Background: Acute myocardial infarction (AMI) is a type of myocardial necrosis caused by acute ischemia or blood flow interruption in the coronary arteries, which poses a serious threat to human health. Activation of cardiac fibroblasts (CFs) and macrophage polarization play a crucial role in the pathogenesis of AMI. This study aims to elucidate the regulatory mechanisms between CFs and macrophage polarization during AMI progression.

Methods: RNA-seq data of AMI were downloaded for analysis. Differential expression analyses were performed on genes encoding secreted proteins, differentially expressed genes in single-cell infarct fibroblasts, and infarct tissues using the Human Protein Atlas (HPA) database. Differentially expressed genes Latent transforming growth factor-β binding protein 3 (LTBP3) and podocan (PODN) were analyzed in single-cell data comparing AMI samples with CFs, followed by receiver operating characteristic (ROC) analysis. LTBP3 and PODN protein expression was examined in a hypoxic cell model. LTBP3 recombinant protein was used to transfect macrophages, and the expression of CD16, CD86, iNOS, MHC-II, CD163, CD206, and arginase (Arg), as well as levels of heparin-binding EGF-like growth factor (HBEGF), interleukin-1 beta (IL-1β), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α), were assessed.

Results: A total of 11 cell types were identified through RNA-seq analysis, and 12 genes co-expressed across three datasets. Notably, LTBP3 and PODN exhibited specifically high expression in myocardial infarction (MI) samples. Both LTBP3 and PODN were significantly upregulated in MI, particularly in CFs (p < 0.05). Western blotting validated the upregulation of LTBP3 in CFs from MI samples compared with normal controls (p < 0.05), while PODN expression showed no significant difference in the AMI cell model relative to controls (p > 0.05). Co-immunoprecipitation (Co-IP) assay demonstrated that LTBP3 interacts with HBEGF. Overexpression of LTBP3 in CFs promoted macrophages polarization toward the M1 phenotype, as indicated by increased levels of M1 markers (CD16, CD86, iNOS, and MHC-II), and decreased levels of M2 markers (CD163, CD206, and Arg) (p < 0.05). Additionally, elevated HBEGF expression in macrophages enhanced the secretion of pro-inflammatory factors IL-1β, IL-6, and TNF-α (p < 0.05). HBEGF knockdown reversed the effects of LTBP3-transfected CFs on macrophage differentiation and mitigated the inflammatory response, as evidenced by reduced IL-1β, IL-6, and TNF-α levels (p < 0.05).

Conclusion: LTBP3 in CFs modulates AMI progression by regulating macrophage polarization.