Abstract 4371035: Myofibroblast Contractile Machinery is Essential for Cardiac Repair after Myocardial Infarction

Abstract

Background: After myocardial infarction (MI), the injured myocardium is repopulated by cardiac fibroblasts (CFs), which rapidly differentiate into contractile cardiac myofibroblasts (CMFs) marked by smooth muscle alpha-actin (SMαA) stress fibers. While CMF contractility is thought to be critical for post-MI repair, its functional significance remains unproven. Our previous work shows SMαA is dispensable for healing, as other actin isoforms compensate to ensure CMF differentiation. Non-muscle myosin IIs (NMMIIs), central mediators of contraction in non-muscle cells, are promising targets for studying CMF function during cardiac repair. Hypothesis: We hypothesize that NMMII-driven contractility allows CMFs to form a supportive cellular lattice that stabilizes the extracellular matrix (ECM) and maintains infarct integrity. Additionally, NMMII influences CMF gene expression and identity via transcriptional and epigenetic mechanisms. Methods: We generated tamoxifen-inducible, CF-specific knockout mice for Myh9 (NMMIIA) and Myh10 (NMMIIB), along with wild-type controls. In vitro studies using isolated CFs examined how NMMII deletion affects CMF function, gene expression, and chromatin state. In vivo , mice underwent MI surgery, followed by histological analysis, cardiac function assessment, and single-cell/nucleus RNA sequencing (sc/snRNAseq). Results: Myh9 deletion, but not Myh10 deletion, disrupted stress fiber formation, impaired CMF proliferation and migration, and destabilized the ECM. Transcriptomic and proteomic analyses revealed major changes in gene and protein expression in Myh9 -deficient CMFs. GSEA showed enrichment of actin cytoskeleton and proliferation pathways in wild-type CMFs, while Myh9 KO CMFs upregulated genes associated with alternative cell fates. Epigenetic profiling (ATACseq, Cut&Tag) showed widespread chromatin remodeling in Myh9 KO CMFs, involving MRTFA-SRF and BAF-PRC2 pathways. Combined Myh9/10 deletion further amplified these effects. In vivo , CF-specific Myh9 and Myh9/10 KO mice exhibited elevated rates of early cardiac rupture and worsened cardiac function post-MI. sc/snRNAseq showed reduced CMF populations and arrested CF differentiation in KO hearts. Conclusion: Our findings establish Myh9 as a critical regulator of CMF differentiation and post-MI cardiac repair, with broad implications for understanding fibroblast plasticity and myocardial healing.