RESUMO
Store-operated Ca(2+) channels (SOCs) are voltage-independent Ca(2+) channels activated upon depletion of the endoplasmic reticulum Ca(2+) stores. Early studies suggest the contribution of such channels to Ca(2+) homeostasis in insulin-secreting pancreatic ß-cells. However, their composition and contribution to glucose-stimulated insulin secretion (GSIS) remains unclear. In this study, endoplasmic reticulum Ca(2+) depletion triggered by acetylcholine (ACh) or thapsigargin stimulated the formation of a ternary complex composed of Orai1, TRPC1, and STIM1, the key proteins involved in the formation of SOCs. Ca(2+) imaging further revealed that Orai1 and TRPC1 are required to form functional SOCs and that these channels are activated by STIM1 in response to thapsigargin or ACh. Pharmacological SOCs inhibition or dominant negative blockade of Orai1 or TRPC1 using the specific pore mutants Orai1-E106D and TRPC1-F562A impaired GSIS in rat ß-cells and fully blocked the potentiating effect of ACh on secretion. In contrast, pharmacological or dominant negative blockade of TRPC3 had no effect on extracellular Ca(2+) entry and GSIS. Finally, we observed that prolonged exposure to supraphysiological glucose concentration impaired SOCs function without altering the expression levels of STIM1, Orai1, and TRPC1. We conclude that Orai1 and TRPC1, which form SOCs regulated by STIM1, play a key role in the effect of ACh on GSIS, a process that may be impaired in type 2 diabetes.
Assuntos
Canais de Cálcio/metabolismo , Sinalização do Cálcio , Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Canais de Cátion TRPC/metabolismo , Acetilcolina/farmacologia , Substituição de Aminoácidos , Animais , Cálcio/metabolismo , Canais de Cálcio/genética , Linhagem Celular Tumoral , Insulina/genética , Secreção de Insulina , Células Secretoras de Insulina/patologia , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Mutação de Sentido Incorreto , Proteína ORAI1 , Ratos , Molécula 1 de Interação Estromal , Canais de Cátion TRPC/genética , Tapsigargina/farmacologiaRESUMO
To what extent hypoxia alters the adenosine (ADO) system and impacts on cardiac function during embryogenesis is not known. Ectonucleoside triphosphate diphosphohydrolase (CD39), ecto-5'-nucleotidase (CD73), adenosine kinase (AdK), adenosine deaminase (ADA), equilibrative (ENT1,3,4), and concentrative (CNT3) transporters and ADO receptors A1, A2A, A2B, and A3 constitute the adenosinergic system. During the first 4 days of development chick embryos were exposed in ovo to normoxia followed or not followed by 6 h hypoxia. ADO and glycogen content and mRNA expression of the genes were determined in the atria, ventricle, and outflow tract of the normoxic (N) and hypoxic (H) hearts. Electrocardiogram and ventricular shortening of the N and H hearts were recorded ex vivo throughout anoxia/reoxygenation ± ADO. Under basal conditions, CD39, CD73, ADK, ADA, ENT1,3,4, CNT3, and ADO receptors were differentially expressed in the atria, ventricle, and outflow tract. In H hearts ADO level doubled, glycogen decreased, and mRNA expression of all the investigated genes was downregulated by hypoxia, except for A2A and A3 receptors. The most rapid and marked downregulation was found for ADA in atria. H hearts were arrhythmic and more vulnerable to anoxia-reoxygenation than N hearts. Despite downregulation of the genes, exposure of isolated hearts to ADO 1) preserved glycogen through activation of A1 receptor and Akt-GSK3ß-GS pathway, 2) prolonged activity and improved conduction under anoxia, and 3) restored QT interval in H hearts. Thus hypoxia-induced downregulation of the adenosinergic system can be regarded as a coping response, limiting the detrimental accumulation of ADO without interfering with ADO signaling.
Assuntos
Adenosina/metabolismo , Coração/embriologia , Hipóxia/embriologia , Hipóxia/metabolismo , Miocárdio/metabolismo , Receptores Purinérgicos P1/metabolismo , 5'-Nucleotidase/genética , 5'-Nucleotidase/metabolismo , Adaptação Fisiológica , Adenosina Quinase/genética , Adenosina Quinase/metabolismo , Animais , Antígenos CD/genética , Antígenos CD/metabolismo , Apirase/genética , Apirase/metabolismo , Embrião de Galinha , Metabolismo Energético , Proteínas de Transporte de Nucleosídeo Equilibrativas/genética , Proteínas de Transporte de Nucleosídeo Equilibrativas/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Glicogênio/metabolismo , Glicogênio Sintase/metabolismo , Quinase 3 da Glicogênio Sintase/metabolismo , Glicogênio Sintase Quinase 3 beta , Coração/fisiopatologia , Hipóxia/genética , Hipóxia/fisiopatologia , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Organogênese , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Mensageiro/metabolismo , Receptores Purinérgicos P1/genética , Transdução de Sinais , Fatores de TempoRESUMO
We previously established that exogenous adenosine (ADO) induces transient arrhythmias in the developing heart via the adenosine A1 receptor (A1AR) and downstream activation of NADPH oxidase/ERK and PLC/PKC pathways. Here, we investigated the mechanisms by which accumulation of endogenous ADO and its derived compound inosine (INO) in the interstitial compartment induce rhythm and conduction troubles. The validated model of the spontaneously beating heart obtained from 4-day-old chick embryos was used. Quantitative RT-PCR showed that enzymes involved in ADO and INO metabolism (CD39, CD73 and eADA) as well as equilibrative (ENT1, -3, -4) and concentrative (CNT3) nucleoside transporters were differentially expressed in atria, ventricle and outflow tract. Inactivation of ENTs by dipyridamole, 1) increased myocardial ADO level, 2) provoked atrial arrhythmias and atrio-ventricular blocks (AVB) in 70% of the hearts, 3) prolonged P wave and QT interval without altering contractility, and 4) increased ERK2 phosphorylation. Blockade of CD73-mediated phosphohydrolysis of AMP to ADO, MEK/ERK pathway inhibition or A1AR inhibition prevented these arrhythmias. Exposure to exogenous INO also caused atrial ectopy associated with AVB and ERK2 phosphorylation which were prevented by A1AR or A2AAR antagonists exclusively or by MEK/ERK inhibitor. Inhibition of ADA-mediated conversion of ADO to INO increased myocardial ADO and decreased INO as expected, but slightly augmented heart rate variability without provoking AVB. Thus, during cardiogenesis, disturbances of nucleosides metabolism and transport, can lead to interstitial accumulation of ADO and INO and provoke arrhythmias in an autocrine/paracrine manner through A1AR and A2AAR stimulation and ERK2 activation.
