Non-phosphorylated Cx43 Research Articles

Molecular remodeling of Cx43, but not structural remodeling, promotes arrhythmias in an arrhythmogenic canine model of nonischemic heart failure

BackgroundBoth gap junctional remodeling and interstitial fibrosis have been linked to impaired electrical conduction velocity (CV) and fatal ventricular arrhythmias in nonischemic heart failure (HF). However, the arrhythmogenic role of the ventricular gap junctional Cx43 in nonischemic HF remains in debate. Here, we assessed this in a newly developed arrhythmogenic canine model of nonischemic HF. Methods and resultsNonischemic HF was induced in canines by combined aortic valve insufficiency and aortic constriction. Left ventricular (LV) myocardium from HF dogs showed similar pathological changes to that of humans. HF dogs had reduced LV function, widened QRS complexes, and spontaneous nonsustained ventricular tachycardia. CV was measured in intact LV epicardium with high-density grid mapping. Total (Cx43-T) and nonphosphorylated Cx43 (Cx43-NP) and histological interstitial fibrosis were assessed from these mapped LV tissues. Longitudinal CV, which was slowed in HF (49 ± 1 vs. 65 ± 2 cm/s in Ctl), was positively correlated with reduced total junctional Cx43 and negatively correlated with markedly increased junctional Cx43-NP (2-fold) in HF. Cx43 dephosphorylation in HF was associated with enhanced colocalization of PP2A at the level of Cx43. Unchanged action potential upstroke and transverse CV were associated with unaltered Cx43 lateralization and interstitial fibrosis in the nonischemic HF canine LV. ConclusionOur unique arrhythmogenic canine model of HF resembles human nonischemic HF (prior to the end stage). Cx43 remodeling occurs prior to the structural remodeling (with lack of fibrosis) in HF and it is crucial in slowed CV and ventricular arrhythmia development. Our findings suggest that altered Cx43 alone is arrhythmogenic and modulation of Cx43 has the anti-arrhythmic therapeutic potential for HF patients.

Glycated Serum Albumin Decreases Connexin 43 Phosphorylation in the Corpus Cavernosum

Background: Glycated serum albumin (GSA) is an early glycosylation product that participates in diabetic vascular complications. This study aimed to examine the role of GSA in early damage to the corpus cavernosum and to identify the involved pathways. Methods: Nine male 8-week-old SD rats weighing 250-300 g were divided into the control (saline vehicle, n=3) and GSA (200 µg/kg, n=6) groups. Corpus cavernosum tissues were harvested. Phosphorylated and non-phosphorylated Cx43, eNOS, PI3K, and Akt were tested by immunohistochemistry and western blotting. Human umbilical vein endothelial cells (HUVECs) overexpressing or not Cx43 were used to analyze the Cx43 phosphorylation site (S368, S262, Y265, S255, and S279/282) by western blotting. Results: The expression of phosphorylated Cx43 in the penis was significantly lower in GSA-treated rats than in controls. The expression levels of p-Cx43, p-eNOS, p-PI3K, and p-Akt were significantly decreased in HUVECs exposed to GSA, both in a dose- and time-dependent manners. The most significant impact on all four proteins was observed with 1 μg/ml of GSA for 12 h. The phosphorylation at the S368, S262, Y265, S255, and S279/282 sites of Cx43 was downregulated to various degrees by GSA, and the S368 site was the most significantly suppressed phosphorylation site compared with the other sites. Conclusions: GSA decreased the expression of phosphorylated Cx43 in the corpus cavernosum of rats. This effect might be related to the decreased phosphorylation of p-eNOS, p-PI3K, and p-Akt, as well as by downregulation of phosphorylation at the S368 site. Funding Statement: This study was funded by the grant from the National Natural Science Foundation of China (No. 81771577 & No. 81401196) Declaration of Interests: All authors declare that they have no competing interests. Ethics Approval Statement: Human umbilical vein endothelial cells (HUVECs) were isolated from human umbilical cords, which were donated under informed consent from the mothers. All the procedures were approved by the Ethics Committee for Clinical Research at Galicia (China) and were performed according to the World Medical Association Declaration of Helsinki. This study was approved by the Institutional Animal Care and Use Committee. All procedures were conducted in accordance with the institutional guidelines.

Anti-arrhythmic Cardiac Phenotype Elicited by Chronic Intermittent Hypoxia Is Associated With Alterations in Connexin-43 Expression, Phosphorylation, and Distribution.

