Date of Graduation

2017

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

Jamal Mustafa

Committee Member

Mark Olfert

Committee Member

Emidio E Pistilli

Abstract

Approximately 9% of the United States population is diagnosed with diabetes mellitus (DM), which is comprised of 2 distinct pathologies: type 1 diabetes mellitus (T1DM) and type 2 diabetes mellitus (T2DM). T1DM, which is caused by insufficient insulin production, affects approximately 5% of diabetic patients, while T2DM results from insulin resistance and affects 95% of all diabetic patients. Within diabetic patients, cardiac complications, such as diabetic cardiomyopathy, are the leading cause of morbidity and mortality. The mitochondrion has been implicated as an underlying factor in the etiology and progression of the cardiac contractile deficits and cardiac failure that accompany DM. The study of cardiac mitochondria is further complicated by the presence of two distinct mitochondrial subpopulations residing within the cardiomyocyte. The pool of mitochondria existing beneath the sarcolemmal membrane are termed the subsarcolemmal mitochondria (SSM), while the group that exists between the myofibrils is called the interfibrillar mitochondria (IFM). Assessment of mitochondrial subpopulations has revealed differential impact to both physiological and pathological stimuli. Specifically, during DM, the IFM are most impacted during T1DM, with the SSM being most impacted under T2DM pathological insult. During DM, proteomic analyses by our laboratory and others reveal decreased abundance of nuclear-encoded mitochondrial proteins essential for processes such as oxidative phosphorylation, fatty acid oxidation and tricarboxylic acid cycle in the subpopulation predominantly impacted by the DM type. Further, our laboratory has previously shown import efficiency to be down in the T1DM IFM, which could play a role in the proteomic dysregulation. Approximately 99% of the mitochondrial proteome is composed of nuclear-encoded proteins imported into the mitochondrion via a complicated mechanism of translocation that coordinates both the outer and inner mitochondrial membranes, thus highlighting the importance of studying the nuclear-encoded mitochondrial protein import process during pathological states. To date, evaluation of the diabetic heart using a highly sensitive echocardiographic analysis software in order to assess subtle changes in left ventricular function prior to overt contractile dysfunction during DM has not been completed. Additionally, the differential proteomic alterations in mitochondrial subpopulations resulting from distinct DM pathologies and the evaluation of inefficient nuclear-encoded mitochondrial protein import due to decrements in a key import constituent in the mitochondrial subpopulation predominantly affected, mitochondrial heat shock protein 70 (mtHsp70), have not been completed. Further, the mechanisms involved in miRNA import into the mitochondrion during DM remains limited. Therefore, the goal of the following studies was to examine subpopulation-specific mitochondrial proteome disruption stemming from inefficient nuclear-encoded mitochondrial protein import and/or increased miRNA influx into the mitochondrion, thus leading to increased contractile dysfunction during DM. T1DM was induced in 6-week-old mice with multiple low-dose (50mg/kg) streptozotocin (STZ) injections for 5 consecutive days. Hyperglycemia was confirmed and echocardiography performed at weeks 1, 3 and 6 post-diabetic onset. Conventional analyses revealed cardiac contractile deficits relative to control at 6-weeks post-T1DM onset. In contrast, short- and long-axis analyses using the speckle-tracking based strain analysis software demonstrated changes in the LV myocardium as early as 1-week post-diabetic onset. These findings show that analysis of myocardial function using speckle-tracking based strain analyses could provide a more precise method for evaluating cardiac contractile dysfunction during the progression of different pathological states. Our laboratory has previously shown that proteomic alterations specific to the T2DM SSM and T1DM IFM occur, potentially due to a decrement in nuclear-encoded mitochondrial protein import. Because mtHsp70, an essential component in the import of nuclear-encoded proteins into the mitochondrion is consistently down during DM, we generated a novel transgenic line with a cardiac-specific overexpression of mtHsp70. We subjected this line to STZ to generate a T1DM mouse model with mtHsp70 overexpression. Further, we utilized the db/db mouse model for T2DM and with a novel ovarian transplantation procedure, we were able to generate an increased abundance of mtHsp70 db/db and control animals, which were approximately 20-weeks-old before hearts were excised and mitochondrial subpopulations isolated. When assessing nuclear-encoded mitochondrial protein import efficiency in the mitochondrial subpopulations during both types of DM, we found decrements to this process in the SSM of T2DM mice and IFM of T1DM mice, which was subsequently restored with mtHsp70 overexpression. Further, alterations to the most impacted mitochondrial subpopulations proteome were noted, with mtHsp70 affording protection. Additionally, we also found mtHsp70 protein content to be down in the T1DM and T2DM human heart. (Abstract shortened by ProQuest.).

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