Integrative multi-omics analysis and experimental validation identify MARK3 as a key ubiquitination-related regulator of cardiac fibroblast activation in heart failure via the TGF-β1/Smad3 pathway

Heart failure (HF) is a complex cardiovascular disease with high heterogeneity. The ubiquitin pathway plays a crucial role in various pathological processes, but its specific mechanisms in HF development and its relationship with the immune microenvironment remain unclear. Bulk RNA-seq datasets (GSE141910, GSE116250) and a single-cell RNA sequencing (scRNA-seq) dataset (GSE222144) were obtained from the Gene Expression Omnibus (GEO) database. Differentially expressed genes (DEGs) analysis, weighted gene co-expression network analysis (WGCNA), and ubiquitination-related gene sets (URGs) were integrated to identify candidate genes. Machine learning algorithms selected key genes and constructed a nomogram prediction model. Immune infiltration (CIBERSORTx), competing endogenous RNA (ceRNA)/transcription factor (TF) regulatory networks, scRNA-seq characterisation, and Mendelian randomisation (MR) analysis were performed. For experimental validation, primary neonatal mouse cardiac fibroblasts (NMCFs) were isolated and treated with 10 µM isoproterenol (ISO) for 24 h. Cell viability (CCK-8), reactive oxygen species (ROS), inflammatory cytokines (TNF-α, IL-1β), fibrotic markers (α-SMA, Collagen I), and expression of the four key genes were assessed by quantitative PCR (qPCR) and Western blot. MARK3 was overexpressed via pcDNA3.1-MARK3 transfection, and a rescue arm with recombinant TGF-β1 (10 ng/mL) was included to interrogate the TGF-β1/Smad3 signalling axis. Twenty ubiquitination-related candidate genes were identified, from which four key genes—CADPS2, DTX4, USP51 and MARK3—were selected by machine learning. The nomogram showed good predictive performance (AUC > 0.7). scRNA-seq revealed that fibroblasts chiefly expressed these genes and exhibited enhanced intercellular communication in HF. MR analysis indicated that low CADPS2 and MARK3 expression and high DTX4 expression may be associated with increased HF risk, whereas the estimate for USP51 was directionally consistent but did not reach statistical significance. Experimentally, ISO significantly impaired NMCF viability, elevated ROS, upregulated TNF-α, IL-1β, α-SMA and Collagen I, downregulated CADPS2 and MARK3, and upregulated DTX4 and USP51, consistent with bioinformatic predictions. MARK3 overexpression markedly attenuated ISO-induced oxidative stress, inflammation and fibrotic trans-differentiation while suppressing TGF-β1 and Smad3 phosphorylation. Exogenous TGF-β1 reversed these protective effects, confirming pathway dependence. This study identifies MARK3 as a protective regulator of cardiac fibroblast activation that functions at least partially through inhibition of the TGF-β1/Smad3 axis, providing a mechanistic basis and candidate therapeutic target for ubiquitination-directed anti-fibrotic strategies in HF.