SIRT4-mediated debutyrylation of SUCLG1 rescues myocardial energy failure in HFpEF: Ginsenoside Rb3 as a novel...

Heart failure with preserved ejection fraction (HFpEF) is a complex cardiovascular disorder characterized by diastolic dysfunction, metabolic dysregulation and limited therapeutic options. Post-translational modifications (PTMs) are key regulators of cardiac metabolism, but the role of butyrylation in HFpEF pathogenesis remains unclear. This study explored the mechanistic role of butyrylation in myocardial energy metabolism of HFpEF and evaluate the therapeutic potential of ginsenoside Rb3 (G-Rb3). A "two-hit" (high-fat diet + L-NAME) mouse model and a phenylephrine (PE)-induced hypertrophic and metabolically stressed cellular model were established. Myocardial PTM screening identified butyrylation as the target for proteomic analysis. G-Rb3 efficacy was evaluated in vivo and in vitro, with mechanistic studies involving Sirtuin 4 (SIRT4) inhibitor and overexpression experiments to confirm its regulatory role. Male mice model displayed earlier and more severe HFpEF phenotypes than females, thus justifying their use in mechanistic studies. Butyrylome analysis revealed hyperbutyrylation of succinate-CoA ligase subunit alpha (SUCLG1) at K90, which impaired its enzymatic function in tricarboxylic acid (TCA) cycle, resulting in reduced succinate and ATP production. SIRT4 was identified as a key regulator of SUCLG1 debutylation, with downregulated SIRT4 expression leading to SUCLG1 hyperbutyrylation. G-Rb3 directly bound SIRT4, reversing SUCLG1 hyperbutyrylation, restoring TCA cycle flux and ATP levels, improving diastolic dysfunction and metabolic abnormalities. These effects were nullified by SIRT4 inhibition, confirming SIRT4 as G-Rb3's critical target. Our study reveals SUCLG1 butyrylation as a novel metabolic regulator in the pathogenesis of HFpEF, orchestrated by SIRT4. G-Rb3, a SIRT4-interacting regulator, rescues this axis, offering a mechanism-based therapy for HFpEF.