RESUMEN
The direct effects of catecholamines on cardiac myocytes may contribute to both normal physiologic adaptation and pathologic remodeling, and may be associated with cellular hypertrophy, apoptosis, and alterations in contractile function. Norepinephrine (NE) signals via alpha- and beta-adrenergic receptors (AR) that are coupled to G-proteins. Pharmacologic studies of cardiac myocytes in vitro demonstrate that stimulation of beta1-AR induces apoptosis which is cAMP-dependent and involves the voltage-dependent calcium influx channel. In contrast, stimulation of beta2-AR exerts an anti-apoptotic effect which appears to be mediated by a pertussis toxin-sensitive G protein. Stimulation of alpha1-AR causes myocyte hypertrophy and may exert an anti-apoptotic action. In transgenic mice, myocardial overexpression of either beta1-AR or G(alpha)s is associated with myocyte apoptosis and the development of dilated cardiomyopathy. Myocardial overexpression of beta2-AR at low levels results in improved cardiac function, whereas expression at high levels leads to dilated cardiomyopathy. Overexpression of wildtype alpha1B-AR does not result in apoptosis, whereas overexpression of G(alpha)q results in myocyte hypertrophy and/or apoptosis depending on the level of expression. Differential activation of the members of the mitogen-activated protein kinase (MAPK) superfamily and production of reactive oxygen species appear to play a key role in mediating the actions of adrenergic pathways on myocyte apoptosis and hypertrophy. This review summarizes current knowledge about the molecular and cellular mechanisms involved in the regulation of cardiac myocyte apoptosis via stimulation of adrenergic receptors and their coupled effector pathways.
Asunto(s)
Agonistas alfa-Adrenérgicos/farmacología , Apoptosis , Miocardio/metabolismo , Norepinefrina/farmacología , Animales , Cardiomiopatía Dilatada/etiología , Proteínas de Unión al GTP Heterotriméricas/genética , Proteínas de Unión al GTP Heterotriméricas/fisiología , Ratones , Ratones Transgénicos , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Modelos Biológicos , Miocardio/citología , Especies Reactivas de Oxígeno/metabolismo , Receptores Adrenérgicos alfa/genética , Receptores Adrenérgicos alfa/metabolismo , Receptores Adrenérgicos beta/genética , Receptores Adrenérgicos beta/metabolismoRESUMEN
Oxidative stress stimulates both growth and apoptosis in cardiac myocytes in vitro. We investigated whether oxidative stress mediates hypertrophy and apoptosis in cyclically stretched ventricular myocytes. Neonatal rat ventricular myocytes cultured on laminin-coated silastic membranes were stretched cyclically (1 Hz) at low (nominal 5%) and high (nominal 25%) amplitudes for 24 hours. Stretch caused a graded increase in superoxide anion production as assessed by superoxide dismutase (SOD)-inhibitable cytochrome c reduction or electron paramagnetic resonance spectroscopy. The role of reactive oxygen species (ROS) was assessed using the cell-permeable SOD/catalase mimetics Mn(II/III)tetrakis(1-methyl-4-peridyl) (MnTMPyP) and EUK-8. Stretch-induced increases in protein synthesis ((3)H-leucine incorporation) and cellular protein content were completely inhibited by MnTMPyP (0.05 mmol/L) at both low and high amplitudes of stretch. In contrast, while MnTMPyP inhibited basal atrial natriuretic factor (ANF) mRNA expression, the stretch-induced increase in ANF mRNA expression was not inhibited by MnTMPyP. In contrast to hypertrophy, only high-amplitude stretch increased myocyte apoptosis, as reflected by increased DNA fragmentation on gel electrophoresis and an approximately 3-fold increase in the number of TUNEL-positive myocytes. Similarly, only high-amplitude stretch increased the expression of bax mRNA. Myocyte apoptosis and bax expression stimulated by high-amplitude stretch were inhibited by MnTMPyP. Both low- and high-amplitude stretch caused rapid phosphorylation of ERK1/2, while high-, but not low-, amplitude stretch caused phosphorylation of JNKs. Activation of both ERK1/2 and JNKs was ROS-dependent. Thus, cyclic strain causes an amplitude-related increase in ROS, associated with differential activation of kinases and induction of hypertrophic and apoptotic phenotypes.
