Connexin43 Remodeling Caused by Inhibition of Plakophilin-2 Expression in Cardiac Cells (original) (raw)
Related papers
Journal of Cellular and Molecular Medicine, 2009
Arrhythmogenic right ventricular cardiomyopathy (ARVC) is a hereditary cardiomyopathy characterized by fibro-fatty replacement of myocardial tissue, ventricular tachycardia, syncope, and cardiac arrest . ARVC affects between 0.02% and 0.1% of the population, and is a leading cause of sudden cardiac death in the third and fourth decade of life . Familial ARVC presentations have been mapped to 9 chromosomal loci, and identified gene mutations are predominantly those of cell-to-cell adhesion proteins, specifically, proteins comprising the desmosome .
Restricted distribution of connexin40, a gap junctional protein, in mammalian heart
Circulation Research, 1994
Connexin40 (Cx40) is a member of the connexin family of gap junction proteins. Its mRNA, abundant in lung, is also present in mammalian heart, although in lower amount. Rabbit antipeptide antibodies directed to the COOH terminus (residues 335 to 356) of rat Cx40 were characterized to investigate the distribution of Cx40 in rat and guinea pig cardiac tissues. The affinity-purified antibodies detect specifically a major protein (Mr, 40000) in immunoblots of total extracts from rat lung and rat and guinea pig heart. In sections of guinea pig atrial tissue treated for immunofluorescence, a strong labeling associated with myocytes was seen with a distribution consistent with that of intercalated disks. The results of immunoelectron microscopy carried out with guinea pig atrial tissue showed that epitopes recognized by these antibodies were exclusively associated with gap junctions. These results, added to those of control experiments, demonstrate that antibodies 335-356 are specific for Cx40. Doublelabeling experiments carried out with lung sections using anti-factor VIII and anti-Cx40 antibodies suggest that Cx40 is expressed in blood vessel endothelial cells. In guinea pig and rat heart sections, investigated using both immunofluorescence and immunoperoxidase techniques, a signal was also found to be associated with vascular walls. In guinea pig heart, only atrial myocytes are Cx4O-positive. No labeling was detected in ventricular myocytes, including those of the His bundle and the bundle branches, which otherwise do express connexin43 (Cx43). In rat heart Cx4O -expressing myocytes are localized in the conduction system, ie, the His bundle, the bundle branches, and the Purkinje fibers. Cx43 is not detected either in the His bundle or in the proximal parts of the bundle branches, and consequently, Cx4O is the first connexin demonstrated in this region of the rat conduction system. Cx40 was not detected in the working ventricular myocytes. Doublelabeling experiments carried out with hen anti-Cx43 antibodies and rabbit anti-Cx4O antibodies demonstrated that, in tissues expressing both Cx43 and Cx4O, these two connexins were localized in the same immunoreactive sites. A few sites, however, appear to contain only one or the other of these two connexins. (Circ Res. 1994;74:839-851.)
