Thomas Desplantez

Gap junction channels and cardiac impulse propagation.

The role of gap junction channels on cardiac impulse propagation is complex. This review focuses on the differential expression of connexins in the heart and the biophysical properties of gap junction channels under normal and disease conditions. Structural determinants of impulse propagation have been gained from biochemical and immunocytochemical studies performed on tissue extracts and intact cardiac tissue. These have defined the distinctive connexin coexpression patterns and relative levels in different cardiac tissues. Functional determinants of impulse propagation have emerged from electrophysiological experiments carried out on cell pairs. The static properties (channel number and conductance) limit the current flow between adjacent cardiomyocytes and thus set the basic conduction velocity. The dynamic properties (voltage-sensitive gating and kinetics of channels) are responsible for a modulation of the conduction velocity during propagated action potentials. The effect is moderate and depends on the type of Cx and channel. For homomeric-homotypic channels, the influence is small to medium; for homomeric-heterotypic channels, it is medium to strong. Since no data are currently available on heteromeric channels, their influence on impulse propagation is speculative. The modulation by gap junction channels is most prominent in tissues at the boundaries between cardiac tissues such as sinoatrial node-atrial muscle, atrioventricular node-His bundle, His bundle-bundle branch and Purkinje fibers-ventricular muscle. The data predict facilitation of orthodromic propagation.

Electrical Propagation in Synthetic Ventricular Myocyte Strands From Germline Connexin43 Knockout Mice

To characterize the role of connexin43 (Cx43) as a determinant of cardiac propagation, we synthesized strands and pairs of ventricular myocytes from germline Cx43−/− mice. The amount of Cx43, Cx45, and Cx40 in gap junctions was analyzed by immunohistochemistry and confocal microscopy. Intercellular electrical conductance, gj, was measured by the dual-voltage clamp technique (DVC), and electrical propagation was assessed by multisite optical mapping of transmembrane potential using a voltage-sensitive dye. Compared with wild-type (Cx43+/+) strands, immunoreactive signal for Cx43 was reduced by 46% in Cx43+/− strands and was absent in Cx43−/− strands. Cx45 signal was reduced by 46% in Cx43+/− strands and to the limit of detection in Cx43−/− strands, but total Cx45 protein levels measured in immunoblots of whole cell homogenates were equivalent in all genotypes. Cx40 was detected in ≈ 2% of myocytes. Intercellular conductance, gj, was reduced by 32% in Cx43+/− cell pairs and by 96% in Cx43−/− cell pairs. The symmetrical dependence of gj on transjunctional voltage and properties of single-channel recordings indicated that Cx45 was the only remaining connexin in Cx43−/− cells. Propagation in Cx43−/− strands was very slow (2.1 cm/s versus 52 cm/s in Cx43+/+) and highly discontinuous, with simultaneous excitation within and long conduction delays (2 to 3 ms) between individual cells. Propagation was abolished by 1 mmol/L heptanol, indicating residual junctional coupling. In summary, knockout of Cx43 in ventricular myocytes leads to very slow conduction dependent on the presence of Cx45. Electrical field effect transmission does not contribute to propagation in synthetic strands.

Intracellular Domains of Mouse Connexin26 and -30 Affect Diffusional and Electrical Properties of Gap Junction Channels

