New insights on the cardiac safety factor: Unraveling the relationship between conduction velocity and robustness of propagation.

Cardiac conduction disturbances are linked with arrhythmia development. The concept of safety factor (SF) has been derived to describe the robustness of conduction, but the usefulness of this metric has been constrained by several limitations. For example, due to the difficulty of measuring the necessary input variables, SF calculations have only been applied to synthetic data. Moreover, quantitative validation of SF is lacking; specifically, the practical meaning of particular SF values is unclear, aside from the fact that propagation failure (i.e., conduction block) is characterized by SF < 1. This study aims to resolve these limitations for our previously published SF formulation and explore its relationship to relevant electrophysiological properties of cardiac tissue. First, HL-1 cardiomyocyte monolayers were grown on multi-electrode arrays and the robustness of propagation was estimated using extracellular potential recordings. SF values reconstructed purely from experimental data were largely between 1 and 5 (up to 89.1% of sites characterized). This range is consistent with values derived from synthetic data, proving that the formulation is sound and its applicability is not limited to analysis of computational models. Second, for simulations conducted in 1-, 2-, and 3-dimensional tissue blocks, we calculated true SF values at locations surrounding the site of current injection for sub- and supra-threshold stimuli and found that they differed from values estimated by our SF formulation by <10%. Finally, we examined SF dynamics under conditions relevant to arrhythmia development in order to provide physiological insight. Our analysis shows that reduced conduction velocity (Θ) caused by impaired intrinsic cell-scale excitability (e.g., due to sodium current a loss-of-function mutation) is associated with less robust conduction (i.e., lower SF); however, intriguingly, Θ variability resulting from modulation of tissue scale conductivity has no effect on SF. These findings are supported by analytic derivation of the relevant relationships from first principles. We conclude that our SF formulation, which can be applied to both experimental and synthetic data, produces values that vary linearly with the excess charge needed for propagation. SF calculations can provide insights helpful in understanding the initiation and perpetuation of cardiac arrhythmia.

Cardiac Cx43, Cx40 and Cx45 co-assembling: involvement of connexins epitopes in formation of hemichannels and Gap junction channels.

BACKGROUND: This review comes after the International Gap Junction Conference (IGJC 2015) and describes the current knowledge on the function of the specific motifs of connexins in the regulation of the formation of gap junction channels. Moreover the review is complemented by a summarized description of the distinct contribution of gap junction channels in the electrical coupling. RESULTS: Complementary biochemical and functional characterization on cell models and primary cells have improved our understanding on the oligomerization of connexins and the formation and the electrical properties of gap junction channels. Studies mostly focused cardiac connexins Cx43 and Cx40 expressed in myocytes, while Cx45 and Cx30.2 have been less investigated, for which main findings are reviewed to highlight their critical contribution in the formation of gap junction channels for ensuring the orchestrated electrical impulse propagation and coordination of atrial and ventricular contraction and heart function, whereas connexin dysfunction and remodeling are pro-arrhythmic factors. Common and specific motifs of residues identified in different domain of each type of connexin determine the connexin homo- and hetero-oligomerization and the channels formation, which leads to specific electrical properties. CONCLUSIONS: These motifs and the resulting formation of gap junction channels are keys to ensure the tissue homeostasis and function in each connexin expression pattern in various tissues of multicellular organisms. Altogether, the findings to date have significantly improved our understanding on the function of the different connexin expression patterns in healthy and diseased tissues, and promise further investigations on the contribution in the different types of connexin.

Functional consequences of co-expressing connexin40 or connexin45 with connexin43 on intercellular electrical coupling.

The functional characteristics of the co-expression of connexin43, connexin40, and connexin45 proteins in human myocardium are thought to play an important role in governing normal propagation of the cardiac electrical impulse and in generating the myocardial substrate for some arrhythmias and conduction disturbances. A rat liver epithelial cell line, that endogenously expresses connexin43, was used to induce also expression of connexin40 or connexin45 after stable transfection using an inducible ecdysone system. Electrical coupling was estimated from measurement of the input resistance of transfected cells using an intracellular microelectrode to inject current and record changes to membrane potential. However, varied expression of the transfected connexin40 or connexin45 did not change electrical coupling, although connexin43/40 co-expression led to better coupling than connexin43/45 co-expression. Quantification of endogenous connexin43 expression, at both mRNA and protein levels, showed that it was altered in a manner dependent on the transfected connexin isotype. The data using rat liver epithelial cells indicate an increased electrical coupling upon expression of connexin40 and connexin43 but decreased coupling with connexin45 and connexin43 co-expression.

Engineering cardiac cell junctions in vitro to study the intercalated disc.

This review article discusses a recent work using engineered cardiac cells to study the function of the intercalated disc putting emphasis on mechanical and electrical coupling.

Characterisation of connexin expression and electrophysiological properties in stable clones of the HL-1 myocyte cell line.

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.

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(+/+) (WT(GFP)) murine ventricular myocytes were patterned using microlithographic techniques. At the interface between pairs of WT(GFP) 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 WT(GFP) 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 WT(GFP) myocytes assures excitation of Cx43(-/-) cells. This explains the previously reported undisturbed contractility in tissues with spatially heterogeneous downregulation of Cx43 expression.

