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Evaluation of frequency, type, and function of gap junctions between skeletal myoblasts overexpressing connexin43 and cardiomyocytes: relevance to cell transplantation

Heart Science Centre, National Heart & Lung Institute, Imperial College London, Harefield Hospital, London, UK

²Correspondence: Cellular Electrophysiology, Heart Science Centre, National Heart & Lung Institute, Imperial College London, Harefield Hospital, London UB9 6JH, UK. E-mail: m.stagg{at}imperial.ac.uk

SPECIFIC AIMS

Arrhythmias after skeletal myoblast (SM) transplantation are likely to be the consequence of the absence of or inappropriate intercellular communication between the host cardiomyocytes and transplanted cells. In this study, we quantified the intercellular communication between adult cardiomyocytes (CM) and SMs in co-culture. We also overexpressed connexin 43 (Cx43) in SMs and tested the hypothesis that such overexpression directly enhances intercellular communication with CMs, thus being potentially beneficial in preventing the arrhythmias observed after SMs transplantation.

PRINCIPAL FINDINGS

1. A Cx43 overexpressing C₂C₁₂ cell line was generated and characterized
The full-length cDNA of rat Cx43 was subcloned into the bicistronic expression vector pIRES2-EGFP. C₂C₁₂ cells, which stably expressed both Cx43 and green fluorescent protein (SM^Cx43), were generated by transfection followed by clonal selection. Control cells were either wild-type SMs (SM^WT) or cells transfected by pIRES2-EGFP subcloned with cDNA of GFP (SM^GFP). Cx43 overexpression was confirmed by Western blotting and immunofluorescence. The SM^Cx43 clone used in these experiments was found to have 5-fold the amount of Cx43 compared with SM^WT.

2. Cx43 overexpression increases gap junctional conductance in monocultures
We compared the steady-state gap junctional conductance (G_j) of SM^WT cell line with a SM^GFP and SM^Cx43 in monoculture. The G_j was unaffected by GFP expression alone, with a G_j in SM^WT of 35.4 ± 7.7 nS (n=11) and SM^GFP of 29.7 ± 7.5 nS (n=16) compared with 184.0 ± 68.0 nS (n=9, P<0.01) in the SM^Cx43. Overexpression of Cx43 in our SM^Cx43 clonal cell line increased G_j in monocultures.

3. In co-culture conditions, Cx43 was detected by immunofluorescence between cardiomyocytes and SM^Cx43
Cx43-labeled co-cultures of the different SMs (wild-type, overexpressing both Cx43 and GFP or overexpressing GFP alone) with CMs were examined by confocal microscopy to determine if gap junctions were formed between the two cell types (Fig. 1 ). Such junctions were found, albeit infrequently, in the co-cultures of CMs and SM^Cx43 (CM-SM^Cx43) but were never detected in the co-culture of CMs and SM^WT (CM-SM^WT). Gap junctions consisting of very few channels would not be easily detected by immunofluorescence but would still allow electrical coupling to occur and could be detected by the more sensitive electrophysiological method of assessing steady-state gap junctional conductance in the co-cultures.

View larger version (42K): [in this window] [in a new window]   Figure 1. A) Confocal micrograph showing the presence of Cx43 labeling (red label, arrows) at the interface between a transfected skeletal myoblast overexpressing Cx43 (SMCx43), which is green due to eGFP expression, and an adult cardiomyocyte (CM) indicating the presence of gap junctions between the two cell types. Nuclei, stained with DAPI, show up as blue. B) No such junctions were observed between SMs expressing only eGFP (SMGFP) and CMs or between wild-type SMs and CMs (not shown). Scale bars = 50 µm. Adjacent micrographs show the difference in thickness between the SM (green) and the CM.

