RESUMO
BACKGROUND: Two isoforms of Phosphoinositide 3-kinase (PI3K), p110γ and p110δ, are predominantly expressed in leukocytes and represent attractive therapeutic targets for the treatment of allergic asthma. The study aim was to assess the impact of administration of an inhaled PI3Kγδ inhibitor (AZD8154) in a rat model of asthma. METHODS: Firstly, we checked that the tool compound, AZD8154, inhibited rat PI3K γ & δ kinases using rat cell-based assays. Subsequently, a time-course study was conducted in a rat model of asthma to assess PI3K activity in the lung and how it is temporally associated with other key transcription pathways and asthma like features of the model. Finally, the impact on lung dosed AZD8154 on target engagement, pathway specificity, airway inflammation and lung function changes was assessed. RESULTS: Data showed that AZD8154 could inhibit rat PI3K γ & δ isoforms and, in a rat model of allergic asthma the PI3K pathway was activated in the lung. Intratracheal administration of AZD8154 caused a dose related suppression PI3K pathway activation (reduction in pAkt) and unlike after budesonide treatment, STAT and NF-κB pathways were not affected by AZD8154. The suppression of the PI3K pathway led to a marked inhibition of airway inflammation and reduction in changes in lung function. CONCLUSION: These data show that a dual PI3Kγδ inhibitor suppress key features of disease in a rat model of asthma to a similar degree as budesonide and indicate that dual PI3Kγδ inhibition may be an effective treatment for people suffering from allergic asthma.
Assuntos
Asma , Modelos Animais de Doenças , Animais , Asma/tratamento farmacológico , Asma/metabolismo , Ratos , Masculino , Classe Ib de Fosfatidilinositol 3-Quinase/metabolismo , Ratos Sprague-Dawley , Inibidores de Fosfoinositídeo-3 Quinase/farmacologia , Inibidores de Fosfoinositídeo-3 Quinase/uso terapêutico , Pulmão/efeitos dos fármacos , Pulmão/metabolismo , Pulmão/patologia , Pulmão/enzimologia , Relação Dose-Resposta a Droga , Inibidores de Proteínas Quinases/farmacologia , Classe I de Fosfatidilinositol 3-Quinases/antagonistas & inibidores , Classe I de Fosfatidilinositol 3-Quinases/metabolismo , Antiasmáticos/farmacologia , Ovalbumina/toxicidadeRESUMO
Cardiac hypertrophy is necessary for the heart to accommodate an increase in workload. Physiological, compensated hypertrophy (e.g. with exercise) is reversible and largely due to cardiomyocyte hypertrophy. Pathological hypertrophy (e.g. with hypertension) is associated with additional features including increased fibrosis and can lead to heart failure. RAF kinases (ARAF/BRAF/RAF1) integrate signals into the extracellular signal-regulated kinase 1/2 cascade, a pathway implicated in cardiac hypertrophy, and activation of BRAF in cardiomyocytes promotes compensated hypertrophy. Here, we used mice with tamoxifen-inducible cardiomyocyte-specific BRAF knockout (CM-BRAFKO) to assess the role of BRAF in hypertension-associated cardiac hypertrophy induced by angiotensin II (AngII; 0.8 mg/kg/d, 7 d) and physiological hypertrophy induced by phenylephrine (40 mg/kg/d, 7 d). Cardiac dimensions/functions were measured by echocardiography with histological assessment of cellular changes. AngII promoted cardiomyocyte hypertrophy and increased fibrosis within the myocardium (interstitial) and around the arterioles (perivascular) in male mice; cardiomyocyte hypertrophy and interstitial (but not perivascular) fibrosis were inhibited in mice with CM-BRAFKO. Phenylephrine had a limited effect on fibrosis but promoted cardiomyocyte hypertrophy and increased contractility in male mice; cardiomyocyte hypertrophy was unaffected in mice with CM-BRAFKO, but the increase in contractility was suppressed and fibrosis increased. Phenylephrine induced a modest hypertrophic response in female mice and, in contrast with the males, tamoxifen-induced loss of cardiomyocyte BRAF reduced cardiomyocyte size, had no effect on fibrosis and increased contractility. The data identify BRAF as a key signalling intermediate in both physiological and pathological hypertrophy in male mice, and highlight the need for independent assessment of gene function in females.