Remodeling of the cellular distribution of gap junctions formed mainly by connexin-43 (Cx43) can be related to the increased incidence of cardiac arrhythmias. It has been shown that adaptation to chronic intermittent hypobaric hypoxia (IHH) attenuates the incidence and severity of ischemic and reperfusion ventricular arrhythmias and increases the proportion of anti-arrhythmic n-3 polyunsaturated fatty acids (n−3 PUFA) in heart phospholipids. Wistar rats were exposed to simulated IHH (7,000 m, 8-h/day, 35 exposures) and compared with normoxic controls (N). Cx43 expression, phosphorylation, localization and n−3 PUFA proportion were analyzed in left ventricular myocardium. Compared to N, IHH led to higher expression of total Cx43, its variant phosphorylated at Ser368 [p-Cx43(Ser368)], which maintains “end to end” communication, as well as p-Cx43(Ser364/365), which facilitates conductivity. By contrast, expression of non-phosphorylated Cx43 and p-Cx43(Ser278/289), attenuating intercellular communication, was lower in IHH than in N. IHH also resulted in increased expression of protein kinase A and protein kinase G while casein kinase 1 did not change compared to N. In IHH group, which exhibited reduced incidence of ischemic ventricular arrhythmias, Cx43 and p-Cx43(Ser368) were more abundant at “end to end” gap junctions than in N group and this difference was preserved after acute regional ischemia (10 min). We further confirmed higher n-3 PUFA proportion in heart phospholipids after adaptation to IHH, which was even further increased by ischemia. Our results suggest that adaptation to IHH alters expression, phosphorylation and distribution of Cx43 as well as cardioprotective n-3PUFA proportion suggesting that the anti-arrhythmic phenotype elicited by IHH can be at least partly related to the stabilization of the “end to end” conductivity between cardiomyocytes during brief ischemia.

Spatial Heterogeneity of Cx43 is an Arrhythmogenic Substrate of Polymorphic Ventricular Tachycardias during Compensated Cardiac Hypertrophy in Rats.

Ventricular remodeling increases the propensity of ventricular tachyarrhythmias and sudden death in patients. We studied the mechanism underlying these fatal arrhythmias, electrical and structural cardiac remodeling, as well as arrhythmogeneity during early, compensated hypertrophy in a rat model of chronic pressure overload. Twenty-six Wistar rats were subjected to transverse aortic constriction (TAC) (n = 13) or sham operation (n = 13). Four weeks postoperative, echo- and electrocardiography was performed. Epicardial (208 or 455 sites) and transmural (30 sites) ventricular activation mapping was performed on Langendorff perfused hearts. Subsequently, hearts were processed for (immuno)histological and molecular analyses. TAC rats showed significant hypertrophy with preserved left ventricular (LV) function. Epicardial conduction velocity (CV) was similar, but more dispersed in TAC. Transmural CV was slowed in TAC (37.6 ± 2.9 cm s(-1)) compared to sham (58.5 ± 3.9 cm s(-1); P < 0.01). Sustained polymorphic ventricular tachycardias were induced from LV in 8/13 TAC and in 0/13 sham rats. During VT, electrical activation patterns showed variable sites of earliest epicardial activation and altering sites of functional conduction block. Wandering epicardial reentrant activation was sporadically observed. Collagen deposition was significantly higher in TAC compared to sham, but not different between arrhythmogenic and non-arrhythmogenic TAC animals. Connexin43 (Cx43) expression was heterogeneous with a higher prevalence of non-phosphorylated Cx43 in arrhythmogenic TAC animals. In TAC rats with compensated cardiac hypertrophy, dispersion of conduction correlated to arrhythmogenesis, an increased heterogeneity of Cx43, and a partial substitution with non-phosphorylated Cx43. These alterations may result in the increased vulnerability to polymorphic VTs.

Expression of Connexin 43 in the Myocardium in Sudden Unexplained Nocturnal Death Syndrome

