RESUMEN
This review summarizes the current knowledge on essential vitamins B1, B2, B3, and B5. These B-complex vitamins must be taken from diet, with the exception of vitamin B3, that can also be synthetized from amino acid tryptophan. All of these vitamins are water soluble, which determines their main properties, namely: they are partly lost when food is washed or boiled since they migrate to the water; the requirement of membrane transporters for their permeation into the cells; and their safety since any excess is rapidly eliminated via the kidney. The therapeutic use of B-complex vitamins is mostly limited to hypovitaminoses or similar conditions, but, as they are generally very safe, they have also been examined in other pathological conditions. Nicotinic acid, a form of vitamin B3, is the only exception because it is a known hypolipidemic agent in gram doses. The article also sums up: (i) the current methods for detection of the vitamins of the B-complex in biological fluids; (ii) the food and other sources of these vitamins including the effect of common processing and storage methods on their content; and (iii) their physiological function.
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Avitaminosis , Complejo Vitamínico B , Humanos , Tiamina , Vitamina A , Vitamina KRESUMEN
Objectives: Many studies indicate the involvement of transient receptor potential (TRP) channels in the development of heart hypertrophy. However, the data is often conflicted and has originated in animal models. Here, we provide systematic analysis of TRP channels expression in human failing myocardium. Methods and results: Left-ventricular tissue samples were isolated from explanted hearts of NYHA III-IV patients undergoing heart transplants (n = 43). Quantitative real-time PCR was performed to assess the mRNA levels of TRPC, TRPM and TRPV channels. Analysis of functional, clinical and biochemical data was used to confirm an end-stage heart failure diagnosis. Compared to myocardium samples from healthy donor hearts (n = 5), we detected a distinct increase in the expression of TRPC1, TRPC5, TRPM4 and TRPM7, and decreased expression of TRPC4 and TRPV2. These changes were not dependent on gender, clinical or biochemical parameters, nor functional parameters of the heart. We detected, however, a significant correlation of TRPC1 and MEF2c expression. Conclusions: The end-stage heart failure displays distinct expressional changes of TRP channels. Our findings provide a systematic description of TRP channel expression in human heart failure. The results highlight the complex interplay between TRP channels and the need for deeper analysis of early stages of hypertrophy and heart failure development.
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Insuficiencia Cardíaca/fisiopatología , Trasplante de Corazón/efectos adversos , Canales de Potencial de Receptor Transitorio/análisis , Análisis de Varianza , Femenino , Insuficiencia Cardíaca/sangre , Insuficiencia Cardíaca/complicaciones , Trasplante de Corazón/métodos , Humanos , Masculino , Persona de Mediana Edad , Proteínas Serina-Treonina Quinasas/análisis , Proteínas Serina-Treonina Quinasas/sangre , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Estadísticas no Paramétricas , Canales Catiónicos TRPC/análisis , Canales Catiónicos TRPC/sangre , Canales Catiónicos TRPM/análisis , Canales Catiónicos TRPM/sangre , Canales de Potencial de Receptor Transitorio/sangre , Canales de Potencial de Receptor Transitorio/farmacologíaRESUMEN
BACKGROUND: Sphingosine-1-phosphate plays vital roles in cardiomyocyte physiology, myocardial ischemia-reperfusion injury, and ischemic preconditioning. The function of the cardiomyocyte sphingosine-1-phosphate receptor 1 (S1P1) in vivo is unknown. METHODS AND RESULTS: Cardiomyocyte-restricted deletion of S1P1 in mice (S1P1 (α) (MHCC) (re)) resulted in progressive cardiomyopathy, compromised response to dobutamine, and premature death. Isolated cardiomyocytes from S1P1 (α) (MHCC) (re) mice revealed reduced diastolic and systolic Ca(2+) concentrations that were secondary to reduced intracellular Na(+) and caused by suppressed activity of the sarcolemmal Na(+)/H(+) exchanger NHE-1 in the absence of S1P1. This scenario was successfully reproduced in wild-type cardiomyocytes by pharmacological inhibition of S1P1 or sphingosine kinases. Furthermore, Sarcomere shortening of S1P1 (α) (MHCC) (re) cardiomyocytes was intact, but sarcomere relaxation was attenuated and Ca(2+) sensitivity increased, respectively. This went along with reduced phosphorylation of regulatory myofilament proteins such as myosin light chain 2, myosin-binding protein C, and troponin I. In addition, S1P1 mediated the inhibitory effect of exogenous sphingosine-1-phosphate on ß-adrenergic-induced cardiomyocyte contractility by inhibiting the adenylate cyclase. Furthermore, ischemic precondtioning was abolished in S1P1 (α) (MHCC) (re) mice and was accompanied by defective Akt activation during preconditioning. CONCLUSIONS: Tonic S1P1 signaling by endogenous sphingosine-1-phosphate contributes to intracellular Ca(2+) homeostasis by maintaining basal NHE-1 activity and controls simultaneously myofibril Ca(2+) sensitivity through its inhibitory effect on adenylate cyclase. Cardioprotection by ischemic precondtioning depends on intact S1P1 signaling. These key findings on S1P1 functions in cardiac physiology may offer novel therapeutic approaches to cardiac diseases.
