RESUMEN
There is a debate on whether H1-histamine receptors can alter contractility in the mammalian heart. We studied here a new transgenic mouse model where we increased genetically the cardiac level of the H1-histamine receptor. We wanted to know if histamine could augment or decrease contractile parameters in mice with cardiac-specific overexpression of human H1-histamine receptors (H1-TG) and compared these findings with those in littermate wild-type mice (WT). In H1-TG mice, we studied the presence of H1-histamine receptors by autoradiography of the atrium and ventricle using [3H]mepyramine. The messenger RNA for human H1-histamine receptors was present in the heart from H1-TG and absent from WT. Using in situ hybridization, we noted mRNA for the human H1-histamine receptor in cardiac cells from H1-TG. We noted that histamine (1 nM-10 µM) in paced (1 Hz) left atrial preparations from H1-TG, exerted at each concentration of histamine initially reduced force of contraction and then raised contractile force. Likewise, in spontaneously beating left atrial preparations from H1-TG, we noted that histamine led to a transient reduction in the spontaneous beating rate followed by an augmentation in the beating rate. The negative inotropic and chronotropic and the positive inotropic effects on histamine in isolated atrial muscle strips from H1-TG were attenuated by the H1-histamine receptor antagonist mepyramine. Histamine failed to exert an increased force or reduce the heartbeat in atrial preparations from WT. We concluded that stimulation of H1-histamine-receptors can decrease and then augment contractile force in the mammalian heart and stimulation of H1-histamine receptors exerts a negative chronotropic effect. SIGNIFICANCE STATEMENT: We made novel transgenic mice with cardiomyocyte-specific high expressional levels of the human H1-histamine receptor to contribute to the clarification of the controversy on whether H1-histamine receptors increase or decrease contractility and beating rate in the mammalian heart. From our data, we conclude that stimulation of H1-histamine receptors first decrease and then raise contractile force in the mammalian heart but exert solely negative chronotropic effects.
Asunto(s)
Histamina , Contracción Miocárdica , Humanos , Ratones , Animales , Ratones Transgénicos , Histamina/farmacología , Pirilamina/farmacología , Corazón , Receptores Histamínicos , Atrios Cardíacos , Frecuencia Cardíaca , Receptores Histamínicos H1/genética , MamíferosRESUMEN
Calsequestrin (CSQ2) is the main Ca2+-binding protein in the sarcoplasmic reticulum of the mammalian heart. In order to understand the function of calsequestrin better, we compared two age groups (young: 4-5 months of age versus adult: 18 months of age) of CSQ2 knock-out mice (CSQ2(-/-)) and littermate wild-type mice (CSQ2(+/+)). Using echocardiography, in adult mice, the basal left ventricular ejection fraction and the spontaneous beating rate were lower in CSQ2(-/-) compared to CSQ2(+/+). The increase in ejection fraction by ß-adrenergic stimulation (intraperitoneal injection of isoproterenol) was lower in adult CSQ2(-/-) versus adult CSQ2(+/+). After hypoxia in vitro (isolated atrial preparations) by gassing the organ bath buffer with 95% N2, force of contraction in electrically driven left atria increased to lower values in young CSQ2(-/-) than in young CSQ2(+/+). In addition, after global ischemia and reperfusion (buffer-perfused hearts according to Langendorff; 20-min ischemia and 15-min reperfusion), the rate of tension development was higher in young CSQ2(-/-) compared to young CSQ2(+/+). Finally, we evaluated signs of inflammation (immune cells, autoantibodies, and fibrosis). However, whereas no immunological alterations were found between all investigated groups, pronounced fibrosis was found in the ventricles of adult CSQ2(-/-) compared to all other groups. We suggest that in young mice, CSQ2 is important for cardiac performance especially in isolated cardiac preparations under conditions of impaired oxygen supply, but with differences between atrium and ventricle. Lack of CSQ2 leads age dependently to fibrosis and depressed cardiac performance in echocardiographic studies.
