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Cardiovascular Research Advance Access first published online on March 31, 2008
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Cardiovascular Research, doi:10.1093/cvr/cvn084
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Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2008. For permissions please email: journals.permissions@oxfordjournals.org

Generation of reentrant arrhythmias by dominant-negative inhibition of connexin43 in rat cultured myocyte monolayers

Takuo Nakagami1,2, Hideo Tanaka1,*, Ping Dai1, Shien-Fong Lin3, Takuji Tanabe1, Hiroki Mani1, Katsuji Fujiwara1, Hiroaki Matsubara2 and Tetsuro Takamatsu1

1 Department of Pathology and Cell Regulation, Kyoto Prefectural University of Medicine Graduate School of Medical Science, Kawaramachi Hirokoji, Kamigyo-Ku, Kyoto 602-8566, Japan
2 Department of Cardiovascular Medicine, Kyoto Prefectural University of Medicine Graduate School of Medical Science, Kyoto, Japan
3 Krannert Institute of Cardiology, Indiana University School of Medicine, Indianapolis, IN, USA

* Corresponding author. Tel: +81 75 251 5322; fax: +81 75 251 5353. E-mail address: hideotan{at}koto.kpu-m.ac.jp

Received 11 January 2008; revised 27 February 2008; accepted 17 March 2008

Time for primary review: 29 days


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Supplementary material
 Funding
 References
 
Aims: Alteration of connexin43 (Cx43)-mediated intercellular communication is known to promote susceptibility to ventricular tachyarrhythmias. However, the precise mechanism of the altered Cx43 responsible for arrhythmogenesis remains unclear. We sought to understand changes in impulse propagation of ventricular myocytes under dominant-negative (DN) inhibition of Cx43 in the development of arrhythmias.

Methods and results: Intercellular communication was inhibited in confluent monolayers of neonatal rat cultured myocytes by an adenoviral vector-mediated gene transfer for DNCx43-fused red fluorescence protein (RFP). A high-resolution, macro-zoom fluorescence imaging system was used to visualize both the fluo4- and RFP-fluorescence intensities as measures of Ca2+ transient propagation and distribution of DNCx43 inhibition, respectively, in the myocyte monolayers. DNCx43 inhibition of the monolayers resulted in not only a significant slowing of Ca2+ transient propagation velocity, but also a preferential emergence of spiral-wave reentrant arrhythmias elicited by rapid pacing. Detailed observations on the development of spiral waves revealed that the gene-transferred myocyte monolayers exhibited regional slowing of propagation and subsequent generation of wave break, resulting in reentrant arrhythmias. Furthermore, DNCx43-RFP-transferred monolayers showed higher fluorescence intensity of RFP at the break point than at the surrounding myocardium, indicating a culprit role of DNCx43 inhibition in the genesis of spiral reentry.

Conclusion: The present results indicate that regional heterogeneity in gap-junctional communication promotes, in addition to slowing of conduction velocity, susceptibility to reentrant tachyarrhythmias.

KEYWORDS Connexin43; Dominant-negative mutation; Myocytes; Spiral wave; Arrhythmia (mechanisms)


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 Supplementary material
 Funding
 References
 
Gap junctions play an indispensable role in electrical coupling of the cardiomyocytes for efficient impulse propagation in the heart.1,2 Numerous studies have revealed that gap-junction proteins are redistributed in diseased hearts. In particular, connexin43 (Cx43), a major gap-junction protein in the ventricular myocardium shows altered abundance and subcellular localization in diseased myocardium, such as infarct border zones,3,4 hypertrophied hearts,5,6 and failing hearts.7 Such topological changes in Cx43, referred to as gap-junction remodelling, are regarded as an important substrate for arrhythmias.8 However, little is known regarding how Cx43 remodelling promotes susceptibility to arrhythmias because no direct correlation has been established for the altered expression or function of Cx43 to the corresponding conduction abnormality in the cardiac tissue.

