Fibroblast Growth Factor-2 Decreases Metabolic Coupling and Stimulates Phosphorylation as Well as Masking of Connexin43 Epitopes in Cardiac Myocytes

Abstract

Cardiac gap junction (GJ) channels, composed of connexins, allow electrical and metabolic couplings between cardiomyocytes, properties important for coordinated action of the heart as well as tissue homeostasis and control of growth and differentiation. Fibroblast growth factor-2 (FGF-2) is an endogenous growth-promoting protein, believed to participate in the short- and long-term responses of the heart to injury. We have examined short-term effects of FGF-2 on cardiac myocyte GJ-mediated metabolic coupling, using cultures of neonatal rat cardiomyocytes. FGF-2 decreased coupling between cardiomyocytes assessed by scrape dye loading as well as microinjection and dye transfer within 30 minutes of administration. Genistein blocked the effects of FGF-2. To determine the mechanism, we next assessed the effect of FGF-2 on expression, distribution, and phosphorylation of connexin43 (Cx43), which is a major cardiomyocyte connexin. FGF-2 did not affect Cx43 mRNA or protein accumulation and synthesis, and it did not change Cx43 localization at sites of intercellular contact as assessed by immunostaining with a polyclonal anti-Cx43 antibody raised against a synthetic peptide containing residues 346 to 363 of Cx43. FGF-2, however, decreased staining intensity at sites of intermyocyte contact when a monoclonal anti-Cx43 antibody was used, suggesting a localized masking of epitope(s) recognized by the monoclonal but not the polyclonal antibody. These epitopes appear to reside within residues 261 to 270 of Cx43, as indicated by full quenching of monoclonal antibody staining with synthetic peptides. In addition, FGF-2 induced a more than twofold increase in Cx43 phosphorylation. Phosphoamino acid analysis indicated increased phosphorylation of Cx43 on serine residues. Although tyrosine phosphorylation of Cx43 was not detected in either treated or control cells, a fraction of Cx43 was immunoprecipitated with anti-phosphotyrosine-specific antibodies in FGF-2-treated myocytes, suggesting interaction (and hence coprecipitation) with phosphotyrosine-containing protein(s). In conclusion, we have identified Cx43 and intercellular communication as targets of FGF-2-triggered and tyrosine phosphorylation-dependent signal transduction in cardiac myocytes. It is suggested that phosphorylation of Cx43 on serine induced by FGF-2 contributes to decreased metabolic coupling between cardiomyocytes.

Phosphorylation of serine 262 in the gap junction protein connexin-43 regulates DNA synthesis in cell-cell contact forming cardiomyocytes

“…Our previous studies suggested that mitotic stimulation may result in a PKCmediated phosphorylation of Cx43 on S262 (Doble et al, 1996;Doble et al, 2000a). Whether Cx43 phosphorylation at any one site, including S262, can affect its ability to inhibit DNA synthesis is not known.…”

“…Neonatal rat cardiomyocytes were prepared from 1-day-old Sprague Dawley rat pups (obtained from the Central Animal Care Facility at the University of Manitoba) as previously described (Doble et al, 1996;Doble et al, 2000b). Our procedures followed the guidelines of the Canadian Council on Animal Care and were approved by the local Animal Care Committee of the National Research Council of Canada.…”

“…Our previous studies provided evidence for a link between mitogenic stimulation of neonatal cardiomyocytes and effects on Cx43. Fibroblast growth factor 2, FGF2, stimulates cardiomyocyte proliferation (Kardami, 1990;Pasumarthi et al, 1996) as well as protein kinase C (PKC; ε subtype)-dependent additional serine phosphorylation of Cx43 (Doble et al, 1996;Doble et al, 2000b). In contrast, transforming growth factor β, a factor that cancels out the mitogenic effect of FGF2 on cardiomyocytes (Kardami, 1990), prevented the FGF2-induced Cx43 phosphorylation (Kardami, 1998).…”

Connexins as Substrates for Protein Kinases and Phosphoprotein Phosphatases

“…The most frequently used experimental approaches to demonstrate that a particular Cx is a phosphoprotein include metabolic labeling of cultured cells with 32 P followed by immunoprecipitation and alkaline phosphatase treatment, phosphoamino acid analysis (Sáez et al 1986;Takeda et al, 1989;Musil et al, 1990;Crow et al, 1990;Sáez et al, 1990;Lau et al, 1992;Goldberg and Lau, 1993;Kurata and Lau, 1994;Doble et al, 1996;Warn-Cramer et al, 1996;Mikalsen et al, 1997;Cheng and Louis, 1999) or two-dimensional phosphopeptide mapping (Sáez et al, 1990;Kurata and Lau, 1994;Díez et al, 1995;Loo et al, 1995;WarnCramer et al, 1996;Berthoud et al, 1997;Díez et al, 1998;Kanemitsu et al, 1998) in vitro phosphorylation assays using fusion proteins or synthetic peptides containing the putative phosphorylation site(s) and purified protein kinases (Sáez et al, 1990;Loo et al, 1995;WarnCramer et al, 1996;Berthoud et al, 1997;Kanemitsu et al, 1998;Shah et al, 2002;O'Brien et al, 2004;Ouyang et al, 2005;Yogo et al, 2006;Alev et al, 2008;Morel et al, 2010); treatment of cultured cells with specific protein kinase or phosphoprotein phosphatase activators or inhibitors to alter 32 P incorporation or the immunoblot pattern of connexins (Lau et al, 1992;Husøy et al, 1993;Guan et al, 1996;Berthoud et al, 1997;…”

“…Phosphoamino acid analysis indicated that hepatocyte Cx32 and Cx43 in uninfected fibroblasts were phosphorylated on seryl residues (Takeda et al, 1989;Crow et al, 1990;Sáez et al, 1990), but Cx43 was also phosphorylated in tyrosyl residues in RSV-transformed fibroblasts (Crow et al, 1990). Using metabolic labeling with 32 P, other studies described that EGF-induced phosphorylation of Cx43 on serine residues in T51B cells through activation of mitogen-activated protein kinase (MAPK) (Lau et al, 1992;WarnCramer et al, 1996), FGF-2 induced phosphorylation of Cx43 in cardiomyocytes (Doble et al, 1996), tyrosine phosphorylation of Cx43 in early passage hamster embryo fibroblast (Mikalsen et al, 1997), phosphorylation of Cx56 by PKC and Cx49 by casein kinase 1 (CK1) in lens fiber cells (Berthoud et al, 1997;Cheng and Louis, 1999). In some cases, the specific phosphorylation site has been identified in reconstituted connexons expressed in Xenopus laevis oocytes.…”