Dact1 involvement in the cytoskeletal arrangement of cardiomyocytes in atrial fibrillation by regulating cx43

Abstract Objective: To determine the role of the dishevelled binding antagonist of beta catenin 1 (DACT1) in the cytoskeletal arrangement of cardiomyocytes in atrial fibrillation (AF). Methods: The DACT1 expression and its associations with the degree of fibrosis and β-catenin in valvular disease patients were analyzed by immunohistochemistry and Masson's staining. DACT1 was overexpressed in the atrial myocyte cell line (HL-1) and the cardiac cell line (H9C2) by adenoviral vectors. Alterations in the fibrous actin (F-actin) content and organization and the expression of β-catenin were detected by flow cytometry, immunofluorescence, and Western blotting. Additionally, the association of DACT1 with gap junctions connexin 43 (Cx43) was detected by immunohistochemistry, immunofluorescence, and Western blotting. Results: Decreased cytoplasmic DACT1 expression in the myocardium was associated with AF (P=0.037) and a high degree of fibrosis (weak vs. strong, P=0.028; weak vs. very strong, P=0.029). A positive association was observed between DACT1 and β-catenin expression in clinical samples (P=0.028, Spearman's rho=0.408). Furthermore, overexpression of DACT1 in HL-1 and H9C2 cells induced an increase in β-catenin and subsequent partial colocalization of DACT1 and β-catenin. In addition, F-actin content and organization were enhanced. Interestingly, DACT1 was positively correlated with the Cx43 expression in clinical samples (P=0.048, Spearman's rho=0.370) and changed the Cx43 distribution in cardiac cell lines. Conclusion: DACT1 proved to be a novel AF-related gene by regulating Cx43 via cytoskeletal organization induced by β-catenin accumulation in cardiomyocytes. DACT1 could thus serve as a potential therapeutic marker for AF.

Expression of Cx43 and Cx45 in Cardiomyocytes of an Overworked Rat Model / 法医学杂志

Objective To study the effect of overwork stress response on the expression of connexin 43（Cx43） and connexin 45（Cx45） in cardiomyocytes and on cardiac function. Methods The experimental animals were divided into control group, overworked 1-month group and overworked 2-month group. A overworked rat model was established by forcing swimming of overworked group. The expressions of Cx43 and Cx45 in myocardial tissues of experimental animals were detected by Western blotting, while the corresponding myocardial tissues were stained with hematoxylin-eosin （HE） staining and Masson's staining, then histologically observed. Results Western blotting results showed that, compared with the control group, Cx43 expression in myocardial tissues of overworked rats decreased while Cx45 expression increased. HE staining and Masson's staining results showed that hypertrophy, rupture and interstitial fiber tissue hyperplasia were observed in myocardial fibers of overworked rats. Conclusion Overwork stress response may affect cardiac function as an independent factor and may even cause heart failure or arrhythmias and lead to death.

Gestational Protein Restriction Increases Cardiac Connexin 43 mRNA levels in male adult rat offspring / Restrição Proteica Gestacional Aumenta Níveis de RNAm de Conexina 43 no Coração de Filhotes Machos Adultos

Abstract Background: The dietary limitation during pregnancy influences the growth and development of the fetus and offspring and their health into adult life. The mechanisms underlying the adverse effects of gestational protein restriction (GPR) in the development of the offspring hearts are not well understood. Objectives: The aim of this study was to evaluate the effects of GPR on cardiac structure in male rat offspring at day 60 after birth (d60). Methods: Pregnant Wistar rats were fed a normal-protein (NP, 17% casein) or low-protein (LP, 6% casein) diet. Blood pressure (BP) values from 60-day-old male offspring were measured by an indirect tail-cuff method using an electro sphygmomanometer. Hearts (d60) were collected for assessment of connexin 43 (Cx43) mRNA expression and morphological and morphometric analysis. Results: LP offspring showed no difference in body weight, although they were born lighter than NP offspring. BP levels were significantly higher in the LP group. We observed a significant increase in the area occupied by collagen fibers, a decrease in the number of cardiomyocytes by 104 µm2, and an increase in cardiomyocyte area associated with an increased Cx43 expression. Conclusion: GPR changes myocardial levels of Cx43 mRNA in male young adult rats, suggesting that this mechanism aims to compensate the fibrotic process by the accumulation of collagen fibers in the heart interstitium.

