RESUMO
BACKGROUND: It has been previously demonstrated that the maintenance of ischemic acidic pH or the delay of intracellular pH recovery at the onset of reperfusion decreases ischemic-induced cardiomyocyte death. OBJECTIVE: To examine the role played by nitric oxide synthase (NOS)/NO-dependent pathways in the effects of acidic reperfusion in a regional ischemia model. METHODS: Isolated rat hearts perfused by Langendorff technique were submitted to 40 min of left coronary artery occlusion followed by 60 min of reperfusion (IC). A group of hearts received an acid solution (pH = 6.4) during the first 2 min of reperfusion (AR) in absence or in presence of l-NAME (NOS inhibitor). Infarct size (IS) and myocardial function were determined. In cardiac homogenates, the expression of P-Akt, P-endothelial and inducible isoforms of NOS (P-eNOS and iNOS) and the level of 3-nitrotyrosine were measured. In isolated cardiomyocytes, the intracellular NO production was assessed by confocal microscopy, under control and acidic conditions. Mitochondrial swelling after Ca2+ addition and mitochondrial membrane potential (Δψ) were also determined under control and acidosis. RESULTS: AR decreased IS, improved postischemic myocardial function recovery, increased P-Akt and P-eNOS, and decreased iNOS and 3-nitrotyrosine. NO production increased while mitochondrial swelling and Δψ decreased in acidic conditions. l-NAME prevented the beneficial effects of AR. CONCLUSIONS: Our data strongly supports that a brief acidic reperfusion protects the myocardium against the ischemia-reperfusion injury through eNOS/NO-dependent pathways.
Assuntos
Óxido Nítrico , Animais , Concentração de Íons de Hidrogênio , Óxido Nítrico/metabolismo , Masculino , Ratos , Ratos Wistar , Óxido Nítrico Sintase Tipo III/metabolismo , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/patologia , Traumatismo por Reperfusão Miocárdica/metabolismo , Traumatismo por Reperfusão Miocárdica/patologia , Traumatismo por Reperfusão Miocárdica/prevenção & controle , Traumatismo por Reperfusão Miocárdica/tratamento farmacológico , NG-Nitroarginina Metil Éster/farmacologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Óxido Nítrico Sintase Tipo II/metabolismo , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Isquemia Miocárdica/metabolismo , Isquemia Miocárdica/patologia , Óxido Nítrico Sintase/metabolismoRESUMO
Systolic Ca2+ transients are shaped by the concerted summation of Ca2+ sparks across cardiomyocytes. At high pacing rates, alterations of excitation-contraction coupling manifest as pro-arrhythmic Ca2+ alternans that can be classified as concordant or discordant. Discordance is ascribed to out-of-phase alternation of local Ca2+ release across the cell, although the triggers and consequences of this phenomenon remain unclear. Rat ventricular cardiomyocytes were paced at increasing rates. A discordance index (SD of local alternans ratios) was developed to quantify discordance in confocal recordings of Ca2+ transients. Index values were significantly increased by rapid pacing, and negatively correlated with Ca2+ transient amplitude change, indicating that discordance is an important contributor to the negative Ca2+ transient-frequency relationship. In addition, the largest local calcium transient in two consecutive transients was measured to build a potential "best release" profile, which quantitatively confirmed discordance-induced Ca2+ release impairment (DICRI). Diastolic Ca2+ homeostasis was also observed to be disrupted by discordance, as late Ca2+ release events elicited instability of resting Ca2+ levels. Finally, the effects of two RyR2 inhibitors (VK-II-86 and dantrolene) were tested. While both compounds inhibited Ca2+ wave generation, only VK-II-86 augmented subcellular discordance. Discordant Ca2+ release is a quantifiable phenomenon, sensitive to pacing frequency, and impairs both systolic and diastolic Ca2+ homeostasis. Interestingly, RyR2 inhibition can induce discordance, which should be considered when evaluating pharmacological RyR2 modulators for clinical use.
