Behavior of embryonic chick heart cells in culture. 2. Cellular responses to epidermal growth factor and other growth signals

Abstract

Muscle cell-enriched primary cell cultures were prepared from 8-day embryonic chick heart ventricles (74% of these cells showed positive staining with anti-cardiac myosin antibody). To determine if Epidermal Growth Factor (EGF) affects cardiac muscle cells, immunostaining and autoradiography were performed to find the Muscle Cell Labeling Index (MLI). MLI represents the proportion of cardiac myosin-positive cells that specifically incorporated [ 3H]thymidine. The MLI for EGF-treated cells was 51%. Controls in Serum-free Nutrient Medium (SFNM) had a MLI of 34.5%. Combinations of growth signals also were tested. EGF, IGF-I (Insulin-like Growth Factor-I), or PDGF (Platelet-derived Growth Factor) alone increased [ 3H]thymidine incorporation in the cells. Adding IGF-I or PDGF simultaneously with EGF enhanced the response of the cells to EGF by increasing [ 3H]thymidine incorporation. TGF-β (Transforming Growth Factor-β) alone was shown to have an inhibitory effect on [ 3H]thymidine incorporation, and when TGF-β was added together with EGF, it attenuated the stimulatory effect of EGF on [ 3H]thymidine incorporation. Phorbol 12-Myristate 13-Acetate (PMA), a tumor promoter, alone had no effect on [ 3H]thymidine incorporation, but its addition suppressed the stimulatory effect of EGF when they were added simultaneously in the presence of 5% FBS. Developmental response of the heart cells to growth signals also was tested. Heart cells from 18-day embryos were used to test the effect of insulin and EGF. Although both insulin and EGF increased [ 3H]thymidine incorporation in heart cells from 8-day embryos, different responses to insulin and EGF occurred with heart cells from 18-day embryos. Whereas the heart cells from 18-day embryos still responded to EGF by increasing [ 3Hjthymidine incorporation, they did not show a response to insulin as measured by [ 3H]thymidine incorporation, suggesting that the loss of response of the heart cells to growth signals may occur at the receptor level. Further studies show that EGF, TGF-α. aFGF, and PDGF increased the total numbers of heart cells, and that aFGF and PDGF also increased the percentages of heart muscle cells.