Assuntos
5'-Nucleotidase/metabolismo , Proteínas de Transporte de Nucleosídeo Equilibrativas/metabolismo , Sistema de Condução Cardíaco/metabolismo , Sistema de Condução Cardíaco/fisiopatologia , Inosina/metabolismo , Receptor A1 de Adenosina/metabolismo , Receptor A2A de Adenosina/metabolismo , Adenosina/metabolismo , Adenosina/farmacologia , Adenosina Desaminase/metabolismo , Animais , Arritmias Cardíacas/genética , Arritmias Cardíacas/metabolismo , Embrião de Galinha , Ativação Enzimática , Regulação da Expressão Gênica , Coração/embriologia , Átrios do Coração/metabolismo , Ventrículos do Coração/metabolismo , Inosina/farmacologia , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Miocárdio/metabolismo , Fosforilação/efeitos dos fármacos , Receptor A1 de Adenosina/genética , Receptor A2A de Adenosina/genética , Transdução de Sinais/efeitos dos fármacosRESUMO
Although the activation of the A(1)-subtype of the adenosine receptors (A(1)AR) is arrhythmogenic in the developing heart, little is known about the underlying downstream mechanisms. The aim of this study was to determine to what extent the transient receptor potential canonical (TRPC) channel 3, functioning as receptor-operated channel (ROC), contributes to the A(1)AR-induced conduction disturbances. Using embryonic atrial and ventricular myocytes obtained from 4-day-old chick embryos, we found that the specific activation of A(1)AR by CCPA induced sarcolemmal Ca(2+) entry. However, A(1)AR stimulation did not induce Ca(2+) release from the sarcoplasmic reticulum. Specific blockade of TRPC3 activity by Pyr3, by a dominant negative of TRPC3 construct, or inhibition of phospholipase Cs and PKCs strongly inhibited the A(1)AR-enhanced Ca(2+) entry. Ca(2+) entry through TRPC3 was activated by the 1,2-diacylglycerol (DAG) analog OAG via PKC-independent and -dependent mechanisms in atrial and ventricular myocytes, respectively. In parallel, inhibition of the atypical PKCζ by myristoylated PKCζ pseudosubstrate inhibitor significantly decreased the A(1)AR-enhanced Ca(2+) entry in both types of myocytes. Additionally, electrocardiography showed that inhibition of TRPC3 channel suppressed transient A(1)AR-induced conduction disturbances in the embryonic heart. Our data showing that A(1)AR activation subtly mediates a proarrhythmic Ca(2+) entry through TRPC3-encoded ROC by stimulating the phospholipase C/DAG/PKC cascade provide evidence for a novel pathway whereby Ca(2+) entry and cardiac function are altered. Thus, the A(1)AR-TRPC3 axis may represent a potential therapeutic target.
Assuntos
Função Atrial , Miocárdio/metabolismo , Receptor A1 de Adenosina/metabolismo , Canais de Cátion TRPC/metabolismo , Função Ventricular , Animais , Western Blotting , Cálcio/metabolismo , Embrião de Galinha , Reação em Cadeia da PolimeraseRESUMO
Whether adenosine, a crucial regulator of the developing cardiovascular system, can provoke arrhythmias in the embryonic/fetal heart remains controversial. Here, we aimed to establish a mechanistic basis of how an adenosinergic stimulation alters function of the developing heart. Spontaneously beating hearts or dissected atria and ventricle obtained from 4-day-old chick embryos were exposed to adenosine or specific agonists of the receptors A(1)AR (CCPA), A(2A)AR (CGS-21680) and A(3)AR (IB-MECA). Expression of the receptors was determined by quantitative PCR. The functional consequences of blockade of NADPH oxidase, extracellular signal-regulated kinase (ERK), phospholipase C (PLC), protein kinase C (PKC) and L-type calcium channel (LCC) in combination with adenosine or CCPA, were investigated in vitro by electrocardiography. Furthermore, the time-course of ERK phosphorylation was determined by western blotting. Expression of A(1)AR, A(2A)AR and A(2B)AR was higher in atria than in ventricle while A(3)AR was equally expressed. Adenosine (100µM) triggered transient atrial ectopy and second degree atrio-ventricular blocks (AVB) whereas CCPA induced mainly Mobitz type I AVB. Atrial rhythm and atrio-ventricular propagation fully recovered after 60min. These arrhythmias were prevented by the specific A(1)AR antagonist DPCPX. Adenosine and CCPA transiently increased ERK phosphorylation and induced arrhythmias in isolated atria but not in ventricle. By contrast, A(2A)AR and A(3)AR agonists had no effect. Interestingly, the proarrhythmic effect of A(1)AR stimulation was markedly reduced by inhibition of NADPH oxidase, ERK, PLC, PKC or LCC. Moreover, NADPH oxidase inhibition or antioxidant MPG prevented both A(1)AR-mediated arrhythmias and ERK phosphorylation. These results suggest that pacemaking and conduction disturbances are induced via A(1)AR through concomitant stimulation of NADPH oxidase and PLC, followed by downstream activation of ERK and PKC with LCC as possible target.