Background: Sudden unexplained nocturnal death syndrome (SUNDS) occurs predominantly in Southeast Asian people including Koreans. SUNDS is problematic for forensic pathologists because the diagnosis depends on the “exclusion of diagnosis.” Moreover, the pathogenesis of SUNDS is still unclear although some cases are known to be intimately related to the Brugada syndrome. Connexin43 (Cx43) is a principal protein of gap junction in adult cardiac myocytes, being distributed to the intercalated discs and phosphorylated in normal condition. Ischemia and hypoxia alter the expression of total Cx43 (tCx43) resulting in redistribution of non-phosphorylated Cx43 (npCx43) to the sarcoplasm or lateral cell borders of cardiac myocytes by continuing dephosphorylation. This study aimed to compare the immunoexpression pattern of Cx43 in the cardiac myocytes of SUNDS and ischemic heart disease (IHD). Methods: The study group included SUNDS victims (SUNDS group, N=26; all males; aged 22-43 years; median age, 30.4 years), had a characteristic premortem history and negative autopsy findings. The control group was individuals who died suddenly due to IHD with severe coronary atherosclerosis, showing no myocardial ischemic change (IHD group, N=24; 22 males and 2 females; aged 35-76 years; median age, 53.5 years). Immunostaining was performed using the ABC method. The primary antibodies were rabbit polyclonal anti-Cx43 antibody (1:2,000, Sigma-Aldrich, St. Louis, MO, USA), which react to both phosphorylated forms of Cx3 and npCx43, and mouse monoclonal anti-npCx43 antibody (1:100, Life Technologies, Frederick, MD, USA), which detect only npCx43. Expression patterns were grouped as no staining (NO), intercalated disc pattern (ID pattern, confined mostly to the intercalated discs), and redistribution pattern (R pattern, extended to the lateral borders of sarcoplasmic membrane). The chi-square test was used to analyze the relationship of tCx43 and npCx43 immunoexpression pattern with SUNDS group and IHD group. A p-value less than 0.05 was considered statistically significant. Results: 1. Expression of tCx43 in the SUNDS and IHD group. The results of tCx43 expression in the cardiac myocytes of the SUNDS and IHD group are summarized: In the SUNDS group, 7/26 cases (26.9%) displayed the NO pattern; 19/26 cases (73.1%), the ID pattern. The R pattern was not observed in the SUNDS group. In the IHD group, 14/24 cases (58.3%) displayed the ID pattern; 10/24 cases (41.7%), the R pattern. The NO pattern was not observed in the IHD group. There was a significant difference in the tCx43 expression between the SUNDS and IHD group (p<0.001). 2. Expression of npCx43 in the SUNDS and IHD group. The results of npCx43 expression in the cardiac myocytes of the SUNDS and IHD group are summarized: In the SUNDS group, 1/26 cases (3.8%) displayed the NO pattern; 23/26 cases (88.5%), the ID pattern; and 2/26 (7.7%), the R pattern. In the IHD group, 7/24 cases (29.2%) displayed the ID pattern; 17/24 cases (70.8%), the R pattern. There was a significant difference in the npCx43 expression between the SUNDS and IHD group (p<0.001). Conclusion: There was a significantly different expression of both tCx43 and npCX43 between the SUNDS and IHD group. A greater reduction in both tCx43 and npCx43 a more delayed redistribution pattern was seen in the myocardium of SUNDS when compared with IHD. In conclusion, these results suggest that the reduced Cx43 expression in SUNDS may be inherent and indicate a risk of arrhythmia.

Modulation of cardiac connexin-43 by omega-3 fatty acid ethyl-ester supplementation demonstrated in spontaneously diabetic rats.

Previous data suggest that type 1 diabetes mellitus leads to the deterioration of myocardial intercellular communication mediated by connexin-43 (Cx43) channels. We therefore aimed to explore Cx43, PKC signaling and ultrastructure in non-treated and omega-3 fatty acid (omega-3) treated spontaneously diabetic Goto-Kakizaki (GK) rats considered as type 2 diabetes model. Four-week-old GK and non-diabetic Wistar-Clea rats were fed omega-3 (200 mg/kg/day) for 2 months and compared with untreated rats. Real-time PCR and immunoblotting were performed to determine Cx43, PKC-epsilon and PKC-delta expression. In situ Cx43 was examined by immunohistochemistry and subcellular alterations by electron microscopy. Omega-3 intake reduced blood glucose, triglycerides, and cholesterol in diabetic rats and this was associated with improved integrity of cardiomyocytes and capillaries in the heart. Myocardial Cx43 mRNA and protein levels were higher in diabetic versus non-diabetic rats and were further enhanced by omega-3. The ratio of phosphorylated (functional) to non-phosphorylated Cx43 was lower in diabetic compared to non-diabetic rats but was increased by omega-3, in part due to up-regulation of PKC-epsilon. In addition, pro-apoptotic PKC-delta expression was decreased. In conclusion, spontaneously diabetic rats at an early stage of disease benefit from omega-3 intake due to its hypoglycemic effect, upregulation of myocardial Cx43, and preservation of cardiovascular ultrastructure. These findings indicates that supplementation of omega-3 may be beneficial also in the management of diabetes in humans.

Anti-arrhythmic effect of acupuncture pretreatment in the rats subjected to simulative global ischemia and reperfusion--involvement of intracellular Ca2+ and connexin 43.

BackgroundThe previous study showed that the cardiac arrhythmias induced by myocardial ischemia and reperfusion were attenuated by the pretreatment of acupuncture; however, the related mechanism is not understood. The present study was therefore designed to determine whether intracellular Ca2+ ([Ca2+]i) and connexin 43 (Cx43) are involved in the mediation of the anti-arrhythmic effect of electro-acupuncture (EA) pretreatment in the rats subjected to simulative global ischemia and reperfusion (SGIR).MethodsSGIR was made in the isolated heart by a low flow perfusion followed by a flow restoration. Four groups of animals are involved in the present study, including normal control group, SGIR group, EA group and EA plus 18 beta-glycyrrhetinic acid (EAG) group. For EA pretreatment, bilateral Neiguan acupoints (PC6) of the rats were stimulated for 30 min once a day in 3 consecutive days. Cx43 antagonist was given to the rats in EAG group 30 minutes before the EA pretreatment. The resting [Ca2+]i concentration, calcium oscillation, the contents of total Cx43 and non-phosphrylated Cx43 and arrhythmia score were compared among different groups.ResultsIn EA group, the arrhythmic score, the resting [Ca2+]i concentration and the number of [Ca2+]i oscillations were all significantly less than those in SGIR group (all P < 0.05), and interestingly, after EA pretreatment, the contents of nonphosphated Cx43 in the EA group were significantly lower than that in SGIR group respectively (P < 0.05). However, when the rats were treated with Cx43 antagonist prior to the EA pretreatment, the protection effects induced by EA pretreatment were reversed.ConclusionsThe results showed that EA pretreatment could produce anti-arrhythmic effect in the rats subjected to SGIR. The anti-arrhythmic effect of EA pretreatment may be due at least partially to the inhibition of SGIR-induced calcium overload and [Ca2+]i oscillations, reduction of non-phosphorylated Cx43 and the enhancement of the corresponding phosphorylated Cx43 in the cardiac cells.Electronic supplementary materialThe online version of this article (doi:10.1186/s12906-015-0521-y) contains supplementary material, which is available to authorized users.

