RESUMEN
The cardiac hormones atrial and brain natriuretic peptides (NPs) counteract the systemic, hypertensive, and hypervolemic actions of angiotensin II (Ang II) via their guanylyl cyclase-A (GC-A) receptor. In the present study, we took advantage of genetically modified mice with conditional, cardiomyocyte (CM)-restricted disruption of GC-A (CM GC-A knockout mice) to study whether NPs can moderate not only the endocrine but also the cardiac actions of Ang II in vivo. Fluorometric measurements of [Ca(2+)](i) transients in isolated, electrically paced adult CMs showed that atrial NP inhibits the stimulatory effects of Ang II on free cytosolic Ca(2+) transients via GC-A. Remarkably, GC-A-deficient CMs exhibited greatly enhanced [Ca(2+)](i) responses to Ang II, which was partly related to increased activation of the Na(+)/H(+)-exchanger NHE-1. Chronic administration of Ang II to control and CM GC-A knockout mice (300 ng/kg body weight per minute via osmotic minipumps during 2 wk) provoked significant cardiac hypertrophy, which was markedly exacerbated in the later genotype. This was concomitant to increased cardiac expression of NHE-1 and enhanced activation of the Ca(2+)/calmodulin-dependent prohypertrophic signal transducers Ca(2+)/calmodulin-dependent kinase II and calcineurin. On the basis of these results, we conclude that NPs exert direct local, GC-A-mediated myocardial effects to antagonize the [Ca(2+)](i)-dependent hypertrophic growth response to Ang II.
Asunto(s)
Angiotensina II/farmacología , Factor Natriurético Atrial/fisiología , Corazón/fisiología , Receptores del Factor Natriurético Atrial/deficiencia , Remodelación Ventricular/fisiología , Angiotensina II/antagonistas & inhibidores , Animales , Factor Natriurético Atrial/genética , Presión Sanguínea/efectos de los fármacos , Señalización del Calcio/fisiología , Ventrículos Cardíacos/efectos de los fármacos , Ratones , Ratones Noqueados , Tamaño de los Órganos , ARN Mensajero/genética , Receptores del Factor Natriurético Atrial/genética , Receptores del Factor Natriurético Atrial/fisiología , Remodelación Ventricular/efectos de los fármacosRESUMEN
The crucial functions of atrial natriuretic peptide (ANP) and endothelial nitric oxide/NO in the regulation of arterial blood pressure have been emphasized by the hypertensive phenotype of mice with systemic inactivation of either the guanylyl cyclase-A receptor for ANP (GC-A-/-) or endothelial nitric-oxide synthase (eNOS-/-). Intriguingly, similar levels of arterial hypertension are accompanied by marked cardiac hypertrophy in GC-A-/-, but not in eNOS-/-, mice, suggesting that changes in local pathways regulating cardiac growth accelerate cardiac hypertrophy in the former and protect the heart of the latter. Our recent observations in mice with conditional, cardiomyocyte-restricted GC-A deletion demonstrated that ANP locally inhibits cardiomyocyte growth. Abolition of these local, protective effects may enhance the cardiac hypertrophic response of GC-A-/- mice to persistent increases in hemodynamic load. Notably, eNOS-/- mice exhibit markedly increased cardiac ANP levels, suggesting that increased activation of cardiac GC-A can prevent hypertensive heart disease. To test this hypothesis, we generated mice with systemic inactivation of eNOS and cardiomyocyte-restricted deletion of GC-A by crossing eNOS-/- and cardiomyocyte-restricted GC-A-deficient mice. Cardiac deletion of GC-A did not affect arterial hypertension but significantly exacerbated cardiac hypertrophy and fibrosis in eNOS-/- mice. This was accompanied by marked cardiac activation of both the mitogen-activated protein kinase (MAPK) ERK 1/2 and the phosphatase calcineurin. Our observations suggest that local ANP/GC-A/cyclic GMP signaling counter-regulates MAPK/ERK- and calcineurin/nuclear factor of activated T cells-dependent pathways of cardiac myocyte growth in hypertensive eNOS-/- mice.
