RESUMO
RATIONALE: Angiotensin (Ang) II-induced apoptosis was reported to be mediated by different signaling molecules. Whether these molecules are either interconnected in a single pathway or constitute different and alternative cascades by which Ang II exerts its apoptotic action, is not known. OBJECTIVE: To investigate in cultured myocytes from adult cat and rat, 2 species in which Ang II has opposite inotropic effects, the signaling cascade involved in Ang II-induced apoptosis. METHODS AND RESULTS: Ang II (1 micromol/L) reduced cat/rat myocytes viability by approximately 40%, in part, because of apoptosis (TUNEL/caspase-3 activity). In both species, apoptosis was associated with reactive oxygen species (ROS) production, Ca(2+)/calmodulin-dependent protein kinase (CaMK)II, and p38 mitogen-activated protein kinase (p38MAPK) activation and was prevented by the ROS scavenger MPG (2-mercaptopropionylglycine) or the NADPH oxidase inhibitor DPI (diphenyleneiodonium) by CaMKII inhibitors (KN-93 and AIP [autocamtide 2-related inhibitory peptide]) or in transgenic mice expressing a CaMKII inhibitory peptide and by the p38MAPK inhibitor, SB202190. Furthermore, p38MAPK overexpression exacerbated Ang II-induced cell mortality. Moreover, although KN-93 did not affect Ang II-induced ROS production, it prevented p38MAPK activation. Results further show that CaMKII can be activated by Ang II or H(2)O(2), even in the presence of the Ca(2+) chelator BAPTA-AM, in myocytes and in EGTA-Ca(2+)-free solutions in the presence of the calmodulin inhibitor W-7 in in vitro experiments. CONCLUSIONS: (1) The Ang II-induced apoptotic cascade converges in both species, in a common pathway mediated by ROS-dependent CaMKII activation which results in p38MAPK activation and apoptosis. (2) In the presence of Ang II or ROS, CaMKII may be activated at subdiastolic Ca(2+) concentrations, suggesting a new mechanism by which ROS reset the Ca(2+) dependence of CaMKII to extremely low Ca(2+) levels.
Assuntos
Angiotensina II/metabolismo , Apoptose , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Cálcio/metabolismo , Miócitos Cardíacos/enzimologia , Estresse Oxidativo , Transdução de Sinais , Animais , Apoptose/efeitos dos fármacos , Benzilaminas/farmacologia , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/antagonistas & inibidores , Calmodulina/antagonistas & inibidores , Calmodulina/metabolismo , Gatos , Sobrevivência Celular , Células Cultivadas , Quelantes/farmacologia , Ácido Egtázico/análogos & derivados , Ácido Egtázico/farmacologia , Ativação Enzimática , Inibidores Enzimáticos/farmacologia , Sequestradores de Radicais Livres/farmacologia , Imidazóis/farmacologia , Camundongos , Camundongos Transgênicos , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/patologia , NADPH Oxidases/antagonistas & inibidores , NADPH Oxidases/metabolismo , Oniocompostos/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Peptídeos/genética , Peptídeos/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Piridinas/farmacologia , Ratos , Ratos Wistar , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/efeitos dos fármacos , Especificidade da Espécie , Sulfonamidas/farmacologia , Fatores de Tempo , Tiopronina/farmacologia , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
The positive inotropic effect produced by Na(+)/K(+)-ATPase inhibition has been used for the treatment of heart failure for over 200 years. Recently, administration of toxic doses of ouabain has been shown to induce cardiac myocyte apoptosis. However, whether prolonged administration of non-toxic doses of ouabain can also promote cardiac myocyte cell death has never been explored. The aim of this study was to assess whether non-toxic doses of ouabain can induce myocyte apoptosis and if so, to examine the underlying mechanisms. For this purpose, cardiac myocytes from rat and cat, two species with different sensitivity to digitalis, were cultured for 24h in the presence or absence of 2 microM (rat) and 25 nm-2 microM ouabain (cat). Cell viability and apoptosis assays showed that ouabain produced, in the rat, a 43+/-5% decrease in cell viability due to apoptosis (enhanced caspase-3 activity, increased Bax/Bcl-2 and TUNEL-positive nuclei) and necrosis (LDH release and trypan blue staining). Similar results were obtained with 25 nM ouabain in the cat. Ouabain-induced reduction in cell viability was prevented by the NCX inhibitor KB-R7943 and by the CaMKII inhibitors, KN93 and AIP. Furthermore, CaMKII overexpression exacerbated ouabain-induced cell mortality which in contrast was reduced in transgenic mice with chronic CaMKII inhibition. However, KN93 failed to affect ouabain-induced inotropy. In addition, whereas ERK(1/2) inhibition with PD-98059 had no effect on cell mortality, PI3K inhibition with wortmannin, exacerbated myocyte death. We conclude that ouabain triggers an apoptotic cascade that involves NCX and CaMKII as a downstream effector. Ouabain simultaneously activates an antiapoptotic cascade involving PI3K/AKT which is however, insufficient to completely repress apoptosis. The finding that KN93 prevents ouabain-induced apoptosis without affecting inotropy suggests the potential use of CaMKII inhibitors as an adjunct to digitalis treatment for cardiovascular disease.
