Regulation of VASP phosphorylation in cardiac myocytes: differential regulation by cyclic nucleotides and modulation of protein expression in diabetic and hypertrophic heart

Abstract

Sartoretto JL, Jin BY, Bauer M, Gertler FB, Liao R, Michel T. Regulation of VASP phosphorylation in cardiac myocytes: differential regulation by cyclic nucleotides and modulation of protein expression in diabetic and hypertrophic heart. Am J Physiol Heart Circ Physiol 297: H1697-H1710, 2009. First published September 4, 2009 doi:10.1152/ajpheart.00595.2009.-Vasodilator-stimulated phosphoprotein (VASP) is a major substrate for cyclic nucleotide-dependent kinases that has been implicated in cardiac pathology, yet many aspects of VASP's molecular regulation in cardiomyocytes are incompletely understood. In these studies, we explored the role of VASP, both in signaling pathways in isolated murine myocytes, as well as in a model of cardiac hypertrophy in VASP null mice. We found that the ␤-adrenergic agonist isoproterenol promotes the rapid and reversible phosphorylation of VASP at Ser157 and Ser239. Forskolin and the cAMP analog 8-(4-chlorophenylthio)-cAMP promote a similar pattern of VASP phosphorylation at both sites. The effects of isoproterenol are blocked by atenolol and by compound H-89, an inhibitor of the cAMP-dependent protein kinase. By contrast, phosphorylation of VASP only at Ser239 is seen following activation of particulate guanylate cyclase by atrial natriuretic peptide, or following activation of soluble guanylate cyclase by sodium nitroprusside, or following treatment of myocytes with cGMP analog. We found that basal and isoproterenol-induced VASP phosphorylation is entirely unchanged in cardiomyocytes isolated from either endothelial or neuronal nitric oxide synthase knockout mice. In cardiomyocytes isolated from diabetic mice, only basal VASP phosphorylation is increased, whereas, in cells isolated from mice subjected to ascending aortic constriction (AAC), we found a significant increase in basal VASP expression, along with an increase in VASP phosphorylation, compared with cardiac myocytes isolated from sham-operated mice. Moreover, there is further increase in VASP phosphorylation in cells isolated from hypertrophic hearts following isoproterenol treatment. Finally, we found that VASP null mice subjected to transverse aortic constriction develop cardiac hypertrophy with a pattern similar to VASP ϩ/ϩ mice. Our findings establish differential receptor-modulated regulation of VASP phosphorylation in cardiomyocytes by cyclic nucleotides. Furthermore, these studies demonstrate for the first time that VASP expression is upregulated in hypertrophied heart. signal transduction; endothelial nitric oxide synthase; neuronal nitric oxide synthase; phosphoprotein VASODILATOR-STIMULATED PHOSPHOPROTEIN (VASP) is the archetypal member of a family of actin-binding proteins termed the ENA/VASP protein family (34). This protein family includes the Drosophila protein Enabled (ENA), its mammalian homolog MENA, the ENA/VASP-like protein Evl, and VASP (13). MENA/VASP proteins are implicated in actin polymerization, and these proteins appear to modulate diverse cellular responses, including cell migration (26). VASP ...

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“…The heart was quickly removed from the chest and retrogradely perfused through the aorta as described (39). Cardiac myocyte isolation methods followed the procedures as described (39), with minor modifications as we have previously reported (22). In brief, enzymatic digestion was initiated by adding collagenase type 2 to the cardiac perfusion solution, followed by the stepwise introduction of CaCl 2 , after which the heart tissue was minced and the cells were dispersed by trituration, following which the cardiac myocytes were allowed to settle, and then washed, pelleted, counted, and plated.…”

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“…Detection of a high degree of autophosphorylation on the overexpressed PKD1 and PKD2 using a phosphospecific antibody (against PKD-pSer916), which reflects the activation state of both isoforms (Matthews et al, 2000), indicated that they were active when transfected into BAECs. In addition, the increased appearance of vasodilatorstimulated phosphoprotein (VASP) phosphorylated at Ser239, a read-out for NO release (Sartoretto et al, 2009), suggested that eNOS phosphorylation on Ser1179 mediated by PKD1 and PKD2 resulted in enzymatic activation and increased NO synthesis. BAECs presented a basal level of eNOS Ser1179 phosphorylation, which indicated this particular site was already phosphorylated in endothelial cells by endogenous serine kinases, including PKD.…”