Am J Physiol Heart Circ Physiol 306: H1324 –H1333, 2014. First published March 7, 2014; doi:10.1152/ajpheart.00653.2013.

Rapid electrical stimulation causes alterations in cardiac intercellular junction proteins of cardiomyocytes Tadamitsu Nakashima,1* Tomoko Ohkusa,1* Yoko Okamoto,1 Masaaki Yoshida,1 Jong-Kook Lee,2 Yoichi Mizukami,3 and Masafumi Yano1 1

Division of Cardiology, Department of Medicine and Clinical Science, Yamaguchi University Graduate School of Medicine, Yamaguchi, Japan; 2Department of Cardiovascular Regenerative Medicine, Osaka University Graduate School of Medicine, Osaka, Japan; and 3Center for Gene Research, Yamaguchi University, Yamaguchi, Japan Submitted 26 August 2013; accepted in final form 28 February 2014

Nakashima T, Ohkusa T, Okamoto Y, Yoshida M, Lee JK, Mizukami Y, Yano M. Rapid electrical stimulation causes alterations in cardiac intercellular junction proteins of cardiomyocytes. Am J Physiol Heart Circ Physiol 306: H1324 –H1333, 2014. First published March 7, 2014; doi:10.1152/ajpheart.00653.2013.—The intercellular junctions contain two complexes, adhesion junctions (AJ) and connexin (Cx) gap junctions (GJs). GJs provide the pathway for intercellular current flow. AJs mediate normal mechanical coupling and play an important role in the stability of GJs. We investigated the effects of rapid electrical stimulation (RES) on cardiac intercellular junctions, especially ␤-catenin and Cx43 alterations. We also studied the effects of ANG II receptor blockade on intercellular junction remodeling. Neonatal rats were euthanized by decapitation, and cardiomyocytes were prepared, cultured, and subjected to RES. We used real-time PCR, western blot analysis, and immunohistochemical methods. Conduction properties were examined by an extracellular potential mapping system. Cx43 protein expression in cardiomyocytes was significantly increased after 60 min. ␤-Catenin expression in the total cell fraction was significantly increased after 30 min. The expression level of ␤-catenin in the nucleus, which functions as a T cell factor/ lymphocyte enhancer binding factor transcriptional activator of Cx43 with its degradation regulated by glycogen synthase kinase-3␤, was dramatically increased after 10 min. Conduction velocity was increased significantly by RES for 60 min. Olmesartan prevented most these effects of RES. We showed an increase of phosphorylated glycogen synthase kinase-3␤, which is phosphorylated by activated MAPKs and inhibits ␤-catenin degradation, was attenuated by olmesartan. The changes in ␤-catenin precede Cx43 GJ remodeling and might play an important role in the formation and stability of GJs. Olmesartan might be a new upstream arrhythmia therapy by modulating intercellular junction remodeling through the ␤-catenin signaling pathway. intercellular junction; adhesion junction; gap junction; ␤-catenin; connexin43 CARDIOMYOCYTES ARE CONNECTED with each other through intercellular junctions, so-called intercalated disks (IDs). The ID contains different junctional complexes [adhesion junctions (AJs; adherens junctions and the desmosome) and gap junction (GJs)]. In the heart, GJs provide the pathway for intercellular current flow and enable coordinated action potential propagation and contraction. GJ channels are constructed from connexins (Cxs). AJs (adherens junctions and the desmosome) play an important role in mediating intercellular mechanical

* T. Nakashima and T. Ohkusa contributed equally to this work. Address for correspondence and present address: T. Ohkusa, Kirameki Project Career Support Center, Kyushu Univ. Hospital, 3-1-1, Maidashi, Higashi-Ku, Fukuoka 812-8582, Japan (e-mail: [email protected]. kyushu-u.ac.jp). H1324

