Localization of proliferating cell nuclear antigen in the developing and mature rat heart cell.
BACKGROUND
The cardiac muscle cell ceases to divide shortly after birth; this cessation is followed by a limited period when DNA synthesis and karyokinesis occur without cytokinesis. The regulation of this process is not known. The purpose of this study is to explore the possible events that could lead to the cessation of cardiac muscle cell division. One protein requisite for DNA synthesis is proliferating cell nuclear antigen (PCNA). This protein is the auxiliary protein of DNA polymerase delta.


METHODS
Rats of fetal age day 18 or days 0, 4, 8, 12, and 16 after birth were obtained. In addition, adult hearts were used for this study. Hearts from the fetal day-18 rats and the day-0 neonatal rats were digested. Cardiac myocytes were isolated and placed in culture for an analysis of DNA synthesis by using tridiated thymidine. Ventricular muscle tissue was isolated from hearts of all ages and frozen 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 cells, it did not have that effect on neonatal cardiac muscle cells in culture. Northern blot analysis revealed that the steady state levels of mRNA for PCNA remained constant from fetal day 18 through day 4 after birth. Steady state levels declined during the second postnatal week and then reached basal levels by day 16. PCNA message was still present in adult heart tissue. By using indirect immunofluorescence and Western blotting, PCNA protein could be located in the nucleus of cardiac muscle cells during the first 2 weeks after birth. At 16 days after birth, the protein was found in the cytoplasm in very low amounts but was not found in the nucleus. The protein was barely detectable by Western blotting in the cytoplasmic fraction from the adult myocardium.


CONCLUSIONS
The results of this study suggest that the PCNA message and protein product declined after birth, but both were present at low levels in the adult myocardium. However, the PCNA protein was not translocated to the nucleus in adult myocardial cells. The events involving PCNA correlated closely with the time period when cell division and then DNA synthesis ceased in these cells.
Marino
T A
TA
Department of Anatomy and Cell Biology, Temple University School of Medicine, Philadelphia, Pennsylvania 19140, USA. marino@astro.ocis.temple.edu
Cao
W
W
Lee
J
J
Courtney
R
R
D000375:Aging / Q000378:metabolism*; D000818:Animals; D000831:Animals, Newborn / Q000254:growth & development / Q000378:metabolism*; D015152:Blotting, Northern; D015153:Blotting, Western; D002478:Cells, Cultured; D005333:Fetus / Q000378:metabolism*; D019084:Fluorescent Antibody Technique, Indirect; D009206:Myocardium / Q000166:cytology / Q000378:metabolism*; D018809:Proliferating Cell Nuclear Antigen / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D014018:Tissue Distribution
D018809:Proliferating Cell Nuclear Antigen