RESUMEN
BACKGROUND: The most common cause of chronic heart failure in the US is secondary or primary dilated cardiomyopathy (DCM). The DCM phenotype exhibits changes in the expression of genes that regulate contractile function and pathologic hypertrophy. However, it is unclear if any of these alterations in gene expression are disease producing or modifying. MATERIALS AND METHODS: One approach to providing evidence for cause-effect of a disease-influencing gene is to quantitatively compare changes in phenotype to changes in gene expression by employing serial measurements in a longitudinal experimental design. We investigated the quantitative relationships between changes in gene expression and phenotype n 47 patients with idiopathic DCM. In endomyocardial biopsies at baseline and 6 months later, we measured mRNA expression of genes regulating contractile function (beta-adrenergic receptors, sarcoplasmic reticulum Ca(2) + ATPase, and alpha- and beta-myosin heavy chain isoforms) or associated with pathologic hypertrophy (beta-myosin heavy chain and atrial natriuretic peptide), plus beta-adrenergic receptor protein expression. Left ventricular phenotype was assessed by radionuclide ejection fraction. RESULTS: Improvement in DCM phenotype was directly related to a coordinate increase in alpha- and a decrease in beta-myosin heavy chain mRNA expression. In contrast, modification of phenotype was unrelated to changes in the expression of beta(1)- or beta(2)-adrenergic receptor mRNA or protein, or to the mRNA expression of sarcoplasmic reticulum Ca(2) + ATPase and atrial natriuretic peptide. CONCLUSION: We conclude that in human DCM, phenotypic modification is selectively associated with myosin heavy chain isoform changes. These data support the hypothesis that myosin heavy chain isoform changes contribute to disease progression in human DCM.
Asunto(s)
Cardiomiopatía Dilatada/genética , Cardiomiopatía Dilatada/patología , Miocardio/metabolismo , Cadenas Pesadas de Miosina/genética , Antihipertensivos/uso terapéutico , Factor Natriurético Atrial/genética , Factor Natriurético Atrial/metabolismo , Biopsia , ATPasas Transportadoras de Calcio/genética , ATPasas Transportadoras de Calcio/metabolismo , Carbazoles/uso terapéutico , Carvedilol , Catecolaminas/metabolismo , Progresión de la Enfermedad , Femenino , Expresión Génica , Ventrículos Cardíacos/diagnóstico por imagen , Ventrículos Cardíacos/fisiopatología , Humanos , Masculino , Metoprolol/uso terapéutico , Persona de Mediana Edad , Fenotipo , Propanolaminas/uso terapéutico , Isoformas de Proteínas , ARN Mensajero/metabolismo , Cintigrafía , Receptores Adrenérgicos beta/genética , Retículo Sarcoplasmático/enzimología , Función Ventricular IzquierdaRESUMEN
BACKGROUND: We have previously demonstrated that changes in myosin heavy chain (MHC) isoforms that occur in failing human hearts resemble the pattern produced in rodent myocardium in response to hypothyroidism. Because thyroid hormone status is usually within normal limits in these patients, we hypothesized that failing/hypertrophied human myocardium might have a defect in thyroid hormone signaling due to alterations in expression of thyroid hormone receptors (TRs). METHODS AND RESULTS: To examine this hypothesis, we used RNase protection assay to measure mRNA levels of TRs in failing left ventricles that exhibited a fetal pattern of gene expression, ie, decreased expression of alpha-MHC with increased beta-MHC expression compared with left ventricles from age-matched controls. We detected expression of TR-alpha(1), -alpha(2), and -beta(1) isoforms in human left ventricles. In failing left ventricles, TR-alpha(1) was downregulated, whereas TR-alpha(2), a splice variant that does not bind thyroid hormone but inhibits responses to liganded TRs, was increased. Expression levels of TR-beta(1) did not differ significantly between the 2 groups. According to linear regression analysis, expression levels of TR-alpha(1) and -alpha(2) were positively and negatively correlated with those of alpha-MHC, respectively. CONCLUSIONS: We conclude that decreases in TR-alpha(1) and increases in TR-alpha(2) may lead to local attenuation of thyroid hormone signaling in the failing human heart and that the resulting tissue-specific hypothyroidism is a candidate for the molecular mechanism that induces fetal gene expression in the failing human ventricle.
