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(Circulation Research. 1996;78:289-301.)
© 1996 American Heart Association, Inc.

Articles

DNA Synthesis in Adult Feline Ventricular Myocytes

Comparison of Hypoxic and Normoxic States

Patricia L. Kozlovskis, Marcel J.D. Smets, William L. Strauss, Robert J. Myerburg

From the Departments of Medicine and Pharmacology (W.L.S.), University of Miami (Fla) School of Medicine.

Abstract Adult mammalian ventricular myocytes are terminally differentiated cells, and the prevailing perception has been that DNA synthesis and repair are not active. We tested the hypothesis that there is potential for DNA synthesis and repair by studying the ability of whole-cell extracts from adult myocytes to incorporate [-³²P]dCTP into damaged plasmids. Left ventricular myocytes were isolated from adult cat hearts by collagenase dissociation. Cells were maintained in room air (control extract, CE) or made ischemic (IE) with N₂ displacement of O₂ and extracted for total protein. The nicked form of the plasmid was produced by exposure to an Fe³⁺/ascorbic acid free radical generating system. Both IE and CE degraded the supercoiled form of the plasmid and incorporated [-³²P]dCTP into the nicked (³²P/DNA mass; CE=2.2, IE=3.0) and linear forms (³²P/DNA mass; CE=28.7, IE=25.2). Exposure of plasmids to UV light did not inhibit incorporation of label. Inhibition studies with the cell extracts suggested a participation of polymerase in myocyte DNA repair/synthesis. Myocyte extract was as active as extract from rapidly growing COS cells at incorporating labeled nucleotides into plasmid DNA. The ability of intact myocytes to incorporate [-³²P]dCTP into endogenous DNA was measured in isolated cells made permeable with saponin. Studies were done in room air or N₂. Permeable cells incorporated [-³²P]dCTP into nuclear DNA, but maximal specific activity of DNA was observed at 15 minutes with ischemia and at 60 minutes with room air control cells (ischemia, 1.34±0.5, 0.86±0.33, 0.60±0.04; air, 1.0, 1.28±0.20, 1.87±0.38, at 15, 30, and 60 minutes, respectively). These data indicate that mammalian adult ventricular myocytes can actively repair and/or synthesize both exogenous and endogenous DNA. A DNA synthetic response to cellular damage may have important pathological and clinical implications.

Key Words: DNA • synthesis • repair • myocyte • ischemia