Asunto(s)
Calcio/metabolismo , Cardiomiopatías/genética , Precondicionamiento Isquémico Miocárdico , Daño por Reperfusión Miocárdica/genética , Miocitos Cardíacos/metabolismo , Receptores de Lisoesfingolípidos/genética , Intercambiadores de Sodio-Hidrógeno/metabolismo , Potenciales de Acción , Adenilil Ciclasas/metabolismo , Animales , Western Blotting , Miosinas Cardíacas/metabolismo , Cardiomiopatías/diagnóstico por imagen , Cardiomiopatías/metabolismo , Proteínas Portadoras/metabolismo , Ecocardiografía , Imagen por Resonancia Magnética , Ratones , Ratones Noqueados , Daño por Reperfusión Miocárdica/metabolismo , Miocitos Cardíacos/efectos de los fármacos , Cadenas Ligeras de Miosina/metabolismo , Fosforilación , Tomografía de Emisión de Positrones , Reacción en Cadena en Tiempo Real de la Polimerasa , Receptores de Lisoesfingolípidos/antagonistas & inhibidores , Sarcómeros/metabolismo , Receptores de Esfingosina-1-Fosfato , Troponina I/metabolismoRESUMEN
Chronic angiotensin-converting enzyme inhibitor (ACEIs) treatment can suppress arrhythmogenesis. To examine whether the effect is more immediate and independent of suppression of pathological remodelling, we tested the antiarrhythmic effect of short-term ACE inhibition in healthy normotensive rats. Wistar rats were administered with enalaprilat (ENA, i.p., 5 mg/kg every 12 h) or vehicle (CON) for 2 weeks. Intraarterial blood pressure in situ was measured in A. carotis. Cellular shortening was measured in isolated, electrically paced cardiomyocytes. Standard 12-lead electrocardiography was performed, and hearts of anaesthetized open-chest rats were subjected to 6-min ischemia followed by 10-min reperfusion to examine susceptibility to ventricular arrhythmias. Expressions of calcium-regulating proteins (SERCA2a, cardiac sarco/endoplasmic reticulum Ca(2+)-ATPase; CSQ, calsequestrin; TRD, triadin; PLB, phospholamban; Thr(17)-PLB-phosphorylated PLB at threonine-17, FKBP12.6, FK506-binding protein, Cav1.2-voltage-dependent L-type calcium channel alpha 1C subunit) were measured by Western blot; mRNA levels of L-type calcium channel (Cacna1c), ryanodine receptor (Ryr2) and potassium channels Kcnh2 and Kcnq1 were measured by qRT-PCR. ENA decreased intraarterial systolic as well as diastolic blood pressure (by 20%, and by 31%, respectively, for both P < 0.05) but enhanced shortening of cardiomyocytes at basal conditions (by 34%, P < 0.05) and under beta-adrenergic stimulation (by 73%, P < 0.05). Enalaprilat shortened QTc interval duration (CON 78 ± 1 ms vs. ENA 72 ± 2 ms; P < 0.05) and significantly decreased the total duration of ventricular fibrillations (VF) and the number of VF episodes (P < 0.05). Reduction in arrhythmogenesis was associated with a pronounced upregulation of SERCA2a (CON 100 ± 20 vs. ENA 304 ± 13; P < 0.05) and complete absence of basal Ca(2+)/calmodulin-dependent phosphorylation of PLB at Thr(17). Short-term ACEI treatment can provide protection against I/R injury-induced ventricular arrhythmias in healthy myocardium, and this effect is associated with increased SERCA2a expression.