Asunto(s)
Calcio , Calsecuestrina , Animales , Calcio/metabolismo , Calsecuestrina/genética , Calsecuestrina/metabolismo , Fibrosis , Atrios Cardíacos/metabolismo , Hipoxia/metabolismo , Isquemia/metabolismo , Mamíferos/metabolismo , Ratones , Ratones Noqueados , Contracción Miocárdica , Retículo Sarcoplasmático/metabolismo , Volumen Sistólico , Función Ventricular IzquierdaRESUMEN
Reversible protein phosphorylation is a posttranslational modification of regulatory proteins involved in cardiac signaling pathways. Here, we focus on the role of protein phosphatase 2A (PP2A) for cardiac gene expression and stress response using a transgenic mouse model with cardiac myocyte-specific overexpression of the catalytic subunit of PP2A (PP2A-TG). Gene and protein expression were assessed under basal conditions by gene chip analysis and Western blotting. Some cardiac genes related to the cell metabolism and to protein phosphorylation such as kinases and phosphatases were altered in PP2A-TG compared to wild type mice (WT). As cardiac stressors, a lipopolysaccharide (LPS)-induced sepsis in vivo and a global cardiac ischemia in vitro (stop-flow isolated perfused heart model) were examined. Whereas the basal cardiac function was reduced in PP2A-TG as studied by echocardiography or as studied in the isolated work-performing heart, the acute LPS- or ischemia-induced cardiac dysfunction deteriorated less in PP2A-TG compared to WT. From the data, we conclude that increased PP2A activity may influence the acute stress tolerance of cardiac myocytes.
Asunto(s)
Isquemia , Miocitos Cardíacos , Proteína Fosfatasa 2 , Sepsis , Animales , Pruebas de Función Cardíaca , Isquemia/metabolismo , Lipopolisacáridos/metabolismo , Ratones , Miocitos Cardíacos/metabolismo , Fosforilación , Proteína Fosfatasa 2/genética , Proteína Fosfatasa 2/metabolismo , Procesamiento Proteico-Postraduccional , Sepsis/metabolismoRESUMEN
As part of our ongoing studies on the potential pathophysiological role of serine/threonine phosphatases (PP) in the mammalian heart, we have generated transgenic mice with cardiac muscle cell-specific overexpression of PP2Acα (PP2A) and PP5 (PP5). For further studies we crossbred PP2A and PP5 mice to obtain PP2AxPP5 double transgenic mice (PP2AxPP5, DT) and compared them with littermate wild-type mice (WT) serving as a control. The mortality of DT mice was greatly enhanced vs. other genotypes. Cardiac fibrosis was noted histologically and mRNA levels of collagen 1α, collagen 3α and fibronectin 1 were augmented in DT. DT and PP2A mice exhibited an increase in relative heart weight. The ejection fraction (EF) was reduced in PP2A and DT but while the EF of PP2A was nearly normalized after ß-adrenergic stimulation by isoproterenol, it was almost unchanged in DT. Moreover, left atrial preparations from DT were less sensitive to isoproterenol treatment both under normoxic conditions and after hypoxia. In addition, levels of the hypertrophy markers atrial natriuretic peptide and B-type natriuretic peptide as well as the inflammation markers interleukin 6 and nuclear factor kappa B were increased in DT. PP2A enzyme activity was enhanced in PP2A vs. WT but similar to DT. This was accompanied by a reduced phosphorylation state of phospholamban at serine-16. Fittingly, the relaxation times in left atria from DT were prolonged. In summary, cardiac co-overexpression of PP2A and PP5 were detrimental to animal survival and cardiac function, and the mechanism may involve dephosphorylation of important regulatory proteins but also fibrosis and inflammation.
Asunto(s)
Glicoproteínas/metabolismo , Proteína Fosfatasa 2C/metabolismo , Sístole/fisiología , Animales , Cardiomiopatías/metabolismo , Fibrosis/metabolismo , Cardiopatías/metabolismo , Humanos , Masculino , Ratones , Ratones Transgénicos , Contracción Miocárdica/fisiología , Miocardio/metabolismo , Miocitos Cardíacos/metabolismo , Fosforilación , Inhibidores de Serina Proteinasa/metabolismo , Sístole/genéticaRESUMEN
Using transgenic (TG) mice that overexpress the human serotonin (5-HT)4a receptor specifically in cardiomyocytes, we wanted to know whether 5-HT can be formed and degraded in the mammalian heart and whether this can likewise lead to inotropic and chronotropic effects in this TG model. We noted that the 5-HT precursor 5-hydroxy-tryptophan (5-HTP) can exert inotropic and chronotropic effects in cardiac preparations from TG mice but not from wild-type (WT) mice; similar results were found in human atrial preparations as well as in intact TG animals using echocardiography. Moreover, by immunohistochemistry we could detect 5-HT metabolizing enzymes and 5-HT transporters in mouse hearts as well as in human atria. Hence, in the presence of an inhibitor of aromatic l-amino acid decarboxylase, the positive inotropic effects of 5-HTP were absent in TG and isolated human atrial preparations, and, moreover, inhibitors of enzymes involved in 5-HT degradation enhanced the efficacy of 5-HT in TG atria. A releaser of neurotransmitters increased inotropy in the isolated TG atrium, and this effect could be blocked by a 5-HT4a receptor antagonist. Fluoxetine, an inhibitor of 5-HT uptake, elevated the potency of 5-HT to increase contractility in the TG atrium. In addition, inhibitors of organic cation and monoamine transporters apparently reduced the positive inotropic potency of 5-HT in the TG atrium. Hence, we tentatively conclude that a local production and degradation of 5-HT in the mammalian heart and more specifically in mammalian myocytes probably occurs. Conceivably, this formation of 5-HT and possibly impaired degradation may be clinically relevant in cases of unexplained tachycardia and other arrhythmias.NEW & NOTEWORTHY The present work suggests that inotropically active serotonin (5-HT) can be formed in the mouse and human heart and probably by cardiomyocytes themselves. Moreover, active degradation of 5-HT seems to occur in the mammalian heart. These findings may again increase the interest of researchers for cardiac effects of 5-HT.