coupling at the ID through linkage to the actin cytoskeleton and desmin filaments, respectively. In adherens junctions, N-cadherin and its cytoplasmic binding proteins, catenin and plakoglobin, are found. The desmosome is made up of desmoglein and desmocollin, and they interact with the linker proteins plakoglobin and plakophilin (PKP). Adherens junctions not only play a critical role in the maintenance of mechanical coupling at the ID but also play an important role in GJ formation and assembly in cultured rat cardiomyocytes (12, 18, 34). Cxs have short half-lives of 1–5 h, reflecting their rapid turnover through synthesis and degradation, and their functions are regulated by phosphorylation (26). Consequently, dynamic turnover of Cxs in the heart may be an important mechanism modulating intercellular coupling and impulse propagation. Recently, we (32) have reported that changes in AJ protein precede Cx43 GJ alterations, and ID remodeling might contribute to arrhythmogenesis during the development of heart failure. Moreover, it has been reported that mutation in proteins of the cardiac desmosome resulted in arrhythmogenic right ventricular cardiomyopathy (ARVC), an inherited disease with sudden cardiac death (28). It has also been reported that mutations linked to familial ARVC are in the gene coding for the desmosomal protein PKP2 (31). However, the relationship between GJs and AJs of cardiomyocytes under the pathological condition is not completely understood, especially under rapid stimulation to cardiomyocytes, the so-called tachycardic condition. The present study was designed to test the following hypothesis: an increase of twitch contraction per unit of time resulting from a sudden increase of heart rate is expected to affect intercellular junction proteins. We studied intercellular junction remodeling, especially that of ␤-catenin, which is one of the component proteins of AJs, and Cx43 alterations caused by rapid electrical stimulation (RES) in cultured rat neonatal cardiomyocytes and whole hearts. In addition, we investigated the effects of ANG II type 1 receptor blockade using olmesartan on intercellular junction remodeling, which is known to be an upstream therapy for lethal arrhythmias due to modulation of the renin-angiotensin-aldosterone system. We used an in vitro cardiomyocyte model exposed RES because of its characteristic features. Previously, we (14) have reported that this in vitro rapid pacing model showed significant Cx43 upregulation, mainly through an upregulated autocrine action of ANG II to activate p38 MAPK and ERK. In the present study, we investigated the effects of RES on stage-dependent changes in intercellular junction protein expressions in cardiomyocytes. The results reveal that changes in AJ protein ␤-catenin precede Cx43 GJ remodeling and might play an important role in the

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formation of GJs. Olmesartan might be an upstream arrhythmia therapy by modulating both AJ and GJ remodeling through the ␤-catenin signaling pathway. MATERIALS AND METHODS

All animal protocols were approved by the Animal Care Committee of Yamaguchi University. This investigation complied with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Pub. No. 85-23, Revised 1996). Cell culture. Neonatal rats (born within 1–2 days) were euthanized by decapitation, and neonatal cardiomyocytes were prepared and cultured according to methods we have previously described (14). Enzymatically dissociated cardiomyocytes were seeded on culture trays at a concentration of 3 ⫻ 105 cells/cm2 and then incubated in Leibovitz L-15 medium (Worthington Biochemical, Lakewood, NJ) supplemented with 5% FBS at 37°C. Cells were grown in random orientation (isotropic growth) to make a confluent layer, and, by day 5, they showed synchronized spontaneous beating with a regular frequency. RES of contraction. Cultured cardiomyocytes on day 5 were transferred to 0.5% serum medium and exposed to RES at 3.0 Hz for up to 60 min. RES was applied by field stimulation via platinum wire electrodes (0.5 mm in diameter) with an interpolar distance of 6.5 cm (for experiments of ID protein expression analysis) or 0.5 cm (for potential mapping experiments) as previously described (14). The stimulation intensity was adjusted to a level ⫻1.3 the threshold for synchronous contraction. Ascorbic acid (250 ␮mol/l), a scavenger of oxidative species, was added to the culture medium (14). We used a specific ANG II type 1 receptor antagonist to elucidate effects on ID remodeling (100 nmol/l olmesartan, a gift from Daiich Sankyo, Tokyo, Japan). We also used specific inhibitors of p38 MAPK [SB-203580 (10 ␮mol/l), Promega, Madison, WI] and ERK [PD98059 (50 ␮mol/l), Promega] to examine effects on glycogen synthase kinase (GSK)-3␤ phosphorylation. These compounds were applied to the culture medium 30 min before the initiation of RES. Langendorff experiments. We used a Langendorff system to examine adult rat whole hearts. Details of the system have been described in our previous reports (27, 32). In brief, isolated rat hearts were continuously perfused on a Langendorff apparatus with modified Krebs-Ringer solution equilibrated with 95% O2 and 5% CO2 (37°C, pH 7.4). Rapid stimulation at 5⬃7 Hz was applied at the center of the anterior left ventricular free wall. Immunohistochemistry and immunoblot analysis. For immunohistochemistry of intercellular junction proteins, rabbit anti-Cx43 antibody (1:100 dilution, Sigma, St. Louis, MO) and mouse anti-␤catenin antibody (1:100 dilution, Invitrogen, Carlsbad, CA) were used. For detection of the nucleus, we used 4=,6-diamidino-2-phenylindole dihydrochloride (1:100 dilution, Calbiochem, San Diego, CA). For detection of the basement membrane to indicate colocalization with Cx43, chicken anti-laminin antibody (1:500 dilution, Abcam, Cambridge, CA) was used. After permeabilization (0.1% Triton X-100), quenching, and blocking (10% goat serum), samples were incubated with the antibody overnight at room temperature. Primary antibodies bound to these antibodies were visualized by FITC-conjugated anti-rabbit IgG (1:200 dilution, Millipore, Billerica, MA) and FITC-conjugated anti-mouse IgG (1:500 dilution, Millipore), Alexa fluor 647-conjugated anti-chicken IgY H&L (1:1,000 dilution, Abcam). We examined sections using a confocal microscope (LSM510, Zeiss). Samples processed without primary antibody served as negative controls. Proportions of these immunoreactive signal were defined as the number of high-signal intensity (⬎90% of the maximum level) pixels divided by the total number of pixels occupied by myocytes (33). Ten individual measurements at different fields were averaged to yield a single value for each culture slide. Collected cells were lysed, and cellular extracts were electrophoresed on SDS-polyacrylamide gels as previously described (14). The

