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.

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.

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.

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.

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.

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).

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.

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.

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.

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.

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.

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.

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.

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.

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.