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Inhibidores de la Enzima Convertidora de Angiotensina/farmacología , Arritmias Cardíacas/fisiopatología , Enalaprilato/farmacología , Contracción Miocárdica/efectos de los fármacos , Miocardio/enzimología , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/genética , Regulación hacia Arriba/efectos de los fármacos , Animales , Arritmias Cardíacas/complicaciones , Arritmias Cardíacas/diagnóstico por imagen , Western Blotting , Canales de Calcio Tipo L/genética , Canales de Calcio Tipo L/metabolismo , Separación Celular , Electrólitos/sangre , Enalaprilato/administración & dosificación , Ventrículos Cardíacos/efectos de los fármacos , Ventrículos Cardíacos/patología , Ventrículos Cardíacos/fisiopatología , Isoproterenol/farmacología , Masculino , Miocardio/patología , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/patología , Tamaño de los Órganos/efectos de los fármacos , Canales de Potasio/genética , Canales de Potasio/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas Wistar , Daño por Reperfusión/complicaciones , Daño por Reperfusión/patología , Daño por Reperfusión/fisiopatología , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/metabolismo , UltrasonografíaRESUMEN
BACKGROUND: Protein phosphatase 5 (PP5) a serine/threonine phosphatase is ubiquitously expressed in mammalian tissues including the heart, but its physiological role in the heart is still unknown. Therefore, we used a transgenic mouse model to get a first insight into the cardiac role of PP5. METHODS AND RESULTS: We generated transgenic mice with cardiac myocyte specific overexpression of PP5. Successful overexpression of PP5 was demonstrated by Western blotting, immunohistochemistry and enhanced arachidonic acid-stimulated protein phosphatase activity in transgenic hearts. Cardiac function was examined on the level of isolated cardiac myocytes, isolated organs and in intact animals. Whereas Ca(2+) transients and cell shortening remained unchanged, L-type Ca(2+) currents were decreased in isolated cardiac myocytes from transgenic mice. Ventricular contractility was reduced in isolated perfused hearts under basal conditions and after ß-adrenergic stimulation. In intact animals, echocardiography revealed increased left ventricular diameters and decreased contractility and invasively measured hemodynamic performance by left ventricular catheterization demonstrated a reduced response to ß-adrenergic stimulation in transgenic mice compared to wild type. CONCLUSIONS: Overexpression of PP5 affected contractility and ß-adrenergic signaling in the hearts of transgenic mice. Taken together, these findings are indicative of a regulatory role of PP5 in cardiac function.
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Contracción Miocárdica/fisiología , Miocitos Cardíacos/enzimología , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fosfoproteínas Fosfatasas/genética , Fosfoproteínas Fosfatasas/metabolismo , Animales , Canales de Calcio Tipo L/fisiología , Regulación Enzimológica de la Expresión Génica/fisiología , Humanos , Ratones , Ratones Transgénicos , Miocitos Cardíacos/citología , Técnicas de Placa-Clamp , Ratas , Receptores Adrenérgicos beta/metabolismo , Transducción de Señal/fisiología , Transgenes/genéticaRESUMEN
The cAMP response element modulator (CREM)alpha is a widely expressed transcriptional repressor that is important for the termination of the T cell immune response and contributes to the abnormal T cell function in patients with systemic lupus erythematosus. We present evidence that APCs of Crem(-/-) mice express increased amounts of the costimulatory molecule CD86 and induce enhanced Ag-dependent and Ag-independent T cell proliferation. Similarly, human APCs in which CREMalpha was selectively suppressed expressed more CD86 on the surface membrane. CREMalpha was found to bind to the CD86 promoter and suppressed its activity. Transfer of APCs from Crem(-/-) mice into naive mice facilitated a significantly stronger contact dermatitis response compared with mice into which APCs from Crem(+/+) mice had been transferred. We conclude that CREMalpha is an important negative regulator of costimulation and APC-dependent T cell function both in vitro and in vivo.
