ORIGINAL ARTICLE

Gap Junction-associated Na+ Influx Is Involved in the Mediation of Ca2+ Transients in Neonatal Rat Ventricular Myocytes Xinxin Yan, PhD, Zheng Zeng, MD, Min Chen, MD, Chen Li, MD, Shu Li, MD, and Dali Luo, PhD

Abstract: Gap junction (GJ) coupling is involved in the regulation of spontaneous Ca2+ transients in neonatal rat ventricular myocytes (NRVMs); however, the underlying mechanism(s) remains incompletely clear. In this study, we investigated Na+ influx mediated by Cx43-associated GJ modulating the spontaneous NRVM excitation. Intracellular Na+ and Ca2+ concentrations ([Na+]i and [Ca2+]i) were assessed using CoroNa Green and Fluo-4 fluorescent probes, respectively, by confocal microscopy. GJ function was evaluated by measuring fluorescence recovery after photobleaching. The results showed that [Na+]i and spontaneous Ca2+ oscillating were significantly decreased by the treatment of NRVMs with GJ uncouplers or adenovirus-mediated Cx43 gene knockdown using short hairpin RNA, while both of the intracellular cation levels were increased by adenovirus-mediated Cx43 overexpression. Neither Na+ channel blocker (tetrodotoxin) nor the GJ uncoupler (Gap27) or Cx43-short hairpin RNA adenovirus could completely suppress the Ca2+ transients, but their combination usage could. In addition, Ca2+ transients disappeared in NRVMs incubated in Na+-free, but not in Ca2+-free medium, in which the remained transients could be further abolished by Gap27. Collectively, our findings suggest that Cx43-associated GJ function is involved in the regulation of Na+ influx into cardiomyocytes, which contributes, at least in part, to triggering spontaneous excitation and regulation of cardiomyocyte automaticity. Key Words: Na+ influx, Ca2+ transients, Connexin43, gap junctions, neonatal rat ventricular myocytes (J Cardiovasc Pharmacol  2015;66:41–49)

INTRODUCTION Gap junctions (GJs) form connecting channels among adjacent cells, allowing intercellular exchange of molecules of up to 1200 Da in size. Each GJ channel is formed of a pair of Received for publication November 18, 2014; accepted February 4, 2015. From the Department of Pharmacology, Capital Medical University, Beijing, China (Li is now with the National Institute for Radiological Protection, China CDC, Beijing, China). Supported by Grants from National Natural Science Foundation (81370339), Beijing National Natural Science Foundation (5122006) and Beijing Key Laboratory of Cardiovascular Diseases Related to Metabolic Disturbance (Z13111000280000). The authors report no conflicts of interest. Reprints: Dali Luo, PhD, Department of Pharmacology, Capital Medical University, Beijing 100069, China (e-mail: [email protected]). Copyright © 2015 Wolters Kluwer Health, Inc. All rights reserved.

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connexons (hemichannels), which are in turn composed of 6 connexin molecules that span the full depth of the membrane.1–3 GJs function as intercellular channels that permit ion flow freely and also facilitate the exchanges of nutrients, metabolites, and small signaling molecules between cells. Consequently, they are crucial for maintaining cellular homeostasis and for cellular biological processes such as gene transcription, growth, development, and differentiation.2,3 In the heart, GJs provide a functional syncytium that allows for instant electrical spreading throughout myocardium, enabling a rhythmic synchronized electrical excitation–contraction coupling.3–5 Multiple studies have demonstrated that disturbances in GJ function may cause cardiac arrhythmias and myocyte apoptosis, 2 common and serious complications of many heart diseases.6–8 Connexin43 (Cx43), one of the 12 members of the connexin family, is expressed predominantly in the ventricular myocardium.3,9 In a previous study, we found that in addition to facilitating Ca2+ spreading among adjacent cells in normal cardiomyocytes, Cx43-associated intercellular communication also affects basal intracellular Ca2+ transients in resting ventricular myocytes.10,11 This suggests that Cx43-mediated intercellular exchange may be involved in the regulation of automaticity in normal cardiomyocytes. Interestingly, several studies have shown that sodium channels also localize to the intercalated disks of isolated ventricular myocytes,12,13 and a nonselective current can be activated by the opening of Cx43 hemichannels in rabbit ventricular myocytes.14 Additionally, a Cx43-associated nonselective current contributes to intracellular Na+ overload in the ischemic heart and during heart failure.14–16 These studies imply a potential correlation between Na+ entry and GJ channel opening in cardiomyocytes, in normal and in pathological conditions. In this study, we investigated whether Cx43 regulates Na+ influx in neonatal rat ventricular myocytes (NRVMs), which exhibit rhythmic spontaneous activity, displayed as Ca2+ transients and contraction. These cells are also convenient for manipulating Cx43 gene expression through viral transduction. We thus examined the effects of enhancing or reducing Cx43 expression with adenoviral transduction on spontaneous Ca2+ transients and intracellular Na+ concentration [Na+]i in NRVMs. We also evaluated the effects of GJ uncoupling and sodium channel blockade in these cells. Our findings suggest that Na+ influx through Cx43-associated GJs is likely involved in the regulation of spontaneous cardiac beating in neonatal ventricular myocytes. www.jcvp.org |

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MATERIALS AND METHODS This study was approved by the Capital Medical University Animal Care and Use Committee, and all studies were conducted in accordance with “Guide for the Care and Use of Laboratory Animals” adopted by the Beijing Government and “Guide for the Care and Use of Laboratory Animals” published by the US National Institutes of Health (publication number 85-23, revised 1996). The proper diethyl ether was used for anesthesia in neonatal rats.