Assuntos
Apoptose/efeitos dos fármacos , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Inibidores Enzimáticos/farmacologia , Miócitos Cardíacos/patologia , Ouabaína/farmacologia , ATPase Trocadora de Sódio-Potássio/antagonistas & inibidores , Animais , Western Blotting , Cálcio/metabolismo , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/antagonistas & inibidores , Gatos , Células Cultivadas , Camundongos , Camundongos Transgênicos , Miócitos Cardíacos/enzimologia , Necrose , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos , Ratos Wistar , Trocador de Sódio e Cálcio/antagonistas & inibidores , Trocador de Sódio e Cálcio/metabolismo , ATPase Trocadora de Sódio-Potássio/metabolismoRESUMO
OBJECTIVES: Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) has been implicated in the regulation of cardiac excitation-contraction coupling (ECC) as well as in apoptotic signaling and adverse remodeling. The goal of the present study is to investigate the role of CaMKII in irreversible ischemia and reperfusion (I/R) injury. METHODS: Isovolumic Langendorff perfused rat hearts were subjected to global no-flow I/R (45 min/120 min), and isolated myocytes were subjected to a protocol of simulated I/R (45 min simulated ischemia/60 min reoxygenation) either in the absence or presence of CaMKII inhibition [KN-93 (KN) or the CaMKII inhibitory peptide (AIP)]. RESULTS: In I/R hearts, an increase in CaMKII activity at the beginning of reperfusion was confirmed by the significantly increased phosphorylation of the Thr(17) site of phospholamban. In the presence of KN, contractile recovery at the end of reperfusion was almost double that of I/R hearts. This recovery was associated with a significant decrease in the extent of infarction, lactate dehydrogenase release (necrosis), TUNEL-positive cells, caspase-3 activity, and an increase in the Bcl-2/Bax ratio (apoptosis). In isolated myocytes, both KN and AIP prevented simulated I/R-induced spontaneous contractile activity and cell mortality. Similar results were obtained when inhibiting the reverse mode Na(+)/Ca(2+) exchanger (NCX) with KB-R7943, sarcoplasmic reticulum (SR) function with ryanodine and thapsigargin, or SR Ca(2+) release with tetracaine. In contrast, overexpression of CaMKII decreased cell viability from 52+/-3% to 26+/-2%. CONCLUSIONS: Taken together, the present findings are the first to establish CaMKII as a fundamental component of a cascade of events integrating the NCX, the SR, and mitochondria that promote cellular apoptosis and necrosis in irreversible I/R injury.
