Date of Graduation

2015

Document Type

Dissertation

Degree Type

PhD

College

School of Medicine

Department

Physiology, Pharmacology & Neuroscience

Committee Chair

John M Hollander

Committee Co-Chair

Stephen E Alway

Committee Member

Albert S Berrebi

Committee Member

Ivan M Olfert

Committee Member

Emidio E Pistilli

Abstract

Cardiac complications such as diabetic cardiomyopathy are the leading cause of morbidity and mortality in patients with diabetes mellitus. Dysfunctional mitochondria, an effect associated with cardiomyopathy, are central in the pathogenesis of type 1 diabetes mellitus. Cardiac mitochondria are comprised of two spatially located mitochondria including mitochondria located beneath the sarcolemma, termed subsarcolemmal mitochondria (SSM) and those located in between myofibrils, termed interfibrillar mitochondria (IFM). Mitochondrial subpopulations have been shown to respond differently to pathological and physiological stimuli as reported in a review published by our laboratory. IFM mitochondria are most impacted in a type 1 diabetic setting. Proteomic alterations in cardiac mitochondria during a diabetic insult reveal impact primarily on IFM on nuclear-encoded mitochondrial proteins, a finding that has been previously reported by our laboratory. Alterations of proteins encoded by the mitochondrial genome have not been observed in our proteomics. Further, regulation of nuclear-encoded proteins by microRNAs (miRNAs) has been previously reported by our laboratory. MiRNAs are 22 nucleotide long post-transcriptional regulators with a 7 nucleotide seeding region specific to complementary sequences in the mRNA. More than 30% of all proteins are regulated by miRNAs and one miRNA has the potential to regulate the expression of multiple proteins. The potential regulation of mitochondrial genome encoded proteins by miRNAs has yet to be investigated in mitochondrial subpopulations during diabetes. Among the altered nuclear encoded proteins in type 1 diabetic IFM is structural protein known as mitofilin. Mitofilin is an inner mitochondrial membrane structural protein, well established for its role in maintaining cristae morphology and structure. It is a central component of the mitochondrial contact site and cristae organizing system (MICOS) complex. Interactions of mitofilin with outer and inner membrane proteins have been reported to be crucial for mitochondrial membrane organization, cristae integrity and inner membrane architecture. Moreover, MICOS has been shown to function in concert with ATP synthase dimers. However, association of mitofilin with ATP synthase subunits is not known. Moreover, literature examining association of mitofilin and regulation of mitochondrial genome by miRNAs in type 1 diabetic insult is sparse. Also, the impact of diabetes mellitus on mitofilin protein interactions, mitochondrial structure and function are currently unclear. It is specifically unknown whether overexpression of mitofilin aids in alleviating complications associated with diabetic cardiomyopathy. The goal of the present studies was to determine novel association of mitofilin and the impact of mitofilin overexpression upon mitochondrial structure and function. Further, regulation of mitochondrial genome by mitochondrial miRNAs (mitomiRs) has been investigated. The overall hypothesis of this application is that alterations of cristae morphology, inner membrane organization and mitochondrial dysfunction observed during type 1 diabetic insult are associated with decrements in mitofilin content as well as translational regulation of mitochondrial encoded proteins due to altered levels of mitochondrial miRNAs (mitomiRs). Type 1 diabetes mellitus was induced in five weeks old mice with multiple low dose injections of streptozotocin (STZ) for five consecutive days. Five weeks post hyperglycemic onset, hearts were excised and mitochondrial subpopulations isolated for further studies. Using a gel based technique, mitochondrial proteins immunoprecipitated with mitofilin were subjected to LC-ESI-MS analysis. Proteins from all electron transport chain complexes, structural proteins and proteins involved in protein import were identified in an immunoprecipitated complex. Association of mitofilin with F0 -ATP synthase subunit b (ATP5F1) was decreased in the diabetic IFM when compared with control. Moreover, interaction of mitofilin with coiled-coil-helix coiled-coil-helix domain 3 (CHCHD3) trended towards decreased in diabetic IFM. A transgenic mouse line overexpressing mitofilin was generated and utilized to investigate the role of mitofilin overexpression in mitochondrial structure and function. 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). No significant changes in mitochondrial dynamic regulating proteins or mitochondrial DNA content were observed. Examination of mitochondrial miRNAs was performed using microarray technology coupled with cross-linking immunoprecipitation and next generation sequencing, we identified a functional pool of mitochondrial microRNAs, termed mitomiRs that are redistributed in spatially-distinct mitochondrial subpopulations in an inverse manner following diabetic insult. (Abstract shortened by UMI.).

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