RESUMO
We examined the effect of specific and local silencing of sodium/hydrogen exchanger isoform 1 (NHE1) with a small hairpin RNA delivered by lentivirus (L-shNHE1) in the cardiac left ventricle (LV) wall of spontaneously hypertensive rats, to reduce cardiac hypertrophy. Thirty days after the lentivirus was injected, NHE1 protein expression was reduced 53.3 ± 3% in the LV of the L-shNHE1 compared with the control group injected with L-shSCR (NHE1 scrambled sequence), without affecting its expression in other organs, such as liver and lung. Hypertrophic parameters as LV weight-to-body weight and LV weight-to-tibia length ratio were significantly reduced in animals injected with L-shNHE1 (2.32 ± 0.5 and 19.30 ± 0.42 mg/mm, respectively) compared with L-shSCR-injected rats (2.68 ± 0.06 and 21.53 ± 0.64 mg/mm, respectively). Histochemical analysis demonstrated a reduction of cardiomyocytes cross-sectional area in animals treated with L-shNHE1 compared with L-shSCR (309,81 ± 20,86 vs. 424,52 ± 21 µm(2), P < 0.05). Echocardiography at the beginning and at the end of the treatment showed that shNHE1 expression for 30 days induced 9% reduction of LV mass. Also, animals treated with L-shNHE1 exhibited a reduced LV wall thickness without changing LV diastolic dimension and arterial pressure, indicating an increased parietal stress. In addition, midwall shortening was not modified, despite the increased wall tension, suggesting an improvement of cardiac function. Chronic shNHE1 expression in the heart emerges as a possible methodology to reduce pathological cardiac hypertrophy, avoiding potentially undesired effects caused from a body-wide inhibition of NHE1.
Assuntos
Cardiomegalia/genética , Cardiomegalia/patologia , Inativação Gênica/fisiologia , Miocárdio/metabolismo , Trocadores de Sódio-Hidrogênio/genética , Animais , Pressão Arterial/genética , Pressão Arterial/fisiologia , Linhagem Celular , Diástole/genética , Diástole/fisiologia , Ecocardiografia/métodos , Células HEK293 , Ventrículos do Coração/metabolismo , Ventrículos do Coração/patologia , Humanos , Masculino , Miocárdio/patologia , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , RNA Interferente Pequeno/genética , Ratos , Ratos Endogâmicos SHR/genética , Ratos Endogâmicos SHR/fisiologia , Trocador 1 de Sódio-HidrogênioRESUMO
Myocardial stretch triggers an angiotensin II-dependent autocrine/paracrine loop of intracellular signals, leading to reactive oxygen species-mediated activation of redox-sensitive kinases. Based on pharmacological strategies, we previously proposed that mineralocorticoid receptor (MR) is necessary for this stretch-triggered mechanism. Now, we aimed to test the role of MR after stretch by using a molecular approach to avoid secondary effects of pharmacological MR blockers. Small hairpin interference RNA capable of specifically knocking down the MR was incorporated into a lentiviral vector (l-shMR) and injected into the left ventricular wall of Wistar rats. The same vector but expressing a nonsilencing sequence (scramble) was used as control. Lentivirus propagation through the left ventricle was evidenced by confocal microscopy. Myocardial MR expression, stretch-triggered activation of redox-sensitive kinases (ERK1/2-p90(RSK)), the consequent Na(+)/H(+) exchanger-mediated changes in pHi (HEPES-buffer), and its mechanical counterpart, the slow force response, were evaluated. Furthermore, reactive oxygen species production in response to a low concentration of angiotensin II (1.0 nmol/L) or an equipotent concentration of epidermal growth factor (0.1 µg/mL) was compared in myocardial tissue slices from both groups. Compared with scramble, animals transduced with l-shMR showed (1) reduced cardiac MR expression, (2) cancellation of angiotensin II-induced reactive oxygen species production but preservation of epidermal growth factor-induced reactive oxygen species production, (3) cancellation of stretch-triggered increase in ERK1/2-p90(RSK) phosphorylation, (4) lack of stretch-induced Na(+)/H(+) exchanger activation, and (5) abolishment of the slow force response. Our results provide strong evidence that MR activation occurs after myocardial stretch and is a key factor to promote redox-sensitive kinase activation and their downstream consequences.