Assuntos
Hipertensão , Miócitos Cardíacos , Feminino , Masculino , Camundongos , Animais , Proteínas Proto-Oncogênicas B-raf/genética , Fenilefrina , Tamoxifeno/farmacologia , Cardiomegalia/genética , FibroseRESUMO
The extracellular signal-regulated kinase 1/2 (ERK1/2) cascade promotes cardiomyocyte hypertrophy and is cardioprotective, with the three RAF kinases forming a node for signal integration. Our aims were to determine if BRAF is relevant for human heart failure, whether BRAF promotes cardiomyocyte hypertrophy, and if Type 1 RAF inhibitors developed for cancer (that paradoxically activate ERK1/2 at low concentrations: the 'RAF paradox') may have the same effect. BRAF was up-regulated in heart samples from patients with heart failure compared with normal controls. We assessed the effects of activated BRAF in the heart using mice with tamoxifen-activated Cre for cardiomyocyte-specific knock-in of the activating V600E mutation into the endogenous gene. We used echocardiography to measure cardiac dimensions/function. Cardiomyocyte BRAFV600E induced cardiac hypertrophy within 10â d, resulting in increased ejection fraction and fractional shortening over 6 weeks. This was associated with increased cardiomyocyte size without significant fibrosis, consistent with compensated hypertrophy. The experimental Type 1 RAF inhibitor, SB590885, and/or encorafenib (a RAF inhibitor used clinically) increased ERK1/2 phosphorylation in cardiomyocytes, and promoted hypertrophy, consistent with a 'RAF paradox' effect. Both promoted cardiac hypertrophy in mouse hearts in vivo, with increased cardiomyocyte size and no overt fibrosis. In conclusion, BRAF potentially plays an important role in human failing hearts, activation of BRAF is sufficient to induce hypertrophy, and Type 1 RAF inhibitors promote hypertrophy via the 'RAF paradox'. Cardiac hypertrophy resulting from these interventions was not associated with pathological features, suggesting that Type 1 RAF inhibitors may be useful to boost cardiomyocyte function.
Assuntos
Cardiomegalia/patologia , Sistema de Sinalização das MAP Quinases/fisiologia , Miócitos Cardíacos/patologia , Proteínas Proto-Oncogênicas B-raf/fisiologia , Animais , Carbamatos/farmacologia , Carbamatos/toxicidade , Cardiomegalia/metabolismo , Tamanho Celular/efeitos dos fármacos , Células Cultivadas , Dimerização , Técnicas de Introdução de Genes , Insuficiência Cardíaca/patologia , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mutação de Sentido Incorreto , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Mutação Puntual , Conformação Proteica/efeitos dos fármacos , Mapeamento de Interação de Proteínas , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas c-raf/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-raf/biossíntese , Ratos , Ratos Sprague-Dawley , Sulfonamidas/farmacologia , Sulfonamidas/toxicidadeRESUMO
Raf kinases signal via extracellular signal-regulated kinases 1/2 (ERK1/2) to drive cell division. Since activating mutations in BRAF (B-Raf proto-oncogene, serine/threonine kinase) are highly oncogenic, BRAF inhibitors including dabrafenib have been developed for cancer. Inhibitors of ERK1/2 signalling used for cancer are cardiotoxic in some patients, raising the question of whether dabrafenib is cardiotoxic. In the heart, ERK1/2 signalling promotes not only cardiomyocyte hypertrophy and is cardioprotective but also promotes fibrosis. Our hypothesis is that ERK1/2 signalling is not required in a non-stressed heart but is required for cardiac remodelling. Thus, dabrafenib may affect the heart in the context of, for example, hypertension. In experiments with cardiomyocytes, cardiac fibroblasts and perfused rat hearts, dabrafenib inhibited ERK1/2 signalling. We assessed the effects of dabrafenib (3 mg/kg/d) on male C57BL/6J mouse hearts in vivo. Dabrafenib alone had no overt effects on cardiac function/dimensions (assessed by echocardiography) or cardiac architecture. In mice treated with 0.8 mg/kg/d angiotensin II (AngII) to induce hypertension, dabrafenib inhibited ERK1/2 signalling and suppressed cardiac hypertrophy in both acute (up to 7 d) and chronic (28 d) settings, preserving ejection fraction. At the cellular level, dabrafenib inhibited AngII-induced cardiomyocyte hypertrophy, reduced expression of hypertrophic gene markers and almost completely eliminated the increase in cardiac fibrosis both in interstitial and perivascular regions. Dabrafenib is not overtly cardiotoxic. Moreover, it inhibits maladaptive hypertrophy resulting from AngII-induced hypertension. Thus, Raf is a potential therapeutic target for hypertensive heart disease and drugs such as dabrafenib, developed for cancer, may be used for this purpose.
