Complement C4a modulates inflammatory response and autophagy after myocardial infarction by regulating PF4 expression: mechanisms and therapeutic potential

Background The complement system plays an important role in myocardial infarction (MI) and heart failure post-MI. However, studies on Complement C4 are virtually nonexistent in the development of MI. Purpose This study aims to investigate the role of complement C4a in AMI. Methods We utilized proteomics data from UK Biobank to perform COX multivariate analysis and a genome-wide Mendelian randomization analysis, identifying activation of C4 system in MI and heart failure post-MI. Blood samples from 376 MI patients were collected for mass spectrometry analysis and ELISA to validate C4a expression. In vitro study, we investigated the expression of C4a and its effects on inflammatory factors and autophagy. Additionally, a SD rat myocardial infarction model was established to further validate the role of C4a in vivo. Results Single-cell sequencing reveals complement system is closely related to in MI. In participants from the UKB-PPP, both observational and genetic studies indicated activation of C4 system had a unique and significant positive correlation with MI and post-MI heart failure among all of complement components. Considering C4a in the blood primarily reflecting of C4 activation. The clinical blood samples from 376 AMI cases showed elevated levels of C4a in the serum of AMI patients, particularly pronounced in those with MI combined with heart failure. C4a expression was significantly increased under hypoxic conditions only in myocardial cells, while no changes were observed in fibroblasts and macrophages. Downregulation of C4a significantly inhibited cell autophagy and had no effect on other cell death model, with the reduction of Beclin 1 and LC3 expression and increased level of autophagy-related protective factor P62. In the SD rat MI model, C4a silencing improved cardiac function and inhibited myocardial cell autophagy as well. Conversely, C4a overexpression produced opposite results. High-throughput sequencing identifies PF4 as a specific downstream gene of C4a in myocardial cells. After PF4 overexpression, the expression of IL-6, TNF-α, Beclin 1, and LC3 proteins decreased while P62 protein increased both in vitro and in vivo. Furthermore, compared to the group with only C4a overexpression, the simultaneous overexpression of C4a and PF4 (salvage experiment) resulted in a significant reduction in myocardial injury and attenuated inflammation and autophagy. Using TRANSFAC, we predicted a potential binding site for STAT2 in the promoter region of PF4. To further validate this, we overexpressed C4a in hypoxic myocardial cells treated with STAT2 agonist. The results indicated that STAT2 may directly regulated PF4 expression and further affect downstream inflammatory cytokines and autophagy proteins. Conclusion Complement C4a plays a critical role in AMI by regulating PF4 expression through STAT2, influencing inflammatory responses and autophagy, and thereby affecting the occurrence and progression of MI.