Faculty Bibliography

There is abundant evidence showing that connexins form gap junctions. Yet this does not exclude the possibility that connexins can exert other functions, separate from that of gap junction (or even a permeable hemichannel) formation. Here, we focus on these noncanonical functions of connexin43 (Cx43), particularly in the heart. We describe two specific examples: the importance of Cx43 on intercellular adhesion, and the role of Cx43 in the function of the sodium channel. We propose that these two functions of Cx43 have important repercussions on the propagation of electrical activity in the heart, irrespective of the presence of permeable gap junction channels. Overall, the gap junction-independent functions of Cx43 in cardiac electrophysiology emerge as an exciting area of future research.

BACKGROUND: Desmosomes and adherens junctions provide mechanical continuity between cardiac cells, whereas gap junctions allow for cell-cell electrical/metabolic coupling. These structures reside at the cardiac intercalated disc (ID). Also at the ID is the voltage-gated sodium channel (VGSC) complex. Functional interactions between desmosomes, gap junctions, and VGSC have been demonstrated. Separate studies show, under various conditions, reduced presence of gap junctions at the ID and redistribution of connexin43 (Cx43) to plaques oriented parallel to fiber direction (gap junction "lateralization"). OBJECTIVE: To determine the mechanisms of Cx43 lateralization, and the fate of desmosomal and sodium channel molecules in the setting of Cx43 remodeling. METHODS: Adult sheep were subjected to right ventricular pressure overload (pulmonary hypertension). Tissue was analyzed by quantitative confocal microscopy and by transmission electron microscopy. Ionic currents were measured using conventional patch clamp. RESULT: Quantitative confocal microscopy demonstrated lateralization of immunoreactive junctional molecules. Desmosomes and gap junctions in lateral membranes were demonstrable by electron microscopy. Cx43/desmosomal remodeling was accompanied by lateralization of 2 microtubule-associated proteins relevant for Cx43 trafficking: EB1 and kinesin protein Kif5b. In contrast, molecules of the VGSC failed to reorganize in plaques discernable by confocal microscopy. Patch-clamp studies demonstrated change in amplitude and kinetics of sodium current and a small reduction in electrical coupling between cells. CONCLUSIONS: Cx43 lateralization is part of a complex remodeling that includes mechanical and gap junctions but may exclude components of the VGSC. We speculate that lateralization results from redirectionality of microtubule-mediated forward trafficking. Remodeling of junctional complexes may preserve electrical synchrony under conditions that disrupt ID integrity.

Gap junctions are essential to the function of multicellular animals, which require a high degree of coordination between cells. In vertebrates, gap junctions comprise connexins and currently 21 connexins are known in humans. The functions of gap junctions are highly diverse and include exchange of metabolites and electrical signals between cells, as well as functions, which are apparently unrelated to intercellular communication. Given the diversity of gap junction physiology, regulation of gap junction activity is complex. The structure of the various connexins is known to some extent; and structural rearrangements and intramolecular interactions are important for regulation of channel function. Intercellular coupling is further regulated by the number and activity of channels present in gap junctional plaques. The number of connexins in cell-cell channels is regulated by controlling transcription, translation, trafficking, and degradation; and all of these processes are under strict control. Once in the membrane, channel activity is determined by the conductive properties of the connexin involved, which can be regulated by voltage and chemical gating, as well as a large number of posttranslational modifications. The aim of the present article is to review our current knowledge on the structure, regulation, function, and pharmacology of gap junctions. This will be supported by examples of how different connexins and their regulation act in concert to achieve appropriate physiological control, and how disturbances of connexin function can lead to disease.

