Mitochondrial Topoisomerase Dysfunction Contributes to Myocardial Mitochondrial Dysfunction and Mitochondrial DNA (mtDNA) Degradation

Abstract

Diabetes promotes mtDNA degradation as an antecedent event to cardiomyocyte cell death and we have reported that mitochondrial topoisomerase dysfunction participates in this pathway. In post mitotic cells, the mitochondrial genome continues to replicate about once a month and resolving the topology of mtDNA replication would be almost insurmountable in the absence of the mitochondrial topoisomerases. It remains unclear how chronic hyperglycemia induces this shift in mitochondrial topoisomerase function from physiologic to pathologic. To that end, we hypothesize that low levels of ROS alter mitochondrial topoisomerase function to accelerate mtDNA damage. DNA relaxation of supercoiled DNA is a hallmark measurement of topoisomerase function. Supercoiled DNA incubated with isolated myocardial mitochondria or purified topoisomerase 1 generated the same isopane pattern of relaxation. In the absence of mitochondrial extracts, H2O2 (125–1000 μM) did not alter DNA relaxation of supercoiled DNA. In contrast, in the presence of myocardial mitochondrial extracts, H2O2 (125–1000 μM) significantly increased DNA relaxation in a dose dependent manner. Similar results were obtained following the DNA cleavage protocol. Using the iron chelator EDTA, we determined that DNA relaxation was not due to a Fenton effect. Following immunoprecipitation using mtTop1 or Topo‐2beta antibodies, mitochondrial extracts retained DNA relaxation function indicating the overlapping presence of functional mitochondrial topoisomerases. Using isolated intact mitochondria, incubation with H2O2 significantly increased mitochondrial superoxide levels; an effect that could be blocked with MitoTempo (mitochondrial specific antioxidant). NOX4 is a member of the NADPH oxidase family, but is unique in its preferential generation of H2O2 over superoxide (O2¾). Others have reported that NOX4 is localized to the outer mitochondrial membrane. Overexpression of NOX4 in H9c2 cells exacerbated intracellular and mitochondrial superoxide levels when incubated in the present of high glucose. Diabetes increases pentose shunt pathway activity, which in turn may fuel mitochondrial localized NOX4 to promote the generation of H2O2 a diffusible ROS species. Collectively our results suggest that increased mitochondrial ROS presentation alters mitochondrial topoisomerase function. Separate from a direct impact of oxidative stress, our studies find that alteration of mitochondrial topoisomerase function accelerates and propagates an increase in mtDNA damage. These findings indicate that the functional state of the mitochondrial topoisomerases participates in the pathophysiology leading to the development of diabetic cardiomyopathy.Support or Funding InformationSupported in part by NIH RO3HD065555101 and TCUS Biomedical Education FundThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.