Co-targeting fibroblast mechanotransduction and cardiomyocyte metabolism wtih a Src family kinase inhibitor as a strategy to treat heart failure

Introduction Cardiac fibrosis is a central determinant of adverse outcomes in heart failure, yet therapies that directly target fibrogenic remodelling remain limited. We previously identified the Src family kinase (SFK) inhibitor PD as a candidate anti-fibrotic compound through a phenotypic screening approach1 (Fig. 1A); however, its therapeutic efficacy and mechanism in heart failure remain unknown. Purpose The purpose of this study is to evaluate the therapeutic potential of PD and elucidate its cellular and molecular mechanisms in a mouse model of pressure overload-induced heart failure. Methods Mice with transverse aortic constriction (TAC) were treated with PD or vehicle. We performed multimodal analyses, including single-nucleus RNA sequencing (snRNA-seq) of heart tissue and RNA velocity to infer state transitions. Complementary in vitro studies were conducted in primary mouse and human cardiac fibroblasts, assessing gene expression, immunostaining, protein signaling, and chromatin profiling by cleavage under target and release using nuclease (CUT&RUN). To examine cardiomyocyte-intrinsic effects, we studied human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) under oxidative stress and evaluated signaling and mitochondrial respiration. Results PD treatment markedly attenuated cardiac remodelling and fibrosis after TAC (Fig. 1B). snRNA-seq with RNA velocity indicated a transcriptional reprogramming of cardiac fibroblasts from activated states toward a quiescent-like phenotype (Fig. 1C). Mechanistically, PD reduced actin polymerisation and suppressed nuclear accumulation of myocardin-related transcription factor A (MRTF-A) in mouse cardiac fibroblasts (Fig. 1D). CUT&RUN analysis demonstrated reduction of serum response factor (SRF)-associated chromatin programs, supporting inhibition of SFK-linked mechanotransduction pathways that sustain fibroblasts activation. Consistently, PD promoted deactivation-associated responses in human cardiac fibroblasts derived from patients with advanced heart failure. Concomitantly, cardiomyocyte snRNA-seq showed enrichment of mitochondrial metabolism-related gene programs with PD treatment. In hPSC-CMs under oxidative stress, PD increased AMPKα Thr172 phosphorylation and enhanced maximal mitochondrial respiratory capacity (Fig. 1E), accompanied by improved contractile performance. PD treatment was also associated with reduced mitochondrial reactive oxygen species reduction and preservation of mitochondrial membrane potential. Conclusion We demonstrate that PD attenuates cardiac remodelling and fibrosis through dual mechanisms: restraining fibroblast activation and enhancing mitochondrial metabolism in cardiomyocytes (Fig. 2). Together, these findings provide evidence for a dual-mechanistic framework for SFK-linked fibrotic remodelling and identify PD as a potential therapeutic candidate for heart failure.Figure1Figure2