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nuclear fraction was prepared as previously described (20) with the NE-PER Nuclear and Cytoplasmic Extraction Reagent kit (Thermo Fisher Scientific, Waltham, MA). The following primary antibodies were used: rabbit anti-Cx43 antibody (1:50,000 dilution, Sigma), mouse anti-␤-catenin antibody (1:50,000 dilution, Invitrogen), mouse anti-GAPDH antibody (1:1,000 dilution, Sigma), mouse anti-histone H1 antibody (1:1,000 dilution, Leinco Technologies, St. Louis, MO), rabbit anti-GSK-3␤ antibody (1:1,000 dilution), and phosphorylated (p-)GSK-3␤ (Ser9) antibody (1:1,000 dilution, Cell Signaling, Beverly, MA). For secondary antibodies, the following were used: anti-rabbit IgG horseradish peroxidase-conjugated antibody (1: 1,000 dilution, Promega, Madison, WI) and anti-mouse IgG horseradish peroxidase-conjugated antibody (1:1,000 dilution, GE Healthcare, Buckinghamshire, UK). The amount of protein recognized by antibodies was quantified by means of an ECL Western Blotting Detection System (GE Healthcare) and SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Scientific, Rockford, IL). The quantitation value of Cx43 was normalized to the value of endogenous GAPDH. All data were normalized to the value of cardiomyocytes cultured in one control dish, which was set to a value of 1. Cx43 mRNA using real-time PCR. Total RNA was isolated from neonate rat cultured cells using the RNeasy Mini kit (Qiagen, Hilden, Germany) and reverse transcribed with Omniscript reverse transcriptase (Qiagen) and oligo-(dT)15 primers (Takara Biotechnology, Shiga, Japan). Levels of real-time PCR products were measured by the QuantiTect SYBR Green Kit (Qiagen) using a Rotor-Gene 6000 (Qiagen). The following primers were used for the amplification of Cx43 and GAPDH: Cx43, sense 5=-CTGGTGACAGAAACAATTCC-3= and antisense 5=GTCGTCGGGGAAATCGAACG-3=; and GAPDH, sense 5=-TGATGGGTGTGAACCACGAG-3= and antisense 5=-GCCCTTCCACGATGCCAAAG-3=. The relative quantitative value was expressed using the comparative threshold cycle (Ct) method (2⫺⌬Ct⫺Cc, where Cc is Ct of GAPDH used as a control gene), and the quantitation value of Cx43 was normalized to the value of endogenous GAPDH. Extracellular potential mapping of propagation. Conduction properties of cultured ventricular myocytes were examined by extracellular potential mapping using a multielectrode array system (MEDP545A and MED64, Alpha MED Scientific, Osaka, Japan) as previously described (14). The 64 planar microelectrodes, each having a size of 50 ⫻ 50 ␮m, were arranged in an 8 ⫻ 8 pattern with an interelectrode distance of 450 ␮m to cover a 3,150 ⫻ 3,150-␮m area beneath cultured cardiomyocytes. All 64 unipolar signals were recorded simultaneously with a common reference from 4 distant electrodes and amplified with high gain (60 dB) and at a lowfrequency (1.0⬃100 Hz) filter setting. Constant stimuli at 3.0 Hz were applied via two lateral electrodes of the recording array to elicit one-way propagation of excitation in the observation area before and after RES. Biphasic pulses of 0.2-ms duration were used for the stimulation with an intensity of 100 –200 ␮A (⫻1.3 the diastolic threshold). The activation time at each recording site was identified by a sharp negative deflection. Conduction velocity was measured by plotting activation time against distance from the stimulation site along a midline from left to right (see Fig. 6). Statistical analysis. Data are presented as means ⫾ SD. Differences in continuously distributed variables between predetermined experimental groups were analyzed using one-way ANOVA followed by Dunnett’s multiple-comparison method with SPSS software (SPSS, Chicago, IL). We did not model experimental design as repeated measures. Differences were taken to be significant at P ⬍ 0.05. RESULTS

