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Interleukin 6 (IL-6), an important component of cardiac microenvironment, favors cardiac repair by improving cardiomyocyte regeneration in different models. This study aimed to investigate the effects of IL-6 on stemness maintenances and cardiac differentiation of mouse embryonic stem cells (mESCs). The mESCs were treated with IL-6 for two days, and then subjected to CCK-8 essay for proliferation analysis and quantitative real-time PCR (qPCR) to evaluate the mRNA expression of genes related to stemness and germinal layers differentiation. Phosphorylation levels of stem cell-related signal pathways were detected by Western blot. siRNA was used to interfere the function of STAT3 phosphorylation. Cardiac differentiation was investigated by the percentage of beating embryoid bodies (EBs) and qPCR analysis of cardiac progenitor markers and cardiac ion channels. IL-6 neutralization antibody was applied to block the endogenous IL-6 effects since the onset of cardiac differentiation (embryonic day of 0, EB0). The EBs were collected on EB7, EB10 and EB15 to investigate the cardiac differentiation by qPCR. On EB15, Western blot was applied to investigate the phosphorylation of several signaling pathways, and immunochemistry staining was adopted to trace the cardiomyocytes. IL-6 antibody was administered for two days (short term) on EB4, EB7, EB10 or EB15, and percentages of beating EBs at late developmental stage were recorded. The results showed that exogenous IL-6 promoted mESCs proliferation and favored maintenances of pluripotency, evidenced by up-regulated mRNA expression of oncogenes (c-fos, c-jun) and stemness markers (oct4, nanog), down-regulated mRNA expression of germ layer genes (branchyury, FLK-1, pecam, ncam, sox17), and increased phosphorylation of ERK1/2 and STAT3. siRNA targeting JAK/STAT3 partially attenuated the effects of IL-6 on cell proliferation and mRNA expression of c-fos and c-jun. During differentiation, long term IL-6 neutralization antibody application decreased the percentage of beating EBs, down-regulated mRNA expression of ISL1, GATA4, α-MHC, cTnT, kir2.1, cav1.2, and declined the fluorescence intensity of cardiac α actinin in EBs and single cell. Long term IL-6 antibody treatment decreased the phosphorylation of STAT3. In addition, short term (2 d) IL-6 antibody treatment starting from EB4 significantly reduced the percentage of beating EBs in late development stage, while short term IL-6 antibody treatment starting from EB10 significantly increased the percentage of beating EBs on EB16. These results suggest that exogenous IL-6 promotes mESCs proliferation and favors stemness maintenance. Endogenous IL-6 regulates mESC cardiac differentiation in a development-dependent manner. These findings provide important basis for the study of microenvironment on cell replacement therapy, as well as a new perspective for understanding the pathophysiology of heart diseases.