Asunto(s)
Ventrículos Cardíacos/patología , Proteínas Proto-Oncogénicas c-bcl-2 , Especies Reactivas de Oxígeno/metabolismo , Animales , Animales Recién Nacidos , Apoptosis/efectos de los fármacos , Factor Natriurético Atrial/genética , Células Cultivadas , Etilenodiaminas/farmacología , Depuradores de Radicales Libres/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Ventrículos Cardíacos/efectos de los fármacos , Ventrículos Cardíacos/metabolismo , Hipertrofia , Leucina/efectos de los fármacos , Leucina/metabolismo , Compuestos Organometálicos/farmacología , Porfirinas/farmacología , Proteínas Proto-Oncogénicas/genética , ARN Mensajero/efectos de los fármacos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Transducción de Señal/efectos de los fármacos , Estrés Mecánico , Superóxidos/metabolismo , Tritio , Proteína X Asociada a bcl-2RESUMEN
Nitric oxide produced by inducible nitric oxide synthase (NOS2) has been implicated in the pathophysiology of chronic myocardial remodeling and failure. We tested the role of NOS2 in left ventricular (LV) remodeling early (1 month) and late (4 months) after myocardial infarction (MI) in mice lacking NOS2. MI size measured 7 days, 1 month, and 4 months after MI was the same in NOS2 knockout (KO) and wild-type (WT) mice. The LV end-diastolic pressure-volume relationship measured by the isovolumic Langendorff technique showed a progressive rightward shift from 1 to 4 months after MI in WT mice. LV developed pressure measured over a range of LV volumes was reduced at 1 and 4 months after MI in WT mice (P<0.05 and P<0.01 versus shams, respectively). In KO mice, the rightward shift was similar to that in WT mice at 1 and 4 months after MI, as was peak LV developed pressure at 1 month after MI. In contrast, at 4 months after MI, peak LV developed pressure in KO mice was higher than in WT mice (P<0.05 versus WT) and similar to that in sham-operated mice. At 1 month after MI, the frequency of terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL)-positive myocytes in the remote myocardium was increased to a similar extent in WT and KO mice. At 4 months after MI, the frequency of apoptotic myocytes was increased in WT mice but not in KO mice (P<0.05 versus WT). Improved contractile function and reduced apoptosis were associated with reduced mortality rate in KO mice at 4 months after MI. Thus, NOS2 does not play an important role in determining infarct size or early LV remodeling during the first month after MI. In contrast, during late (ie, 4 months after MI) remodeling, NOS2 in remote myocardium contributes to decreased contractile function, increased myocyte apoptosis in remote myocardium, and reduced survival.
Asunto(s)
Apoptosis , Contracción Miocárdica , Infarto del Miocardio/fisiopatología , Óxido Nítrico Sintasa/deficiencia , Función Ventricular Izquierda , Animales , Apoptosis/genética , Presión Sanguínea/genética , Peso Corporal/genética , Supervivencia Celular/genética , Progresión de la Enfermedad , Inmunohistoquímica , Etiquetado Corte-Fin in Situ , Técnicas In Vitro , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Contracción Miocárdica/genética , Infarto del Miocardio/patología , Miocardio/enzimología , Miocardio/patología , Óxido Nítrico Sintasa/biosíntesis , Óxido Nítrico Sintasa/genética , Óxido Nítrico Sintasa de Tipo II , Tamaño de los Órganos/genética , Volumen Sistólico/genética , Análisis de Supervivencia , Función Ventricular Izquierda/genéticaRESUMEN
Isolated permeabilized cardiac myocytes have been used in the study of myofilament calcium sensitivity through measurement of the isometric force-pCa curve. Determining this force-pCa relationship in skinned myocytes is relatively expensive and carries a high degree of variability. We therefore attempted to establish an alternative high-throughput method to measure calcium sensitivity in cardiac myocytes. With the use of commercially available software that allows for precise measurement of sarcomere spacing, we measured sarcomere length changes in unloaded skinned cardiac myocytes over a range of calcium concentrations. With the use of this technique, we were able to accurately detect acute increases or decreases in myofilament calcium sensitivity after exposure to 10 mM caffeine or 5 mM 2,3-butanedione monoxime, respectively. This technique allows for the simple and rapid determination of myofilament calcium sensitivity in cardiac myocytes in a reproducible and inexpensive manner and could be used for high-throughput screening of pharmacological agents and/or transgenic mouse models for changes in myofilament calcium sensitivity.
