Autoradiographic Studies and Mitosis of Heart Muscle Cells in Experimental Cardiac Hypertrophy

Transcription

1 Tohoku J. exp. Med., 1970, 102, Autoradiographic Studies and Mitosis of Heart Muscle Cells in Experimental Cardiac Hypertrophy RIKURO SASAKI, TOSHIAKI MORISHITA, SHIZUKA ICHIKAWA and SHOICHI YAMAGATA The Third Department of Medicine,* Tohoku University School of Medicine, Sendai SASAKI, R., MoRISHITA, T., IcHIKAWA, S. and YAMAGATA, S. Autoradiographic Studies and Mitosis of Heart Muscle Cells in Experimental Cardiac Hypertrophy. Tohoku J. exp. Med., 1970, 102 (2), In order to find DNA synthesis and mitosis of heart muscle cells in experimental cardiac hypertrophy, autoradiographic studies after 3H-thymidine administration and evaluation of mitosis after colchicine treatment were made on Goldblatt rats. The labeling index of heart muscle cells and of interstitial cells in 4 hours was % and %, respectively. Concomitant mitosis of labeled heart muscle cells and of interstitial cells was found in 0-0.1% and %, respectively. The mitotic figures were scarcely found after colchicine administration. Though colchicine yields rather ambiguous results, the autoradiographic studies gave definite evidence for cell division of heart muscle cells in experimental cardiac hypertrophy. \autoradiography; mitosis; colchicine; cardiac hypertrophy A series of experiments on division of heart muscle cells in normal rats, including autoradiographic studies and mitosis of heart muscle cells, have been reported.1-5 They give definite evidence of division of heart muscle cells during normal growth. Thus, it appears that heart muscle cells unequivocally divide and increase in number in normal postnatal development of rats. On the contrary, there are conflicting opinions on the division of heart muscle cells in cardiac hypertrophy. It was shown in our recent studies that the number of heart muscle cells and of interstitial cells increased in experimental cardiac hypertrophy; both histometrical estimation of heart muscle cells and chemical determination of DNA content in heart muscle cells supported the above mentioned conclusion.6,7 In the present report autoradiographic studies on the synthesis of DNA after administration of 3H-thymidine and evaluation of mitosis of heart muscle cells after colchicine treatment in experimental cardiac hypertrophy are described. MATERIALS AND METHODS Experimental cardiac hypertrophy was produced in rats according to Goldblatt's method.8 3H-thymidine was given to rats with sustained systolic hypertension 6 to 7 weeks after Goldblatt's procedure. The amount and method of administration of 3H-thymidine, Received for publication, July 4, * Director: Prof. S. Yamagata. 159

2 160 R. Sasaki et al. and the autoradiographic technique were previously described. Similarly, Colcemid was given to rats with sustained hypertension 9 weeks after Goldblatt's procedure. The amount and method of administration of Colcemid were also described elsewhere.3 RESULTS The incorporation of 3H-thymidine into the nuclei of heart muscle cells was shown in Figs Fig. 4 shows the synthesis of DNA in the nucleus of inter stitial cell. In these figures, (A) was focused on nucleus itself and (B) on grains incorporated into the same nucleus. Fig. 5 shows mitosis with concomitant uptake Fig. 1. Section of the myocardium of rat 7 weeks after Goldblatt's procedure showing the incorporation of 3H-thymidine into heart muscle cell nucleus.

3 Autoradiography and Mitosis of Heart Muscle Cells 161 Fig. 2. Section of the myocardium of rat 7 weeks after Goldblatt's procedure showing the incorporation of 3H-thymidine into heart muscle cell nucleus. of 3H-thymidine. In this study, nuclei with more than 10 grains are considered to be labeled ones. The labeling index of heart muscle cells and of interstitial cells in 4 hours was % and %, respectively (Table 1). The mitotic rate of heart muscle cells and of interstitial cells found concomitantly was 0-0.1% and %, respectively. Mitotic figures of heart muscle cells and of interstitial cells after colchicine administration were shown in Figs. 6-9, but the mitotic rate was not estimated because of very rare appearance of mitosis.

4 162 R. Sasaki et al. Fig. 3. Section of the myocardium of rat 7 weeks after Goldblatt's procedure showing the incorporation of 3H-thymidine into heart muscle cell nucleus. DISCUSSION Rumyantsev9 has reported that the incorporation of 3H-thymidine into heart muscle cell nuclei of normal adult mice is only %. It has been thought that this is so small a quantity that the cell division of heart muscle is probably negligible. Pelc10 also found the incorporation of 3H-thymidine into heart muscle cell nculei of normal adult mice and mentioned that it might be nonmitotic incorporation of 3H-thymidine into DNA in non-dividing tissues. There have been no previous attempts to investigate possible incorporation of 3H-thymidine

5 Autoradiography and Mitosis of Heart Muscle Cells 163 Fig. 4. Section of the myocardium of rat 6 weeks after Goldblatt's procedure showing the incorporation of 3H-thymidine into interstitial cell nucleus. into nuclei of heart muscle cells in experimental cardiac hypertrophy, and the concept that heart muscle cells do not divide in cardiac hypertrophy has been persistently maintained. Our study showed that the mean labeling index of heart muscle cells and of interstitial cells in 4 hours was 0.3% and 1.95%, respectively. These appear to be certainly minimal. If, however, the cell division continues with these rates, the increase in number of heart muscle cells and of interstitial cells amounts to approximately 20-30% in several weeks. These figures are well consistent with previously estimated number of cells by bistometrical studies.6 It is unknown

