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.

Mitochondrial dysfunction is a contributor to diabetic cardiomyopathy. Previously, we observed proteomic decrements within the inner mitochondrial membrane (IMM) and matrix of diabetic cardiac interfibrillar mitochondria (IFM) correlating with dysfunctional mitochondrial protein import. The goal of this study was to determine whether overexpression of mitochondria phospholipid hydroperoxide glutathione peroxidase 4 (mPHGPx), an antioxidant enzyme capable of scavenging membrane-associated lipid peroxides in the IMM, could reverse proteomic alterations, dysfunctional protein import, and ultimately, mitochondrial dysfunction associated with the diabetic heart. MPHGPx transgenic mice and controls were made diabetic by multiple low-dose streptozotocin injections and examined after 5 wk of hyperglycemia. Five weeks after hyperglycemia onset, in vivo analysis of cardiac contractile function revealed decreased ejection fraction and fractional shortening in diabetic hearts that was reversed with mPHGPx overexpression. MPHGPx overexpression increased electron transport chain function while attenuating hydrogen peroxide production and lipid peroxidation in diabetic mPHGPx IFM. MPHGPx overexpression lessened proteomic loss observed in diabetic IFM. Posttranslational modifications, including oxidations and deamidations, were attenuated in diabetic IFM with mPHGPx overexpression. Mitochondrial protein import dysfunction in diabetic IFM was reversed with mPHGPx overexpression correlating with protein import constituent preservation. Ingenuity Pathway Analyses indicated that oxidative phosphorylation, tricarboxylic acid cycle, and fatty acid oxidation processes most influenced in diabetic IFM were preserved by mPHGPx overexpression. Specific mitochondrial networks preserved included complex I and II, mitochondrial ultrastructure, and mitochondrial protein import. These results indicate that mPHGPx overexpression can preserve the mitochondrial proteome and provide cardioprotective benefits to the diabetic heart.

Croston TL, Thapa D, Holden AA, Tveter KJ, Lewis SE, Shepherd DL, Nichols CE, Long DM, Olfert IM, Jagannathan R, Hollander JM. Functional deficiencies of subsarcolemmal mitochondria in the type 2 diabetic human heart. Am J Physiol Heart Circ Physiol 307: H54 -H65, 2014. First published April 28, 2014 doi:10.1152/ajpheart.00845.2013.-The mitochondrion has been implicated in the development of diabetic cardiomyopathy. Examination of cardiac mitochondria is complicated by the existence of spatially distinct subpopulations including subsarcolemmal (SSM) and interfibrillar (IFM). Dysfunction to cardiac SSM has been reported in murine models of type 2 diabetes mellitus; however, subpopulationbased mitochondrial analyses have not been explored in type 2 diabetic human heart. The goal of this study was to determine the impact of type 2 diabetes mellitus on cardiac mitochondrial function in the human patient. Mitochondrial subpopulations from atrial appendages of patients with and without type 2 diabetes were examined. Complex I-and fatty acid-mediated mitochondrial respiration rates were decreased in diabetic SSM compared with nondiabetic (P Յ 0.05 for both), with no change in IFM. Electron transport chain (ETC) complexes I and IV activities were decreased in diabetic SSM compared with nondiabetic (P Յ 0.05 for both), with a concomitant decline in their levels (P Յ 0.05 for both). Regression analyses comparing comorbidities determined that diabetes mellitus was the primary factor accounting for mitochondrial dysfunction. Linear spline models examining correlative risk for mitochondrial dysfunction indicated that patients with diabetes display the same degree of state 3 and electron transport chain complex I dysfunction in SSM regardless of the extent of glycated hemoglobin (HbA1c) and hyperglycemia. Overall, the results suggest that independent of other pathologies, mitochondrial dysfunction is present in cardiac SSM of patients with type 2 diabetes and the degree of dysfunction is consistent regardless of the extent of elevated HbA1c or blood glucose levels. mitochondria; diabetes mellitus; diabetic cardiomyopathy DIABETES MELLITUS IS A CONDITION that is becoming epidemic in proportion, with an estimated 330 million to be affected worldwide by the year 2030 (12). Of the individuals diagnosed, type 2 diabetes mellitus accounts for ϳ90 -95% of cases (12), which has been attributed to poor diet and sedentary lifestyles (21). Type 2 diabetes mellitus is characterized by insulin resistance resulting from an imbalance in glucose homeostasis (21). The body does not properly use the insulin produced in response to increased blood glucose levels, and over time the pancreas eventually loses its ability to produce insulin (12).Cardiomyopathies are a leading cause of morbidity and mortality in individuals with diabetes mellitus (18). Mitochondrial dysfunction contributes to the development of cardiovascular complications resulting from type 2 diabetes mellitus (7, 9, 15). The cardiomyocyte possesses spatially distinct subpopulati...

Mitofilin, also known as heart muscle protein, is an inner mitochondrial membrane structural protein that plays a central role in maintaining cristae morphology and structure. It is a critical component of the mitochondrial contact site and cristae organizing system (MICOS) complex which is important for mitochondrial architecture and cristae morphology. Our laboratory has previously reported alterations in mitochondrial morphology and proteomic make-up during type 1 diabetes mellitus, with mitofilin being significantly down-regulated in interfibrillar mitochondria (IFM). The goal of this study was to investigate whether overexpression of mitofilin can limit mitochondrial disruption associated with the diabetic heart through restoration of mitochondrial morphology and function. A transgenic mouse line overexpressing mitofilin was generated and mice injected intraperitoneally with streptozotocin using a multi low-dose approach. Five weeks following diabetes mellitus onset, cardiac contractile function was assessed. Restoration of ejection fraction and fractional shortening was observed in mitofilin diabetic mice as compared to wild-type controls (P<0.05 for both). Decrements observed in electron transport chain (ETC) complexes I, III, IV and V activities, state 3 respiration, lipid peroxidation as well as mitochondria membrane potential in type 1 diabetic IFM were restored in mitofilin diabetic mice (P<0.05 for all). Qualitative analyses of electron micrographs revealed restoration of mitochondrial cristae structure in mitofilin diabetic mice as compared to wild-type controls. Furthermore measurement of mitochondrial internal complexity using flow cytometry displayed significant reduction in internal complexity in diabetic IFM which was restored in mitofilin diabetic IFM (P<0.05). Taken together these results suggest that transgenic overexpression of mitofilin preserves mitochondrial structure, leading to restoration of mitochondrial function and attenuation of cardiac contractile dysfunction in the diabetic heart.