In vitro confluent monolayers of cultured cardiomyocytes are widely used as a simplified two-dimensional tissue model to study the mechanism of arrhythmias because the topology and function of the cells can conveniently be manipulated.9 For example, manipulation of the myocyte alignment10 and co-culture with non-myocytes11,12 resulted in dramatic changes in the propagation patterns leading to arrhythmias. Similarly, manipulation of gap-junction-mediated intercellular communication, if performed in the two-dimensional layers, might elucidate key mechanism(s) for arrhythmogenesis. In practice, our laboratory previously demonstrated in neonatal rat cultured myocytes that dominant-negative (DN) inhibition of Cx43 impaired intercellular coupling and desynchronized Ca2+ transients among the individual cells.13 Subsequently, Kizana et al.14 also demonstrated that mutation of Cx43 resulted in three- to four-fold slowing of conduction velocity of Ca2+ transients in neonatal rat myocyte monolayers. However, it is undetermined whether or not inhibition of gap-junction-mediated impulse conduction generates tachyarrhythmias. This is because even marked reduction of Cx43 expression (by 70–95%) was resistant to development of arrhythmias in cardiac-specific Cx43 conditioning knockout mice in vivo.15–17

We hypothesize that combined visualization of spatial distribution of Cx43 inhibition and patterns of impulse propagation would reveal a culprit mechanism for generation of reentrant tachyarrhythmias on confluent myocyte monolayers. To address this issue, we took a novel approach to imaging both the inhibition of Cx43 gap-junctional function and patterns of impulse propagation of Ca2+ transients. The Cx43-mediated intercellular communication was inhibited by gene transfer of an adenoviral vector for DNCx43-fused red fluorescence protein (Ad.DNCx43.RFP). Arrhythmias were induced by applying rapid pacing to the monolayers. Under these experimental conditions, we analysed both the impulse propagation and distribution of DNCx43 inhibition by measuring Ca2+ indicator fluo4- and RFP-fluorescence intensities, respectively.

The goals of the present study were: (i) to examine whether inhibition of Cx43-mediated gap-junctional communication slows conduction and augments susceptibility to arrhythmias in monolayers of cultured myocytes, and (ii) if so, how the impulse propagation is altered spatiotemporally during the development of arrhythmias with reference to Cx43 functions. We provide direct evidence that DNCx43 slows impulse propagation and that regionally inhomogeneous DNCx43 inhibition promotes susceptibility to reentrant tachyarrhythmias in myocyte monolayers.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 Supplementary material
 Funding
 References
 
This study conforms to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996) and was conducted under the approval of the Animal Care Committee of Kyoto Prefectural University of Medicine.

2.1 Creation of recombinant adenoviral vector
We used a DNA construct containing CMV promoter upstream of a rat mutant Cx43 with a deletion of amino acids 130–13713 and with a monomeric red fluorescent protein (mRFP1) provided by Dr R.Y. Tsien18 fused with its carboxyl terminus to produce recombinant adenoviruses (Ad.DNCx43.RFP). A control virus was constructed with LacZ following the CMV promoter. Recombinant adenoviruses were generated with the AdEasy System (Stratagene) and purified using double cesium chloride gradients. Viral titres were determined by using Adeno-X Rapid Titer Kit (BD Biosciences Clontech, Palo Alto, CA, USA). The titres of viral stocks were determined by immunostaining of infected AD293 cells after 48 h with antibodies directed against the viral hexon protein.

2.2 Cell culture
Primary cultures of 2-day-old neonatal ventricular myocytes were prepared from Wistar rats as described previously.13 Briefly, myocytes dissociated with collagenase (type II; Worthington) were resuspended in Dulbecco's modified Eagle's medium containing 10% heat-inactivated foetal bovine serum, 100 U/mL penicillin, 100 µg/mL streptomycin, and 0.1 mM BrdU, and differentially pre-plated in two 45 min steps to reduce the fibroblast content. Approximately 1.0 x 106 cardiomyocytes were plated in each 35 mm polystyrene tissue-culture dish (Iwaki Co.). Myocytes cultured for 4 days on the dishes were grown in random orientation to make a confluent monolayer. Unless otherwise specified, cells were infected with the virus 48 h after isolation. After 1 h application of the myocytes to the virus, cells were incubated with virus-free medium, in which serum was reduced to 2% to inhibit proliferation of non-cardiac cells. Experiments were performed with the monolayers showing spatially synchronized, spontaneous beating of the myocytes with regular frequency at 4 days after plating under phase-contrast microscopy (IX-50; Olympus).

