DOUBLE STRAND DNA BREAKS IN C57Bl AND mdx MICE CARDIOMYOCYTES AFTER DYNAMICAL STRESS
V. M. Mikhailov, I. V. Vezhenkova
Institute of Cytology RAS, St. Petersburg;
e-mail: vmikhailov@mail.cytspb.rssi.ru
The survival of cardiac myocytes under different physiological and pathological conditions presents pressing
problem. mdx mice cardiac myocytes are a promising model of cell survival under condition of oxidative
stress. Our early results have shown that some part of mdx mice cardiomyocytes is in early stage of apoptosis
(Kazakov, Mikhailov, 2001). But the development of cell death with loss of apoptotical cardiac myocytes occurs
only after dynamical stress (bathing during 5 min) (Mikhailov et al., 2001). DNA endonuclease activity in the
myocardium and low level of cardiac myocytes death during usual being of mdx mice allowed us to suggest
DNA repair to be involved in the survival of mdx mice cardiac myocytes (Mikhailov et al., 2003). To confirm
the suggestion we have studied the dynamics of formation and elimination of double strand DNA breaks in mdx
myocardium cells after 5 min bathing at 12 °N. To visualise double strand DNA breaks formation cell nuclei
were stained by monoclonal antibodies to phosphorylated H2Ax histone and to mouse PAP. Double staining
with monoclonal anti-H2Ax antibodies and monoclonal anti-α-actin antibodies were used to separate cardiac
myocytes from other myocardial cell types. The results showed that during 40 min after stress the deal of
H2Ax-positive nuclei in mdx myocardium cells grew up to 41.7 ± 11.4 % as compared with the initial control
level of 6.7 ± 0.2 %. The number of H2Ax-positive nuclei in these cells decreased after 24 h to 5.7 ± 0.2 %. The
quantity of tagged myocardium cell nuclei in C57Bl/6 mice after stress was negligible and did not go beyond
0.01 %. Dynamical stress also induced the increase in the rate of 3H-Thymidine incorporation by mdx mice cardiac
myocytes from 0.3 ± 0.3 up to 2.9 ± 0.5 %. There was not change in the rate of 3H-Thymidine incorporation
by cardiac myocytes in C57Bl/6 mice. The numbers of labelled nuclei before and after stress were 0.2 and
0.3 %, correspondingly. The number of 3H-Thymidine labelled mdx cardiac myocytes fell down up to
0.4 ± 0.2 % within 24 h after stress; the level of labelled C57Bl/6 cardiac myocytes did not change. We have
concluded that 3H-Thymidine incorporation into cardiac myocytes nuclei and staining of these nuclei by monoclonal
antiboies phosphorylated H2Ax histone after stress demonstrate rather DNA repair than cardiomyocytes
entry into the cell cycle.
Key words: double strand DNA breaks, C57Bl and mdx mice, differentiation, repair, dynamical stress
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