Asunto(s)
Expresión Génica , Cardiopatías/genética , Receptores de Hormona Tiroidea/genética , Transducción de Señal/genética , Adulto , Factor Natriurético Atrial/genética , Femenino , Proteínas Fetales/genética , Humanos , Complejo Mayor de Histocompatibilidad/genética , Masculino , Persona de Mediana Edad , ARN Mensajero/biosíntesis , Receptores de Hormona Tiroidea/biosíntesisRESUMEN
BACKGROUND: The regulation of angiotensin II receptors and the two major subtypes (AT1 and AT2) in chronically failing human ventricular myocardium has not been previously examined. METHODS AND RESULTS: Angiotensin II receptors were measured by saturation binding of 125I-[Sar1,Ile8]angiotensin II in crude membranes from nonfailing (n = 19) and failing human left ventricles with idiopathic dilated cardiomyopathy (IDC; n = 31) or ischemic cardiomyopathy (ISC; n = 21) and membranes from a limited number of right ventricles in each category. The AT1 and AT2 fractions were determined by use of an AT1-selective antagonist, losartan. beta-Adrenergic receptors were also measured by binding of 125I-iodocyanopindolol with the beta 1 and beta 2 fractions determined by use of a beta 1-selective antagonist, CGP20712A, AT1 but not AT2 density was significantly decreased in the combined (IDC + ISC) failing left ventricles (nonfailing: AT1 4.66 +/- 0.48, AT2 2.73 +/- 0.39; failing: AT1 3.20 +/- 0.29, AT2 2.70 +/- 0.33 fmol/mg protein; mean +/- SE). The decrease in AT1 density was greater in the IDC than in the ISC left ventricles (IDC: 2.73 +/- 0.40, P < .01; ISC: 3.89 +/- 0.39 fmol/mg protein, P = NS versus nonfailing). beta 1 but not beta 2 density was decreased in the failing left ventricles. AT1 density was correlated with beta 1 density in all left ventricles (r = .43). AT1 density was also decreased in IDC right ventricles. In situ reverse transcription-polymerase chain reaction in sections of nonfailing and failing ventricles indicated that AT1 mRNA was present in both myocytes and nonmyocytes. CONCLUSIONS: AT1 receptors are selectively downregulated in failing human ventricles, similar to the selective downregulation of beta 1 receptors. The relative lack of AT1 downregulation in ISC hearts may be related to differences in the degree of ventricular dysfunction.
Asunto(s)
Cardiomiopatía Dilatada/metabolismo , Insuficiencia Cardíaca/metabolismo , Miocardio/metabolismo , Receptores de Angiotensina/biosíntesis , Adulto , Angiotensina II/análogos & derivados , Angiotensina II/metabolismo , Membrana Celular/metabolismo , Regulación hacia Abajo , Femenino , Insuficiencia Cardíaca/patología , Ventrículos Cardíacos , Humanos , Cinética , Masculino , Miocardio/patología , Reacción en Cadena de la Polimerasa , Ensayo de Unión Radioligante , Receptor de Angiotensina Tipo 1 , Receptor de Angiotensina Tipo 2 , Receptores Adrenérgicos beta 1/metabolismo , Receptores Adrenérgicos beta 2/metabolismo , Valores de ReferenciaRESUMEN
In both cell culture based model systems and in the failing human heart, beta-adrenergic receptors ( beta-AR) undergo agonist-mediated down-regulation. This decrease correlates closely with down-regulation of its mRNA, an effect regulated in part by changes in mRNA stability. Regulation of mRNA stability has been associated with mRNA-binding proteins that recognize A + U-rich elements within the 3'-untranslated regions of many mRNAs encoding proto-oncogene and cytokine mRNAs. We demonstrate here that the mRNA-binding protein, AUF1, is present in both human heart and in hamster DDT1-MF2 smooth muscle cells and that its abundance is regulated by beta-AR agonist stimulation. In human heart, AUF1 mRNA and protein was significantly increased in individuals with myocardial failure, a condition associated with increases in the beta-adrenergic receptor agonist norepinephrine. In the same hearts, there was a significant decrease (approximately 50%) in the abundance of beta1-AR mRNA and protein. In DDT1-MF2 cells, where agonist-mediated destabilization of beta2-AR mRNA was first described, exposure to beta-AR agonist resulted in a significant increase in AUF1 mRNA and protein (approximately 100%). Conversely, agonist exposure significantly decreased (approximately 40%) beta2-adrenergic receptor mRNA abundance. Last, we demonstrate that AUF1 can be immunoprecipitated from polysome-derived proteins following UV cross-linking to the 3'-untranslated region of the human beta1-AR mRNA and that purified, recombinant p37AUF1 protein also binds to beta1-AR 3'-untranslated region mRNA.