2.3 Dye-transfer experiments
To evaluate DN effects of Cx43 in Ad.DNCx43.RFP-infected myocytes, Lucifer yellow dye (LY) contained in a glass capillary (Femtotips; Eppendorff) was injected into myocytes with a microinjector (Transjector 5246, Eppendorff). Fluorescence imaging of the cell cluster was conducted in combination with the DIC imaging by a conventional fluorescence microscope (IX-50; Olympus) before and after the dye injection. Cell images were acquired by a cooled CCD camera (iXon; Andor).

2.4 Loading of the cardiomyocytes with fluo4
Initiation and propagation of the electrical impulses were imaged via fluo4-fluorescence changes in intracellular Ca2+ transients. Cultured myocytes showing spatiotemporally synchronous, regular beating were used for the Ca2+ imaging. Cells were incubated for 15 min at 37°C with fluo-4/AM (Dojindo, Japan) at 5.5 µM in HEPES-buffered Tyrode's solution consisting of (in mM) NaCl 135, KCl 5.4, CaCl2 1.8, MgCl2 1, NaH2PO4 0.33, HEPES 5, and glucose 5 (pH 7.4, adjusted with NaOH). After washout of fluo-4/AM with Tyrode's solution, the culture dishes were placed on a microscope stage equipped with a motorized X-Y linear stage (MMC-2, Chuo Precision Industrial, Japan) and thermoregulator under continuous superfusion with warmed Tyrode's solution (33°C in the dish) bubbled with 100% O2.

2.5 Imaging of Ca2+ transient propagation and dominant-negative inhibition of connexin43
We constructed an imaging system incorporating macro-zoom, dual-fluorescent microscope (MVX10; Olympus) equipped with zooming function ranging from the cellular level (x4) to the tissue level (x0.63) with high temporal resolution at 455 frames/s (see Supplementary material online, Figure S1). Fluorescence signals for both the fluo4 fluorescence (Ca2+ transients) and RFP fluorescence (DNCx43) were acquired by a high-resolution CCD camera on an acquisition system (MiCAM02, Brainvision, Japan). For calcium imaging, three pieces of emitters of blue LED light (CCS, Japan) equipped with an interference filter (480 ± 20 nm) were evenly exposed to the centre of the dish to excite fluo4 within the cardiomyocytes. The fluo4-fluorescence intensity was detected through a dichroic mirror of 505 nm and an emission filter >510 nm. Ca2+ imaging (macro area, x0.63) was conducted on areas measuring 17 x 11 mm (96 x 64 pixels) at an acquisition rate of 455 frames/s. Expression of DNCx43 was assessed by fluorescence of emitted RFP signals of >610 nm on excitation at 545 ± 10 nm by a halogen light source (100 mW; Olympus). Cellular images (micro area, x4) of both the fluo4 and RFP were depicted (2.8 x 1.9 mm; 192 x 128 pixels) from an orderly arranged nine different areas of each sample. All the images were transferred to a computer for storage and analysis.

2.6 Induction of spiral-wave reentrant arrhythmias
To evoke excitation of the monolayers, point electrical stimulation was applied to the rims of the myocyte layers with a bipolar Ag–AgCl2 electrode (3 mm in diameter). The electrode was positioned in the vertical orientation ~5 mm from the edge of the dish. Trains of biphasic pulses (10 ms; x1.5 the diastolic threshold) with fixed frequencies were applied for 7 s by programmed pacing (Tellus Image, Japan) to drive the myocytes with a progressive increase of stimulation frequency from 1 to 9 Hz until failure of 1:1 capture or development of spiral waves. Spiral waves were identified when the wavefronts of Ca2+ transients showed self-sustaining rotations at least three times independently of pacing.

2.7 Data analysis
The propagation velocity of Ca2+ transients (CaVprop) was measured from the X–t image scanned along a straight line from the pacing site to the leading edge of the wavefront in the X–Y movie, unless otherwise specified. The durations of Ca2+ transients were evaluated at 50 and 80% relaxation (CaTD50 and CaTD80, respectively). Expression of RFP was evaluated by mean values of RFP fluorescence intensity in square areas of 192 x 128 pixels (2.8 x 1.9 mm). Data were stored, displayed, and analysed using MiCAM02 acquisition/analysing system (Brainvision). Quantitative data were expressed as mean ± SD. Statistical analyses were conducted by ANOVA. A value of P < 0.05 was regarded as statistically significant.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 Supplementary material
 Funding
 References
 