Assuntos
Miocárdio/metabolismo , Receptores de Mineralocorticoides/metabolismo , Trocadores de Sódio-Hidrogênio/metabolismo , Animais , Vetores Genéticos , Coração/fisiologia , Lentivirus , Masculino , Mitocôndrias/metabolismo , RNA Interferente Pequeno/metabolismo , Ratos , Ratos Wistar , Espécies Reativas de Oxigênio/metabolismo , Trocador 1 de Sódio-HidrogênioRESUMO
The stretch of cardiac muscle increases developed force in two phases. The first phase, which occurs rapidly, constitutes the well-known Frank-Starling mechanism and it is generally attributed to enhanced myofilament responsiveness to Ca(2+). The second phase or slow force response (SFR) occurs gradually and is due to an increase in the calcium transient amplitude as a result of a stretch-triggered autocrine/paracrine mechanism. We previously showed that Ca(2+) entry through reverse Na(+)/Ca(2+) exchange underlies the SFR, as the final step of an autocrine/paracrine cascade involving release of angiotensin II/endothelin, and a Na(+)/H(+) exchanger (NHE-1) activation-mediated rise in Na+. In the present review we mainly focus on our three latest contributions to the understanding of this signalling pathway triggered by myocardial stretch: 1) The finding that an increased production of reactive oxygen species (ROS) from mitochondrial origin is critical in the activation of the NHE-1 and therefore in the genesis of the SFR; 2) the demonstration of a key role played by the transactivation of the epidermal growth factor receptor; and 3) the involvement of mineralocorticoid receptors (MR) activation in the stretch-triggered cascade leading to the SFR. Among these novel contributions, the critical role played by the MR is perhaps the most important one. This finding may conceivably provide a mechanistic explanation to the recently discovered strikingly beneficial effects of MR antagonism in humans with cardiac hypertrophy and failure.
Assuntos
Comunicação Autócrina/fisiologia , Contração Miocárdica/fisiologia , Miocárdio/metabolismo , Comunicação Parácrina/fisiologia , Receptores de Mineralocorticoides/metabolismo , Angiotensina II/metabolismo , Angiotensina II/fisiologia , Cálcio/metabolismo , Cardiomegalia/metabolismo , Doenças Cardiovasculares/fisiopatologia , Insuficiência Cardíaca/metabolismo , Insuficiência Cardíaca/prevenção & controle , Humanos , Espécies Reativas de Oxigênio/metabolismo , Trocador de Sódio e Cálcio/metabolismo , Trocadores de Sódio-Hidrogênio/metabolismo , Hormônios Tireóideos/metabolismoRESUMO
Myocardial stretch elicits a rapid increase in developed force, which is mainly caused by an increase in myofilament calcium sensitivity (Frank-Starling mechanism). Over the ensuing 10-15 min, a second gradual increase in force takes place. This slow force response to stretch is known to be the result of an increase in the calcium transient amplitude and constitutes the in vitro equivalent of the Anrep effect described 100 years ago in the intact heart. In the present review, we will update and discuss what is known about the Anrep effect as the mechanical counterpart of autocrine/paracrine mechanisms involved in its genesis. The chain of events triggered by myocardial stretch comprises 1) release of angiotensin II, 2) release of endothelin, 3) activation of the mineralocorticoid receptor, 4) transactivation of the epidermal growth factor receptor, 5) increased formation of mitochondria reactive oxygen species, 6) activation of redox-sensitive kinases upstream myocardial Na(+)/H(+) exchanger (NHE1), 7) NHE1 activation, 8) increase in intracellular Na(+) concentration, and 9) increase in Ca(2+) transient amplitude through the Na(+)/Ca(2+) exchanger. We will present the experimental evidence supporting each of the signaling steps leading to the Anrep effect and its blunting by silencing NHE1 expression with a specific small hairpin interference RNA injected into the ventricular wall.