To evaluate the influence of intracellular domains of connexin (Cx) on channel transfer properties, we analyzed mouse connexin (Cx) Cx26 and Cx30, which show the most similar amino acid sequence identities within the family of gap junction proteins. These connexin genes are tightly linked on mouse chromosome 14. Functional studies were performed on transfected HeLa cells stably expressing both mouse connexins. When we examined homotypic intercellular transfer of microinjected neurobiotin and Lucifer yellow, we found that gap junctions in Cx30-transfected cells, in contrast to Cx26 cells, were impermeable to Lucifer yellow. Furthermore, we observed heterotypic transfer of neurobiotin between Cx30-transfectants and HeLa cells expressing mouse Cx30.3, Cx40, Cx43 or Cx45, but not between Cx26 transfectants and HeLa cells of the latter group. The main differences in amino acid sequence between Cx26 and Cx30 are located in the presumptive cytoplasmic loop and C-terminal region of these integral membrane proteins. By exchanging one or both of these domains, using PCR-based mutagenesis, we constructed Cx26/30 chimeric cDNAs, which were also expressed in HeLa cells after transfection. Homotypic intercellular transfer of injected Lucifer yellow was observed exclusively with those chimeric constructs that coded for both cytoplasmic domains of Cx26 in the Cx30 backbone polypeptide chain. In contrast, cells transfected with a construct that coded for the Cx26 backbone with the Cx30 cytoplasmic loop and C-terminal region did not show transfer of Lucifer yellow. Thus, Lucifer yellow transfer can be conferred onto chimeric Cx30 channels by exchanging the cytoplasmic loop and the C-terminal region of these connexins. In turn, the cytoplasmic loop and C-terminal domain of Cx30 prevent Lucifer yellow transfer when swapped with the corresponding domains of Cx26. In chimeric Cx30/Cx26 channels where the cytoplasmic loop and C-terminal domains had been exchanged, the unitary channel conductance was intermediate between those of the parental channels. Moreover, the voltage sensitivity was slightly reduced. This suggests that these cytoplasmic domains interfere directly or indirectly with the diffusivity, the conductance and voltage gating of the channels.

Adenoviral Expression of IKs Contributes to Wavebreak and Fibrillatory Conduction in Neonatal Rat Ventricular Cardiomyocyte Monolayers

Previous studies have shown that the gating kinetics of the slow component of the delayed rectifier K+ current (IKs) contribute to postrepolarization refractoriness in isolated cardiomyocytes. However, the impact of such kinetics on arrhythmogenesis remains unknown. We surmised that expression of IKs in rat cardiomyocyte monolayers contributes to wavebreak formation and facilitates fibrillatory conduction by promoting postrepolarization refractoriness. Optical mapping was performed in 44 rat ventricular myocyte monolayers infected with an adenovirus carrying the genomic sequences of KvLQT1 and minK (molecular correlates of IKs) and 41 littermate controls infected with a GFP adenovirus. Repetitive bipolar stimulation was applied at increasing frequencies, starting at 1 Hz until loss of 1:1 capture or initiation of reentry. Action potential duration (APD) was significantly shorter in IKs-infected monolayers than in controls at 1 to 3 Hz (P<0.05), whereas differences at higher pacing frequencies did not reach statistical significance. Stable rotors occurred in both groups, with significantly higher rotation frequencies, lower conduction velocities, and shorter action potentials in the IKs group. Wavelengths in the latter were significantly shorter than in controls at all rotation frequencies. Wavebreaks leading to fibrillatory conduction occurred in 45% of the IKs reentry episodes but in none of the controls. Moreover, the density of wavebreaks increased with time as long as a stable source sustained the fibrillatory activity. These results provide the first demonstration that IKs-mediated postrepolarization refractoriness can promote wavebreak formation and fibrillatory conduction during pacing and sustained reentry and may have important implications in tachyarrhythmias.

Connexin43 ablation in foetal atrial myocytes decreases electrical coupling, partner connexins, and sodium current

AIMS Remodelling and regional gradients in expression of connexins (Cx) are thought to contribute to atrial electrical dysfunction and atrial fibrillation. We assessed the effect of interaction between Cx43, Cx40, and Cx45 on atrial cell-to-cell coupling and inward Na current (I(Na)) in engineered pairs of atrial myocytes derived from wild-type mice (Cx43(+/+)) and mice with genetic ablation of Cx43 (Cx43(-/-)). METHODS AND RESULTS Cell pairs were engineered by microcontact printing from atrial Cx43(+/+) and Cx43(-/-) murine myocytes (1 day before birth, 3-5 days in culture). Dual and single voltage clamp were used to measure intercellular electrical conductance, g(j), and its dependence on transjunctional voltage, V(j), single gap junction channel conductances, and I(Na). 3D reconstructions of Cx43, Cx40, and Cx45 immunosignals in gap junctions were made from confocal slices. Full genetic Cx43 ablation produced a decrease in immunosignals of Cx40 to 62 ± 10% (mean ± SE; n= 17) and Cx45 to 66 ± 8% (n= 16). G(j) decreased from 80 ± 9 nS (Cx43(+/+), n= 17) to 24 ± 2 nS (Cx43(-/-), n= 35). Single channel analysis showed a shift in the main peak of the channel histogram from 49 ± 1.7 nS (Cx43(+/+)) to 67 ± 1.8 nS (Cx43(-/-)) with a second minor peak appearing at 27 ± 1.5 pS. The dependence of g(j) on V(j) decreased with Cx43 ablation. Importantly, peak I(Na) decreased from -350 ± 44 pA/pF (Cx43(+/+)) to -154 ± 28 pA/pF (Cx43(-/-)). CONCLUSIONS The dependence of Cx40, Cx45, and I(Na) on Cx43 expression indicates a complex interaction between connexins and I(Na) in the atrial intercalated discs that is likely to be of relevance for arrhythmogenesis.