Asunto(s)
Calcio/fisiología , Corazón/fisiología , Contracción Miocárdica/fisiología , Animales , Calcio/farmacología , Masculino , Contracción Miocárdica/efectos de los fármacos , Ratas , Ratas WistarRESUMEN
Osteopontin (OPN), an extracellular matrix protein, is expressed in the myocardium with hypertrophy and failure. We tested the hypothesis that OPN plays a role in left ventricular (LV) remodeling after myocardial infarction (MI). Accordingly, OPN expression and LV structural and functional remodeling were determined in wild-type (WT) and OPN knockout (KO) mice 4 weeks after MI. Northern analysis showed increased OPN expression in the infarcted region, peaking 3 days after MI and gradually decreasing over the next 28 days. In the remote LV, OPN expression was biphasic, with peaks at 3 and 28 days. In situ hybridization and immunohistochemical analyses showed increased OPN mRNA and protein primarily in the interstitium. Infarct size, heart weight, and survival were similar in KO and WT mice after MI (P=NS), whereas the lung wet weight/dry weight ratio was increased in the KO mice (P<0.005 versus sham-operated mice). Peak LV developed pressure was reduced to a similar degree after MI in the KO and WT mice. The number of terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL)-positive myocytes was similar in KO and WT mice after MI. In contrast, post-MI LV chamber dilation was approximately twice as great in KO versus WT mice (P<0.001). Myocyte length increased after MI in WT mice (P<0.001) but not in KO mice. Electron microscopy showed increased collagen content in WT mice after MI but not in KO mice after MI. Type I collagen content was increased approximately 3-fold and approximately 7-fold in remote and infarcted regions, respectively, of WT hearts after MI but not in KO hearts (P<0.01 versus WT hearts). Likewise, Northern analyses showed increased collagen I(alpha(1)) mRNA after MI in remote regions of WT hearts but not in KO hearts. Thus, increased OPN expression plays an important role in regulating post-MI LV remodeling, at least in part, by promoting collagen synthesis and accumulation.
Asunto(s)
Colágeno/metabolismo , Dilatación Patológica/fisiopatología , Infarto del Miocardio/metabolismo , Sialoglicoproteínas/deficiencia , Remodelación Ventricular , Animales , Volumen Cardíaco , Tamaño de la Célula , Colágeno/genética , Colágeno/ultraestructura , Dilatación Patológica/etiología , Dilatación Patológica/patología , Modelos Animales de Enfermedad , Inmunohistoquímica , Hibridación in Situ , Etiquetado Corte-Fin in Situ , Técnicas In Vitro , Pulmón/patología , Ratones , Ratones Endogámicos , Ratones Noqueados , Infarto del Miocardio/complicaciones , Infarto del Miocardio/fisiopatología , Miocardio/metabolismo , Miocardio/patología , Miocardio/ultraestructura , Tamaño de los Órganos , Osteopontina , ARN Mensajero/metabolismo , Sialoglicoproteínas/genética , Sialoglicoproteínas/metabolismo , Tasa de Supervivencia , Disfunción Ventricular Izquierda/etiología , Disfunción Ventricular Izquierda/fisiopatologíaRESUMEN
BACKGROUND: Myocardial infarction (MI) promotes deleterious remodeling of the myocardium, resulting in ventricular dilation and pump dysfunction. We examined whether supplementing infarcted myocardium with skeletal myoblasts would (1) result in viable myoblast implants, (2) attenuate deleterious remodeling, and (3) enhance in vivo and ex vivo contractile performance. METHODS AND RESULTS: Experimental MI was induced by 1-hour coronary ligation followed by reperfusion in adult male Lewis rats. One week after MI, 10(6) myoblasts were injected directly into the infarct region. Three groups of animals were studied at 3 and 6 weeks after cell therapy: noninfarcted control (control), MI plus sham injection (MI), and MI plus cell injection (MI+cell). In vivo cardiac function was assessed by maximum exercise capacity testing and ex vivo function was determined by pressure-volume curves obtained from isolated, red cell-perfused, balloon-in-left ventricle (LV) hearts. MI and MI+cell hearts had indistinguishable infarct sizes of approximately 30% of the LV. At 3 and 6 weeks after cell therapy, 92% (13 of 14) of MI+cell hearts showed evidence of myoblast graft survival. MI+cell hearts exhibited attenuation of global ventricular dilation and reduced septum-to-free wall diameter compared with MI hearts not receiving cell therapy. Furthermore, cell therapy improved both post-MI in vivo exercise capacity and ex vivo LV systolic pressures. CONCLUSIONS: Implanted skeletal myoblasts form viable grafts in infarcted myocardium, resulting in enhanced post-MI exercise capacity and contractile function and attenuated ventricular dilation. These data illustrate that syngeneic myoblast implantation after MI improves both in vivo and ex vivo indexes of global ventricular dysfunction and deleterious remodeling and suggests that cellular implantation may be beneficial after MI.