Assuntos
Antineoplásicos/farmacologia , Fibrose/tratamento farmacológico , Hipertensão/tratamento farmacológico , Imidazóis/farmacologia , Oximas/farmacologia , Animais , Cardiomegalia/fisiopatologia , Modelos Animais de Doenças , Hipertensão/fisiopatologia , Camundongos Endogâmicos C57BL , Miocárdio/patologia , Miócitos Cardíacos/metabolismo , Ratos , Transdução de Sinais/efeitos dos fármacos , Remodelação Ventricular/efeitos dos fármacosRESUMO
Reactive oxygen species (ROS) play a key role in development of heart failure but, at a cellular level, their effects range from cytoprotection to induction of cell death. Understanding how this is regulated is crucial to develop novel strategies to ameliorate only the detrimental effects. Here, we revisited the fundamental hypothesis that the level of ROS per se is a key factor in the cellular response by applying different concentrations of H2O2 to cardiomyocytes. High concentrations rapidly reduced intracellular ATP and inhibited protein synthesis. This was associated with activation of AMPK which phosphorylated and inhibited Raptor, a crucial component of mTOR complex-1 that regulates protein synthesis. Inhibition of protein synthesis by high concentrations of H2O2 prevents synthesis of immediate early gene products required for downstream gene expression, and such mRNAs (many encoding proteins required to deal with oxidant stress) were only induced by lower concentrations. Lower concentrations of H2O2 promoted mTOR phosphorylation, associated with differential recruitment of some mRNAs to the polysomes for translation. Some of the upregulated genes induced by low H2O2 levels are cytoprotective. We identified p21Cip1/WAF1 as one such protein, and preventing its upregulation enhanced the rate of cardiomyocyte apoptosis. The data support the concept of a "redox rheostat" in which different degrees of ROS influence cell energetics and intracellular signalling pathways to regulate mRNA and protein expression. This sliding scale determines cell fate, modulating survival vs death.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Apoptose , Regulação da Expressão Gênica , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , Transdução de Sinais , Serina-Treonina Quinases TOR/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Animais Recém-Nascidos , Apoptose/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Citoproteção/efeitos dos fármacos , Doxorrubicina/farmacologia , Ativação Enzimática/efeitos dos fármacos , Genes Precoces , Peróxido de Hidrogênio/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Miócitos Cardíacos/efeitos dos fármacos , Oxirredução , Fosforilação/efeitos dos fármacos , Polirribossomos/metabolismo , Biossíntese de Proteínas/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos Sprague-Dawley , Transdução de Sinais/efeitos dos fármacos , Estresse Fisiológico/efeitos dos fármacosRESUMO
OBJECTIVES: Potassium-based depolarizing St Thomas' Hospital cardioplegic solution No 2 administered as intermittent, oxygenated blood is considered as a gold standard for myocardial protection during cardiac surgery. However, the alternative concept of polarizing arrest may have beneficial protective effects. We hypothesize that polarized arrest with esmolol/adenosine/magnesium (St Thomas' Hospital Polarizing cardioplegic solution) in cold, intermittent oxygenated blood offers comparable myocardial protection in a clinically relevant animal model. METHODS: Twenty anaesthetized young pigs, 42 ± 2 (standard deviation) kg on standardized tepid cardiopulmonary bypass (CPB) were randomized (10 per group) to depolarizing or polarizing cardiac arrest for 60 min with cardioplegia administered in the aortic root every 20 min as freshly mixed cold, intermittent, oxygenated blood. Global and local baseline and postoperative cardiac function 60, 120 and 180 min after myocardial reperfusion was evaluated with pressure-conductance catheter and strain by Tissue Doppler Imaging. Regional tissue blood flow, cleaved caspase-3 activity, GRK2 phosphorylation and mitochondrial function and ultrastructure were evaluated in myocardial tissue samples. RESULTS: Left ventricular function and general haemodynamics did not differ between groups before CPB. Cardiac asystole was obtained and maintained during aortic cross-clamping. Compared with baseline, heart rate was increased and left ventricular end-systolic and end-diastolic pressures decreased in both groups after weaning. Cardiac index, systolic pressure and radial peak systolic strain did not differ between groups. Contractility, evaluated as dP/dtmax, gradually increased from 120 to 180 min after declamping in animals with polarizing cardioplegia and was significantly higher, 1871 ± 160 (standard error) mmHg/s, compared with standard potassium-based cardioplegic arrest, 1351 ± 70 mmHg/s, after 180 min of reperfusion (P = 0.008). Radial peak ejection strain rate increased and the load-independent variable preload recruitable stroke work was increased with polarizing cardioplegia after 180 min, 64 ± 3 vs 54 ± 2 mmHg (P = 0.018), indicating better preserved left ventricular contractility with polarizing cardioplegia. Phosphorylation of GRK2 in myocardial tissue did not differ between groups. Fractional cytoplasmic volume in myocytes was reduced in hearts arrested with polarizing cardioplegia, indicating reduction of cytoplasmic oedema. CONCLUSIONS: Polarizing oxygenated blood cardioplegia with esmolol/adenosine/magnesium offers comparable myocardial protection and improves contractility compared with the standard potassium-based depolarizing blood cardioplegia.