Asunto(s)
Atrios Cardíacos/metabolismo , Miocitos Cardíacos/metabolismo , Serotonina/metabolismo , 5-Hidroxitriptófano/metabolismo , 5-Hidroxitriptófano/farmacología , Animales , Inhibidores de Descarboxilasas de Aminoácidos Aromáticos/farmacología , Descarboxilasas de Aminoácido-L-Aromático/metabolismo , Cardiotónicos/farmacología , Relación Dosis-Respuesta a Droga , Proteínas de Transporte de Nucleósido Equilibrativas/metabolismo , Femenino , Atrios Cardíacos/efectos de los fármacos , Atrios Cardíacos/enzimología , Frecuencia Cardíaca , Humanos , Preparación de Corazón Aislado , Masculino , Ratones Transgénicos , Monoaminooxidasa/metabolismo , Inhibidores de la Monoaminooxidasa/farmacología , Contracción Miocárdica , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/enzimología , Proteínas de Transporte de Catión Orgánico/metabolismo , Receptores de Serotonina 5-HT4/genética , Receptores de Serotonina 5-HT4/metabolismo , Serotoninérgicos/farmacología , Proteínas de Transporte de Serotonina en la Membrana Plasmática/metabolismo , Transducción de SeñalRESUMEN
Dopamine can exert effects in the mammalian heart via five different dopamine receptors. There is controversy whether dopamine receptors increase contractility in the human heart. Therefore, we have generated mice that overexpress the human D1-dopamine receptor in the heart (D1-TG) and hypothesized that dopamine increases force of contraction and beating rate compared to wild-type mice (WT). In D1-TG hearts, we ascertained the presence of D1-dopamine receptors by autoradiography using [3H]SKF 38393. The mRNA for human D1-dopamine receptors was present in D1-TG hearts and absent in WT. We detected by in-situ-hybridization mRNA for D1-dopamine receptors in atrial and ventricular D1-TG cardiomyocytes compared to WT but also in human atrial preparations. We noted that in the presence of 10 µM propranolol (to antagonize ß-adrenoceptors), dopamine alone and the D1- and D5-dopamine receptor agonist SKF 38393 (0.1-10 µM cumulatively applied) exerted concentration- and time-dependent positive inotropic effects and positive chronotropic effects in left or right atrial preparations from D1-TG. The positive inotropic effects of SKF 38393 in left atrial preparations from D1-TG led to an increased rate of relaxation and accompanied by and probably caused by an augmented phosphorylation state of the inhibitory subunit of troponin. In the presence of 0.4 µM propranolol, 1 µM dopamine could increase left ventricular force of contraction in isolated perfused hearts from D1-TG. In this model, we have demonstrated a positive inotropic and chronotropic effect of dopamine. Thus, in principle, the human D1-dopamine receptor can couple to contractility in the mammalian heart.