Assuntos
Interleucina-6 , Células-Tronco Embrionárias Murinas , Animais , Camundongos , Diferenciação Celular , Proteínas Proto-Oncogênicas c-fos , RNA MensageiroRESUMO
BACKGROUND: Alzheimer's disease (AD) is a persistent neuropathological injury that manifests via neuronal/synaptic death, age spot development, tau hyperphosphorylation, neuroinflammation, and apoptosis. Synapsin 1 (SYN1), a neuronal phosphoprotein, is believed to be responsible for the pathology of AD. OBJECTIVE: This study aimed to elucidate the exact role of SYN1 in ameliorating AD and its potential regulatory mechanisms. METHODS: The AD dataset GSE48350 was downloaded from the GEO database, and SYN1 was focused on differential expression analysis and Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses. After establishing an AD rat model, they were treated with RNAi lentivirus to trigger SYN1 overexpression. The amelioration of SYN1 in AD-associated behavior was validated using multiple experiments (water maze test and object recognition test). SYN1's repairing effect on the important factors in AD was confirmed by detecting the concentration of inflammatory factors (interleukin (IL)-6, IL-1ß, tumor necrosis factor (TNF)-α), neurotransmitters (acetylcholine (ACh), dopamine (DA), and 5-hydroxytryptophan (5-HT)) and markers of oxidative stress (glutathione (GSH), malondialdehyde (MDA), reactive oxygen species (ROS)). Molecular biology experiments (qRT-PCR and western blot) were performed to examine AD-related signaling pathways after SYN1 overexpression. RESULTS: Differential expression analysis yielded a total of 545 differentially expressed genes, of which four were upregulated and 541 were downregulated. The enriched pathways were basically focused on synaptic functions, and the analysis of the protein- protein interaction network focused on the key genes in SYN1. SYN1 significantly improved the spatial learning and memory abilities of AD rats. This enhancement was reflected in the reduced escape latency of the rats in the water maze, the significantly extended dwell time in the third quadrant, and the increased number of crossings. Furthermore, the results of the object recognition test revealed reduced time for rats to explore familiar and new objects. After SYN1 overexpression, the cAMP signaling pathway was activated, the phosphorylation levels of the CREB and PKA proteins were elevated, and the secretion of neurotransmitters such as ACh, DA, and 5-HT was promoted. Furthermore, oxidative stress was suppressed, as supported by decreased levels of MDA and ROS. Regarding inflammatory factors, the levels of IL-6, IL-1ß, and TNF-α were significantly reduced in AD rats with SYN1 overexpression. CONCLUSION: SYN1 overexpression improves cognitive function and promotes the release of various neurotransmitters in AD rats by inhibiting oxidative stress and inflammatory responses through cAMP signaling pathway activation. These findings may provide a theoretical basis for the targeted diagnosis and treatment of AD.
Assuntos
Doença de Alzheimer , Disfunção Cognitiva , Ratos , Animais , Doença de Alzheimer/complicações , Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Sinapsinas/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Peptídeos beta-Amiloides/metabolismo , Doenças Neuroinflamatórias , Serotonina/metabolismo , Disfunção Cognitiva/genética , Estresse Oxidativo , Fator de Necrose Tumoral alfa/metabolismo , Interleucina-6/metabolismo , Neurotransmissores , Modelos Animais de DoençasRESUMO
The study aims to investigate the effects of cardiac fibroblast (CF) paracrine factors on murine embryonic stem cells (ESCs). Conditioned mediums from either neonatal cardiac fibroblasts (ConM-NCF) or adult cardiac fibroblasts (ConM-ACF) were diluted by 1:50 and 1:5, respectively, to investigate whether these conditioned mediums impact murine ESCs distinctly with RT-real time PCR techniques, cell proliferation essay, ELISA and by counting percentage of beating embryoid bodies (EBs) during ESCs differentiation. The data showed that the paracrine ability of CFs changed dramatically during development, in which interleukin 6 (IL6) increased with maturation. ConM-NCF 1:50 and ConM-NCF 1:5 had opposite effects on the pluripotent markers, although they both reduced mouse ESC proliferation. ConM-ACF 1:50 promoted ESCs pluripotent markers and proliferation, while ConM-ACF 1:5 exerted negative effects. All CF-derived conditioned mediums inhibited cardiac differentiation, but with distinguishable features: ConM-NCF 1:50 slightly decreased the early cardiac differentiation without altering the maturation tendency or cardiac specific markers in EBs at differentiation of day 17; ConM-ACF 1:50 had more significant inhibitory effects on early cardiac differentiation than ConM-NCF 1:50 and impeded cardiac maturation with upregulation of cardiac specific markers. In addition, IL6 neutralization antibody attenuated positive effect of ConM-ACF 1:50 on ESCs proliferation, but had no effects on ConM-NCF 1:50. Long-term IL6 neutralization reduced the percentage of beating EBs at early developmental stage, but did not alter the late cardiac differentiation. Taken together, both the quality and quantity of factors and cytokines secreted by CFs are critical for the ESC fate. IL6 could be a favorable cytokine for ESC pluripotency and the early cardiac differentiation.