PURPOSE OF REVIEW: Connexins are the pore forming subunits of gap junction channels. They are essential for cardiac action potential propagation. Connexins are modified at the transcriptional or posttranslational levels under pathological states such as cardiac hypertrophy or ischemia, thus contributing to the arrhythmogenic substrate. However, the relation between nucleotide substitutions in the connexin gene and the occurrence of cardiac arrhythmias remains largely unexplored. RECENT FINDINGS: Recent studies have reported an association between nucleotide substitutions in the connexin40 (Cx40) and connexin43 (Cx43) genes (GJA5 and GJA1, respectively) and cardiac arrhythmias. Of note, however, germline mutations in Cx43 are considered causative of oculodentodigital dysplasia, a pleiotropic syndrome wherein cardiac manifestations are notoriously absent. SUMMARY: Here, we review some of the current knowledge on the association between cardiac connexins and inherited arrhythmias.

Introduction: Progressive cardiac conduction disturbance (PCCD) is a hereditary disorder of the His-Purkinje system that often leads to complete heart block, pacemaker implantation, or sudden death. Although mutations in genes SCN5A, SCN1B and TRPM4 have been identified in some PCCD pedigrees, large scale studies of its clinical features, prognosis, and genetic basis are not available. We studied a population of 63 Japanese PCCD patients (47 probands and 16 family members; male 37, female 26) without underlying structural heart diseases. Despite the assumption that PCCD predominates in the elderly, the age of onset of the proband showed a wide-range distribution (59.6+/-23.7 years) with two peaks in the 2nd and 6th decade of life. Patients were followed for a variable period of time (0.3 to 33 years; 11.6+/-10.0 years). Progressive aggravation of the conduction disturbance and cardiac dysfunction with LVEF<35% was evident in 46% and 33% of patients, respectively. Pacemaker or ICD was implanted in 44 patients (70%). Six patients, three with device therapy and three without, died suddenly. Genetic screening revealed mutations in SCN5A (coding for Nav1.5; n=9) and in GJA5 (coding for connexin40; n=1). Heterologous expression of the GJA5 mutant in N2A cells resulted in marked reduction of junctional conductance and diffuse localization of Cx40-immunoreactive proteins in the vicinity of the plasma membrane without formation of gap junctions. The proband of the GJA5 mutation and his mother died at the age of 10 and 30, respectively, indicating an early-onset and malignant variant of PCCD. Moreover, mutations were identified in LMNA(n=9), the gene encoding lamin A/C, an inner nuclear membrane protein involved in a number of reported cases of dilated cardiomyopathy. The electrophysiological defects preceded the cardiac dysfunction in all 9 LMNA carriers and in 2 family members, presumably due to an age-dependent enhancement of apoptosis in cells of the conduction system. These data show that the PCCD is a heterogeneous disease that can be associated with defects in the amino acid sequence of integral membrane proteins involved in cell excitability and/or action potential propagation. A separate group may represent a prodromal stage of lamin A/C-related dilated cardiomyopathy. Methods: N/A Results:N/A Conclusions: N/A

BACKGROUND: Reduced expression of connexin 43 (Cx43) and sodium channel (Nav1.5) and increased expression of collagen (fibrosis) are important determinants of impulse conduction in the heart. OBJECTIVE: To study the importance and interaction of these factors at very low Cx43 expression, inducible Cx43 knockout mice with and without inducible ventricular tachycardia (VT) were compared through electrophysiology and immunohistochemistry. METHODS: Cx43(CreER(T)/fl) mice were induced with tamoxifen and killed after 2 weeks. Epicardial activation mapping was performed on Langendorff-perfused hearts, and arrhythmia vulnerability was tested. Mice were divided into arrhythmogenic (VT+; n = 13) and nonarrhythmogenic (VT-; n = 10) animals, and heart tissue was analyzed for Cx43, Nav1.5, and fibrosis. RESULTS: VT+ mice had decreased Cx43 expression with increased global, but not local, heterogeneity of Cx43 than did VT- mice. Nav1.5-immunoreactive protein expression was lower in VT+ than in VT- mice, specifically at sites devoid of Cx43. Levels of fibrosis were similar between VT- and VT+ mice. QRS duration was increased and epicardial activation was more dispersed in VT+ mice than in VT- mice. The effective refractory period was similar between the 2 groups. Premature stimulation resulted in a more severe conduction slowing in VT+ than in VT- hearts in the right ventricle. Separate patch-clamp experiments in isolated rat ventricular myocytes confirmed that the loss of Cx43 expression correlated with the decreased sodium current amplitude. CONCLUSIONS: Global heterogeneity in Cx43 expression and concomitant heterogeneous downregulation of sodium-channel protein expression and sodium current leads to slowed and dispersed conduction, which sensitizes the heart for ventricular arrhythmias

