Platelet-derived growth factor induces cellular growth in cultured chick ventricular myocytes

Objectives: Platelet-derived growth factor (PDGF) stimulates growth in various types of cells, but little is known about its effect on cardiac myocytes. Therefore, we examined whether PDGF had a direct effect on cardiac myocytes and investigated their intracellular signaling pathways. Methods: A primary culture of chick embryonic (Hamburger and Hamilton stage 36) ventricular myocytes was prepared. Cellular growth was estimated by 3-(4,5- dimethylthiozol-2-yl)-2,5-diphenyltetrazolium bromide assay and 5-bromo-2'- deoxyuridine incorporation assay. The number of PDGF binding sites was measured by binding assay. Induction of c-fos mRNA was analyzed by Northern blot analysis. The binding activity of activator protein (AP)-1 was examined by electrophoretic mobility shift assay. The activation of mitogen-activated protein kinase (MAPK) and signal transducers and activators of transcription (STATs) was analyzed by Western blot analysis, immunoprecipitation, and immunocytochemistry. Furthermore, intracellular Ca²⁺ concentration ([Ca²⁺](i)) was measured with indo-1 and L-type Ca²⁺ channel current (I(Ca)) was recorded with the patch clamp technique. Results: PDGF-AB and - BB, but not PDGF-AA, increased viable cell number (5 ng/ml of PDGF-AA, -AB, - BB: 101±4%, 115*±4%, 122*±4%, respectively, n=4, *P<0.05) and DNA synthesis (104±11%, 202*±18%, 295*±25%, respectively, n =4, *P<0.05). Scatchard analysis demonstrated that the maximal number of PDGF-AA, -AB, -BB binding sites was 5±1, 63±12, 126±24 fmol/10⁶ cells, respectively. PDGF- BB provoked induction of c-fos mRNA and increases in binding activity to the AP-1 site. PDGF-BB also induced tyrosine phosphorylation and nuclear translocation of MAPK. The c-fos induction, the increased AP-1 binding activity and the acceleration of DNA synthesis were all attenuated by genistein (100 μM) or MAPK kinase inhibitor (10 or 50 μM PD98059). Interestingly, protein kinase C inhibitor (250 nM calphostin C) attenuated the increases of AP-1 binding activity to some extent, but did not inhibit the c-fos induction at all. The phosphorylation states of STATs were not significantly affected by PDGF-BB. PDGF-BB did not alter [Ca²⁺](i) or I(Ca). Conclusions: We conclude that PDGF can exert direct effects on embryonic cardiac myocytes and induce their growth. MAPK cascade may play an important role in the PDGF-induced embryonic myocardial growth.