Assuntos
Células-Tronco Embrionárias , Células-Tronco Embrionárias Murinas , Animais , Fibroblastos , Coração , Camundongos , Comunicação ParácrinaRESUMO
BACKGROUND: Little is known about the clinical significance of upper esophageal sphincter (UES) motility disorders and their association with the treatment response of type II achalasia. None of the three versions of the Chicago Classification of Esophageal Motility Disorders has defined UES abnormality metrics or their function. UES abnormalities exist in some patients and indicate a clinically significant problem in patients with achalasia. AIM: To demonstrate the manometric differentiation on high-resolution esophageal manometry between subjects with abnormal UES and normal UES, and the association between UES type and the treatment response of type II achalasia. METHODS: In total, 498 consecutive patients referred for high-resolution esophageal manometry were analyzed retrospectively. The patients were divided into two groups, those with normal and abnormal UES function. UES parameters were analyzed after determining lower esophageal sphincter (LES) function. Patients with type II achalasia underwent pneumatic dilation for treatment. Using mixed model analyses, correlations between abnormal UES and treatment response were calculated among subjects with type II achalasia. RESULTS: Of the 498 consecutive patients, 246 (49.40%) were found to have UES abnormalities. Impaired relaxation alone was the most common UES abnormality (52.85%, n = 130). The incidence rate of type II achalasia was significantly higher in subjects with abnormal UES than those with normal UES (9.77% vs 2.58%, P = 0.01). After pneumatic dilation, LES resting pressure, LES integrated relaxation pressure, and UES residual pressure were significantly decreased (41.91 ± 9.20 vs 26.18 ± 13.08, 38.94 ± 10.28 vs 16.71 ± 5.65, and 11.18 ± 7.93 vs 5.35 ± 4.77, respectively, P < 0.05). According to the Eckardt score, subjects with type II achalasia and abnormal UES presented a significantly poorer treatment response than those with normal UES (83.33% vs 0.00%, P < 0.05). CONCLUSION: Impaired relaxation alone is the most common UES abnormality. The incidence of type II achalasia is associated with abnormal UES. Type II achalasia with abnormal UES has a poorer treatment response, which is a potentially prognostic indicator of treatment for this disease.
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This study aimed to examine the functional role of microRNA-20 (miR-20) and its potential target, Kir6.1, in ischemic myocardiocytes. The expression of miR-20 was detected by real-time PCR. Myocardiocytes were stained with terminal deoxynucleotidyl transferase dUTP nick end labeling (TU-NEL) reagent for apoptosis evaluation. Western blotting was used to detect the Kir6.1 protein in ischemic myocardiocytes transfected with miR-20 mimics or inhibitors. Luciferase reporter gene assay was performed to confirm the targeting effect of miR-20 on KCNJ8. The results showed that miR-20 was remarkably down-regulated, while the KATP subunit Kir6.1 was significantly up-regulated, during myocardial ischemia. The miR-20 overexpression promoted the apoptosis of ischemic myocardiocytes, but showed no such effect on normal cells. Under ischemic condition, myocardiocytes transfected with miR-20 mimics expressed less Kir6.1. On the contrary, inhibiting miR-20 increased the expression of Kir6.1 in the cells. Co-transfection of miR-20 mimics with the KCNJ8 3'-UTR plasmid into HEK293 cells consistently produced less luciferase activity than transfection of the plasmid alone. It was concluded that miR-20 may regulate myocardiac ischemia by targeting KATP subunit Kir6.1 to accelerate the cell apoptosis. Therefore miR-20 may serve as a therapeutic target for myocardial ischemic disease.
