ABSTRACT: Introduction: Hypertrophic Cardiomyopathy (HCM) is a cardiac disorder characterized by an increased interstitial fibrosis and Extracellular Matrix (ECM) remodeling. Cardiac fibroblasts (CFs) have a crucial role in ECM remodeling as well as influencing contractility. There is accumulating evidence that the phenotype of CFs is disease-specific. Here, we investigate the transcriptome signature of CFs in Hypertrophic obstructive Cardiomyopathy (HOCM) patients and its potential functional significance. Methods: Primary CFs were isolated from myectomy specimens of 12 clinically phenotyped HOCM patients and 3 controls. RNA libraries were prepared from cell isolates for subsequent whole transcriptome sequencing. Differential expression analysis was conducted using the Tuxedo pipeline. Pathway and Gene Ontology (GO) enrichment were performed using Protein-protein interaction network analysis and functional clustering were performed using Cytoscape StringApp. Expression of candidate genes and proteins was validated using real time quantitative PCR, Immunocytochemistry, immunohistochemistry, cytokines/chemokines profiling and western blotting, in patient-derived CFs extracts, CFs-conditioned media, and myocardial tissue sections. Results: Whole transcriptome analysis identified 265 significant differentially expressed genes (DEGs) (q-value ≤ 0.05 and log fold-change ≥ 1.5) in HOCM fibroblasts compared to controls. The most significant GO terms identified were associated with ECM organization and inflammatory response. The most significant GO terms identified were associated with ECM organization and inflammatory response, with circos plot analysis further highlighting pathway-specific gene overlap within inflammatory and structural signaling clusters. The DEGs encompassed gene families such as collagens, proteases, fibulins, inflammatory and signaling cytokines, integrins and signaling receptors and kinases. Notably, MYC was upregulated alongside chemokine ligands and receptors, including CXCR4, CXCL3, CXCL8, CCL2, CCL4 and CCL11, highlighting a potential MYC-linked chemokine signaling axis within the inflammatory HOCM-CFs phenotype. The protein expression of selected ‘extracellular Matrix organization’ and ‘inflammatory response’ genes confirmed the transcriptome results. Conclusion: Transcriptomic profiling of patient-derived HOCM-CFs identified candidate genes and pathways associated with inflammatory signaling, ECM remodeling, and altered cell–cell and cell–matrix communication. These findings show that advanced HOCM-CFs acquire an inflammatory-remodeling phenotype, with the involvement of ECM genes other than collagens.