6 164 R. Sasaki et al. Fig. 5. Section of the myocardium of rat 6 weeks after Goldblatt's procedure showing interstitial cell nucleus in mitotic state with concomitant 3H-thymidine incorporation. to what extent the heart muscle cells continue to increase. Though the exact mechanism is unknown, the cell division of heart muscle cells ceases at or about 10 weeks after Goldblatt's procedure. This was confirmed by our previous similar experiment performed 10 weeks after Goldblatt's procedure, which failed to demonstrate 3H-thymidine incorporation into heart muscle nuclei. It is very likely that heart muscle cells begin to divide soon after elevation of blood pressure and cease to divide within several weeks in this experiment. Therefore, it is important at what period after Goldblatt's procedure the experiment is performed, and when the incorporation of 3H-thymidine is to be evaluated. This is the reason why

7 Autoradiography and Mitosis of Heart Muscle Cells 165 TABLE 1. Labeling indices and mitotic rate of cells found concomitantly on autographic specimens 3H-thymidine was given 6-7 weeks after the procedure in the present study. It is very interesting that the mitotic rates of heart muscle cells and of interstitial cells found concomitantly on the autoradiographic specimens are quite similar to the rates of 3H-thymidine incorporation into nuclei. These findings strongly suggest that heart muscle cells divide after DNA synthesis. Because of considerably low rates of 3H-thymidine incorporation, however, DNA synthetic time and mitotic time were not calculated in this experiment according to the previously described method.5 Evaluation on mitotic rates of heart muscle cells after colchicine administra tion yielded poor results. Mitotic figures were found more scarcely compared with autographic specimens without colchicine. Probable reason of this failure might be that the amount of colchicine was inadequate or that its administration after Goldblatt's procedure (9 weeks) was too retarded, because the cell division of heart muscle was presumably very scanty at that time. Though colchicine administration yielded insufficient results, it seems that the autoradiographic studies give definite evidence to division of heart muscle cells in experimental cardiac hypertrophy. References 1) Sasaki, R., Watanabe, Y., Morishita, T. & Yamagata, S. Estimation of the cell number of heart muscles in normal rats. Tohoku J. exp. Med., 1968, 95, ) Sasaki, R., Watanabe, Y., Morishita, T. & Yamagata, S. Determination of deoxyribonucleic acid content of heart muscle and myocardial growth in normal rats. Tohoku J. exp. Med., 1968, 95, ) Sasaki, R., Morishita, T. & Yamagata, S. Mitosis of heart muscle cells in normal rats. Tohoku J. exp. Med., 1968, 96, ) Sasaki, R., Morishita, T. & Yamagata, S. Autoradiographic studies on heart muscle cells in normal rats. Tohoku J. exp. Med., 1970, 100, ) Sasaki, R., Morishita, T. & Yamagata, S. A method for determining deoxyri bonucleic acid synthetic time and mitotic time of heart muscle by autoradiographic technique. Tohoku J. exp. Med., 1970, 100, ) Sasaki, R., Morishita, T. & Yamagata, S. Estimation of the cell number of heart muscle in experimental cardiac hypertrophy. Tohoku J. exp. Med., 1970, 101, ) Sasaki, R., Morishita, T. & Yamagata, S. Determination of deoxyribonucleic acid

8 166 R. Sasaki et al. content of heart muscle in experimental cardiac hypertrophy. Tohoku J. exp. Med., 1970, 101, ) Goldblatt, H., Lynch, J., Hanzal, R.F. & Summerville, W.W. Studies on experimental hypertension. I. The production of persistent elevation of systolic blood pressure by means of renal ischemia. J. exp. Med., 1934, 59, ) Rumyantsev, P.P. Autographic study on the synthesis of DNA, RNA, and proteins in normal cardiac muscle cells and those changed by experimental injury. Folia histochem. cytochem. 1966, 4, ) Pelc, S.R. Labelling of DNA and cell division in so-called non-dividing tissues. J. Cell Biol. 1964, 22, Fig. 6. Section of the myocardium of rat 9 weeks after Goldblatt's procedure showing a mitotic figure of the nucleus of a heart muscle cell. Chromatin appears as clustered rods and granules. Fig. 7. Section of the myocardium of rat 9 weeks after Goldblatt's procedure showing a mitotic figure of the nucleus of a heart muscle cell. Chromatin appears as clustered rods and granules. Fig. 8. Section of the myocardium of rat 9 weeks after Goldlbatt's procedure showing a mitotic figure of the nucleus of an interstitial cell. Chromatin appears as clumps of coarse granules. Fig. 9. Section of the myocardium of rat 9 weeks after Goldblatt's procedure showing a mitotic figure of the nucleus of an interstitial cell. Chromatin appears as clumps of coarse granules.

9 Autoradiography and Mitosis of Heart Muscle Cells 167