Antiarrhythmic peptide AAP10 prevents arrhythmias induced by protein kinase C activation in rabbit left ventricular wedges.

As the mechanisms underlying PKC activation induced arrhythmias are not yet fully verified, we investigated the role of gap junctions in arrhythmias induced by PKC activation.Arterially-perfused rabbit left ventricular preparations were randomly assigned to perfusion with phorbol ester (PMA) or in combination with AAP10. Transmural ECG as well as action potentials from both endocardium and epicardium were simultaneously recorded throughout all experiments. Changes in connexin43 (Cx43) and nonphosphorylated Cx43 on S368 were measured by Western blot analysis.In the PMA group, the QT interval was shortened, the interval from the peak to the end of the electrocardiographic T wave (Tp-e) and induced nonsustained ventricular tachycardia (VT) were increased, and the expressions of Cx43 and nonphosphorylated Cx43 on S368 were decreased compared with the control group. Compared with the PMA group, without significant changes in the QT interval and the expression of nonphosphorylated connexin43 on S368, Tp-e and induced VT decreased and the expression of Cx43 increased in the AAP10 group.AAP10 can prevent PMA-induced rabbit ventricular arrhythmias by attenuating the increase of Tp-e and the decrease of expression of Cx43. These data suggest that increasing gap junction coupling prevents arrhythmias induced by protein kinase C activation.

Uremic toxin p-cresol induces disassembly of gap junctions of cardiomyocytes

High serum levels of p-cresol have been associated with cardiovascular diseases. This study investigated the effects of p-cresol on gap junctions in neonatal cultured cardiomyocytes. p-Cresol reduced the spontaneous contraction rates of cardiomyocytes, and caused irregular cardiomyocyte beating. Junctional connexin 43 (Cx43) plaques became smaller in size and the gap junction intercellular communication (GJIC) impaired. Moreover, p-cresol increased intracellular Ca+2 levels, and induced Ca+2-dependent protein kinase Cα (PKCα) activation. p-Cresol decreased P1 and P2 Cx43 levels, and increased non-phosphorylated S368-Cx43 levels. The above changes as well as Cx43 disassembly and GJIC decrease induced by p-cresol were prevented by the BAPTA-AM or PKCα inhibitor Gö6976. These results suggest that PKCα mediates p-cresol-induced gap junction disassembly and GJIC dysfunction via S368-Cx43 serine dephosphorylation. This hypothesis was further confirmed in H9c2 cells by siRNA approach. SiRNA knockdown of PKCα prevented p-cresol-induced increase in nonphosphorylated Cx43. This finding supports the association of p-cresol and cardiovascular diseases.

Increased susceptibility to ischemia-induced ventricular tachyarrhythmias in depressed rats: Involvement of reduction of connexin 43

Connexin 43 (Cx43) has been reported to contribute to the occurrence of ventricular arrhythmias during myocardial ischemia (MI). In this study, we investigated the expression of Cx43 and the incidences of ventricular tachyarrhythmias [i.e., ventricular tachycardia (VT) and ventricular fibrillation (VF)] during acute MI in chronic mild stress (CMS) in rats. Male Sprague-Dawley (SD) control and CMS rats were assigned into a sham operation (SO) group and a MI group. Ventricular tachyarrhythmias were assessed and Cx43 protein expression was measured by Western blotting. During 30-min ischemia, the incidences of VT (7/12, 58.3%) and VF (5/12, 41.7%) in the CMS-MI group were significantly decreased compared with those in the control-MI group (12/12, 100.0% and 11/12, 91.7%; P<0.05). The amount of total Cx43 of the CMS-SO group was significantly decreased to approximately 50% compared with that of the control-SO group (P<0.05). The 30-min ischemia did not result in a significant change in the amount of total Cx43 (total Cx43 is defined as the non-phosphorylated Cx43 and phosphorylated Cx43) compared to that of the SO group in CMS rats (P>0.05). The amount of non-phosphorylated Cx43 in the CMS-MI group was markedly increased compared to that of the CMS-SO group (P<0.05), suggesting that the relative amount of phosphorylated Cx43 was significantly decreased in CMS rats. The gap junctional permeability in the CMS-MI group (50.4±4.9%) was significantly decreased compared with the normal non-ischemic value in the CMS-SO group (100%). The present study suggested that the incidence of ischemia-induced ventricular tachyarrhythmias was markedly increased in depressed rats, which may be associated with the reduction of Cx43 protein expression in the ventricle of depressed rats.