Resumo Fundamento: A limitação dietética durante a gravidez influencia o crescimento e desenvolvimento do feto e da prole e sua saúde na vida adulta. Os mecanismos subjacentes dos efeitos adversos da restrição proteica gestacional (RPG) no desenvolvimento dos corações da prole não são bem compreendidos. Objetivos: Avaliar os efeitos da RPG sobre a estrutura cardíaca em filhotes machos de ratas aos 60 dias após o nascimento (d60). Métodos: Ratos fêmeas Wistar grávidas foram alimentadas com uma dieta de proteína normal (PN, 17% caseína) ou de baixa proteína (BP, caseína 6%). Os valores de pressão arterial (PA) de descendentes do sexo masculino de 60 dias de idade foram medidos por meio de um método indireto de manguito de cauda usando um eletro esfigmomanômetro. Os corações (d60) foram coletados para avaliação da expressão de RNAm da conexina 43 (Cx43) e análise morfológica e morfométrica. Resultados: A prole BP não mostrou diferença no peso corporal, embora tenha nascido mais leve do que a prole PN. Os níveis de PA foram significativamente mais altos no grupo BP. Observou-se um aumento significativo na área ocupada pelas fibras colágenas, diminuição do número de cardiomiócitos em 104 µm2 e aumento da área de cardiomiócitos associada ao aumento da expressão de Cx43. Conclusão: A RPG altera os níveis miocárdicos de RNAm de Cx43 em ratos adultos jovens, sugerindo que este mecanismo visa compensar o processo fibrótico pelo acúmulo de fibras de colágeno no interstício cardíaco.

Type 2 Diabetes Induces Prolonged P-wave Duration without Left Atrial Enlargement

Prolonged P-wave duration has been observed in diabetes. However, the underlying mechanisms remain unclear. The aim of this study was to elucidate the possible mechanisms. A rat model of type 2 diabetes mellitus (T2DM) was used. P-wave durations were obtained using surface electrocardiography and sizes of the left atrium were determined using echocardiography. Cardiac inward rectifier K+ currents (I(k1)), Na+ currents (I(Na)), and action potentials were recorded from isolated left atrial myocytes using patch clamp techniques. Left atrial tissue specimens were analyzed for total connexin-40 (Cx40) and connexin-43 (Cx43) expression levels on western-blots. Specimens were also analyzed for Cx40 and Cx43 distribution and interstitial fibrosis by immunofluorescent and Masson trichrome staining, respectively. The mean P-wave duration was longer in T2DM rats than in controls; however, the mean left atrial sizes of each group of rats were similar. The densities of I(k1) and I(Na) were unchanged in T2DM rats compared to controls. The action potential duration was longer in T2DM rats, but there was no significant difference in resting membrane potential or action potential amplitude compared to controls. The expression level of Cx40 protein was significantly lower, but Cx43 was unaltered in T2DM rats. However, immunofluorescent labeling of Cx43 showed a significantly enhanced lateralization. Staining showed interstitial fibrosis was greater in T2DM atrial tissue. Prolonged P-wave duration is not dependent on the left atrial size in rats with T2DM. Dysregulation of Cx40 and Cx43 protein expression, as well as fibrosis, might partly account for the prolongation of P-wave duration in T2DM.

ß-ARRESTIN/CONNEXIN 43 (Cx43) complex anchors ERKS outside the nucleus: a prerequisite for bisphosphonate antiapoptotic effect mediated by Cx43/ERK in osteocytes / El complejo ß-ARRESTINA/CONEXINA 43 (Cx43) retiene ERKS fuera del núcleo: un paso necesario para el efecto anti-apoptótico de los bisfosfonatos mediado por Cx34/ERK en osteocitos

Bisphosphonates (BPs) anti-fracture efficacy may be due in part to inhibition of osteocyte apoptosis. This effect requires opening of connexin (Cx) 43 hemichannels and phosphorylation of the extracellular signal regulated kinases (ERKs). However, unlike ERK activation by other stimuli, the Cx43/ERK pathway activated by BPs does not result in nuclear ERK accumulation. Instead, the anti-apoptotic effect of BPs depends on phosphorylation of cytoplasmic ERK targets and is abolished by forced nuclear retention of ERKs. We now report that ERKs and the scaffolding protein ß-arrestin co-immuno-precipitate with Cx43 in MLO-Y4 osteocytic cells and that the BP alendronate increases this association. Moreover, ERK2 fused to red fluorescent protein (ERK2-RFP) co-localizes with Cx43 fused to green fluorescent protein outside the nucleus in cells untreated or treated with alendronate. Alendronate does not induce ERK nuclear accumulation in cells transfected with wild type ß-arrestin (wtARR) or vector control, whereas it does in cells expressing a dominant negative ß-arrestin mutant (dnARR) consisting of the ß-arrestin-clathrin binding domain that competes with endogenous ß-arrestin for binding to clathrin. Alendronate activates ERKs in dnARRtransfected cells as effectively as in cells transfected with wtARR, demonstrating that dnARR only interferes with subcellular localization but not with activation of ERKs by BPs. Further, whereas alendronate inhibits apoptosis in cells expressing wtARR or vector control, it is ineffective in cells expressing dnARR. Thus, BPs induce the formation of a complex comprising Cx43, ß-arrestin, and clathrin, which directs ERKs outside the nucleus and is indispensable for osteocyte survival induced by BPs. (AU)

