Consequences of Endogenous Mitochondrial Oxidative Stress in the Heart
For many years, it has been proposed that oxidative stress within the mitochondria causes mutations to the mitochondrial genome, resulting in changes in copy number per single cell. Ultimately, this compensatory increase in mtDNA, coupled with an increase in mutations, has been suggested to play a major role in the pathobiology of aging. However, evidence for this phenomenon is somewhat controversial. Importantly, oxidative stress can vary between individual cells; therefore, the overall goal of this project is to determine if oxidative stress causes both an altered copy number for mtDNA in singlecardiomyocytes and develop a methodology for sequencing mtDNA from single cells compared to normal controls. In this study, the constitutive Sod2 knockout mouse model was used. All mice were treated daily with the SOD2 mimetic, Euk-189. For the first time, echocardiography was performed on constitutive Sod2 nullizygous mice. Sod2 mice were shown to have impaired systolic function, as demonstrated by significantly lower ejection fraction and fractional shortening values. Additionally, these mice show severe cardiac fibrosis and decreased FECH activity in cardiac tissue. The long-term goal of this project will be to carry out next-generation sequencing on single cardiomyocytes isolated from both Sod2 knockout and wild-type mice. However, a necessary first step is to develop the methodology for both isolating single cardiomyocytes and to quantitate mtDNA copy number for each single cell. We have been successful in accomplishing both these goals. The final outcome of these experiments will be to determine mtDNA sequences for multiple mtDNA genomes from single cells, and we hypothesize that mice undergoing endogenous oxidative stress will have accumulated more DNA mutations and deletions than wild-type mice. These data will ultimately inform us as to the role of mitochondrial DNA mutations in cardiac dysfunction.
Mitochondrial Oxidative Stress, Laboratory and Basic Science Research, Life Sciences
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