Effects of RES on Cx43 expression. Figure 1A shows hematoxylin and eosin staining and confocal images of Cx43/ laminin immunolabeling of cultured cardiomyocytes. Cx43containing GJs were visualized as aggregates of bright punctate fluorescent domains around the cell perimeter of the cell

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Fig. 1. Confocal images of connexin (Cx)43 immunolabeling in cultured cardiomyocytes. A: representative immunofluorescence images of Cx43. Anti-laminin antibody was used to determine the basement membrane and then merged with Cx43 immunolabeling. B: proportion of the total cell area occupied by Cx43-immunoreactive signals (means ⫾ SD, n ⫽ 9). Solid bars, without (⫺) olmesartan (OS); open bars, with (⫹) OS; NS, not significant. **P ⬍ 0.01 vs. 0 min (baseline).

membrane at the abutment with neighboring cells. RES for 60 min resulted in a homogeneous increase of Cx43-immunoreactive signals without affecting their distribution pattern. Quantitative analysis revealed a significant increase in the Cx43-positive area by RES (1.4 ⫾ 0.2% at 60 min, n ⫽ 9, P ⬍ 0.01 vs. 0 min; Fig. 1B). The expression of Cx43 mRNA in cultured cardiomyocytes was also significantly increased by RES up to 60 min (by 1.2 ⫾ 0.1-fold, n ⫽ 9, P ⬍ 0.01 vs. 0 min; Fig. 2A). In the immunoblot analysis (Fig. 2B), the Cx43 antibody recognized three bands migrating between 40 and 43 kDa. The amount of Cx43 protein was increased gradually by RES, and the change reached a statistical significance at 60 min (by 1.5 ⫾ 0.3-fold, n ⫽ 9, P ⬍ 0.01 vs. 0 min). In whole heart experiments, the protein expression of Cx43 was also increased by RES, and the change reached a statistical significance at 60 min (by 1.9 ⫾ 0.4-fold, n ⫽ 9, P ⬍ 0.01 vs. 0 min; Fig. 2C). Effects of RES on ␤-catenin expression. Figure 3, A–C, shows confocal images of ␤-catenin immunolabeling in cultured cardiomyocytes. RES for 60 min resulted in a homogeneous cell surface increase of ␤-catenin-immunoreactive signals. Interestingly, ␤-catenin expression at the nucleus was

gradually increased by RES after 10 min. Quantitative analysis revealed a significant increase in the cell surface ␤-cateninpositive area by RES (2.7 ⫾ 0.6% at 30 min and 2.9 ⫾ 0.9% at 60 min, n ⫽ 9, P ⬍ 0.01 vs. 0 min; Fig. 3D). The amount of ␤-catenin protein in the total cell fraction of cardiomyocytes was significantly increased after 30 min of RES and thereafter (by 1.5 ⫾ 0.3-fold at 30 min and by 1.5 ⫾ 0.3-fold at 60 min, n ⫽ 9, P ⬍ 0.05 vs. 0 min; Fig. 4A). The amount of ␤-catenin protein in the whole heart was also significantly increased after 30 min of RES and thereafter (by 1.6 ⫾ 0.2-fold at 30 min and by 1.8 ⫾ 0.-fold at 60 min, n ⫽ 9, P ⬍ 0.05 and P ⬍ 0.01 vs. 0 min, respectively; Fig. 4B). Effects of RES on nuclear ␤-catenin expression. To clarify the upregulation of ␤-catenin expression in cardiomyocytes and in the whole heart, we investigated ␤-catenin expression in the nuclear fraction. Nuclear ␤-catenin functions as a T cell factor/lymphocyte enhancer binding factor (TCF/LEF) transcriptional activator, and Cx43 is known to be one of the TCF/LEF target genes. Interestingly, nuclear ␤-catenin expression in cardiomyocyates was remarkably increased by RES for 10 min and thereafter compared with baseline (by 1.4 ⫾ 0.2-fold at 10 min, n ⫽ 9, P ⬍ 0.05 vs. 0 min; Fig. 5B). In

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Fig. 2. Quantitative analysis of Cx43 mRNA and protein levels in cultured cardiomyocytes and whole hearts exposed to rapid electrical stimulation (RES). A: Cx43 mRNA levels in cultured cardiomyocytes estimated by real-time PCR. The amount of Cx43 mRNA was normalized to GAPDH (means ⫾ SD, n ⫽ 9). Solid bars, without OS; open bars, with OS. **P ⬍ 0.01 vs. 0 min (baseline). B: Cx43 protein levels in cultured cardiomyocytes estimated by Western blot analysis. C: Cx43 protein levels in the whole heart estimated by Western blot analysis. Representative data are shown. The amount of Cx43 was normalized to GAPDH (means ⫾ SD, n ⫽ 9). Solid bars, without OS; open bars, with OS. **P ⬍ 0.01 vs. 0 min (baseline).