Assuntos
Canais KATP/metabolismo , MicroRNAs/metabolismo , Isquemia Miocárdica/genética , Subunidades Proteicas/metabolismo , Regiões 3' não Traduzidas/genética , Animais , Apoptose/genética , Linhagem Celular , Regulação para Baixo/genética , Canais KATP/genética , Camundongos , MicroRNAs/genética , Isquemia Miocárdica/patologia , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Subunidades Proteicas/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
Thymosin ß4 (Tß4) is a key factor in cardiac development, growth, disease, epicardial integrity, blood vessel formation and has cardio-protective properties. However, its role in murine embryonic stem cells (mESCs) proliferation and cardiovascular differentiation remains unclear. Thus we aimed to elucidate the influence of Tß4 on mESCs. Target genes during mESCs proliferation and differentiation were detected by real-time PCR or Western blotting, and patch clamp was applied to characterize the mESCs-derived cardiomyocytes. It was found that Tß4 decreased mESCs proliferation in a partial dose-dependent manner and the expression of cell cycle regulatory genes c-myc, c-fos and c-jun. However, mESCs self-renewal markers Oct4 and Nanog were elevated, indicating the maintenance of self-renewal ability in these mESCs. Phosphorylation of STAT3 and Akt was inhibited by Tß4 while the expression of RAS and phosphorylation of ERK were enhanced. No significant difference was found in BMP2/BMP4 or their downstream protein smad. Wnt3 and Wnt11 were remarkably decreased by Tß4 with upregulation of Tcf3 and constant ß-catenin. Under mESCs differentiation, Tß4 treatment did not change the expression of cardiovascular cell markers α-MHC, PECAM, and α-SMA. Neither the electrophysiological properties of mESCs-derived cardiomyocytes nor the hormonal regulation by Iso/Cch was affected by Tß4. In conclusion, Tß4 suppressed mESCs proliferation by affecting the activity of STAT3, Akt, ERK and Wnt pathways. However, Tß4 did not influence the in vitro cardiovascular differentiation.
Assuntos
Proliferação de Células/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Células-Tronco Embrionárias Murinas/efeitos dos fármacos , Miócitos Cardíacos/efeitos dos fármacos , Timosina/farmacologia , Animais , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/genética , Diferenciação Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , MAP Quinases Reguladas por Sinal Extracelular/genética , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno/genética , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Camundongos , Células-Tronco Embrionárias Murinas/citologia , Células-Tronco Embrionárias Murinas/metabolismo , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , Proteína Homeobox Nanog/genética , Proteína Homeobox Nanog/metabolismo , Fator 3 de Transcrição de Octâmero/genética , Fator 3 de Transcrição de Octâmero/metabolismo , Técnicas de Patch-Clamp , Cultura Primária de Células , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas c-fos/genética , Proteínas Proto-Oncogênicas c-fos/metabolismo , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Fator de Transcrição STAT3/genética , Fator de Transcrição STAT3/metabolismo , Transdução de SinaisRESUMO
Cirrhotic rats show higher expression levels of hepatic RhoA and Rho-kinase than normal healthy rats, and the activation of this signaling pathway leads to portal hypertension. Sodium ferulate (SF) has been shown to decrease the production of geranylgeranyl pyrophosphate (GGPP), a substance essential for RhoA activation. In the present study, to investigate the effects of SF on fibrosis, portal hypertension and the RhoA/Rho-kinase pathway, hepatic cirrhosis was induced in rats by bile duct ligation. Liver function and fibrogenesis-related biochemical parameters, the hepatic hydroxyproline content, the pathological characteristics of the liver sections and the levels of hepatic α-smooth muscle actin (α-SMA; by immunohistochemistry) were analyzed to assess effects of SF on hepatic fibrosis. In addition, hepatic RhoA, Rho-kinase and endothelial nitric oxide synthase (eNOS) expression was examined by immunohistochemistry. Apoptosis in the SF-treated and SF + GGPP-treated rat primary hepatic stellate cells (HSCs) and a human stellate cell line (LX-2) was examined by flow cytometry. Intrahepatic resistance and responsiveness to the α1-adrenoceptor agonist, methoxamine, were investigated by in situ liver perfusion. Treatment with SF did not affect fibrosis-related biochemical parameters or the hydroxyproline content; however, SF reduced the histological evidence of fibrosis and hepatocyte damage. The SF-treated rats had a significantly lower expression of α-SMA and Rho-kinase, as well as an increased hepatic eNOS content; however, SF did not affect RhoA expression. The SF-treated HSCs had a significantly increased apoptotic rate compared to the untreated rats. Following the addition of GGPP, the rate apoptotic rate decreased. SF reduced basal intrahepatic resistance and the responsiveness of hepatic vascular smooth muscle to methoxamine. Therefore, our data demonstrate that SF reduces fibrogenesis, decreases portal pressure in cirrhotic rats and inhibits the activation of the RhoA/Rho-kinase signaling pathway.
