In this study, we demonstrated that pyroptosis of MCF is involved in the development of VMC and depends on activation of the NLRP3 signaling pathway. Furthermore, We found that pyroptosis promotes the production of CXCL4 by activating the Wnt/β-Catenin pathway, which in turn promotes myocardial fibrosis by activating the PI3K/AKT pathway.
VMC is an acute inflammatory disease of the heart caused by viral infection, which is the main cause of sudden cardiac death in children and adolescents[28–30]. About 21% of patients with acute VMC will progress to dilated cardiomyopathy, leading to heart failure and even sudden death[25]. During this period, the heart occurred pyroptosis and is infiltrated by immune cells, which subsequently causes the activation of myocardial fibroblasts, leading to cardiac fibrosis[31]. However, the precise molecular mechanisms underlying the association between pyroptosis and VMC development need to be investigated further. Numerous studies have shown that the occurrence of cardiovascular diseases is often accompanied by the increase of AngII, and the level of AngII is positively correlated with cardiac fibrosis. AngII is a polypeptide produced by the hydrolysis of angiotensin Ⅰ by angiotensin converting enzyme. It effects cardiovascular function and is specifically involved in the course of disease. Studies have shown that AngII can cause pyroptosis of cells[32, 33]. In this study we demonstrated that during the development of VMC, elevated AngII induces pyroptosis of MCF resulting in the production of CXCL4, which subsequently activates MCF and promotes cardiac fibrosis.
Pyroptosis is a programmed necrosis of inflammatory cells mediated by inflammasome, accompanied by the release of several inflammatory cytokines including IL-1β and IL-18 from the cell[34, 35]. The inflammasome is a multiprotein complex composed of pattern recognition receptors, adaptor ASC, and Cysteine aspartic protease cysteine proteinogen-1(Caspase-1) that recognizes pathogen-associated molecular patterns (PAMPs) or host-derived danger signaling molecules (DAMPs) and activates Caspase-1[36, 37]. Activated Caspase-1 enables the precursors of IL-1β and IL-18 to transform into mature IL-1β and IL-18, and eventually these inflammatory cytokines are released into the extracellular membrane through pores formed by GSDMD-N to induce inflammatory responses. NLRP3 inflammasome is a critical component of pyroptosis induction[38]. In our study, we demonstrated that VMC can induce pyroptosis of myocardial fibroblasts by activating the NLRP3 inflammasome. Using the NLRP3 inhibitor MCC950 and NLRP3−/− mice, we further found that pyroptosis of myocardial fibroblasts is induced by activation of NLRP3 inflammasome and that inhibition of pyroptosis is beneficial for VMC. Furthermore, we also found that AngII treatment of myocardial fibroblasts can cause pyroptosis through in vitro experiment but MCC950 treatment or transfection of NLRP3 siRNA can inhibit pyroptosis. Therefore, inhibiting NLRP3 inflammasome activation may be beneficial in viral myocarditis. Our study provides new perspectives on the regulation of fibrosis in myocarditis and suggests that pyroptosis of MCF promotes myocardial fibrosis. Therefore, targeting the NLRP3 inflammasome may be a novel treatment for VMC.
In the development of myocarditis, cardiac fibrosis occurs and myocardial fibroblasts are the main cells that cause cardiac fibrosis[16, 39]. When cardiac fibroblasts are activated, they produce large amounts of collagens, and as collagen is deposited, the heart eventually develops fibrosis. Cardiac fibrosis, which causes arrhythmias and abnormal wound healing, eventually leads to heart failure, is a common pathological phenomenon of heart disease[40, 41]. Myocardial fibroblast is an important part of the heart and plays an indispensable role in the physiological and pathological conditions of the heart. When the body is affected by acute myocardial injury, various inflammatory cytokines and pro-fibrotic factors stimulate the activation of myocardial fibroblasts and produce extracellular matrix (ECM), matrix metalloproteinases (MMP) and tissue inhibitors of metalloproteinases (TIMPs). Poor cardiac fibrosis can lead to the disorder of myocardial cell arrangement, myocardial hypertrophy, cytoskeletal rearrangement, collagen deposition, which poses a major threat to heart health[42]. In this study, we found that the occurrence of VMC is accompanied by cardiac fibrosis, which can be reduced by inhibiting pyroptosis of MCF.
CXCL4 is a multifunctional chemokine involved in a variety of biological responses, including the regulation of immune response and angiogenesis. CXCL4 not only has proinflammatory function but also promotes fibrosis[43–45]. In myeloproliferative tumors, hematopoietic deficiency of CXCL4 inhibits fibrosis. CXCL4 knockout alleviates cardiac fibrosis in a model of fibrosis caused by transverse aortic contraction. Our study shows that pyroptosis activates the Wnt pathway of MCF to produce CXCL4, which has a profibrotic effect. Transcriptome sequencing analysis showed that CXCL4 treatment activated the PI3K/AKT signaling pathway of MCF. In order to verify whether the PI3K/AKT signaling pathway is related to the profibrotic function of CXCL4, and then pretreatment of MCF with PI3K inhibitors, it was found that PI3K inhibitors inhibited the profibrotic effect of CXCL4. Together, our study shows that pyroptosis of MCF produces CXCL4 by activating the Wnt/β-Catenin pathway. In addition, further studies have shown that CXCL4 promotes fibrosis through PI3K/AKT pathway
In conclusion, our results suggest that the vital role of regulation of the NLRP3 inflammasome in the development of viral myocarditis. Inhibition of MCF pyroptosis caused by activation of NLRP3 can reduce heart damage. In addition, pyroptosis induced MCF to secrete CXCL4 by activating the Wnt/β-Catenin pathway, which promote cardiac fibrosis through activating the PI3K/AKT signaling pathway. NLRP3 may be considered a potential therapeutic target for myocarditis treatment via inhibition of CXCL4 secretion through the Wnt/β-Catenin pathway and inhibition of fibrosis through the PI3K/AKT pathway.