Control of cardiac gene transcription by fibroblast growth factors

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Abstract
Skeletal α‐actin (SkA) is representative of the cardiac genes that are expressed at high levels in embryonic myocardium, downregulated after birth, and reactivated by trophic signals including basic fibroblast growth factor (FGF‐2) and type β transforming growth factors (TGFβ). To investigate the molecular basis for cardiac‐restricted and growth factor‐induced SkA transcription, we have undertaken a mutational analysis of the SkA promoter in neonatal ventricular myocytes, with emphasis on the role of three nominal serum response elements. Serum response factor (SRF) and the bifunctional factor YY1 are the predominant cardiac proteins contacting the proximal SRE (SRE1). Mutations of SRE1 that prevent recognition by SRF and YY1, or SRF alone, virtually abolish SkA transcription; mutation of distal SREs was ineffective. A mutation which selectively abrogates YY1 binding increases expression, substantiating the predicted role of YY1 as an inhibitor of SRF effects. SkA transcription requires combinatorial action of SRE1 with consensus sites for Sp1 and the SV40 enhancer binding protein, TEF‐1. As an isolated motif, SRE1 can confer responsiveness to both FGF‐2 and TGFβ to a heterologous promoter. Whether TEF‐1 binding sites likewise can function as FGF response elements is unknown. Molecular dissection of mechanisms that govern the differentiated cardiac phenotype has largely been undertaken to date in neonatal ventricular myocytes, as the adult ventricular myocyte has been refractory to conventional procedures for gene transfer. To circumvent expected limitations of other methods, we have used replication‐deficient adenovirus to achieve efficient gene transfer to adult cardiac cells in culture. Adult rat ventricular myocytes were infected, 24 h after plating, with adenovirus type 5 containing a CMV‐IE promoter‐driven lacZ reporter gene, and were assayed for the presence of β‐galactosidase 48 h after infection. The frequency of lacZ+ rod‐shaped myocytes was half‐maximal at 4 ×105 PFU, and approached 90% at 1 × 108 PFU. Uninfected cells and cells infected with lacZ− virus remained colorless. The β‐galactosidase activity concurred with the proportion of lacZ+ cells and was contingent on the presence of exogenous lacZ gene. Thus, adult ventricular myocytes are amenable to efficient gene transfer with recombinant adenovirus. We have constructed virus conferring luciferase activity driven by the SkA promoter (Ad5/SkA/luc) to test for potential developmental control of growth factor responses in cardiac muscle. In adult ventricular myocytes, the construct remains inducible by TGFβ, but little 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 differentiated ventricular muscle cells. The relative uniformity for gene transfer by adenovirus should facilitate tests to determine the impact of FGF receptors and FGF signaling proteins upon the endogenous genes and gene products of virally modified adult ventricular muscle cells.

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