Assuntos
Ácidos Cumáricos/farmacologia , Cirrose Hepática Biliar/tratamento farmacológico , Pressão na Veia Porta/efeitos dos fármacos , Quinases Associadas a rho/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismo , Animais , Apoptose/efeitos dos fármacos , Ductos Biliares/fisiopatologia , Células Estreladas do Fígado/efeitos dos fármacos , Células Estreladas do Fígado/metabolismo , Hipertensão Portal/complicações , Hipertensão Portal/tratamento farmacológico , Hipertensão Portal/fisiopatologia , Imuno-Histoquímica , Fígado/metabolismo , Cirrose Hepática Biliar/complicações , Cirrose Hepática Biliar/fisiopatologia , Masculino , Óxido Nítrico Sintase Tipo III/genética , Óxido Nítrico Sintase Tipo III/metabolismo , Fosfatos de Poli-Isoprenil/metabolismo , Ratos , Ratos Wistar , Transdução de Sinais , Quinases Associadas a rho/genética , Proteína rhoA de Ligação ao GTP/genéticaRESUMO
This study is to explore a new method of investigating molecular basis for electrophysiological properties of early fetal cardiomyocytes. Single embryonic cardiomyocytes of mouse early developmental heart (E10.5) were obtained by a collagenase B digestion approach. After recording spontaneous action potential using whole cell patch clamp technique, the single cell was picked by a glass micropipette, followed by a standard RT-PCR to explore the expression levels of several ion channel genes. Three phenotypes of cardiomyocytes were demonstrated with distinct properties: ventricular-like, atrial-like, and pacemaker-like action potentials. Ventricular-like and atrial-like cells were characterized with much negative maximum diastolic potential (MDP) and a higher V(max) (maximum velocity of depolarization) compared to pacemaker-like cells. MDP of ventricular-like cells was the most negative. In parallel, stronger expression of SCN5a, SCN1b and Kir2.1 were observed in ventricular-like and atrial-like cells compared to that of pacemaker-like cells, where Kir2.1 in ventricular-like cells was the most abundant. Cardiomyocytes with distinct electrophysiological properties had distinct gene expression pattern. Single cell RT-PCR combined with patch clamp technique could serve as a precise detector to analyze the molecular basis of the special electrophysiological characteristics of cardiomyocytes.
Assuntos
Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/fisiologia , Canal de Sódio Disparado por Voltagem NAV1.5/metabolismo , Canais de Potássio Corretores do Fluxo de Internalização/metabolismo , Subunidade beta-1 do Canal de Sódio Disparado por Voltagem/metabolismo , Animais , Fenômenos Eletrofisiológicos , Feminino , Feto , Masculino , Camundongos , Canal de Sódio Disparado por Voltagem NAV1.5/genética , Técnicas de Patch-Clamp , Canais de Potássio Corretores do Fluxo de Internalização/genética , Reação em Cadeia da Polimerase em Tempo Real , Subunidade beta-1 do Canal de Sódio Disparado por Voltagem/genéticaRESUMO
Cavernous transformation of the portal vein (CTPV) is not quite common in adults, and cases with CTPV and acute liver abscess are lacking. We report a patient with CTPV inducing extrahepatic and intrahepatic obstruction, finally leading to acute liver abscess due to bile duct infection. We aim to find out the possible relationship between CTPV and acute liver abscess. A 45-year-old female patient was admitted to our hospital for recurrent upper abdominal pain and distension for one year, aggravated with fever for three years. A diagnosis of CTPV and liver abscess was made by 16-slice computed tomography. Effective antibiotics and drainage were used for this patients, and she was eventually cured. When treating patients with CTPV, extrahepatic and intrahepatic obstruction, one should be aware of the presence of acute liver abscess, and empirical antibiotics might be valuable.