addition, nuclear ␤-catenin expression in the whole heart was remarkably increased by RES for 10 min and thereafter compared with baseline (by 1.6 ⫾ 0.4-fold at 10 min, n ⫽ 9, P ⬍ 0.05 vs. 0 min; Fig. 5C). Effects of RES on the propagation of excitation. We investigated functional manifestation by the multielectrode extracellular potential mapping system. A clear local electrogram composed of a positive deflection followed by a negative deflection was recorded from most (⬎90%) of the 64 electrodes (Fig. 6A). Evoked spikes were propagated throughout cultured cardiomyocytes and also synchronized. Isochrone maps of activation time showed almost uniform propagation of excitation from left to right both before (control; Fig. 6B) and after RES for 60 min (Fig. 6C). The activation times after RES at the respective recording sites were much shorter than those of the control, indicating RES-induced acceleration of propagation. Figure 7 shows pooled data of conduction velocity. In the absence of olmesartan, the values of conduction velocity at 60 min after RES (25.7 ⫾ 4.0 cm/s, n ⫽ 6) were significantly higher than those of the control (19.1 ⫾ 4.0 cm/s, n ⫽ 6, P ⬍ 0.05). In the presence of olmesartan, RES did not affect conduction velocity; values at 60 min after RES (19.0 ⫾ 1.7 cm/s, n ⫽ 5) were similar to those of the control (18.5 ⫾ 2.4 cm/s, n ⫽ 5). Effects of RES on GSK-3␤. We next examined GSK-3␤, which modulates Cx43 expression through the Wnt/␤-catenin signaling pathway. GSK-3␤ is known to be phosphorylated by activated MAPKs and inhibits ␤-catenin degradation (13). We (14) have also reported that ERK and p38 MAPK of MAPKs are activated by RES. Total GSK-3␤ was unchanged, but p-GSK-3␤ showed a rapid significant increase in response to RES at 10 min (by 1.7 ⫾ 0.5-fold, n ⫽ 13, P ⬍ 0.01 vs. 0 min; Fig. 8A). These results suggest that p-GSK-3␤ might be increased by RES through activated MAPKs.

Prevention of RES-induced ␤-catenin/Cx43 upregulation and the p-GSK-3␤ increase by olmesartan or MAPK inhibitors. If ␤-catenin upregulation is the result of an inhibition of degradation by p-GSK-3␤ due to RES-induced activated p38 or ERK MAPKs, the increase of RES-induced GSK-3␤ phosphorylation and ␤-catenin/Cx43 upregulation should be prevented by pharmacological blockade of ANG II type 1 receptors. First, we checked the concentration-inhibition effect of olmesartan on Cx43 and ␤-catenin protein expression in cultured cardiomyocytes. As shown in Fig. 9, olmesartan antagonized RES-induced upregulation of Cx43 (Fig. 9A) and ␤-catenin (Fig. 9B) protein expression in a dose-dependent manner. As shown in Figs. 1–5, baseline levels of Cx43 and ␤-catenin were unaffected by treatment of cardiomyocytes with olmesartan (100 nmol/l). In immunohistochemistry experiments, RES for 60 min in the presence of olmesartan did not cause significant changes in the Cx43 and ␤-catenin positive area (Figs. 1 and 3). In the presence of olmesartan, RES for 60 min did not cause significant increases in Cx43 mRNA (Fig. 2A), Cx43 protein (Fig. 2B), and ␤-catenin expression in both total cell and nuclear fractions (Figs. 4 and 5B). Moreover, olmesartan attenuated the increase in conduction velocity of cardiomyocytes exposed to RES for 60 min (Figs. 6D and 7). Interestingly, the p-GSK-3␤ response to RES for 10 min was abolished by olmesartan (Fig. 8B). In addition, the p-GSK-3␤ response to RES for 10 min was also inihibited by SB-203580 (p38 MAPK inhibitor) and PD-98059 (ERK inhibitor; Fig. 8, C and D). These results indicate effective prevention of RES-induced ␤-catenin/Cx43 upregulation and the p-GSK-3␤ increase by the ANG II type 1 receptor blocker olmesartan or inhibitors of p38 MAPK and ERK.

