RESUMO
Although primary causes of Alzheimer's and vascular dementia are unknown, the importance of preceding vascular lesions is widely accepted. Furthermore, there is strong evidence for the involvement of autoimmune mechanisms. Here, we report the presence of agonistic autoantibodies directed at adrenergic receptors in the circulation of patients with mild to moderate Alzheimer's and vascular dementia. In 59% of these patients, agonistic autoantibodies against the α(1) -adrenergic receptor and the ß(2) -adrenergic receptor were identified. The majority of positive patients (66%) contained both types of autoantibodies in combination. In a control group of patients with neurological impairments others than Alzheimer's and vascular dementia, only 17% were found to harbour these autoantibodies. The autoantibodies to the α(1) -adrenergic receptor interacted preferably with the extracellular loop1 of the receptor. They were further studied in IgG preparations from the column regenerate of a patient who underwent immunoadsorption. The α(1) -adrenergic receptor autoantibodies specifically bound to the extracellular loop1 peptide of the receptor with an apparent EC(50) value of 30 nm. They mobilized intracellular calcium in a clonal cell line expressing the human form of the α(1) -adrenergic receptor. Our data support the notion that autoimmune mechanisms play a significant role in the pathogenesis of Alzheimer's and vascular dementia. We suggest that agonistic autoantibodies to the α(1) -adrenergic and the ß(2) -adrenergic receptor may contribute to vascular lesions and increased plaque formation.
Assuntos
Doença de Alzheimer/imunologia , Autoanticorpos/imunologia , Demência Vascular/imunologia , Receptores Adrenérgicos alfa 1/imunologia , Receptores Adrenérgicos beta 2/imunologia , Idoso , Idoso de 80 Anos ou mais , Animais , Cálcio/sangue , Cálcio/metabolismo , Células Cultivadas , Feminino , Humanos , Imunoglobulina G/análise , Imunoglobulina G/sangue , Masculino , Pessoa de Meia-Idade , Placa Aterosclerótica/imunologia , Placa Aterosclerótica/patologia , RatosRESUMO
Calcium-dependent mechanisms and the renin angiotensin system (RAS) are critically involved in the hypertrophic growth of the myocardium. The calcium/calmodulin-dependent protein kinase II (CaMKII) is a ubiquitous mediator in calcium signaling and modulates calcium handling and growth mechanisms in cardiomyocytes. Here we present data on expression of cardiac isoforms of CaMKIIdelta, the dominant form in the myocardium, in compensatory hypertrophy of stroke-prone spontaneously hypertensive rats (SHRSP) compared to the normotensive Wistar-Kyoto (WKY) control strain. Cardiac hypertrophy in SHRSP was documented by an increased heart weight/body weight ratio (HW/BW) of 31% (p < 0.05) and a more than six-fold elevated atrial natriuretic factor (ANF) transcript level (p < 0.05). Compensatory hypertrophic growth in SHRSP produced a specific phenotype of CaMKIIdelta isoforms characterized by increased transcript levels of the embryonic/neonatal isoform delta4 (48%, p < 0.05) and the isoform delta9 (31%, p < 0.05) with no changes in delta2 and delta3. Inhibition of angiotensin converting enzyme (ACE) by cilazapril completely regressed myocardial hypertrophy, normalized ANF transcript levels, and restored the normal phenotype of CaMKIIdelta by reducing transcripts for delta4 and delta9 to levels present in WKY controls. Our data suggest the importance of specific changes in the CaMKII isoform composition for growth processes in the myocardium.
Assuntos
Inibidores da Enzima Conversora de Angiotensina/farmacologia , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Cardiomegalia/genética , Cardiomegalia/metabolismo , Cilazapril/farmacologia , Miocárdio/enzimologia , Animais , Fator Natriurético Atrial/genética , Fator Natriurético Atrial/metabolismo , Peso Corporal/efeitos dos fármacos , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina , Proteínas Quinases Dependentes de Cálcio-Calmodulina/genética , Cardiomegalia/patologia , Predisposição Genética para Doença , Técnicas In Vitro , Isoenzimas/metabolismo , Masculino , Miocárdio/patologia , Tamanho do Órgão/efeitos dos fármacos , Fenótipo , Ratos , Ratos Endogâmicos SHR/genética , Ratos Endogâmicos WKY , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Acidente Vascular Cerebral/genética , Transcrição GênicaRESUMO
S100A1, a Ca(2+) binding protein of the EF-hand type, is preferentially expressed in myocardial tissue and has been found to colocalize with the sarcoplasmic reticulum (SR) and the contractile filaments in cardiac tissue. Because S100A1 is known to modulate SR Ca(2+) handling in skeletal muscle, we sought to investigate the specific role of S100A1 in the regulation of myocardial contractility. To address this issue, we investigated contractile properties of adult cardiomyocytes as well as of engineered heart tissue after S100A1 adenoviral gene transfer. S100A1 gene transfer resulted in a significant increase of unloaded shortening and isometric contraction in isolated cardiomyocytes and engineered heart tissues, respectively. Analysis of intracellular Ca(2+) cycling in S100A1-overexpressing cardiomyocytes revealed a significant increase in cytosolic Ca(2+) transients, whereas in functional studies on saponin-permeabilized adult cardiomyocytes, the addition of S100A1 protein significantly enhanced SR Ca(2+) uptake. Moreover, in Triton-skinned ventricular trabeculae, S100A1 protein significantly decreased myofibrillar Ca(2+) sensitivity ([EC(50%)]) and Ca(2+) cooperativity, whereas maximal isometric force remained unchanged. Our data suggest that S100A1 effects are cAMP independent because cellular cAMP levels and protein kinase A-dependent phosphorylation of phospholamban were not altered, and carbachol failed to suppress S100A1 actions. These results show that S100A1 overexpression enhances cardiac contractile performance and establish the concept of S100A1 as a regulator of myocardial contractility. S100A1 thus improves cardiac contractile performance both by regulating SR Ca(2+) handling and myofibrillar Ca(2+) responsiveness.
Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Ventrículos do Coração/metabolismo , Contração Miocárdica/fisiologia , Miocárdio/metabolismo , Citoesqueleto de Actina/fisiologia , Animais , Cálcio/metabolismo , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/farmacologia , ATPases Transportadoras de Cálcio/metabolismo , Células Cultivadas , AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Expressão Gênica , Técnicas de Transferência de Genes , Ventrículos do Coração/citologia , Humanos , Líquido Intracelular/metabolismo , Contração Isométrica/efeitos dos fármacos , Contração Isométrica/fisiologia , Contração Miocárdica/efeitos dos fármacos , Miocárdio/citologia , Coelhos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes de Fusão/farmacologia , Proteínas S100 , Retículo Sarcoplasmático/metabolismo , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático , SuínosRESUMO
Myocardial hypertrophy is characterized by abnormal intracellular Ca2+ handling and decreased contractile performance. Ca2+/calmodulin-dependent protein kinase II (CaMKII) phosphorylates numerous Ca2+ handling proteins and thus can regulate intracellular Ca2+ homeostasis directly. We therefore investigated whether differential expression of CaMKII isoforms occurs with cardiac hypertrophy which might promote an abnormal intracellular Ca2+ homeostasis. We further investigated the potential influence of angiotensin (Ang) II on CaMKII expression levels. Hearts from adult Spontaneously Hypertensive Rats (SHR) and hearts from two transgenic rat models with Ang II-dependent hypertension were studied. The expression of the cardiac CaMKII isoforms delta2, delta3, delta4 and delta9 was determined by RT-PCR and immunoblot methods. Rats transgenic for the mouse Ren-2 gene (mrTGR), SHR and controls were studied at the age of 6 months and rats transgenic for the human renin-angiotensin system (hrTGR) from postnatal day 1 to week 8. SHR and mrTGR had an increased heart/body weight ratio (26 and 25%) compared with controls (p < 0.05). SHR hearts showed significantly increased mRNA levels of delta4 and delta9 (p < 0.05) with no change for delta2 and delta3. mrTGR hearts had a significantly increased delta4 and a significantly decreased delta3 transcript level (p < 0.05) with no change for delta2 and delta9. hrTGR hearts developed severe hypertrophy (42%) after postnatal day 14. The neonatal delta2, delta3 and delta4 isoform expression levels were higher (30-100%) compared with SD controls. The levels decreased with increasing age and equalized to controls at week 8, except for delta4 which started to increase after week 4 (p < 0.05). CaMKIIdelta protein levels of all cardiac hypertrophy models were increased in sarcoplasmic reticulum preparations (50-120%) compared with controls (p < 0.05) while the cytosolic levels remained unchanged. Thus, CaMKIIdelta isoforms are differentially expressed in cardiac hypertrophy. The fetal delta4 isoform was constantly expressed. CaMKIIdelta adopts the fetal phenotype independent of the type of hypertrophic stimulus. The observed alterations of CaMKIIdelta isoform patterns may affect intracellular Ca2+ homeostasis and thus contribute to the abnormal contractile phenotype of cardiac hypertrophy.
Assuntos
Proteínas Quinases Dependentes de Cálcio-Calmodulina/química , Cardiomegalia , Fatores Etários , Angiotensinas/genética , Animais , Animais Geneticamente Modificados , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina , Humanos , Immunoblotting , Masculino , Miocárdio/citologia , Miocárdio/metabolismo , Fenótipo , Isoformas de Proteínas , RNA Mensageiro/metabolismo , Ratos , Ratos Endogâmicos SHR , Ratos Wistar , Renina/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Retículo Sarcoplasmático/metabolismoRESUMO
Recognition of significance of nitric oxide synthases (NOS) in cardiovascular regulations has led to intensive research and development of therapies focused on NOS as potential therapeutic targets. However, the NOS isoform profile of cardiac tissue and subcellular localization of NOS isoforms remain a matter of debate. The aim of this study was to investigate the localization of an inducible NOS isoform (NOS2) in cardiomyocytes. Employing a novel immunocytochemical technique of a catalyzed reporter deposition system with tyramide and electron microscopical immunocytochemistry complemented with Western blotting and RT-PCR, we detected NOS2 both in rat neonatal and adult cultured cardiomyocytes and in the normal myocard of adult rats as well as in the human myocard of patients with dilative cardiomyopathy. NOS2 was targeted predominantly to a particulate component of the cardiomyocyte--along contractile fibers, in the plasma membrane including T-tubules, as well as in the nuclear envelope, mitochondria and Golgi complex. Our results point to an involvement of NOS2 in maintaining cardiac homeostasis and contradict to the notion that NOS2 is expressed in cardiac tissue only in response to various physiological and pathogenic factors. NOS2 targeting to mitochondria and contractile fibers suggests a relationship of NO with contractile function and energy production in the cardiac muscle.
