CONTROL OF CARDIAC GENE-TRANSCRIPTION BY FIBROBLAST GROWTH-FACTORS

Skeletal alpha-actin (SkA) is representative of the cardiac genes that are expressed at high levels in embryonic myocardium, downregulated a fter birth, and reactivated by trophic signals including basic fibrobl ast growth factor (FGF-2) and type beta transforming growth factors (T GF beta). To investigate the molecular basis for cardiac-restricted an d growth factor-induced SkA transcription, we have undertaken a mutati onal analysis of the SkA promoter in neonatal ventricular myocytes, wi th emphasis on the role of three nominal serum response elements. Seru m response factor (SRF) and the bifunctional factor YY1 are the predom inant cardiac proteins contacting the proximal SRE (SRE1). Mutations o f SRE1 that prevent recognition by SRF and YY1, or SRF alone, virtuall y abolish SkA transcription; mutation of distal SREs was ineffective. A mutation which selectively abrogates YY1 binding increases expressio n, substantiating the predicted role of YY1 as an inhibitor of SRF eff ects. SkA transcription requires combinatorial action of SRE1 with con sensus sites for Spl and the SV40 enhancer binding protein, TEF-1. As an isolated motif, SRE1 can confer responsiveness to both FGF-2 and TG F beta to a heterologous promoter. Whether TEF-1 binding sites likewis e can function as FGF response elements is unknown. Molecular dissecti on of mechanisms that govern the differentiated cardiac phenotype has largely been undertaken to date in neonatal ventricular myocytes, as t he adult ventricular myocyte has been refractory to conventional proce dures for gene transfer. To circumvent expected limitations of other m ethods, we have used replication-deficient adenovirus to achieve effic ient gene transfer to adult cardiac cells in culture, Adult rat ventri cular myocytes were infected, 24 h after plating, with adenovirus type 5 containing a CMV-IE promoter-driven lacZ reporter gene, and were as sayed for the presence of beta-galactosidase 48 h after infection. The frequency of lacZ(+) rod-shaped myocytes was half-maximal at 4 x 10(5 ) PFU, and approached 90% at 1 x 10(8) PFU. Uninfected cells and cells infected with lacZ(-) virus remained colorless. The beta-galactosidas e activity concurred with the proportion of lacZ(+) cells and was cont ingent on the presence of exogenous lacZ gene. Thus, adult ventricular myocytes are amenable to efficient gene transfer with recombinant ade novirus. We have constructed virus conferring luciferase activity driv en by the SkA promoter (Ad5/SkA/luc) to test for potential development al control of growth factor responses in cardiac muscle. In adult vent ricular myocytes, the construct remains inducible by TGF beta, but lit tle or no response is seen to FGF-2 or FGF-1, which is consistent with prior reports that the FGF receptor is downregulated in terminally di fferentiated ventricular muscle cells. The relative uniformity for gen e transfer by adenovirus should facilitate tests to determine the impa ct of FGF receptors and FGF signaling proteins upon the endogenous gen es and gene products of virally modified adult ventricular muscle cell s. (C) 1994 Wiley-Liss, Inc.