Faculty Bibliography

Intercellular communication is essential for proper cardiac function. Mechanical and electrical activity need to be synchronized so that the work of individual myocytes transforms into the pumping function of the organ. Mechanical continuity is provided by desmosomes and adherens junctions, while gap junctions provide a pathway for passage of ions and small molecules between cells. These complexes preferentially reside at the site of end-end contact between myocytes, within the intercalated disc. Recognition that some forms of arrhythmogenic cardiomyopathy are caused by mutations in desmosomal protein genes has galvanized interest in the biology of the desmosome and its interactions with other junctional molecules. This review presents the cellular and molecular biology of the desmosome, current knowledge on the relation of desmosomal mutations and disease phenotypes, and an overview of the molecular pathophysiology of arrhythmogenic right ventricular cardiomyopathy. Clinical experience and results from cellular and animal models provide insights into the intercalated disc as a functional unit and into the basic substrates that underlie pathogenesis and arrhythmogenesis of arrhythmogenic right ventricular cardiomyopathy

OBJECTIVE: The gap junction protein connexin37 (Cx37) plays an important role in cell-cell communication in the vasculature. A C1019T Cx37 gene polymorphism, encoding a P319S substitution in the regulatory C terminus of Cx37 (Cx37CT), correlates with arterial stenosis and myocardial infarction in humans. This study was designed to identify potential binding partners for Cx37CT and to determine whether the polymorphism modified this interaction. METHODS AND RESULTS: Using a high-throughput phage display, we retrieved 2 binding motifs for Cx37CT: WHK ... [K,R]XP ... and FHK ... [K,R]XXP ... , the first being more common for Cx37CT-319P and the second more common for Cx37CT-319S. One of the peptides (WHRTPRLPPPVP) showed 77.7% homology with residues 843 to 854 of endothelial nitric oxide synthase (eNOS). In vitro binding of this peptide or of the homologous eNOS sequence to both Cx37CT isoforms was confirmed by cross-linking and surface plasmon resonance. Electrophysiological analysis of Cx37 single channel activity in transfected N2a cells showed that eNOS-like and eNOS(843-854) increased the frequency of events with conductances higher than 300 pS. We demonstrated that eNOS coimmunoprecipitated with Cx37 in a mouse endothelial cell (EC) line (bEnd.3), human primary ECs, and a human EC line transfected with Cx37-319P or Cx37-319S. Cx37 and eNOS colocalized at EC membranes. Moreover, a dose-dependent increase in nitric oxide production was observed in ECs treated with Cx37 antisense. CONCLUSIONS: Overall, our data show for the first time a functional and specific interaction between eNOS and Cx37. This interaction may be relevant for the control of vascular physiology both in health and in disease

The intercalated disk (ID) is a complex structure that electromechanically couples adjoining cardiac myocytes into a functional syncitium. The integrity of the disk is essential for normal cardiac function, but how the diverse elements are assembled into a fully integrated structure is not well understood. In this study, we examined the assembly of new IDs in primary cultures of adult rat cardiac myocytes. From 2 to 5 days after dissociation, the cells flatten and spread, establishing new cell-cell contacts in a manner that recapitulates the in vivo processes that occur during heart development and myocardial remodeling. As cells make contact with their neighbors, transmembrane adhesion proteins localize along the line of apposition, concentrating at the sites of membrane attachment of the terminal sarcomeres. Cx43 gap junctions and ankyrin-G, an essential cytoskeletal component of voltage gated sodium channel complexes, were secondarily recruited to membrane domains involved in cell-cell contacts. The consistent order of the assembly process suggests that there are specific scaffolding requirements for integration of the mechanical and electrochemical elements of the disk. Defining the relationships that are the foundation of disk assembly has important implications for understanding the mechanical dysfunction and cardiac arrhythmias that accompany alterations of ID architecture

AIMS: Hearts of mice expressing K258stop in place of connexin43 (Cx43) protein were subjected to acute myocardial infarction in order to assess the importance of Cx43 regulation on infarct size and arrhythmia susceptibility. This mutation K258stop prevents chemical regulation of Cx43 channels, including by low intracellular pH. METHODS AND RESULTS: Langendorff-perfused hearts of mice harbouring one Cx43 knockout (KO) allele and one K258stop or Cx43 allele (K258stop/KO; Cx43/KO as control) were subjected to 1 h of ischaemia and 4 h of reperfusion by reversibly occluding the left anterior descending (LAD) coronary artery. Inducibility of ventricular tachyarrhythmias (VTs) was tested by applying an endocardial burst-pacing protocol during LAD occlusion. Separately, time course and the extent of acidification-induced closure of gap junction channels were tested by dual-voltage clamp. Infarct volume (as per cent of area at risk) was significantly larger in K258stop/KO hearts compared with Cx43/KO controls (42.2 +/- 3 vs. 30.4 +/- 1.7%, P = 0.004, n = 8 each). During LAD occlusion, K258stop/KO hearts had a higher incidence of pacing-induced VT and a higher frequency of occurrence of spontaneous premature ventricular beats. The occurrence of ventricular arrhythmias was also significantly larger in the K258stop/KO hearts during reperfusion. In separate experiments, we demonstrated reduced sensitivity to acidification-induced uncoupling in cell pairs obtained from K258stop/KO hearts. CONCLUSION: Loss of the regulatory domain of Cx43 leads to an increase in infarct size and increased susceptibility to arrhythmias following acute coronary occlusion