Assuntos
Miocárdio/enzimologia , Óxido Nítrico Sintase/metabolismo , Animais , Animais Recém-Nascidos , Western Blotting , Cardiomiopatia Dilatada/enzimologia , Células Cultivadas , Imunofluorescência , Complexo de Golgi/enzimologia , Humanos , Microscopia Imunoeletrônica , Mitocôndrias Cardíacas/enzimologia , Fibras Musculares Esqueléticas/enzimologia , Miocárdio/ultraestrutura , Óxido Nítrico Sintase Tipo II , Membrana Nuclear/enzimologia , Reação em Cadeia da Polimerase , Ratos , Ratos WistarRESUMO
The site-specific phospholamban phosphorylation was studied with respect to the interplay of cAMP- and Ca(2+)signaling in neonatal rat cardiomyocytes. To elucidate the signal pathway(s) for the activation of Ca(2+)/calmodulin-dependent protein kinase (CaMKII) we studied Thr17 phosphorylation of phospholamban in dependence of Ca(2+)channel activation by S(-)-Bay K8644 and in dependence of the depletion of the sarcoplasmic reticulum Ca(2+)stores by ryanodine or thapsigargin in the absence or presence of beta -adrenergic stimulation. The isoproterenol (0.1 microM)-induced Thr17 phosphorylation was potentiated 2.5-fold in presence of 1 microM S(-)-Bay K8644. Interestingly, S(-)-Bay K8644 alone was also able to induce Thr17 phosphorylation in a dose- and time-dependent fashion. Ryanodine (1.0 microM) reduced both the isoproterenol (0.1 microM) and S(-)-Bay K8644-(1 microM) mediated Thr17 phosphorylation by about 90%. Thapsigargin (1 microM) diminished the S(-)-Bay K8644 and isoproterenol-associated Thr17 phosphorylation by 53.5+/-6.3% and 92. 5+/-11.1%, respectively. Ser16 phosphorylation was not affected under these conditions. KN-93 reduced the Thr17 phosphorylation by S(-)-Bay K8644 and isoproterenol to levels of 1.1+/-0.3% and 8.6+/-2. 1%, respectively. However, the effect of KN-93 was attenuated (47. 8+/-3.6%) in isoproterenol prestimulated cells. Protein phosphatase inhibition by okadaic acid increased exclusively the Ser16 phosphorylation. In summary, our results reflect a cross-talk between beta -adrenoceptor stimulation and intracellular Ca(2+)at the level of CaMKII-mediated phospholamban phosphorylation in neonatal rat cardiomyocytes. We report conditions which exclusively produce Thr17 or Ser16 phosphorylation. We postulate that Ca(2+)transport systems of the sarcoplasmic reticulum are critical determinants for the activation of CaMKII that catalyzes phosphorylation of phospholamban.
Assuntos
Agonistas Adrenérgicos beta/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Miocárdio/citologia , Treonina/metabolismo , Éster Metílico do Ácido 3-Piridinacarboxílico, 1,4-Di-Hidro-2,6-Dimetil-5-Nitro-4-(2-(Trifluormetil)fenil)/farmacologia , Animais , Animais Recém-Nascidos , Western Blotting , Cálcio/metabolismo , Agonistas dos Canais de Cálcio/farmacologia , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Células Cultivadas , AMP Cíclico/metabolismo , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/farmacologia , Ionóforos/farmacologia , Isoproterenol/farmacologia , Ácido Okadáico/farmacologia , Fosforilação , Ratos , Ratos Wistar , Rianodina/farmacologia , Retículo Sarcoplasmático/metabolismo , Serina/metabolismo , Transdução de Sinais , Tapsigargina/farmacologia , Fatores de TempoRESUMO
BACKGROUND: In adult human heart, both beta(1)- and beta(2)-adrenergic receptors mediate hastening of relaxation; however, it is unknown whether this also occurs in infant heart. We compared the effects of stimulation of beta(1)- and beta(2)-adrenergic receptors on relaxation and phosphorylation of phospholamban and troponin I in ventricle obtained from infants with tetralogy of Fallot. METHODS AND RESULTS: Myocardium dissected from the right ventricular outflow tract of 27 infants (age range 21/2 to 35 months) with tetralogy of Fallot was set up to contract 60 times per minute. Selective stimulation of beta(1)-adrenergic receptors with (-)-norepinephrine (NE) and beta(2)-adrenergic receptors with (-)-epinephrine (EPI) evoked phosphorylation of phospholamban (at serine-16 and threonine-17) and troponin I and caused concentration-dependent increases in contractile force (-log EC(50) [mol/L] NE 5.5+/-0.1, n=12; EPI 5.6+/-0.1, n=13 patients), hastening of the time to reach peak force (-log EC(50) [mol/L] NE 5.8+/-0.2; EPI 5.8+/-0.2) and 50% relaxation (-log EC(50) [mol/L] NE 5.7+/-0.2; EPI 5.8+/-0.1). Ventricular membranes from Fallot infants, labeled with (-)-[(125)I]-cyanopindolol, revealed a greater percentage of beta(1)- (71%) than beta(2)-adrenergic receptors (29%). Binding of (-)-epinephrine to beta(2)-receptors underwent greater GTP shifts than binding of (-)-norepinephrine to beta(1)-receptors. CONCLUSIONS: Despite their low density, beta(2)-adrenergic receptors are nearly as effective as beta(1)-adrenergic receptors of infant Fallot ventricle in enhancing contraction, relaxation, and phosphorylation of phospholamban and troponin I, consistent with selective coupling to G(s)-protein.
Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Subunidades alfa Gs de Proteínas de Ligação ao GTP/metabolismo , Miocárdio/metabolismo , Receptores Adrenérgicos beta 1/metabolismo , Receptores Adrenérgicos beta 2/metabolismo , Tetralogia de Fallot/metabolismo , Troponina I/metabolismo , Pré-Escolar , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Epinefrina/metabolismo , Feminino , Guanosina Trifosfato/metabolismo , Ventrículos do Coração/citologia , Ventrículos do Coração/fisiopatologia , Humanos , Lactente , Masculino , Contração Miocárdica , Miocárdio/patologia , Fosforilação , Serina/metabolismo , Treonina/metabolismoRESUMO
delta-isoforms of the Ca(2+)/calmodulin-dependent protein kinase type II (CaMKII) are considered to substantially influence cardiac functions. However, no data exist on the expression of these isoforms in the mouse heart. We analyzed the transcript pattern of non-neuronally expressed delta-isoforms in heart and skeletal muscle of adult mice by RT-PCR. For members of the delta-CaMKII subclass with both variable domains (subclass II), weak transcriptional expression of isoforms delta(2) and delta(3) was found in the heart. In skeletal muscle no delta(3)-specific transcript was detectable. In cardiac tissue, stronger signals result from amplifications of delta(9) and from members of the subclass I lacking the second variable domain. Western blotting was performed using a subclass II-specific antibody. In murine cardiac and skeletal muscle tissue a delta-CaMKII protein pattern was obtained similar to that described for rat. To gain insight into the expression of delta-CaMKII during the earliest steps of cardiogenic differentiation, we analyzed the transcript pattern of murine embryonic stem cell-derived cardiomyocytes in various differentiation stages. Reproducible RT-PCR signals could be obtained for delta(6) and delta(10), both belonging to the delta-CaMKII subclass I. Transcripts for delta(6) were ubiquitously expressed, whereas transcripts for delta(10) were detectable in increasing amounts after 7-10 days of the onset of cardiogenic differentiation. Our results point to a differentiation-dependent expression of the two delta-CaMKII subclasses, and also to differences in the expression of individual members of subclass I during the early stages of cardiogenic differentiation.
Assuntos
Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Diferenciação Celular , Regulação da Expressão Gênica no Desenvolvimento , Regulação Enzimológica da Expressão Gênica , Miocárdio/enzimologia , Células-Tronco/citologia , Animais , Sequência de Bases , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina , Proteínas Quinases Dependentes de Cálcio-Calmodulina/genética , Primers do DNA , Embrião de Mamíferos/citologia , Camundongos , Camundongos Endogâmicos , Músculo Esquelético/enzimologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Ca(2+)/calmodulin-dependent protein kinases II (CaMKII) have important functions in regulating cardiac excitability and contractility. In the present study, we examined whether CaMKII regulated the transient outward K(+) current (I(to)) in whole-cell patch-clamped human atrial myocytes. We found that a specific CaMKII inhibitor, KN-93 (20 micromol/L), but not its inactive analog, KN-92, accelerated the inactivation of I(to) (tau(fast): 66.9+/-4.4 versus 43.0+/-4.4 ms, n=35; P<0.0001) and inhibited its maintained component (at +60 mV, 4.9+/-0.4 versus 2.8+/-0.4 pA/pF, n = 35; P<0. 0001), leading to an increase in the extent of its inactivation. Similar effects were observed by dialyzing cells with a peptide corresponding to CaMKII residues 281 to 309 or with autocamtide-2-related inhibitory peptide and by external application of the calmodulin inhibitor calmidazolium, which also suppressed the effects of KN-93. Furthermore, the phosphatase inhibitor okadaic acid (500 nmol/L) slowed I(to) inactivation, increased I(sus), and inhibited the effects of KN-93. Changes in [Ca(2+)](i) by dialyzing cells with approximately 30 nmol/L Ca(2+) or by using the fast Ca(2+) buffer BAPTA had opposite effects on I(to). In BAPTA-loaded myocytes, I(to) was less sensitive to KN-93. In myocytes from patients in chronic atrial fibrillation, characterized by a prominent I(sus), KN-93 still increased the extent of inactivation of I(to). Western blot analysis of atrial samples showed that delta-CaMKII expression was enhanced during chronic atrial fibrillation. In conclusion, CaMKII control the extent of inactivation of I(to) in human atrial myocytes, a process that could contribute to I(to) alterations observed during chronic atrial fibrillation.
