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SUMMARY Chronic metabolic diseases have been linked to molecular signatures of mitochondrial dysfunction. Nonetheless, molecular remodeling of the transcriptome, proteome, and/or metabolome does not necessarily translate to functional consequences that confer physiologic phenotypes. The work here aims to bridge the gap between molecular and functional phenomics by developing and validating a multiplexed assay platform for comprehensive assessment of mitochondrial energy transduction. The diagnostic power of the platform stems from a modified version of the creatine kinase energetic clamp technique, performed in parallel with multiplexed analyses of dehydrogenase activities and ATP synthesis rates. Together, these assays provide diagnostic coverage of the mitochondrial network at a level approaching that gained by molecular “-omics” technologies. Application of the platform to a comparison of skeletal muscle versus heart mitochondria reveals mechanistic insights into tissue-specific distinctions in energy transfer efficiency. This platform opens exciting opportunities to unravel the connection between mitochondrial bioenergetics and human disease.

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.

SUMMARY Acyl CoA metabolites derived from the catabolism of carbon fuels can react with lysine residues of mitochondrial proteins, giving rise to a large family of post-translational modifications (PTMs). Mass spectrometry-based detection of thousands of acyl-PTMs scattered throughout the proteome has established a strong link between mitochondrial hyperacylation and cardiometabolic diseases; however, the functional consequences of these modifications remain uncertain. Here, we use a comprehensive respiratory diagnostics platform to evaluate three disparate models of mitochondrial hyperacylation in the mouse heart caused by genetic deletion of malonyl CoA decarboxylase (MCD), SIRT5 demalonylase and desuccinylase, or SIRT3 deacetylase. In each case, elevated acylation is accompanied by marginal respiratory phenotypes. Of the >60 mitochondrial energy fluxes evaluated, the only outcome consistently observed across models is a ~15% decrease in ATP synthase activity. In sum, the findings suggest that the vast majority of mitochondrial acyl PTMs occur as stochastic events that minimally affect mitochondrial bioenergetics.

Several human diseases have been found to be caused by mitochondrial DNA (mtDNA) mutations. Pathogenic mutated (mut) mtDNAs are usually "heteroplasmic," coexisting intracellularly with wild-type (wt) mtDNAs. For some mtDNA mutations, cells have normal levels of respiratory chain function unless the percentage of mut-mtDNA is very high. Although progress in understanding the molecular basis of mitochondrial diseases has been remarkable, the heterogeneity of mut-mtDNA distribution, even among cells of the same tissue, makes it difficult to clearly delineate the relationships between mtDNA mutations, gene dosage, and clinical phenotypes. In a search for screening methods for idennfying cultured cells with deficient mitochondrial function, we incubated living cells harboring mut-mtDNAs with dihydrorhodamine 123 (DHR123), an uncharged, noduorescent agent that can be converted by oxidation to the fluorescent laser dye rhodamine 123 (R123). Bright mitochondrial staining was observed in cells that respired normally. Fluorescence was significantly reduced in cells with mitochondrial respiratory chain dysfunction resulting from very high levels of mut-mtDNAs. The data show that DHR123 is useful for assessing mitochondrial function in single cells, and can be used for isolating viable, respiratory chain-deficient cells from heterogeneous cultures. ( J Hisrochem Cyrochem 44: 571-579, 19%)

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The objective of this study was to investigate the effect of chemical structure, ion concentration, and ion type on the release rate of biologically available ions useful for remineralization from microcapsules with ion permeable membranes. A heterogeneous polymerization technique was utilized to prepare microcapsules containing either an aqueous solution of K₂HPO₄, Ca(NO₃)₂, or NaF. Six different polyurethane-based microcapsule shells were prepared and characterized based on ethylene glycol, butanediol, hexanediol, octanediol, triethylene glycol, and bisphenol A structural units. Ion release profiles were measured as a function of initial ion concentration within the microcapsule, ion type, and microcapsule chemical structure. The rate of ion release increased with initial concentration of ion stored in the microcapsule over a range of 0.5-3.0M. The monomer used in the synthesis of the membrane had a significant effect on ion release rates at 3.0 M salt concentration. At 1.0 M, the ethylene glycol released ions significantly faster than the hexanediol-, octanediol-, and butanediol-based microcapsules. Ion release was fastest for fluoride and slowest for phosphate for the salts used in this study. It was concluded that the microcapsules are capable of releasing calcium, phosphate, and fluoride ions in their biologically available form.