Bnip3 promotes the release of mitochondria-derived extracellular vesicles in cardiac fibroblasts to exacerbate inflammation response after myocardial infarction

Abstract

Abstract Background Cardiac fibroblasts (CFs) play a crucial role in tissue repair and remodeling following myocardial infarction (MI). Furthermore, CFs possess the ability to sense signals of myocardial injury and secrete cytokines, thereby eliciting a pro-inflammatory response during the early post-MI phase. Our previous studies have demonstrated that CFs located in the infarcted region release small extracellular vesicles (sEVs) abundant in damaged mitochondria, termed "mt-sEVs". These mt-sEVs are internalized by macrophages, prompting the secretion of IL-6 and IL-1β via NLRP3/Caspase-1 pathway. Nonetheless, the precise mechanisms underlying the promotion of mt-sEVs release remain elusive. Objective This study aimed to investigate the pivotal genes involved in mediating the release of CFs-derived mt-sEVs, with potential implications for post-MI therapeutic strategies. Methods Primary CFs were cultured for 24 hours in sugar-free medium with 1% oxygen (OGD) in vitro. Conditioned medium was subjected to ultracentrifugation to isolate sEVs. The presence of damaged mitochondrial components within the sEVs was confirmed using Mito-tracker and nanoflowmetry. Subcellular structures were visualized via transmission electron microscopy (TEM). Protein interactions were validated through co-immunoprecipitation (co-IP) assays. CF-specific gene knockout mice were generated and subjected to MI surgery. The proportion of F4/80+/CD86+ cells was quantified using flow cytometry. Cardiac function was evaluated using echocardiography, and the extent of cardiac fibrosis was assessed via Masson staining. Results Re-analysis of single-cell RNA sequencing data (GSE153480) unveiled a significant upregulation of the mitophagy receptor Bnip3 in CF sub-populations following MI, and the up-regulated trend was also observed in OGD-CFs. Deletion of Bnip3 hindered the expression of LC3 and its co-localization with mitochondria. TEM observed extensive fusion of mitophagosomes with multivesicular bodies in OGD-CFs. Fluorescence signal detected by nano flowmetry significantly decreased in Bnip3 knockdown sEVs incubated with Mito-tracker. Bnip3 deletion in CFs resulted in a significant downregulation of the sEVs release key factor Rab27a, concomitant with a striking increase in Rab27a ubiquitination. Predicted binding sites of both Bnip3 and Rab27a with the deubiquitinase Usp7 were validated through co-IP assays. Immunofluorescence staining demonstrated the co-localization of Usp7 with Bnip3 in OGD-CFs. CF-specific deletion of Bnip3 reduced the proportion of M1 macrophages, reversed left ventricular dysfunction, and mitigated myocardial fibrosis in post-MI hearts. Conclusion Collectively, our study reveals that Bnip3-mediated mitochondrial secretory autophagy in CFs was significantly activated to drive the release of mt-sEVs. Additionally, Bnip3 interacts with Usp7 to deubiquitinate Rab27a, promoting the release of mt-sEVs in CFs, exacerbating the inflammatory response after MI.Graphical abstract