Materials Fluo-4/AM, CoroNa Green (C36676) and 6-carboxyfluorescein diacetate (6-CFDA) were obtained from Molecular Probes (Invitrogen Inc, Carlsbad, CA). Tetrodotoxin (TTX), nifedipine, BAPTA/AM, carbenoxolone (CBX), heptanol, Gap27, and KB-R7943 were purchased from Sigma-Aldrich (St Louis, MO). They were solubilized in different solvents, and an equal amount of solvent is used in control experiments. Sprague-Dawley rats (2 days old) were obtained from the Experimental Animal Center of Capital Medical University (Beijing, China). All the other reagents, unless otherwise indicated, were purchased from Sigma-Aldrich. The antibodies for p-Cx43, Cx43, and GAPDH were purchased from Santa Cruz Biotechnology, Inc (Santa Cruz, CA).

Isolation and Culture of NRVMs NRVMs were isolated by enzymatic digestion with 0.1% trypsin and 0.03% collagenase, as described previously.11 After removing cardiac fibroblasts, NRVMs were plated onto 35-mm dishes at a density of 1 · 106 cells per milliliter in Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum (Hyclone, Australian), 100 units per milliliter penicillin/streptomycin, and 0.1 mM 5-bromo-2deoxyuridine to inhibit fibroblast proliferation.

described.11 Experiments were performed at room temperature (22–248C).

FRAP Measurement

NRVMs were loaded with the dye 6-CFDA (7 mg/mL), a specific dye known to permeate GJ after hydrolysis to 6-carboxyfluorescein for 5 minutes at room temperature, and then were washed 3 times for 10 minutes. Fluorescence recovery after photobleaching (FRAP) was measured by the laser scanning microscope as previously described.11,17 Briefly, a cell adjacent to other cells was selected, and its fluorescence was photobleached by strong laser pulses (488 nm) with 30 scans for a total duration of 15 and 20 seconds, reaching almost complete and approximately 60% photobleaching in NRVMs. The fluorescence intensity was recorded at excitation 488 nm and emission 570 nm in the bleached cells every 2–10 seconds over a period of 7 minutes before and after photobleaching. In each experiment, an isolated bleached cell and a labeled isolated cell, left unbleached, served as reference controls for the loss of fluorescence due to repeated scanning and dye leakage. After correction and normalization, fluorescence was plotted over time to generate fluorescence recovery curves. Analysis allows us to obtain 2 values by equations: the recovery degree (It/I) and the recovery speed {comparative fluorescence intensity recovery rate {(CFIRR) = [(It 2 I0)/(I 2 I0) · 100%]/t} as reported with minor modifications.17

Cell Infection With Adenovirus

All the NRVMs were used after 48-hour culture. Cardiomyocytes were loaded with 10 mM CoroNa Green (CoroNa) for 45 minutes at 378C followed by 3 washes with HEPES-buffered physiological saline solution (HBSS) (Na+ 135, KCl 5, MgCl2 1, CaCl2 1.8, HEPES 10, glucose 11, pH7.4) (in millimolars). After these cells were mounted on the laser confocal microscopy (Leica SP5) equipped with ·63 oil immersion objective (NA 1.4), their basal fluorescence was recorded with excitation wave 492 nm and emission wave 516 nm, and then they were washed with HEPES buffer with Ca2+ but without Na+ medium substituted with N-Methyl-D-glucamine (NMDG) 135 mM for 20 minutes at room temperature (22–248C). The changes in the fluorescence intensity with time course were collected by a series scanning, and the [Na+]i was expressed as F/F0, where F0 stands for the mean basal fluorescence obtained from 3 times of scanning.

The construction of adenovirus was carried out by Invitrogen Inc (Shanghai, China). Cx43 coding region was amplified by PCR reaction. The sequences for the pair of primers are 50 -CCGCTCGAGGCCACCATGGGTGACTGGAGTGCCTTGGG-30 and 50 -CCGGAATTCTTAAATCTCCAGGTCATCAGGCCGA-30 . Then, the PCR products were digested by XhoI and EcoRI and cloned into pIRES2-EGFP vector. The Cx43 overexpression plasmids pGJA1-IRES-EGFP (wt-Cx43) was confirmed by sequencing and subcloned into pAD/CMV/V5-DEST vector by gateway reconstitution technique to make pAd-JX-GJA1IRES2-EGFP adenovirus construct. To make the Cx43 knockdown plasmids, the complementary sequences 5 0 -TGCTGGATTCGCGTCTTCTTGTTGTCGTTTTGGCCACTGACTGACGACAACAAAGACGCGAATC-30 and 5 0 -CCTGGATTCGCGTCTTTGTTGTCGTCAGTCAGTGGCCAAAACGACAACAAGAAGACGCGAATCC-3 0 were verified to avoid the off-target silencing and inserted into pcDNA 6.2-GW/EmGFP miRNA vector using BLOCK-iT Pol II miR RNAi Expression Vector Kit. After evaluation of the knockdown effects, it was also subcloned into pAD/CMV/V5DEST vector. NRVMs were transfected with adenovirus construct of wt-Cx43 or Cx43-shRNA gene for 48 hours and then were examined for the relationship of Cx43 expression with Ca2+ transients.

