Type II diabetes increases mitochondrial DNA mutations in the left ventricle of the Goto-Kakizaki diabetic rat.

Mitochondrial dysfunction has a significant role in the development of diabetic cardiomyopathy. Mitochondrial oxidant stress has been accepted as the singular cause of mitochondrial DNA (mtDNA) damage as an underlying cause of mitochondrial dysfunction. However, separate from a direct effect on mtDNA integrity, diabetic-induced increases in oxidant stress alter mitochondrial topoisomerase function to propagate mtDNA mutations as a contributor to mitochondrial dysfunction. Both glucose-challenged neonatal cardiomyocytes and the diabetic Goto-Kakizaki (GK) rat were studied. In both the GK left ventricle (LV) and in cardiomyocytes, chronically elevated glucose presentation induced a significant increase in mtDNA damage that was accompanied by decreased mitochondrial function. TTGE analysis revealed a number of base pair substitutions in the 3' end of COX3 from GK LV mtDNA that significantly altered the protein sequence. Mitochondrial topoisomerase DNA cleavage activity in isolated mitochondria was significantly increased in the GK LV compared with Wistar controls. Both hydroxycamptothecin, a topoisomerase type 1 inhibitor, and doxorubicin, a topoisomerase type 2 inhibitor, significantly exacerbated the DNA cleavage activity of isolated mitochondrial extracts indicating the presence of multiple functional topoisomerases in the mitochondria. Mitochondrial topoisomerase function was significantly altered in the presence of H2O2 suggesting that separate from a direct effect on mtDNA, oxidant stress mediated type II diabetes-induced alterations of mitochondrial topoisomerase function. These findings are significant in that the activation/inhibition state of the mitochondrial topoisomerases will have important consequences for mtDNA integrity and the well being of the diabetic myocardium.

Chronically elevated glucose compromises myocardial mitochondrial DNA integrity by alteration of mitochondrial topoisomerase function.

Mitochondrial dysfunction has a significant role in the development and complications of diabetic cardiomyopathy. Mitochondrial dysfunction and mitochondrial DNA (mtDNA) mutations are also associated with different types of cancer and neurodegenerative diseases. The goal of this study was to determine if chronically elevated glucose increase in mtDNA damage contributed to mitochondrial dysfunction and identify the underlying basis for mtDNA damage. H9c2 myotubes (a cardiac-derived cell line) were studied in the presence of 5.5, 16.5, or 33.0 mM glucose for up to 13 days. Tests of mitochondria function (Complex I and IV activity and ATP generation) were all significantly depressed by elevated media glucose. Intramitochondrial superoxide and intracellular superoxide levels were transiently increased during the experimental period. AnnexinV binding (a marker of apoptosis) was significantly increased after 7 and 13 days of high glucose. Thirteen days of elevated glucose significantly increased mtDNA damage globally and across the region encoding for the three subunits of cytochrome oxidase. Using mitochondria isolated from cells chronically exposed to elevated glucose, we observed significant increases in topoisomerase-linked DNA cleavage. Mitochondria-dependent DNA cleavage was significantly exacerbated by H(2)O(2) and that immunoprecipitation of mitochondrial extracts with a mtTOP1 antibody significantly decreased DNA cleavage, indicating that at least part of this activity could be attributed to mtTOP1. We conclude that even mild increases in glucose presentation compromised mitochondrial function as a result of a decline in mtDNA integrity. Separate from a direct impact of oxidative stress on mtDNA, ROS-induced alteration of mitochondrial topoisomerase activity exacerbated and propagated increases in mtDNA damage. These findings are significant in that the activation/inhibition state of the mitochondrial topoisomerases will have important consequences for mitochondrial DNA integrity and the well being of the myocardium.