Asunto(s)
Trasplante de Células , Ventrículos Cardíacos/patología , Infarto del Miocardio/terapia , Animales , Supervivencia de Injerto , Ventrículos Cardíacos/fisiopatología , Masculino , Actividad Motora/fisiología , Músculo Esquelético/citología , Contracción Miocárdica , Infarto del Miocardio/mortalidad , Infarto del Miocardio/patología , Ratas , Ratas Endogámicas Lew , Tasa de Supervivencia , Sístole/fisiología , Factores de TiempoRESUMEN
We examined the relative roles of the mitogen-activated protein kinases (MAPK) in mediating the alpha1-adrenergic receptor (alpha1-AR) stimulated hypertrophic phenotype in adult rat ventricular myocytes (ARVM). Norepinephrine (NE; 1 microM) in the presence of the beta -AR antagonist propranolol (Pro; 2 microM) caused activation of Ras (>six-fold), MAPK/ERK kinase 1 and 2 (MEK1/2, >10-fold) and extracellular signal-regulated kinases 1 and 2 (ERK1/2, approximately 30-fold) within 5 min, as determined by kinase activity assays and Western blots using phospho-specific antibodies. Conversely, p38 and c-Jun amino-terminal kinases (JNK) were not activated by NE/Pro. Activated MEK1/2 signals remained detectable at 2 h, and activated ERK1/2 remained detectable at 48 h. The alpha1-AR selective inhibitor prazosin (100 nM) completely inhibited the NE/Pro-stimulated activation of Ras, MEK1/2 and ERK1/2. The MEK inhibitor PD98059 caused a concentration-dependent inhibition of NE/Pro-stimulated protein synthesis (as assessed by [3H]leucine incorporation and cellular protein accumulation) and ERK1/2 activation, with approximately 50% inhibition at a concentration between 10 and 50 microM, which is consistent with the known IC50 values of PD98059 for MEK1 (4 microM) and MEK2 (50 microM). Thus, these data show that alpha1-AR stimulated hypertrophy in ARVM is dependent on the MEK1/2-ERK1/2 signaling pathway.
Asunto(s)
Ventrículos Cardíacos/metabolismo , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Receptores Adrenérgicos alfa 1/metabolismo , Agonistas alfa-Adrenérgicos/farmacología , Antagonistas Adrenérgicos beta/farmacología , Animales , Cardiomegalia/metabolismo , Células Cultivadas , Activación Enzimática , Flavonoides/farmacología , Ventrículos Cardíacos/citología , Proteínas Quinasas JNK Activadas por Mitógenos , MAP Quinasa Quinasa 1 , MAP Quinasa Quinasa 2 , Masculino , Proteína Quinasa 1 Activada por Mitógenos/antagonistas & inhibidores , Proteína Quinasa 3 Activada por Mitógenos , Quinasas de Proteína Quinasa Activadas por Mitógenos/antagonistas & inhibidores , Proteínas Quinasas Activadas por Mitógenos/antagonistas & inhibidores , Norepinefrina/farmacología , Prazosina/farmacología , Propranolol/farmacología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-raf/metabolismo , Ratas , Ratas Sprague-Dawley , Proteínas Quinasas p38 Activadas por Mitógenos , Proteínas ras/metabolismoRESUMEN
Norepinephrine (NE) causes hypertrophic growth of cardiac myocytes via stimulation of alpha1-adrenergic receptors (alpha1-AR). Reactive oxygen species (ROS) can act as signaling molecules for cell growth. Accordingly, we tested the hypothesis that ROS mediate alpha1-AR-stimulated hypertrophic growth in adult rat ventricular myocytes (ARVM). NE increased the level of intracellular ROS as assessed by lucigenin chemiluminescence or cytochrome c reduction, and this effect was prevented by the superoxide dismutase (SOD)-mimetic MnTMPyP. NE also caused the induction of MnSOD mRNA. alpha1-AR stimulation with NE (1 microM) in the presence of propranolol (2 microM) for 48-96 h caused a hypertrophic growth phenotype characterized by a 36+/-3% increase in 3H-leucine incorporation, a 49+/-14% increase in protein accumulation, a six-fold induction of atrial natriuretic peptide mRNA, actin filament reorganization, and the induction of MnSOD mRNA. These responses were all prevented by pretreatment with the alpha1-AR-selective antagonist prazosin (100 n M) or the SOD-mimetics MnTMPyP (50 microM) and Euk-8 (100 microM). MnTMPyP had no effect on alpha1-AR-stimulated 3H-inositol phosphate turnover or the hypertrophic phenotype caused by the protein kinase C activator phorbol-12-myristate-13-acetate. Thus, ROS play a critical role in mediating the hypertrophic growth response to alpha1-AR-stimulation in ARVM.
