LOCALIZATION OF PROLIFERATING CELL NUCLEAR ANTIGEN IN THE DEVELOPING AND MATURE RAT-HEART CELL

Background: The cardiac muscle cell ceases to divide shortly after bir th; this cessation is followed by a limited period when DNA synthesis and karyokinesis occur without cytokinesis. The regulation of this pro cess is not known. The purpose of this study is to explore the possibl e events that could lead to the cessation of cardiac muscle cell divis ion, One protein requisite for DNA synthesis is proliferating cell nuc lear antigen (PCNA), This protein is the auxiliary protein of DNA poly merase delta. Methods: Rats of fetal age day 18 or days 0, 4, 8, 12, a nd 16 after birth were obtained, In addition, adult hearts were used f or this study, Hearts from the fetal day-18 rats and the day-0 neonata l rats were digested, Cardiac myocytes were isolated and placed in cul ture for an analysis of DNA synthesis by using tridiated thymidine, Ve ntricular muscle tissue was isolated from hearts of all ages and froze n in liquid nitrogen for Northern and Western blot analyses. Results: Tridiated thymidine analysis revealed that, although serum stimulation significantly increased the number of labeled fetal cardiac muscle ce lls, it did not have that effect on neonatal cardiac muscle cells in c ulture. Northern blot analysis revealed that the steady state levels o f mRNA for PCNA remained constant from fetal day 18 through day 4 afte r birth, Steady state levels declined during the second postnatal week and then reached basal levels by day 16. PCNA message was still prese nt in adult heart tissue. By using indirect immunofloursecence and Wes tern blotting, PCNA protein could be located in the nucleus of cardiac muscle cells during the first 2 weeks after birth, At 16 days after b irth, the protein was found in the cytoplasm in very low amounts but w as not found in the nucleus, The protein was barely detectable by West ern blotting in the cytoplasmic fraction from the adult myocardium. Co nclusions: The results of this study suggest that the PCNA message and protein product declined after birth, but both were present at low le vels in the adult myocardium, However, the PCNA protein was not transl ocated to the nucleus in adult myocardial cells. The events involving PCNA correlated closely with the time period when cell division and th en DNA synthesis ceased in these cells. (C) 1996 Wiley-Liss, Inc.