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Quantification of Mitochondrial DNA Lesions in Cultured H9c2 Cardiomyocytes Subjected to Oxidative Stress
Author(s) -
RodríguezMuñoz Adlín,
BarretoTorres Giselle,
SotoHernández Jessica,
AyalaPeña Sylvette,
Javadov Sabzali,
TorresRamos Carlos
Publication year - 2015
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.29.1_supplement.977.13
Subject(s) - mitochondrial dna , oxidative stress , mitochondrion , microbiology and biotechnology , dna damage , nuclear dna , biology , polymerase chain reaction , dna , oxidative phosphorylation , real time polymerase chain reaction , dna repair , gene , biochemistry
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 H 2 O 2 followed by DNA isolation and QPCR analysis. Compared to untreated cells, cells treated with 75, 100 and 200 µM of H 2 O 2 showed a significant 60%, 80% and 90% reduction in the amplification of a mtDNA target respectively. These results indicate that the QPCR assay can be applied to detect mtDNA damage in rat cardiomyocytes after acute oxidative stress. Future experiments involve: (1) the determination of the repair kinetics of mtDNA damage and (2) the effects of mtDNA lesions in mitochondrial function. Supported by NIH grants SC1HL118669 and G12RR03051.