Assuntos
Arritmias Cardíacas/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Coração/embriologia , NADPH Oxidases/metabolismo , Proteína Quinase C/metabolismo , Receptor A1 de Adenosina/metabolismo , Transdução de Sinais , Fosfolipases Tipo C/metabolismo , Adenosina/farmacologia , Animais , Arritmias Cardíacas/genética , Canais de Cálcio Tipo L/metabolismo , Átrios do Coração/efeitos dos fármacos , Átrios do Coração/metabolismo , Ventrículos do Coração/efeitos dos fármacos , Ventrículos do Coração/metabolismo , Camundongos , Modelos Biológicos , Fosforilação/efeitos dos fármacos , RNA Mensageiro/genética , Receptor A1 de Adenosina/genéticaRESUMO
The ability of the developing myocardium to tolerate oxidative stress during early gestation is an important issue with regard to possible detrimental consequences for the fetus. In the embryonic heart, antioxidant defences are low, whereas glycolytic flux is high. The pro- and antioxidant mechanisms and their dependency on glucose metabolism remain to be explored. Isolated hearts of 4-day-old chick embryos were exposed to normoxia (30 min), anoxia (30 min), and hyperoxic reoxygenation (60 min). The time course of ROS production in the whole heart and in the atria, ventricle, and outflow tract was established using lucigenin-enhanced chemiluminescence. Cardiac rhythm, conduction, and arrhythmias were determined. The activity of superoxide dismutase, catalase, gutathione reductase, and glutathione peroxidase as well as the content of reduced and oxidized glutathione were measured. The relative contribution of the ROS-generating systems was assessed by inhibition of mitochondrial complexes I and III (rotenone and myxothiazol), NADPH oxidases (diphenylene iodonium and apocynine), and nitric oxide synthases (N-monomethyl-L-arginine and N-iminoethyl-L-ornithine). The effects of glycolysis inhibition (iodoacetate), glucose deprivation, glycogen depletion, and lactate accumulation were also investigated. In untreated hearts, ROS production peaked at 10.8 ± 3.3, 9 ± 0.8, and 4.8 ± 0.4 min (means ± SD; n = 4) of reoxygenation in the atria, ventricle, and outflow tract, respectively, and was associated with arrhythmias. Functional recovery was complete after 30-40 min. At reoxygenation, 1) the respiratory chain and NADPH oxidases were the main sources of ROS in the atria and outflow tract, respectively; 2) glucose deprivation decreased, whereas glycogen depletion increased, oxidative stress; 3) lactate worsened oxidant stress via NADPH oxidase activation; 4) glycolysis blockade enhanced ROS production; 5) no nitrosative stress was detectable; and 6) the glutathione redox cycle appeared to be a major antioxidant system. Thus, the glycolytic pathway plays a predominant role in reoxygenation-induced oxidative stress during early cardiogenesis. The relative contribution of mitochondria and extramitochondrial systems to ROS generation varies from one region to another and throughout reoxygenation.
Assuntos
Glicólise/fisiologia , Coração/fisiopatologia , Hipóxia/metabolismo , Mitocôndrias Cardíacas/metabolismo , Miocárdio/metabolismo , NADPH Oxidases/metabolismo , Estresse Oxidativo/fisiologia , Traumatismo por Reperfusão/metabolismo , Animais , Embrião de Galinha , Complexo I de Transporte de Elétrons/antagonistas & inibidores , Complexo I de Transporte de Elétrons/metabolismo , Complexo III da Cadeia de Transporte de Elétrons/antagonistas & inibidores , Complexo III da Cadeia de Transporte de Elétrons/metabolismo , Glicogênio/metabolismo , Glicólise/efeitos dos fármacos , Coração/efeitos dos fármacos , Hipóxia/tratamento farmacológico , Hipóxia/fisiopatologia , Iodoacetatos/farmacologia , Lactatos/metabolismo , Metacrilatos/farmacologia , Mitocôndrias Cardíacas/efeitos dos fármacos , Mitocôndrias Cardíacas/fisiologia , NADPH Oxidases/antagonistas & inibidores , NADPH Oxidases/fisiologia , NG-Nitroarginina Metil Éster/farmacologia , Ornitina/análogos & derivados , Ornitina/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Traumatismo por Reperfusão/tratamento farmacológico , Traumatismo por Reperfusão/fisiopatologia , Rotenona/farmacologia , Tiazóis/farmacologiaRESUMO
Activation of the Janus Kinase 2/Signal Transducer and Activator of Transcription 3 (JAK2/STAT3) pathway is known to play a key role in cardiogenesis and to afford cardioprotection against ischemia-reperfusion in adult. However, involvement of JAK2/STAT3 pathway and its interaction with other signaling pathways in developing heart transiently submitted to anoxia remains to be explored. Hearts isolated from 4-day-old chick embryos were submitted to anoxia (30 min) and reoxygenation (80 min) with or without the antioxidant MPG, the JAK2/STAT3 inhibitor AG490 or the PhosphoInositide-3-Kinase (PI3K)/Akt inhibitor LY-294002. Time course of phosphorylation of STAT3α(tyrosine705) and Reperfusion Injury Salvage Kinase (RISK) proteins [PI3K, Akt, Glycogen Synthase Kinase 3beta (GSK3beta), Extracellular signal-Regulated Kinase 2 (ERK2)] was determined in homogenate and in enriched nuclear and cytoplasmic fractions of the ventricle. STAT3 DNA-binding was determined. The chrono-, dromo- and inotropic disturbances were also investigated by electrocardiogram and mechanical recordings. Phosphorylation of STAT3α(tyr705) was increased by reoxygenation, reduced (~50%) by MPG or AG490 but not affected by LY-294002. STAT3 and GSK3beta were detected both in nuclear and cytoplasmic fractions while PI3K, Akt and ERK2 were restricted to cytoplasm. Reoxygenation led to nuclear accumulation of STAT3 but unexpectedly without DNA-binding. AG490 decreased the reoxygenation-induced phosphorylation of Akt and ERK2 and phosphorylation/inhibition of GSK3beta in the nucleus, exclusively. Inhibition of JAK2/STAT3 delayed recovery of atrial rate, worsened variability of cardiac cycle length and prolonged arrhythmias as compared to control hearts. Thus, besides its nuclear translocation without transcriptional activity, oxyradicals-activated STAT3α can rapidly interact with RISK proteins present in nucleus and cytoplasm, without dual interaction, and reduce the anoxia-reoxygenation-induced arrhythmias in the embryonic heart.
