DNA synthesis in adult feline ventricular myocytes. Comparison of hypoxic and normoxic states.
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 [alpha-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 [alpha-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 delta 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 [alpha-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 [alpha-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.
Kozlovskis
P L
PL
Department of Medicine, University of Miami School of Medicine, FL 33101, USA.
Smets
M J
MJ
Strauss
W L
WL
Myerburg
R J
RJ
D000818:Animals; D002415:Cats; D015687:Cell Hypoxia; D002478:Cells, Cultured; D004260:DNA Repair; D004261:DNA Replication; D006352:Heart Ventricles / Q000473:pathology; D010957:Plasmids / Q000502:physiology / Q000528:radiation effects; D014466:Ultraviolet Rays; D016276:Ventricular Function