Assuntos
Função Atrial , Proteínas Quinases Dependentes de Cálcio-Calmodulina/fisiologia , Canais de Potássio/fisiologia , Potássio/fisiologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Benzilaminas/farmacologia , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina , Proteínas Quinases Dependentes de Cálcio-Calmodulina/antagonistas & inibidores , Células Cultivadas , Inibidores Enzimáticos/farmacologia , Humanos , Ativação do Canal Iônico/efeitos dos fármacos , Ativação do Canal Iônico/fisiologia , Transporte de Íons/efeitos dos fármacos , Transporte de Íons/fisiologia , Pessoa de Meia-Idade , Bloqueadores dos Canais de Potássio , Transdução de Sinais/fisiologia , Sulfonamidas/farmacologiaRESUMO
In the heart, Ca2+/calmodulin-dependent protein kinase II is critically involved in the regulation of Ca2+ homeostasis. Previously the predominant expression of a subclass of Ca2+/calmodulin-dependent protein kinase II delta-subunit, containing a second variable domain, was demonstrated in cardiac tissue. Here we report on the expression pattern of the non-neuronal members of this delta-subunit subclass, delta 2, delta 3, delta 4, and delta 9 in the developing heart of the rat. By semiquantitative RT-PCR isoform delta 3 was shown to be typically expressed in the heart, whereas delta 4 was expressed in skeletal muscle of adult rat. From embryonic day 14 up to the adult state of rat ventricular muscle, amounts of delta 9 transcripts remained unchanged, transcript levels of isoforms delta 2 and delta 3 were significantly increased, whereas level of delta 4 transcript was significantly decreased. Immunoblotting, using an antibody recognizing specifically those delta-isoforms containing the second variable domain, revealed three separated protein signals at about 59 kDa, 58 kDa, and 56 kDa. The immunoreaction at about 59 kDa, corresponding to the predicted molecular mass of delta 4, was dramatically diminished, whereas a significant increase in the signal at about 58 kDa was assumed to represent an increase in isoform delta 3. The protein signal at about 56 kDa, close to the predicted molecular mass of isoform delta 2, was high in the embryonic heart and significantly decreased after birth. Our data suggest the predominant expression of isoform delta 2 in the embryonic heart, establish delta 3 to be the typical isoform in the adult heart and define the skeletal muscle form delta 4 to be characteristic for fetal and neonatal stages of the heart.
Assuntos
Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Regulação Enzimológica da Expressão Gênica , Isoenzimas/metabolismo , Miocárdio/enzimologia , Animais , Sequência de Bases , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina , Proteínas Quinases Dependentes de Cálcio-Calmodulina/genética , Primers do DNA , Coração/embriologia , Coração/crescimento & desenvolvimento , Isoenzimas/genética , RatosRESUMO
BACKGROUND: Recent studies of beta-adrenergic receptor (beta-AR) subtype signaling in in vitro preparations have raised doubts as to whether the cAMP/protein kinase A (PKA) signaling is activated in the same manner in response to beta2-AR versus beta1-AR stimulation. METHODS AND RESULTS: The present study compared, in the intact dog, the magnitude and characteristics of chronotropic, inotropic, and lusitropic effects of cAMP accumulation, PKA activation, and PKA-dependent phosphorylation of key effector proteins in response to beta-AR subtype stimulation. In addition, many of these parameters and L-type Ca2+ current (ICa) were also measured in single canine ventricular myocytes. The results indicate that although the cAMP/PKA-dependent phosphorylation cascade activated by beta1-AR stimulation could explain the resultant modulation of cardiac function, substantial beta2-AR-mediated chronotropic, inotropic, and lusitropic responses occurred in the absence of PKA activation and phosphorylation of nonsarcolemmal proteins, including phospholamban, troponin I, C protein, and glycogen phosphorylase kinase. However, in single canine myocytes, we found that beta2-AR-stimulated increases in both ICa and contraction were abolished by PKA inhibition. Thus, the beta2-AR-directed cAMP/PKA signaling modulates sarcolemmal L-type Ca2+ channels but does not regulate PKA-dependent phosphorylation of cytoplasmic proteins. CONCLUSIONS: These results indicate that the dissociation of beta2-AR signaling from cAMP regulatory systems is only apparent and that beta2-AR-stimulated cAMP/PKA signaling is uncoupled from phosphorylation of nonsarcolemmal regulatory proteins involved in excitation-contraction coupling.
Assuntos
AMP Cíclico/fisiologia , Coração/efeitos dos fármacos , Contração Miocárdica/efeitos dos fármacos , Miocárdio/metabolismo , Processamento de Proteína Pós-Traducional , Receptores Adrenérgicos beta 2/fisiologia , Sistemas do Segundo Mensageiro/fisiologia , Sulfonamidas , Citoesqueleto de Actina/metabolismo , Adrenérgicos/farmacologia , Animais , Proteínas de Ligação ao Cálcio/metabolismo , Cardiotônicos/farmacologia , AMP Cíclico/análogos & derivados , AMP Cíclico/farmacologia , Proteínas Quinases Dependentes de AMP Cíclico/fisiologia , Cães , Imidazóis/farmacologia , Isoquinolinas/farmacologia , Relaxamento Muscular/efeitos dos fármacos , Norepinefrina/farmacologia , Fosforilase Quinase/metabolismo , Fosforilase a/metabolismo , Fosforilase b/metabolismo , Fosforilação/efeitos dos fármacos , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Receptores Adrenérgicos beta 1/efeitos dos fármacos , Receptores Adrenérgicos beta 1/fisiologia , Receptores Adrenérgicos beta 2/efeitos dos fármacos , Reserpina/farmacologia , Sarcolema/metabolismo , Sistemas do Segundo Mensageiro/efeitos dos fármacos , Tionucleotídeos/farmacologia , Troponina C/metabolismoRESUMO
Phospholamban is a critical regulator of sarcoplasmic reticulum Ca2+-ATPase and myocardial contractility. To determine the extent of cross signaling between Ca2+ and cAMP pathways, we have investigated the beta-adrenergic-induced phosphorylation of Ser16 and Thr17 of phospholamban in perfused rat hearts using antibodies recognizing phospholamban phosphorylated at either position. Isoproterenol caused the dose-dependent phosphorylation of Ser16 and Thr17 with strikingly different half-maximal values (EC50 = 4.5 +/- 1.6 and 28. 2 +/- 1.4 nmol/l, respectively). The phosphorylation of Ser16 induced by isoproterenol, forskolin, or 3-isobutyl-1-methylxanthine correlated to increased cardiac relaxation (r = 0.96), whereas phosphorylation of Thr17 did not. Elevation of extracellular Ca2+ did not induce phosphorylation at Thr17; only in the presence of a submaximal dose of isoproterenol, phosphorylation at Thr17 increased eightfold without additional effects on relaxation rate. Thr17 phosphorylation was partially affected by ryanodine and was completely abolished in the presence of 1 micromol/l verapamil or nifedipine. The data indicate that 1) phosphorylation of phospholamban at Ser16 by cAMP-dependent protein kinase is the main regulator of beta-adrenergic-induced cardiac relaxation definitely preceding Thr17 phosphorylation and 2) the beta-adrenergic-mediated phosphorylation of Thr17 by Ca2+-calmodulin-dependent protein kinase required influx of Ca2+ through the L-type Ca2+ channel.
Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Contração Miocárdica/fisiologia , Receptores Adrenérgicos beta/metabolismo , Agonistas Adrenérgicos beta/farmacologia , Animais , Calcimicina/farmacologia , Cálcio/metabolismo , Cálcio/farmacologia , Canais de Cálcio/fisiologia , Canais de Cálcio Tipo L , Proteínas de Ligação ao Cálcio/química , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , ATPases Transportadoras de Cálcio/metabolismo , AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Relação Dose-Resposta a Droga , Ionóforos/farmacologia , Isoproterenol/farmacologia , Contração Miocárdica/efeitos dos fármacos , Miocárdio/química , Miocárdio/enzimologia , Técnicas de Cultura de Órgãos , Fosforilação , Ratos , Receptores Adrenérgicos beta/fisiologia , Retículo Sarcoplasmático/química , Retículo Sarcoplasmático/enzimologia , Serina/metabolismo , Treonina/metabolismoRESUMO
It is still a matter of debate, whether decreased protein expression of SERCA 2a and phospholamban (PLB), or alterations in the phosphorylation state of PLB are responsible for the reduced SERCA 2a function in failing human myocardium. Thus, in membrane preparations from patients with terminal heart failure due to idiopathic dilated cardiomyopathy (NYHA IV. heart transplants) and control hearts (NF), SERCA 2a activity was measured with an NADH coupled assay with as well as without stimulation with protein kinase A (PKA). The protein expression of SERCA 2a, PLB and calsequestrin as well as the phosphorylation status of PLB (Back-phosphorylation technique: Serine-16-PLB specific antibody) were analysed using Western blotting technique and specific antibodies. In NF, the maximal activity (Vmax) and the Ca(2+)-sensitivity of SERCA 2a activity were significantly higher compared to NYHA IV. Protein expression of SERCA 2a, PLB and calsequestrin were unchanged, whereas both, the phosphorylation status of PLB as well as serine-16-PLB-phosphorylation, were significantly reduced in NYHA IV. After stimulation with PKA only the Ca(2+)-sensitivity, but not Vmax increased concentration-dependently. Therefore, in human myocardium, the Ca(2+)-sensitivity but not the Vmax of SERCA 2a is regulated by cAMP-dependent phosphorylation of phospholamban at position serine-16. Threonine-17-PLB-phosphorylation or direct phosphorylation of SERCA 2a may be candidates for regulation of maximal SERCA 2a activity in human myocardium.
Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , ATPases Transportadoras de Cálcio/metabolismo , Cálcio/metabolismo , Insuficiência Cardíaca/metabolismo , Miocárdio/metabolismo , Adulto , Western Blotting , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Densitometria , Relação Dose-Resposta a Droga , Eletroforese em Gel de Poliacrilamida , Feminino , Humanos , Cinética , Masculino , Pessoa de Meia-Idade , Radioisótopos de Fósforo/metabolismo , FosforilaçãoRESUMO
Despite its importance for the regulation of heart function, little is known about the isoform expression of the multifunctional Ca2+/calmodulin-dependent protein kinase (CaMKII) in human myocardium. In this study, we investigated the spectrum of CaMKII isoforms delta2, delta3, delta4, delta8, and delta9 in human striated muscle tissue. Isoform delta3 is characteristically expressed in cardiac muscle. In skeletal muscle, specific expression of a new isoform termed delta11 is demonstrated. Complete sequencing of human delta2 cDNA, representing all common features of the investigated CaMKII subclass, revealed its high homology to the corresponding rat cDNA. Comparative semiquantitative reverse transcription-polymerase chain reaction analyses from left ventricular tissues of normal hearts and from patients suffering from dilated cardiomyopathy showed a significant increase in transcript levels of isoform delta3 relative to the expression of glyceraldehyde-3-phosphate dehydrogenase in diseased hearts (101. 6+/-11.0% versus 64.9+/-9.9% in the nonfailing group; P<0.05, n=6). Transcript levels of the other investigated cardiac CaMKII isoforms remained unchanged. At the protein level, by using a subclass-specific antibody, we observed a similar increase of a delta-CaMKII-specific signal (7.2+/-1.0 versus 3.8+/-0.7 optical density units in the nonfailing group; P<0.05, n=4 through 6). The diseased state of the failing hearts was confirmed by a significant increase in transcript levels for atrial natriuretic peptide (292. 9+/-76.4% versus 40.1+/-3.2% in the nonfailing group; P<0.05, n=3 through 6). Our data characterize for the first time the delta-CaMKII isoform expression pattern in human hearts and demonstrate changes in this expression pattern in heart failure.