Assuntos
Bloqueadores dos Canais de Cálcio , Sinalização do Cálcio , Miócitos Cardíacos , Canal de Liberação de Cálcio do Receptor de Rianodina , Animais , Arritmias Cardíacas/metabolismo , Cálcio/metabolismo , Bloqueadores dos Canais de Cálcio/farmacologia , Acoplamento Excitação-Contração , Miócitos Cardíacos/metabolismo , Ratos , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Retículo SarcoplasmáticoRESUMO
This commentary analyzes the possible effects of lightness-a typical attribute of modern (liquid) society, according to Bauman-on the way we are doing science. We share our opinion in an attempt to discern whether some unwanted practices that may affect our scientific results (such as technology misuse, bonus rewards, publishing under pressure, or indolence for getting accurate results) can be attributed, at least partially, to the liquid characteristic of modern society. We also examine whether the different systems that support science favor these actions, conspiring against what should be the primary goal of science: the search for truth. We finally consider several aspects that should be taken into account to rescue science from the intrusion of weightless actions.
RESUMO
Sepsis is associated with cardiac dysfunction, which is at least in part due to cardiomyocyte apoptosis. However, the underlying mechanisms are far from being understood. Using the colon ascendens stent peritonitis mouse model of sepsis (CASP), we examined the subcellular mechanisms that mediate sepsis-induced apoptosis. Wild-type (WT) CASP mice hearts showed an increase in apoptosis respect to WT-Sham. CASP transgenic mice expressing a CaMKII inhibitory peptide (AC3-I) were protected against sepsis-induced apoptosis. Dantrolene, used to reduce ryanodine receptor (RyR) diastolic sarcoplasmic reticulum (SR) Ca2+ release, prevented apoptosis in WT-CASP. To examine whether CaMKII-dependent RyR2 phosphorylation mediates diastolic Ca2+ release and apoptosis in sepsis, we evaluated apoptosis in mutant mice hearts that have the CaMKII phosphorylation site of RyR2 (Serine 2814) mutated to Alanine (S2814A). S2814A CASP mice did not show increased apoptosis. Consistent with RyR2 phosphorylation-dependent enhancement in diastolic SR Ca2+ release leading to mitochondrial Ca2+ overload, mitochondrial Ca2+ retention capacity was reduced in mitochondria isolated from WT-CASP compared to Sham and this reduction was absent in mitochondria from CASP S2814A or dantrolene-treated mice. We conclude that in sepsis, CaMKII-dependent RyR2 phosphorylation results in diastolic Ca2+ release from SR which leads to mitochondrial Ca2+ overload and apoptosis.
Assuntos
Apoptose/fisiologia , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Miócitos Cardíacos/metabolismo , Fosforilação/fisiologia , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Sepse/metabolismo , Animais , Cálcio/metabolismo , Sinalização do Cálcio/fisiologia , Proteínas de Ligação ao Cálcio/metabolismo , Modelos Animais de Doenças , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mitocôndrias/metabolismo , Retículo Sarcoplasmático/metabolismoRESUMO
In different pathological situations, cardiac cells undergo hyperosmotic stress and cell shrinkage. This change in cellular volume has been associated with contractile dysfunction and cell death. However, the intracellular mechanisms involved in hyperosmotic stress-induced cell death have not been investigated in depth in adult cardiac myocytes. Given that osmotic stress has been shown to promote endoplasmic reticulum stress (ERS), a recognized trigger for apoptosis, we examined whether hyperosmotic stress triggers ERS in adult cardiac myocytes and if so whether this mechanism mediates hyperosmotic stress-induced cell death. Adult rat cardiomyocytes cultured overnight in a hypertonic solution (HS) containing mannitol as the osmolite, showed increased expression of ERS markers, GRP78, CHOP and cleaved-Caspase-12, compared with myocytes in isotonic solution (IS), suggesting that hyperosmotic stress induces ERS. In addition, HS significantly reduced cell viability and increased TUNEL staining and the expression of active Caspase-3, indicative of apoptosis. These effects were prevented with the addition of the ERS inhibitor, 4-PBA, indicating that hyperosmotic stress-induced apoptosis is mediated by ERS. Hyperosmotic stress-induced apoptosis was also prevented when cells were cultured in the presence of a Ca2+-chelating agent (EGTA) or the CaMKII inhibitor (KN93), suggesting that hyperosmotic stress-induced ERS is mediated by a Ca2+ and CaMKII-dependent mechanism. Similar results were observed when hyperosmotic stress was induced using glucose as the osmolite. We conclude that hyperosmotic stress promotes ERS by a CaMKII-dependent mechanism leading to apoptosis of adult cardiomyocytes. More importantly, we demonstrate that hyperosmotic stress-triggered ERS contributes to hyperglycemia-induced cell death.