Cardiac connexins Cx43 and Cx45: formation of diverse gap junction channels with diverse electrical properties.

AbstractHeLa cells expressing rat connexin43 (Cx43) and/or mouse Cx45 were studied with the dual voltage-clamp technique. Different types of cell pairs were established and their gap junction properties determined, i.e. the dependence of the instantaneous and steady-state conductances (gj,inst, gj,ss) on the transjunctional voltage (Vj) and the kinetics of inactivation of the gap junction current (Ij). Pairs of singly transfected cells showed homogeneous behaviour at both Vj polarities. Homotypic Cx43-Cx43 and Cx45-Cx45 cell pairs yielded distinct symmetrical functions gj,inst=f(Vj) and gj,ss=f(Vj). Heterotypic Cx43-Cx45 preparations exhibited asymmetric functions gj,inst=f(Vj) and gj,ss=f(Vj) suggesting that connexons Cx43 and Cx45 gate with positive and negative Vj, respectively. Preparations containing a singly (Cx43 or Cx45) or doubly (Cx43/45) transfected cell showed quasi-homogeneous behaviour at one Vj polarity and heterogeneous behaviour at the other polarity. The former yielded Boltzmann parameters intermediate between those of Cx43-Cx43, Cx45-Cx45 and Cx43-Cx45 preparations; the latter could not be explained by homotypic and heterotypic combinations of homomeric connexons. Each pair of doubly transfected cells (Cx43/Cx45) yielded unique functions gj,inst=f(Vj) and gj,ss=f(Vj). This can not be explained by combinations of homomeric connexons. We conclude that Cx43 and Cx45 form homomeric-homotypic, homomeric-heterotypic channels as well as heteromeric-homotypic and heteromeric-heterotypic channels. This has implications for the impulse propagation in specific areas of the heart.

Cell-to-cell coupling in engineered pairs of rat ventricular cardiomyocytes: relation between Cx43 immunofluorescence and intercellular electrical conductance

Gap junctions are composed of connexin (Cx) proteins, which mediate intercellular communication. Cx43 is the dominant Cx in ventricular myocardium, and Cx45 is present in trace amounts. Cx43 immunosignal has been associated with cell-to-cell coupling and electrical propagation, but no studies have directly correlated Cx43 immunosignal to electrical cell-to-cell conductance, g(j), in ventricular cardiomyocyte pairs. To assess the correlation between Cx43 immunosignal and g(j), we developed a method to determine both parameters from the same cell pair. Neonatal rat ventricular cardiomyocytes were seeded on micropatterned islands of fibronectin. This allowed formation of cell pairs with reproducible shapes and facilitated tracking of cell pair locations. Moreover, cell spreading was limited by the fibronectin pattern, which allowed us to increase cell height by reducing the surface area of the pattern. Whole cell dual voltage clamp was used to record g(j) of cell pairs after 3-5 days in culture. Fixation of cell pairs before removal of patch electrodes enabled preservation of cell morphology and offline identification of patched pairs. Subsequently, pairs were immunostained, and the volume of junctional Cx43 was quantified using confocal microscopy, image deconvolution, and three-dimensional reconstruction. Our results show a linear correlation between g(j) and Cx43 immunosignal within a range of 8-50 nS.

Functional differences between human Cx37 polymorphic hemichannels.