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AIM: To study the effects and mechanisms by which hyposmotic challenge modulate function of L-type calcium current (I(Ca,L)) in rat ventricular myocytes. METHODS: The whole-cell patch-clamp techniques were used to record I(Ca,L) in rat ventricular myocytes. RESULTS: Hyposmotic challenge(â¼220 mosmol/L) induced biphasic changes of I(Ca,L), a transient increase followed by a sustained decrease. I(Ca,L) increased by 19.1%±6.1% after short exposure (within 3 min) to hyposmotic solution. On the contrary, long hyposmotic challenge (10 min) decreased I(Ca,L) to 78.1%±11.0% of control, caused the inactivation of I(Ca,L), and shifted the steady-state inactivation curve of I(Ca,L) to the right. The decreased I(Ca,L) induced by hyposmotic swelling was reversed by isoproterenol or protein kinase A (PKA) activator foskolin. Hyposmotic swelling also reduced the stimulated I(Ca,L) by isoproterenol or foskolin. PKA inhibitor H-89 abolished swelling-induced transient increase of I(Ca,L), but did not affect the swelling-induced sustained decrease of I(Ca,L). NO donor SNAP and protein kinase G (PKG) inhibitor Rp-8-Br-PET-cGMPS did not interfere with swelling-induced biphasic changes of I(Ca,L). Protein kinase C (PKC) activator PMA decreased I(Ca,L) and hyposmotic solution with PMA reverted the decreased I(Ca,L) by PMA. PKC inhibitor BIM prevented the swelling-induced biphasic changes of I(Ca,L). CONCLUSION: Hyposmotic challenge induced biphasic changes of I(Ca,L), a transient increase followed by a sustained decrease, in rat ventricular myocytes through PKC pathway, but not PKG pathway. PKA system could be responsible for the transient increase of I(Ca,L) during short exposure to hyposmotic solution.
Assuntos
Canais de Cálcio Tipo L/fisiologia , Ventrículos do Coração/citologia , Ativação do Canal Iônico/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Proteína Quinase C/metabolismo , Animais , Canais de Cálcio Tipo L/metabolismo , Técnicas de Cultura de Células , Células Cultivadas , Colforsina/farmacologia , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de GMP Cíclico/metabolismo , Feminino , Ventrículos do Coração/efeitos dos fármacos , Ventrículos do Coração/enzimologia , Ventrículos do Coração/metabolismo , Isoproterenol/farmacologia , Potenciais da Membrana/efeitos dos fármacos , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/enzimologia , Concentração Osmolar , Técnicas de Patch-Clamp , Inibidores de Proteínas Quinases/farmacologia , Ratos , Ratos Sprague-Dawley , Transdução de SinaisRESUMO
In our studies, we have applied a novel tool, microelectrode arrays (MEA), to investigate the electrophysiological properties of murine embryonic hearts in vitro. The electrical signals were recorded from the areas of the heart adhering to the 60 MEA electrodes, being called field potentials (FPs). As an extracelluar recording, the waveform of the FP appeared similar to a reversed action potential obtained from single cell by whole cell current clamp and the FP duration was comparable with the action potential duration. To study propagation of spontaneous electrical activity, we have compared the occurrence time of FPs recorded from different electrodes. It is shown that there was already an apparent A-V delay [(50.21+/-9.7) ms] at day 9.5 post coitum (E9.5) when heart was still tubular-like and atrium and ventricle were not separated anatomically, while occurence of FP at different electrodes of ventricular area were almost synchronous. Further, we looked into the modulation of spontaneous electrical activity during cardiac development: at E9.5 of embryonic development, 1 mumol/L of isoproterenol (Iso) increased beating frequency by (34.04+/-7.31)%, shortened the A-V delay by (20.00+/-6.44) % and prolonged FP duration. In contrast, 1 mumol/L of carbachol (CCh) slowed down beating frequency by (42.32+/-5.36) %, A-V conduction by (26.00+/-4.81) % and shortened FP duration; however at late stage (E16.5), the regulatory effect of Iso and CCh was strengthened. Therefore we conclude that cardiac conduction system is already established at E9.5 when the four-chambered heart is not formed yet and the regulation of spontaneous activity by sympathetic and para-sympathetic system is gradually matured during cardiac development.