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Células Presentadoras de Antígenos/inmunología , Antígeno B7-2/inmunología , Modulador del Elemento de Respuesta al AMP Cíclico/inmunología , Regulación de la Expresión Génica/inmunología , Animales , Células Presentadoras de Antígenos/metabolismo , Antígeno B7-2/genética , Antígeno B7-2/metabolismo , Modulador del Elemento de Respuesta al AMP Cíclico/genética , Modulador del Elemento de Respuesta al AMP Cíclico/metabolismo , Dermatitis por Contacto/inmunología , Dermatitis por Contacto/patología , Citometría de Flujo , Expresión Génica/inmunología , Humanos , Activación de Linfocitos/inmunología , Ratones , Ratones Transgénicos , Análisis de Secuencia por Matrices de Oligonucleótidos , Regiones Promotoras Genéticas , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Linfocitos T/inmunologíaRESUMEN
BACKGROUND: Chronic stimulation of the beta(1)-adrenoceptor (beta(1)AR) plays a crucial role in the pathogenesis of heart failure; however, underlying mechanisms remain to be elucidated. The regulation by transcription factors cAMP response element-binding protein (CREB) and cyclic AMP response element modulator (CREM) represents a fundamental mechanism of cyclic AMP-dependent gene control possibly implicated in beta(1)AR-mediated cardiac deterioration. METHODS AND RESULTS: We studied the role of CREM in beta(1)AR-mediated cardiac effects, comparing transgenic mice with heart-directed expression of beta(1)AR in the absence and presence of functional CREM. CREM inactivation protected from cardiomyocyte hypertrophy, fibrosis, and left ventricular dysfunction in beta(1)AR-overexpressing mice. Transcriptome and proteome analysis revealed a set of predicted CREB/CREM target genes including the cardiac ryanodine receptor, tropomyosin 1alpha, and cardiac alpha-actin as altered on the mRNA or protein level along with the improved phenotype in CREM-deficient beta(1)AR-transgenic hearts. CONCLUSIONS: The results imply the regulation of genes by CREM as an important mechanism of beta(1)AR-induced cardiac damage in mice.
Asunto(s)
Cardiomegalia/genética , Cardiomegalia/fisiopatología , Modulador del Elemento de Respuesta al AMP Cíclico/metabolismo , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Receptores Adrenérgicos beta 1/metabolismo , Animales , Cardiomegalia/metabolismo , AMP Cíclico/metabolismo , Electroforesis en Gel Bidimensional , Perfilación de la Expresión Génica , Espectrometría de Masas , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Análisis de Secuencia por Matrices de Oligonucleótidos , Proteómica , ARN Mensajero/metabolismo , Receptores Adrenérgicos beta 1/genética , Función Ventricular Izquierda/fisiologíaRESUMEN
Cardiac-specific overexpression of the catalytic subunit of protein phosphatase type 1 (PP1) in mice results in hypertrophy, depressed contractility, propensity to heart failure, and premature death. To further address the role of PP1 in heart function, PP1 mice were crossed with mice that overexpress a functional COOH-terminally truncated form of PP1 inhibitor-2 (I-2(140)). Protein phosphatase activity was increased in PP1 mice but was normalized in double transgenic (DT) mice. The maximal rates of contraction (+dP/dt) and of relaxation (-dP/dt) were reduced in catheterized PP1 mice but normalized in DT mice. Similar contractile abnormalities were observed in isolated, perfused work-performing hearts and in whole animals by means of echocardiography. The increased absolute and relative heart weights observed in PP1 mice were normalized in DT mice. Histological analyses indicated that PP1 mice had significant cardiac fibrosis, which was absent in DT mice. Furthermore, PP1 mice exhibited an age-dependent increase in mortality, which was abrogated in DT mice. These results indicate that I-2 overexpression prevents the detrimental effects of PP1 overexpression in the heart and further underscore the fundamental role of PP1 in cardiac function. Therefore, PP1 inhibitors such as I-2 could offer new therapeutic options to ameliorate the deleterious effects of heart failure.