Effect of antiarrhythmic peptide on ventricular arrhythmia induced by lysophosphatidic acid

To investigate the effect and potential mechanism of lysophosphatidic acid (LPA) and antiarrhythmic peptide (AAP10) on rabbit ventricular arrhythmia. Twenty-four rabbits were randomly divided into three groups (n = 8 each): control group, LPA group and AAP10 + LPA group. Using arterially perfused rabbit ventricular wedge preparations, transmural ECG and action potentials from both endocardium and epicardium were simultaneously recorded in the whole process of all experiments with two separate floating microeletrodes. The incidence of ventricular arrhythmia post S1S2 stimulation was recorded. Protein levels of nonphosphorylated Cx43 and total Cx43 were evaluated by Western blot. The distribution of nonphosphorylated Cx43 was observed by confocal immunofluorescence microscopy. Compared with the control group, the QT interval, endocardial action potential duration, transmural repolarization dispersion (TDR) and incidence of ventricular arrhythmia were significantly increased and nonphosphorylated Cx43 expression was significantly upregulated in the LPA group. Compared with the LPA group, cotreatment with AAP10 can reduce the QT interval, endocardial action potential duration, TDR and incidence of ventricular arrhythmia (25.0% vs 62.5%, P < 0.01) and downregulate nonphosphorylated Cx43. LPA could promote the arrhythmia possibly by upregulating nonphosphorylated Cx43 and subsequent gap junction transmission inhibition. Gap junction enhancer AAP10 could attenuate the pro-arrhythmic effect of LPA probably by downregulating myocardial nonphosphorylated Cx43 expression.

English

Aconitine, a strong poisonous type of alkaloid, has a pharmaceutical effect in stimulating the membranes of cardiomyocyte. However, other effects of aconitine on the Connexin43 (Cx43) and PKC&alpha; expression&nbsp;on cardiomyocyte are unknown. In this study, we investigated whether aconitine also mediates the phosphorylation status of Cx43 and PKC&alpha;&nbsp;in cultured ventricular myocytes of neonatal rats. The band intensity of phosphorylated Cx43 and nonphosphorylated Cx43 in cultured and aconitine-treated cardiomyocytes were determined by Western blot analysis. The changes in phosphorylation status occurring in PKC&alpha; in cultures were revealed by quantitative immunofluorescent microscopy. A decreased band intensity (0.37&plusmn;0.04) of phosphorylated Cx43 (P-Cx43) and a concomitant increased band intensity (3.56 &plusmn; 0.65)&nbsp;of nonphosphorylated Cx43 (NP-Cx43) were found, compared to the controls (1.00&nbsp;for P-Cx43 and NP-Cx43). It also revealed that, after aconitine treatment, the amount of phosphorylated PKC&alpha; (P-PKC&alpha;) decreased significantly. Similar changes were revealed in phosphorylation status occurring in PKC&alpha; in the cultures under the same treatment conditions. These observations suggest that aconitine not only induces dephosphorylation of Cx43, but also alters expression of P-PKC&alpha; in cultured cardiomyocytes. &nbsp; Key words:&nbsp;Aconitine, cardiomyocyte, connexin 43 (Cx43), protein kinase C-&alpha; (PKC&alpha;), protein phosphorylation.

Short-Duration Therapeutic Hypothermia Causes Prompt Connexin43 Gap Junction Remodeling in Isolated Rabbit Hearts

Whether connexin43 gap junctions (Cx43 GJs) and spatial heterogeneity of conduction velocity (CV) restitutions are altered in hearts during moderate (MH; 33°C) and severe (SH; 30°C) hypothermia remains unclear. Using an optical mapping system, ventricular CV was evaluated by S₁ pacing in 29 Langendorff-perfused isolated rabbit hearts at baseline (37°C, n=9), 30-min MH (n=6), 30-min SH (n=9), and rewarming (R, 30-min SH followed by 30-min 37°C, n=5). After CV evaluation, myocardium was collected to measure the level and distribution of non-phosphorylated (NP-Cx43) and total (T-Cx43) Cx43 by immunoblotting and immunoconfocal microscopy. In 6 additional hearts, Cx43 GJ remodeling was evaluated at 30-min SH with (n=3) or without (n=3) pretreatment of a protein kinase C (PKC) inhibitor. CV slowing and spatial heterogeneities of CV restitutions were enhanced in MH and SH hearts. NP-Cx43 was downregulated in MH (P=0.002) and SH (P<0.001) hearts. NP-Cx43 levels among 4 different ventricular sites became inhomogeneous in MH (P=0.017) and SH (P=0.046) hearts. However, T-Cx43 levels were unchanged. The percentages of lateralized NP- and T-Cx43 were increased in MH, SH, and R hearts. Pretreatment of PKC inhibitor attenuated SH-induced NP-Cx43 lateralization (P=0.0495). Short-duration (30 min) therapeutic hypothermia causes prompt Cx43 GJs remodeling, in which the PKC pathway is involved. Rewarming abolished hypothermia-induced conduction disturbance, while Cx43 GJs lateralization did not completely recover.