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Potenciais de Ação/fisiologia , Coração Fetal/fisiologia , Sistema de Condução Cardíaco/embriologia , Microeletrodos , Animais , Fenômenos Eletrofisiológicos , Sistema de Condução Cardíaco/fisiologia , Técnicas In Vitro , CamundongosRESUMO
We isolated mouse embryonic cardiomyocytes derived from timed-pregnant females at different periods and used patch-clamp technique to investigate the muscarinic cholinergic modulation of pacemaker current I(f) in different developmental stages. In early development stage (EDS), muscarinic agonist carbachol (CCh) significantly decreased the magnitude of the pacemaker current I(f) but had no effect in late development stage (LDS). Forskolin (a direct adenylate cyclase activator) and IBMX (a non-selective phosphodiesterase inhibitor) increased I(f) in both EDS and LDS cells. Interestingly, although both forskolin and IBMX increased basal I(f), their effects on CCh-inhibited I(f) were different. Forskolin did not reverse the inhibitory action of CCh until intermediate development stage (IDS). In contrast, IBMX reversed the inhibitory action of CCh on I(f) in EDS but not in IDS. It is suggested that a decrease in intracellular cAMP is a possible mechanism for CCh to modulate I(f). During the EDS and IDS CCh controls the cytoplasmic cAMP level by different pathways: In EDS, CCh modulates I(f) possibly by activating PDE which accelerates the breakdown of cAMP, but in IDS possibly by inhibiting adenylate cyclase (AC) which then reduces the synthesis of cAMP.
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Coração/embriologia , Agonistas Muscarínicos/farmacologia , Miócitos Cardíacos/fisiologia , Marca-Passo Artificial , Receptores Muscarínicos/metabolismo , Animais , Carbacol/farmacologia , Colforsina/metabolismo , Colforsina/farmacologia , Feminino , Coração/fisiologia , Camundongos , Miócitos Cardíacos/efeitos dos fármacos , Inibidores de Fosfodiesterase/metabolismo , Inibidores de Fosfodiesterase/farmacologia , GravidezRESUMO
AIM: To investigate the muscarinic regulation of L-type calcium current (I(Ca-L)) during development. METHODS: The whole cell patch-clamp technique was used to record II(Ca-L) in mice embryonic cardiomyocytes at different stages (the early developmental stage, EDS; the intermediate developmental stage, IDS; and the late developmental stage, LDS). Carbachol (CCh) was used to stimulate M-receptor in the embryonic cardiomyocytes of mice. RESULTS: The expression of I(Ca-L) density did not change in different developmental stages (P>0.05). There was no difference in the sensitivity of I(Ca-L) to CCh during development (P>0.05). This inhibitory action of CCh was mediated by inhibition of cyclic AMP since 8-bromo-cAMP completely reversed the muscarinic inhibitory action. IBMX, a non-selective inhibitor of phosphodiesterase (PDE), reversed the inhibitory action of M-receptor on I(Ca-L) current by 71.2 %+/-9.2 % (n=8) and 11.3 %+/-2.5 % (n=9) in EDS and LDS respectively. However forskolin, an agonist of adenylyl cyclase (AC), reversed the action of CCh by 14.5 %+/-3.5 % (n=5) and 82.7 %+/-10.4 % (n=7) in EDS and LDS respectively. CONCLUSION: The inhibitory action of CCh on I(Ca-L) current was mediated in different pathways: in EDS, the inhibitory action of M-receptor on I(Ca-L) channel mainly depended on the stimulation of PDE. However, in LDS, the regulation by M-receptor on I(Ca-L) channel mainly depended on the inactivation of AC.