Assuntos
Apoptose , Estresse do Retículo Endoplasmático , Hiperglicemia , Miócitos Cardíacos/patologia , Animais , Apoptose/efeitos dos fármacos , Butilaminas/farmacologia , Sinalização do Cálcio , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Hiperglicemia/induzido quimicamente , Masculino , Manitol , Pressão Osmótica , Cultura Primária de Células , Ratos , Ratos WistarRESUMO
Background: Hypothyroidism, the most common endocrine disease, induces cardiac electrical remodeling that creates a substrate for ventricular arrhythmias. Recent studies report that high thyrotropin (TSH) levels are related to cardiac electrical abnormalities and increased mortality rates. The aim of the present work was to investigate the direct effects of TSH on the heart and its possible causative role in the increased incidence of arrhythmia in hypothyroidism. Methods: A new rat model of central hypothyroidism (low TSH levels) was created and characterized together with the classical propylthiouracil-induced primary hypothyroidism model (high TSH levels). Electrocardiograms were recorded in vivo, and ionic currents were recorded from isolated ventricular myocytes in vitro by the patch-clamp technique. Protein and mRNA were measured by Western blot and quantitative reverse transcription polymerase chain reaction in rat and human cardiac myocytes. Adult human action potentials were simulated in silico to incorporate the experimentally observed changes. Results: Both primary and central hypothyroidism models increased the L-type Ca2+ current (ICa-L) and decreased the ultra-rapid delayed rectifier K+ current (IKur) densities. However, only primary but not central hypothyroidism showed electrocardiographic repolarization abnormalities and increased ventricular arrhythmia incidence during caffeine/dobutamine challenge. These changes were paralleled by a decrease in the density of the transient outward K+ current (Ito) in cardiomyocytes from animals with primary but not central hypothyroidism. In vitro treatment with TSH for 24 hours enhanced isoproterenol-induced spontaneous activity in control ventricular cells and diminished Ito density in cardiomyocytes from control and central but not primary hypothyroidism animals. In human myocytes, TSH decreased the expression of KCND3 and KCNQ1, Ito, and the delayed rectifier K+ current (IKs) encoding proteins in a protein kinase A-dependent way. Transposing the changes produced by hypothyroidism and TSH to a computer model of human ventricular action potential resulted in enhanced occurrence of early afterdepolarizations and arrhythmia mostly in primary hypothyroidism, especially under ß-adrenergic stimulation. Conclusions: The results suggest that suppression of repolarizing K+ currents by TSH underlies most of the electrical remodeling observed in hypothyroidism. This work demonstrates that the activation of the TSH-receptor/protein kinase A pathway in the heart is responsible for the cardiac electrical remodeling and arrhythmia generation seen in hypothyroidism.