Assuntos
Citoplasma/metabolismo , Quinase 3 da Glicogênio Sintase/metabolismo , Traumatismo por Reperfusão Miocárdica/metabolismo , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais/fisiologia , Animais , Embrião de Galinha , Ensaio de Desvio de Mobilidade Eletroforética , Glicogênio Sintase Quinase 3 beta , Immunoblotting , Traumatismo por Reperfusão Miocárdica/embriologia , Traumatismo por Reperfusão Miocárdica/fisiopatologia , Técnicas de Cultura de Órgãos , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
We have previously reported in the early septating embryonic heart that electromechanical disturbances induced by anoxia-reoxygenation are distinct in atria, ventricle, and outflow tract, and are attenuated in ventricle by opening of mitochondrial K(ATP) (mitoK(ATP)) channels. Here, we assessed the regional activation of mitogen-activated protein kinases (MAPKs) ERK, p38, and JNK in response to anoxia-reoxygenation and H(2)O(2). Hearts isolated from 4-day-old chick embryos were subjected to 30-min anoxia and 60-min reoxygenation or exposed to H(2)O(2) (50 microM-1 mM). The temporal pattern of activation of ERK, p38, and JNK in atria, ventricle, and outflow tract was determined using immunoblotting and/or kinase assay. The effect of the mitoK(ATP) channel opener diazoxide (50 microM) on JNK phosphorylation was also analyzed. Under basal conditions, total ERK and JNK were homogeneously distributed within the heart, whereas total p38 was the lowest in outflow tract. The phosphorylated/total form ratio of each MAPK was similar in all regions. Phosphorylation of ERK increased in atria and ventricle at the end of reoxygenation without change in outflow tract. Phosphorylation of p38 was augmented by anoxia in the three regions, and returned to basal level at the end of reoxygenation except in the outflow tract. JNK activity was not altered by anoxia-reoxygenation in atria and outflow tract. In ventricle, however, the diazoxide-inhibitable peak of JNK activity known to occur during reoxygenation was not accompanied by a change in phosphorylation level. H(2)O(2) over 500 microM impaired cardiac function, phosphorylated ERK in all the regions and p38 in atria and outflow tract, but did not affect JNK phosphorylation. At a critical stage of early cardiogenesis, anoxia, reoxygenation, exogenous H(2)O(2) and opening of mitoK(ATP) channels can subtly modulate ERK, p38, and JNK pathways in a region-specific manner.
Assuntos
Coração/embriologia , Peróxido de Hidrogênio/farmacologia , Hipóxia/enzimologia , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Miocárdio/enzimologia , Estresse Oxidativo/efeitos dos fármacos , Animais , Embrião de Galinha , Diazóxido/farmacologia , Relação Dose-Resposta a Droga , Ativação Enzimática , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Coração/efeitos dos fármacos , Hipóxia/embriologia , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Fosforilação , Canais de Potássio/agonistas , Canais de Potássio/metabolismo , Fatores de Tempo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
The hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are expressed in pacemaker cells very early during cardiogenesis. This work aimed at determining to what extent these channels are implicated in the electromechanical disturbances induced by a transient oxygen lack which may occur in utero. Spontaneously beating hearts or isolated ventricles and outflow tracts dissected from 4-day-old chick embryos were exposed to a selective inhibitor of HCN channels (ivabradine 0.1-10microM) to establish a dose-response relationship. The effects of ivabradine on electrocardiogram, excitation-contraction coupling and contractility of hearts submitted to anoxia (30min) and reoxygenation (60min) were also determined. The distribution of the predominant channel isoform, HCN4, was established in atria, ventricle and outflow tract by immunoblotting. Intrinsic beating rate of atria, ventricle and outflow tract was 164+/-22 (n=10), 78+/-24 (n=8) and 40+/-12bpm (n=23, mean+/-SD), respectively. In the whole heart, ivabradine (0.3microM) slowed the firing rate of atria by 16% and stabilized PR interval. These effects persisted throughout anoxia-reoxygenation, whereas the variations of QT duration, excitation-contraction coupling and contractility, as well as the types and duration of arrhythmias were not altered. Ivabradine (10microM) reduced the intrinsic rate of atria and isolated ventricle by 27% and 52%, respectively, whereas it abolished activity of the isolated outflow tract. Protein expression of HCN4 channels was higher in atria and ventricle than in the outflow tract. Thus, HCN channels are specifically distributed and control finely atrial, ventricular and outflow tract pacemakers as well as conduction in the embryonic heart under normoxia and throughout anoxia-reoxygenation.
Assuntos
Antiarrítmicos/farmacologia , Arritmias Cardíacas/prevenção & controle , Benzazepinas/farmacologia , Canais de Cátion Regulados por Nucleotídeos Cíclicos/antagonistas & inibidores , Sistema de Condução Cardíaco/efeitos dos fármacos , Coração/efeitos dos fármacos , Hipóxia/tratamento farmacológico , Oxigênio/metabolismo , Animais , Arritmias Cardíacas/embriologia , Arritmias Cardíacas/metabolismo , Arritmias Cardíacas/fisiopatologia , Relógios Biológicos/efeitos dos fármacos , Western Blotting , Embrião de Galinha , Canais de Cátion Regulados por Nucleotídeos Cíclicos/metabolismo , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Eletrocardiografia , Acoplamento Excitação-Contração/efeitos dos fármacos , Coração/embriologia , Coração/fisiopatologia , Átrios do Coração/efeitos dos fármacos , Átrios do Coração/metabolismo , Sistema de Condução Cardíaco/embriologia , Sistema de Condução Cardíaco/metabolismo , Sistema de Condução Cardíaco/fisiopatologia , Frequência Cardíaca/efeitos dos fármacos , Ventrículos do Coração/efeitos dos fármacos , Ventrículos do Coração/metabolismo , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização , Hipóxia/embriologia , Hipóxia/metabolismo , Hipóxia/fisiopatologia , Ivabradina , Contração Miocárdica/efeitos dos fármacos , Canais de Potássio/metabolismo , Fatores de Tempo , Técnicas de Cultura de TecidosRESUMO
UNLABELLED: L-Type Ca(2+) and K(ATP) Channels in Pacing-Induced Cardioprotection. AIMS: The L-type Ca(2+) channel, the sarcolemmal (sarcK(ATP)), and mitochondrial K(ATP) (mitoK(ATP)) channels are involved in myocardial preconditioning. We aimed at determining to what extent these channels can also participate in pacing-induced cardioprotection. METHODS: Hearts of 4-day-old chick embryos were paced in ovo during 12 hour using asynchronous intermittent ventricular stimulation at 110% of the intrinsic rate. Sham operated and paced hearts were then submitted in vitro to anoxia (30 minutes) and reoxygenation (60 minutes). These hearts were exposed to L-type Ca(2+) channel agonist Bay-K-8644 (BAY-K) or blocker verapamil, nonselective K(ATP) channel antagonist glibenclamide (GLIB), mitoK(ATP) channel agonist diazoxide (DIAZO), or antagonist 5-hydroxydecanoate. Electrocardiogram, electromechanical delay (EMD) reflecting excitation-contraction (E-C) coupling, and contractility were determined. RESULTS: Under normoxia, heart rate, QT duration, conduction, EMD, and ventricular shortening were similar in sham and paced hearts. During reoxygenation, arrhythmias ceased earlier and ventricular EMD recovered faster in paced hearts than in sham hearts. In sham hearts, BAY-K (but not verapamil), DIAZO (but not 5-hydroxydecanoate) or GLIB accelerated recovery of ventricular EMD, reproducing the pacing-induced protection. By contrast, none of these agents further ameliorated recovery of the paced hearts. CONCLUSION: The protective effect of chronic asynchronous pacing at near physiological rate on ventricular E-C coupling appears to be associated with subtle activation of L-type Ca(2+) channel, inhibition of sarcK(ATP) channel, and/or opening of mitoK(ATP) channel.