Asunto(s)
Regulación de la Expresión Génica , Ribonucleoproteína Heterogénea-Nuclear Grupo D , Miocardio/metabolismo , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/metabolismo , Receptores Adrenérgicos beta/fisiología , Secuencia de Bases , Cartilla de ADN/química , Proteínas de Unión al GTP/fisiología , Insuficiencia Cardíaca/metabolismo , Ribonucleoproteína Nuclear Heterogénea D0 , Humanos , Datos de Secuencia Molecular , Músculo Liso/metabolismo , Polirribosomas/metabolismo , Polirribosomas/efectos de la radiación , Proto-Oncogenes Mas , ARN Mensajero/química , Proteínas Recombinantes , Transducción de Señal , Rayos UltravioletaRESUMEN
Heart failure in humans is characterized by alterations in myocardial adrenergic signal transduction, the most prominent of which is down-regulation of beta 1-adrenergic receptors. We tested the hypothesis that down-regulation of beta 1-adrenergic receptors in the failing human heart is related to decreased steady-state levels of beta 1 receptor mRNA. Due to the extremely low abundance of beta 1 receptor mRNA, measurements were possible only by quantitative polymerase chain reaction (QPCR) or by RNase protection methods. Because the beta 1 receptor gene is intronless and beta 1 receptor mRNA abundance is low, QPCR yielded genomic amplification in total RNA, and mRNA measurements had to be performed in poly (A)(+)-enriched RNA. By QPCR the concentration of beta 1 receptor mRNA varied from 0.34 to 7.8 x 10(7) molecules/microgram poly(A)(+)-enriched RNA, and the assay was sensitive to 16.7 zeptomol. Using 100-mg aliquots of left ventricular myocardium obtained from organ donors (nonfailing ventricles, n = 12) or heart transplant recipients (failing ventricles, n = 13), the respective beta 1 mRNA levels measured by QPCR were 4.2 +/- 0.7 x 10(7)/micrograms vs. 2.10 +/- 0.3 x 10(7)/micrograms (P = 0.006). In these same nonfailing and failing left ventricles the respective beta 1-adrenergic receptor densities were 67.9 +/- 6.9 fmol/mg vs. 29.6 +/- 3.5 fmol/mg (P = 0.0001). Decreased mRNA abundance in the failing ventricles was confirmed by RNase protection assays in total RNA, which also demonstrated a 50% reduction in beta 1 message abundance. We conclude that down-regulation of beta 1 receptor mRNA contributes to down-regulation of beta 1 adrenergic receptors in the failing human heart.
