体的选择性抑制剂MCC950的腹膜内给药,导致血清肌酸激酶同工酶水平降低,心肌肌钙蛋白I,乳酸脱氢酶,白细胞介素-18,白细胞介素-1β,预防炎症细胞的浸润,CVB3感染下心功能的改善。过表达内源性钙蛋白酶抑制剂calpastatin的转基因小鼠(Tg-CAST小鼠)不仅表现出减少的细胞凋亡,炎症,纤维化,和增强心脏功能,但也抑制NLRP3炎性体和焦亡。使用PD151746选择性抑制钙蛋白酶-1通过下调NLRP3炎性体保护体外心肌细胞免受CVB3感染,因此,保存的细胞活力。机械上,我们发现,在CVB3治疗后,线粒体功能障碍先于炎症反应,使用线粒体靶向抗氧化剂(mito-TEMPO)消除线粒体活性氧(ROS),可以概括Tg-CAST小鼠的表型.此外,钙蛋白酶-1激活的促进或抑制在体外调节线粒体呼吸链。Mito-TEMPO逆转钙蛋白酶-1介导的NLRP3炎症激活和细胞死亡。我们还发现线粒体钙蛋白酶-1在CVB3刺激后增加,激活NLRP3炎性体并导致细胞死亡。此外,在CVB3治疗后,ATP合酶-α(ATP5A1)被发现是钙蛋白酶-1的切割靶标。使用ATP5A1-小干扰RNA下调ATP5A1受损的线粒体功能,细胞活力下降,并诱导NLRP3炎性体激活。总之,CVB3感染诱导钙蛋白酶-1在线粒体中积累,并导致随后的ATP5A1裂解,线粒体ROS过度生产,线粒体功能受损,最终引起NLRP3炎性体激活并诱导细胞凋亡。因此,我们的发现确立了钙蛋白酶在病毒性心肌炎中的作用,并揭示了其作用的潜在机制。钙蛋白酶似乎是治疗病毒性心肌炎的有希望的靶标。
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.