Cardiovascular disease is the primary cause of mortality for individuals with type 2 diabetes mellitus. During the diabetic condition, cardiovascular dysfunction can be partially attributed to molecular changes in the tissue, including alterations in microRNA (miRNA) interactions. MiRNAs have been reported in the mitochondrion and their presence may influence cellular bioenergetics, creating decrements in functional capacity. In this study, we examined the roles of Argonaute 2 (Ago2), a protein associated with cytosolic and mitochondrial miRNAs, and Polynucleotide Phosphorylase (PNPase), a protein found in the inner membrane space of the mitochondrion, to determine their role in mitochondrial miRNA import. In cardiac tissue from human and mouse models of type 2 diabetes mellitus, Ago2 protein levels were unchanged while PNPase protein expression levels were increased; also, there was an increase in the association between both proteins in the diabetic state. MiRNA-378 was found to be significantly increased in db/db mice, leading to decrements in ATP6 levels and ATP synthase activity, which was also exhibited when overexpressing PNPase in HL-1 cardiomyocytes and in HL-1 cells with stable miRNA-378 overexpression (HL-1-378). To assess potential therapeutic interventions, flow cytometry evaluated the capacity for targeting miRNA-378 species in mitochondria through antimiR treatment, revealing miRNA-378 level-dependent inhibition. Our study establishes PNPase as a contributor to mitochondrial miRNA import through the transport of miRNA-378, which may regulate bioenergetics during type 2 diabetes mellitus. Further, our data provide evidence that manipulation of PNPase levels may enhance the delivery of antimiR therapeutics to mitochondria in physiological and pathological conditions.

Enhanced sensitivity in echocardiographic analyses may allow for early detection of changes in cardiac function beyond the detection limits of conventional echocardiographic analyses, particularly in a small animal model. The goal of this study was to compare conventional echocardiographic measurements and speckle-tracking based strain imaging analyses in a small animal model of type 1 diabetes mellitus. Conventional analyses revealed differences in ejection fraction, fractional shortening, cardiac output, and stroke volume in diabetic animals relative to controls at 6-weeks post-diabetic onset. In contrast, when assessing short- and long-axis speckle-tracking based strain analyses, diabetic mice showed changes in average systolic radial strain, radial strain rate, radial displacement, and radial velocity, as well as decreased circumferential and longitudinal strain rate, as early as 1-week post-diabetic onset and persisting throughout the diabetic study. Further, we performed regional analyses for the LV and found that the free wall region was affected in both the short- and long-axis when assessing radial dimension parameters. These changes began 1-week post-diabetic onset and remained throughout the progression of the disease. These findings demonstrate the use of speckle-tracking based strain as an approach to elucidate cardiac dysfunction from a global perspective, identifying left ventricular cardiac regions affected during the progression of type 1 diabetes mellitus earlier than contractile changes detected by conventional echocardiographic measurements.

Nanomaterial production is expanding as new industrial and consumer applications are introduced. Nevertheless, the impacts of exposure to these compounds are not fully realized. The present study was designed to determine whether gestational nano-sized titanium dioxide exposure impacts cardiac and metabolic function of developing progeny. Pregnant Sprague-Dawley rats were exposed to nano-aerosols (~10 mg/m, 130- to 150-nm count median aerodynamic diameter) for 7-8 nonconsecutive days, beginning at gestational Physiological and bioenergetic effects on heart function and cardiomyocytes across three time points, fetal (gestational), neonatal (4-10 days), and young adult (6-12 wk), were evaluated. Functional analysis utilizing echocardiography, speckle-tracking based strain, and cardiomyocyte contractility, coupled with mitochondrial energetics, revealed effects of nano-exposure. Maternal exposed progeny demonstrated a decrease in E- and A-wave velocities, with a 15% higher E-to-A ratio than controls. Myocytes isolated from exposed animals exhibited ~30% decrease in total contractility, departure velocity, and area of contraction. Bioenergetic analysis revealed a significant increase in proton leak across all ages, accompanied by decreases in metabolic function, including basal respiration, maximal respiration, and spare capacity. Finally, electron transport chain complex I and IV activities were negatively impacted in the exposed group, which may be linked to a metabolic shift. Molecular data suggest that an increase in fatty acid metabolism, uncoupling, and cellular stress proteins may be associated with functional deficits of the heart. In conclusion, gestational nano-exposure significantly impairs the functional capabilities of the heart through cardiomyocyte impairment, which is associated with mitochondrial dysfunction. Cardiac function is evaluated, for the first time, in progeny following maternal nanomaterial inhalation. The findings indicate that exposure to nano-sized titanium dioxide (nano-TiO) during gestation negatively impacts cardiac function and mitochondrial respiration and bioenergetics. We conclude that maternal nano-TiO inhalation contributes to adverse cardiovascular health effects, lasting into adulthood.