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176


The year in arrhythmias--2009: part I

Olshansky, Brian; Delmar, Mario; Tomaselli, Gordon F
PMID: 20185116
ISSN: 1556-3871
CID: 113840

Ordered assembly of the adhesive and electrochemical connections within newly formed intercalated disks in primary cultures of adult rat cardiomyocytes

Geisler, Sarah B; Green, Kathleen J; Isom, Lori L; Meshinchi, Sasha; Martens, Jeffrey R; Delmar, Mario; Russell, Mark W
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
PMCID:2868981
PMID: 20467587
ISSN: 1110-7251
CID: 113838

Cx43 CT domain influences infarct size and susceptibility to ventricular tachyarrhythmias in acute myocardial infarction

Maass, Karen; Chase, Sharon E; Lin, Xianming; Delmar, Mario
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
PMCID:2777952
PMID: 19620131
ISSN: 1755-3245
CID: 113845

Phosphorylation of connexin43 on serine 306 regulates electrical coupling

Procida, Kristina; Jorgensen, Lone; Schmitt, Nicole; Delmar, Mario; Taffet, Steven M; Holstein-Rathlou, Niels-Henrik; Nielsen, Morten Schak; Braunstein, Thomas Hartig
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
PMCID:2803062
PMID: 19879542
ISSN: 1556-3871
CID: 113842

Plakophilins: multifunctional scaffolds for adhesion and signaling

Bass-Zubek, Amanda E; Godsel, Lisa M; Delmar, Mario; Green, Kathleen J
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
PMCID:3091506
PMID: 19674883
ISSN: 1879-0410
CID: 113843

Loss of plakophilin-2 expression leads to decreased sodium current and slower conduction velocity in cultured cardiac myocytes

Sato, Priscila Y; Musa, Hassan; Coombs, Wanda; Guerrero-Serna, Guadalupe; Patino, Gustavo A; Taffet, Steven M; Isom, Lori L; Delmar, Mario
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
PMCID:2742576
PMID: 19661460
ISSN: 1524-4571
CID: 113844

Novel pharmacophores of connexin43 based on the "RXP" series of Cx43-binding peptides

Verma, Vandana; Larsen, Bjarne Due; Coombs, Wanda; Lin, Xianming; Spagnol, Gaelle; Sorgen, Paul L; Taffet, Steven M; Delmar, Mario
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
PMCID:2737470
PMID: 19556520
ISSN: 1524-4571
CID: 113846

Reentry in Cardioversion: 'We can see it from here...' [Editorial]

Berenfeld, Omer; Delmar, Mario
PMCID:2630526
PMID: 19121807
ISSN: 1556-3871
CID: 113847

Loss of the carboxyl-terminal domain of connexin43 increases susceptibility to ventricular tachyarrhythmias during acute myocardial infarction in mice [Meeting Abstract]

Maass K.; Chase S.E.; Delmar M.
Introduction: Connexin43 (Cx43) gap junction channels close during myocardial infarction (MI). To assess the importance of Cx43 regulation on arrhythmia susceptibility, mice in which the coding region of Cx43 was replaced with a mutation lacking most of the carboxyl-terminal domain (K258stop) were subjected to MI. This mutation has been shown to prevent chemical regulation of Cx43 channels by low intracellular pH in vitro. Due to reduced viability of homozygous K258stop mice, studies were carried out in animals harboring one Cx43 knockout allele and one K258stop or Cx43 allele, respectively (i.e., K258stop/KO; Cx43/KO). Methods: Langendorff-perfused hearts (n=12 per group) were subjected to 1 hour of ischemia and 4 hours of reperfusion by reversibly occluding the left anterior descending (LAD) coronary artery. Hearts were monitored for spontaneous ventricular tachyarrhythmias (VT) and for inducibility of VT by endocardial burst pacing near the apex of the left ventricle (3 x 18 S1 stimuli; 80, 60, 40 and 20ms cycle length; 2.5 times threshold) 15, 30, 45 and 60 minutes after the onset of LAD occlusion. Results: 45 minutes after the onset of LAD occlusion, VT could be induced by at least one pacing frequency in 81.8% of K258stop/KO hearts. The number increased to 100% at 60 minutes. For Cx43/KO hearts only 41.7% and 75% of the hearts developed VT at 45 min and 60 min, respectively. The average number of VT events elicited (regardless of burst pacing frequency) was significantly higher in the K258stop/KO hearts (2.64 +/- 0.56 v. 0.83 +/- 0.39 at 45min; p: 0.014; 3.18 +/- 0.55 v.1.67 +/- 0.47 at 60min; p= 0.047). VT episodes were also of longer duration in the K258stop/KO group. During reperfusion, K258stop/KO hearts showed a higher incidence of spontaneous VT (85.7% v. 42.9% of hearts) and increased numbers of episodes (7.14 +/- 2.27 v. 1.57 +/- 0.95; p=0.043). Conclusions: Loss of the regulatory domain of Cx43 leads to an increased susceptibility to arrhythmias following acute coronary occlusion. Whether similar results would be obtained when Cx43 channels remain open but structurally intact remains to be determined
EMBASE:70393494
ISSN: 1547-5271
CID: 131859

Characterization of the molecular phenotype of two arrhythmogenic right ventricular cardiomyopathy (ARVC)-related plakophilin-2 (PKP2) mutations

Joshi-Mukherjee, Rosy; Coombs, Wanda; Musa, Hassan; Oxford, Eva; Taffet, Steven; Delmar, Mario
BACKGROUND: Arrhythmogenic right ventricular cardiomyopathy (ARVC) has been linked to mutations in desmosomal proteins, including plakophilin-2 (PKP2). Little is known about the changes in cellular function and structure that follow expression of ARVC-relevant PKP2 mutations. OBJECTIVE: The purpose of this study was to investigate the function and distribution of an ARVC-relevant PKP2 mutant where arginine at position 79 was replaced by a stop codon (R79x). METHODS: Results were compared with those obtained with mutation 179fs (frameshift at position 179). Mutant constructs were introduced by adenoviral infection into neonatal rat ventricular myocytes in culture. RESULTS: Both mutant proteins failed to preferentially localize to sites of cell-cell apposition. Their expression did not disrupt localization of endogenous PKP2, connexin-43 (Cx43), or desmoplakin (DP). However, we observed reduced abundance of Cx43 after R79x expression. Early truncation of PKP2 at position 79 also prevented its physical interaction with both DP and Cx43. Finally, R79x expression correlated with loss of expression of HSP90, a protein relevant to cardiomyocyte apoptosis. CONCLUSION: These results provide the first observations of the cellular/molecular phenotype consequent to these PKP2 mutations and give insight into the possible cellular substrates that lead to ARVC
PMCID:2636742
PMID: 19084810
ISSN: 1556-3871
CID: 113848