Asunto(s)
Ratones Transgénicos , Contracción Miocárdica , Receptores de Dopamina D1 , Animales , Receptores de Dopamina D1/metabolismo , Receptores de Dopamina D1/genética , Humanos , Contracción Miocárdica/efectos de los fármacos , Masculino , Dopamina/metabolismo , Ratones , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/efectos de los fármacos , Agonistas de Dopamina/farmacología , Miocardio/metabolismo , 2,3,4,5-Tetrahidro-7,8-dihidroxi-1-fenil-1H-3-benzazepina/farmacología , ARN Mensajero/metabolismo , ARN Mensajero/genética , Atrios Cardíacos/metabolismo , Atrios Cardíacos/efectos de los fármacos , Corazón/efectos de los fármacos , Corazón/fisiología , Ratones Endogámicos C57BL , Frecuencia Cardíaca/efectos de los fármacosRESUMEN
Pleiotropic effects of serotonin (5-HT) in the cardiovascular system are well documented. However, it remains to be elucidated, whether 5-HT is present in adult mammalian cardiomyocytes. To address this issue, we investigated the levels of 5-HT in blood, plasma, platelets, cardiac tissue, and cardiomyocytes from adult mice and for comparison in human right atrial tissue. Immunohistochemically, 5-HT was hardly found in mouse cardiac tissue, but small amounts could be detected in renal preparations, whereas adrenal preparations revealed a strong positive immunoreaction for 5-HT. Using a sensitive HPLC detection system, 5-HT was also detectable in the mouse heart and human atrium. Furthermore, we could identify 5-HT in isolated cardiomyocytes from adult mice. These findings were supported by detection of the activity of 5-HT-forming enzymes-tryptophan hydroxylase and aromatic L-amino acid decarboxylase-in isolated cardiomyocytes from adult mice and by inhibition of these enzymes with p-chlorophenylalanine and 3-hydroxybenzyl hydrazine. Addition of the first intermediate of 5-HT generation, that is 5-hydroxytryptophan, enhanced the 5-HT level and inhibition of monoamine oxidase by tranylcypromine further increased the level of 5-HT. Our findings reveal the presence and synthesis of 5-HT in cardiomyocytes of the mammalian heart implying that 5-HT may play an autocrine and/or paracrine role in the heart.
Asunto(s)
Miocitos Cardíacos/metabolismo , Serotonina/metabolismo , 5-Hidroxitriptófano/farmacología , Glándulas Suprarrenales/citología , Glándulas Suprarrenales/metabolismo , Animales , Inhibidores de Descarboxilasas de Aminoácidos Aromáticos , Descarboxilasas de Aminoácido-L-Aromático/metabolismo , Plaquetas/metabolismo , Células Cultivadas , Fenclonina/farmacología , Atrios Cardíacos/citología , Humanos , Hidrazinas/farmacología , Ratones , Miocardio/citología , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/enzimología , Serotonina/biosíntesis , Serotonina/sangre , Triptófano/metabolismo , Triptófano Hidroxilasa/antagonistas & inhibidores , Triptófano Hidroxilasa/metabolismoRESUMEN
In the neonatal mammalian heart, the role of ryanodine receptor (=Ca(2+) release channel)-mediated sarcoplasmic reticulum (SR) Ca(2+) release for excitation-contraction coupling is still a matter of debate. Using an adenoviral system, we overexpressed separately the junctional SR proteins triadin, junctin, and calsequestrin, which are probably involved in regulation of ryanodine receptor function. Infection of neonatal rat cardiac myocytes with triadin, junctin, or calsequestrin viruses, controlled by green fluorescent protein expression, resulted in an increased protein level of the corresponding transgenes. Measurement of Ca(2+) transients of infected cardiac myocytes revealed unchanged peak amplitudes under basal conditions but with overexpression of calsequestrin and triadin caffeine-releasable SR Ca(2+) content was increased. Our results demonstrate that an increased expression of triadin or calsequestrin is associated with an increased SR Ca(2+) storage but unchanged Ca(2+) signaling in neonatal rat cardiac myocytes. This is consistent with an ancillary role of the sarcoplasmic reticulum in excitation-contraction coupling in the developing mammalian heart.