Intercalated disc structures have conventionally been considered to be independent. Recent work shows that molecules initially thought of as belonging to one complex can actually affect another. Here, I focus on the cross-talk between connexin43 (Cx43, 'the gap junction protein') and the sodium channel complex and, conversely, on ankyrin-G (AnkG, a 'component of the sodium channel complex') and gap junctions. I speculate as to the possibility that one molecule affects the function of the other by regulating its trafficking into the intercalated disc

BACKGROUND: -Progressive familial heart block type I (PFHBI) is a hereditary arrhythmia characterized by progressive conduction disturbances in the His-Purkinje system. PFHBI has been linked to genes such as SCN5A that influence cardiac excitability, but not to genes that influence cell-to-cell communication. Our goal was to explore whether nucleotide substitutions in genes coding for connexin proteins would associate with clinical cases of PFHBI and if so, to establish a genotype-cell phenotype correlation for that mutation. METHODS AND RESULTS: -We screened 156 probands afflicted with PFHBI. In addition to 12 sodium channel mutations, we found a germline GJA5 (connexin40; Cx40) mutation (Q58L) in an afflicted family. Heterologous expression of Cx40-Q58L in connexin-deficient neuroblastoma cells resulted in marked reduction of junctional conductance (Cx40-WT: 22.2+/-1.7 nS, n=14; Cx40-Q58L: 0.56+/-0.34 nS, n=14; p<0.001) and diffuse localization of immunoreactive proteins in the vicinity of the plasma membrane without formation of gap junctions. Heteromeric co-transfection of Cx40-WT and Cx40-Q58L resulted in homogenous distribution of proteins in the plasma membrane rather than in membrane plaques in about 50% of cells; well-defined gap junctions were observed in other cells. Junctional conductance values correlated with the distribution of gap junction plaques. CONCLUSIONS: -Mutation Cx40-Q58L impairs gap junction formation at cell-cell interfaces. This is the first demonstration of a germline mutation in a connexin gene that associates with inherited ventricular arrhythmias, and emphasizes the importance of Cx40 in normal propagation in the specialized conduction system

BACKGROUND: Sodium channel alpha-subunits in ventricular myocytes (VMs) segregate either to the intercalated disc, or to lateral membranes, where they associate with region-specific molecules. OBJECTIVE: To determine the functional properties of sodium channels as a function of their location in the cell. METHODS: Local sodium currents were recorded from adult rodent VMs and Purkinje cells using the cell-attached macropatch configuration. Electrodes were placed either in the cell midsection (M), or cell end (area originally occupied by the intercalated disc; ID). Channels were identified as TTX-sensitive (TTX-S) or TTX-resistant (TTX-R) by application of 100 nM TTX. RESULTS: Average peak-current amplitude was larger in ID than M, and largest at site of contact between attached cells. TTX-S channels were found only in M region of VMs, and not in Purkinje myocytes. TTX-R channels were found in M and ID, but their biophysical properties differed depending on recording location. Sodium current in rat VMs was upregulated by TNF-alpha. The magnitude of current increase was largest in M, but this difference was abolished by 100 nM TTX. CONCLUSIONS: Our data suggest that: a) a large fraction of TTX-R (likely Na(v)1.5) channels in the M region of VMs are inactivated at normal resting potential, leaving most of the burden of excitation to TTX-R channels in the ID; b) cell-cell adhesion increases functional channel density at ID. c) TTX-S (likely non-Na(v)1.5) channels make a minimal contribution to sodium current under control conditions, but represent a functional reserve that can be upregulated by exogenous factors