Confocal Ca2+ Transients and Waves Imaging

Western Blot

Loading of the myocytes with Fluo-4 and the measurement of Ca2+ transients were performed as previously

For the whole cell lysate, NRVMs were lysed in RIPA buffer containing 1 mM phenylmethanesulfonyl fluoride and

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Cytosolic Na+ Measurement

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Gap-mediated Na+ Influx Regulates Automaticity

complete protease inhibitor cocktail. Lysates were boiled for 5 minutes, resolved on a 10% SDS-PAGE gel and transferred to PVDF membrane. Membranes were blocked with 5% nonfat milk powder in tris-buffered saline containing 0.1% (vol/vol) Tween20 for 60 minutes at room temperature. AntiCx43 (rabbit polyclonal immunoglobulin G: mapping between amino acids 233-382 of Cx43 C-terminus of human origin) and anti-GAPDH (mouse monoclonal immunoglobulin G1 FL of GAPDH of rabbit) antibodies were used overnight at 48C at dilution of 1:1000, 1:1000, and 1:1500, respectively. The immunoblotted membrane was then incubated with horseradish peroxidase-conjugated secondary antibody for 1 hour and immunoreactive bands were detected by using enhanced chemiluminescence.

hairpin RNA (Cx43-shRNA), for silencing. Western blot analysis revealed a substantial titer-dependent reduction in Cx43 protein expression 48 hours after Cx43-shRNA adenoviral transduction and an approximately 3-fold increase in Cx43 expression in NRVMs transduced with wt-Cx43 (Fig. 1A). Corresponding with the changes in Cx43 expression, cells treated with wt-Cx43 displayed an increase in the rate of spontaneous Ca2+ transients, whereas cells treated with Cx43-shRNA displayed a decrease in the rate of spontaneous Ca2+ transients, compared with vector or scrambled controls (Figs. 1A, B). We next examined the effects of a variety of GJ antagonists, including heptanol, carbenoxolone (CBX), and Gap27, a specific GJ uncoupler that mimics the short sequences in the second extracellular loop of Cx43 and directly interacts with exposed GJ channels.18 As shown in Figures 1C, D, heptanol (2 mM, for 2 minutes) completely abolished the spontaneous Ca2+ transients. Similarly, CBX (30 mM, for 20 minutes) and Gap27 (400 mM, for 30 minutes) also substantially suppressed the Ca2+ transients in NRVMs. These results indicate that changes in Cx43 expression and function robustly affect the rate of global Ca2+ spiking, suggesting an important role of Cx43-associated GJs in the regulation of automaticity in NRVMs.

Statistical Analysis

Results are presented as mean 6 SEM. Differences between data sets were considered significant when P , 0.05. Statistical analysis was performed by one-way analysis of variance of repeated measures followed by Student’s t-test as appropriate.

RESULTS Cx43-mediated Regulation of in NRVMs

Ca2+

Transients Ca2+

To investigate the role of Cx43 in regulating transients, we monitored spontaneous Ca2+ transients in NRVMs after transduction with adenovirus carrying the wild-type rat Cx43 gene (wt-Cx43), for overexpression, or rat Cx43-short

Cx43-mediated Regulation of Na+ Influx in NRVMs To determine whether Na+ influx, a trigger of action potentials in excitable cells, is also affected by Cx43associated GJ function in myocytes, we examined real-time [Na+]i changes using the Na+ probe CoroNa Green by confocal microscopy. Unlike the fluctuations in internal Ca2+

FIGURE 1. Effects of modulating Cx43 expression on spontaneous Ca2+ transients in NRVMs. A, Cx43 expression and Ca2+ transients in NRVMs were assessed by Western blot and laser confocal microscopy, respectively. Cx43 overexpression and silencing were performed by adenovirus infection (MOI = 30 or 10, 20, and 40). B, Changes in global Ca2+ transients in response to manipulation of Cx43 expression in NRVMs. C, Traces represent Ca2+ transients in NRVMs treated without or with 2-mM heptanol for 2 minutes. D, Bar graph of the Ca2+ transients in NRVMs in the different groups treated with control (Con), 2 mM heptanol for 2 minutes, 30 mM CBX for 20 minutes, or 400 mM Gap27 for 30 minutes. Statistical data from 3 to 5 independent determinations (n = 48–76 cells for each bar) show the effect of the drugs on the Ca2+ transients. **P , 0.01 versus Con. Copyright © 2015 Wolters Kluwer Health, Inc. All rights reserved.