Asunto(s)
Proteínas de Unión al Calcio/metabolismo , Calcio/metabolismo , Acoplamiento Excitación-Contracción/fisiología , Transporte Iónico/fisiología , Miocitos Cardíacos/metabolismo , Retículo Sarcoplasmático/efectos de los fármacos , Retículo Sarcoplasmático/metabolismo , Adenoviridae , Animales , Animales Recién Nacidos , Cafeína/farmacología , Señalización del Calcio/fisiología , Proteínas de Unión al Calcio/genética , Calsecuestrina/genética , Calsecuestrina/metabolismo , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Acoplamiento Excitación-Contracción/efectos de los fármacos , Regulación de la Expresión Génica , Vectores Genéticos , Corazón/efectos de los fármacos , Corazón/fisiología , Transporte Iónico/efectos de los fármacos , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Contracción Miocárdica/efectos de los fármacos , Contracción Miocárdica/fisiología , Miocitos Cardíacos/citología , Miocitos Cardíacos/efectos de los fármacos , Ratas , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Transducción GenéticaRESUMEN
Histamine is metabolized by several enzymes in vitro and in vivo. The relevance of this metabolism in the mammalian heart in vivo is unclear. However, histamine can exert positive inotropic effects (PIE) and positive chronotropic effects (PCE) in humans via H2-histamine receptors. In transgenic mice (H2-TG) that overexpress the human H2 receptor in cardiomyocytes but not in wild-type littermate mice (WT), histamine induced PIE and PCE in isolated left or right atrial preparations. These H2-TG were used to investigate the putative relevance of histamine degrading enzymes in the mammalian heart. Histidine, the precursor of histamine, increased force of contraction (FOC) in human atrial preparations. Moreover, histamine increased the phosphorylation state of phospholamban in human atrium. Here, we could detect histidine decarboxylase (HDC) and histamine itself in cardiomyocytes of mouse hearts. Moreover, our data indicate that histamine is subject to degradation in the mammalian heart. Inhibition of the histamine metabolizing enzymes diamine oxidase (DAO) and monoamine oxidase (MAO) shifted the concentration response curves for the PIE in H2-TG atria to the left. Moreover, activity of histamine metabolizing enzymes was present in mouse cardiac samples as well as in human atrial samples. Thus, drugs used for other indication (e.g. antidepressants) can alter histamine levels in the heart. Our results deepen our understanding of the physiological role of histamine in the mouse and human heart. Our findings might be clinically relevant because we show enzyme targets for drugs to modify the beating rate and force of the human heart.
RESUMEN
Serotonin (5-HT) exerts pleiotropic effects in the human cardiovascular system. Some of the effects are thought to be mediated via 5-HT(4) receptors, which are expressed in the human atrium and in ventricular tissue. However, a true animal model to study these receptors in more detail has been hitherto lacking. Therefore, we generated, for the first time, a transgenic (TG) mouse with cardiac myocyte-specific expression of the human 5-HT(4) receptor. RT-PCR and immunohistochemistry revealed expression of the receptor at the mRNA and protein levels. Stimulation of isolated cardiac preparations by isoproterenol increased phospholamban phosphorylation at Ser(16) and Thr(17) sites. 5-HT increased phosphorylation only in TG mice but not in wild-type (WT) mice. Furthermore, 5-HT increased contractility in isolated perfused hearts from TG mice but not WT mice. These effects of 5-HT could be blocked by the 5-HT(4) receptor-selective antagonist GR-125487. An intravenous infusion of 5-HT increased left ventricular contractility in TG mice but not in WT mice. Similarly, the increase in contractility by 5-HT in isolated cardiomyocytes from TG mice was accompanied by and probably mediated through an increase in L-type Ca(2+) channel current and in Ca(2+) transients. In intact animals, echocardiography revealed an inotropic and chronotropic effect of subcutaneously injected 5-HT in TG mice but not in WT mice. In isolated hearts from TG mice, spontaneous polymorphic atrial arrhythmias were noted. These findings demonstrate the functional expression of 5-HT(4) receptors in the heart of TG mice, and a potential proarrhythmic effect in the atrium. Therefore, 5-HT(4) receptor-expressing mice might be a useful model to mimic the human heart, where 5-HT(4) receptors are present and functional in the atrium and ventricle of the healthy and failing heart, and to investigate the influence of 5-HT in the development of cardiac arrhythmias and heart failure.
Asunto(s)
Corazón/fisiología , Contracción Miocárdica/fisiología , Miocardio/metabolismo , Miocitos Cardíacos/metabolismo , Receptores de Serotonina 5-HT4/metabolismo , Análisis de Varianza , Animales , Western Blotting , Ecocardiografía , Inmunohistoquímica , Ratones , Ratones Transgénicos , Fosforilación , Receptores de Serotonina 5-HT4/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal/fisiologíaRESUMEN
As part of our ongoing studies on the potential pathophysiological role of serine/threonine phosphatases (PP) in the mammalian heart, we have generated mice with cardiac-specific overexpression of PP2Cß (PP2C-TG) and compared them with littermate wild type mice (WT) serving as a control. Cardiac fibrosis was noted histologically in PP2C-TG. Collagen 1a, interleukin-6 and the natriuretic peptides ANP and BNP were augmented in PP2C-TG vs. WT (p < 0.05). Left atrial preparations from PP2C-TG were less resistant to hypoxia than atria from WT. PP2C-TG maintained cardiac function after the injection of lipopolysaccharide (LPS, a model of sepsis) and chronic isoproterenol treatment (a model of heart failure) better than WT. Crossbreeding of PP2C-TG mice with PP2A-TG mice (a genetic model of heart failure) resulted in double transgenic (DT) mice that exhibited a pronounced increase of heart weight in contrast to the mild hypertrophy noted in the mono-transgenic mice. The ejection fraction was reduced in PP2C-TG and in PP2A-TG mice compared with WT, but the reduction was the highest in DT compared with WT. PP2A enzyme activity was enhanced in PP2A-TG and DT mice compared with WT and PP2C-TG mice. In summary, cardiac overexpression of PP2Cß and co-overexpression of both the catalytic subunit of PP2A and PP2Cß were detrimental to cardiac function. PP2Cß overexpression made cardiac preparations less resistant to hypoxia than WT, leading to fibrosis, but PP2Cß overexpression led to better adaptation to some stressors, such as LPS or chronic ß-adrenergic stimulation. Hence, the effect of PP2Cß is context sensitive.