Assuntos
Arritmias Cardíacas/metabolismo , Remodelamento Atrial/fisiologia , Hipotireoidismo/metabolismo , Miócitos Cardíacos/metabolismo , Tireotropina/metabolismo , Potenciais de Ação , Animais , Antitireóideos/toxicidade , Arritmias Cardíacas/etiologia , Arritmias Cardíacas/fisiopatologia , Bexaroteno/toxicidade , Cálcio/metabolismo , Simulação por Computador , Modelos Animais de Doenças , Suscetibilidade a Doenças , Eletrocardiografia , Humanos , Hipotireoidismo/complicações , Hipotireoidismo/fisiopatologia , Isoproterenol/farmacologia , Canal de Potássio KCNQ1/efeitos dos fármacos , Canal de Potássio KCNQ1/genética , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Miócitos Cardíacos/efeitos dos fármacos , Técnicas de Patch-Clamp , Propiltiouracila/toxicidade , RNA Mensageiro/metabolismo , Ratos , Canais de Potássio Shal/efeitos dos fármacos , Canais de Potássio Shal/genética , Tireotropina/farmacologiaRESUMO
Hemodynamic collapse and myocardial dysfunction are among the major causes of death in severe sepsis. The purpose of this study was to assess the role played by toll-like receptor 4 and by the NLRP3 inflammasome in the cardiac dysfunction that occurs after high-grade polymicrobial sepsis. We performed the colon ascendens stent peritonitis (CASP) surgery in Tlr4, Nlrp3, and caspase-1 mice. We also assessed for the first time the electrical heart function in the colon ascendens stent peritonitis (CASP) model. The QJ interval was increased in wild-type C57BL/6J mice after CASP when compared with sham controls, a result paralleled by an increase in the cardiac action potential (AP) duration (APD). The decreases in ejection fraction (EF), left ventricle end diastolic volume, stroke volume, and cardiac output found after CASP were similar among all groups of mice. Similar heart response was found when Nlrp3 mice were submitted to high-grade cecal ligation and puncture. Despite developing cardiac dysfunction similar to wild types after CASP, Nlrp3 mice had reduced circulating levels of interleukin (IL)-1ß, IL-6 and tumor necrosis factor-α. Our results demonstrate that the genetic ablation of Tlr4, Nlrp3, and caspase-1 does not prevent the cardiac dysfunction, despite preventing the increase in pro-inflammatory cytokines, indicating that these are not feasible targets to therapy in high-grade sepsis.
Assuntos
Caspase 1/metabolismo , Colo/metabolismo , Cardiopatias/metabolismo , Interleucina-1beta/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Peritonite/complicações , Peritonite/metabolismo , Receptor 4 Toll-Like/metabolismo , Animais , Ecocardiografia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Função Ventricular Esquerda/fisiologiaRESUMO
In different pathological situations, cardiac cells undergo hyperosmotic stress (HS) and cell shrinkage. This change in cellular volume has been associated with contractile dysfunction and cell death. Given that nitric oxide (NO) is a well-recognized modulator of cardiac contractility and cell survival, we evaluated whether HS increases NO production and its impact on the negative inotropic effect observed during this type of stress. Superfusing cardiac myocytes with a hypertonic solution (HS: 440 mOsm) decreased cell volume and increased NO-sensitive DAF-FM fluorescence compared with myocytes superfused with an isotonic solution (IS: 309 mOsm). When cells were exposed to HS in addition to different inhibitors: L-NAME (NO synthase inhibitor), nitroguanidine (nNOS inhibitor), and Wortmannin (eNOS inhibitor) cell shrinkage occurred in the absence of NO release, suggesting that HS activates nNOS and eNOS. Consistently, western blot analysis demonstrated that maintaining cardiac myocytes in HS promotes phosphorylation and thus, activation of nNOS and eNOS compared to myocytes maintained in IS. HS-induced nNOS and eNOS activation and NO production were also prevented by AMPK inhibition with Dorsomorphin (DORSO). In addition, the HS-induced negative inotropic effect was exacerbated in the presence of either L-NAME, DORSO, ODQ (guanylate cyclase inhibitor), or KT5823 (PKG inhibitor), suggesting that NO provides contractile support via a cGMP/PKG-dependent mechanism. Our findings suggest a novel mechanism of AMPK-dependent NO release in cardiac myocytes with putative pathophysiological relevance determined, at least in part, by its capability to reduce the extent of contractile dysfunction associated with hyperosmotic stress.