Asunto(s)
Cardiomegalia/prevención & control , Miocardio/enzimología , Proteína Fosfatasa 1/metabolismo , Proteínas/metabolismo , Animales , Cardiomegalia/diagnóstico por imagen , Cardiomegalia/metabolismo , Cardiomegalia/fisiopatología , Fibrosis , Ratones , Ratones Endogámicos C3H , Ratones Transgénicos , Contracción Miocárdica , Proteína Fosfatasa 1/antagonistas & inhibidores , Proteína Fosfatasa 1/genética , Proteína Fosfatasa 2/genética , Proteína Fosfatasa 2/metabolismo , Proteínas/genética , Factores de Tiempo , Ultrasonografía , Función Ventricular Izquierda , Presión VentricularRESUMEN
AIMS: The progression of human heart failure is associated with increased protein phosphatase 1 (PP1) activity, which leads to a higher dephosphorylation of cardiac regulatory proteins such as phospholamban. In this study, we tested the hypothesis whether the inhibitor-2 (I-2) of PP1 can mediate cardiac protection by inhibition of PP1 activity. METHODS AND RESULTS: We induced pressure overload by transverse aortic constriction (TAC) for 28 days in transgenic (TG) mice with heart-directed overexpression of a constitutively active form of I-2 (TG(TAC)) and wild-type littermates (WT(TAC)). Both groups were compared with sham-operated mice. TAC treatment resulted in comparable ventricular hypertrophy in both groups. However, TG(TAC) exhibited a higher atrial mass and an enhanced ventricular mRNA expression of beta-myosin heavy chain. The increased afterload was associated with the development of focal fibrosis in TG. Consistent with signs of overt heart failure, fractional shortening and diastolic function were impaired in TG(TAC) as revealed by Doppler echocardiography. The contractility was reduced in catheterized banded TG mice, which is in line with a depressed shortening of isolated myocytes. This is due to profoundly abnormal cytosolic Ca(2+) transients and a reduced stimulation of phosphorylation of phospholamban (PLB)(Ser16) after TAC in TG mice. Moreover, administration of isoproterenol was followed by a blunted contractile response in isolated myocytes of TG(TAC) mice. CONCLUSION: These results suggest that cardiac-specific overexpression of a constitutively active form of I-2 is deleterious for cardiac function under conditions of pressure overload. Thus, the long-term inhibition of PP1 by I-2 is not a therapeutic option in the treatment of heart failure.
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Insuficiencia Cardíaca/enzimología , Miocardio/enzimología , Proteína Fosfatasa 1/metabolismo , Proteínas/metabolismo , Agonistas Adrenérgicos beta/farmacología , Animales , Aorta/cirugía , Señalización del Calcio , Proteínas de Unión al Calcio/metabolismo , Cardiomegalia/enzimología , Cardiotónicos/farmacología , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Fibrosis , Atrios Cardíacos/enzimología , Insuficiencia Cardíaca/diagnóstico por imagen , Insuficiencia Cardíaca/tratamiento farmacológico , Insuficiencia Cardíaca/fisiopatología , Ventrículos Cardíacos/enzimología , Ligadura , Ratones , Ratones Transgénicos , Contracción Miocárdica , Miocardio/patología , Fosforilación , Proteínas/genética , Ultrasonografía , Disfunción Ventricular Izquierda/enzimologíaRESUMEN
BACKGROUND: Survivin inhibits apoptosis and regulates cell division in many organs, but its function in the heart is unknown. METHODS AND RESULTS: We show that cardiac-specific deletion of survivin resulted in premature cardiac death. The underlying cause was a dramatic reduction in total cardiomyocyte numbers as determined by a stereological method for quantification of cells per organ. The resulting increased hemodynamic load per cell led to progressive heart failure as assessed by echocardiography, magnetic resonance imaging, positron emission tomography, and invasive catheterization. The reduction in total cardiomyocyte number in alpha-myosin heavy chain (MHC)-survivin(-/-) mice was due to an approximately 50% lower mitotic rate without increased apoptosis. This occurred at the expense of DNA accumulation because survivin-deficient cardiomyocytes displayed marked DNA polyploidy indicative of consecutive rounds of DNA replication without cell division. Survivin small interfering RNA knockdown in neonatal rat cardiomyocytes also led to polyploidization and cell cycle arrest without apoptosis. Adenoviral overexpression of survivin in cardiomyocytes inhibited doxorubicin-induced apoptosis, induced DNA synthesis, and promoted cell cycle progression. The phenotype of the alphaMHC-survivin(-/-) mice also allowed us to determine the minimum cardiomyocyte number sufficient for normal cardiac function. In human cardiomyopathy, survivin was potently induced in the failing heart and downregulated again after hemodynamic support by a left ventricular assist device. Its expression positively correlated with the mean cardiomyocyte DNA content. CONCLUSIONS: We suggest that the ontogenetically determined cardiomyocyte number may be an independent factor in the susceptibility to cardiac diseases. Through its profound impact on both cardiomyocyte replication and apoptosis, survivin may emerge as a promising new target for myocardial regeneration.