Asunto(s)
Corazón/efectos de los fármacos , Miocardio/patología , Especies Reactivas de Oxígeno/metabolismo , Receptores Adrenérgicos alfa 1/efectos de los fármacos , Citoesqueleto de Actina/efectos de los fármacos , Citoesqueleto de Actina/ultraestructura , Actinas/metabolismo , Animales , Factor Natriurético Atrial/biosíntesis , Factor Natriurético Atrial/genética , División Celular/efectos de los fármacos , Inducción Enzimática/efectos de los fármacos , Etilenodiaminas/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Ventrículos Cardíacos/efectos de los fármacos , Ventrículos Cardíacos/metabolismo , Ventrículos Cardíacos/patología , Hipertrofia , Fosfatos de Inositol/metabolismo , Miocardio/metabolismo , Norepinefrina/farmacología , Compuestos Organometálicos/farmacología , Porfirinas/farmacología , Prazosina/farmacología , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Ratas , Receptores Adrenérgicos alfa 1/metabolismo , Transducción de Señal , Superóxido Dismutasa/biosíntesis , Superóxido Dismutasa/genéticaRESUMEN
We tested the hypothesis that left ventricular (LV) remodeling late after myocardial infarction (MI) is associated with myocyte apoptosis in myocardium remote from the infarcted area and is related temporally to LV dilation and contractile dysfunction. One, four, and six months after MI caused by coronary artery ligation, LV volume and contractile function were determined using an isovolumic balloon-in-LV Langendorff technique. Apoptosis and nuclear morphology were determined by terminal deoxynucleotidyl transferase-mediated nick end-labeling (TUNEL) and Hoechst 33258 staining. Progressive LV dilation 1-6 mo post-MI was associated with reduced peak LV developed pressure (LVDP). In myocardium remote from the infarct, there was increased wall thickness and expression of atrial natriuretic peptide mRNA consistent with reactive hypertrophy. There was a progressive increase in the number of TUNEL-positive myocytes from 1 to 6 mo post-MI (2.9-fold increase at 6 mo; P < 0. 001 vs. sham). Thus LV remodeling late post-MI is associated with increased apoptosis in myocardium remote from the area of ischemic injury. The frequency of apoptosis is related to the severity of LV dysfunction.
Asunto(s)
Apoptosis , Corazón/fisiopatología , Hemodinámica/fisiología , Infarto del Miocardio/fisiopatología , Función Ventricular Izquierda/fisiología , Animales , Presión Sanguínea , Peso Corporal , Diástole , Técnicas In Vitro , Masculino , Ratones , Contracción Miocárdica , Infarto del Miocardio/patología , Miocardio/patología , Tamaño de los Órganos , Sístole , Factores de TiempoRESUMEN
Sympathetic nervous system activity to the myocardium is increased in patients with heart failure. It is now appreciated that norepinephrine (NE), the primary sympathetic neurotransmitter, can exert direct adverse effects on cardiac myocytes and might thereby contribute to pathological remodeling, a chronic process which leads to progressive left ventricular (LV) chamber dilation and loss of contractile function. The demonstration of apoptosis in failing human hearts has led to the thesis that continuing loss of viable myocytes is a mechanism for progressive myocardial failure. For many years it has been appreciated that chronic exposure to catecholamines can exert a toxic effect on the myocardium. In vitro studies in cultured cardiac myocytes show that tonic exposure to NE increases the number of apoptotic myocytes via stimulation of the beta-adrenergic receptor (beta-AR) pathway. Interestingly, a beta1-AR selective antagonist completely prevented NE-stimulated apoptosis, whereas a beta2-AR selective antagonist increased the amount of apoptosis, suggesting that beta1- versus beta2-AR may couple to different signaling pathways. In rats, isoproterenol infusion for as little as 12 hours increased the frequency of terminal deoxynucleotidyltransferase-mediated nick end-labeling (TUNEL)-positive myocytes. Likewise, mice that overexpress beta1-AR or G alpha s in the myocardium develop left ventricular dilation, contractile dysfunction and apoptosis. Although the link between apoptosis and myocardial failure remains to be proven, these in vitro and in vivo observations provide a rational mechanism by which beta-AR antagonists may help to prevent or slow LV remodeling and failure in patients.
Asunto(s)
Apoptosis/efectos de los fármacos , Insuficiencia Cardíaca/etiología , Norepinefrina/metabolismo , Receptores Adrenérgicos beta/metabolismo , Disfunción Ventricular Izquierda/etiología , Remodelación Ventricular/efectos de los fármacos , Agonistas Adrenérgicos beta/farmacología , Animales , Animales Recién Nacidos , Regulación de la Expresión Génica , Insuficiencia Cardíaca/tratamiento farmacológico , Insuficiencia Cardíaca/metabolismo , Humanos , Isoproterenol/farmacología , Ratones , Ratones Transgénicos , Norepinefrina/efectos adversos , Ratas , Receptores Adrenérgicos beta/efectos de los fármacosRESUMEN
Increased sympathetic nerve activity to the myocardium is a central feature in patients with heart failure. Norepinephrine, the primary transmitter of the sympathetic nervous system, signals via binding to alpha- and beta-adrenergic receptors (AR) that are coupled to G-proteins. Pharmacologic studies of cardiac myocytes in vitro demonstrate that beta-AR can stimulate apoptosis. Likewise, in transgenic mice overexpression of beta 1-AR or G alpha s is associated with myocyte apoptosis and the development of dilated cardiomyopathy. Whereas beta 1-AR stimulate apoptosis in vitro and in vivo, beta 2-AR may either stimulate or inhibit apoptosis and myocardial failure depending on the level of expression. Receptors coupling to Gi and Gq may also be able to mediate or modulate apoptosis and the development of myocardial failure, suggesting the potential for interactions between the beta-AR system and numerous remodeling stimuli that act through Gi or Gq signaling pathways. It appears likely that the mitogen-activated protein kinase superfamily plays a key role in mediating the actions of adrenergic pathways on myocyte apoptosis. These observations suggest that the adrenergic nervous system plays an important role in the regulation of myocyte apoptosis, and may thus contribute to the development of myocardial failure.
