Abstract
In response to hormones and mechanical stretch, neonatal rat ventricular myocytes exhibit a hypertrophic response that is characterized by induction of cardiac-specific genes and increased myocardial cell size. Hypertrophic stimuli also activate mitogen-activated protein kinase (MAPK), an enzyme thought to play a central role in the regulation of cell growth and differentiation. To determine if MAPK activation is sufficient for acquisition of the molecular and morphological features of cardiac hypertrophy we compared four agonists that stimulate G protein-coupled receptors. Whereas phenylephrine and endothelin transactivate cardiac-specific promoter/luciferase reporter genes, increase atrial natriuretic factor (ANF) expression, and promote myofilament organization, neither carbachol nor ATP induces these responses. Interestingly, all four agonists activate both the p42 and the p44 isoforms of MAPK. Furthermore, the kinetics of MAPK activation are not different for the hypertrophic agonist phenylephrine and the nonhypertrophic agonist carbachol. Transient transfection of myocytes with dominant-interfering mutants of p42 and p44 MAPK failed to block phenylephrine-induced ANF expression, although Ras-induced gene expression was inhibited by expression of the mutant MAPK constructs. Moreover, PD 098059, an inhibitor of MAPK kinase, blocked phenylephrine-stimulated MAPK activity but not ANF reporter gene expression. Thus, MAPK activation is not sufficient for G protein receptor-mediated induction of cardiac cell growth and gene expression and is apparently not required for transcriptional activation of the ANF gene.
Highlights
In response to hormones and mechanical stretch, neonatal rat ventricular myocytes exhibit a hypertrophic response that is characterized by induction of cardiacspecific genes and increased myocardial cell size
It has been proposed that the signaling pathways utilized by G protein-coupled receptors and tyrosine kinase receptors converge at mitogen-activated protein kinase (MAPK) and that MAPK plays a central role in cardiac hypertrophy [15]
We compared the abilities of four G protein-coupled receptor agonists to induce promoter activities of two cardiac-specific genes in neonatal rat ventricular myocytes
Summary
Cell Culture—Neonatal ventricular myocytes were prepared from hearts of 1–3-day-old Sprague-Dawley rats as described previously [18, 19]. Freshly isolated ventricular myocytes at a density of 1 ϫ 106/ml of medium were transfected by electroporation with 10 g of the ANF/luciferase reporter gene and 3 g of cytomegalovirus/-galactosidase. MAPK Assays and Immunodetection—After plating, myocardial cells were washed and maintained in serum-free medium for 24 h prior to experimentation. Neonatal rat ventricular myocytes were transfected with an ANF promoter/luciferase (upper panel) or an MLC-2 promoter/luciferase (lower panel) reporter gene and incubated for 48 h with either no drug, 10 nM ET, 100 M phenylephrine (with 2 M propranolol to block -adrenergic receptors), 300 M carbachol, or 50 M ATP. After preincubation of the gel for 30 min at 4 °C in 20 mM HEPES, 2 mM dithiothreitol, 10 mM MgCl2, pH 7.6, in situ phosphorylation of MBP was performed in the same buffer containing 6.7 Ci/ml [␥-32P]ATP at 30 °C for 2 h. After extensive washing in 5% trichloroacetic acid, 10% sodium pyrophosphate, gels were dried, exposed to film, and phosphate incorporation quantitated by radioanalytic scanning of gels
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