Acetyl-CoA synthetase 2 maintains cytosolic acetyl-CoA homeostasis to preserve mitochondrial integrity and attenuate cardiac dysfunction under chronic β-adrenergic stress.

Heart failure is often accompanied by metabolic remodeling; however, the contribution of compartment-specific acetyl-CoA homeostasis to mitochondrial dysfunction remains unclear. Here, we identify acetyl-CoA synthetase 2 (ACSS2) as an important regulator of cytosolic acetyl-CoA homeostasis and mitochondrial integrity under chronic β-adrenergic stress. Chronic isoproterenol stimulation induced heart failure with reduced ejection fraction in mice and selectively suppressed myocardial ACSS2 expression, resulting in depletion of cytosolic acetyl-CoA without altering total cellular levels. Similar changes were observed in H9c2 cardiomyoblasts exposed to prolonged β-adrenergic stimulation. Genetic deletion of ACSS2 recapitulated this metabolic disturbance, leading to mitochondrial structural remodeling and impaired oxidative respiration without evidence of altered mitochondrial biogenesis. Mechanistically, ACSS2 deficiency was associated with compartment-specific alterations in protein acetylation, characterized by reduced cytosolic acetylation and increased mitochondrial acetylation. Mitochondrial dysfunction was reversible, as long-term supplementation with butyrate restored mitochondrial respiratory capacity. Conversely, cardiomyocyte-targeted ACSS2 overexpression preserved cytosolic acetyl-CoA levels and was associated with improved mitochondrial respiratory function and attenuated cardiac dysfunction in vivo under chronic β-adrenergic stress. Together, these findings suggest that ACSS2-dependent cytosolic acetyl-CoA homeostasis contributes to the maintenance of mitochondrial quality and cardiac resilience, highlighting the importance of metabolic compartmentalization in heart failure pathophysiology.