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Fibroblast growth factor-2 (FGF-2), administered to the isolated rat heart by perfusion and under constant pressure, is protective against ischemia-reperfusion (I-R). Here we have investigated whether FGF-2 cardioprotection: (a) is dependent on flow modulation; (b) is linked to effects on contractility; (c) is mediated by protein kinase C (PKC); and (d) is linked to PKC and/or mitogen activated protein kinase (MAPK) associated with the sarcolemma. The isolated rat heart was used as a model. Under conditions of constant flow FGF-2 induced significant improvement in recovery of contractile function during I-R. Under constant perfusion pressure, FGF-2 induced a negative inotropic effect (15% decrease in developed pressure). Chelerythrine, a specific PKC inhibitor, prevented both the FGF-2-induced negative inotropic effect before ischemia, and cardioprotection during I-R. FGF-2 induced a chelerythrine-preventable, five-fold increase in sarcolemmal calcium-independent PKC activity. It also increased the association of PKC subtypes -epsilon and -delta with sarcolemmal membranes, detected by Western blotting, as well as, for PKC delta, by immunolocalization. FGF-2 increased the association of PKC epsilon with the membrane fraction of adult cardiomyocyte in culture, confirming that it can affect PKC signaling in cardiomyocytes directly and in a manner similar to its effects in situ. Finally, FGF-2 induced increased active MAPK at sarcolemmal as well as cytosolic sites. Active sarcolemmal MAPK remained elevated when the FGF-2-induced protection was prevented by chelerythrine. In conclusion, we have provided evidence that cardioprotection by FGF-2 is independent of flow modulation. PKC activation mediates both the FGF-2-induced negative inotropic effect before ischemia and the cardioprotective effect assessed during reperfusion, suggesting a cause and effect relationship. Furthermore, FGF-2 cardioprotection is linked to targeting of sarcolemmal sites by calcium-independent PKC.

Basic fibroblast growth factor (bFGF) has been implicated in the changes in gene expression that, under pathological conditions such as ischemia or volume overload, lead to adult cardiomyocyte hypertrophy. In many tissues, one of the first events following cell activation by growth factors is an enhancement of the intracellular free calcium concentration, generated by fluxes from internal storage compartments and through channels of the plasma membrane. The present study was undertaken to determine whether cardiac myocytes isolated from adult rat ventricles express Ca2+-permeable channels activated by bFGF. Using the cell-attached mode of the patch-clamp technique, we observed that bFGF (from 0.1-10 nM) induced an increase of fast burst openings, mediated by Ca2+-permeable channels with low conductance (15 pS) and voltage-independence. Inside-out patch-clamp experiments revealed that inositol 1,4,5-trisphosphate (5 microM) enhanced the opening of Ca2+-permeable channels with similar properties as the bFGF-induced channels, indicating that IP3 may be a second messenger of this process. Confocal fluorescence imaging of intracellular free calcium provided direct evidence that bFGF induced an increase of cytoplasmic and nucleoplasmic free Ca2+ concentrations which were generated, in part, by Ca2+ influx through the plasma membrane. In conclusion, this study supports the presence, in the plasma membrane of adult cardiac myocytes, of messenger-activated calcium channels which could play key roles in the calcium-dependent pathways that are activated in response to growth factors., (Copyright 1997 Academic Press Limited.)

Basic fibroblast growth factor (bFGF) is a potent mitogen for many cell lineages including fetal cardiomyocytes. Furthermore, bFGF has been shown to modify gene expression, in vitro, in adult nonproliferative ventricular myocytes. This effect is suspected to be partly responsible for the genetic modifications that occur in vivo under pathophysiological conditions such as ischemia or pressure overload and that lead to myocardial hypertrophy. However, little is known about the first steps of the molecular mechanisms that take place soon after cell activation by bFGF. In this study, using biochemical and electrophysiological approaches, we have established, on cardiomyocytes cultured from neonatal rat ventricles, that (i) differentiated beating cells express at least two classes of bFGF-receptors having high and low affinity (Kd = 10 +/- 2 pM and 1 +/- 0.5 nM); (ii) the stimulation of these bFGF receptors promotes an increase in the beating frequencies of cultured cardiomyocytes (40 +/- 10%); (iii) bFGF provokes the activation of poorly specific and voltage-independent calcium channels (12pS); (iv) inositol 1,4,5-trisphosphate enhances similar bFGF-induced Ca2+ currents and is therefore suspected to be a second messenger triggering this activation. These results support the presence, in cultured cardiomyocytes, of new calcium channels whose activation after bFGF binding may be partly responsible for the cell response to this growth factor.