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Tamanho Celular , Cardiopatias/enzimologia , Contração Miocárdica , Miócitos Cardíacos/enzimologia , Óxido Nítrico/metabolismo , Pressão Osmótica , Adaptação Fisiológica , Animais , GMP Cíclico/metabolismo , Proteínas Quinases Dependentes de GMP Cíclico/metabolismo , Ativação Enzimática , Guanilato Ciclase/metabolismo , Cardiopatias/patologia , Cardiopatias/fisiopatologia , Masculino , Miócitos Cardíacos/patologia , Óxido Nítrico Sintase Tipo I/metabolismo , Óxido Nítrico Sintase Tipo III/metabolismo , Fosforilação , Ratos Wistar , Transdução de SinaisRESUMO
The force-frequency relationship (FFR) is an important intrinsic regulatory mechanism of cardiac contractility. However, a decrease (negative FFR) or no effect (flat FFR) on contractile force in response to an elevation of heart rate is present in the normal rat or in human heart failure. Reactive oxygen species (ROS) can act as intracellular signaling molecules activating diverse kinases as calcium-calmodulin-dependent protein kinase II (CaMKII) and p-38 MAP kinase (p-38K). Our aim was to elucidate the intracellular molecules implicated in the FFR of isolated rat ventricular myocytes. The myocytes were field-stimulated via two-platinum electrodes. Sarcomere length was recorded with a video camera. Ca2+ transients and intracellular pHi were recorded by epifluorescence. Increasing frequency from 0.5 to 3 Hz decreased cell shortening without changes in pHi. This negative FFR was changed to positive FFR when the myocytes were pre-incubated with the ROS scavenger MPG, the NADPH oxidase blocker apocynin, or by inhibiting mitochondrial ROS production with 5-HD. Similar results were obtained when the cells were pre-incubated with the CaMKII blocker, KN-93, or the p-38K inhibitor, SB-202190. Consistently, the levels of phosphorylation of p-38K and the oxidation of CaMKII were significantly higher at 2 Hz than at 0.5 Hz. Despite the presence of positive inotropic effect during stimulation frequency enhancement, Ca2+ transient amplitudes were reduced in MPG- and SB-202190-treated myocytes. In conclusion, our results indicate that the activation of the intracellular pathway involving ROS-CaMKII-p-38K contributes to the negative FFR of rat cardiomyocytes, likely by desensitizing the response of contractile myofilaments to Ca2+.
Assuntos
Contração Miocárdica/fisiologia , Miócitos Cardíacos/metabolismo , Miofibrilas/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Animais , Cálcio/metabolismo , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Ventrículos do Coração/metabolismo , Masculino , Ratos , Ratos WistarRESUMO
Diabetes mellitus (DM) encompasses a multitude of secondary disorders, including heart disease. One of the most frequent and potentially life threatening disorders of DM-induced heart disease is ventricular tachycardia (VT). Here we show that toll-like receptor 2 (TLR2) and NLRP3 inflammasome activation in cardiac macrophages mediate the production of IL-1ß in DM mice. IL-1ß causes prolongation of the action potential duration, induces a decrease in potassium current and an increase in calcium sparks in cardiomyocytes, which are changes that underlie arrhythmia propensity. IL-1ß-induced spontaneous contractile events are associated with CaMKII oxidation and phosphorylation. We further show that DM-induced arrhythmias can be successfully treated by inhibiting the IL-1ß axis with either IL-1 receptor antagonist or by inhibiting the NLRP3 inflammasome. Our results establish IL-1ß as an inflammatory connection between metabolic dysfunction and arrhythmias in DM.