Assuntos
Proteínas Quinases Dependentes de Cálcio-Calmodulina/biossíntese , Proteínas Quinases Dependentes de Cálcio-Calmodulina/química , Cardiomiopatia Dilatada/enzimologia , Miocárdio/enzimologia , Adulto , Sequência de Aminoácidos , Sequência de Bases , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina , Proteínas Quinases Dependentes de Cálcio-Calmodulina/genética , Humanos , Pessoa de Meia-Idade , Dados de Sequência Molecular , Músculo Esquelético/enzimologia , Miocárdio/patologia , Isoformas de Proteínas/biossíntese , Isoformas de Proteínas/genéticaRESUMO
BACKGROUND: Catecholamines hasten cardiac relaxation through beta-adrenergic receptors, presumably by phosphorylation of several proteins, but it is unknown which receptor subtypes are involved in human ventricle. We assessed the role of beta1- and beta2-adrenergic receptors in phosphorylating proteins implicated in ventricular relaxation. METHODS AND RESULTS: Right ventricular trabeculae, obtained from freshly explanted hearts of patients with dilated cardiomyopathy (n=5) or ischemic cardiomyopathy (n=5), were paced at 60 bpm. After measurement of the contractile and relaxant effects of epinephrine (10 micromol/L) or zinterol (10 micromol/L), mediated through beta2-adrenergic receptors, and of norepinephrine (10 micromol/L), mediated through beta1-adrenergic receptors, tissues were freeze clamped. We assessed phosphorylation of phospholamban, troponin I, and C-protein, as well as specific phosphorylation of phospholamban at serine 16 and threonine 17. Data did not differ between the 2 disease groups and were therefore pooled. Epinephrine, zinterol, and norepinephrine increased contractile force to approximately the same extent, hastened the onset of relaxation by 15+/-3%, 5+/-2%, and 20+/-3%, respectively, and reduced the time to half-relaxation by 26+/-3%, 21+/-3%, and 37+/-3%. These effects of epinephrine, zinterol, and norepinephrine were associated with phosphorylation (pmol phosphate/mg protein) of phospholamban 14+/-3, 12+/-4, and 12+/-3; troponin I 40+/-7, 33+/-7, and 31+/-6; and C-protein 7.2+/-1.9, 9.3+/-1.4, and 7.5+/-2.0. Phosphorylation of phospholamban occurred at both Ser16 and Thr17 residues through both beta1- and beta2-adrenergic receptors. CONCLUSIONS: Norepinephrine and epinephrine hasten human ventricular relaxation and promote phosphorylation of implicated proteins through both beta1- and beta2-adrenergic receptors, thereby potentially improving diastolic function.
Assuntos
Agonistas de Receptores Adrenérgicos beta 2 , Agonistas Adrenérgicos beta/uso terapêutico , Cardiomiopatia Dilatada/tratamento farmacológico , Ventrículos do Coração/efeitos dos fármacos , Isquemia Miocárdica/tratamento farmacológico , Vasodilatadores/uso terapêutico , Adolescente , Adulto , Cálcio/farmacologia , Proteínas de Ligação ao Cálcio/metabolismo , Cardiomiopatia Dilatada/metabolismo , Proteínas de Transporte/metabolismo , Epinefrina/uso terapêutico , Feminino , Ventrículos do Coração/metabolismo , Humanos , Masculino , Pessoa de Meia-Idade , Isquemia Miocárdica/metabolismo , Norepinefrina/uso terapêutico , Fosforilação , Assistência Terminal/métodos , Troponina I/metabolismoRESUMO
Forskolin and dibutyryl cyclic adenosine monophosphate (cAMP) stimulate force of contraction independent of beta-adrenoceptor stimulation. We studied their effects on force of contraction and phosphorylation of regulatory proteins in isolated electrically driven trabeculae carneae from failing human ventricles. The phosphorylation state of the regulatory protein phospholamban was studied because its phosphorylation usually faithfully follows contractility. For comparison, the phosphorylation state of the inhibitory subunit of troponin was studied. The phosphorylation state was inferred from in vitro phosphorylation of homogenates with cAMP-dependent protein kinase in the presence of radioactive gamma[32P]ATP Proteins were separated by electrophoresis, and radioactivity in the proteins of interest was quantified. The maximal positive inotropic effects occurred at 30 microM forskolin and were attenuated in comparison with the maximal effects to dibutyryl cAMP (1 mM). Both forskolin and dibutyryl cAMP enhanced phospholamban phosphorylation. However, phospholamban phosphorylation in intact trabeculae treated with 30 microM forskolin and 1 mM dibutyryl cAMP was comparable. It is suggested that phospholamban phosphorylation can be dissociated from inotropy at least in isolated trabeculae from failing human hearts.