Asunto(s)
Factor Natriurético Atrial/farmacología , Miocardio/patología , Óxido Nítrico Sintasa/genética , Óxido Nítrico Sintasa/fisiología , Animales , Presión Sanguínea , Northern Blotting , Western Blotting , GMP Cíclico/metabolismo , Eliminación de Gen , Genotipo , Ventrículos Cardíacos/patología , Hipertensión/patología , Hipertrofia , Ratones , Ratones Noqueados , Ratones Transgénicos , Óxido Nítrico Sintasa de Tipo II , Óxido Nítrico Sintasa de Tipo III , Fenotipo , Fosforilación , ARN Mensajero/metabolismo , Transducción de SeñalRESUMEN
To dissect the tissue-specific functions of atrial natriuretic peptide (ANP), we recently introduced loxP sites into the murine gene for its receptor, guanylyl cyclase-A (GC-A), by homologous recombination (tri-lox GC-A). For either smooth-muscle or cardiomyocyte-restricted deletion of GC-A, floxed GC-A mice were mated to transgenic mice expressing Cre-recombinase under the control of the smooth-muscle SM22 or the cardiac alphaMHC promoter. As shown in these studies, Cre-mediated recombination of the floxed GC-A gene fully inactivated GC-A function in a cell-restricted manner. In the present study we show that alphaMHC-Cre, but not SM22-Cre, with high frequency generates genomic recombinations of the floxed GC-A gene segments which were transmitted to the germline. Alleles with partial or complete deletions were readily recovered from the next generation, after segregation of the Cre-transgene. We took advantage of this strategy to generate a new mouse line with global, systemic deletion of GC-A. Doppler-echocardiographic and physiological studies in these mice demonstrate for the first time the tremendous impact of ANP/GC-A dysfunction on chronic blood volume homeostasis.
Asunto(s)
Factor Natriurético Atrial/metabolismo , Volumen Sanguíneo/genética , Silenciador del Gen , Guanilato Ciclasa/metabolismo , Hipertensión/genética , Modelos Animales , Receptores del Factor Natriurético Atrial/metabolismo , Animales , Presión Sanguínea , Southern Blotting , Cartilla de ADN , Ecocardiografía Doppler , Componentes del Gen , Guanilato Ciclasa/genética , Corazón/fisiología , Integrasas/metabolismo , Ratones , Ratones Transgénicos , Proteínas de Microfilamentos/genética , Proteínas Musculares/genética , Músculo Liso/metabolismo , Cadenas Pesadas de Miosina/genética , Cadenas Pesadas de Miosina/metabolismo , Regiones Promotoras Genéticas/genética , Receptores del Factor Natriurético Atrial/genética , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Cardiac hypertrophy is a common and often lethal complication of arterial hypertension. Atrial natriuretic peptide (ANP) has been postulated to exert local antihypertrophic effects in the heart. Thus, a loss of function of the ANP receptor guanylyl cyclase-A (GC-A) might contribute to the increased propensity to cardiac hypertrophy, although a causative role in vivo has not been definitively demonstrated. To test whether local ANP modulates cardiomyocyte growth, we inactivated the GC-A gene selectively in cardiomyocytes by homologous loxP/Cre-mediated recombination. Thereby we have circumvented the systemic, hypertensive phenotype associated with germline inactivation of GC-A. Mice with cardiomyocyte-restricted GC-A deletion exhibited mild cardiac hypertrophy, markedly increased mRNA expression of cardiac hypertrophy markers such as ANP (fivefold), alpha-skeletal actin (1.7-fold), and beta-myosin heavy chain (twofold), and increased systemic circulating ANP levels. Their blood pressure was 7-10 mmHg below normal, probably because of the elevated systemic levels and endocrine actions of ANP. Furthermore, cardiac hypertrophic responses to aortic constriction were enhanced and accompanied by marked deterioration of cardiac function. This phenotype is consistent with a local function of the ANP/GC-A system to moderate the molecular program of cardiac hypertrophy.