RESUMEN
The concept of endothelium-derived relaxing factor (EDRF) implies that nitric oxide (NO) produced by NO synthase (NOS) in the endothelium in response to vasorelaxants such as acetylcholine (ACh) acts on the underlying vascular smooth muscle cells (VSMC) inducing vascular relaxation. The EDRF concept was derived from experiments on denuded blood vessel strips and, in frames of this concept, VSMC were regarded as passive recipients of NO from endothelial cells. However, it was later found that VSMC express NOS by themselves, but the principal question remained unanswered, is the NO generation by VSMC physiologically relevant? We hypothesized that the destruction of the vascular wall anatomical integrity by rubbing off the endothelial layer might increase vascular superoxides that, in turn, reduced the NO bioactivity as a relaxing factor. To test our hypothesis, we examined ACh-induced vasorelaxation under protection against oxidative stress and found that superoxide scavengers restored vasodilatory responses to ACh in endothelium-deprived blood vessels. These findings imply that VSMC can release NO in amounts sufficient to account for the vasorelaxatory response and challenge the concept of the obligatory role of endothelial cells in the relaxation of arterial smooth muscle.
Asunto(s)
Arterias/fisiología , Endotelio Vascular/fisiología , Músculo Liso Vascular/fisiología , Vasodilatación , Acetilcolina/farmacología , Animales , Arterias/efectos de los fármacos , Arterias/metabolismo , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/metabolismo , Depuradores de Radicales Libres/farmacología , Masculino , Músculo Liso Vascular/efectos de los fármacos , Músculo Liso Vascular/metabolismo , Óxido Nítrico/metabolismo , Óxido Nítrico Sintasa/metabolismo , Óxido Nítrico Sintasa de Tipo III/metabolismo , Estrés Oxidativo , Superóxidos/metabolismoRESUMEN
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.
Asunto(s)
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
About half of the cardiac serine/threonine phosphatase activity is due to the activity of protein phosphatase type 1 (PP1). The activity of PP1 can be inhibited by an endogenous protein for which the expression inhibitor-2 (I-2) has been coined. We have previously described a transgenic mouse overexpressing a truncated form of I-2. Here, we have described and initially characterized several founders that overexpress the non-truncated (i.e., full length) I-2 in the mouse heart (TG) and compared them with non-transgenic littermates (WT). The founder with the highest overexpression of I-2 displayed under basal conditions no difference in contractile parameters (heart rate, developed tension, and its first derivate) compared to WT. The relative level of PP1 inhibition was similar in mice overexpressing the non-truncated as well as the truncated form of I-2. For comparison, we overexpressed I-2 by an adenoviral system in several cell lines (myocytes from a tumor-derived cell line (H9C2), neonatal rat cardiomyocytes, smooth muscle cells from rat aorta (A7R5)). We noted gene dosage-dependent staining for I-2 protein in infected cells together with reduced PP1 activity. Finally, I-2 expression in neonatal rat cardiomyocytes led to an increase of Ca2+ transients by about 60%. In summary, we achieved immunologically confirmed overexpression of wild-type I-2 in cardiovascular cells which was biochemically able to inhibit PP1 in the whole heart (using I-2 transgenic mice) as well as in isolated cells including cardiomyocytes (using I-2 coding virus) indirectly underscoring the importance of PP1 for cardiovascular function.