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Cardiopatías/patología , Corazón/fisiología , Proteínas Asociadas a Microtúbulos/fisiología , Miocardio/patología , Miocitos Cardíacos/patología , Proteínas de Neoplasias/fisiología , Animales , Recuento de Células , Tamaño de la Célula , Células Cultivadas , Cardiopatías/fisiopatología , Humanos , Proteínas Inhibidoras de la Apoptosis , Masculino , Ratones , Ratones Noqueados , Proteínas Asociadas a Microtúbulos/análisis , Miocardio/citología , Miocitos Cardíacos/citología , Proteínas de Neoplasias/análisis , Proteínas Represoras , Survivin , Regulación hacia ArribaRESUMEN
Triadin is involved in the regulation of cardiac excitation-contraction coupling. However, the extent of its contribution to the regulation of sarcoplasmic reticulum (SR) Ca release remains unclear, because overexpression of triadin in single-transgenic mice was associated with the downregulation of its homologous protein, junctin. In the present study, this problem was circumvented by cross-breeding of mice with heart-directed overexpression of triadin and junctin (JxT). This resulted in a stable approximately threefold expression of total triadin but unchanged junctin protein. Transgenic mice exhibited cardiac hypertrophy and structural abnormalities of myofibrils. Measurement of cardiac function by echocardiography and edge detection in myocytes revealed an impaired relaxation in JxT mice. The stimulation of beta-adrenergic receptors resulted in a depressed contractility and an impaired relaxation in catheterized hearts and myocytes of JxT mice. The use of a maximum stimulation frequency (5 Hz) was associated with both a lower shortening and relengthening in isolated myocytes of JxT mice. The contractile effects in JxT myocytes were paralleled by similar changes of the intracellular Ca concentration ([Ca](i)) peak amplitude and Ca transient decay kinetics at basal conditions, under administration of isoproterenol, and with high-frequency stimulation. Finally, we found a higher caffeine-induced [Ca](i) peak amplitude in JxT myocytes. Our data show that the stable expression of triadin, independent of junctin expression, resulted in cardiac hypertrophy, prolonged basal relaxation, a depressed response to beta-adrenergic agonists, and altered Ca transients. Thus the maintenance of triadin expression is essential for normal SR Ca cycling and contractile function.
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Señalización del Calcio/fisiología , Calcio/metabolismo , Proteínas Portadoras/metabolismo , Corazón/fisiología , Proteínas Musculares/metabolismo , Contracción Miocárdica/fisiología , Miocitos Cardíacos/fisiología , Miocitos Cardíacos/ultraestructura , Animales , Proteínas Portadoras/genética , Células Cultivadas , Perros , Ratones , Ratones Transgénicos , Proteínas Musculares/genéticaRESUMEN
The transcription factor cAMP response element (CRE)-binding protein (CREB, Creb1) plays a critical role in regulating gene expression in response to activation of the cAMP-dependent signaling pathway, which is implicated in the pathophysiology of heart failure. Using the Cre-loxP system, we generated mice with a cardiomyocyte-specific inactivation of CREB and studied in this model whether CREB is critical for cardiac function. CREB-deficient mice were viable and displayed neither changes in cardiac morphology nor alterations of basal or isoproterenol-stimulated left ventricular function in vivo or of important cardiac regulatory proteins. Since CREB was proposed as a negative regulator of cardiomyocyte apoptosis by enhancing the expression of the antiapoptotic protein Bcl-2, we analyzed the fragmentation of DNA, the activity of caspases 3/7 and the expression of Bcl-2 and did not observe any differences between CREB-deficient and CREB-normal hearts. Our results suggest that the presence of CREB is not critical for normal cardiac function in mice.
