Quantification of Mitochondrial DNA Lesions in Cultured H9c2 Cardiomyocytes Subjected to Oxidative Stress

Oxidative stress plays a crucial role in the pathogenesis of cardiovascular diseases such as myocardial infarction, hypertrophy and heart failure. ROS can induce a variety of DNA lesions in nucleus and mitochondria. However, quantitative analyses of these DNA lesions require methodical approaches that can discriminate between nuclear and mitochondrial DNA (mtDNA) lesions. In this study, we aimed to apply a PCR-based (QPCR) assay for the quantification of mtDNA lesions in cultured cardiomyocytes subjected to oxidative stress. In this assay, lesions that blocks the progression of the PCR polymerase result in decreased amplification of the target sequence, giving a relative measure of the lesions present. H9c2 embryonic rat cardiomyocytes cultures were treated for 1 h with increasing concentrations of H2O2 followed by DNA isolation and QPCR analysis. Compared to untreated cells, cells treated with 75, 100 and 200 µM of H2O2 showed a significant 60%, 80% and 90% reduction in the amplification of a mtDNA targe...