Assuntos
Diabetes Mellitus Experimental/imunologia , Interleucina-1beta/imunologia , Macrófagos/imunologia , Miócitos Cardíacos/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/imunologia , Taquicardia Ventricular/imunologia , Receptor 2 Toll-Like/imunologia , Potenciais de Ação , Animais , Antirreumáticos/farmacologia , Arritmias Cardíacas/etiologia , Arritmias Cardíacas/imunologia , Arritmias Cardíacas/metabolismo , Cálcio/metabolismo , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Caspase 1/metabolismo , Diabetes Mellitus Experimental/complicações , Diabetes Mellitus Experimental/metabolismo , Inflamassomos/antagonistas & inibidores , Proteína Antagonista do Receptor de Interleucina 1/farmacologia , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Camundongos , Camundongos Transgênicos , Contração Miocárdica , Miócitos Cardíacos/imunologia , Proteína 3 que Contém Domínio de Pirina da Família NLR/antagonistas & inibidores , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Potássio/metabolismo , Receptores de Interleucina-1/antagonistas & inibidores , Receptores de Interleucina-1/genética , Receptores de Interleucina-1/imunologia , Taquicardia Ventricular/etiologia , Taquicardia Ventricular/metabolismo , Receptor 2 Toll-Like/genéticaRESUMO
Some cardiac non-genomic effects of aldosterone (Ald) are reported to be mediated through activation of the classic mineralocorticoid receptor (MR). However, in the last years, it was proposed that activation of the novel G protein-coupled receptor GPR30 mediates certain non-genomic effects of Ald. The aim of this study was to elucidate if the sodium/bicarbonate cotransporter (NBC) is stimulated by Ald and if the activation of GPR30 mediates this effect. NBC activity was evaluated in rat cardiomyocytes perfused with HCO3(-)/CO2 solution in the continuous presence of HOE642 (sodium/hydrogen exchanger blocker) during recovery from acidosis using intracellular fluorescence measurements. Ald enhanced NBC activity (% of ΔJHCO3(-); control: 100±5.82%, n=7 vs Ald: 151.88±11.02%, n=5; P<0.05), which was prevented by G15 (GPR30 blocker, 90.53±7.81%, n=7). Further evidence for the involvement of GPR30 was provided by G1 (GPR30 agonist), which stimulated NBC (185.13±18.28%, n=6; P<0.05) and this effect was abrogated by G15 (124.19±10.96%, n=5). Ald- and G1-induced NBC stimulation was abolished by the reactive oxygen species (ROS) scavenger MPG and by the NADPH oxidase inhibitor apocynin. In addition, G15 prevented Ald- and G1-induced ROS production. Pre-incubation of myocytes with wortmannin (PI3K-AKT pathway blocker) prevented Ald- or G1-induced NBC stimulation. In summary, Ald stimulates NBC by GPR30 activation, ROS production and AKT stimulation.
Assuntos
Aldosterona/farmacologia , Miocárdio/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Simportadores de Sódio-Bicarbonato/metabolismo , Animais , Fator de Crescimento Epidérmico/farmacologia , Receptores ErbB/metabolismo , Concentração de Íons de Hidrogênio , Espaço Intracelular/metabolismo , Masculino , Modelos Biológicos , Fosforilação/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos Wistar , Espécies Reativas de Oxigênio/metabolismo , Receptores de Mineralocorticoides/metabolismo , Ativação Transcricional/efeitos dos fármacosRESUMO
AIMS: Cardiomyocyte swelling occurs in multiple pathological situations and has been associated with contractile dysfunction, cell death, and enhanced propensity to arrhythmias. We investigate whether hypotonic swelling promotes nitric oxide (NO) release in cardiomyocytes, and whether it impacts on swelling-induced contractile dysfunction. METHODS AND RESULTS: Superfusing rat cardiomyocytes with a hypotonic solution (HS; 217 mOsm), increased cell volume, reduced myocyte contraction and Ca(2+) transient, and increased NO-sensitive 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate (DAF-FM) fluorescence. When cells were exposed to HS + 2.5 mM of the NO synthase inhibitor l-NAME, cell swelling occurred in the absence of NO release. Swelling-induced NO release was also prevented by the nitric oxide synthase 1 (NOS1) inhibitor, nitroguanidine, and significantly reduced in NOS1 knockout mice. Additionally, colchicine (inhibitor of microtubule polymerization) prevented the increase in DAF-FM fluorescence induced by HS, indicating that microtubule integrity is necessary for swelling-induced NO release. The swelling-induced negative inotropic effect was exacerbated in the presence of either l-NAME, nitroguandine, the guanylate cyclase inhibitor, ODQ, or the PKG inhibitor, KT5823, suggesting that NOS1-derived NO provides contractile support via a cGMP/PKG-dependent mechanism. Indeed, ODQ reduced Ca(2+) wave velocity and both ODQ and KT5823 reduced the HS-induced increment in ryanodine receptor (RyR2, Ser2808) phosphorylation, suggesting that in this context, cGMP/PKG may contribute to preserve contractile function by enhancing sarcoplasmic reticulum Ca(2+) release. CONCLUSIONS: Our findings suggest a novel mechanism for NO release in cardiomyocytes with putative pathophysiological relevance determined, at least in part, by its capability to reduce the extent of contractile dysfunction associated with hypotonic swelling.