4. Cx43 overexpression increased the number of cell-cell pairs, which display gap junction formation
Formation of gap junctional conductance, detected by recording currents with the dual whole cell patch clamp method, occurred in 22% of CM-SM^WT (n=73) and was significantly more frequent in CM-SM^Cx43 (48% n=71, P<0.001). The presence of increased Cx43 in the SMs therefore enhanced the formation of gap junctional conductance in vitro.

5. Cx43 overexpression in SMs increased gap junctional conductance measured in co-cultures with CMs
In the cell pairs, which had demonstrated G_j, the steady-state G_j determined at +10 mV was significantly greater in CM-SM^Cx43 compared with CM-SM^WT (Fig. 2 B). G_j was 14.8 ± 2.0 nS in CM-SM^WT (n=12) compared with 29.7 ± 4.3 nS in CM-SM^Cx43 (n=21, P<0.05). At any given voltage both the instantaneous and steady-state current was greater in CM-SM^Cx43 than in CM-SM^WT. Macroscopic gap junctional conductances ranged from 3.8 to 32.0 nS in CM-SM^WT and from 5.8 to 94.1 nS in CM-SM^Cx43. The larger variability in the measured G_j of the CM-SM^Cx43 group compared with the CM-SM^WT may represent the variability in functional availability of the overexpressed Cx43 protein for gap junction formation. A two-way Boltzmann function was used to fit the steady-state conductance vs. transjunctional voltage relationship. We observed a good degree of symmetry in the shape of the Boltzmann curves fitted to both CM-SM^WT and CM-SM^Cx43, which would indicate homotypic gap junction channel formation. This is most likely the pairing of Cx43 hemichannels from the CMs with endogenous Cx43 in the case of CM-SM^WT and to both endogenous and overexpressed Cx43 in the case of CM-SM^Cx43 cell-cell pairs.

View larger version (17K): [in this window] [in a new window]   Figure 2. Steady-state gap junctional conductance measurement of cells in co-culture between cardiomyocytes (CMs) and either wild-type skeletal myoblasts (CM-SMWT) or skeletal myoblasts overexpressing Cx43 (CM-SMCx43). A) Representative figures of the current recorded in cell two (I2) in response to a voltage change in cell 1 (V1) to measure Gj at ±10 mV. B) Steady-state measurement of Gj at +10 mV in CM-SMCx43 (n=21) was greater than CM-SMWT (n=12, **P<0.01).

CONCLUSIONS AND SIGNIFICANCE

We found that overexpression of Cx43 enhanced the frequency of gap junction formation and the amplitude of gap junctional conductance between SMs and CMs (Fig. 3 ). The previous observation that connexin43 overexpression has anti-arrhythmic effects in co-cultures can therefore be explained by changes at molecular and functional levels leading to increased gap junctional conductance.

View larger version (33K): [in this window] [in a new window]   Figure 3. Schematic diagram of the experimental design and principal findings of the current study.

Previous studies using immunofluorescence techniques could not detect connectivity between CMs and SMs in vivo. Similarly, in our experiments we were unable to detect gap junctions by immunofluorescence in CM-SM^WT in vitro. Nonetheless, there were gap junctions present as the dual whole cell patch clamp technique determined a 22% connectivity between CM-SM^WT in vitro. This could be explained by the more accurate and quantitative assessment of gap junction formation using electrophysiological recording compared with imaging techniques alone. This finding is important when considering published data comparing in vivo and in vitro assessment of gap junction formation between SMs and CMs.

With gene therapy able to provide enhanced gap junctional conductance between transplanted SMs and CMs, there is scope to further investigate the proposed benefits of this increased cell-to-cell communication. In the present study we have shown that overexpressing Cx43 brings about enhanced gap junction formation and function in pairs of SMs and CMs. The gap junctions formed have normal properties. These findings suggest that overexpressing Cx43 in SMs leads to enhanced cell-to-cell communication with CMs and should be considered as a possible strategy to improve cell integration in the myocardium during cell transplantation.

FOOTNOTES

¹ These authors have contributed equally to the work presented in this manuscript.

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-5088fje;

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