POINT MUTATIONS IN RAT MUSCLE AND HEART mtDNA





J. W. Pak, F. Vang, and J. M. Aiken

Department of Animal Health and Biomedical Science, 1656 Linden Drive, University of Wisconsin-Madison, Madison, WI 53706



The recent identification of an age-dependent accumulation of specific point mutations in the control region of human mtDNA implicates involvement of point mutations in mitochondrial dysfunction. The control region is the least conserved area of mitochondrial genome and is believed to be highly susceptible to oxidative damage. Immunohistochemical studies using a monoclonal anti-8OHG antibody demonstrated that oxidative damage was concurrent with ETS abnormalities in muscles. The majority of these ETS abnormal fibers exhibited a ragged red phenotype (COX-/SDH++). There is a strong correlation between mtDNA deletion mutations and electron transport system (ETS) abnormal regions of muscle fibers in aging rats. The deletion breakpoints frequently occur around the two origins of DNA synthesis indicating mutational hot spots in the major arc region. The goal of this study is to determine whether mtDNA point mutations accumulate to detectable levels in rat skeletal muscle and heart. To investigate the accumulation of age-related point mutations, the control region and the major arc region of the Fischer 344 x Brown Norway F1 hybrid rat mtDNA were PCR-amplified and directly sequenced from laser-captured single ETS abnormal muscle fibers or cardiomyocytes and normal cells in the 5- and 36-month old rats. The sequence analysis revealed a nucleotide insertion (15423A) in the control regions in both ragged red and wild-type phenotypes and in both tissue types, suggesting it is strain-specific, compared to the published rat mtDNA sequence (Rattus norvegicus). A number of strain-specific nucleotide sequence changes were also found in the major arc regions of all mtDNA sequenced. However, nucleotide differences were observed in the coding regions of deletion-containing mtDNA genomes from cardiomyocytes, suggesting an association between age and point mutations.




Key words: Point mutation, mtDNA, rat, skeletal muscle, heart







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