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.

Abstract-To characterize the role of connexin43 (Cx43) as a determinant of cardiac propagation, we synthesized strands and pairs of ventricular myocytes from germline Cx43 Ϫ/Ϫ mice. The amount of Cx43, Cx45, and Cx40 in gap junctions was analyzed by immunohistochemistry and confocal microscopy. Intercellular electrical conductance, g j , 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 ϩ/ϩ ) strands, immunoreactive signal for Cx43 was reduced by 46% in Cx43 ϩ/Ϫ strands and was absent in Cx43 Ϫ/Ϫ strands. Cx45 signal was reduced by 46% in Cx43 ϩ/Ϫ strands and to the limit of detection in Cx43 Ϫ/Ϫ strands, but total Cx45 protein levels measured in immunoblots of whole cell homogenates were equivalent in all genotypes. Cx40 was detected in Ϸ 2% of myocytes. Intercellular conductance, g j , was reduced by 32% in Cx43 ϩ/Ϫ cell pairs and by 96% in Cx43 Ϫ/Ϫ cell pairs. The symmetrical dependence of g j on transjunctional voltage and properties of single-channel recordings indicated that Cx45 was the only remaining connexin in Cx43Ϫ/Ϫ cells. Propagation in Cx43 Ϫ/Ϫ strands was very slow (2.1 cm/s versus 52 cm/s in Cx43 ϩ/ϩ ) 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. Key Words: Cx43 Ⅲ Cx45 Ⅲ very slow electrical propagation Ⅲ discontinuous propagation Ⅲ heptanol C onnexin (Cx) proteins form intercellular channels enabling the intercellular exchange of ions and small molecules. 1 In the heart, they facilitate rapid, coordinated electrical excitation, a prerequisite for normal rhythmic contraction. Three different connexins, Cx43, Cx40, and Cx45, are expressed in heart. 2,3 Cx43, the most abundant connexin, is found in ventricular and atrial myocardium. Cx40 is expressed in atrial tissue and the cardiac conduction system. Although Cx45 expression in working ventricular myocardium is modest compared with Cx43, Cx45 is vital for early embryogenesis and cardiac development. 4,5 Cx45 is also expressed in the sinoatrial and atrioventricular nodes. It colocalizes with Cx40 in the conduction system, 6 and with Cx43 in ventricular myocardium. 7 The three cardiac connexins form channels with unique properties. 8 Although multiple connexins are coexpressed in cardiac tissues, their interactions and contributions to electrical or metabolic function are not understood completely. Cx43 and Cx45 likely form heteromeric/heterotypic channels, 9 -12 which may fulfill distinct functions. Cardiac diseases that lead to arrhythmias are associated with gap junction remodeling. [13][14][15] Therefore, know...

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HeLa cells expressing rat connexin43 (Cx43) and/or mouse Cx45 were studied with the dual voltageclamp 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 (g j,inst , g j,ss ) on the transjunctional voltage (V j ) and the kinetics of inactivation of the gap junction current (I j ). Pairs of singly transfected cells showed homogeneous behaviour at both V j polarities. Homotypic Cx43-Cx43 and Cx45-Cx45 cell pairs yielded distinct symmetrical functions g j,inst =f(V j ) and g j,ss =f(V j ). Heterotypic Cx43-Cx45 preparations exhibited asymmetric functions g j,inst =f(V j ) and g j,ss =f(V j ) suggesting that connexons Cx43 and Cx45 gate with positive and negative V j , respectively. Preparations containing a singly (Cx43 or Cx45) or doubly (Cx43/45) transfected cell showed quasihomogeneous behaviour at one V j 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 g j,inst =f(V j ) and g j,ss =f(V j ). This can not be explained by combinations of homomeric connexons. We conclude that Cx43 and Cx45 form homomerichomotypic, 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.

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.

Abstract-Previous studies have shown that the gating kinetics of the slow component of the delayed rectifier K ϩ 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 (PϽ0.05), 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. Key Words: I Ks , postrepolarization refractoriness Ⅲ wavebreak Ⅲ gene expression D uring ventricular and atrial fibrillation, conduction of the electrical wavefront is characterized by complex patterns of propagation, including reentry, wavefront fragmentation (wavebreak), and wavelet formation. 1 A wavebreak occurs if the stimulatory efficacy of a wavefront does not suffice to excite all the tissue downstream. The free shoulder of a broken wave is then prone to curl and give rise to a rotor. 2 To date, the molecular mechanisms of wavebreaks leading to fibrillatory conduction remain poorly understood.Studies in single guinea pig myocytes showed that slow recovery of excitability during diastole was, in part, a consequence of the slow gating kinetics of the delayed rectifier potassium outward current I K . 3,4 Shortly after these studies were published, I K was found to be the result of the activation of 2 outward currents: I Kr and the I Ks . 5 Given the large amounts of I Ks present in guinea pig myocytes 6,7 and the slow deactivation kinetics of this current, 8 we surmise that I Ks is a likely candidate to have an important role in regulating excitability during the diastolic interval, ie, postrepolarization refractorin...

McCain ML, Desplantez T, Geisse NA, Rothen-Rutishauser B, Oberer H, Parker KK, Kleber AG. Cell-to-cell coupling in engineered pairs of rat ventricular cardiomyocytes: relation between Cx43 immunofluorescence and intercellular electrical conductance. Am J Physiol Heart Circ Physiol 302: H443-H450, 2012. First published November, 11, 2011 doi:10.1152/ajpheart.01218.2010.-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, gj, in ventricular cardiomyocyte pairs. To assess the correlation between Cx43 immunosignal and gj, 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 gj 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 gj and Cx43 immunosignal within a range of 8 -50 nS.

The role of Ca2+ entry in determining the electrical properties of cerebellar Purkinje cell (PC) dendrites and somata was investigated in cerebellar slice cultures. Immunohistofluorescence demonstrated the presence of at least three distinct types of Ca2+ channel proteins in PCs: the α1A subunit (P/Q type Ca2+ channel), the α1G subunit (T type) and the α1E subunit (R type). In PC dendrites, the response started in 66 % of cases with a slow depolarization (50 ± 15 ms) triggering one or two fast (∼1 ms) action potentials (APs). The slow depolarization was identified as a low‐threshold non‐P/Q Ca2+ AP initiated, most probably, in the dendrites. In 16 % of cases, this response propagated to the soma to elicit an initial burst of fast APs. Somatic recordings revealed three modes of discharge. In mode 1, PCs display a single or a short burst of fast APs. In contrast, PCs fire repetitively in mode 2 and 3, with a sustained discharge of APs in mode 2, and bursts of APs in mode 3. Removal of external Ca2+ or bath applications of a membrane‐permeable Ca2+ chelator abolished repetitive firing. Tetraethylammonium (TEA) prolonged dendritic and somatic fast APs by a depolarizing plateau sensitive to Cd2+ and to ω‐conotoxin MVII C or ω‐agatoxin TK. Therefore, the role of Ca2+ channels in determining somatic PC firing has been investigated. Cd2+ or P/Q type Ca2+ channel‐specific toxins reduced the duration of the discharge and occasionallyinduced the appearance of oscillations in the membrane potential associated with bursts of APs. In summary, we demonstrate that Ca2+ entry through low‐voltage gated Ca2+ channels, not yet identified, underlies a dendritic AP rarelyeliciting a somatic burst of APs whereas Ca2+ entry through P/Q type Ca2+ channels allowed a repetitive firing mainly by inducing a Ca2+‐dependent hyperpolarization.