Assuntos
Ponte Cardiopulmonar/métodos , Parada Cardíaca Induzida/métodos , Parada Cardíaca/cirurgia , Animais , Modelos Animais de Doenças , SuínosRESUMO
OBJECTIVES: Lung deflation and inflation during cardiac surgery with cardiopulmonary bypass contributes to pulmonary dysfunction postoperatively. Theophylline treatment for lung diseases has traditionally been thought to act by phosphodiesterase inhibition; however, increasing evidence has suggested other plausible mechanisms. We investigated the effects of deflation and reinflation on signaling pathways (p38-mitogen-activated protein kinase [MAPK], extracellular signal-regulated kinase 1 and 2 [ERK1/2], and Akt) and whether theophylline influences the deflation-induced lung injury and associated signaling. METHODS: Isolated rat lungs were perfused (15 mL/min) with deoxygenated rat blood in bicarbonate buffer and ventilated. After 20 minutes' equilibration, the lungs were deflated (60 minutes, aerobic perfusion 1.5 mL/min), followed by reinflation (60 minutes, anaerobic reperfusion 15 mL/min). Compliance, vascular resistance, and kinase phosphorylation were assessed during deflation and reinflation. The effects of SB203580 (50 µM), a p38-MAPK inhibitor, and theophylline (0.083 mM [therapeutic] or 3 mM [supratherapeutic]) on physiology and signaling were studied. RESULTS: Deflation reduced compliance by 44% compared with continuously ventilated lungs. p38-MAPK and Akt phosphorylation increased (three to fivefold) during deflation and reinflation, and ERK1/2 phosphorylation increased (approximately twofold) during reinflation. SB203580 had no effect on lung physiology or ERK1/2 and Akt activation. Both theophylline doses increased cyclic adenosine monophosphate, but only 3 mM theophylline improved compliance. p38-MAPK phosphorylation was not affected by theophylline; 0.083 mM theophylline inhibited reinflation-induced ERK1/2 phosphorylation (72%±3%); and 3 mM theophylline inhibited Akt phosphorylation during deflation (75%±5%) and reinflation (87%±4%). CONCLUSIONS: Lung deflation and reinflation stimulates differential p38-MAPK, ERK1/2, and Akt activation, suggesting a role in lung injury during cardiopulmonary bypass. However, p38-MAPK was not involved in the compromised compliance. A supratherapeutic theophylline dose protected lungs against deflation-induced injury and was associated with inhibition of phosphoinositide 3-kinase/Akt rather than phosphodiesterase.