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concentration ([Ca2+]i) that can be monitored with Fluo-4 at the micromolar scale at millisecond resolution, fast changes in the fluorescence intensity of [Na+]i, such as during the overshoot of spontaneous action potentials after the opening of sodium channels cannot be monitored using currently available Na+ probes.19,20 Thus, we assessed the changes in the fluorescence intensity of CoroNa Green and compared them with those of the paralleled controls. NRVMs were incubated in HBSS in the presence or absence of tetrodotoxin (TTX), a sodium channel blocker,21 or the extracellular NaCl that was replaced with N-methyl-D-glucamine (NMDG; 135 mM). A basal Na+ fluorescence was observed in cells incubated with Na+-containing medium in either the presence or absence of external Ca2+ (Fig. 2A). This basal fluorescence was significantly reduced or barely detectable in Na+-free medium (data not shown), demonstrating a dependence on extracellular Na+ but not Ca2+. TTX (10 mM, for 6 minutes), as well as the GJ uncouplers heptanol (2 mM, for 2 minutes) and Gap27 (300 mM, for 30 minutes), significantly attenuated CoroNa Green fluorescence in normal Na+-containing medium (Figs. 2A–C), suggesting an inhibition of Na+ entry or an inhibition of Na+ excretion by the GJ uncouplers. Because [Na+]i was much lower than extracellular [Na+], and the rate and amplitude of the Ca2+ transients were also simultaneously lowered by inhibition of GJ function, a decrease in Na+ influx was more likely to have contributed to the reduction in CoroNa Green fluorescence. Subsequently, we examined [Na+]i levels in NRVMs transduced with Cx43-shRNA or wt-Cx43 virus for 48 hours and found that the basal CoroNa Green fluorescence was also significantly enhanced in wt-Cx43–treated cells and decreased in Cx43-shRNA–treated cells, compared with cells transduced with vector or scramble control (Figs. 3A–C). The increase in basal CoroNa Green fluorescence in wt-Cx43– treated cells was completely abolished in Na+-free medium, confirming the contribution of Na+ influx in the GJ-mediated increase in [Na+]i. Heptanol (2 mM, for 2 minutes) reduced

the [Na+]i to a similar level in all groups (Figs. 3A, B). This provides addition evidence that alteration in Na+ influx through manipulating Cx43-associated GJs is likely related to the spontaneous electrical activity in NRVMs. Next, we examined the possibility that the changes in [Na+]i induced by GJ manipulation were secondary to the changes in [Ca2+]i. The effect of nifedipine, an inhibitor of L-type Ca2+ channels, on the basal CoroNa Green signal was measured. Although this drug, like the GJ uncouplers (Fig. 1), dramatically inhibited Ca2+ oscillations in a dose-dependent manner (Fig. 3E), nifedipine did not mimic the effects of heptanol, Gap27, or Cx43-shRNA adenovirus on the basal [Na+]i (Figs. 3A, B and D) and on the GJ permeability assessed by FRAP (Figs. 3F–H).11 Thus, in agreement with the finding that Na+ entry is independent of internal Ca2+ change,22 the data suggest that the effect of GJ inhibition on Na+ influx is independent of its effect on Ca2+ transients, that is, a decrease in internal Ca2+ does not affect Na+ entry or GJ permeability in these settings.

Correlation Between Na+ Influx and Ca2+ Transients Mediated by Cx43-Associated Gap Junctions in NRVMs It is well known that Na+ currents through sodium channels and GJ-mediated intercellular communication are critical for initiating cardiac action potentials and propagation of the electrical signal throughout myocardium, in which the Ca2+ influx or the Ca2+ transients is mainly through the activation of voltage-gated Ca2+ channels. However, it is not clear whether Na+ flux mediated by GJ channels is also able to trigger excitations in cardiomyocytes.23,24 The preceding results demonstrate that in addition to being modulated by sodium channels (Fig. 2), [Na+]i and Ca2+ transients in cardiomyocytes are accordingly regulated by Cx43-associated GJ function and expression level (Figs. 1–3),10 suggesting an intrinsic relationship between them. Therefore, to examine

FIGURE 2. Effects of modulating Cx43 function on basal Na+ fluorescence intensity in NRVMs. A, [Na+]i fluorescence images were recorded in NRVMs in cells treated with vehicle, TTX, heptanol, or Gap27 using laser confocal microscopy. B, Traces of [Na+]i fluorescence intensity recorded for 1 minute in NRVMs before and after treatment with vehicle, 10 mM TTX for 6 minutes, 2 mM heptanol for 2 minutes, or 300 mM Gap27 for 30 minutes. C, Data were obtained from 6 to 8 independent determinations for each treatment. **P , 0.01 versus Con.

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Gap-mediated Na+ Influx Regulates Automaticity

FIGURE 3. Effects of modulating Cx43 expression on [Na+]i fluorescence intensity in NRVMs. A, [Na+]i fluorescence images were captured by laser confocal microscopy in NRVMs transduced with vector, Cx43-shRNA, or wt-Cx43 virus, and treated with heptanol or nifedipine. B, Traces of [Na+]i fluorescence intensity in NRVMs recorded for 1 minute in cells transduced with vector, Cx43-shRNA, or wt-Cx43 virus, before and after treatment with vehicle, 2 mM heptanol, or 1 mM nifedipine for 2 minutes. C, Statistical data showed that [Na+]i fluorescence intensity was decreased in the Cx43-shRNA group but increased in the wt-Cx43 group. ##P , 0.01 versus vector. D, Data showing that [Na+]i fluorescence intensity was decreased in the 3 groups treated with heptanol. *P , 0.05 versus Con, **P , 0.01 versus Con. (E) Traces of Ca2+ transients in NRVMs before and after nifedipine (1 mM) or heptanol (2 mM) treatment. Statistical data show that the effect of nifedipine on Ca2+ transients was dose-dependent. F, Traces of bleached cells showing 6-CFDA transfer through GJs among interconnected NRVMs monitored with the FRAP method. I, I0, It, and It1/2 represent the fluorescence intensity initially, after bleaching, recovery at 400 seconds, and at the time at 50% recovery, respectively. Effects of heptanol (2 mM, 2 minutes) and nifedipine (1 mM, 2 minutes) on fluorescence recovery in bleached cells are indicated as a percentage of 2 parameters, It/I (G) and CFIRR (H), which represent the degree and speed of fluorescence recovery after normalization with control values. A rescue effect was found in both heptanol- and nifedipine-treated cells.