Assuntos
Citoesqueleto/fisiologia , Miócitos Cardíacos/fisiologia , Óxido Nítrico/metabolismo , Osmorregulação , Animais , GMP Cíclico/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Contração Miocárdica , Óxido Nítrico Sintase Tipo I/metabolismo , Ratos WistarRESUMO
Tachycardia promotes cell death and cardiac remodeling, leading to congestive heart failure. However, the underlying mechanism of tachycardia- or rapid pacing (RP)-induced cell death remains unknown. Myocyte loss by apoptosis is recognized as a critical factor in the progression to heart failure and simulation of tachycardia by RP has been shown to increase the intracellular levels of at least two potentially proapoptotic molecules, Ca(2+) and reactive oxygen species (ROS). However, whether these molecules mediate tachycardia- or RP-induced cell death has yet to be determined. The aim of this study was to examine the subcellular mechanisms underlying RP-induced apoptosis. For this purpose rat ventricular myocytes were maintained quiescent or paced at 0.5, 5 and 8Hz for 1hr. RP at 5 and 8Hz decreased myocyte viability by 58±3% and 75±6% (n=24), respectively, compared to cells maintained at 0.5Hz, and increased caspase-3 activity and Bax/Bcl-2 ratio, indicative of apoptosis. RP-induced cell death and apoptosis were prevented when pacing protocols were conducted in the presence of either the ROS scavenger, MPG, or nifedipine to reduce Ca(2+) entry or the CaMKII inhibitors, KN93 and AIP. Consistently, myocytes from transgenic mice expressing a CaMKII inhibitory peptide (AC3-I) were protected against RP-induced cell death. Interestingly, tetracaine and carvedilol used to reduce ryanodine receptor (RyR) diastolic Ca(2+) release, and ruthenium red used to prevent Ca(2+) entry into the mitochondria prevented RP-induced cell death, whereas PI3K inhibition with Wortmannin exacerbated pacing-induced cell mortality. We conclude that CaMKII activation and ROS production are involved in RP-induced apoptosis. Particularly, our results suggest that CaMKII-dependent posttranslational modifications of the cardiac ryanodine receptor (RyR) leading to enhanced diastolic Ca(2+) release and mitochondrial Ca(2+) overload could be the underlying mechanism involved. We further show that RP simultaneously activates a protective cascade involving PI3K/AKT signaling which is however, insufficient to completely suppress apoptosis.
Assuntos
Apoptose , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Taquicardia/metabolismo , Androstadienos/farmacologia , Animais , Morte Celular , Sobrevivência Celular , Camundongos , Camundongos Transgênicos , Miócitos Cardíacos/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos , Transdução de Sinais/efeitos dos fármacos , WortmaninaRESUMO
BACKGROUND: Digitalis-induced Na(+) accumulation results in an increase in Ca(2+)(i) via the Na(+)/Ca(2+) exchanger, leading to enhanced sarcoplasmic reticulum (SR) Ca(2+) load, responsible for the positive inotropic and toxic arrhythmogenic effects of glycosides. A digitalis-induced increase in Ca(2+)(i) could also activate calcium-calmodulin kinase II (CaMKII), which has been shown to have proarrhythmic effects. Here, we investigate whether CaMKII underlies digitalis-induced arrhythmias and the subcellular mechanisms involved. METHODS AND RESULTS: In paced rat ventricular myocytes (0.5 Hz), 50 µmol/L ouabain increased contraction amplitude by 160 ± 5%. In the absence of electric stimulation, ouabain promoted spontaneous contractile activity and Ca(2+) waves. Ouabain activated CaMKII (p-CaMKII), which phosphorylated its downstream targets, phospholamban (PLN) (Thr17) and ryanodine receptor (RyR) (Ser2814). Ouabain-induced spontaneous activity was prevented by inhibiting CaMKII with 2.5 µmol/L KN93 but not by 2.5 µmol/L of the inactive analog, KN92. Similar results were obtained using the CaMKII inhibitor, autocamtide-2 related inhibitory peptide (AIP) (1 to 2.5 µmol/L), and in myocytes from transgenic mice expressing SR-targeted AIP. Consistently, CaMKII overexpression exacerbated ouabain-induced spontaneous contractile activity. Ouabain was associated with an increase in SR Ca(2+) content and Ca(2+) spark frequency, indicative of enhanced SR Ca(2+) leak. KN93 suppressed the ouabain-induced increase in Ca(2+) spark frequency without affecting SR Ca(2+) content. Similar results were obtained with digoxin. In vivo, ouabain-induced arrhythmias were prevented by KN93 and absent in SR-AIP mice. CONCLUSIONS: These results show for the first time that CaMKII mediates ouabain-induced arrhythmic/toxic effects. We suggest that CaMKII-dependent phosphorylation of the RyR, resulting in Ca(2+) leak from the SR, is the underlying mechanism involved.