Asunto(s)
Apoptosis , Insuficiencia Cardíaca/etiología , Miocardio/metabolismo , Receptores Adrenérgicos beta/metabolismo , Transducción de Señal , Agonistas Adrenérgicos beta/farmacología , Animales , Apoptosis/efectos de los fármacos , Células Cultivadas , Proteínas de Unión al GTP/metabolismo , Insuficiencia Cardíaca/metabolismo , Insuficiencia Cardíaca/fisiopatología , Humanos , Isoproterenol/farmacología , Sistema de Señalización de MAP Quinasas , Ratones , Norepinefrina/farmacología , Ratas , Receptores Adrenérgicos beta 1/metabolismo , Receptores Adrenérgicos beta 2/metabolismoRESUMEN
BACKGROUND: beta-Adrenergic receptor (beta-AR) stimulation increases apoptosis in adult rat cardiac (ventricular) myocytes (ARVMs) via activation of adenylyl cyclase. beta(2)-ARs may couple to a G(i)-mediated signaling pathway that can oppose the actions of adenylyl cyclase. METHODS AND RESULTS: In ARVMs, beta-AR stimulation for 24 hours increased the number of apoptotic cells as measured by flow cytometry. beta-AR-stimulated apoptosis was abolished by the beta(1)-AR-selective antagonist CGP 20712A (P<0.05 versus beta-AR stimulation alone) but was potentiated by the beta(2)-AR-selective antagonist ICI 118,551 (P<0.05 versus beta-AR stimulation alone). The muscarinic agonist carbachol also prevented beta-AR-stimulated apoptosis (P<0.05 versus beta-AR stimulation alone), whereas pertussis toxin potentiated the apoptotic action of beta-AR stimulation (P<0.05 versus beta-AR stimulation alone) and prevented the antiapoptotic action of carbachol. CONCLUSIONS: In ARVMs, stimulation of beta(1)-ARs increases apoptosis via a cAMP-dependent mechanism, whereas stimulation of beta(2)-ARs inhibits apoptosis via a G(i)-coupled pathway. These findings have implications for the pathophysiology and treatment of myocardial failure.
Asunto(s)
Toxina de Adenilato Ciclasa , Apoptosis/efectos de los fármacos , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/antagonistas & inhibidores , Corazón/efectos de los fármacos , Miocardio/citología , Toxina del Pertussis , Receptores Adrenérgicos beta 1/fisiología , Receptores Adrenérgicos beta 2/fisiología , Sistemas de Mensajero Secundario/efectos de los fármacos , Factores de Virulencia de Bordetella/farmacología , Agonistas Adrenérgicos beta/farmacología , Antagonistas Adrenérgicos beta/farmacología , Animales , Carbacol/farmacología , Cardiotónicos/farmacología , AMP Cíclico/metabolismo , Imidazoles/farmacología , Isoproterenol/farmacología , Masculino , Agonistas Muscarínicos/farmacología , Prazosina/farmacología , Propanolaminas/farmacología , Ratas , Ratas Sprague-Dawley , Receptores Adrenérgicos beta 1/efectos de los fármacos , Receptores Adrenérgicos beta 2/efectos de los fármacosRESUMEN
Neuregulins are a family of growth-promoting peptides known to be important in neural and mesenchymal tissue development. Targeted disruption of neuregulin (NRG)-1 or one of two of its cognate receptors, ErbB2 or ErbB4, results in embryonic lethality because of failure of the heart to develop. Although expression of NRGs and their receptors declines after midembryogenesis, both ErbB2 and ErbB4 are present in cardiac myocytes, and NRG-1 expression remains inducible in primary cultures of coronary microvascular endothelial cells from adult rat ventricular muscle. In neonatal rat ventricular myocytes, a soluble NRG-1, recombinant human glial growth factor-2, increased [(3)H]phenylalanine uptake and induced expression of atrial natriuretic factor (ANF) and sarcomeric F-actin polymerization. The effect of NRG-1 on [(3)H]phenylalanine uptake and sarcomeric F-actin polymerization was maximal at 20 ng/ml but declined at higher concentrations. NRG-1 activated p42/p44 mitogen-activated protein kinase (MAPK) [extracellular signal-regulated kinase (ERK)-2/ERK1] and ribosomal S6 kinase (RSK)-2 (90-kDa ribosomal S6 kinase), both of which could be inhibited by the MAPK/ERK kinase-1 antagonist PD-098059. NRG-1 also activated 70-kDa ribosomal S6 kinase, which was inhibited by either rapamycin or wortmannin. Activation of these pathways exhibited the same "biphasic" response to increasing NRG-1 concentrations. Wortmannin and LY-294002 blocked sarcomeric F-actin polymerization but not [(3)H]phenylalanine uptake or ANF expression, whereas PD-098059 consistently blocked both [(3)H]phenylalanine uptake and ANF expression but not actin polymerization. In contrast, rapamycin inhibited [(3)H]phenylalanine uptake and F-actin polymerization but not ANF expression. Thus NRG-ErbB signaling triggers multiple nonredundant pathways in postnatal ventricular myocytes.
