Highlights d Mitochondria lacking CrAT and Sirt3 are susceptible to extreme protein acetylation d Hyperacetylation is accompanied by disturbances in redox balance and insulin action d Hyperacetylation does not affect mitochondrial respiration and enhances fat oxidation d Sirt3 flux and acetyl-lysine turnover promote a fuel switch from fat to glucose

Rationale: Circumstantial evidence links the development of heart failure to post-translational modifications of mitochondrial proteins, including lysine acetylation (Kac). Nonetheless, direct evidence that Kac compromises mitochondrial performance remains sparse. Objective: This study sought to explore the premise that mitochondrial Kac contributes to heart failure by disrupting oxidative metabolism. Methods and Results: A dual knockout (DKO) mouse line with deficiencies in carnitine acetyltransferase (CrAT) and sirtuin 3 (Sirt3), enzymes that oppose Kac by buffering the acetyl group pool and catalyzing lysine deacetylation, respectively, was developed to model extreme mitochondrial Kac in cardiac muscle, as confirmed by quantitative acetyl-proteomics. The resulting impact on mitochondrial bioenergetics was evaluated using a respiratory diagnostics platform that permits comprehensive assessment of mitochondrial function and energy transduction. Susceptibility of DKO mice to heart failure was investigated using transaortic constriction (TAC) as a model of cardiac pressure overload. The mitochondrial acetyl-lysine landscape of DKO hearts was elevated well beyond that observed in response to pressure overload or Sirt3 deficiency alone. Relative changes in the abundance of specific acetylated lysine peptides measured in DKO versus Sirt3 KO hearts were strongly correlated. A proteomics comparison across multiple settings of hyperacetylation revealed ∼86% overlap between the populations of Kac peptides affected by the DKO manipulation as compared to experimental heart failure. Despite the severity of cardiac Kac in DKO mice relative to other conditions, deep phenotyping of mitochondrial function revealed a surprisingly normal bioenergetics profile. Thus, of the >120 mitochondrial energy fluxes evaluated, including substrate-specific dehydrogenase activities, respiratory responses, redox charge, mitochondrial membrane potential and electron leak, we found minimal evidence of oxidative insufficiencies. Similarly, DKO hearts were not more vulnerable to dysfunction caused by TAC-induced pressure overload. Conclusions: The findings challenge the premise that hyperacetylation per se threatens metabolic resilience in the myocardium by causing broad-ranging disruption to mitochondrial oxidative machinery.