BACKGROUND: Phosphorylation is a key regulatory event in controlling the function of the cardiac gap junction protein connexin43 (Cx43). Three new phosphorylation sites (S296, S297, S306) have been identified on Cx43; two of these sites (S297 and S306) are dephosphorylated during ischemia. The functional significance of these new sites is currently unknown. OBJECTIVE: The purpose of this study was to examine the role of S296, S297, and S306 in the regulation of electrical intercellular communication. METHODS: To mimic constitutive dephosphorylation, serine was mutated to alanine at the three sites and expressed in HeLa cells. Electrical coupling and single channel measurements were performed by double patch clamp. Protein expression levels were assayed by western blotting, localization of Cx43, and phosphorylation of S306 by immunolabeling. Free hemichannels were assessed by biotinylation. RESULTS: Macroscopic conductance in cells expressing S306A was reduced to 57% compared to wild type (WT), whereas coupling was not significantly changed in cells expressing either S296A or S297A. S306A-expressing cells displayed similar protein and free hemichannel abundance compared to WT Cx43, whereas the fractional area of plaques in cell-to-cell interfaces was increased. However, single channel measurements showed a WT Cx43 main state conductance of 119 pS, whereas the main state conductance of S306A channels was reduced to 95 pS. Furthermore, channel gating was affected in S306A channels. CONCLUSION: Lack of phosphorylation at serine 306 results in reduced coupling, which can be explained by reduced single channel conductance. We suggest that dephosphorylation of S306 partly explains the electrical uncoupling seen in myocardial ischemia

Armadillo family proteins known as plakophilins have been characterized as structural components of desmosomes that stabilize and strengthen adhesion by enhancing attachments with the intermediate filament cytoskeleton. However, plakophilins and their close relatives are emerging as versatile scaffolds for multiple signaling and metabolic processes that not only facilitate junction dynamics but also more globally regulate diverse cellular activities. While perturbation of plakophilin functions contribute to inherited diseases and cancer pathogenesis, the functional significance of the multiple PKP isoforms and the mechanisms by which their behaviors are regulated remain to be elucidated

RATIONALE: Plakophilin-2 (PKP2) is an essential component of the cardiac desmosome. Recent data show that it interacts with other molecules of the intercalated disc. Separate studies show preferential localization of the voltage-gated sodium channel (Na(V)1.5) to this region. OBJECTIVE: To establish the association of PKP2 with sodium channels and its role on action potential propagation. METHODS AND RESULTS: Biochemical, patch clamp, and optical mapping experiments demonstrate that PKP2 associates with Na(V)1.5, and that knockdown of PKP2 expression alters the properties of the sodium current, and the velocity of action potential propagation in cultured cardiomyocytes. CONCLUSIONS: These results emphasize the importance of intermolecular interactions between proteins relevant to mechanical junctions, and those involved in electric synchrony. Possible relevance to the pathogenesis of arrhythmogenic right ventricular cardiomyopathy is discussed

Gap junction pharmacology is a nascent field. Previous studies have identified molecules that enhance intercellular communication, and may offer potential for innovative antiarrhythmic therapy. However, their specific molecular target(s) and mechanism(s) of action remain unknown. Previously, we identified a 34-aa peptide (RXP-E) that binds the carboxyl terminal domain of Cx43 (Cx43CT) and prevents cardiac gap junction closure and action potential propagation block. These results supported the feasibility of a peptide-based pharmacology to Cx43, but the structure of the core active element in RXP-E, an essential step for pharmacological development, remained undefined. Here, we used a combination of molecular modeling, surface plasmon resonance, nuclear magnetic resonance and patch-clamp strategies to define, for the first time, a unique ensemble of pharmacophores that bind Cx43CT and prevent closure of Cx43 channels. Two particular molecules are best representatives of this family: a cyclized heptapeptide (called CyRP-71) and a linear octapeptide of sequence RRNYRRNY. These 2 small compounds offer the first structural platform for the design of Cx43-interacting gap junction openers. Moreover, the structure of these compounds offers an imprint of a region of Cx43CT that is fundamental to gap junction channel function