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Fig. 3. Confocal images of ␤-catenin immunolabeling in cultured cardiomyocytes. A: representative immunofluorescence images of ␤-catenin (green) before (0 min) and after RES with or without OS. B: magnified representative immunofluorescence images of ␤-catenin (green) and nuclei (blue) after 10 min of RES. Red arrows indicate the nuclei. The merged images are the immunofluorescence images of ␤-catenin (green) merged with immunofluorescence images of nuclei (blue). C: representative volume images of nuclei using three-dimensional imaging. D: proportion of the total cell area occupied by ␤-catenin-immunoreactive signals (means ⫾ SD, n ⫽ 9). Solid bars, without OS; open bars, with OS. **P ⬍ 0.01 vs. 0 min (baseline).

DISCUSSION

The novel finding of this study is that RES caused alterations in cardiac intercellular proteins and changes of ␤-catenin preceded GJ remodeling of cardiomyocytes. Blockade of ANG II type 1 receptors might play an important role in the modulation of intercellular junction remodeling through a part of the Wnt/␤-catenin signaling pathway. The increase of nuclear ␤-catenin precedes Cx43 GJ remodeling and might enhance propensity to arrhythmias. The ANG II type 1 receptor blocker olmesartan affected intercellular junction protein remodeling and might alter the electrophysiological properties of cardiomyocytes exposed to RES. ID remodeling and arrhythmogenesis. Changes in electrical coupling through the ID of cardiomyocytes play a critical role in arrhythmogenesis in the diseased heart (2). Abnormal mechanical coupling through AJs (adherens junctions and the desmosome) occurs in several diseases of the myocardium (10, 15, 25, 30), and AJ remodeling is one of the important factors of arrhythmogenic substrates. Some studies (3, 7, 18) of GJs and AJs in cultured cardiomyocytes have reported that GJs are not necessary for the establish-

ment of cell AJs at the ID. In addition, Gutstein et al. (9) also reported that the GJ is not necessary for the organization of AJs and associated proteins in the cardiac ID. On the other hand, it is well known that AJs play an important role in GJ formation and assembly in cultured cardiomyocytes (12, 18, 34). However, the relationship between GJs and AJs of cardiomyocytes is not completely understood, especially under pathological conditions. There are some reports suggesting that there is a molecular crosstalk between AJs and GJs. Some investigators have reported the importance of maintaining cell junction integrity. McKoy et al. (22) showed that a mutation in the plakoglobin gene leads to ARVC. Oxford et al. (24) reported that loss of PKP2 expression led to a decrease in Cx43 content and a decrease in dye coupling between cardiomyocytes. Moreover, there are some reports (17, 19) that have suggested that abnormalities in the cadherin/catenin complex in heart disease may be an underlying mechanism leading to the establishment of the arrhythmogenic substrate by destabilizing GJs. Based on these reports, AJs play an important role in the regulation of GJs.

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Fig. 4. Quantitative analysis of ␤-catenin protein levels in the total cell fraction in cultured cardiomyocytes (A) and in whole hearts (B) exposed to RES. Representative data are shown. The amount of ␤-catenin was normalized to GAPDH (means ⫾ SD, n ⫽ 9). Solid bars, without OS; open bars, with OS. *P ⬍ 0.05 and **P ⬍ 0.01 vs. 0 min (baseline).

We (32) have previously identified a mechanism for lethal ventricular arrhythmias resulting from alterations in the cardiac ID. Our study (32) using the UM-X7.1 cardiomyopathic hamster model revealed that changes in ␤-catenin AJ protein precede Cx43 GJ alterations, ID remodeling might contribute to ventricular arrhythmogenesis during the development of

heart failure, and an ANG II type 1 receptor blocker might be a new upstream therapy for lethal arrhythmia by regulating ID remodeling. In addition, we (14) have also reported, using in vitro experiments with cultured cardiomyocytes, that shortterm RES causes upregulation of Cx43 through an activated autocrine action of ANG II to activate ERK and p38 MAPKs.

Fig. 5. Quantitative analysis of nuclear ␤-catenin protein levels in cultured cardiomyocytes and in whole hearts exposed to RES. A: localization of anti-histone H1 antibody, which was used as a specific marker of the nuclear fraction, in different protein fractions of cardiomyocytes. The different protein fractions (30 ␮g/lane) were subjected to immunoblot analysis. A strong signal was observed in the nuclear fraction. Fractions are indicated as follows: cytosolic (C), total membrane fraction (M), total fraction (T), and nuclear fraction (N). B: quantitative analysis of nuclear ␤-catenin protein levels in cultured cardiomyocytes. Representative data are shown. C: quantitative analysis of nuclear ␤-catenin protein levels in the whole heart. Representative data are shown. The amount of ␤-catenin was normalized to GAPDH (means ⫾ SD, n ⫽ 9). Solid bars, without OS; open bars, with OS. *P ⬍ 0.05 and **P ⬍ 0.01 vs. 0 min (baseline). AJP-Heart Circ Physiol • doi:10.1152/ajpheart.00653.2013 • www.ajpheart.org

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Fig. 6. Multielectrode extracellular potential mapping during the propagation of excitation. A: representative electrograms recorded from the 64 terminals with an image of cultured cardiomyocytes stained by hematoxylin and eosin including microelectrodes, each having a size of 50 ⫻ 50 ␮m. Arrows show stimulus artifacts. ST, stimulation point. B and C: isochrone maps of activation time in a culture dish before (control) and after RES for 60 min in the absence of OS. D: isochrone map of activation time in another culture dish after RES for 60 min in the presence of 100 nmol/l OS. *Stimulation points.