Asunto(s)
Insuficiencia Cardíaca/metabolismo , Miocardio/metabolismo , ARN Mensajero/metabolismo , Receptores Adrenérgicos beta 1/biosíntesis , Elementos sin Sentido (Genética) , Secuencia de Bases , ADN/aislamiento & purificación , ADN/metabolismo , Cartilla de ADN , Insuficiencia Cardíaca/cirugía , Trasplante de Corazón , Trasplante de Corazón-Pulmón , Humanos , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa/métodos , ARN Mensajero/biosíntesis , ARN Mensajero/aislamiento & purificación , Receptores Adrenérgicos beta 2/biosíntesis , Valores de ReferenciaRESUMEN
Weekly injections of the catecholamine depleting agent 6-hydroxydopamine (6-OHDA) were used to denervate rabbit hearts chemically. Analyses of morphology and beta-adrenergic receptor density were made at 1, 2, and 4 weeks. Changes resulting from subacute and chronic inflammatory processes were evident by light microscopy after 1 week. At that time, electron microscopy revealed marked increases in collagen, large myocytic vacuolizations in myocytes, widened gap junctions, and myofibrillar degeneration and dropout. Receptor density was marginally increased at 2 weeks but was decreased (p less than .05) at 4 weeks (maximal [3H]dihydroalprenolol (DHA) binding in fmol/mg: 69.6 +/- 5.4 in controls vs 49.2 +/- 5.1 in 6-OHDA-treated animals). Basal, isoproterenol-stimulated and F- -stimulated adenylate cyclase activities were decreased in the 6-OHDA-treated group at 4 weeks. We conclude that administration of 6-OHDA may cause severe myocardial damage, and that this process may involve loss of some functional components of the cell membrane.
Asunto(s)
Hidroxidopaminas/farmacología , Adenilil Ciclasas/metabolismo , Animales , Cardiomiopatías/patología , Dihidroalprenolol/metabolismo , Femenino , Corazón/efectos de los fármacos , Hidroxidopaminas/toxicidad , Miocardio/enzimología , Miocardio/patología , Norepinefrina/metabolismo , Oxidopamina , Conejos , Simpatectomía QuímicaRESUMEN
We tested the hypothesis that cardiac histamine release mediates subacute doxorubicin (DXR) cardiotoxicity in rabbits. New Zealand white rabbits given DXR 20 mg/kg i.v. over 30 min developed myocardial damage 24 h later that was similar to that observed in humans. In isolated heart preparations, DXR produced dose-related cardiac histamine release at DXR concentrations of 1, 5, and 25 micrograms/ml given as 1-min exposures. Prior exposure of isolated hearts to 10 microM cromolyn sodium completely prevented histamine release from 5 micrograms/ml DXR. Pretreatment of animals with cromolyn produced significant protection against DXR-mediated subacute cardiotoxicity. We conclude that the release of cardiac histamine may be involved in the pathogenesis of anthracycline cardiotoxicity.
Asunto(s)
Cardiopatías/inducido químicamente , Liberación de Histamina/efectos de los fármacos , Animales , Antibióticos Antineoplásicos , Cromolin Sódico/farmacología , Doxorrubicina/sangre , Doxorrubicina/toxicidad , Femenino , Cardiopatías/metabolismo , Naftacenos/toxicidad , Conejos , Factores de TiempoRESUMEN
We tested the hypothesis that anthracycline-induced cardiac and renal damage is mediated by vasoactive substances. A 1-minute exposure to 5 micrograms per ml. of doxorubicin (DXR, Adriamycin) produced cardiac histamine release in isolated rabbit hearts. Under conditions in which histamine uptake and metabolism were impaired, the administration of DXR, 2 mg. per kg., over 1 minute was associated with elevations in arterial histamine and catecholamines. The chronic weekly administration of DXR produced severe cardiac and renal damage. The administration of combined histaminic and adrenergic blockade with diphenhydramine, cimetidine, phentolamine, and propranolol (DCPP) pre- and immediately post-DXR resulted in near total protection against DXR-mediated cardiac damage and prevented the majority of the renal lesions. The combined administration of diphenhydramine, cimetidine, phentolamine, and propranolol did not appear to be acting by mechanisms other than blockade of vasoactive amine receptors as cardiac uptake of DXR and the DXR antitumor response were not altered by diphenhydramine, cimetidine, phentolamine, and propranolol. This study demonstrates that anthracycline-associated cardiac and renal toxicity may be mediated by vasoactive substances and that anthracycline cardiomyopathy is potentially preventable.