Assuntos
Acoplamento Excitação-Contração , Mecanorreceptores/metabolismo , Mecanotransdução Celular , Modelos Cardiovasculares , Força Muscular , Contração Miocárdica , Miocárdio/metabolismo , Reflexo de Estiramento , Animais , Comunicação Autócrina , Cálcio/metabolismo , Proteínas de Transporte de Cátions/genética , Proteínas de Transporte de Cátions/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Comunicação Parácrina , Interferência de RNA , Trocador 1 de Sódio-Hidrogênio , Trocadores de Sódio-Hidrogênio/genética , Trocadores de Sódio-Hidrogênio/metabolismoRESUMO
The increase in myocardial reactive oxygen species after epidermal growth factor receptor transactivation is a crucial step in the autocrine/paracrine angiotensin II/endothelin receptor activation leading to the slow force response to stretch (SFR). Since experimental evidence suggests a link between angiotensin II or its AT1 receptor and the mineralocorticoid receptor (MR), and MR transactivates the epidermal growth factor receptor, we thought to determine whether MR activation participates in the SFR development in rat myocardium. We show here that MR activation is necessary to promote reactive oxygen species formation by a physiological concentration of angiotensin II (1 nmol l(-1)), since an increase in superoxide anion formation of ~50% of basal was suppressed by blocking MR with spironolactone or eplerenone. This effect was also suppressed by blocking AT1, endothelin (type A) or epidermal growth factor receptors, by inhibiting NADPH oxydase or by targeting mitochondria, and was unaffected by glucocorticoid receptor inhibition. All interventions except AT1 receptor blockade blunted the increase in superoxide anion promoted by an equipotent dose of endothelin-1 (1 nmol l(-1)) confirming that endothelin receptors activation is downstream of AT1. Similarly, an increase in superoxide anion promoted by an equipotent dose of aldosterone (10 nmol l(-1)) was blocked by spironolactone or eplerenone, by preventing epidermal growth factor receptor transactivation, but not by inhibiting glucocorticoid receptors or protein synthesis, suggesting non-genomic MR effects. Combination of aldosterone plus endothelin-1 did not increase superoxide anion formation more than each agonist separately. We found that aldosterone increased phosphorylation of the redox-sensitive kinases ERK1/2-p90RSK and the NHE-1, effects that were eliminated by eplerenone or by preventing epidermal growth factor receptor transactivation. Finally, we provide evidence that the SFR is suppressed by MR blockade, by preventing epidermal growth factor receptor transactivation or by scavenging reactive oxygen species, but it is unaffected by glucocorticoid receptor blockade or protein synthesis inhibition. Our results suggest that MR activation is a necessary step in the stretch-triggered reactive oxygen species-mediated activation of redox-sensitive kinases upstream NHE-1.
Assuntos
Coração/fisiologia , Músculo Liso/fisiologia , Contração Miocárdica/fisiologia , Receptores de Mineralocorticoides/fisiologia , Aldosterona/farmacologia , Angiotensina II/metabolismo , Animais , Endotelina-1/farmacologia , Receptores ErbB/metabolismo , Técnicas In Vitro , Masculino , Mitocôndrias Cardíacas/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Músculos Papilares/fisiologia , Ratos , Ratos Wistar , Receptores de Endotelina/metabolismo , Receptores de Mineralocorticoides/metabolismo , Proteínas Quinases S6 Ribossômicas 90-kDa/metabolismo , Transdução de Sinais , Trocadores de Sódio-Hidrogênio/metabolismo , Estresse Mecânico , Superóxidos/metabolismoRESUMO
A reduction in the risk of coronary heart disease has been associated to moderate red wine consumption. We tested whether a nonalcoholic red wine extract would open mitochondrial K(ATP) channels in guinea pig myocytes. The opening of mitochondrial K(ATP) channels was assessed by endogenous flavoprotein fluorescence. Red wine extract (100 µg·mL(-1)) increased flavoprotein oxidation (10.9% ± 1.2%, n = 20). This effect was prevented by the mitochondrial K(ATP) channel blocker, 5-hydroxydecanoate (500 µmol·L(-1); 0.3% ± 1.1%, n = 13), confirming the hypothesis that red wine extract opens mitochondrial K(ATP) channels.