Based on our previous reports, in the present study, we focused on investigating stage-dependent changes in ␤-catenin and Cx43 protein expression in cardiomyocytes exposed to RES. Interestingly, our results showed that the expression of ␤-catenin was significantly increased before the increase of Cx43 expression. These results suggest that an increase of ␤-catenin precedes Cx43 GJ remodeling and might result in an arrhyth-

Fig. 7. Effects of RES on conduction velocity. Conduction velocity from left to right was measured along the midline in the observation area at 0, 30, and 60 min after RES. Values (means ⫾ SD) obtained in the absence (; n ⫽ 6) and presence of 100 nmol/l OS (Œ; n ⫽ 5) plotted against time after the initiation of RES. *P ⬍ 0.05 vs. 0 min (baseline).

mogenic substrate of cardiomyocytes exposed to short-term RES. The ␤-catenin precedence is consistent with our previous report of ID remodeling in UM-X7.1 cardiomyopathic hamsters. Based on our results and those of previous reports (22, 24, 28, 31, 32), ␤-catenin might play an important role in the formation and maintenance of GJs. Alterations in ␤-catenin expression and Cx43 GJ remodeling during RES. It is well known that the cadherin/catenin complex plays an important role not only in mechanical regulation but also in the signal transduction pathway that responds to extracellular signals (4). ␤-Catenin especially contributes to the adhesion processes with cadherin association, and it also participates in the Wnt/Wingless signaling cascade (5, 6, 23). The best-characterized Wnt signaling pathway is the canonical Wnt/␤-catenin pathway (21). When Wnt proteins bind to transmembrane receptors of the Frizzled family, cytoplasmic ␤-catenin is stabilized, enters the nucleus, and acts as a transcriptional activator of the TCF/LEF family. Ai et al. (1) has reported that Wnt signaling is an important modulator of Cx43-dependent intercellular coupling. Interestingly, Cx43 is well known to be one of the TCF/LEF target genes. Under physiological conditions, in the absence of Wnt signals, ␤-catenin is ubiquinated and degraded by GSK-3␤ in the proteosome (13, 16). GSK-3␤ is a negative regulator of protein synthesis and negatively regulates downstream signaling mechanisms of the Wnt/␤-catenin pathway (11). Interestingly, in our experiments, the expression level of ␤-catenin at the nucleus, which functions as a TCF/LEF transcriptional activa-

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Fig. 8. A: changes of total glycogen synthase kinase (GSK)-3␤ and phosphorylated (p-)GSK-3␤ in response to RES. B: prevention of RES-induced activation of p-GSK-3␤ by OS. C: prevention of RES-induced activation of p-GSK-3␤ by SB-203580 (p38 MAPK inhibitor). D: prevention of RES-induced activation of p-GSK-3␤ by PD-98059 (ERK inhibitor).Values were normalized to GAPDH (means ⫾ SD, n ⫽ 7⬃13). Œ, p-GSK-3␤; , total GSK-3␤. **P ⬍ 0.01 vs. 0 min (baseline).

tor of Cx43, was dramatically increased by RES for 10 min, resulting in an upregulation of Cx43 in cardiomyocytes. We (27, 32) have previously reported reduced changes in the TCF/LEF DNA-␤-catenin binding complex of the nucleus during the development of heart failure, resulting in a decrease in the expression of Cx43 mRNA/protein and in the initiation/ perpetuation of lethal arrhythmias. In addition, reduced ␤-catenin-dependent transcription via TCF/LEF transcriptional factors has been suggested to be the molecular mechanism behind ARVC caused by the loss of desmoplakin (8). The response of the Cx43 promoter to ␤-catenin-dependent transactivation suggests that alterations in nuclear ␤-catenin expression affect Cx43 gene/protein expression in cardiomyocytes. These results propose that AJ and GJ remodeling results, at least in part, in an important arrhythmogenic substrate.

