Semester

Summer

Date of Graduation

2010

Document Type

Dissertation

Degree Type

PhD

College

School of Medicine

Department

Physiology, Pharmacology & Neuroscience

Committee Chair

John M. Hollander.

Abstract

Cardiovascular complications leading to heart failure are the leading cause of death amongst both type 1 and type 2 diabetic patients. Increased reactive oxygen species (ROS) generation due to hyperglycemia and an enhanced fatty acid environment has been suggested to lead to mitochondrial dysfunction. Mitochondria are particularly susceptible to oxidative damage because the inner mitochondrial membrane (IMM) is the main site of ROS generation. Mitochondrial dysfunction is complicated by the fact that two spatially-distinct subpopulations of mitochondria reside in the heart. The subsarcolemmal mitochondria (SSM) are located at the cell periphery, while the interfibrillar mitochondria (IFM) situate along the myofibrils. It has been suggested that these two populations of mitochondria respond differently to various pathologies and physiological stimuli based upon their location in the cardiomyocyte. The goal of the following studies was to examine whether mitochondria located in different areas of the cell respond differently with type 1 and type 2 diabetes mellitus (DM). Type 1 DM was induced in 8 week old mice with a multiple low dose (50mg/kg) injection of Streptozotocin (STZ) administered for five days. Five weeks post hyperglycemia onset, hearts were excised and mitochondrial subpopulations were isolated. Db/db (BKS.Cg-m +/+ Leprdb /J) mice were utilized as a model of type 2 DM and aged 18 weeks before hearts were extracted and mitochondrial subpopulations isolated. In the type 1 diabetic hearts, flow cytometry analysis of mitochondrial morphology indicated a decrease in size and internal complexity in the IFM with no changes in the SSM. On the other hand, in the type 2 diabetic hearts, the SSM size and complexity were decreased. Further, mitochondrial electron transport chain (ETC) activities and respiration were significantly decreased in the type 1 diabetic IFM while significantly decreased in SSM from type 2 diabetic hearts. Enhanced oxidative damage, assessed through lipid peroxidation by-products and nitrotyrosine formation, was specific to the IFM in the type 1 diabetic heart but enhanced in the SSM of the type 2 diabetic heart. Proteomic assessment revealed altered proteomic profiles in the type 1 diabetic IFM; however, changes were greater in the SSM of the type 2 diabetic heart. These data suggest for the first time, that depending on their location within the cell, mitochondria respond differently with type 1 and type 2 diabetic insults. The mitochondria located between the myofibrils are more dysfunctional in the type 1 diabetic heart; however, the mitochondria located beneath the sarcolemma are more dysfunctional in the type 2 diabetic heart. This emphasizes the importance of incorporating spatial location when examining mitochondrial dysfunction in the diabetic heart. Further, because the IMM is particularly susceptible to oxidative damage, we examined whether overexpression of a mitochondrially-targeted antioxidant enzyme, mitochondrial phospholipid hydroperoxide glutathione peroxidase (mPHGPx), provides protection in the diabetic heart. MPHGPx transgenic mice and controls were made diabetic through multiple low dose injections of STZ. Five weeks following hyperglycemia onset, in vivo analysis of cardiac contractile function revealed significantly decreased ejection fraction and fractional shortening in the diabetic heart, which was reversed with mPHGPx overexpression. MPHGPx overexpression in the diabetic heart increased mitochondrial ETC complex I, III, and IV activities in the diabetic IFM, with no differences on the SSM. Enhanced hydrogen peroxide production and lipid peroxidation were significantly attenuated in the diabetic IFM with overexpression of mPHGPx, with no changes in the SSM. MPHGPx is a unique antioxidant in that it has the ability to insert into the IMM and directly scavenge lipid hydroperoxides. Therefore, we assessed a phospholipid within the IMM essential for proper mitochondrial function, cardiolipin, and found preservation of cardiolipin in the diabetic IFM with overexpression of mPHGPx. These results indicate that mPHGPx overexpression provides cardioprotective benefits to the diabetic heart. Further, cardioprotection is associated with enhanced IMM function and preservation of a key IMM constituent, cardiolipin. These findings provide further rationale for the use of mPHGPx as a potential mitochondrially-targeted therapeutic that is capable of providing protection in the diabetic heart.

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