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Canais de Cálcio Tipo L/efeitos dos fármacos , Carbacol/farmacologia , Coração/embriologia , Agonistas Muscarínicos/farmacologia , Miócitos Cardíacos/fisiologia , 1-Metil-3-Isobutilxantina/farmacologia , 8-Bromo Monofosfato de Adenosina Cíclica/farmacologia , Animais , Canais de Cálcio Tipo L/metabolismo , Colforsina/farmacologia , Feminino , Camundongos , Miócitos Cardíacos/efeitos dos fármacos , Técnicas de Patch-Clamp , Inibidores de Fosfodiesterase/farmacologiaRESUMO
To explore the electrophysiological characteristics of embryonic cardiomyocytes, single embryonic cardiomyocytes were obtained from mice at different periods by a collegenase B digestion approach, whole cell patch clamp recording technique was used to record I(f) and I(Ca-L), and spontaneous action potential was also recorded. The morphological and spontaneous contractile properties of the isolated cells appeared to be typical embryonic cardiomyocytes when the cells were assessed by phase-contrast microscope. Whole cell recording of isolated cells is easily performed by the whole cell patch clamp technique. Elelctrophysiological properties of I(f) and I(Ca-L) from embryonic cardiomyocytes have been proved to be similar to those from adult pacemaker cells or cardiomyocytes. The established method of isolation is simple, stable, effective and reliable. It allows to obtain as early as 8.5-day embryonic myocytes. The electrophysiological recording of embryonic cardiomyocytes will provide a useful model for exploring the electrophysiological characteristics of embryonic cardiomyocytes and the possible mechanism underlying some heart diseases.
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Separação Celular/métodos , Miócitos Cardíacos/citologia , Miócitos Cardíacos/fisiologia , Potenciais de Ação/fisiologia , Animais , Células Cultivadas , Eletrofisiologia , Embrião de Mamíferos , Camundongos , Técnicas de Patch-ClampRESUMO
The hyperpolarization-activated current (I(f)) plays an important role in determining the spontaneous rate of cardiac pacemaker cells. The automatic rhythmicity also exists in working cells of embryonic heart, therefore we studied developmental changes in functional expression and beta-adrenergic regulation of I(f) in embryonic mouse heart. The expression of I(f) is high in early developmental stage (EDS) (10.5 d after coitus) ventricular myocytes, low in intermediate developmental stage (IDS) (13.5 d) atrial or ventricular myocytes and even lower in late developmental stage (LDS) (16.5 d) atrial or ventricular myocytes, indicating that these cells of the EDS embryonic heart have some properties of pacemaker cells. Beta-adrenergic agonist isoproterenol (ISO) stimulates I(f) in LDS but not in EDS cardiomyocytes, indicating that the beta-adrenergic regulation of I(f) is not mature in EDS embryonic heart. But forskolin (a direct activator of adenylate cyclase) and 8-Br-cAMP (a membrane-permeable analogue of cAMP) increase the amplitude of I(f) in EDS cells, indicating that adenylate cyclase and cAMP function fairly well at early stage of development. Furthermore, the results demonstrate that I(f) is modulated by phosphorylation via cAMP dependent PKA both in EDS and LDS cells.