Cell cycle dependent regulation of gap junction coupling and apoptosis in GFSHR-17 granulosa cells

Recent results have shown that the level of gap junction coupling could modulate the induction of apoptotic reactions. We previously observed that 1H-[1,2, 4]Oxadiazole[4,3-a]quinoxalin-1-one (ODQ), a block- er of guanylyl cyclase, inhibited gap junction coupling and thereby promoted activation of characteristic apoptotic reactions such as chromatin condensation, DNA strand breaking, and formation of blebs in GFSHR-17 granulosa cells, the in vitro model for granulosa cells of the maturing ovular follicle. In the present report, we focus on the effects of ODQ with respect to the cell cycle in GFSHR-17 granulosa cells. In synchronised GFSHR-17 granulosa cells, the double whole-cell patch-clamp technique revealed that gap junction conductance in mitotic cells was reduced in comparison to cells in interphase. This reduction of gap junction conductance correlated with a reduction of non-phosphorylated Cx43 in mitotic cells. We compared the stimulation of apoptotic reactions by ODQ between cells in mitosis and in interphase. We observed that the induction of both chromatin condensation and DNA strand breaking by ODQ was increased in mitotic cells, as compared to cells in interphase. The effects of ODQ were not observed in He-La cells that do not express connexins. The results in- dicate that reduction of gap junction coupling in mitotic GFSHR-17 granulosa cells depends on phosphor- rylation of Cx43 and raises the sensitivity to stimulation of apoptosis. We propose that gap junction coupling is involved in regulation of apoptosis of granulosa cells in maturing ovular follicle.

Regulation of gap-junction protein connexin 43 by β-adrenergic receptor stimulation in rat cardiomyocytes

beta-adrenergic receptor (beta-AR) agonists are among the most potent factors regulating cardiac electrophysiological properties. Connexin 43 (Cx43), the predominant gap-junction protein in the heart, has an indispensable role in modulating cardiac electric activities by affecting gap-junction function. The present study investigates the effects of short-term stimulation of beta-AR subtypes on Cx43 expression and gap junction intercellular communication (GJIC) function. The level of Cx43 expression in neonatal rat cardiomyocytes (NRCM) was detected by a Western blotting assay. The GJIC function was evaluated by scrape loading/dye transfer assay. Stimulation of beta-AR by the agonist isoproterenol for 5 min induces the up-regulation of nonphosphorylated Cx43 protein level, but not total Cx43. Selective beta(2)-AR inhibitor ICI 118551, but not beta(1)-AR inhibitor CGP20712, could fully abolish the effect. Moreover, pretreatment with both protein kinase A inhibitor H89 and G(i) protein inhibitor pertussis toxin also inhibited the isoproterenol-induced increase of nonphosphorylated Cx43 expression. Isoproterenol-induced up-regulation of nonphosphorylated Cx43 is accompanied with enhanced GJIC function. Taken together, beta(2)-AR stimulation increases the expression of nonphosphorylated Cx43, thereby enhancing the gating function of gap junctions in cardiac myocytes in both a protein kinase A- and G(i)-dependent manner.Acta Pharmacologica Sinica (2009) 30: 928-934; doi: 10.1038/aps.2009.92.

Dephosphorylation of cardiomyocyte Cx43 is associated with myocardial ischemia and reperfusion injury

Objective Myocardial ischemia/reperfusion(I/R) injury is the leading cause of death in the world. However, the details of the mechanism of its pathophysiology are still unknown. The present study was designed to investigate the role of connexin 43(Cx43) in acute models of myocardial I/R injury. Methods Male C57BL/6 mice were subjected to myocardial ischemia(45 min) followed by reperfusion(4 hrs) in vivo. The whole operation was monitored using a two-lead ECG. Hearts were harvested and the level of protein was assessed by western blot analysis. Haematoxylin and Eosin(HE) staining was used to detect the extent of neutrophil infiltration. The expression level of IL-6 was detected by ELISA. Results A murine myocardial I/R injury model was constructed successfully. Phosphorylated Cx43 decreased 83. 45% while non-phosphorylated Cx43 increased 1.62- fold in the myocardium after I/R injury. Neutrophil infiltration and the expression of the inflammatory cytokine IL-6 increased in the myocardium following I/R. Conclusion During myocardial I/R injury, cardiomyocyte Cx43 is dephosphorylated, and this may be associated with an inflammatory response.