Asunto(s)
Cardiomegalia/inducido químicamente , Cardiomegalia/patología , Miocardio/patología , Neurregulina-1 , Actinas/metabolismo , Animales , Animales Recién Nacidos , Factor Natriurético Atrial/genética , Células Cultivadas , Activación Enzimática , Inhibidores Enzimáticos/farmacología , Expresión Génica/efectos de los fármacos , Ventrículos Cardíacos , Humanos , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Quinasas de Proteína Quinasa Activadas por Mitógenos/fisiología , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Proteínas Quinasas Activadas por Mitógenos/fisiología , Neurregulina-1/farmacología , Fosfatidilinositol 3-Quinasas/metabolismo , Fosfatidilinositol 3-Quinasas/fisiología , Precursores de Proteínas/genética , Ratas , Proteínas Recombinantes/farmacología , Proteínas Quinasas S6 Ribosómicas/metabolismo , Proteínas Quinasas S6 Ribosómicas/fisiología , Sarcómeros/metabolismoRESUMEN
Increased production of nitric oxide (NO) after induction of the cytokine-inducible isoform of nitric oxide synthase (iNOS or NOS2) in cardiac myocytes and other parenchymal cells within the heart may in addition to contributing to myocyte contractile dysfunction also contribute to the induction of programmed cell death (apoptosis). To investigate the mechanism(s) by which increased NO production leads to apoptosis, we examined the role of NO in primary cultures of neonatal rat ventricular myocytes (NRVMs) after induction by the cytokines interleukin-1beta (IL-1beta) and interferon gamma (IFNgamma) or exposure to the exogenous NO donor S-nitroso-N-acetylcysteine (SNAC) or peroxynitrite (ONOO(-)). Both SNAC (1 mmol/L) and ONOO(-) (100 micromol/L), but not their respective controls (ie, N-acetylcysteine and pH-inactivated ONOO(-)), induced apoptosis in confluent, serum-starved NRVMs at 48 hours. Similarly, incubation of NRVMs with IL-1beta and IFNgamma for 48 hours resulted in an increase in iNOS expression, nitrite production, and programmed cell death. Both the cytokine-induced nitrite accumulation and myocyte apoptosis could be completely prevented by the nonselective NOS inhibitor L-nitroarginine (3 mmol/L) or the specific iNOS inhibitor 2-amino-5, 6-dihydro-6-methyl-4H-1,3-thiazine (AMT, 100 micromol/L). NO-mediated myocyte apoptosis was not attenuated by the inhibition of soluble guanylyl cyclase with ODQ, nor could apoptosis be induced by the incubation of NRVMs with 1 mmol/L 8-bromo-cGMP, a cell-permeant cGMP analogue. However, NO-mediated apoptosis was significantly attenuated by the superoxide dismutase mimetic and ONOO(-) scavenger Mn(III)tetrakis (4-benzoic acid) porphyrin (MnTBAP, 100 micromol/L). NO/ONOO(-)-mediated apoptosis was associated with increased expression of Bax with no change in Bcl-2 mRNA abundance. Furthermore, apoptotic cell death was also confirmed in adult rat ventricular myocytes (ARVMs) when grown in heteroculture with IL-1beta- and IFNgamma-treated rat cardiac microvascular endothelial cells. Therefore, cytokine-induced apoptosis in NRVMs and ARVMs is mediated by iNOS induction, ONOO(-), and associated with an increase in Bax levels.