Assuntos
Estimulação Cardíaca Artificial/métodos , Modelos Animais de Doenças , Hipóxia Fetal/metabolismo , Hipóxia Fetal/prevenção & controle , Traumatismo por Reperfusão Miocárdica/metabolismo , Miocárdio/metabolismo , Canais de Potássio/metabolismo , Animais , Embrião de Galinha , Humanos , Precondicionamento Isquêmico Miocárdico/métodos , Traumatismo por Reperfusão Miocárdica/embriologia , Traumatismo por Reperfusão Miocárdica/prevenção & controle , Resultado do TratamentoRESUMO
INTRODUCTION: The spatio-temporal pattern of arrhythmias in the embryonic/fetal heart subjected to a transient hypoxic or hypothermic stress remains to be established. METHODS AND RESULTS: Spontaneously beating hearts or isolated atria, ventricles, and conotruncus from 4-day-old chick embryos were subjected in vitro to 30-minute anoxia and 60-minute reoxygenation. Hearts were also submitted to 30-minute hypothermia (0-4 degrees C) and 60-minute rewarming. ECG disturbances and alterations of atrial and ventricular electromechanical delay (EMD) were systematically investigated. Baseline functional parameters were stable during at least 2 hours. Anoxia induced tachycardia, followed by bradycardia, atrial ectopy, first-, second-, and third-degree atrio-ventricular blocks and, finally, transient electromechanical arrest after 6.8 minutes, interquartile ranges (IQR) 3.1-16.2 (n = 8). Reoxygenation triggered also Wenckebach phenomenon and ventricular escape beats. At the onset of reoxygenation QT, PR, and ventricular EMD increased by 68%, 70%, and 250%, respectively, whereas atrial EMD was not altered. No fibrillations, no ventricular ectopic beats, and no electromechanical dissociation were observed. Arrhythmic activity of the isolated atria persisted throughout anoxia and upon reoxygenation, whereas activity of the isolated ventricles abruptly ceased after 5 minutes of anoxia and resumed after 5 minutes of reoxygenation. During hypothermia-rewarming, cardiac activity stopped at 17.9 degrees C, IQR 16.2-20.6 (n = 4) and resumed at the same temperature with no arrhythmias. All preparations fully recovered after 40 minutes of reoxygenation or rewarming. CONCLUSION: In the embryonic heart, arrhythmias mainly originated in the sinoatrial tissue and resembled those observed in the adult heart. Furthermore, oxygen readmission was by far more arrhythmogenic than rewarming and the chronotropic, dromotropic, and inotropic effects were fully reversible.
Assuntos
Arritmias Cardíacas/embriologia , Arritmias Cardíacas/fisiopatologia , Coração/embriologia , Coração/fisiopatologia , Hipotermia/fisiopatologia , Hipóxia/fisiopatologia , Oxigênio/uso terapêutico , Reaquecimento/métodos , Animais , Embrião de Galinha , Galinhas , Modelos Animais de Doenças , Sistema de Condução Cardíaco/embriologia , Sistema de Condução Cardíaco/fisiopatologia , Hipotermia/complicações , Hipotermia/terapia , Hipóxia/complicações , Hipóxia/terapia , Técnicas In VitroRESUMO
Although exogenous serotonin at the hypoglossal motor nucleus (HMN) activates the genioglossus muscle, endogenous serotonin plays a minimal role in modulating genioglossus activity in awake and sleeping rats (Sood S, Morrison JL, Liu H, and Horner RL. Am J Respir Crit Care Med 172: 1338-1347, 2005). This result therefore implies that medullary raphe neurons also play a minimal role in the normal physiological control of the HMN, but this has not yet been established because raphe neurons release other excitatory neurotransmitters onto respiratory motoneurons in addition to serotonin. This study tests the hypothesis that inhibition of medullary raphe serotonergic neurons with 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) suppresses genioglossus and diaphragm activities in awake and sleeping rats. Ten rats were implanted with electrodes to record sleep-wake states and genioglossus and diaphragm activities. Microdialysis probes were also implanted into the nucleus raphe obscurus (NRO). Experiments in 10 anesthetized and vagotomized rats were also performed using the same methodology. In anesthetized rats, microdialysis perfusion of 0.1 mM 8-OH-DPAT into the NRO decreased genioglossus activity by 60.7+/-9.0% and diaphragm activity by 13.3+/-3.4%. Diaphragm responses to 7.5% CO2 were also significantly reduced by 8-OH-DPAT. However, despite the robust effects observed in anesthetized and vagotomized rats, there was no effect of 0.1 mM 8-OH-DPAT on genioglossus or diaphragm activities in conscious rats awake or asleep. The results support the concept that endogenously active serotonergic medullary raphe neurons play a minimal role in modulating respiratory motor activity across natural sleep-wake states in freely behaving rodents. This result has implications for pharmacological strategies aiming to manipulate raphe neurons and endogenous serotonin in obstructive sleep apnea.