Assuntos
Doença das Coronárias/prevenção & controle , Mitocôndrias Cardíacas/metabolismo , Miócitos Cardíacos/metabolismo , Canais de Potássio/agonistas , Vinho/análise , Animais , Células Cultivadas , Ácidos Decanoicos/farmacologia , Flavoproteínas/metabolismo , Cobaias , Hidroxiácidos/farmacologia , Cinética , Mitocôndrias Cardíacas/efeitos dos fármacos , Miócitos Cardíacos/efeitos dos fármacos , Oxirredução , Extratos Vegetais/metabolismo , Bloqueadores dos Canais de Potássio/farmacologia , Canais de Potássio/química , Vinho/efeitos adversosRESUMO
The use of antagonists of the mineralocorticoid receptor in the treatment of myocardial hypertrophy and heart failure has gained increasing importance in the last years. The cardiac Na(+)/H(+) exchanger (NHE-1) upregulation induced by aldosterone could account for the genesis of these pathologies. We tested whether aldosterone-induced NHE-1 stimulation involves the transactivation of the epidermal growth factor receptor (EGFR). Rat ventricular myocytes were used to measure intracellular pH with epifluorescence. Aldosterone enhanced the NHE-1 activity. This effect was canceled by spironolactone or eplerenone (mineralocorticoid receptor antagonists), but not by mifepristone (glucocorticoid receptor antagonist) or cycloheximide (protein synthesis inhibitor), indicating that the mechanism is mediated by the mineralocorticoid receptor triggering nongenomic pathways. Aldosterone-induced NHE-1 stimulation was abolished by the EGFR kinase inhibitor AG1478, suggesting that is mediated by transactivation of EGFR. The increase in the phosphorylation level of the kinase p90(RSK) and NHE-1 serine703 induced by aldosterone was also blocked by AG1478. Exogenous epidermal growth factor mimicked the effects of aldosterone on NHE-1 activity. Epidermal growth factor was also able to increase reactive oxygen species production, and the epidermal growth factor-induced activation of the NHE-1 was abrogated by the reactive oxygen species scavenger N-2-mercaptopropionyl glycine, indicating that reactive oxygen species are participating as signaling molecules in this mechanism. Aldosterone enhances the NHE-1 activity via transactivation of the EGFR, formation of reactive oxygen species, and phosphorylation of the exchanger. These results call attention to the consideration of the EGFR as a new potential therapeutic target of the cardiovascular pathologies involving the participation of aldosterone.
Assuntos
Aldosterona/farmacologia , Receptores ErbB/metabolismo , Miócitos Cardíacos/efeitos dos fármacos , Trocadores de Sódio-Hidrogênio/efeitos dos fármacos , Animais , Células Cultivadas , Receptores ErbB/genética , Modelos Animais , Miócitos Cardíacos/metabolismo , Fosforilação/fisiologia , Distribuição Aleatória , Ratos , Ratos Wistar , Espécies Reativas de Oxigênio/metabolismo , Sensibilidade e Especificidade , Transdução de Sinais/efeitos dos fármacos , Trocadores de Sódio-Hidrogênio/metabolismo , Superóxidos/metabolismo , Ativação TranscricionalRESUMO
Myocardial stretch induces a biphasic force response: a first abrupt increase followed by a slow force response (SFR), believed to be the in vitro manifestation of the Anrep effect. The SFR is due to an increase in Ca²âº transient of unclear mechanism. We proposed that Naâº/H⺠exchanger (NHE-1) activation is a key factor in determining the contractile response, but recent reports challenged our findings. We aimed to specifically test the role of the NHE-1 in the SFR. To this purpose small hairpin interference RNA capable of mediating specific NHE-1 knockdown was incorporated into a lentiviral vector (l-shNHE1) and injected into the left ventricular wall of Wistar rats. Injection of a lentiviral vector expressing a nonsilencing sequence (scramble) served as control. Myocardial NHE-1 protein expression and function (the latter evaluated by the recovery of pH(i) after an acidic load and the SFR) were evaluated. Animals transduced with l-shNHE1 showed reduced NHE-1 expression (45 ± 8% of controls; P < 0.05), and the presence of the lentivirus in the left ventricular myocardium, far from the site of injection, was evidenced by confocal microscopy. These findings correlated with depressed basal pH(i) recovery after acidosis [(max)dpH(i)/dt 0.055 ± 0.008 (scramble) vs. 0.009 ± 0.004 (l-shNHE1) pH units/min, P < 0.05], leftward shift of the relationship between J(Hâº) (H⺠efflux corrected by the intrinsic buffer capacity), and abolishment of SFR (124 ± 2 vs. 101 ± 2% of rapid phase; P < 0.05) despite preserved ERK1/2 phosphorylation [247 ± 12 (stretch) and 263 ± 23 (stretch l-shNHE1) % of control; P < 0.05 vs. nonstretched control], well-known NHE-1 activators. Our results provide strong evidence to propose NHE-1 activation as key factor in determining the SFR to stretch.