Assuntos
Ponte Cardiopulmonar/efeitos adversos , Lesão Pulmonar/etiologia , Pulmão/enzimologia , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Perfusão/métodos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Animais , Citoproteção , Ativação Enzimática , Técnicas In Vitro , Pulmão/efeitos dos fármacos , Pulmão/patologia , Pulmão/fisiopatologia , Complacência Pulmonar , Lesão Pulmonar/enzimologia , Lesão Pulmonar/patologia , Lesão Pulmonar/fisiopatologia , Lesão Pulmonar/prevenção & controle , Masculino , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , Inibidores de Fosfodiesterase/farmacologia , Fosforilação , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Ratos Wistar , Transdução de Sinais/efeitos dos fármacos , Teofilina/farmacologia , Fatores de Tempo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
In response to pathophysiological stresses, cardiac myocytes undergo hypertrophic growth or apoptosis. Multiple signalling pathways have been implicated in these responses and among them, kinases such as mitogen-activated protein kinases (MAPKs) and Akt. However, the distinction between signalling pathways originally believed to be specific for either hypertrophy, apoptosis or cell survival is fading. The existing data, coming from different experimental systems, often are conflicting. In this study, we sought to compare aspects of intracellular signalling activated by diverse stimuli in a single experimental system, adult rat cardiac myocytes. Furthermore, we assessed the role of these stimuli in eliciting a particular cell phenotype, i.e. whether they promote hypertrophy, cell survival or apoptosis. The results demonstrate that the hypertrophic agonist phenylephrine is the most potent activator of MAPKs/mitogen and stress- activated kinase MSK1, although its effect on Akt phosphorylation is relatively minor. The pro-apoptotic concentration of H2O2 activates strongly both MAPKs and PI3K/Akt pathways. Insulin-like growth factor-1 has a minimal effect on phosphorylation of MAPKs/MSK1, but it is a potent activator of Akt. In conclusion, hypertrophic, pro-survival or apoptotic stimuli operate through the same signalling pathways with different time course and amplitude of kinase activation. Thus, to determine the effect of different stimuli on cell fate, it is important to assess signalling pathways as a network and not as a single pathway.
Assuntos
Peróxido de Hidrogênio/farmacologia , Fator de Crescimento Insulin-Like I/farmacologia , Miócitos Cardíacos/efeitos dos fármacos , Transdução de Sinais , Animais , Apoptose/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Miócitos Cardíacos/metabolismo , Fenilefrina/farmacologia , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Levosimendan is a cardiovascular drug for the treatment of acute and decompensated heart failure. The current weight of evidence on the cardioprotective effects of levosimendan originates from whole heart models and there is no information on the mechanism whereby signalling pathways are activated. In the present study, we investigated the effect of levosimendan on ischaemia/reperfusion injury and the underlying mechanism in cardiac myocytes. Pretreatment with levosimendan reversed the effects of ischaemia and ischaemia/reperfusion on cell viability and enhanced phosphorylation of Akt, p38-mitogen activated protein kinase (MAPK) and extracellular signal-regulated kinases 1/2 (ERK1/2). Inhibitors of these kinases and the blocker of the mitochondrial K(ATP) channels, 5-hydroxydecanoate, completely abolished the protection afforded by levosimendan. Levosimendan stimulated the phosphorylation of Akt, ERK1/2 and p38-MAPK with different kinetics and the activation of these pathways was dependent on the opening of the mitochondrial K(ATP) channels and the production of oxygen free radicals. The levosimendan-induced phosphorylation of ERK1/2 and Akt was reduced by inhibitors of epidermal growth factor receptor and Src. On the other hand, inhibition of the protein kinase A (PKA) pathway reduced phosphorylation of p38-MAPK. Furthermore, p38-MAPK was activated when a phosphodiesterase inhibitor or a selective PKA activator was used. Overall, our results suggest that levosimendan regulates the wiring of the natural salvaging pathways to execute the prosurvival signals. This network includes Akt, ERK1/2 and p38-MAPK. Opening of mitochondrial K(ATP) channels and the subsequent production of oxygen free radicals, the epidermal growth factor receptor/Src, and the cAMP/PKA pathways seem to mediate this response.
Assuntos
Cardiotônicos/farmacologia , Citoproteção/efeitos dos fármacos , Hidrazonas/farmacologia , Miócitos Cardíacos/citologia , Miócitos Cardíacos/efeitos dos fármacos , Piridazinas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Animais , Masculino , Proteína Quinase 1 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Oxigênio/metabolismo , Fosforilação/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos , Ratos Wistar , Traumatismo por Reperfusão/metabolismo , Traumatismo por Reperfusão/patologia , Simendana , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Oxidative stress promotes cardiac myocyte death and has been implicated in the pathogenesis of many cardiovascular diseases. Bcl-2 family proteins are key regulators of the apoptotic response, while their functions can be regulated by post-transcriptional modifications including phosphorylation, dimerization or proteolytic cleavage. This study used adult cardiac myocytes to test the hypothesis that activation of specific kinase signalling pathways by oxidative stress may modulate either the expression or the phosphorylation of Bcl-2, with the resulting effect of a decrease or increase in its anti-apoptotic function. Stimulation of cardiac myocytes with 0.2 mM H(2)O(2), which induces apoptosis, resulted in a marked down-regulation of Bcl-2 protein simultaneously with an increase in its phosphorylation. Inhibition of p38-MAPK resulted in attenuation of Bcl-2 phosphorylation, whereas inhibition of ERK1/2, JNKs or PI-3-K had no effect. These data suggest that activation of p38 MAPK by oxidative stress results in the phosphorylation and degradation of Bcl-2 and the inactivation of its anti-apoptotic activity.