Assuntos
Diafragma/fisiologia , Bulbo/fisiologia , Núcleos da Rafe/fisiologia , Serotonina/fisiologia , Língua/fisiologia , Inconsciência/fisiopatologia , 8-Hidroxi-2-(di-n-propilamino)tetralina/farmacologia , Animais , Pressão Sanguínea/efeitos dos fármacos , Pressão Sanguínea/fisiologia , Dióxido de Carbono/farmacologia , Diafragma/efeitos dos fármacos , Eletroencefalografia , Nervo Hipoglosso/efeitos dos fármacos , Nervo Hipoglosso/fisiologia , Masculino , Bulbo/efeitos dos fármacos , Neurônios Motores/efeitos dos fármacos , Neurônios Motores/fisiologia , Neurotransmissores/fisiologia , Núcleos da Rafe/efeitos dos fármacos , Ratos , Ratos Wistar , Agonistas do Receptor de Serotonina/farmacologia , Língua/efeitos dos fármacos , VagotomiaRESUMO
In vivo exposure to chronic hypoxia is considered to be a cause of myocardial dysfunction, thereby representing a deleterious condition, but repeated aeration episodes may exert some cardioprotection. We investigated the possible role of ATP-sensitive potassium channels in these mechanisms. First, rats (n = 8/group) were exposed for 14 days to either chronic hypoxia (CH; 10% O(2)) or chronic hypoxia with one episode/day of 1-hr normoxic aeration (CH+A), with normoxia (N) as the control. Second, isolated hearts were Langendorff perfused under hypoxia (10% O(2), 30 min) and reoxygenated (94% O(2), 30 min) with or without 3 microM glibenclamide (nonselective K(+)(ATP) channel-blocker) or 100 microM diazoxide (selective mitochondrial K(+)(ATP) channel-opener). Blood gasses, hemoglobin concentration, and plasma malondialdehyde were similar in CH and CH+A and in both different from normoxic (P < 0.01), body weight gain and plasma nitrate/nitrite were higher in CH+A than CH (P < 0.01), whereas apoptosis (number of TUNEL-positive nuclei) was less in CH+A than CH (P < 0.05). During in vitro hypoxia, the efficiency (ratio of ATP production/pressure x rate product) was the same in all groups and diazoxide had no measurable effects on myocardial performance, whereas glibenclamide increased end-diastolic pressure more in N and CH than in CH+A hearts (P < 0.05). During reoxgenation, efficiency was markedly less in CH with respect to N and CH+A (P < 0.0001), and ratex pressure product remained lower in CH than N and CH+A hearts (P < 0.001), but glibenclamide or diazoxide abolished this difference. Glibenclamide, but not diazoxide, decreased vascular resistance in N and CH (P < 0.005 and < 0.001) without changes in CH+A. We hypothesize that cardioprotection in chronically hypoxic hearts derive from cell depolarization by sarcolemmal K(+)(ATP) blockade or from preservation of oxidative phosphorylation efficiency (ATP turnover/myocardial performance) by mitochondrial K(+)(ATP) opening. Therefore K(+)(ATP) channels are involved in the deleterious effects of chronic hypoxia and in the cardioprotection elicited when chronic hypoxia is interrupted with short normoxic aeration episodes.
Assuntos
Coração/efeitos dos fármacos , Coração/fisiopatologia , Hipóxia/fisiopatologia , Miocárdio/patologia , Canais de Potássio/metabolismo , Animais , Apoptose/fisiologia , Doença Crônica , Glibureto/farmacologia , Marcação In Situ das Extremidades Cortadas , Masculino , Reperfusão Miocárdica , Técnicas de Cultura de Órgãos , Estresse Oxidativo/fisiologia , Bloqueadores dos Canais de Potássio/farmacologia , Canais de Potássio/efeitos dos fármacos , Ratos , Ratos Sprague-DawleyRESUMO
AIMS: It is well established that dysfunction of voltage-dependent ion channels results in arrhythmias and conduction disturbances in the foetal and adult heart. However, the involvement of voltage-insensitive cationic TRPC (transient receptor potential canonical) channels remains unclear. We assessed the hypothesis that TRPC channels play a crucial role in the spontaneous activity of the developing heart. METHODS AND RESULTS: TRPC isoforms were investigated in isolated hearts obtained from 4-day-old chick embryos. Using RT-PCR, western blotting and co-immunoprecipitation, we report for the first time that TRPC1, 3, 4, 5, 6, and 7 isoforms are expressed at the mRNA and protein levels and that they can form a macromolecular complex with the α1C subunit of the L-type voltage-gated calcium channel (Cav1.2) in atria and ventricle. Using ex vivo electrocardiograms, electrograms of isolated atria and ventricle and ventricular mechanograms, we found that inhibition of TRPC channels by SKF-96365 leads to negative chrono-, dromo-, and inotropic effects, prolongs the QT interval, and provokes first- and second-degree atrioventricular blocks. Pyr3, a specific antagonist of TRPC3, affected essentially atrioventricular conduction. On the other hand, specific blockade of the L-type calcium channel with nifedipine rapidly stopped ventricular contractile activity without affecting rhythmic electrical activity. CONCLUSIONS: These results give new insights into the key role that TRPC channels, via interaction with the Cav1.2 channel, play in regulation of cardiac pacemaking, conduction, ventricular activity, and contractility during cardiogenesis.