3.1 Propagation of Ca2+ transients in confluent monolayers of the cultured myocytes
Figure 1 shows a representative example of Ca2+ dynamics captured at 2.2 ms intervals in a confluent monolayer of ventricular myocytes without gene transfer. The tissue exhibited homogeneous wavefront propagation of Ca2+ transients from the pacing site (arrowhead on the left side) to the opposite (right) side at pacing frequency of 1 Hz (Figure 1A), showing a stable configuration of Ca2+ transients (Figure 1B; see also Supplementary movie 1). Detailed analyses revealed that the Ca2+ transients propagated in a concentric fashion and with a constant CaVprop of 244 mm/s in the monolayer as shown in the corresponding isochronal map (Figure 1C) and line-scan (X–t) image (Figure 1D), respectively. Such uniform propagation was observed at excitation frequencies of up to 8 Hz. Similar observations were made in all 15 non-transducted samples examined, where Ca2+ transients propagated regularly without failure of 1:1 capturing or arrhythmias. We confirmed that CaVprop was significantly reduced in a frequency-dependent manner (see Supplementary material online, Figure S2A) with homogeneous wavefront propagation (see Supplementary material online, Figure S2B). Moreover, waveforms of Ca2+ transients were also modulated by frequency: the higher the frequency, the shorter the durations of Ca2+ transients (see Supplementary material online, Figure S2C) without beat-to-beat alternans or failure of excitation (see Supplementary material online, Figure S2D). Thus, the non-transducted myocyte monolayers exhibited spatially uniform excitability and conductivity, showing no evidence of heterogeneity in intracellular Ca2+ handlings even at high-frequency pacing.


Figure 1
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  		Figure 1 Ca2+ dynamics in the confluent monolayer of neonatal rat cardiomyocytes without the gene transfer. (A) Sequential X–Y images (from 1 to 4) of Ca2+ transient propagation (illustrated every 15.4 ms) elicited by 1 Hz stimulation at the point indicated by the arrowhead. The vertical colour bar denotes relative concentration of intracellular Ca2+: black, quiescent phase; red, peak of the Ca2+ transient, i.e. early activation phase. Inset above the image 1 is a schematic illustration of pacing electrode applied to the 35 mm-culture dish. (B) Representative Ca2+ transients depicted from the square region in the X–Y image on the left in response to electrical stimuli (red vertical lines in the right panel). (C) An isochronal map corresponding to the Ca2+ transient propagation in (A). Isochronal lines are drawn at intervals of 8.8 ms. (D) Line-scan (X–t) image scanned in a straight line along the direction of the wavefront in (A). See also Supplementary material online, Movie 1.

 
3.2 Dominant-negative inhibition of the myocytes using adenoviral vector
DNCx43 inhibition of the intercellular communication was confirmed for the Ad.DNCx43.RFP-infected myocytes by dye transfer experiments (Figure 2A). The myocytes showing no RFP fluorescence (NT) displayed transfer of the LY to the neighbouring myocytes, whereas the RFP-positive cells (DN) failed to transfer the dye to their neighbours. These observations, confirmed also in three other samples, indicate that the RFP-positive cells show remarkable inhibition of gap-junctional communication. In addition, expression of DNCx43 was dependent on the multiplicity of infection (MOI), as illustrated by the RFP fluorescence (Figure 2B,a). The per cent RFP-positive cells was positively correlated with MOI; the higher the MOI, the greater the percentage of cells showing RFP (Figure 2B,b), indicating that the DNCx43 expression of the myocytes can be manipulated by MOI. We confirmed that cardiomyocytes were randomly distributed in the dish with no discernible differences in cell shape or density among the samples irrespective of the gene transfer (data not shown).


Figure 2
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  		Figure 2 Dominant-negative (DN) inhibition of intercellular communication by gene transfer of Ad.DNCx43.RFP. (A) Dye transfer experiments for confirmation of DN inhibition of Cx43 gap-junction-mediated intercellular communication. Transmitted light images (trans) are on the left panels. In paired cells without gene transfer (NT), Lucifer yellow dye (LY) transferred to the neighbouring myocytes, whereas the RFP-positive cells (RFP) failed to transfer the dye to their neighbours. Scale bars = 100 µm. (B) Fluorescence images of DNCx43.RFP expression in the confluent monolayers at various MOIs (a) and per cent RFP-positive area over the total area from 15 samples for MOI at 0.38, 14 at 1.5, 10 at 6, and 8 at 24 (b). Scale bars = 100 µm.