whether Na+ influx mediated by Cx43-associated GJs contributes to the pacemaking activity in NRVMs, we measured the Ca2+ transients of myocytes incubated in Na+-free HEPES buffer for 1 minute or after exposure to TTX to eliminate Copyright © 2015 Wolters Kluwer Health, Inc. All rights reserved.

the involvement of Na+ influx through sodium channels. The experiments were performed on monolayer cultures of NRVMs and on single cells as the latter exhibit faster transients likely because more hemichannels are exposed to the www.jcvp.org |

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extracellular space.10 As expected, in Na+-free medium, the spontaneous coordinated Ca2+ transients were completely disappeared in both monolayer and single cells (Figs. 4A, B), but the inhibition of sodium channels with 10 mM or 30 mM TTX (data not shown) for 6 minutes only partially suppressed the synchronized Ca2+ oscillations in monolayer and the Ca2+ transients in single NRVMs. The residual Ca2+ transients after TTX treatment could be abolished by the addition of Gap27 or heptanol at concentrations similar to that used in the preceding experiments (Figs. 4C, D). Conversely, the residual Ca2+ transients after Gap27 treatment were further abolished by TTX treatment (10 mM, data not shown). As Na+/Ca2+ exchange (NCX), an important mechanism for Na+ and Ca2+ transport across the membrane, is also inhibited in Na+-free medium, we then used NCX antagonist KB-R7943 (10 mM) to treat myocytes for 10 minutes before collection of Ca2+ transients to exclude the possibility of NCX involvement. As previously reported,22 inhibition of NCX did not affect the spontaneous Ca2+ oscillations in NRVMs incubated in normal HBSS (Figs. 4E, F), and also did not recover the abolishment effect of Na+-free medium on Ca2+ transients (data not shown), demonstrating that the elimination of Ca2+ transients in Na+-free buffer are unlikely due to the blockade of NCX. In contrast to the effect of Na+-free medium, cells incubated in Ca2+-free medium with 0.2 mM EGTA for 10 minutes exhibited Ca2+ oscillations with lowered amplitude and frequency. The addition of BAPTA (200 mM, for 10 minutes) to chelating [Ca2+]i or Gap27 completely abolished the residual Ca2+ transients, and washout with Ca2+-containing medium was able to restore the Ca2+ transients in both

monolayer and single cells (Fig. 5A). Similar to the effects on basal Ca2+ transients, the basal [Na+]i was not different between cells incubated in Ca2+-containing and Ca2+-free medium but was significantly decreased by Gap27 or heptanol treatment. In addition, the basal CoroNa Green signal was not significantly changed upon addition of BAPTA in the monolayer (Fig. 5B), and similar results were also obtained using single cells (data not shown).

DISCUSSION This study demonstrates that Na+ influx mediated by Cx43-associated GJs is involved in the regulation of global Ca2+ transients or pacemaking in cultured neonatal ventricular myocytes. Spontaneous Ca2+ oscillations in cultured neonatal ventricular myocytes are characteristic electrical patterns arising from membrane depolarization; however, the mechanisms underlying the rhythmic activity are not clear.25,26 It is well known that Na+ influx through sodium channels in the plasma membrane is essential for the genesis of action potentials and excitation–contraction coupling. Among the several subtypes of sodium channels found in cardiomyocytes,27,28 Nav1.5 is the predominant sodium channel distributed in ventricular myocytes,29 and TTX is commonly used as a nonspecific sodium channel blocker. Here, we used TTX to block plasma membrane sodium channels and Na+-free HBSS buffer to eliminate extracellular Na+ influx. Spontaneous Ca2+ oscillations in both monolayer and single NRVMs were robustly suppressed by TTX treatment and completely disappeared in Na+-free medium (Fig. 4), confirming the critical role of Na+ entry in cardiomyocyte excitation. Notably, GJ uncouplers suppressed

FIGURE 4. Effects of extracellular Na+ and sodium channel inhibition on Ca2+ transients in NRVMs. A, images of Ca2+ transients in NRVMs were recorded by laser confocal microscopy in regular HEPES buffer and in HEPES buffer with NMDG instead of Na+ for a period of 20 minutes. B, Traces represent spontaneous Ca2+ transients in both monolayer and single NRVMs incubated in HEPES buffer with or without Na+. C, Traces of spontaneous Ca2+ transients in NRVMs incubated in HEPES buffer, then treated with 10 mM TTX for 6 minutes followed by the addition of 300 mM Gap27 for 30 minutes or 2 mM heptanol for 2 minutes. D, Data were obtained from 6 to 8 independent determinations for each treatment. **P , 0.01 versus Con, ##P , 0.01 versus TTX. E, Traces represent Ca2+ transients in NRVMs treated without or with 10 mM KB-R7943 for 10 minutes. F, Bar graph shows the statistical data obtained from the measurement of Ca2+ transients of NRVMs in E.