Assuntos
Sistema de Condução Cardíaco/metabolismo , Coração/embriologia , Canais de Cátion TRPC/metabolismo , Aminoquinolinas/farmacologia , Aminoquinolinas/toxicidade , Animais , Bloqueio Atrioventricular/induzido quimicamente , Bloqueio Atrioventricular/metabolismo , Western Blotting , Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio Tipo L/efeitos dos fármacos , Canais de Cálcio Tipo L/metabolismo , Embrião de Galinha , Relação Dose-Resposta a Droga , Eletrocardiografia , Técnicas Eletrofisiológicas Cardíacas , Regulação da Expressão Gênica no Desenvolvimento , Coração/efeitos dos fármacos , Sistema de Condução Cardíaco/efeitos dos fármacos , Sistema de Condução Cardíaco/embriologia , Frequência Cardíaca , Contração Miocárdica , Nifedipino/farmacologia , Reação em Cadeia da Polimerase , Pirazóis/farmacologia , Pirazóis/toxicidade , RNA Mensageiro/metabolismo , Canais de Cátion TRPC/antagonistas & inibidores , Canais de Cátion TRPC/genética , Fatores de Tempo , Função VentricularRESUMO
Whether the response of the fetal heart to ischemia-reperfusion is associated with activation of the c-Jun N-terminal kinase (JNK) pathway is not known. In contrast, involvement of the sarcolemmal L-type Ca2+ channel (LCC) and the mitochondrial KATP (mitoKATP) channel has been established. This work aimed at investigating the profile of JNK activity during anoxia-reoxygenation and its modulation by LCC and mitoK(ATP) channel. Hearts isolated from 4-day-old chick embryos were submitted to anoxia (30 min) and reoxygenation (60 min). Using the kinase assay method, the profile of JNK activity in the ventricle was determined every 10 min throughout anoxia-reoxygenation. Effects on JNK activity of the LCC blocker verapamil (10 nM), the mitoK(ATP) channel opener diazoxide (50 microM) and the blocker 5-hydroxydecanoate (5-HD, 500 microM), the mitochondrial Ca2+ uniporter (MCU) inhibitor Ru360 (10 microM), and the antioxidant N-(2-mercaptopropionyl) glycine (MPG, 1 mM) were determined. In untreated hearts, JNK activity was increased by 40% during anoxia and peaked fivefold relative to basal level after 30-40 min reoxygenation. This peak value was reduced by half by diazoxide and was tripled by 5-HD. Furthermore, the 5-HD-mediated stimulation of JNK activity during reoxygenation was abolished by diazoxide, verapamil or Ru360. MPG had no effect on JNK activity, whatever the conditions. None of the tested pharmacological agents altered JNK activity under basal normoxic conditions. Thus, in the embryonic heart, JNK activity exhibits a characteristic pattern during anoxia and reoxygenation and the respective open-state of LCC, MCU and mitoKATP channel can be a major determinant of JNK activity in a ROS-independent manner.
Assuntos
Cálcio/metabolismo , Coração/embriologia , Hipóxia/enzimologia , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Canais de Potássio/metabolismo , Animais , Canais de Cálcio/metabolismo , Galinhas , Estabilidade Enzimática , Ventrículos do Coração/embriologia , Ventrículos do Coração/enzimologia , Técnicas In Vitro , Ativação do Canal Iônico , Modelos Biológicos , Sarcolema/metabolismoRESUMO
Whereas previous studies have shown that opening of the mitochondrial ATP-sensitive K(+) (mitoK(ATP)) channel protects the adult heart against ischemia-reperfusion injury, it remains to be established whether this mechanism also operates in the developing heart. Isolated spontaneously beating hearts from 4-day-old chick embryos were subjected to 30 min of anoxia followed by 60 min of reoxygenation. The chrono-, dromo-, and inotropic disturbances, as well as alterations of the electromechanical delay (EMD), reflecting excitation-contraction (E-C) coupling, were investigated. Production of reactive oxygen species (ROS) in the ventricle was determined using the intracellular fluorescent probe 2',7'-dichlorofluorescin (DCFH). Effects of the specific mitoK(ATP) channel opener diazoxide (Diazo, 50 microM) or the blocker 5-hydroxydecanoate (5-HD, 500 microM), the nitric oxide synthase (NOS) inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME, 50 microM), the antioxidant N-(2-mercaptopropionyl)glycine (MPG, 1 mM), and the PKC inhibitor chelerythrine (Chel, 5 microM) on oxidative stress and postanoxic functional recovery were determined. Under normoxia, the baseline parameters were not altered by any of these pharmacological agents, alone or in combination. During the first 20 min of postanoxic reoxygenation, Diazo doubled the peak of ROS production and, interestingly, accelerated recovery of ventricular EMD and the PR interval. Diazo-induced ROS production was suppressed by 5-HD, MPG, or L-NAME, but not by Chel. Protection of ventricular EMD by Diazo was abolished by 5-HD, MPG, L-NAME, or Chel, whereas protection of the PR interval was abolished by L-NAME exclusively. Thus pharmacological opening of the mitoK(ATP) channel selectively improves postanoxic recovery of cell-to-cell communication and ventricular E-C coupling. Although the NO-, ROS-, and PKC-dependent pathways also seem to be involved in this cardioprotection, their interrelation in the developing heart can differ markedly from that in the adult myocardium.
