Semester

Spring

Date of Graduation

2012

Document Type

Dissertation

Degree Type

PhD

College

College of Physical Activity and Sport Sciences

Department

Exercise Physiology

Committee Chair

John M Hollander

Abstract

Cardiac complications, including diabetic cardiomyopathy, are the leading cause of morbidity and mortality within diabetes mellitus. Mitochondrial dysfunction has been suggested as an underlying factor in the initiation and progression of the pathology. Cardiac mitochondria are characterized as two spatially distinct subpopulations within the myocyte including mitochondria located beneath the sarcolemma, termed subsarcolemmal mitochondria (SSM), and those located between myofibrils, termed interfibrillar mitochondria (IFM). Mitochondrial subpopulations have been shown to respond differently to physiological and pathological stimuli with the IFM being the most impacted in a type 1 diabetic setting. Proteomic evaluations within various models of diabetes have highlighted dynamic alterations of the mitochondrial proteome as a consequence of the pathology. To date, no studies have identified how the proteomes of mitochondrial subpopulations are differentially impacted during a type 1 diabetic insult. Further, the mechanisms involved in diabetes-driven mitochondrial proteomic alterations remain limited. Therefore, the goal of the present studies was to determine whether subpopulation-specific proteomes were altered with type 1 diabetes mellitus. Further, we sought to identify mechanisms involved in mitochondrial proteomic dysregulation prevalent within diabetic cardiomyopathy. Type 1 diabetes mellitus was induced in 8 week old mice with multiple low dose (50mg/kg) injections of streptozotocin (STZ) administered for 5 days. Five weeks post hyperglycemic onset, hearts were excised and mitochondrial subpopulations were isolated. Proteomic analyses revealed that the proteome of diabetic IFM was significantly dysregulated compared to control with no changes within diabetic SSM compared to control. Further, nuclear-encoded mitochondrial protein import was significantly decreased in the diabetic IFM, which correlated with decreased abundance of essential protein import constituent mitochondrial heat shock protein 70 (MtHsp70). Because greater than 99% of proteins are of nuclear encoded origin and must be imported into the mitochondria, decrements to the import process may prove to be a novel mechanism of dysfunction within the diabetic IFM. The inner mitochondrial membrane (IMM), the location of essential mitochondrial complexes including import translocases of the inner membrane (TIM), has been shown to be particularly prone to diabetes induced oxidative damage. Reduction in oxidative damage within various pathologies has been shown to have beneficial effects upon mitochondrial functionality. Therefore, we overexpressed antioxidant mitochondrial phospholipid hydroperoxide glutathione peroxidase (mPHGPx) to assess its impact upon the mitochondrial import process and proteomic makeup during a diabetic insult. Remarkably, nuclear-encoded mitochondrial protein import was corrected within the diabetic mPHGPx IFM, which correlated with restitution of a large proportion of mitochondrial proteins negatively impacted by diabetes mellitus including those involved in oxidative phosphorylation, the tricarboxylic acid cycle, fatty acid oxidation, and mitochondrial protein import. MPHGPx is capable of scavenging mitochondrial lipid hydroperoxides specifically in mitochondrial membranes and hydrogen peroxide to a lesser extent. Overexpression of the antioxidant preserved or enhanced the protein content of essential mitochondrial import constituents translocase of the outer membrane 20 (Tom20), Tim23, Tim50, and MtHsp70 in mPHGPX diabetic IFM. Therefore, we believe proteomic correction within mPHGPx diabetic IFM may be a consequence of preservation of mitochondrial protein import machinery. These findings support the rationale for the use of mPHGPx as a mitochondrial-targeted therapeutic capable of protection in the diabetic heart. Additional mechanisms of mitochondrial proteomic dysregulation within diabetes mellitus may exist including alterations to the transcriptional/translational regulators, microRNAs (miRNAs). Therefore, we performed a broad scale miRNA analysis on control and STZ-treated mouse hearts 5 weeks post diabetic onset to determine the effect of diabetes mellitus on miRNA modulation. Twenty nine miRNAs were shown to be dysregulated within the diabetic heart including miRNA-141 (miR-141), which was enhanced by 5 fold. miRNA targeting analyses (targetscan.org) revealed miR-141 likely to regulated Slc25a3, the mitochondrial inorganic phosphate carrier. Slc25a3 is essential for ATP production as it acts as conduit for inorganic phosphate to pass from the cytoplasm into the matrix, providing phosphate to fuel the ATP synthase. IFM Slc25a3 protein content was decreased in the diabetic IFM, which correlated with decreased ATP synthesis rates. Similarly, overexpression of miR-141 decreased Slc25a3 protein content and ATP synthase activity within HEK293 cells. These findings show, for the first time, miRNA modulation within diabetes mellitus has a direct impact upon mitochondrial proteomic makeup as well as mitochondrial functionality. Further, miR-141 ablation may provide a protective benefit to mitochondria and subsequent cardiac function during a type 1 diabetic insult. Taken together, the studies highlighted above prove that mitochondrial proteomic dysregulation prevalent within diabetes mellitus is a complicated process involving spatially distinct mitochondrial subpopulations and multiple mechanisms of action. Targeted therapeutics aimed at the correction of one or more of these mechanisms may provide cardiac benefit from diabetic induced dysfunction via preservation of the mitochondrial proteome.

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