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Gap-mediated Na+ Influx Regulates Automaticity

FIGURE 5. Effects of extracellular Ca2+ depletion on Ca2+ transients and [Na+]i fluorescence in NRVMs. A, Typical traces of spontaneous Ca2+ transients in monolayer and single NRVMs in regular Ca2+-containing medium or Ca2+-free medium with 0.2 mM EGTA before and after BAPTA/AM or Gap27 treatment, followed by the addition of extracellular Ca2+. B, Traces of [Na+]i fluorescence intensity in NRVMs in regular Ca 2+ -containing medium or Ca2+-free medium with 0.2 mM EGTA before and after BAPTA, Gap27, or heptanol treatment.

the Ca2+ transients and the [Na+]i accordingly in these cells, whereas the Ca2+ channel blocker nifedipine only inhibited the Ca2+ signals but not the internal Na+ abundance (Fig. 3). These findings suggest that GJ-mediated Na+ and/or Ca2+ influx are likely involved in the regulation of spontaneous coordinated Ca2+ spiking. The dramatic changes in intracellular cation activities produced by manipulating Cx43 expression provides further evidence for the role of GJs in the regulation of Ca2+ and Na+ influx, ie, cardiomyocyte automaticity (Figs. 1–3), which is in good agreement with Jansen JA’s study that showed a reduction in sodium current amplitude following decreased Cx43 expression in adult ventricular myocytes.30 It should be noted that high concentrations of Gap27, CBX or TTX, or modulating Cx43 expression alone did not completely abolish the spontaneous Ca2+ transients (Fig. 1), whereas the combination of TTX, which inhibits Na+ entry through plasma membrane sodium channels, and Gap27, which blocks the exchange of molecules through GJ channels could (Fig. 4). In addition, although Na+-free medium fully stopped Ca2+ oscillations, cells incubated in Ca2+-free medium still oscillated, which could be terminated by Copyright © 2015 Wolters Kluwer Health, Inc. All rights reserved.

chelating the intracellular Ca2+ with BAPTA or addition of Gap27, in both monolayer and single cells (Fig. 5). This suggests that most oscillating Ca2+ is mobilized from the internal stores in cardiomyocytes, and that GJ sensitive Na+ entrance in the absence of extracellular Ca2+ plays a role in the cardiomyocyte excitation. Additionally, it is interesting to note that sodium channels are found to co-localize with Cx43-containing hemichannels or GJs in the intercalated disks of cardiomyocytes,12,13,31 and a close correlation between the opening of GJ channels and Na+ influx have been showed.32 For examples, hemichannel opening contributes to Na+ and Ca2+ overload during metabolic inhibition in isolated myocytes,33 and in rabbit ventricular myocytes a Cx43 hemichannel-mediated nonselective Na+ current is found.14 Our findings that inhibitions of GJ function or Cx43 expression suppress the basal [Na+]i, while overexpressing Cx43 enhances basal [Na+]i further supports the correlation between Na+ entrance and Cx43-associated GJ function in cardiomyocytes. Furthermore, our finding extends that the GJ-based Na+ activity is involved in the regulation of basal Ca2+ transients, ie, the spontaneous automyticity, in normal NRVMs. www.jcvp.org |

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Here, the monolayer NRVMs that are relevant to myocyte function in intact cardiac cells and the single cells, which should mostly express hemichannels, behaved similarly in response to GJ inhibition or deficiency in Cx43 expression (Figs. 4 and 5).10 Moreover, Lucifer Yellow, a GJpermeable and membrane-impermeable dye, enters NRVMs at resting state, and its uptake is also accordingly changed due to GJ inhibition or alteration in Cx43 expression in both single and monolayer cells.10 Thus, these data indicate that a basal activity of hemichannels also exists in NRVMs, which is sufficient to trigger electrical excitation. Other studies in muscle cells34,35 and nonmuscle cells36,37 also demonstrate that a functional hemichannel open ensures releases of second messengers or transfer a relevant quantities of Ca2+ to the extracellular milieu under physiological conditions. Under pathophysiological conditions such as cardiomyocytes ischemia and cellular stress, however, dysregulated hemichannel properties or exaggerated hemichannel activity have been found to link with arrhythmias and cell homeostatic imbalances and even cell death.38–40 Importantly, GJ intervention seems to be effective in prevention of cell death and arrhythmia. For instances, in myocardial infarction, persistent opening of GJs leading to increased sodium current and hypercontractility aggravated heart injury and dysfunction, for which GJ uncouplers can be effective as preventative approach.41–44 In addition, acetylcholine can activate the function of GJs by sustaining the Cx43 protein level, leading to inhibition of the lethal arrhythmia that follows myocardial infarction.45 Therefore, our results together with all these reports demonstrate that in addition to synchronizing electrical and mechanical coupling among cardiomyocytes in the ventricle during each heartbeat, Cx43-associated GJs also play a role in the regulations of basal [Na+]i as already described.46 Importantly, this GJ-mediated Na+ influx or Ca2+ influx or both is involved in the genesis of spontaneous electrical activity in neonatal cardiomyocytes. These findings suggest that Na+ entrance through GJs and hemichannels is more likely contributing to the excitation (Figs. 1–5), because (1) Ca2+ transients could be abolished by nifedipine but not TTX except for combining with Gap27, (2) [Na+]i was accordingly changed with the manipulations of GJ function or expression levels but was not changed upon nifedipine exposure, (3) Ca2+ transients disappeared in the absence of external Na+ but not Ca2+, and (4) it has been found that sodium channel is distributed to the discs and regulates a Na+ current different from that of ordinary sodium channels in cardiomyocytes.46 Thus, in this regard, Na+ signaling seems more complicated than we originally understood; it may be distinct in the regulation of cardiac function because of different entrance passages in physiological settings. As such, sodium channel blockers may be not effective, whereas GJ modulator may work sometimes in prevention of some arrhythmias in pathophysiological conditions.44–46

genesis of spontaneous NRVM automaticity. This study may provide evidence for the mechanism(s) underlying, at least partly, GJ regulating cardiac rhythmicity in physiological conditions, which may also be relevant to its alterations in GJ function and/or Cx43 abundance leading to arrhythmias found in pathological settings. REFERENCES