Assuntos
Glicina/análogos & derivados , Coração/embriologia , Hipóxia/metabolismo , Proteínas de Membrana/metabolismo , Traumatismo por Reperfusão Miocárdica/metabolismo , Óxido Nítrico/metabolismo , Proteína Quinase C/metabolismo , Animais , Antiarrítmicos/farmacologia , Nó Atrioventricular/fisiologia , Embrião de Galinha , Galinhas , Ácidos Decanoicos/farmacologia , Diazóxido/farmacologia , Inibidores Enzimáticos/farmacologia , Sequestradores de Radicais Livres/farmacologia , Glicina/farmacologia , Hidroxiácidos/farmacologia , Miocárdio/metabolismo , NG-Nitroarginina Metil Éster/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/fisiologia , Oxigênio/farmacologia , Canais de Potássio , Proteína Quinase C/antagonistas & inibidores , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/fisiologia , Compostos de Sulfidrila/farmacologia , Vasodilatadores/farmacologiaRESUMO
The developing cardiovascular system is known to operate normally in a hypoxic environment. However, the functional and ultrastructural recovery of embryonic/fetal hearts subjected to anoxia lasting as long as hypoxia/ischemia performed in adult animal models remains to be investigated. Isolated spontaneously beating hearts from Hamburger-Hamilton developmental stages 14 (14HH), 20HH, 24HH, and 27HH chick embryos were subjected in vitro to 30 or 60 min of anoxia followed by 60 min of reoxygenation. Morphological alterations and apoptosis were assessed histologically and by transmission electron microscopy. Anoxia provoked an initial tachycardia followed by bradycardia leading to complete cardiac arrest, except for in the youngest heart, which kept beating. Complete atrioventricular block appeared after 9.4 +/- 1.1, 1.7 +/- 0.2, and 1.6 +/- 0.3 min at stages 20HH, 24HH, and 27HH, respectively. At reoxygenation, sinoatrial activity resumed first in the form of irregular bursts, and one-to-one atrioventricular conduction resumed after 8, 17, and 35 min at stages 20HH, 24HH, and 27HH, respectively. Ventricular shortening recovered within 30 min except at stage 27HH. After 60 min of anoxia, stage 27HH hearts did not retrieve their baseline activity. Whatever the stage and anoxia duration, nuclear and mitochondrial swelling observed at the end of anoxia were reversible with no apoptosis. Thus the embryonic heart is able to fully recover from anoxia/reoxygenation although its anoxic tolerance declines with age. Changes in cellular homeostatic mechanisms rather than in energy metabolism may account for these developmental variations.
Assuntos
Coração/embriologia , Coração/fisiopatologia , Hipóxia/fisiopatologia , Oxigênio/farmacologia , Recuperação de Função Fisiológica , Animais , Bradicardia/fisiopatologia , Embrião de Galinha , Espaço Extracelular/química , Espaço Extracelular/metabolismo , Coração/efeitos dos fármacos , Bloqueio Cardíaco/fisiopatologia , Frequência Cardíaca/efeitos dos fármacos , Concentração de Íons de Hidrogênio , Técnicas In Vitro , Ácido Láctico/biossíntese , Taquicardia/fisiopatologia , Fatores de TempoRESUMO
BACKGROUND/AIM: Excitation-contraction coupling is modulated by nitric oxide (NO) which otherwise has either beneficial or detrimental effects on myocardial function during hypoxia-reoxygenation. This work aimed at characterizing the variations of electromechanical delay (EMD) induced by anoxia-reoxygenation within the developing heart and determining whether atrial and ventricular EMD are modulated by NO to the same extent. METHODS: Hearts of 4 or 4.5-day-old chick embryos were excised and submitted in vitro to normoxia (45 min), anoxia (30 min) and reoxygenation (60 min). Electrocardiogram and atrial and ventricular contractions were simultaneously recorded throughout experiment. Anoxia-reoxygenation-induced chrono-, dromo-and inotropic disturbances and changes in EMD in atrium (EMDa) and ventricle (EMDv) were investigated in control hearts and in hearts exposed to 0.1, 1, 10, 50 and 100 microM of DETA-NONOate (a NO donating agent) or to 50 microM of L-NAME (a NOS inhibitor). RESULTS: Under normoxia, heart rate, PR interval, ventricular shortening velocity, EMDa and EMDv were similar in control, L-NAME-treated and DETA-NONOate-treated hearts. Under anoxia, cardiac activity became markedly erratic within less than 10 min in all groups. At the onset of reoxygenation, EMDv was increased by about 300% with respect to the preanoxic value while EMDa did not vary significatively. Compared to control conditions, L-NAME or DETA-NONOate had no influence on the negative chrono-, dromo- and inotropic effects induced by anoxia-reoxygenation. However, L-NAME prolonged EMDv during anoxia and delayed EMDv recovery during reoxygenation while 100 microM DETA-NONOate had the opposite effects. EMDa was neither affected by NOS inhibitor nor NO donor. At the end of reoxygenation, all the investigated parameters returned to their basal values. CONCLUSION: This work provides evidence that a NO-dependent pathway is involved in regulation of the ventricular excitation-contraction coupling in the anoxic-reoxygenated developing heart.
Assuntos
Ventrículos do Coração/patologia , Coração/embriologia , Óxido Nítrico/química , Animais , Embrião de Galinha , Galinhas , Eletrocardiografia , Eletrofisiologia/métodos , Átrios do Coração/embriologia , Ventrículos do Coração/embriologia , Hipóxia/metabolismo , Técnicas In Vitro , Contração Miocárdica , Miocárdio/metabolismo , Miocárdio/patologia , NG-Nitroarginina Metil Éster/farmacologia , Óxido Nítrico/metabolismo , Compostos Nitrosos/farmacologia , Oxigênio/metabolismo , Traumatismo por Reperfusão , Transdução de Sinais , Fatores de TempoRESUMO
Zebrafish and Xenopus have become popular model organisms for studying vertebrate development of many organ systems, including the heart. However, it is not clear whether the single ventricular hearts of these species possess any equivalent of the specialized ventricular conduction system found in higher vertebrates. Isolated hearts of adult zebrafish (Danio rerio) and African toads (Xenopus laevis) were stained with voltage-sensitive dye and optically mapped in spontaneous and paced rhythms followed by histological examination focusing on myocardial continuity between the atrium and the ventricle. Spread of the excitation wave through the atria was uniform with average activation times of 20 +/- 2 and 50 +/- 2 ms for zebrafish and Xenopus toads, respectively. After a delay of 47 +/- 8 and 414 +/- 16 ms, the ventricle became activated first in the apical region. Ectopic ventricular activation was propagated significantly more slowly (total ventricular activation times: 24 +/- 3 vs. 14 +/- 2 ms in zebrafish and 74 +/- 14 vs. 35 +/- 9 ms in Xenopus). Although we did not observe any histologically defined tracts of specialized conduction cells within the ventricle, there were trabecular bands with prominent polysialic acid-neural cell adhesion molecule staining forming direct myocardial continuity between the atrioventricular canal and the apex of the ventricle; i.e., the site of the epicardial breakthrough. We thus conclude that these hearts are able to achieve the apex-to-base ventricular activation pattern observed in higher vertebrates in the apparent absence of differentiated conduction fascicles, suggesting that the ventricular trabeculae serve as a functional equivalent of the His-Purkinje system.