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

Objectives: Platelet-derived growth factor (PDGF) stimulates growth in vari ous types of cells, but little is known about its effect on cardiac myocyte s. Therefore, we examined whether PDGF had a direct effect on cardiac myocy tes and investigated their intracellular signaling pathways. Methods: A pri mary culture of chick embryonic (Hamburger and Hamilton stage 36) ventricul ar myocytes was prepared. Cellular growth was estimated by 3-(4,5-dimethylt hiozol-2-yl)-2,5-diphenyltetrazolium bromide assay and 5-bromo-2'-deoxyurid ine incorporation assay. The number of PDGF binding sites was measured by b inding assay. Induction of c-fos mRNA was analyzed by Northern blot analysi s. The binding activity of activator protein (AP)-1 was examined by electro phoretic 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 immunocyt ochemistry. Furthermore, intracellular Ca2+ concentration ([Ca2+](i)) was m easured with indo-1 and L-type Ca2+ channel current (I-Ca) was recorded wit h the patch clamp technique. Results: PDGF-AB and -BB, but not PDGF-AA, inc reased 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 demons trated that the maximal number of PDGF-AA, -AB, -BB binding sites was 5+/-1 , 63+/-12, 126+/-24 fmol/10(6) cells, respectively. PDGF-BB provoked induct ion 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 accelerat ion of DNA synthesis were all attenuated by genistein (100 mu M) or MAPK ki nase inhibitor (10 or 50 mu M PD98059). Interestingly, protein kinase C inh ibitor (250 nM calphostin C) attenuated the increases of AP-1 binding activ ity to some extent, but did not inhibit the c-fos induction at all. The pho sphorylation states of STATs were not significantly affected by PDGF-BB. PD GF-BB did not alter [Ca2+](i) or I-Ca. Conclusions: We conclude that PDGF c an exert direct effects on embryonic cardiac myocytes and induce their grow th. MAPK cascade may play an important role in the PDGF-induced embryonic m yocardial growth. (C) 1999 Elsevier Science B.V. All rights reserved.