P-1 Remodeling of Myocardial Gap Junctions and its Correlation with Cardiac Contractility in Diabetic Rats

not available at time of printing. Poster Sessions 1: Heart Failure/Myocardial Diseases P-1 Remodeling of Myocardial Gap Junctions and its Correlation with Cardiac Contractility in Diabetic Rats Dong-Feng Yeih1, Pen-Chih Liao1, Ho-Tsung Hsin1, Kuo-Chin Chen1, Ai-Hsian Li1, Yung-Zu Tseng2. 1Department of Cardiology, Far-Eastern Memorial Hospital, Taiwan, 2Department of Cardiology, National Taiwan University, Taiwan Introduction: Gap junctions play an essential role in coupling adjacent cardiomyocytes, ensuring organized propagation of action potential and synchronizing beating in cardiomyocytes. However, it remains unclear that temporal remodeling of myocardial gap junctions and its relation to cardiac contractility in diabetic rats. Methods: Male Wistar rats, weighing 250 to 300 grams, were randomized to injection with either vehicle or streptozotocin (50mg, IP). Invasive hemodynamic studies with simultaneous recordings of left ventricular pressure and aortic flow signals were done at 4, 8 and 12 weeks after injection. Maximal systolic elastance (Emax) and maximum theoretical flow (Qmax) were assessed by curve fitting techniques; protein expression of non-phosphorylated Cx43 (P0) and total amount of Cx43 (T) in the left ventricle by western blotting analysis. Results: Emax was significantly lower in diabetic rats than in controls and Emax was significantly depressed with time in diabetic groups. On the other hand, Qmax was preserved and cardiac output was maintained in diabetic rats. Protein expression of total Cx43 was persistently attenuated in diabetic groups, while P0 was enhanced at 8 and 12 weeks after injection. The ratio of P0 to T was significantly increased with time, which correlated well with the declines in Emax in diabetic rats. Conclusions: Temporal remodeling of myocardial gap junctions, with persistently lower total Cx43 and gradually increased non-phosphorylated Cx43 and the ratio of P0 to T, occurred in the evolution of systolic dysfunction in diabetic rats. P-2 Quantification of Left Ventricular Diastolic Dynamics in Normal and Diseased Myocardium by Speckle-Tracking Echocardiography Kaoru Dohi1, Katsuya Onishi2, Takeshi Takamura1, Emiyo Sugiura1, Hiroshi Nakajima1, Kazuhide Ichikawa1, Masaki Tanabe1, Hiroya Tamada1, Masatoshi Miyahara1, Mashio Nakamura1, Masaaki Ito1. 1Department of Cardiology, Mie University Graduate School of Medicine, Japan, 2Department of Molecular and Laboratory Medicine, Mie University Graduate School of Medicine, Japan Background: The present study aimed to quantify left ventricular (LV) diastolic dynamics and to elucidate their relations to systolic properties in normal and diseased myocardium. Methods: 50 patients with hypertensive LV hypertrophy (LVH: EF 61±8%, QRS 96±16ms), 50 patients with dilated cardiomyopathy (DCM: EF 31±10%, QRS 121±32ms), and 50 normal controls (Control: EF 5±6%, QRS 89±9ms) had echo-study with speckle-tracking strain and strain rate imaging. To quantify LV diastolic relaxation and segmental synchrony, global peak relaxation rate (PRR) during early diastole and standard deviation (SD) of the 18 segmental time-to-PRR (TPRR-SD) were calculated from apical 4-, 2-, and long axis views. To elucidate the relations between diastolic and systolic properties, global peak systolic strain (PSS) and SD of the segmental time-to-peak PSS (TPSS-SD) were also evaluated. Results: LV relaxation and diastolic synchrony were significantly impaired in LVH and was more prominent in DCM (PRR: 0.8±0.3* 1/s in LVH, 0.5±0.2*# 1/s in DCM, and 1.2±0.3 1/s in Control, TPRR-SD: 55±16*ms in LVH, 79±35*# ms in DCM, and 42±13ms in Control, *p < 0.05 vs. Control and #p < 0.05 vs. LVH, respectively). There were strong correlations between PSS and PRR, and TPRR-SD and TPSS-SD (r = 0.83 and r = 0.83, p < 0.05, respectively). Conclusion: Speckle-tracking echocardiography quantified LV diastolic dynamics and exhibited the strong relation to systolic properties.

Expression of connexin 43 in ovarian cancer and its relationship with chemoresistance