Taken together, our findings demonstrate that Na+ influx mediated by Cx43-associated GJs is involved in the regulation of basal intracellular Na+ activity and also in the

1. Sohl G, Willecke K. Gap junctions and the connexin protein family. Cardiovasc Res. 2004;62:228–232. 2. Kumar NM, Gilula NB. The gap junction communication channel. Cell. 1996;84:381–388. 3. Giepmans BN. Gap junctions and connexin-interacting proteins. Cardiovasc Res. 2004;62:233–245. 4. Severs NJ, Coppen SR, Dupont E, et al. Gap junction alterations in human cardiac disease. Cardiovasc Res. 2004;62:368–377. 5. Sakamoto A. Electrical and ionic abnormalities in the heart of cardiomyopathic hamsters: in quest of a new paradigm for cardiac failure and lethal arrhythmia. Mol Cell Biochem. 2004;259:183–187. 6. Mehta PP, Perez-Stable C, Nadji M, et al. Suppression of human prostate cancer cell growth by forced expression of connexin genes. Dev Genet. 1999;24:91–110. 7. Mehta PP, Bertram JS, Loewenstein WR. Growth inhibition of transformed cells correlates with their junctional communication with normal cells. Cell. 1986;44:187–196. 8. Rose B, Mehta PP, Loewenstein WR. Gap-junction protein gene suppresses tumorigenicity. Carcinogenesis. 1993;14:1073–1075. 9. Severs NJ, Bruce AF, Dupont E, et al. Remodelling of gap junctions and connexin expression in diseased myocardium. Cardiovasc Res. 2008;80: 9–19. 10. Li C, Meng QL, Yu XF, et al. Regulatory effect of connexin43 on basal Ca2+ signaling in rat ventricular myocytes. PLoS One. 2012;7:e36165. 11. Kang M, Lin N, Li C, et al. Cx43 phosphorylation on S279/282 and intercellular communication are regulated by IP3/IP3 receptor signaling. Cell Commun Signal. 2014;12:58. 12. Westenbroek RE, Bischoff S, Fu Y, et al. Localization of sodium channel subtypes in mouse ventricular myocytes using quantitative immunocytochemistry. J Mol Cell Cardiol. 2013;64:69–78. 13. Kucera JP, Rohr S, Rudy Y. Localization of sodium channels in intercalated disks modulates cardiac conduction. Circ Res. 2002;91: 1176–1182. 14. Kondo RP, Wang SY, John AS, et al. Metabolic inhibition activates a non-selective current through connexin hemichannels in isolated ventricular myocytes. J Mol Cell Cardiol. 2000;32:1859–1872. 15. Imahashi K, Kusuoka H, Hashimoto K, et al. Intracellular sodium accumulation during ischemia as the substrate for reperfusion injury. Circ Res. 1999;84:1401–1406. 16. Valdivia CR, Chu WW, Pu J, et al. Increased late sodium current in myocytes from a canine heart failure model and from failing human heart. J Mol Cell Cardiol. 2005;38:475–483. 17. Luo D, Yang D, Lan X, et al. Nuclear Ca2+ sparks and waves mediated by inositol 1,4,5-trisphosphate receptors in neonatal rat cardiomyocytes. Cell Calcium. 2008;43:165–174. 18. Evans WH, Leybaert L. Mimetic peptides as blockers of connexin channel-facilitatd intercellular communication. Cell Commun Adhes. 2007;14:265–273. 19. Damkier HH, Aalkjaer C, Praetorius J. Na+-dependent HCO3- import by the slc4a10 gene product involves Cl- export. J Biol Chem. 2010;285: 26998–27007. 20. Zhang J, Liu H, Sun J, et al. Arabidopsis fatty acid desaturase FAD2 is required for salt tolerance during seed germination and early seedling growth. PLoS One. 2012;7:e30355. 21. Zimmer T. Effects of tetrodotoxin on the mammalian cardiovascular system. Mar Drugs. 2010;8:741–762. 22. Satoh H, Ginsburg KS, Qing K, et al. KB-R7943 block of Ca2+ influx via Na+/Ca2+ exchange does not alter twitches or glycoside inotropy but prevents Ca2+ overload in rat ventricular myocytes. Circulation. 2000; 101:1441–1446. 23. Desplantez T, Dupont E, Severs NJ, et al. Gap junction channels and cardiac impulse propagation. J Membr Biol. 2007;218:13–28.

