Weighted gene co-expression network reveals CCND1 as a promising biomarker and therapeutic target for myocardial fibrosis in hypertrophic cardiomyopathy

BACKGROUND: Myocardial fibrosis represents a critical prognostic indicator of poor outcomes in hypertrophic cardiomyopathy (HCM), frequently predisposing patients to lethal ventricular arrhythmias and progressive heart failure. However, the upstream molecular mechanisms driving cardiac fibrogenesis in HCM remain largely unresolved. This study aimed to identify key fibrotic pathways and regulatory genes in HCM patients using weighted gene co-expression network analysis (WGCNA). METHODS AND RESULTS: We quantified the severity of myocardial fibrosis using late gadolinium enhancement (LGE) on cardiac magnetic resonance (CMR) imaging and integrated these imaging data with transcriptomic co-expression networks to identify key modules associated with HCM-related fibrosis. WGCNA identified four fibrosis-associated modules containing 251 candidate genes (defined by gene significance > 0.5 and module membership > 0.8). Functional enrichment analysis revealed that activation of the TGF-β1 pathway and cell proliferation-related pathways significantly contributes to HCM fibrogenesis. We subsequently identified 323 differentially expressed genes (DEGs; 103 up-regulated and 220 down-regulated) between HCM patients with LGE and healthy controls. Integrating WGCNA candidate genes with the DEGs yielded 9 overlapping genes. Spearman correlation and receiver operating characteristic (ROC) curve analyses demonstrated that the expression levels of MYH11, C10orf71, CCND1, JMJD4, and SNAP47 positively correlated with the extent of myocardial fibrosis (LGE%). Following validation in a transverse aortic constriction (TAC) mouse model, elevated CCND1 expression in cardiac fibroblasts emerged as a prominent feature of HCM-related fibrosis. In vitro assays in neonatal rat cardiac fibroblasts confirmed that CCND1 expression increases upon TGF-β1 stimulation. Notably, treatment with methotrexate (MTX), a CCND1 inhibitor, significantly reduced CCND1 expression and suppressed fibroblast activation following TGF-β1 stimulation. CONCLUSIONS: To our knowledge, this is the first study to integrate CMR imaging with WGCNA to identify key pathways and hub genes associated with myocardial fibrosis in HCM patients. Our findings suggest that TGF-β1 and cell proliferation-related pathways contribute to fibrogenesis in HCM. We identify the cell-cycle-related gene CCND1 as a potential fibrotic marker in HCM, and our preliminary preclinical data suggest that its inhibitor, MTX, may warrant further investigation as a candidate therapeutic agent targeting myocardial fibrosis.