To examine the expression of protein kinase C (PKC), connexin 43 (Cx43) and non-phosphorylated Cx43 in ovarian cancer, and discuss the role of phosphorylated of Cx43 in chemoresistance in ovarian cancer. We examined the expression of Cx43, non-phosphorylated Cx43 and PKC in ovarian cancer tissue by immunohistochemistry, and compared their expression in chemosensitivity group and chemoresistance group. Cisplatin resistant ovarian cancer cell line SKOV3/DDP cells were treated by staurosporine (a kind of PKC inhibitors). Then expression of Cx43, nonphosphorylated Cx43 and PKC were tested. Meanwhile, we tested chemosensitivity of SKOV3/DDP cells by ATP bioluminescence tumor chemosensitivity assay (ATP-TCA). (1) Immunohistochemically, the rates of positive expression of Cx43 and non-phosphorylated Cx43 were 54%, 14% respectively in the chemoresistance group, which were 83%, 59% in the chemosensitivity group respectively (P < 0.05). The rate of positive expression of PKC in 28 chemoresistance ovarian cancer cases (64%) was higher than that in 29 chemosensitivity cases (31%, P < 0.05). Both of them were significantly lower in chemoresistance group than in chemosensitivity group (P < 0.05). In addition, the expression of PKC was negatively correlated with the expression of Cx43 and non-phosphorylated Cx43. The correlation coefficients were -0.626 and -0.714, respectively (P < 0.05). (2) Immunohistochemically, PKC was down regulated, and Cx43 and non-phosphorylated Cx43 were up regulated in SKOV3/DDP cells after staurosporine treatment. The longer the staurosporine worked, the more expression of Cx43 was. (3) By ATP-TCA, SKOV3/DDP cells were resistant to paclitaxel and cisplatin. The tumor growth inhibition was higher in the group of paclitaxel or cisplatin combined staurosporine than in the group of paclitaxel or cisplatin alone. The sensitivity was intermediate in the group combined with low concentration staurosporine (1 x 10(-8) mol/L), and the sensitivity was high in the group combined with high concentration staurosporine (1 x 10(-7) mol/L). Phosphorylation of Cx43 caused by PKC leads to decrease in the expression of Cx43. This effect makes ovarian cancer cells less chemosensitive. Phosphorylation of Cx43 caused by PKC can be inhibited by staurosporine.

Abstract 1525: Atrial Rhythm Acutely Modulates Connexin43 Expression and Phosphorylation State in Human Atrial Fibrillation

Background: Adjacent areas of heterogenous conduction in the atria form a substrate for atrial fibrillation (AF). Connexin proteins (Cx) underlie conduction between myocytes; conduction is modulated by connexin expression and phosphorylation. Here we tested the hypothesis that left atrial (LA) expression and/or phosphorylation of Cx43 is distinctly regulated by atrial rhythm. Methods: Cx43 expression and phosphorylation was evaluated in LA tissue homogenates from 3 groups: cardiac surgery patients with history of AF presenting for surgery in AF (AFAF, n=8); history of AF but in sinus rhythm (AFSR, n=8); patients with no history of AF (CTL, n=8). Groups were matched for age, LA size, EF (&gt;50%), CAD, valve disease, and hypertension. Results: Western blot analysis revealed increased total Cx43 expression (t-Cx43) in AFAF (2.03±0.38) compared to AFSR (0.85±0.15, p=0.01) and SR (0.98±0.16, p=0.02) groups. Blots re-probed with an antibody detecting non-phosphorylated Cx43 at S368 (Np-Cx43) revealed a significant decrease in the Np-Cx43/t-Cx43 ratio, indicating increased S368 phosphorylation in the AFAF group (by 23% in AFAF vs. AFSR, p=0.046; by 12% in AFAF vs. CTL, p=0.051). Subcellular compartmentation of t-Cx43 was assessed in 3 samples per group using tissue fractionation and immunoblotting. Cytoskeletal t-Cx43 levels were increased in AFAF vs. AFSR and vs. CTL, ANOVA, p=0.016); t-Cx43 expression was not different between groups in membrane, cytoplasmic or nuclear fractions. Confocal microscopy revealed greater Cx43 lateralization in both AF groups compared to CTL. Conclusion: Our results (increased Cx43 expression and phosphorylation) suggest that AF rhythm at the time of surgery , more than the history of AF, has a distinct impact on LA Cx43 remodeling. Further the dynamic increase restricted to the cytoskeletal compartment suggests that cytoskeletal changes in AF may modulate connexin trafficking to the cell surface. This may affect the anisotropic properties of atrial conduction. Additional electrophysiological studies are needed to determine the functional contribution of lateralized Cx. Our study provides the first evidence linking acute rhythm status with regulation of Cx43 expression and phosphorylation in human atria.

Effects of streptozotocin-induced diabetes on connexin43 mRNA and protein expression in ventricular muscle

Abnormal QT prolongation with the associated arrhythmias is a significant predictor of mortality in diabetic patients. Gap junctional intercellular communication allows electrical coupling between heart muscle cells. The effects of streptozotocin (STZ)-induced diabetes mellitus on the expression and distribution of connexin 43 (Cx43) in ventricular muscle have been investigated. Cx43 mRNA expression was measured in ventricular muscle by quantitative PCR. The distribution of total Cx43, phosphorylated Cx43 (at serine 368) and non-phosphorylated Cx43 was measured in ventricular myocytes and ventricular muscle by immunocytochemistry and confocal microscopy. There was no significant difference in Cx43 mRNA between diabetic rat ventricle and controls. Total and phosphorylated Cx43 were significantly increased in ventricular myocytes and ventricular muscle and dephosphorylated Cx43 was not significantly altered in ventricular muscle from diabetic rat hearts compared to controls. Disturbances in gap junctional intercellular communication, which in turn may be attributed to alterations in balance between total, phosphorylated and dephosporylated Cx43, might partly underlie prolongation of QRS and QT intervals in diabetic heart.