Temporal dynamics of the multi-omic response reveals the modulation of macrophage subsets post-myocardial infarction.

Aims: Macrophage-mediated inflammatory response plays a pivotal role in the pathological process of myocardial infarction (MI). Although single-cell RNA sequencing (scRNA-seq) shed light on the differentiation and function of macrophage subsets post-MI, it failed to capture protein-level changes, limiting comprehensive assessment.

Methods: To address this limitation, first, we analyzed the scRNA-seq data from ArrayExpress, after comprehensive quality control, normalization, dimensionality reduction, and clustering analyses, macrophage subsets were systematically classified and annotated using established computational pipelines. Then, these computational classifications were validated through rigorous animal experiments. Finally, we integrated proteomic data from our experimental model and scRNA-seq to analyze the effect of protein modification changes in macrophage subsets on prognosis post-MI.

Results: A total of seven macrophage clusters were identified and annotated, including Spp1Mac, MHCIIMac, Ltc4sMac, Lyve1Mac, DcnMac, Ly6c1Mono and Stmn1Mac. Spp1Mac and Ltc4sMac, linked to leukocyte migration and chemotaxis, peaked at 3 days. MHCIIMac and and DcnMac, involved in antigen processing and presentation and collagen fibril organization, peaked at 7 days. Lyve1Mac reduced on days 3 and 7, and by day 28 returned to baseline levels. By integrating scRNA-seq and proteomics, there 78, 126, and 64 differentially expressed proteins (DEPs) in macrophages were obtained at 3, 7 and 28 days, respectively. At 3 days, DEPs were linked to inflammation, protein processing and maturation. At 7 days, DEPs were enriched in regulation of actin filament organization and cellular response to interferon-gamma. At 28 days, DEPs exhibited inhibitory effects on inflammation, cell apoptosis and migration. The post-translational modifications (PTMs) of DEPs involved phosphorylation, acetylation and lactylation, and these DEPs are mainly associated with inflammation, fibrosis and cytoskeletal organization, including Marcks, Anxa2, Vim, Dcn, Tpm3 and so on.

Conclusion: By integrating scRNAseq and proteomics data, this study provides a comprehensive overview of heterogeneity and dynamic protein level changes of macrophages post-MI, and characterizes the PTMs in macrophages, and lays the groundwork for developing targeted interventions tailored to specific cell types and time points post-MI.