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CONCLUSIONS

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J Cardiovasc Pharmacol   Volume 66, Number 1, July 2015

Gap-mediated Na+ Influx Regulates Automaticity

24. Daniel A, Luciana RG, Huan H, et al. Trypanosomacruzi induces changes in cardiac connexin43 expression. Microbes Infect. 2008;10:21–28. 25. Viatchenko-Karpinski S, Fleischmann BK, Liu Q, et al. Intracellular Ca2+ oscillations drive spontaneous contractions in cardiomyocytes during early development. Proc Natl Acad Sci U S A. 1999;96:8259–8264. 26. Vinogradova TM, Lyashkov AE, Zhu W, et al. High basal protein kinase A-dependent phosphorylation drives rhythmic internal Ca2+ store oscillations and spontaneous beating of cardiac pacemaker cells. Circ Res. 2006;98:505–514. 27. Lei M, Jones SA, Liu J, et al. Requirement of neuronal- and cardiac-type sodium channels for murine sinoatrial node pacemaking. J Physiol. 2004; 559:835–848. 28. Maier SKG, Westenbroek RE, McCormick KA, et al. Distinct subcellular localization of different sodium channel a and b subunits in single ventricular myocytes from mouse heart. Circulation. 2004;109:1421–1427. 29. Yao L, Fan P, Jiang Z, et al. Nav1. 5-dependent persistent Na+ influx activates CaMKII in rat ventricular myocytes and N1325S mice. Am J Physiol Cell Physiol. 2011;301:C577–C586. 30. Jansen JA, Noorman M, Musa H, et al. Reduced heterogeneous expression of Cx43 results in decreased Nav1.5 expression and reduced sodium current that accounts for arrhythmia vulnerability in conditional Cx43 knockout mice. Heart Rhythm. 2012;9:600–607. 31. Rhett JM, Ongstad EL, Jourdan J, et al. Cx43 associates with Na(v)1.5 in the cardiomyocyte perinexus. J Membr Biol. 2012;245:411–422. 32. Ebihara L, Korzyukov Y, Kothari S, et al. Cx46 hemichannels contribute to the sodium leak conductance in lens fiber cells. Am J Physiol Cell Physiol. 2014;306:C506–C513. 33. Li FH, Sugishita K, Su Z, et al. Activation of connexin-43 hemichannels can elevate [Ca2+]i and [Na+]i in rabbit ventricular myocytes during metabolic inhibition. J Mol Cell Cardiol. 2001;33:2145–2155. 34. John S, Cesario D, Weiss JN. Gap junctional hemichannels in the heart. Acta Physiol Scand. 2003;179:23–31. 35. Verma V, Hallett MB, Leybaert L, et al. Perturbing plasma membrane hemichannels attenuates calcium signalling in cardiac cells and HeLa cells expressing connexins. Eur J Cell Biol. 2009;88:79–90.

36. Burra S, Nicolella DP, Francis WL, et al. Dendritic processes of osteocytes are mechanotransducers that induce the opening of hemichannels. Proc Natl Acad Sci U S A. 2010;107:13648–13653. 37. Anselmi F, Hernandez VH, Crispino G, et al. ATP release through connexin hemichannels and gap junction transfer of second messengers propagate Ca2+ signals across the inner ear. Proc Natl Acad Sci U S A. 2008;105:18770–18775. 38. Shintani IK, Uemura K, Yoshida K. Hemichannels in cardiomyocytes open transiently during ischemia and contribute to reperfusion injury following brief ischemia. Am J Physiol Heart Circ Physiol. 2007;293: H1714–H1720. 39. Chi Y, Gao K, Li K, et al. Purinergic control of AMPK activation by ATP released through connexin 43 hemichannels-pivotal roles in hemichannel-mediated cell injury. J Cell Sci. 2014;127:1487–1499. 40. Patel D, Gemel J, Xu Q, et al. Atrial fibrillation-associated connexin40 mutants make hemichannels synergistically form gap junction channels novel properties. FEBS Lett. 2014;588:1458–1464. 41. Lundy SD, Murphy SA, Dupras SK, et al. Cell-based delivery of dATP via gap junctions enhances cardiac contractility. J Mol Cell Cardiol. 2014;72:350–359. 42. Rodriguez SA, García DD, Ruiz MM, et al. Enhanced effect of gap junction uncouplers on macroscopic electrical properties of reperfused myocardium. J Physiol. 2004;559:245–257. 43. Lewandowski R, Procida K, Vaidyanathan R, et al. RXP-E: a connexin43-binding peptide that prevents action potential propagation block. Circ Res. 2008;103:519–526. 44. Sun B, Qi X, Jiang J. Heptanol decreases the incidence of ischemiainduced ventricular arrhthmias through altering electrophysiological properties and connexin43 in rat hearts. Biomed Rep. 2014;2: 349–353. 45. Ando M, Katare RG, Kakinuma Y, et al. Efferent vagal nerve stimulation protects heart against ischemia-induced arrhythmias by preserving connexin43 protein. Circulation. 2005;112:164–170. 46. Delmar M. Connexin43 regulates sodium current; ankyrin-G gap junctions: the intercalated disc exchanger. Cardiovasc Res. 2012;93:220–222.

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Gap Junction-associated Na+ Influx Is Involved in the Mediation of Ca2+ Transients in Neonatal Rat Ventricular Myocytes.

Gap junction (GJ) coupling is involved in the regulation of spontaneous Ca(2+) transients in neonatal rat ventricular myocytes (NRVMs); however, the u...
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