Diabetic cardiomyopathy is associated with increased risk of heart failure in type 1 diabetic patients. Mitochondrial dysfunction is suggested as an underlying contributor to diabetic cardiomyopathy. Cardiac mitochondria are characterized by subcellular spatial locale, including mitochondria located beneath the sarcolemma, subsarcolemmal mitochondria (SSM), and mitochondria situated between the myofibrils, interfibrillar mitochondria (IFM). The goal of this study was to determine whether type 1 diabetic insult in the heart influences proteomic make-up of spatially distinct mitochondrial subpopulations and to evaluate the role of nuclear encoded mitochondrial protein import. Utilizing multiple proteomic approaches (iTRAQ and two-dimensional-differential in-gel electrophoresis), IFM proteomic make-up was impacted by type 1 diabetes mellitus to a greater extent than SSM, as evidenced by decreased abundance of fatty acid oxidation and electron transport chain proteins. Mitochondrial phosphate carrier and adenine nucleotide translocator, as well as inner membrane translocases, were decreased in the diabetic IFM ( P < 0.05 for both). Mitofilin, a protein involved in cristae morphology, was diminished in the diabetic IFM ( P < 0.05). Posttranslational modifications, including oxidations and deamidations, were most prevalent in the diabetic IFM. Mitochondrial heat shock protein 70 (mtHsp70) was significantly decreased in diabetic IFM ( P < 0.05). Mitochondrial protein import was decreased in the diabetic IFM with no change in the diabetic SSM ( P < 0.05). Taken together, these results indicate that mitochondrial proteomic alterations in the type 1 diabetic heart are more pronounced in the IFM. Further, proteomic alterations are associated with nuclear encoded mitochondrial protein import dysfunction and loss of an essential mitochondrial protein import constituent, mtHsp70, implicating this process in the pathogenesis of the diabetic heart.

Background Cardiomyocytes are rich in mitochondria which are situated in spatially-distinct subcellular regions including those under the plasma membrane, subsarcolemmal mitochondria; and those between the myofibrils, interfibrillar mitochondria. We previously observed subpopulation-specific differences in mitochondrial proteomes following diabetic insult. The objective of this study was to determine whether mitochondrial genome-encoded proteins are regulated by microRNAs inside the mitochondrion and whether subcellular spatial location or diabetes mellitus influences the dynamics. Methods and Results Using microarray technology coupled with cross-linking immunoprecipitation and next generation sequencing, we identified a pool of mitochondrial microRNAs, termed mitomiRs that are redistributed in spatially-distinct mitochondrial subpopulations in an inverse manner following diabetic insult. Redistributed mitomiRs displayed distinct interactions with the mitochondrial genome requiring specific stoichiometric associations with RISC constituents argonaute-2 (Ago2) and fragile X mental retardation–related protein 1 (FXR1) for translational regulation. In the presence of Ago2 and FXR1, redistribution of mitomiR-378 to the IFM following diabetic insult led to down regulation of mitochondrially-encoded F0 component ATP6. Next generation sequencing analyses identified specific transcriptome and mitomiR sequences associated with ATP6 regulation. Overexpression of mitomiR-378 in HL-1 cells resulted in its accumulation in the mitochondrion and down-regulation of functional ATP6 protein, while antagomir blockade restored functional ATP6 protein and cardiac pump function. Conclusions We propose mitomiRs can translationally regulate mitochondrially-encoded proteins in spatially-distinct mitochondrial subpopulations during diabetes mellitus. The results reveal the requirement of RISC constituents in the mitochondrion for functional mitomiR translational regulation and provide a connecting link between diabetic insult and ATP synthase function.

Hollander JM, Thapa D, Shepherd DL. Physiological and structural differences in spatially distinct subpopulations of cardiac mitochondria: influence of cardiac pathologies.

Lysine acetylation is a reversible posttranslational modification and is particularly important in the regulation of mitochondrial metabolic enzymes. Acetylation uses acetyl-CoA derived from fuel metabolism as a cofactor, thereby linking nutrition to metabolic activity. In the present study, we investigated how mitochondrial acetylation status in the heart is controlled by food intake and how these changes affect mitochondrial metabolism. We found that there was a significant increase in cardiac mitochondrial protein acetylation in mice fed a long-term high-fat diet and that this change correlated with an increase in the abundance of the mitochondrial acetyltransferase-related protein GCN5L1. We showed that the acetylation status of several mitochondrial fatty acid oxidation enzymes (long-chain acyl-CoA dehydrogenase, short-chain acyl-CoA dehydrogenase, and hydroxyacyl-CoA dehydrogenase) and a pyruvate oxidation enzyme (pyruvate dehydrogenase) was significantly upregulated in high-fat diet-fed mice and that the increase in long-chain and short-chain acyl-CoA dehydrogenase acetylation correlated with increased enzymatic activity. Finally, we demonstrated that the acetylation of mitochondrial fatty acid oxidation proteins was decreased after GCN5L1 knockdown and that the reduced acetylation led to diminished fatty acid oxidation in cultured H9C2 cells. These data indicate that lysine acetylation promotes fatty acid oxidation in the heart and that this modification is regulated in part by the activity of GCN5L1. Recent research has shown that acetylation of mitochondrial fatty acid oxidation enzymes has greatly contrasting effects on their activity in different tissues. Here, we provide new evidence that acetylation of cardiac mitochondrial fatty acid oxidation enzymes by GCN5L1 significantly upregulates their activity in diet-induced obese mice.

Baseler WA, Thapa D, Jagannathan R, Dabkowski ER, Croston TL, Hollander JM. miR-141 as a regulator of the mitochondrial phosphate carrier (Slc25a3) in the type 1 diabetic heart.