Assuntos
Arritmias Cardíacas/induzido quimicamente , Sinalização do Cálcio/efeitos dos fármacos , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Cardiotônicos/toxicidade , Frequência Cardíaca/efeitos dos fármacos , Ventrículos do Coração/efeitos dos fármacos , Miócitos Cardíacos/efeitos dos fármacos , Ouabaína/toxicidade , Animais , Antiarrítmicos/farmacologia , Arritmias Cardíacas/diagnóstico , Arritmias Cardíacas/fisiopatologia , Arritmias Cardíacas/prevenção & controle , Benzilaminas/farmacologia , Cálcio/metabolismo , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/metabolismo , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/antagonistas & inibidores , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/genética , Estimulação Cardíaca Artificial , Células Cultivadas , Eletrocardiografia , Ativação Enzimática , Ventrículos do Coração/enzimologia , Ventrículos do Coração/fisiopatologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Transgênicos , Contração Miocárdica/efeitos dos fármacos , Miócitos Cardíacos/enzimologia , Peptídeos/genética , Peptídeos/metabolismo , Peptídeos/farmacologia , Fosforilação , Inibidores de Proteínas Quinases/farmacologia , Ratos , Ratos Wistar , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Retículo Sarcoplasmático/efeitos dos fármacos , Retículo Sarcoplasmático/enzimologia , Trocador de Sódio e Cálcio/metabolismo , Sulfonamidas/farmacologia , Fatores de Tempo , TransfecçãoRESUMO
The Na(+)/H(+) exchanger (NHE-1) plays a key role in pH(i) recovery from acidosis and is regulated by pH(i) and the ERK1/2-dependent phosphorylation pathway. Since acidosis increases the activity of Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) in cardiac muscle, we examined whether CaMKII activates the exchanger by using pharmacological tools and highly specific genetic approaches. Adult rat cardiomyocytes, loaded with the pH(i) indicator SNARF-1/AM were subjected to different protocols of intracellular acidosis. The rate of pH(i) recovery from the acid load (dpH(i)/dt)-an index of NHE-1 activity in HEPES buffer or in NaHCO(3) buffer in the presence of inhibition of anion transporters-was significantly decreased by the CaMKII inhibitors KN-93 or AIP. pH(i) recovery from acidosis was faster in CaMKII-overexpressing myocytes than in overexpressing beta-galactosidase myocytes (dpH(i)/dt: 0.195+/-0.04 vs. 0.045+/-0.010 min(-)(1), respectively, n=8) and slower in myocytes from transgenic mice with chronic cardiac CaMKII inhibition (AC3-I) than in controls (AC3-C). Inhibition of CaMKII and/or ERK1/2 indicated that stimulation of NHE-1 by CaMKII was independent of and additive to the ERK1/2 cascade. In vitro studies with fusion proteins containing wild-type or mutated (Ser/Ala) versions of the C-terminal domain of NHE-1 indicate that CaMKII phosphorylates NHE-1 at residues other than the canonical phosphorylation sites for the kinase (Ser648, Ser703, and Ser796). These results provide new mechanistic insights and unequivocally demonstrate a role of the already multifunctional CaMKII on the regulation of the NHE-1 activity. They also prove clinically important in multiple disorders which, like ischemia/reperfusion injury or hypertrophy, are associated with increased NHE-1 and CaMKII.