 
3.3 Slowing of Ca2+ transient propagation by dominant-negative inhibition of connexin43
In genetically Cx43-engineered mouse hearts, moderate to severe (by 70–95%) reduction of Cx43 expression is required to develop conduction delay and sudden arrhythmic death.15–17 To investigate to what extent DN inhibition of Cx43 modulates Ca2+ transient propagation in the myocyte monolayers, we analysed CaVprop in preparations infected with Ad.DNCx43.RFP. As expected, DN inhibition of Cx43 resulted in a significant reduction of CaVprop. The values for CaVprop measured at 3 Hz pacing were significantly decreased in a MOI-dependent manner (Figure 3A). Such negative dromotropic effects of DNCx43 inhibition were not due to the adenovirus per se because no significant difference was observed in CaVprop between the tissues with and without expression of LacZ at MOI of 0.38 (Figure 3A). At the higher MOI of 0.38, some samples showed somewhat irregular wavefronts, i.e. regional slowing of Ca2+ transient propagation (Figure 3B; right) when compared with monolayers of NT and at lower MOI of 0.09 (Figure 3B; left and centre, respectively). Furthermore, at MOIs of 1.5 or higher, the monolayer failed to show planar wavefronts of conduction in the dish, and there were multiple areas exhibited spontaneous excitation showing asynchronous fragmented waves (see Supplementary material online, Figure S3 and Movie 2). These observations indicate that spatial uniformity of Ca2+ transient propagation as well as CaVprop is closely correlated with the extent of DN inhibition of Cx43 function. Since coordinated propagation was absent at MOIs higher than 0.38, DN samples at MOIs of 0.38 and lower were used for induction of arrhythmias.


Figure 3
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  		Figure 3 Slowing of CaVprop in myocyte monolayers by DN inhibition of Cx43. (A) The bar graphs and error bars represent mean values of CaVprop and their SDs, respectively, measured at 3 Hz pacing for non-transducted (NT, n = 16) samples, LacZ-transferred ones (LacZ, n = 5), and DNCx43. RFP-transferred ones (DN) at MOIs of 0.09 (n = 15), 0.38 (n = 11), and 1.5 and higher (n = 22). At DN of 1.5 and higher CaVprop was unmeasurable due to the multiple, fragmented excitations. *P < 0.05. (B) Representative isochronal maps of Ca2+ transient propagation at 3 Hz pacing (8.8 ms intervals) for NT, DN0.09 and DN0.38 samples. Each area in (B) is 17 x 11 mm.

 
3.4 Induction of spiral-wave reentry by dominant-negative inhibition of connexin43
It has been known that rapid pacing can induce reentrant activity in myocyte monolayers.9,10 We found that in addition to the slowing of CaVprop, DN inhibition of Cx43 produced spiral-wave reentrant arrhythmias by high-frequency pacing. Figure 4A shows representative sequential images (a) and isochronal map (b) of spiral-wave reentry induced by 7 Hz pacing on a DNCx43 monolayer at MOI of 0.38 (see also Supplementary material online, Movie 3). This pattern of circus movements of impulse was quite distinct from the uniform propagation observed in non-transducted samples (cf. Figure 1). Although non-transducted samples were resistant to the induction of such circus movements by rapid stimulation up to 7 Hz, reentrant propagations were preferentially induced on DNCx43 samples by pacing at the equivalent frequencies of 7 Hz or lower. Pooled data indicated that spirals were induced more frequently at higher MOI (Figure 4B). Notably, however, even at the higher MOI of 0.38, no significant difference was detected for CaVprop at 3 Hz pacing between tissues with and without spiral-wave reentry (Figure 4C), although somewhat inhomogeneous wavefronts were noted, as observed in samples at MOI of 0.38 (Figure 3B). This indicates that slowing of CaVprop in itself would not lead to development of ventricular tachyarrhythmias.


