Abstract Body (Do not enter title and authors here): Introduction: Elevated resting heart rate (RHR) is an established independent risk factor for cardiovascular disease. Cardiomyocyte (CM) metabolic flexibility is critical for cardiac regeneration. Our preliminary data indicated that moderate heart rate reduction (HRR), achievable with clinically available antiarrhythmic drugs, promotes cardiac regeneration and exhibits a strong correlation with myocardial metabolic pattern. However, how HRR regulates energy metabolism of CMs and the detailed molecular mechanism remains unclear.
Aims: This study aims to establish genetic models of heart rate reduction (HRR) independent of drugs, to elucidate the mechanisms by which pure HRR regulates cardiomyocyte metabolism and cardiac regeneration.
Methods and Results: Modulation of connexin 30 (Cx30) and Visinin-like protein 1(Vsnl1) in sinoatrial node was used to regulate heart rate. Both transgenic mouse models exhibited stable 10-20% HRR without cardiac functional impairment. Consistent with previous findings using pharmacological HRR, genetic HRR similarly enhanced cardiac regeneration post-myocardial injury. Conversely, elevated HR impaired the cardiac regenerative capacity in mice. Integrative analysis using PET-CT, small animal optical imaging, Seahorse metabolic assays, and key metabolic enzyme profiling demonstrated that genetic HRR enhanced glucose metabolism, upregulated key glycolytic enzymes, and activated the pentose phosphate pathway (PPP). Further, targeted metabolomics unexpectedly revealed increased fumarate levels within the tricarboxylic acid (TCA) cycle. This elevation was mediated by transketolase (TKT) in the PPP via the inhibition of fumarate hydratase (FH) expression. Mechanistically, co-immunoprecipitation (Co-IP) with disuccinimidyl suberate (DSS) crosslinking demonstrated that elevated fumarate reacts with specific cysteine residues on pyruvate kinase M2 (PKM2), forming S-(2-succinyl) cysteine (S-2SC). This succination—a novel post-translational modification of PKM2—induced its tetramer-to-dimer transition and subsequent nuclear translocation. Nuclear PKM2 then facilitated CM cell cycle re-entry and proliferation.
Conclusions: Collectively, our findings demonstrated that genetically-induced heart rate reduction promotes cardiac regeneration by driving metabolic rewiring towards fumarate accumulation. This elevated fumarate subsequently induces PKM2 succination, a novel post-translational modification, which reactivates the CM cell cycle.