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DNA content of isolated rat heart cells during postnatal development: Quantitative cytochemistry of mono-and binucleated myocytes
DNA CONTENT OF ISOLATED RAT HEART CELLS DURING POSTNATAL DEVELOPMENT: QUANTITATIVE CYTOCHEMISTRY OF MONO-AND BINTJCLEATED MYOCYTES. Frederick H. Kasten, Boris Kudriavtsev and Pave1 P. Rumyantsev. Institute of Cytology, Academy of Sciences, Leningrad, USSR and Louisiana State Univ. Med. Ctr. New Orleans, LA 70119 The relative DNA content of individual nuclei from isolated rat heart cells was measured during the early and late postnatal periods when multinucleated myocytes develop. Ventricles from l-day to 2-month rats were enzymatically dissociated into single-cell suspensions. After fixing and staining for DNA in the fluorescent-Feulgen reaction (Histochemie 1:466, 1959; Tsitologia 16:256, 1974), nuclear DNA was measured by cytofluorometry. At birth, myocytes are small, largely mononucleated, have 2C DNA, and exhibit DNA synthesis. Binucleation occurs rapidly and reaches a maximum of 75-80% in 7-10 days. Cells increase in size and DNA synthesis ceases by 2 weeks. At least 95% of nuclei in mono-and binucleated myocytes are 2C and the remainder are polyploid. Single nuclear DNA values of 303 double nuclei do not differ significantly from each other. Since myocytes become binucleated through incomplete cytokinesis at mitosis (In Vitro 8:128, 1972), our results indicate that there is a well-ordered segregation of chromosomes to yield a balanced genetic complement in each nucleus of binucleated myocytes. (Supported by an Interacademy Award to F.H.K. from the National Academy of Sciences-USA.)
m CYTOCHFMISTRY OF T-TUBULES IN CULTURED RAT CARDIOMYOCYTES. Frederick H. Kasten and Wolfgang Sehulze. Central Institute for Heart-Circulation Research, Academy of Sciences, BerlinBuch, GDR and Louisiana State Univ. Med. Ctr., New Orleans, LA 70119 Neonatal rat ventricular cells were grown in cell culture and examined for the presence of the T-tubular system using sensitive EM enzymatic techniques. Beating myocytes were maiin tained for 4 and 8 days in vitro, fixed in cold glutaraldehyde, incubated for thiamin pyrrophosphatase (TPPase) and adenosine triphosphatase (ATPase), post-fixed in osmic acid, stained with uranyl acetate, embedded, and sectioned en face for transmission EM. Enzymatic activity was demonstrated in specialized elements of the sarcoplasmic reticulum, including lateral vesicles and subsarcolemmal cisternae or couplings. The terminal elements were seen as diads or triads in association with T-tubules. These T-tubular components were detected in both 4-and 8-day cultures, especially in transverse sections in the cell interior and at the surface. Up to now, only primative beginnings of T-tubules were seen by EM in neonatal heart cultures (Cell Tissue Res. 203:173, 1979). The present demonstration of the T-tubular system by ultrastructural cytochemistry completes the repertoire of cardiac organelles and specialized structures associated with cultivated cardiomyocytes and reaffirms the use of this in vitro model for biochemical and physiological studies. (Supported by an Interacademy Award and Senior Fogarty Fellowship to F.H.K. from the National Academy of Sciences-USA and the NIH.)
m CYTOCHFMISTRY OF T-TUBULES IN CULTURED RAT CARDIOMYOCYTES. Frederick H. Kasten and Wolfgang Sehulze. Central Institute for Heart-Circulation Research, Academy of Sciences, BerlinBuch, GDR and Louisiana State Univ. Med. Ctr., New Orleans, LA 70119 Neonatal rat ventricular cells were grown in cell culture and examined for the presence of the T-tubular system using sensitive EM enzymatic techniques. Beating myocytes were maiin tained for 4 and 8 days in vitro, fixed in cold glutaraldehyde, incubated for thiamin pyrrophosphatase (TPPase) and adenosine triphosphatase (ATPase), post-fixed in osmic acid, stained with uranyl acetate, embedded, and sectioned en face for transmission EM. Enzymatic activity was demonstrated in specialized elements of the sarcoplasmic reticulum, including lateral vesicles and subsarcolemmal cisternae or couplings. The terminal elements were seen as diads or triads in association with T-tubules. These T-tubular components were detected in both 4-and 8-day cultures, especially in transverse sections in the cell interior and at the surface. Up to now, only primative beginnings of T-tubules were seen by EM in neonatal heart cultures (Cell Tissue Res. 203:173, 1979). The present demonstration of the T-tubular system by ultrastructural cytochemistry completes the repertoire of cardiac organelles and specialized structures associated with cultivated cardiomyocytes and reaffirms the use of this in vitro model for biochemical and physiological studies. (Supported by an Interacademy Award and Senior Fogarty Fellowship to F.H.K. from the National Academy of Sciences-USA and the NIH.)