Assuntos
Bucladesina/farmacologia , Proteínas de Ligação ao Cálcio/metabolismo , Colforsina/farmacologia , Insuficiência Cardíaca/fisiopatologia , Contração Miocárdica/efeitos dos fármacos , Adolescente , Adulto , Idoso , Cardiotônicos , Feminino , Humanos , Técnicas In Vitro , Masculino , Pessoa de Meia-Idade , FosforilaçãoRESUMO
The intracellular mechanisms underlying the action of the endogenous vasodilators such as NO/EDRF, adenosine, and prostacyclin acting through cGMP and cAMP, respectively, are not well understood. One important action of cyclic nucleotides in smooth muscle relaxation is to lower the cytosolic Ca2+ concentration by enhanced sequestration into the sarcoplasmic reticulum. The present study was undertaken to elucidate the potential role of phosphorylation of phospholamban, the regulator of sarcoplasmic reticulum Ca2+ pump, for the control of coronary vascular tone by NO/EDRF, adenosine, and prostacyclin. Phospholamban was identified in pig coronary artery preparations by immunofluorescence microscopy, Western blotting and in vitro phosphorylation. Segments of pig coronary artery, with either intact or denuded endothelium, were precontracted with prostaglandin F2alpha (PGF2alpha). In endothelium-denuded preparations 3-morpholinosydnonimine (SIN-1), 5'-N-ethylcarboxiamidoadenosine (NECA), and iloprost (ILO) caused both relaxation and phospholamban phosphorylation with the potency: SIN-1 > NECA > ILO. The regulatory myosin light chain was significantly dephosphorylated only by SIN-1. In endothelium-intact pig coronary artery, L-NAME caused additional vasoconstriction and a decrease in phospholamban phosphorylation, while phosphorylation of myosin light chain remained unchanged. An inverse relationship between phospholamban phosphorylation and vessel tone was obtained. Our findings demonstrate significant phospholamban phosphorylation during coronary artery relaxation evoked by NO, prostacyclin, and adenosine receptor activation. Because of the close correlation between phosphorylation of phospholamban and vessel relaxation, we propose that phospholamban phosphorylation is an important mechanism by which endogenous vasodilators, especially endothelial NO/EDRF, control coronary vascular smooth muscle tone.
Assuntos
Adenosina/fisiologia , Proteínas de Ligação ao Cálcio/metabolismo , Vasos Coronários/fisiologia , Epoprostenol/fisiologia , Óxido Nítrico/fisiologia , Animais , Artérias/metabolismo , Artérias/fisiologia , Vasos Coronários/metabolismo , Técnicas In Vitro , Relaxamento Muscular/fisiologia , Tono Muscular , Cadeias Leves de Miosina/metabolismo , Fosforilação , SuínosRESUMO
Despite their important role in controlling the cardiac Ca2+ homeostasis, presence and functions of individual isoforms of the multifunctional Ca2+/calmodulin-dependent protein kinase in the heart are not well studied. Here we report on expression of isoforms of the delta class in two differentiation states of the embryonic rat heart-derived cell line H9c2 compared to adult rat heart. Reverse transcription coupled polymerase chain reaction analysis revealed specific expression patterns of four variants of the delta class (delta B, delta C, delta 4, delta 9) in adult rat heart, H9c2 myoblasts, and skeletal muscle-like H9c2 myotubes. delta C was identified as a common isoform with higher amounts in H9c2 cells and the prominent one in myoblasts. In contrast, expression of delta 9 accompanied cardiac as well as skeletal muscle differentiation. Expression of delta B, however, was representative for differentiated cardiac muscle, whereas delta 4 expression coincided with differentiation into the skeletal muscle-like state. Our results demonstrate differentiation-dependent isoform expression of the delta class of the multifunctional Ca2+/calmodulin-dependent protein kinase of muscle. The identification of cardiac target proteins for this kinase, e.g. the alpha 1-subunit of the L-type Ca2+ channel, the sarcoplasmic reticulum Ca(2+)-ATPase, phospholamban and the ryanodine receptor define H9c2 myoblasts as a suitable model system for further functional characterization of the identified cardiac delta isoforms.
Assuntos
Proteínas Quinases Dependentes de Cálcio-Calmodulina/genética , Isoenzimas/genética , Miocárdio/enzimologia , Animais , Diferenciação Celular/genética , Linhagem Celular , Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Regulação Enzimológica da Expressão Gênica , Coração/embriologia , Músculos/citologia , Músculos/embriologia , Músculos/enzimologia , Miocárdio/citologia , RatosRESUMO
We investigated the effects of beta-adrenergic stimulation on the activity of the endogenous cardiac sarcoplasmic reticulum Ca2+/calmodulin-dependent protein kinase (SRCaM kinase) in Langendorff-perfused rat hearts. We found that isoproterenol induced generation of autonomous (Ca2+-independent) SRCaM kinase activity to 28 +/- 4.4% of the total activity. Moreover, dephosphorylation of the autonomous SRCaM kinase with protein phosphatase 2A resulted in an enzyme that was again dependent on Ca2+ and calmodulin for its activity. Activation of SRCaM kinase was coupled to phospholamban phosphorylation and activation of the cAMP-signaling system. Our results suggest that the cardiac SRCaM kinase is activated in response to beta-adrenoceptor stimulation. This activation stimulates autophosphorylation at its regulatory domain and converts it to an active Ca2+-independent species that may be the basis for potentiation of Ca2+ transients in the heart.