Asunto(s)
Apoptosis/fisiología , Citocinas/farmacología , Miocardio/patología , Óxido Nítrico Sintasa/fisiología , Animales , Apoptosis/efectos de los fármacos , Células Cultivadas , Inducción Enzimática , Corazón/fisiología , Nitratos/metabolismo , Óxido Nítrico Sintasa de Tipo II , Ratas , Ratas Sprague-Dawley , Transducción de Señal/efectos de los fármacosRESUMEN
Oxidative stress has been implicated in the pathophysiology of myocardial failure. We tested the hypothesis that inhibition of endogenous antioxidant enzymes can regulate the phenotype of cardiac myocytes. Neonatal rat ventricular myocytes in vitro were exposed to diethyldithiocarbamic acid (DDC), an inhibitor of cytosolic (Cu, Zn) and extracellular superoxide dismutase (SOD). DDC inhibited SOD activity and increased intracellular superoxide in a concentration-dependent manner. A low concentration (1 micromol/L) of DDC stimulated myocyte growth, as demonstrated by increases in protein synthesis, cellular protein, prepro-atrial natriuretic peptide, and c-fos mRNAs and decreased sarcoplasmic reticulum Ca(2+)ATPase mRNA. These actions were all inhibited by the superoxide scavenger Tiron (4,5-dihydroxy-1,3-benzene disulfonic acid). Higher concentrations of DDC (100 micromol/L) stimulated myocyte apoptosis, as evidenced by DNA laddering, characteristic nuclear morphology, in situ terminal deoxynucleotidyl transferase-mediated nick end-labeling (TUNEL), and increased bax mRNA expression. DDC-stimulated apoptosis was inhibited by the SOD/catalase mimetic EUK-8. The growth and apoptotic effects of DDC were mimicked by superoxide generation with xanthine plus xanthine oxidase. Thus, increased intracellular superoxide resulting from inhibition of SOD causes activation of a growth program and apoptosis in cardiac myocytes. These findings support a role for oxidative stress in the pathogenesis of myocardial remodeling and failure.
Asunto(s)
Apoptosis/fisiología , Fibras Musculares Esqueléticas/patología , Miocardio/patología , Superóxido Dismutasa/antagonistas & inhibidores , Animales , Animales Recién Nacidos , Cardiomegalia/enzimología , Cardiomegalia/genética , Cardiomegalia/patología , División Celular/fisiología , Membrana Celular/fisiología , Células Cultivadas , Quelantes/farmacología , Ditiocarba/farmacología , Regulación Enzimológica de la Expresión Génica , Hipertrofia , Etiquetado Corte-Fin in Situ , Técnicas In Vitro , Fibras Musculares Esqueléticas/efectos de los fármacos , Fibras Musculares Esqueléticas/enzimología , Miocardio/enzimología , Estrés Oxidativo/fisiología , Fenotipo , Ratas , Superóxido Dismutasa/genética , Superóxidos/metabolismoRESUMEN
-The clinical efficacy of anthracycline antineoplastic agents is limited by a high incidence of severe and usually irreversible cardiac toxicity, the cause of which remains controversial. In primary cultures of neonatal and adult rat ventricular myocytes, we found that daunorubicin, at concentrations /=10 micromol/L induced necrotic cell death within 24 hours, with no changes characteristic of apoptosis. To determine whether reactive oxygen species play a role in daunorubicin-mediated apoptosis, we monitored the generation of hydrogen peroxide with dichlorofluorescein (DCF). However, daunorubicin (1 micromol/L) did not increase DCF fluorescence, nor were the antioxidants N-acetylcysteine or the combination of alpha-tocopherol and ascorbic acid able to prevent apoptosis. In contrast, dexrazoxane (10 micromol/L), known clinically to limit anthracycline cardiac toxicity, prevented daunorubicin-induced myocyte apoptosis, but not necrosis induced by higher anthracycline concentrations (>/=10 micromol/L). The antiapoptotic action of dexrazoxane was mimicked by the superoxide-dismutase mimetic porphyrin manganese(II/III)tetrakis(1-methyl-4-peridyl)porphyrin (50 micromol/L). The recognition that anthracycline-induced cardiac myocyte apoptosis, perhaps mediated by superoxide anion generation, occurs at concentrations well below those that result in myocyte necrosis, may aid in the design of new therapeutic strategies to limit the toxicity of these drugs.
Asunto(s)
Antibióticos Antineoplásicos/toxicidad , Apoptosis/efectos de los fármacos , Fármacos Cardiovasculares/farmacología , Daunorrubicina/toxicidad , Corazón/efectos de los fármacos , Razoxano/farmacología , Animales , Células Cultivadas , Relación Dosis-Respuesta a Droga , Necrosis , Estrés Oxidativo , Ratas , Ratas Sprague-Dawley , Superóxidos/metabolismoRESUMEN
It is now apparent that NO is produced in the myocardium. There it plays a central role in normal myocardial physiology. In addition, NO has the ability to exert whether beneficial or deleterious effects on the structure and function of the myocardium. At low, "physiologic" concentrations, NO may protect from deleterious stimuli such as mechanical stress and norepinephrine. At higher, "pathologic" concentrations, NO may cause the loss of myocytes. The mechanisms by which NO exerts these contrasting effects may involve decreases and increases in oxidative stress, respectively. A better understanding of the role NO plays in the development and progression of myocardial failure may lead to new treatment strategies.