Assuntos
Apoptose , Miócitos Cardíacos/metabolismo , Estresse Oxidativo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Animais , Apoptose/efeitos dos fármacos , Células Cultivadas , Relação Dose-Resposta a Droga , Ativação Enzimática , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Peróxido de Hidrogênio/toxicidade , Imidazóis/farmacologia , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Masculino , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/enzimologia , Miócitos Cardíacos/patologia , Oxidantes/toxicidade , Estresse Oxidativo/efeitos dos fármacos , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-bcl-2/genética , Piridinas/farmacologia , RNA Mensageiro/metabolismo , Ratos , Ratos Wistar , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Gq-protein-coupled receptor (GqPCR) signalling is associated with the induction of cardiac myocyte hypertrophy, which is characterized by an increase in expression of immediate early genes via activation of pre-existing transcription factors. Here, we explore the role of MSK1 and MAPK signalling pathways in the regulation of the immediate early gene c-jun. The results provide further support for the role of MSK1 in cardiac myocyte hypertrophy and indicate that PE activates distinct signalling mechanisms which culminate with a complex activation of c-jun. ERK1/2 and JNKs are the principal kinases responsible for phosphorylation of c-Jun, whereas c-jun mRNA and protein up-regulation by PE is mediated by multiple signalling pathways that include MSK1, ERK1/2, p38-MAPK and JNKs. These signalling mechanisms seem to be critical to the phenotypic changes of cardiac myocytes in response to hypertrophic stimulation.
Assuntos
Sistema de Sinalização das MAP Quinases/fisiologia , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Miócitos Cardíacos/citologia , Miócitos Cardíacos/enzimologia , Proteínas Proto-Oncogênicas c-jun/metabolismo , Proteínas Quinases S6 Ribossômicas 90-kDa/metabolismo , Animais , Cardiomegalia/enzimologia , Crescimento Celular , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Masculino , Proteína Quinase 1 Ativada por Mitógeno/genética , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/genética , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Miócitos Cardíacos/efeitos dos fármacos , Fenilefrina/farmacologia , Fosforilação , RNA Mensageiro/metabolismo , Ratos , Regulação para CimaRESUMO
The contractile cells in the heart (the cardiac myocytes) are terminally differentiated. In response to pathophysiological stresses, cardiac myocytes undergo hypertrophic growth or apoptosis, responses associated with the development of cardiac pathologies. There has been much effort expended in gaining an understanding of the stimuli which promote these responses, and in identifying the intracellular signaling pathways which are activated and potentially involved. These signaling pathways presumably modulate gene and protein expression to elicit the end-stage response. For the regulation of gene expression, the signal may traverse the cytoplasm to modulate nuclear-localized transcription factors as occurs with the mitogen-activated protein kinase or protein kinase B/Akt cascades. Alternatively, the signal may promote translocation of transcription factors from the cytoplasm to the nucleus as is seen with the calcineurin/NFAT and JAK/STAT systems. We present an overview of the principal signaling pathways implicated in the regulation of gene expression in cardiac myocyte pathophysiology, and summarize the current understanding of these pathways, the transcription factors they regulate and the changes in gene expression associated with the development of cardiac pathologies. Finally, we discuss how intracellular signaling and gene expression may be integrated to elicit the overall change in cellular phenotype.