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Proteína de Unión a Elemento de Respuesta al AMP Cíclico/fisiología , Miocitos Cardíacos/citología , Animales , Apoptosis , Proteínas Reguladoras de la Apoptosis/análisis , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/deficiencia , Fragmentación del ADN , Corazón/fisiología , Ratones , Ratones Noqueados , Miocardio/patología , Miocitos Cardíacos/química , Miocitos Cardíacos/metabolismo , Tasa de Supervivencia , Factores de Transcripción , Función Ventricular IzquierdaRESUMEN
OBJECTIVE: Protein phosphatase 1 (PP1) has been implicated in the control of cardiac function. Cardiac specific overexpression of the catalytic subunit, PP1c, results in hypertrophy and depressed contractility. METHODS: To further address the role of PP1, transgenic mice (TG) were generated that overexpress in heart a functional COOH-terminally truncated form (amino acids 1-140) of the PP1 inhibitor-2 (I-2(140)). RESULTS: The TG hearts show increased levels of I-2(140) mRNA as well as protein and activity. No increase in absolute or relative heart weight was observed, nor any changes in gross pathology or increase in morbidity or mortality in the TG mice. Immunohistochemical and biochemical analyses revealed that expression of the I-2(140) protein is confined to cardiomyocytes where it is mainly localized in the cytosol. The total protein phosphatase (PP) activity was reduced by 80% in TG hearts as compared to wild-type littermates (WT). The PP1c mRNA level was the same in TG and WT, while the protein level was increased by approximately 7-fold in TG animals. The maximal rates of contraction (+dP/dt) and of relaxation (-dP/dt) were increased by 32% and 40%, respectively, in the intact catheterized TG mice compared to WT. However, the maximal contractile response to beta-adrenergic agonists was comparable in hearts from TG and WT mice. In isolated cardiomyocytes of TG mice, Ca2+transient amplitude was increased by 50% under basal conditions and by 60% upon rapid caffeine application. The phospholamban (PLB) protein level was unchanged whereas the basal phosphorylation of PLB at Ser(16) was significantly increased in TG hearts. CONCLUSION: These results indicate that I-2(140) overexpression results in decreased PP1 activity and enhanced contractility in the heart, underscoring the fundamental role of PP1 in cardiac function.
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Cardiomegalia/enzimología , Contracción Miocárdica/fisiología , Miocardio/enzimología , Fosfoproteínas Fosfatasas/metabolismo , Proteínas/genética , Animales , Northern Blotting/métodos , Calcio/metabolismo , Canales de Calcio/metabolismo , Ingeniería Genética , Inmunohistoquímica/métodos , Ratones , Ratones Transgénicos , Miocardio/metabolismo , Perfusión , Fosfoproteínas Fosfatasas/antagonistas & inhibidores , Proteína Fosfatasa 1 , Proteínas/metabolismo , Retículo Sarcoplasmático/metabolismoRESUMEN
The transcriptional activation mediated by cAMP-response element (CRE) and transcription factors of the CRE-binding protein (CREB)/CRE modulator (CREM) family represents an important mechanism of cAMP-dependent gene regulation possibly implicated in detrimental effects of chronic beta-adrenergic stimulation in end-stage heart failure. We studied the cardiac role of CREM in transgenic mice with heart-directed expression of CREM-IbDeltaC-X, a human cardiac CREM isoform. Transgenic mice displayed atrial enlargement with atrial and ventricular hypertrophy, developed atrial fibrillation, and died prematurely. In vivo hemodynamic assessment revealed increased contractility of transgenic left ventricles probably due to a selective up-regulation of SERCA2, the cardiac Ca(2+)-ATPase of the sarcoplasmic reticulum. In transgenic ventricles, reduced phosphorylation of phospholamban and of the CREB was associated with increased activity of serine-threonine protein phosphatase 1. The density of beta(1)-adrenoreceptor was increased, and messenger RNAs encoding transcription factor dHAND and small G-protein RhoB were decreased in transgenic hearts as compared with wild-type controls. Our results indicate that heart-directed expression of CREM-IbDeltaC-X leads to complex cardiac alterations, suggesting CREM as a central regulator of cardiac morphology, function, and gene expression.