Asunto(s)
Miocardio/metabolismo , Proteína Fosfatasa 1/metabolismo , Proteínas/genética , Adenoviridae/genética , Animales , Línea Celular , Corazón/fisiología , Masculino , Ratones Transgénicos , Miocitos del Músculo Liso/metabolismo , Proteínas/metabolismo , Ratas WistarRESUMEN
The concept of endothelium-derived relaxing factor (EDRF) put forward in 1980 by Furchgott and Zawadzki implies that nitric oxide (NO) produced by NO synthase (NOS) in the endothelium diffuses to the underlying vascular smooth muscle, where it modulates vascular tone as well as vascular smooth muscle cell (VSMC) proliferation by increasing cGMP formation with subsequent activation of cGMP-dependent protein kinase. According to this concept, VSMC do not express NOS by themselves. This attractive, simple scheme is now under considerable debate. To address this issue, we designed this study with the use of a novel supersensitive immunocytochemical technique of signal amplification with tyramide and electron microscopic immunogold labeling complemented with Western blotting, as in our recent studies demonstrating NOS in the myocardial and skeletal muscles. We provide the first evidence that, in contrast to the currently accepted view, VSMC in various blood vessels express all three NOS isoforms depending on the blood vessel type. These findings suggest an alternative mechanism by which local NOS expression may modulate vascular functions in an endothelium-independent manner.
Asunto(s)
Músculo Liso Vascular/enzimología , Óxido Nítrico Sintasa/análisis , Animales , Vasos Sanguíneos/enzimología , Western Blotting , Humanos , Inmunohistoquímica/métodos , Masculino , Microscopía Fluorescente , Óxido Nítrico Sintasa/inmunología , Isoformas de Proteínas/análisis , Isoformas de Proteínas/inmunología , Porcinos , Tiramina/químicaRESUMEN
A general problem in immunocytochemistry is the development of a reliable multiple immunolabeling method with primary antibodies originating from the same host species. Here, we briefly outline different approaches intended to close this technological gap and focus on multiple immunolabeling with monoclonal primary antibodies. To this end, we generated a basic universal protocol for the use of secondary antibodies selectively recognizing different isotypes/subclasses of monoclonal primary antibodies. This approach is widely applicable and offers a simple procedure for simultaneously detecting two or more antigens.
Asunto(s)
Anticuerpos/inmunología , Antígenos/análisis , Antígenos/inmunología , Técnica del Anticuerpo Fluorescente/métodos , Animales , Anticuerpos/química , Anticuerpos/clasificación , Especificidad de Anticuerpos , Color , HumanosRESUMEN
The incidence of liver diseases has increased over the past few years. For this reason, the consequences of induced nitric oxide (NO) synthesis in liver damages warrant further studies. To address this issue, we investigated the expression of key enzymes engaged in the control of NO signaling in the rat liver after carbon tetrachloride (CCl4) intoxication and subsequent regeneration. CCl4 intoxication resulted in up-regulation of the entire NO signal transduction machinery. Expression patterns of arginase, soluble guanylyl cyclase and cyclic nucleotide phosphodiesterase revealed striking parallels with that of NO synthase (NOS). Co-expression of the major components of the l-arginine-NO-cGMP signaling cascade both in hepatocytes and in nonparenchymal cells indicates an autocrine rather than a paracrine fashion of NO signaling in the liver. Up-regulation of NOS after CCl4 intoxication fell behind the oxidative stress and was found to be associated with the initiation of parenchymal regeneration implying a beneficial effect of NO.
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Arginina/fisiología , GMP Cíclico/fisiología , Hígado/efectos de los fármacos , Óxido Nítrico/fisiología , Transducción de Señal/fisiología , Animales , Tetracloruro de Carbono/toxicidad , Hígado/metabolismo , Hígado/patología , Regeneración Hepática , Masculino , Óxido Nítrico Sintasa/genética , ARN Mensajero/análisis , Ratas , Ratas WistarRESUMEN
OBJECTIVE: Junctin is a major transmembrane protein in cardiac junctional sarcoplasmic reticulum, which forms a quaternary complex with the ryanodine receptor (Ca(2+) release channel), triadin, and calsequestrin. METHODS: To better understand the role of junctin in excitation-contraction coupling in the heart, we generated transgenic mice with targeted overexpression of junctin to mouse heart, using the alpha-MHC promoter to drive protein expression. RESULTS: The protein was overexpressed 10-fold in mouse ventricles and overexpression was accompanied by cardiac hypertrophy (19%). The levels of two other junctional SR-proteins, the ryanodine receptor and triadin, were reduced by 32% and 23%, respectively. However, [3H]ryanodine binding and the expression levels of calsequestrin, phospholamban and SERCA2a remained unchanged. Cardiomyocytes from junctin-overexpressing mice exhibited impaired relaxation: Ca(2+) transients decayed at a slower rate and cell relengthening was prolonged. Isolated electrically stimulated papillary muscles from junctin-overexpressing hearts exhibited prolonged mechanical relaxation, and echocardiographic parameters of relaxation were prolonged in the living transgenic mice. The amplitude of caffeine-induced Ca(2+) transients was lower in cardiomyocytes from junctin-overexpressing mice. The inactivation kinetics of L-type Ca(2+) channel were prolonged in junctin-overexpressing cardiomyocytes using Ca(2+) or Ba(2+) as charge carriers. CONCLUSION: Our data provide evidence that cardiac-specific overexpression of junctin is accompanied by impaired myocardial relaxation with prolonged Ca(2+) transient kinetics on the cardiomyocyte level.