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.

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.

Influence of v5/6-His tag on the properties of gap junction channels composed of connexin43, connexin40 or connexin45.

HeLa cells expressing wild-type connexin43, connexin40 or connexin45 and connexins fused with a V5/6-His tag to the carboxyl terminus (CT) domain (Cx43-tag, Cx40-tag, Cx45-tag) were used to study connexin expression and the electrical properties of gap junction channels. Immunoblots and immunolabeling indicated that tagged connexins are synthesized and targeted to gap junctions in a similar manner to their wild-type counterparts. Voltage-clamp experiments on cell pairs revealed that tagged connexins form functional channels. Comparison of multichannel and single-channel conductances indicates that tagging reduces the number of operational channels, implying interference with hemichannel trafficking, docking and/or channel opening. Tagging provoked connexin-specific effects on multichannel and single-channel properties. The Cx43-tag was most affected and the Cx45-tag, least. The modifications included (1) V(j)-sensitive gating of I(j) (V(j), gap junction voltage; I(j), gap junction current), (2) contribution and (3) kinetics of I(j) deactivation and (4) single-channel conductance. The first three reflect alterations of fast V(j) gating. Hence, they may be caused by structural and/or electrical changes on the CT that interact with domains of the amino terminus and cytoplasmic loop. The fourth reflects alterations of the ion-conducting pathway. Conceivably, mutations at sites remote from the channel pore, e.g., 6-His-tagged CT, affect protein conformation and thus modify channel properties indirectly. Hence, V5/6-His tagging of connexins is a useful tool for expression studies in vivo. However, it should not be ignored that it introduces connexin-dependent changes in both expression level and electrophysiological properties.

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.

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 (I(Ks)) contribute to postrepolarization refractoriness in isolated cardiomyocytes. However, the impact of such kinetics on arrhythmogenesis remains unknown. We surmised that expression of I(Ks) 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 I(Ks)) 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 I(Ks)-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 I(Ks) 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 I(Ks) 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 I(Ks)-mediated postrepolarization refractoriness can promote wavebreak formation and fibrillatory conduction during pacing and sustained reentry and may have important implications in tachyarrhythmias.

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.

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

HeLa 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.

K+ channel activation and low-threshold Ca2+ spike of rat cerebellar Purkinje cells in vitro.

Using the whole-cell configuration of the patch-clamp recording method, we analyzed the role of K+ conductances in determining the characteristics of the dendritically-initiated low-threshold Ca+ spike (LTS) recorded at the somatic level of rat cerebellar Purkinje cells (PCs) in slice cultures. Blockade of tetra-ethyl-ammonium-(TEA)- and 4-aminopyridine-(4-AP)-sensitive K+ channels increased the amplitude of the LTS. This effect was prominent with 4-AP, which promotes the fast-decaying component of the LTS. Surprisingly, a shortening of the LTS was induced by the blockade of K+ channel activity instead of a broadening of spikes as generally observed. We propose that, when propagating to the soma, TEA- and 4-AP-sensitive K+ channel activity affects the electrical properties of dendrites such that the LTS is attenuated and slowed down.

Control of the propagation of dendritic low-threshold Ca(2+) spikes in Purkinje cells from rat cerebellar slice cultures.

To investigate the ionic mechanisms controlling the dendrosomatic propagation of low-threshold Ca(2+) spikes (LTS) in Purkinje cells (PCs), somatically evoked discharges of action potentials (APs) were recorded under current-clamp conditions. The whole-cell configuration of the patch-clamp method was used in PCs from rat cerebellar slice cultures. Full blockade of the P/Q-type Ca(2+) current revealed slow but transient depolarizations associated with bursts of fast Na(+) APs. These can occur as a single isolated event at the onset of current injection, or repetitively (i.e. a slow complex burst). The initial transient depolarization was identified as an LTS Blockade of P/Q-type Ca(2+) channels increased the likelihood of recording Ca(2+) spikes at the soma by promoting dendrosomatic propagation. Slow rhythmic depolarizations shared several properties with the LTS (kinetics, activation/inactivation, calcium dependency and dendritic origin), suggesting that they correspond to repetitively activated dendritic LTS, which reach the soma when P/Q channels are blocked. Somatic LTS and slow complex burst activity were also induced by K(+) channel blockers such as TEA (2.5 x 10(-4) M) charybdotoxin (CTX, 10(-5) M), rIberiotoxin (10(-7) M), and 4-aminopyridine (4-AP, 10(-3) M), but not by apamin (10(-4) M). In the presence of 4-AP, slow complex burst activity occurred even at hyperpolarized potentials (-80 mV). In conclusion, we suggest that the propagation of dendritic LTS is controlled directly by 4-AP-sensitive K(+) channels, and indirectly modulated by activation of calcium-activated K(+) (BK) channels via P/Q-mediated Ca(2+) entry. The slow complex burst resembles strikingly the complex spike elicited by climbing fibre stimulation, and we therefore propose, as a hypothesis, that dendrosomatic propagation of the LTS could underlie the complex spike.