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Proteínas de Unión al ADN/fisiología , Miocardio/metabolismo , Factores de Transcripción/fisiología , Animales , Fibrilación Atrial , Cardiomegalia , Modulador del Elemento de Respuesta al AMP Cíclico , Proteínas de Unión al ADN/genética , Regulación de la Expresión Génica , Humanos , Ratones , Ratones Transgénicos , Mortalidad , Miocardio/química , Miocardio/patología , Fosforilación , Isoformas de Proteínas , Factores de Transcripción/genética , Disfunción VentricularRESUMEN
Reversible protein phosphorylation is an essential regulatory mechanism in many cellular functions. In contrast to protein kinases, the role and regulation of protein phosphatases has remained ambiguous. To address this issue, we generated transgenic mice that overexpress the catalytic subunit alpha of protein phosphatase 2A (PP2A) (PP2Acalpha) in the heart driven by the alpha-myosin heavy chain promoter. Overexpression of the PP2Acalpha gene in the heart led to increased levels of the transgene both at RNA and protein levels. This was accompanied by a significant increase of PP2A enzyme activity in the myocardium. Morphological analysis revealed isles of necrosis and fibrosis. The phosphorylation state of phospholamban, troponin inhibitor, and eukaryotic elongation factor 2 was reduced significantly. The expression of junctional (calsequestrin) and free SR proteins (SERCA and phospholamban) was not altered. Whereas no increase in morbidity or mortality was noted, transgenic mice developed cardiac hypertrophy and reduced contractility of the heart, as well as cardiac dilatation as shown by biplane echocardiography. Taken together, these findings are indicative of the fundamental role of PP2A in cardiac function and imply that disturbances in protein phosphatases expression and activity may cause or aggravate the course of cardiac diseases.
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Corazón/fisiología , Fosfoproteínas Fosfatasas/química , Actinas/genética , Animales , Northern Blotting , Western Blotting , Calcio/metabolismo , Dominio Catalítico , Células Cultivadas , ADN Complementario/metabolismo , Dobutamina/farmacología , Relación Dosis-Respuesta a Droga , Ecocardiografía , Fibrosis , Cardiopatías/metabolismo , Procesamiento de Imagen Asistido por Computador , Inmunohistoquímica , Ratones , Ratones Transgénicos , Miocardio/metabolismo , Fosfoproteínas Fosfatasas/metabolismo , Fosforilación , Regiones Promotoras Genéticas , Proteína Fosfatasa 2 , ARN/metabolismo , Receptores Adrenérgicos beta/metabolismo , TransgenesRESUMEN
Congestive heart failure is the common endpoint of various cardiac diseases representing a leading cause of cardiovascular mortality in Western countries. Characteristic functional alterations of the failing heart are explained by expressional changes of myocardial regulatory proteins; however, little is known about underlying mechanisms regulating cardiac gene expression in the failing heart. Here, we address the specific role of transcription factor CREM for cardiac function in CREM mutant mice with complete inactivation of the CREM gene. We show that CREM mutant mice display distinct alterations of cardiac function resembling characteristic functional defects of the failing heart. Left ventricular hemodynamic assessment of CREM mutant mice revealed impairment of both cardiac contraction and relaxation in basal state, as well as a decreased responsiveness to beta-adrenergic stimulation. The diminished cardiac contractile performance was associated with a selective down-regulation of beta1-adrenergic receptors and a decreased ventricular expression of SERCA, the Ca2+-ATPase of the sarcoplasmic reticulum. The cardiac phenotype of CREM mutant mice provides the first evidence that CREM represents an important key regulator of cardiac gene expression, which is essential for normal left ventricular contractile performance and response to beta-adrenoreceptor stimulation.