Figure 4
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  		Figure 4 Demonstration of spiral reentry in a DNCx43 myocyte monolayer elicited at 7 Hz pacing. (A, a) Sequential X–Y images of the Ca2+ transient propagation depicted every 22 ms. The red area and the white arrows denote wavefronts and the direction of propagation, respectively. One full rotation of the spiral reentry is illustrated from the panels on the upper left (6758.4 ms) to the lower right (6890.4 ms). (b) Isochronal map (drawn at 8.8 ms intervals) for a stable single-loop reentry. The white arrows indicate the direction of the wavefronts. *The core of the reentry. Each area shown is 17 x 11 mm. (B) Comparison of spiral-wave inducibility (per cent of the monolayers over the total number of myocyte monolayers) for the spiral reentry among samples of non-transduction (NT, n = 16) and with DN inhibition at MOIs of 0.09 (n = 15) and 0.38 (n = 11) by rapid pacing up to 8 Hz. (C) Comparison of CaVprop in tissues with (SP+, n = 4) and without (SP–, n = 7) spiral reentry at MOI of 0.38 at 3 Hz pacing. See also Supplementary material online, Movie 3.

 
3.5 Regional inhibition of DNCx43 in genesis of spiral-wave reentry
By detailed observation of the wavefronts during the development of the spiral-wave reentry (Figure 5A), we found that DNCx43-myocyte monolayers exhibited regional slowing of Ca2+ transient propagation (a) and subsequent generation of regional conduction block, i.e. wave break (b, c) that resulted in reentrant arrhythmias (Figure 5B, see also Supplementary material online, Movie 4). The line-scan (X–t) images at the leading-edge (i.e. faster) wavefront revealed a constant CaVprop (see ‘fast line’ in Figure 6A), whereas CaVprop at the delayed wavefront (i.e. the ‘slow line’) exhibited regional depression along the path of the propagation. Such a regional difference in CaVprop was more remarkable at higher frequency of pacing (Figure 6B), as evident in the ratio of CaVprop between these two regions, i.e. CaVprop(fast/slow) in Figure 6C. A similar series of events was observed in six other DNCx43 monolayers, three of which were at MOI of 0.38, and the other three, at 0.09. Moreover, as shown in Figure 7, the RFP-fluorescence intensity of the break point (BP) indicated that the area of the wave break (indicated by ‘b’) showed stronger expression of RFP than the surrounding regions (‘a’, ‘c’ and ‘rest’). Such spatial heterogeneity of the DNCx43 expression was unexpected because we carefully plated cells on the dishes and applied the adenoviruses homogeneously. In addition, no difference was observed in the cell shape and density between the area of wave break and the rest (data not shown). Thus, spatial heterogeneity of DNCx43 would be incidental due to the nature of gene-transfer effects. From seven other samples, we also confirmed that regional inhibition of Cx43 is responsible for spiral reentry. Pooled data from the eight samples showing spiral waves revealed that RFP-fluorescence intensity was significantly higher at the wave-BP than that at the rest, i.e. surrounding area (Figure 8A). In addition, the mean per cent RFP-positive area was larger at the wave-BP than at the rest (Figure 8B). Thus, regionally heterogeneous Cx43 inhibition combined with the slowing of CaVprop produced wave break and eventually led to reentrant arrhythmias.


Figure 5
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  		Figure 5 Sequential images of impulse propagation in the development of wave break and spiral-wave reentry elicited by 6 Hz pacing. (A) Three different sequences of snapshot images in regional slowing of the wavefront (a), development of wave break (b), and reentry of the impulses (b and c). The time of each frame is shown below the each image. *The region of wave break and //the block of the impulse propagation. Arrows indicate the directions of the wavefronts. The corresponding isochronal maps (16.0 ms interval) are attached on the right panels. (B) Sequential images of the development of spiral-wave reentry shown every 22 ms. The area of each image is 17 x 11 mm. Arrowheads are sites of electrical pacing. See also Supplementary material online, Movie 4.

 


Figure 6
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  		Figure 6 Analysis of inhomogeneous wavefronts of Ca2+ transient propagation in DNCx43 monolayer identical to Figure 5B. (A) Line-scan (X–t) images of propagating Ca2+ transients at the fast line (at the leading-edge) and the slow line (at the depressed region) drawn from the point of pacing at three different frequencies. Scan lines for the X–t images are shown on the top panel. For clarity, localized conduction slowing is indicated by white lines along the wavefronts. (B) Frequency-dependent slowing of CaVprop measured from the line-scan images along the fast- and slow-lines. (C) Frequency-dependent changes in the CaVprop(fast/slow) ratio, i.e. ratio of CaVprop at the fast line to CaVprop at the slow line.

