Single-cell transcriptomics landscape decodes cardiac fibroblast heterogeneity and reveals PDK4-driven profibrotic activation during cardiac fibrosis

Cardiac fibroblasts act as a key driver of myocardial fibrosis across various heart diseases, but the recently recognized heterogeneity within fibroblast populations has hindered mechanistic understanding. This study aimed to investigate the heterogeneity of cardiac fibroblasts and the underlying mechanisms in both healthy and diseased hearts. A total of 236,748 cardiac fibroblasts from single-cell transcriptomics data from 73 samples were integrated, clustered and annotated, followed by a comprehensive analysis of cell composition, RNA velocity, differential gene expression as well as Non-Negative Matrix Factorization (NMF) generated gene expression programs. Based on these results, we conducted immunoblotting, metabolomics analysis, Co-IP, ChIP-qPCR to examine the role of PDK4 during profibrotic activation in neonatal mouse primary cardiac fibroblasts. We also performed immunofluorescence, flow cytometry and echocardiography to evaluate the effect of PDK4 inhibition in MI mice. Eight major populations of cardiac fibroblasts were identified and divided into two lineages. Twelve meta-programs (MPs) of transcriptional profiles of cardiac fibroblasts were uncovered by NMF analysis and revealed a strong association between PDK4-mediated glycolysis and Extracellular Matrix (ECM) deposition. Subsequently, experiments in primary cardiac fibroblasts of neonatal mouse indicated that PDK4 promoted lactate production and Histone H3 lactylation, which in turn, increased TGF-β1 expression and ECM synthesis. In addition, inhibition of PDK4 could alleviate cardiac fibrosis and improve cardiac function in MI mice. Collectively, our study delineates cardiac fibroblast heterogeneity after myocardial infarction and identifies PDK4 as a key regulator of profibrotic activation. Our findings support a mechanistic model in which PDK4-mediated lactate production and histone lactylation are linked to expression of TGF-β1 and ECM synthesis in cardiac fibroblasts. Together with the antifibrotic effects of fibroblast-targeted PDK4 knockdown by AAV9 in vivo, these results position PDK4 as a mechanistically relevant and potentially actionable therapeutic target for myocardial fibrosis.