La efectividad de los bisfosfonatos (BPs) en la prevención de fracturas puede deberse en parte a la inhibición de la apoptosis de osteocitos. Este efecto depende de la apertura de hemicanales de conexina (Cx) 43 y la fosforilación de quinasas reguladas por señales extracelulares (ERKs). Sin embargo, a diferencia de la activación de ERKs debida a otros estímulos, la vía de señalización Cx43/ERK activada por BPs no conlleva la acumulación de ERKs en el núcleo. El efecto anti-apoptótico de los BPs depende de la fosforilación de blancos citoplasmáticos de ERKs y es inhibido cuando las quinasas son retenidas en el núcleo. En este estudio hemos demostrado que ERKs y la proteína "scaffolding" ß-arrestina co-inmunoprecipitan con Cx43 en células osteocíticas MLO-Y4 y que alendronato aumenta esta asociación. Más aún, ERK2 fusionada a la proteína roja fluorescente (ERK2-RFP) co-localiza con Cx43 fusionada con la proteína verde fluorescente fuera del núcleo en células tratadas con vehículo o alendronato. Alendronato no indujo la acumulación nuclear de ERK en células transfectadas con ß-arrestina nativa (wtARR) o con un vector control, pero si lo hizo en células que expresan una forma dominante negativa de ß-arrestina (dnARR), consistente en el dominio de interacción entre ß-arrestina y clatrina, y que compite con ß-arrestina endógena por la unión a clatrina. Alendronato activa ERKs con la misma eficiencia en células transfectadas con dnARR o wtARR, demostrando que dnARR sólo interfiere con la localización subcelular de ERKs, pero no con su activación inducida por los BPs. Más aún, mientras alendronato inhibe apoptosis en células que expresan wtARR o vector control, es inefectivo en células que expresan dnARR. En conclusión, los BPs inducen la formación de un complejo que incluye Cx43, ß-arrestina y clatrina, el cual retiene ERKs fuera del núcleo y es indispensable para la sobrevida de los osteocitos inducida por estas drogas. (AU)

A spatial model showing differences between juxtacrine and paracrine mutual oocyte-granulosa cells interactions.

The bidirectional communication between oocytes and granulosa cells are mediated by several factors via a local feedback loop(s). The current model was carried out to study the spatial mutual interaction of porcine denuded oocytes and granulosa cells either in direct contact (juxtacrine) or paracrine co-culture using transwell system. Transwell 0.4 µm polyester membrane inserts were used to permit oocytes-granulosa cells paracrine communication with a distance of 2 mm between them in co-culture. Oocytes were cultured with granulosa cells in a defined basic maturation medium for 44 h. In results, oocyte secreted factors (OSFs; GDF9 and BMP15) temporal expression showed progressive decrement by the end of culture in case of direct contact with granulosa cells while it was increased progressively in the paracrine co-culture groups. However, oocytes that were cultured in direct contact showed a significant increase in blastocyst development after parthenogenetic activation than the paracrine co-cultured ones (20% vs. 11.5%, respectively). By the end of culture, granulosa cell count in direct contact showed a significant decrease than the indirect co-culture group (1.2 × 105 cell/mL vs. 2.1 × 105 cell/mL, respectively). Steroids (P4 and E2) and steriodogenesis enzymes mRNA levels showed significant temporal alterations either after 22 h and 44 h of IVM in both juxtacrine and paracrine co-culture systems (P ≤ 0.05). CX43 was much more highly expressed in the granulosa of the direct contact group than the indirect co-culture group. These results indicate the difference in mutual communication between oocytes and granulosa cells that were cocultured either in direct contact (juxtacrine) or with a short distance (paracrine) and propose a new paradigm to study different ovarian follicular cells interaction.