 


Figure 7
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  		Figure 7 Regionally heterogeneous expression of DNCx43.RFP. (A) An isochronal map (16.0 ms interval) of the monolayer with spiral waves identical to Figure 5A,b (left panel). Nine square ROIs (2.8 x 1.9 mm for each) on the identical image (right panel) denote the areas at which RFP fluorescence intensities were measured for analysis. (B) RFP fluorescence images at ROIs labelled in (a–c) in (A). RFP-positive cells are more highly distributed in population in area (b) than in the surrounding areas (a and c). (C) Bar graphs showing mean fluorescence intensities of the nine areas shown in (A). (D) Comparison of mean RFP-fluorescence intensity on the area (b) and the rest.

 


Figure 8
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  		Figure 8 High intensity of RFP fluorescence at the area of wave break from 8 samples. (A) Comparison of the RFP fluorescence intensities between the area of break points (BPs; 2.8 x 1.9 mm) and the rest of the whole area of observation (rest; 17 x 11 mm). (B) Comparison of the per cent RFP-positive area between the area of BP and the rest.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
Supplementary material
 Funding
 References
 
4.1 Experimental design for DNCx43 monolayer models
Although it has been suggested that remodelling of Cx43 contributes to arrhythmogenesis,3,7,8 precise mechanism(s) underlying how Cx43 gap-junctional dysfunction leads to generation of arrhythmias remain unresolved. This is because no effective method has been available to simultaneously detect the propagation patterns and respective Cx43 functions. In the present study, we took a novel approach that directly visualizes Ca2+ transient propagation and DN inhibition of Cx43 gap-junctional communication in order to address the link between Cx43 and arrhythmias. We used myocyte monolayers gene-transferred with adenoviral vector for DNCx43.RFP to manipulate and topologically detect inhibition of Cx43 gap-junctional communication, where impulse propagation can be regulated simply by MOI. Fluo4-fluorescence Ca2+ transients were measured as an indicator of excitability and conductivity with a high-speed CCD camera because small voltage-sensitive dye signals in the two-dimensional samples are beyond analytic limitation of CCD camera but can be detected only by contact photo-diode arrays.9,10 In our case, the CCD camera was essential to detect precise distribution of DNCx43RFP signals because of its high spatial resolution.

4.2 Slowing of Ca2+ transient propagation by DNCx43 inhibition
The present study confirmed that Cx43 gap junctions are an important determinant for conductivity of Ca2+ transients because CaVprop was modulated by extent of DNCx43 inhibition: higher MOI samples showed stronger DNCx43 inhibition and slower CaVprop. In addition, Cx43 gap-junctional communication was found to be indispensable for impulse propagation because extreme DN inhibition of Cx43 (at MOI of 1.5 or higher) abolished effective propagation of Ca2+ transients, showing multifocal fragmented activities. Thus, our gene-transfer approach can modulate CaVprop and further disrupt conduction by inhibition of Cx43-mediated intercellular communication.

The present observations further indicate that Cx43-mediatied conduction slowing is essential for arrhythmogenesis. This is because reentrant arrhythmias were more preferentially induced in the monolayers with stronger inhibition of Cx43 than with weaker inhibition. However, DN inhibition of Cx43-mediated communication in itself seemed to be inadequate for generation of arrhythmias, because CaVprop was apparently not different between the inducible and non-inducible monolayers (Figure 4C).