Assuntos
Benzilaminas/farmacologia , Proteínas Quinases Dependentes de Cálcio-Calmodulina/antagonistas & inibidores , Traumatismo por Reperfusão Miocárdica/enzimologia , Traumatismo por Reperfusão Miocárdica/patologia , Miocárdio/enzimologia , Miocárdio/patologia , Sulfonamidas/farmacologia , Animais , Apoptose/efeitos dos fármacos , Cálcio/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Células Cultivadas , L-Lactato Desidrogenase/metabolismo , Masculino , Miócitos Cardíacos/enzimologia , Necrose , Perfusão , Fosforilação , Ratos , Retículo Sarcoplasmático/metabolismo , Trocador de Sódio e Cálcio/antagonistas & inibidores , Tioureia/análogos & derivados , Tioureia/farmacologia , Fatores de TempoRESUMO
BACKGROUND: The flat or negative force frequency relationship (FFR) is a hallmark of the failing heart. Either decreases in SERCA2a expression, increases in Na(+)/Ca(2+) exchanger (NCX) expression or elevated Na(+)(i) have been independently proposed as mediators of the negative FFR. METHODS AND RESULTS: To determine whether each one of these mechanisms is sufficient to account for the negative FFR of the failing heart or on the contrary, various mechanisms, acting in concert are required. SERCA2a was pharmacologically inhibited with thapsigargin (TG) or cyclopiazonic acid (CPA) or by using siRNA technology; Na(+)(i) was increased with either ouabain (Oua) or monensin and NCX protein was overexpressed by gene transfer (Ad.NCX), to mimic in nonfailing cat myocytes the phenotype of the failing heart and examine their effect on the FFR. The positive FFR of healthy myocytes remained unaffected after either SERCA2a inhibition, Na(+)(i) elevation, or NCX overexpression. However, the combination of TG + Oua, Oua + Ad.NCX, or TG + Ad.NCX, converted the positive FFR to negative. Moreover, the FFR became negative at lower frequencies, when the 3 interventions were combined. CONCLUSIONS: Ca(2+) handling has to be altered at several levels to explain the negative FFR of the failing heart. These anomalies in Ca(2+) homeostasis acting in synergy have additive effects.
Assuntos
Cálcio/metabolismo , Insuficiência Cardíaca/metabolismo , Líquido Intracelular/metabolismo , Miocárdio/patologia , Miócitos Cardíacos/metabolismo , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Animais , Western Blotting , ATPases Transportadoras de Cálcio/antagonistas & inibidores , Gatos , Células Cultivadas , Modelos Animais de Doenças , Inibidores Enzimáticos/farmacologia , Corantes Fluorescentes , Expressão Gênica , Insuficiência Cardíaca/patologia , Indóis/farmacologia , Miocárdio/metabolismo , Miócitos Cardíacos/patologia , RNA Interferente Pequeno/genética , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/antagonistas & inibidores , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/genética , Sódio/metabolismo , Tapsigargina/farmacologiaRESUMO
The octapeptide angiotensin II (ANG II) can modulate cardiac contractility and is increased in heart failure, where contractile function is impaired. In rat cardiac myocytes, 1 microM of ANG II produces a negative inotropic effect (NIE) (24.6 +/- 5% reduction). However, the subcellular signaling involved in this effect remains elusive. We examined the mechanisms and signaling events involved in the reduction in contractile function induced by the peptide in indo-1-loaded rat cardiomyocytes. The results showed that the NIE of ANG II was not associated with a parallel decrease in the intracellular Ca2+ transient, indicating that a decrease in myofilament responsiveness to Ca2+ underlies the reduction in contractility. We assessed the role of PKC, tyrosine kinases, reactive oxygen species (ROS), and mitogen-activated protein kinases (MAPKs) in the NIE of the peptide. Pretreatment of cells with the NAD(P)H oxidase inhibitor diphenyleneiodonium chloride or with the superoxide scavenger 4,5-dihydroxy-1,3-benzene-disulfonic acid did not affect the ANG II-induced NIE. Moreover, ANG II-induced ROS production, after 20 min of incubation with the peptide, could not be detected with the use of either the fluorophore 5-(6)-chloromethyl-2',7'-dichlorodihydrofluorecein diacetate or lucigenin-enhanced chemiluminescence. In contrast, the ANG II-induced NIE was abrogated by the inhibitors of PKC (calphostin C), tyrosine kinase (genistein), and p38 MAPK (SB-202190). Furthermore, the NIE was significantly exacerbated (60 +/- 10% reduction) by p38 MAPK overexpression. These results exclude the participation of ROS in the NIE of the peptide and point to PKC and tyrosine kinase as upstream mediators. Furthermore, they reveal p38 MAPK as the putative effector of the reduction in myofilament responsiveness to Ca2+ and the decrease in contractility induced by the peptide.