Changes of HCN4, Cx43 Expression in the Sinoatrial Node of Electric Shock Death / 法医学杂志

OBJECTIVE@#To investigate the expression of hyperpolarization-activated cyclic nucleotide-gated cation channel 4 (HCN4) and connexin43 (Cx43) in the sinoatrial node of electric shock death.@*METHODS@#As experimental group, 34 cases of electric shock death who had definite current mark evidence were selected from pathology department of Xuzhou Medical College from 2010 to 2013. As the control group, 20 cases of fatal severe craniocerebral injury in traffic accidents were chosen. The expressions of HCN4 and Cx43 in the sinoatrial node were observed by immunohistochemical technology.@*RESULTS@#HCN4 positive cells expressed in the cell membrane and cytoplasm of the sinoatrial node. Cx43 positive cells expressed in the cell membrane and cytoplasm of T cells and myocardial cells. The expression of HCN4 was significantly higher than that of the control group (P < 0.05) and the expression of Cx43 was significantly lower than that of the control group (P < 0.05).@*CONCLUSION@#The changes of HCN4 and Cx43 expressions in the sinoatrial node illustrate electric shock death might be related to the abnormalities of cardiac electrophysiology and conduction.

Forensic pathological significance of immunohistochemical study with CX43 in rats on early myocardial ischaemia / 法医学杂志

OBJECTIVE@#To explore one of evidence for pathologic diagnosis of early myocardial ischaemia.@*METHODS@#Rats were ligated of the left coronary artery according to a previously documented technique, and heart tissue was sampled at different ischaemia time. The expression of CX43 in myocardial cell was detected by Immunohistochemistry.@*RESULTS@#It is showed that the distribution and amount of CX43 positive staining in each group of the myocardial ischaemia was different from that of the control group.@*CONCLUSION@#The changes of CX43 detected by Immunohistochemical methods may be helpful for the diagnosis of early myocardial ischaemia, but further pathologic investigation and research is necessary.

Immunohistochemical study of Cx43 dephosphorylation in human left ventricular myocardium suffered by acute ischemia / 法医学杂志

OBJECTIVE@#This study was performed to detect the phosphorylation state of Cx43 in human left ventricular myocardium among sudden deaths caused by acute myocardial ischemia (AMI) especially sudden coronary death (SCD) and control groups. And then evaluate the significance of these findings in diagnosing the early pathological changes of acute myocardial ischemia.@*METHODS@#Immunohistochemistry (IHC) SP techniques were adopted to detect the phosphorylation state of Cx43 in the left ventricular myocardium samples of 45 deceased, which classified as group I--SCD, group II & III (other two groups of AMI) and Group IV & V (two control groups, sudden death caused by lethal acute cranio-cerebral injury or pathologic intracranial hemorrhage). In addition, we selected anti-Pan-Cadherin (construction protein of adherent junctions on the intercalated disc) and PHA-E+L/Bio, to detect the integration of myocardial mechanical coupling and membranes (applying affinityhistochemistry, AHC) respectively.@*RESULTS@#(1) Phosphorylated Cx43 positive staining was almost invisible in Group I, II and III or scattered in sarcoplasm in few samples; but it was assembling at the IDs clearly in group IV and V. (2) Strongly positive staining of Pan-Cadherin could be observed at the IDs and (3) integrated myocardial membranes were found in all samples.@*CONCLUSION@#These findings suggested that compared with the control groups, the integration of myocardial mechanical coupling and membranes did not alter in AMI. But Cx43, the key protein of electrical coupling on myocardial gap junctions, occurred dephosphorylation remarkably in AMI. Thus applying IHC techniques to detect the Cx43 dephosphorylation in human left ventricular myocardium maybe useful to recognize the onset of arrhythmia in AMI, especially in SCD whose myocardium without apparent morphological changes.

Correlation study between the connexin43 and the myocardial ischemia in rabbit / 法医学杂志

OBJECTIVE@#To study the correlation between quantity of the connexin43 express and ischemia time in rabbit.@*METHODS@#Left coronary arteries were ligated to create early myocardial ischemia during distinct time, HBFP staining was used in the ischemia spot and SP method was used to show the expression of connexin43 and the ischemia are were measured by the computer image analysis.@*RESULTS@#Negative correlation was observed between the connexin43 quantity and the ischemia time. The significant was difference.@*CONCLUSION@#With ischemia time raised, expression of connexin43 was descented.