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Proteínas de Unión al Calcio , Cardiomegalia/metabolismo , Proteínas Portadoras/genética , Proteínas de la Membrana , Oxigenasas de Función Mixta , Proteínas Musculares/genética , Contracción Miocárdica , Miocitos Cardíacos/metabolismo , Animales , Calcio/metabolismo , Canales de Calcio Tipo L/metabolismo , Proteínas Portadoras/metabolismo , Tamaño de la Célula , Ecocardiografía Doppler , Estimulación Eléctrica , Péptidos y Proteínas de Señalización Intracelular , Ratones , Ratones Transgénicos , Proteínas Musculares/metabolismo , Relajación Muscular , Músculos Papilares , Canal Liberador de Calcio Receptor de Rianodina/metabolismoRESUMEN
Potential ortho- and pathophysiological roles for nitric oxide synthases (NOS) in cardiac functions have been and are continuing to be described. However, cellular signaling mechanisms controlling nitric oxide (NO) production in the heart remain obscure. The aim of this study was to investigate signaling mechanisms involved in regulation of NOS expression and NO generation in cardiomyocytes. Using immunocytochemical methods in conjunction with western blotting, we have found that cultured neonatal rat cardiomyocytes express constitutively all three NOS isoforms targeted predominantly to the particulate component of cardiomyocytes - mitochondria and along contractile fibers, as well as along plasma membrane including T-tubules. Biochemical assay of NO generation has shown that exposure of cultured neonatal rat cardiac cells to isoproterenol (beta-adrenergic stimulation), iloprost [stable prostaglandin I(2) (PGI(2)) analogue], as well as inflammatory cytokines and dibutyryl adenosine-3',5'-monophosphate (db-cAMP), resulted in a marked up-regulation of NOS expression by cardiomyocytes. In db-cAMP-stimulated cells, inhibition of protein kinase A (PKA) and protein kinase C (PKC) reduced immunolabeling of NOS and concomitantly lowered NO production. Taken together, these data point to an involvement of beta-adrenergic mechanisms, cytokine and PGI(2) receptors, adenylyl cyclase, PKA, and PKC in the control of NO generation and expression of NOS in rat cardiomyocytes.
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Regulación Enzimológica de la Expresión Génica , Isoenzimas/metabolismo , Miocitos Cardíacos/enzimología , Óxido Nítrico Sintasa/metabolismo , Animales , Animales Recién Nacidos , Bucladesina/farmacología , Células Cultivadas , Isoenzimas/genética , Miocitos Cardíacos/citología , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/fisiología , Óxido Nítrico/metabolismo , Óxido Nítrico Sintasa/genética , Nitritos/metabolismo , Inhibidores de Proteínas Quinasas/metabolismo , Ratas , Acetato de Tetradecanoilforbol/farmacologíaRESUMEN
Lack of standardisation and inaccuracy in HER2 test results may adversely influence patient evaluation and therapy selection. In the present study we applied immunohistochemistry (IHC) using the A0485 and CB11 antibodies and fluorescence in situ hybridisation (FISH) for detection of HER2 in 74 routinely processed breast carcinoma specimens. The rapidity of cellular proliferation was assessed by standardised AgNOR analysis and compared with HER2 status. Protein over-expression was found in 30/74 cases by A0485 and in 20/74 by CB11 antibodies, while amplification was detected in 22/74 carcinomas by FISH. Twenty-seven of 74 tumours were high-level AgNOR expressors (mean AgNOR area >3.369 microm2), 19 of which revealed amplification. The highest concordance between results was achieved by FISH and CB11-IHC (97%), the concordance between FISH and A0485-IHC was 89 and 84% between FISH and AgNOR quantity, respectively. The overall concordance between A0485 and CB11-IHC was 85% with 10 incongruent cases, all scored 2+ by A0485 and 0/1+ by CB11. Eight of the discordant tumours were non-amplified by FISH and 7 were low AgNOR-expressors. Our results indicate that using CB11 antibody, a nearly complete agreement can be achieved between HER2 IHC and FISH in diagnostic paraffin material. Moreover, in 2+ positive IHC cases, the AgNOR analysis may represent an additional tool to select patients as candidates for Herceptin therapy due to the strong negative predictor value.