Effects of the ANG II receptor blocker olmesartan on ␤-catenin expression. In this study, olmesartan improved intercellular junction remodeling, attenuated the increase of RES-induced ␤-catenin/Cx43 upregulation, inhibited the phosphorylation of GSK-3␤, and decreased conduction velocity in cultured cardiomyocytes. Although we cannot establish the precise mechanisms of olmesartan in the regulation of nuclear ␤-catenin expression, here we try to discuss the possible mechanisms of olmesartan on the attenuation of RES-induced ␤-catenin upregulation. We (14) have previously reported that short-term RES causes an upregulted autocrine action of ANG II to activate p38 MAPK and ERK. In addition, ␤-catenin degradation is negatively regulated by active GSK-3␤ (13, 16). Interestingly, it has been reported that GSK-3␤ is phosphorylated by p38

Fig. 9. Concentration-inhibition curves for OS on the increase of Cx43 expression at 60 min after RES (A) and the increase of ␤-catenin expression at 30 min after RES (B) in cultured cardiomyocytes. Values are means ⫾ SE; n ⫽ 6.

AJP-Heart Circ Physiol • doi:10.1152/ajpheart.00653.2013 • www.ajpheart.org

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MAPK, and this phosphorylation leads to the inactivation of GSK-3␤ (29), resulting in an inhibition of protein degradation and an increase of protein synthesis/expression, e.g., hypertrophy (11). Our data also showed that p38 MAPK and ERK inhibitors attenuated the phosphorylation of GSK-3␤. If activated p38 MAPK phosphorylates GSK-3␤ to inhibit ␤-catenin degradation, the TCF/LEF transcriptional factor is activated to increase Cx43 mRNA/protein expression. The ANG II receptor blocker olmesartan might then be useful to decrease the phosphorylation of GSK-3␤ by MAPK and increase the degradation of ␤-catenin, resulting in an attenuation of Cx43 upregulation. The nature of the signaling interactions between ANG IIMAPKs-GSK3␤-␤-catenin in several pathological conditions awaits further studies. Study limitations. For the experiments using cultured cardiomyocytes, we used neonatal, not adult, rat ventricular myocytes in our experiments. The reasons why are as follows: we should get a sufficient amount of protein to investigate alterations in qualitative and quantitative intercellular junction proteins of pure/simplified cardiomyocytes using protein purification and Western methods. Moreover, we needed monolayer cultured cardiomyocytes to examine cell-to-cell intercellular communication and propagation properties using a multielectrode array system. In the case of whole heart experiments, some other structural and molecular factors might modulate intercellular junction remodeling. Thus, we used cultured neonatal rat ventricular myocytes to simplify the experiments. Conclusions. In the present study, we identified the precedence of changes in AJ protein ␤-catenin during the development of GJ remodeling. Alterations in AJ protein might play an important role in the formation and stability of GJs in cardiomyocytes exposed to RES. Furthermore, we investigated one of the underlying mechanisms of the effects of an ANG II type 1 receptor blocker on ␤-catenin expression. ANG II receptor blockers might be a new upstream therapy for lethal arrhythmia by modulating ID remodeling. ACKNOWLEDGMENTS Olmesartan was kindly provided by Daiich Sankyo (Tokyo, Japan). GRANTS This work was supported in part by Japan Society for the Promotion of Scient KAKENHI Grant C23591081 (to T. Ohkusa). DISCLOSURES No conflicts of interest, financial or otherwise, are declared by the author(s). AUTHOR CONTRIBUTIONS Author contributions: T.N. and T.O. conception and design of research; T.N., T.O., Y.O., J.-K.L., Y.M., and M. Yano performed experiments; T.N., T.O., J.-K.L., and Y.M. analyzed data; T.N., T.O., M. Yoshida, and M. Yano interpreted results of experiments; T.N. and T.O. prepared figures; T.N., T.O., M. Yoshida, J.-K.L., Y.M., and M. Yano drafted manuscript; T.N., T.O., M. Yoshida, J.-K.L., Y.M., and M. Yano edited and revised manuscript; T.N., T.O., M. Yoshida, J.-K.L., Y.M., and M. Yano approved final version of manuscript. REFERENCES 1. Ai Z, Fisher A, Spry DC, Brown AM, Fishman GI. Wnt-1 regulation of connexin43 in cardiac myocytes. J Clin Invest 105: 161–171, 2000. 2. Akar FG, Tomaselli GF. Conduction abnormalities in nonischemic dilated cardiomyopathy: basic mechanisms and arrhythmic consequences. Trends Cardiovasc Med 15: 259 –264, 2005.

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Rapid electrical stimulation causes alterations in cardiac intercellular junction proteins of cardiomyocytes.

The intercellular junctions contain two complexes, adhesion junctions (AJ) and connexin (Cx) gap junctions (GJs). GJs provide the pathway for intercel...
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