Assuntos
Mecanorreceptores/metabolismo , Força Muscular , Contração Miocárdica , Músculos Papilares/metabolismo , Interferência de RNA , Trocadores de Sódio-Hidrogênio/metabolismo , Acidose/metabolismo , Acidose/fisiopatologia , Animais , Regulação para Baixo , Concentração de Íons de Hidrogênio , Injeções Intramusculares , Masculino , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Músculos Papilares/fisiopatologia , Fosforilação , RNA Interferente Pequeno/administração & dosagem , Ratos , Ratos Wistar , Transdução de Sinais , Trocador 1 de Sódio-Hidrogênio , Trocadores de Sódio-Hidrogênio/genética , Fatores de TempoRESUMO
Cardiac Na(+)/H(+) exchanger (NHE1) hyperactivity is a central factor in cardiac remodeling following hypertension, myocardial infarction, ischemia-reperfusion injury, and heart failure. Treatment of these pathologies by inhibiting NHE1 is challenging because specific drugs that have been beneficial in experimental models were associated with undesired side effects in clinical practice. In the present work, small interference RNA (siRNA) produced in vitro to specifically silence NHE1 (siRNA(NHE1)) was injected once in vivo into the apex of the left ventricular wall of mouse myocardium. After 48 h, left ventricular NHE1 protein expression was reduced in siRNA(NHE1)-injected mice compared with scrambled siRNA by 33.2 ± 3.4% (n = 5; P < 0.05). Similarly, NHE1 mRNA levels were reduced by 20 ± 2.0% (n = 4). At 72 h, siRNA(NHE1) spreading was evident from the decrease in NHE1 expression in three portions of the myocardium (apex, medium, base). NHE1 function was assessed based on maximal velocity of intracellular pH (pH(i)) recovery (dpH(i)/dt) after an ammonium prepulse-induced acidic load. Maximal dpH(i)/dt was reduced to 14% in siRNA(NHE1)-isolated left ventricular papillary muscles compared with scrambled siRNA. In conclusion, only one injection of naked siRNA(NHE1) successfully reduced NHE1 expression and activity in the left ventricle. As has been previously suggested, extensive NHE1 expression reduction may indicate myocardial spread of siRNA molecules from the injection site through gap junctions, providing a valid technique not only for further research into NHE1 function, but also for consideration as a potential therapeutic strategy.
Assuntos
Proteínas de Transporte de Cátions/genética , Inativação Gênica/efeitos dos fármacos , Coração/efeitos dos fármacos , Coração/fisiologia , RNA Interferente Pequeno/farmacologia , Trocadores de Sódio-Hidrogênio/genética , Animais , Soluções Tampão , Proteínas de Transporte de Cátions/efeitos dos fármacos , Células HEK293 , Humanos , Concentração de Íons de Hidrogênio , Imunoquímica , Injeções , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Miocárdio/metabolismo , Músculos Papilares/efeitos dos fármacos , RNA Interferente Pequeno/administração & dosagem , RNA Interferente Pequeno/biossíntese , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Trocador 1 de Sódio-Hidrogênio , Trocadores de Sódio-Hidrogênio/efeitos dos fármacos , Técnicas de Cultura de Tecidos , Função Ventricular Esquerda/genética , Função Ventricular Esquerda/fisiologiaRESUMO
BACKGROUND/AIMS: Flow restoration to ischemic myocardium reduces infarct size (IS), but it also promotes reperfusion injury. A burst of reactive oxygen species (ROS) and/or NHE-1 reactivation were proposed to explain this injury. Our study was aimed to shed light on this unresolved issue. METHODS: Regional infarction (40 min-ischemia/2 hs-reperfusion) was induced in isolated and perfused rat hearts. Maximal doses of N-(2-mercaptopropionyl)-glycine (MPG 2mmol/L, ROS scavenger), cariporide (10µmol/L, NHE-1 inhibitor), or sildenafil (1µmol/L, phosphodiesterase5A inhibitor) were applied at reperfusion onset. Their effects on IS, myocardial concentration of thiobarbituric acid reactive substances (TBARS), ERK1/2, p90(RSK), and NHE-1 phosphorylation were analyzed. RESULTS: All treatments decreased IS â¼ 50% vs. control. No further protection was obtained by combining cariporide or MPG with sildenafil. Myocardial TBARS increased after infarction and were decreased by MPG or cariporide, but unaffected by sildenafil. In line with the fact that ROS induce MAPK-mediated NHE-1 activation, myocardial infarction increased ERK1/2, p90(RSK), and NHE-1 phosphorylation. MPG and cariporide cancelled these effects. Sildenafil did not reduce the phosphorylated ERK1/2-p90(RSK) levels but blunted NHE-1 phosphorylation suggesting a direct dephosphorylating action. CONCLUSIONS: 1) Reperfusion injury would result from ROS-triggered MAPK-mediated NHE-1 phosphorylation (and reactivation) during reperfusion; 2) sildenafil protects the myocardium by favouring NHE-1 dephosphorylation and bypassing ROS generation.