A polymorphism in the human Cx37 gene (C1019T), resulting in a non-conservative amino acid change in the regulatory C-terminus of the Cx37 protein (P319S), has been proposed as a prognostic marker for atherosclerosis. We have recently demonstrated that Cx37 hemichannels control the initiation of atherosclerotic plaque development by regulating ATP-dependent monocyte adhesion in atherosclerosis-susceptible apolipoprotein E-deficient mice. In this study, we have measured the electrical properties of Cx37 hemichannels (HCs) and gap junction channels (GJCs) with voltage-clamp methods. To this end, we have transfected hCx37-P319, hCx37-S319 or empty pIRES-eGFP vector cDNA into communication-deficient HeLa cells. In clones expressing similar levels of Cx37, exposure of single cells to low-Ca(2+) solution induced a voltage-sensitive HC current. The analysis yielded a bell-shaped function g(hc)=f(V(m)) (g(hc): normalized conductance at steady state; V(m): membrane potential) with a maximum around V(m)=-30 mV. The peak g(hc) of Cx37-P319 was 3-fold larger than that of Cx37-S319 HCs. Experiments on cell pairs revealed that Cx37-P319 GJCs exhibited a 1.5-fold larger unitary conductance than Cx37-S319 GJCs. Hence, the larger peak g(hc) of the former may reflect a larger conductance of their HCs. Using the same clones, we found that Cx37-P319 cells released more ATP and were less adhesive than Cx37-S319 cells. The reduction in adhesiveness of Cx37-expressing cells was prevented by extracellular apyrase. We conclude that the differences in biophysical properties between polymorphic HCs may be responsible for inequality in ATP release between Cx37-P319 and Cx37-S319 cells, which results in differential cell adhesion.

Electrical Coupling and Propagation in Engineered Ventricular Myocardium With Heterogeneous Expression of Connexin43

Rationale: Spatial heterogeneity in connexin (Cx) expression has been implicated in arrhythmogenesis. Objective: This study was performed to quantify the relation between the degree of heterogeneity in Cx43 expression and disturbances in electric propagation. Methods and Results: Cell pairs and strands composed of mixtures of Cx43−/− (Cx43KO) or GFP-expressing Cx43+/+ (WTGFP) murine ventricular myocytes were patterned using microlithographic techniques. At the interface between pairs of WTGFP and Cx43KO cells, dual-voltage clamp showed a marked decrease in electric coupling (approximately 5% of WT) and voltage gating suggested the presence of mixed Cx43/Cx45 channels. Cx43 and Cx45 immunofluorescence signals were not detectable at this interface, probably because of markedly reduced gap junction size. Macroscopic propagation velocity, measured by multisite high-resolution optical mapping of transmembrane potential in strands of cells of mixed Cx43 genotype, decreased with an increasing proportion of Cx43KO cells in the strand. A marked decrease in conduction velocity was observed in strands composed of <50% WT cells. Propagation at the microscopic scale showed a high degree of dissociation between WTGFP and Cx43KO cells, but consistent excitation without development of propagation block. Conclusions: Heterogeneous ablation of Cx43 leads to a marked decrease in propagation velocity in tissue strands composed of <50% cells with WT Cx43 expression and marked dissociation of excitation at the cellular level. However, the small residual electric conductance between Cx43 and WTGFP myocytes assures excitation of Cx43−/− cells. This explains the previously reported undisturbed contractility in tissues with spatially heterogeneous downregulation of Cx43 expression.

Characterisation of Connexin Expression and Electrophysiological Properties in Stable Clones of the HL-1 Myocyte Cell Line

Abstract The HL-1 atrial line contains cells blocked at various developmental stages. To obtain homogeneous sub-clones and correlate changes in gene expression with functional alterations, individual clones were obtained and characterised for parameters involved in conduction and excitation-contraction coupling. Northern blots for mRNAs coding for connexins 40, 43 and 45 and calcium handling proteins (sodium/calcium exchanger, L- and T-type calcium channels, ryanodine receptor 2 and sarco-endoplasmic reticulum calcium ATPase 2) were performed. Connexin expression was further characterised by western blots and immunofluorescence. Inward currents were characterised by voltage clamp and conduction velocities measured using microelectrode arrays. The HL-1 clones had similar sodium and calcium inward currents with the exception of clone 2 which had a significantly smaller calcium current density. All the clones displayed homogenous propagation of electrical activity across the monolayer correlating with the levels of connexin expression. Conduction velocities were also more sensitive to inhibition of junctional coupling by carbenoxolone (∼80%) compared to inhibition of the sodium current by lidocaine (∼20%). Electrical coupling by gap junctions was the major determinant of conduction velocities in HL-1 cell lines. In summary we have isolated homogenous and stable HL-1 clones that display characteristics distinct from the heterogeneous properties of the original cell line.