Inhibition of TGFbeta signaling potentiates the FGF-2-induced stimulation of cardiomyocyte DNA synthesis.

OBJECTIVE: Added transforming growth factor beta (TGFbeta) inhibits the proliferation of immature cardiomyocytes. We have now examined the hypothesis that suppression of endogenous TGFbeta signaling will boost the proliferative response (DNA synthesis) of cardiac myocytes to serum and/or to the mitogenic factor fibroblast growth factor-2 (FGF-2). METHODS AND RESULTS: Overexpression of a kinase-deficient TGFbeta type II receptor (TGFbetaRIIDeltaKD) resulted in a 2.8-fold increase in cardiomyocyte DNA synthesis in serum-rich cultures, an effect requiring active FGFR-1 since it was not observed in the presence of excess kinase-deficient FGFR-1. This finding suggested that endogenous TGFbeta-TGFbetaRII suppressed endogenous FGFR-1-mediated signals that stimulate or are permissive for DNA synthesis. TGFbeta had no effect, however, on the FGF-2-induced acute stimulation of extracellular signal regulated kinase1/2. FGF-2, added in the absence or presence of TGFbeta inhibition, elicited a 3- or a 13-fold stimulation of DNA synthesis, respectively, pointing to a synergistic effect. CONCLUSION: Inhibition of TGFbetaRII-transduced signaling upregulates the proliferative response of cardiomyocytes to serum, and greatly potentiates the stimulatory effect of FGF-2. A combinatorial strategy including activation of FGF-2 and inhibition of TGFbeta-triggered signal transduction may be required for maximal stimulation of immature cardiomyocyte DNA synthesis.

High levels of CUG-initiated FGF-2 expression cause chromatin compaction, decreased cardiomyocyte mitosis, and cell death.

Fibroblast growth factor 2 (FGF-2) is a multifunctional mitogen present in CUG-and AUG-initiated forms, referred to as 'hi' and 'lo' FGF-2, respectively. We have used an adenoviral vector to express the predominantly nuclear human 'hi' FGF-2 and examined the relationship between expression levels, mitotic entry, cell number and chromatin compaction of cardiac myocytes, over 1-3 days in culture. At a multiplicity of infection (m.o.i.) of 50, levels of 'hi' FGF-2 (assessed by Western blotting) and mitotic index (fraction of myocyte nuclei staining positive for phosphorylated histone H3) paralleled each other, becoming maximal at 2 days. At 200 m.o.i., maximal expression of 'hi' FGF-2 (approximately double that at 50 m.o.i) was achieved at 2 days and coincided with decreased mitotic index and increased chromatin compaction. At 3 days compaction was maximal, mitotic index was minimal, and cell numbers decreased, accompanied by the appearance of DNA laddering, an indicator of apoptosis. Overall, the lower dose of 'hi' FGF-2 and early time points favored a proliferative phenotype while the higher dose, and later time points, promoted chromatin compaction, inhibition of proliferation and cell death.