cAMP-mediated beta-adrenergic signaling negatively regulates Gq-coupled receptor-mediated fetal gene response in cardiomyocytes.
ABSTRACT The treatment with beta-blockers causes an enhancement of the norepinephrine-induced fetal gene response in cultured cardiomyocytes. Here, we tested whether the activation of cAMP-mediated beta-adrenergic signaling antagonizes alpha(1)-adrenergic receptor (AR)-mediated fetal gene response. To address this question, the fetal gene program, of which atrial natriuretic peptide (ANP) and the beta-isoform of myosin heavy chain are classical members, was induced by phenylephrine (PE), an alpha(1)-AR agonist. In cultured neonatal rat cardiomyocytes, we found that stimulation of beta-ARs with isoproterenol, a beta-AR agonist, inhibited the fetal gene expression induced by PE. Similar results were also observed when cardiomyocytes were treated with forskolin (FSK), a direct activator of adenylyl cyclase, or 8-CPT-6-Phe-cAMP, a selective activator of protein kinase A (PKA). Conversely, the PE-induced fetal gene expression was further upregulated by H89, a selective PKA inhibitor. To evaluate whether these results could be generalized to Gq-mediated signaling and not specifically to alpha(1)-ARs, cardiomyocytes were treated with prostaglandin F(2)alpha, another Gq-coupled receptor agonist, which is able to promote fetal gene expression. This treatment caused an increase of both ANP mRNA and protein levels, which was almost completely abolished by FSK treatment. The capability of beta-adrenergic signaling to regulate the fetal gene expression was also evaluated in vivo conditions by using beta1- and beta2-AR double knockout mice, in which the predominant cardiac beta-AR subtypes are lacking, or by administering isoproterenol (ISO), a beta-AR agonist, at a subpressor dose. A significant increase of the fetal gene expression was found in beta(1)- and beta(2)-AR gene deficient mice. Conversely, we found that ANP, beta-MHC and skACT mRNA levels were significantly decreased in ISO-treated hearts. Collectively, these data indicate that cAMP-mediated beta-adrenergic signaling negatively regulates Gq cascade activation-induced fetal gene expression in cultured cardiomyocytes and that this inhibitory regulation is already operative in the mouse heart under physiological conditions.
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ABSTRACT: The skeletal alpha-actin gene is expressed in fetal rat heart and is induced during norepinephrine (NE)-stimulated hypertrophy in cultures of neonatal rat cardiac myocytes. Here we report that NE positively regulates the human skeletal alpha-actin gene promoter in transiently transfected neonatal rat cardiac myocytes. NE increased expression from the full-length promoter by 2.4-fold. A DNA region required for NE responsiveness but not for tissue-specific expression was located between base pair -2000 and base pair -1300. Distinct regions required for cardiac myocyte expression were located between -1300 to -710 and -153 to -87. None of these elements separately conferred tissue specificity or adrenergic responsiveness on a heterologous promoter, although the intact promoter from -2000 to -36 conferred both when cloned in its correct position and orientation. Additional elements in the basal promoter (-87 to +187) were required for maximal NE responsiveness. The NE induction was mediated by the beta-adrenergic receptor in high-density cultures (3-4 x 10(6) cells per 60-mm dish), as was induction of hypertrophy, contractility, and endogenous skeletal alpha-actin gene expression. The beta-adrenergic agonist isoproterenol was as potent as NE in inducing expression. Furthermore, beta-adrenergic antagonists inhibited the effects on skeletal alpha-actin gene expression but alpha 1-adrenergic antagonists did not. The alpha 1-adrenergic system was intact in these high-density cultures, since the effects of NE on the expression of another contractile protein gene, alpha-myosin heavy chain, were blocked by alpha 1- but not by beta-adrenergic antagonists. In these high-density cultures, cell contact and intermyocardiocyte bridging were prevalent. When cardiac myocytes were plated at a low density, minimizing cell contact, NE induction of skeletal alpha-actin gene expression and hypertrophy was mediated by the alpha 1-adrenoceptor. Factors related to cell communication may influence the pathways mediating NE-regulated gene transcription during cardiac myocyte hypertrophy.Proceedings of the National Academy of Sciences 04/1991; 88(6):2132-6. · 9.74 Impact Factor
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ABSTRACT: Mitogen-activated protein kinase (MAPK) signaling pathways are important regulators of cell growth, proliferation, and stress responsiveness. A family of dual-specificity MAP kinase phosphatases (MKPs) act as critical counteracting factors that directly regulate the magnitude and duration of p38, c-Jun N-terminal kinase (JNK), and extracellular signal-regulated kinase (ERK) activation. Here we show that constitutive expression of MKP-1 in cultured primary cardiomyocytes using adenovirus-mediated gene transfer blocked the activation of p38, JNK1/2, and ERK1/2 and prevented agonist-induced hypertrophy. Transgenic mice expressing physiological levels of MKP-1 in the heart showed (1) no activation of p38, JNK1/2, or ERK1/2; (2) diminished developmental myocardial growth; and (3) attenuated hypertrophy in response to aortic banding and catecholamine infusion. These results provide further evidence implicating MAPK signaling factors as obligate regulators of cardiac growth and hypertrophy and demonstrate the importance of dual-specificity phosphatases as counterbalancing regulatory factors in the heart.Circulation Research 02/2001; 88(1):88-96. · 11.86 Impact Factor
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ABSTRACT: BALB/c mice express abnormally high levels of alpha-skeletal actin in the heart, which may be related to a duplication in the promoter of the alpha-cardiac actin gene. To evaluate the effects of overexpression of the alpha-skeletal actin isoform on cardiac contractile function, we studied these mice using the isolated perfused work-performing murine heart model and measured actin isoform expression in the same hearts. We quantified myocardial contractility from the maximum rate of contraction (+dP/dt) and time to peak pressure and relaxation from -dP/dt and time to half relaxation of left intraventricular pressure. Dot blots of total RNA hybridized against oligonucleotide sequences specific for either alpha-skeletal or alpha-cardiac actin mRNA showed that increased levels of alpha-skeletal actin RNA correlated significantly with increased contractility of hearts from the BALB/c mice (r = .80, n = 15, P < .001). The present study demonstrates a significant functional correlation between alpha-actin isoform content and cardiac contractile function and also that alpha-skeletal actin may promote an increased contractile function in the heart compared with alpha-cardiac actin.Circulation Research 04/1994; 74(4):740-6. · 11.86 Impact Factor