Double-strand breaks of DNA of C57BL and mdx mouse cardiomyocytes after dynamic stress

Abstract

One of the approaches to analysis of survival of cardiomyocytes during oxidative stress can be the use of animals with genetic defects—mdx mice. In mdx mice, disturbance of dystrophine synthesis is known to be accompanied by development of oxydative stress in contractile cells that in turn produces cell death. Earlier we established that dynamic stress leads to the formation of low molecular DNA fragments in the mdx mouse myocardium. It is beyond any doubt that the DNA fragmentation develops via formation of double-strand DNA breaks (DB). To record the dynamics of the appearance and disappearance of DB in the mdx mouse cardiomyocytes after dynamic stress, we used an antibody to the phosphorylated form of the γ-H2Ax histone. In the absence of stress, DB in myocardial cell nuclei are revealed both in C57Bl and in mdx mice. The percentage of cardiomyocyte nuclei with DB in C57Bl and in mdx mice was 0.05 ± 0.07% and 6.7 ± 0.2%, respectively (Table 1). In the C57Bl mice 1 h after dynamic stress the fraction of labeled cardiomyocyte nuclei rose to 1.0 ± 0.02%, while in the mdx mice—to 41.7 ± 11.4% (Table 1). At 24 h after the dynamic stress 5.7 ± 0.2% cardiomyocyte nuclei remained labeled in the mdx mouse myocardium (Table 1), whereas in C57Bl mice no labeled cardiomyocyte nuclei were revealed. One hour after the dynamic stress, 0.3 ± 0.2% of cardiomyocyte nuclei of the C57Bl mice incorporated 3H-thymidine. In the mdx mice, 2.9 ± 0.5% of cardiomyocyte nuclei incorporated 3H-thymidine. At 24 h after the stress and 3H-thymidine administration the percentage of cardiomyocyte nuclei in the mdx mice fell to 0.4 ± 0.2%. In the C57Bl mice primarily labeled nuclei were not revealed. The 3H-thymidine incorporation is not associated with entrance of cardiomyocytes into the mitotic cycle; we consider it as a manifestation of reparative DNA synthesis. We conclude is that the disappearance of DB in DNA from the mdx mouse myocardium 24 h after the dynamic stress is associated both with DNA reparation and the loss of cardiomyocytes.