4.3 Regional heterogeneity of DNCx43 in development of arrhythmias
What would be the culprit determinant augmenting susceptibility to arrhythmias in DNCx43 monolayers? Our results show that regionally suppressed Cx43-mediated gap-junctional communication is essential for the genesis of reentrant arrhythmias by rapid pacing, a well-established procedure to augment electrical instability.9,10 During the development of reentrant arrhythmias in DNCx43 monolayers, we directly observed the following series of events in sequence: regional delay in Ca2+ transient propagation, formation of the wave break, and eventual development of reentrant arrhythmias. Rapid pacing predisposed the monolayers to be more arrhythmogenic because this procedure at higher frequency produced more remarkable regional differences in CaVprop between the two regions (Figure 6B), suggesting a steeper restitution of conduction velocity at the depressed (slow) region than at the dominant (fast) ones, an essential substrate for reentrant tachyarrhythmias.10 Such sequential changes in Ca2+ transient propagation are similar to the previous observation by Bian and Tung,10 who also visualized precise patterns of impulse propagation during the development of reentrant arrhythmias produced by minute manipulation of structural heterogeneity. In our monolayers, structural heterogeneity might have existed due to the random orientation. In this respect, Bub et al.19 speculated that intrinsic structural heterogeneity is responsible for arrhythmogenesis in monolayers of chick embryonic heart cells under pharmacological inhibition of Cx43 by heptanol. Nevertheless, the present data indicate a predominant role of regional heterogeneity in Cx43 gap-junctional communication in arrhythmogenesis because spiral waves were induced not on non-transducted samples, but exclusively on DNCx43 samples with higher incidence at higher MOI. Moreover, the culprit role of regional Cx43 gap-junctional communication was also evidenced by regionally high DNCx43.RFP signals at the region of conduction slowing and wave break. The arrhythomogenic regional inhibition of Cx43 we observed here may be closely related to high-incidence of arrhythmias in the Cx43KO mice induced by regional ischaemia,20–21 chimeric Cx43 knockout mice,22 and various diseased hearts with gap-junctional remodelling.3–8

In addition to the spiral-wave reentrant tachyarrhythmias, DNCx43 monolayers may develop abnormal automaticity. Like the fragmented activities observed in highly DNCx43 inhibited samples, high-degree inhibition of Cx43 gap-junctional communication may well induce abnormal automaticity because such inhibition of intercellular communication would reduce electrotonic effects by the surrounding myocytes, which may generate a localized region of automatic excitation. Further study is required to address whether and how DNCx43 contributes to triggered arrhythmias or automatic tachyarrhythmias.

4.4 Unanswered questions
Since Ca2+ transients are evoked by membrane excitation except in chaotic tachyarrhythmias such as ventricular fibrillation,23 CaVprop can be a surrogate for conductivity of action potentials during regular rhythms at physiological frequency or ventricular tachycardia. We therefore consider that the observed events of Ca2+ transient propagation would in principle reflect the impulse propagation. However, undetermined are the eventual properties of electrical activities during regionally depressed CaVprop or spiral-wave reentrant arrhythmias. In this respect, computer simulations by Quan and Rudy24 demonstrated that reduced cellular coupling markedly increases dispersion of the action potential duration, leading to formation of reentrant circuit. Laurita et al.25 also demonstrated in Langendorff-perfused guinea-pig hearts that restitution of action-potential durations is influenced by cell-to-cell uncoupling. Thus, susceptibility to arrhythmias elicited by impairment of Cx43 gap-junctional communication may be established by the gap-junction-mediated conduction delay and the resultant dispersion in excitability and refractoriness which can be augmented in a frequency-dependent manner. Combined detection of action potentials and Cx43 inhibition with high spatiotemporal resolution would be required to elucidate this possibility.

Finally, we should also note that our in vitro experimental observations would not simply be applicable to the heart in vivo because we used two-dimensional sheets of neonatal myocytes showing random orientation. Application of our present imaging system to the Cx43-mutant hearts tagged with fluorescent protein in vivo would provide more direct information on arrhythmogenicity involving Cx43 remodelling in various disease states.

4.5 Conclusion
The present results provide direct evidence that Cx43 gap junctions are essential determinants for impulse propagation and that regional heterogeneity of Cx43-mediated intercellular communication contributes to generation of reentrant arrhythmias. Our findings provide important insights into understanding the proarrhythmic mechanism in diseased hearts under gap-junctional remodelling.


   Supplementary material
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Supplementary material
Funding
 References
 
Supplementary materials are available at Cardiovascular Research online.


   Funding
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Supplementary material
 Funding
References
 
Grants-in-Aid from Japan Society for the Promotion of Science (B18300218 to T.T. and C18590784 to H.T.) and Core Research for Evolutional Science and Technology (CREST to T.T.).


   Acknowledgement
 
We thank R.Y. Tsien for providing mRFP1 cDNA.

Conflict of interest: none declared.


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Supplementary material
 Funding
 References
 

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