Functional Interactions Between Mechanical Junction Proteins, Connexin43 and the Voltage-Gated Sodium Channel Complex in the Heart.

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 VGSCs have been demonstrated. Separate studies show, under various conditions, decreased abundance of gap junctions at the ID, and redistribution of connexin43 (Cx43) to plaques oriented parallel to fiber direction (gap junction “lateralization”). The mechanisms of Cx43 lateralization, and the fate of desmosomal and VGSC molecules in the setting of Cx43 remodeling, remain understudied. To study remodeling we employed the sheep pulmonary hypertension model. We found that Cx43 lateralization is a part of a complex remodeling that includes mechanical and gap junctions, but may exclude components of VGSC. Cx43/desmosomal remodeling was accompanied by lateralization of two microtubule-associated proteins relevant for Cx43 trafficking: EB1 and the kinesin protein Kif5b. Thus, 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. We then further focused on the importance of the expression of Cx43 and desmosomal protein, PKP2, on the function of VGSCs in the heart. Studies of PKP2 and Cx43 deficiency (PKP2+/- and Cx43-/-) mice models demonstrated that these proteins affect the distribution and function of VGSC. In an attempt to reveal the mechanism for Cx43-mediated regulation of VGSCs we analyzed the ability of Cx43 to stabilize tubulin and for the plus-end of the microtubules to reach its anchoring point at sites rich in the adherens junction protein N-cadherin. Using Cx43 null mice and the immortal cell line of cardiac origin (HL-1) we were able to show that, in the heart, connexin43 regulates the function of VGSCs via its tubulin binding domain. Overall, the data presented in this thesis further illustrate the intimate functional interactions of the proteins residing at the ID and reinforces the idea of these proteins working together as parts of macromolecular complexes.PHDMolecular and Integrative PhysiologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/96169/1/halina_1.pd

A Connexin40 Mutation Associated With a Malignant Variant of Progressive Familial Heart Block Type I

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 with PFHBI. In addition to 12 sodium channel mutations, we found a germ line GJA5 (connexin40 [Cx40]) mutation (Q58L) in 1 family. Heterologous expression of Cx40-Q58L in connexin-deficient neuroblastoma cells resulted in marked reduction of junctional conductance (Cx40-wild type [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 cotransfection of Cx40-WT and Cx40-Q58L resulted in homogenous distribution of proteins in the plasma membrane rather than in membrane plaques in ̃ 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 germ line 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

Remodeling of mechanical junctions and of microtubule-associated proteins accompany cardiac connexin43 lateralization.

<p>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").</p> <p>OBJECTIVE: To determine the mechanisms of Cx43 lateralization, and the fate of desmosomal and sodium channel molecules in the setting of Cx43 remodeling.</p> <p>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.</p> <p>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.</p> <p>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.</p

Remodeling of the cardiac sodium channel, connexin 43, and plakoglobin at the intercalated disk in patients with arrhythmogenic cardiomyopathy.

Background Arrhythmogenic cardiomyopathy (AC) is closely associated with desmosomal mutations in a majority of patients. Arrhythmogenesis in patients with AC is likely related to remodeling of cardiac gap junctions and increased levels of fibrosis. Recently, using experimental models, we also identified sodium channel dysfunction secondary to desmosomal dysfunction. Objective To assess the immunoreactive signal levels of the sodium channel protein NaV1.5, as well as connexin43 (Cx43) and plakoglobin (PKG), in myocardial specimens obtained from patients with AC. Methods Left and right ventricular free wall postmortem material was obtained from 5 patients with AC and 5 controls matched for age and sex. Right ventricular septal biopsies were taken from another 15 patients with AC. All patients fulfilled the 2010 revised Task Force Criteria for the diagnosis of AC. Immunohistochemical analyses were performed using antibodies against Cx43, PKG, NaV1.5, plakophilin-2, and N-cadherin. Results N-cadherin and desmoplakin immunoreactive signals and distribution were normal in patients with AC compared to controls. Plakophilin-2 signals were unaffected unless a plakophilin-2 mutation predicting haploinsufficiency was present. Distribution was unchanged compared to that in controls. Immunoreactive signal levels of PKG, Cx43, and NaV1.5 were disturbed in 74%, 70%, and 65% of the patients, respectively. Conclusions A reduced immunoreactive signal of PKG, Cx43, and NaV1.5 at the intercalated disks can be observed in a large majority of the patients. Decreased levels of Nav1.5 might contribute to arrhythmia vulnerability and, in the future, potentially could serve as a new clinically relevant tool for risk assessment strategies