Assuntos
Cardiomegalia/metabolismo , Expressão Gênica , Miócitos Cardíacos/metabolismo , Proteínas Quinases/metabolismo , Transdução de Sinais/genética , Estresse Fisiológico/metabolismo , Animais , Apoptose/genética , Cardiomegalia/genética , Cardiomegalia/patologia , Cardiomegalia/fisiopatologia , DNA/química , DNA/metabolismo , Humanos , Sistema de Sinalização das MAP Quinases/genética , Miócitos Cardíacos/enzimologia , Miócitos Cardíacos/patologia , Conformação de Ácido Nucleico , Fosfoproteínas Fosfatases/genética , Fosfoproteínas Fosfatases/metabolismo , Proteínas Quinases/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Estresse Fisiológico/complicações , Estresse Fisiológico/genética , Estresse Fisiológico/patologia , Estresse Fisiológico/fisiopatologia , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
cAMP responsive element binding protein (CREB) is a stimulus induced transcription factor with possible relevance for the pathophysiology of the heart. In the present study, we provide evidence that the hypertrophic agonist, phenylephrine (PE), promotes phosphorylation of CREB in adult rat cardiac myocytes through alpha(1)- and beta-adrenergic receptors. PE-induced phosphorylation of CREB was partially inhibited by Ro318220 and H89, which were shown to be potent inhibitors of mitogen- and stress-activated protein kinase-1 (MSK1) activation, implicating the involvement of this kinase in the response. Similar results were obtained when cardiac myocytes were treated with the inhibitors of ERK1/2 and p38 MAPK pathways. In addition, inhibition of protein kinase A by RpcAMP reduced phosphorylation of CREB, suggesting that this pathway is also involved. Furthermore, PE stimulation was accompanied by an increase in CRE-binding activity, which was reduced by drugs that prevented phosphorylation of CREB. An enhanced CBP/phospho-CREB complex formation was also observed, suggesting recruitment of CBP to phosphorylated CREB. These results suggest that PE stimulates phosphorylation and DNA binding activity of CREB in adult rat ventricular myocytes through multiple signaling pathways involving ERK1/2, p38 MAPK, MSK1 and PKA. The same pathways seem to regulate atrial natriuretic peptide (ANF) mRNA expression, a highly conserved marker gene of cardiac hypertrophy, suggesting that the PE-stimulated activation of CREB is likely to play an important role in the hypertrophic response.
Assuntos
Agonistas Adrenérgicos/farmacologia , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/fisiologia , Miócitos Cardíacos/enzimologia , Fenilefrina/farmacologia , Proteínas Quinases S6 Ribossômicas 90-kDa/fisiologia , Agonistas de Receptores Adrenérgicos alfa 1 , Animais , Fator Natriurético Atrial/genética , Proteína de Ligação a CREB , Cardiomegalia/genética , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/imunologia , Proteínas Quinases Dependentes de AMP Cíclico/antagonistas & inibidores , Regulação da Expressão Gênica/efeitos dos fármacos , Imunoprecipitação , Indóis/farmacologia , Isoquinolinas/farmacologia , Proteína Quinase 3 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 3 Ativada por Mitógeno/fisiologia , Miócitos Cardíacos/efeitos dos fármacos , Proteínas Nucleares/imunologia , Proteínas Nucleares/metabolismo , Fosforilação/efeitos dos fármacos , RNA Mensageiro/análise , RNA Mensageiro/metabolismo , Ratos , Receptores Adrenérgicos alfa 1/fisiologia , Receptores Adrenérgicos beta/fisiologia , Proteínas Quinases S6 Ribossômicas 90-kDa/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Sulfonamidas/farmacologia , Transativadores/imunologia , Transativadores/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases p38 Ativadas por Mitógeno/fisiologiaRESUMO
We investigated the activation of mitogen-activated protein kinases (MAPKs) pathways by purinergic stimulation in cardiac myocytes from adult rat hearts. ATPgammaS increased the phosphorylation (activation) of the extracellular signal regulated kinase 1 and 2 (ERK1/2) and p38 MAPK. ERK1/2 and p38 MAPK activation was differential, ERK1/2 being rapid and transient while that of p38 MAPK slow and sustained. Using selective inhibitors, activation of ERK1/2 was shown to involve protein kinase C and MEK1/2 while that of p38 MAPK was regulated by both protein kinase C and protein kinase A. Furthermore, we show that purinergic stimulation induces the phosphorylation of the MAPK downstream target, mitogen- and stress-activated protein kinase 1 (MSK1), in cardiac myocytes. The time course of MSK1 phosphorylation closely follows that of ERK activation. Inhibitors of the ERK and p38 MAPK pathways were tested on the phosphorylation of MSK1 at two different time points. The results suggest that ERKs initiate the response but both ERKs and p38 MAPK are required for the maintenance of the complete phosphorylation of MSK1. The temporal relationship of MSK1 phosphorylation and cPLA2 translocation induced by purinergic stimulation, taken together with previous findings, is an indication that cPLA2 may be a downstream target of MSK1.