S100A6 is a negative regulator of the induction of cardiac genes by trophic stimuli in cultured rat myocytes

S100A6 (calcyclin), a member of the S100 family of EF-hand Ca2+ binding proteins, has been implicated in the regulation of cell growth and proliferation. We have previously shown that S100B, another member of the S100 family, is induced postinfarction and limits the hypertrophic response of surviving cardiac myocytes. We presently report that S100A6 expression is also increased in the periinfarct zone of rat heart postinfarction and in cultured neonatal rat myocytes by treatment with several trophic agents, including platelet-derived growth factor (PDGF), the alpha1-adrenergic agonist phenylephrine (PE), and angiotensin II (AII). Cotransfection of S100A6 in cultured neonatal rat cardiac myocytes inhibits induction of the cardiac fetal gene promoters skeletal alpha-actin (skACT) and beta-myosin heavy chain (beta-MHC) by PDGF, PE, AII, and the prostaglandin F2alpha (PGF2alpha), induction of the S100B promoter by PE, and induction of the alpha-MHC promoter by triiodothyronine (T3). By contrast, S100B cotransfection selectively inhibited only PE induction of skACT and beta-MHC promoters. Fluorescence microscopy demonstrated overlapping intracellular distribution of S100B and S100A6 in transfected myocytes and in postinfarct myocardium but heterodimerization of the two proteins could not be detected by co-immunoprecipitation. We conclude that S100A6 may function as a global negative modulator of differentiated cardiac gene expression comparable to its putative role in cell cycle progression of dividing cells.     

Striated muscle-specific beta(1D)-integrin and FAK are involved in cardiac myocyte hypertrophic response pathway

Alterations in the extracellular matrix occur during the cardiac hypertrophic process. Because integrins mediate cell-matrix adhesion and beta(1D)-integrin (beta1D) is expressed exclusively in cardiac and skeletal muscle, we hypothesized that beta1D and focal adhesion kinase (FAK), a proximal integrin-signaling molecule, are involved in cardiac growth. With the use of cultured ventricular myocytes and myocardial tissue, we found the following: 1) beta1D protein expression was upregulated perinatally; 2) alpha(1)-adrenergic stimulation of cardiac myocytes increased beta1D protein levels 350% and altered its cellular distribution; 3) adenovirally mediated overexpression of beta1D stimulated cellular reorganization, increased cell size by 250%, and induced molecular markers of the hypertrophic response; and 4) overexpression of free beta1D cytoplasmic domains inhibited alpha(1)-adrenergic cellular organization and atrial natriuretic factor (ANF) expression. Additionally, FAK was linked to the hypertrophic response as follows: 1) coimmunoprecipitation of beta1D and FAK was detected; 2) FAK overexpression induced ANF-luciferase; 3) rapid and sustained phosphorylation of FAK was induced by alpha(1)-adrenergic stimulation; and 4) blunting of the alpha(1)-adrenergically modulated hypertrophic response was caused by FAK mutants, which alter Grb2 or Src binding, as well as by FAK-related nonkinase, a dominant interfering FAK mutant. We conclude that beta1D and FAK are both components of the hypertrophic response pathway of cardiac myocytes.     

Photoaffinity labeling of alpha 1-adrenergic receptors of rat heart

The photoaffinity probe [125I]aryl azidoprazosin was used to examine structural aspects of rat left ventricular alpha 1-adrenergic receptor. Autoradiography of sodium dodecyl sulfate-polyacrylamide gel electrophoresis-resolved proteins from photoaffinity-labeled membranes revealed a specifically labeled protein of mass 77 kDa. Adrenergic drugs competed with the photoaffinity probe for binding to the receptor in a manner expected of an alpha 1-adrenergic antagonist. Because the autoradiographic pattern was unaltered by incubating labeled membranes in gel sample buffer containing high concentrations of reducing agents, the binding component of the cardiac alpha 1-adrenergic receptor appears to be a single polypeptide chain. The photoaffinity probe specifically labeled a single protein of approximately 68 kDa in membranes of cardiac myocytes prepared from rat left ventricles. The role played by sulfhydryls in receptor structure and function was also studied. Dithiothreitol (DTT) inhibited [3H]prazosin binding to left ventricular membranes and altered both the equilibrium dissociation constant and maximal number of [3H]prazosin-binding sites but not the ability of the guanine nucleotide guanyl-5'-yl imidodiphosphate to decrease agonist affinity for the receptors. When photoaffinity-labeled membranes were incubated with 40 mM DTT for 30 min at room temperature, two specifically labeled proteins of 77 and 68 kDa were identified. The DTT-induced conversion of the 77-kDa protein to 68 kDa was irreversible with washing, but the effect of DTT on [3H]prazosin binding was reversible. Both 77- and 68-kDa proteins were observed with liver membranes even in the absence of reducing agent. We suggest that the DTT-induced conversion of the 77-kDa protein to 68 kDa is due to enhancement in protease activity by the reductant. These results document that the cardiac alpha 1-adrenergic receptor is a 77-kDa protein, similar in mass to the receptor in liver and other sites. Proteolysis likely accounts for lower Mr forms of this receptor found in cardiac myocytes and in previous publications on hepatic alpha 1-receptors.