Gap junction remodeling and altered connexin43 expression in the failing human heart
Cardiac Cell Biology, 2003
Gap junctions (GJ) are important determinants of cardiac conduction and the evidence has recently emerged that altered distribution of these junctions and changes in the expression of their constituent connexins (Cx) may lead to abnormal coupling between cardiomyocytes and likely contribute to arrhythmogenesis. However, it is largely unknown whether changes in the expression and distribution of the major cardiac GJ protein, Cx43, is a general feature of diverse chronic myocardial diseases or is confined to some particular pathophysiological settings. In the present study, we therefore set out to investigate qualitatively and quantitatively the distribution and expression of Cx43 in normal human myocardium and in patients with dilated (DCM), ischemic (ICM), and inflammatory cardiomyopathies (MYO). Left ventricular tissue samples were obtained at the time of cardiac transplantation and investigated with immunoconfocal and electron microscopy. As compared with the control group, Cx43 labeling in myocytes bordering regions of healed myocardial infarction (ICM), small areas of replacement fibrosis (DCM) and myocardial inflammation (MYO) was found to be highly disrupted instead of being confined to the intercalated discs. In all groups, myocardium distant from these regions showed an apparently normal Cx43 distribution at the intercalated discs. Quantitative immunoconfocal analysis of Cx43 in the latter myocytes revealed that the Cx43 area per myocyte area or per myocyte volume is significantly decreased by respectively 30 and 55% in DCM, 23 and 48% in ICM, and by 21 and 40% in MYO as compared with normal human myocardium. In conclusion, focal disorganization of GJ distribution and down-regulation of Cx43 are typical features of myocardial remodeling that may play an important role in the development of an arrhythmogenic substrate in human cardiomyopathies. (Mol Cell Biochem 242: 135-144, 2003)
“Orphan” Connexin43 in Plakophilin-2 Deficient Hearts Revealed by Volume Electron Microscopy
Frontiers in Cell and Developmental Biology
Previous studies revealed an abundance of functional Connexin43 (Cx43) hemichannels consequent to loss of plakophilin-2 (PKP2) expression in adult murine hearts. The increased Cx43-mediated membrane permeability is likely responsible for excess entry of calcium into the cells, leading to an arrhythmogenic/cardiomyopathic phenotype. The latter has translational implications to the molecular mechanisms of inheritable arrhythmogenic right ventricular cardiomyopathy (ARVC). Despite functional evidence, visualization of these “orphan” (i.e., non-paired in a gap junction configuration) Cx43 hemichannels remains lacking. Immuno-electron microscopy (IEM) remains an extremely powerful tool to localize, with nanometric resolution, a protein within its native structural landscape. Yet, challenges for IEM are to preserve the antigenicity of the molecular target and to provide access for antibodies to reach their target, while maintaining the cellular/tissue ultrastructure. Fixation is important...
Interactions Between Ankyrin-G, Plakophilin-2, and Connexin43 at the Cardiac Intercalated Disc
Circulation Research, 2011
Rationale-The early description of the intercalated disc defined three structures, all of them involved in cell-cell communication: desmosomes, gap junctions and adherens junctions. Current evidence demonstrates that molecules not involved in providing a physical continuum between cells, also populate the intercalated disc. Key among them is the voltage-gated sodium channel (VGSC) complex. An important component of this complex is the cytoskeletal adaptor protein ankyrin-G (AnkG).
F1000 - Post-publication peer review of the biomedical literature, 2000
Rationale-The early description of the intercalated disc defined three structures, all of them involved in cell-cell communication: desmosomes, gap junctions and adherens junctions. Current evidence demonstrates that molecules not involved in providing a physical continuum between cells, also populate the intercalated disc. Key among them is the voltage-gated sodium channel (VGSC) complex. An important component of this complex is the cytoskeletal adaptor protein ankyrin-G (AnkG). Objective-To test the hypothesis that AnkG partners with desmosome and gap junction molecules, and exerts a functional effect on intercellular communication in the heart. Methods and Results-We utilized a combination of microscopy, immunochemistry, patch clamp and optical mapping to assess the interactions between AnkG, plakophilin-2 (PKP2) and Connexin43 (Cx43). Co-immunoprecipitation studies from rat heart lysate demonstrated associations between the three molecules. Using siRNA technology we demonstrated that loss of AnkG expression caused significant changes in subcellular distribution and/or abundance of PKP2 and Cx43, as well as a decrease in intercellular adhesion strength and electrical coupling. Regulation of AnkG and of Na v 1.5 by PKP2 was also demonstrated. Finally, optical mapping experiments in AnkG-silenced cells demonstrated a shift in the minimal frequency at which ratedependence activation block was observed. Conclusions-These experiments support the hypothesis that AnkG is a key functional component of the intercalated disc, at the intersection of three complexes often considered independent: the VGSC, gap junctions and the cardiac desmosome. Possible implications to the pathophysiology of inherited arrhythmias (such as arrhythmogenic right ventricular cardiomyopathy; ARVC) are discussed.
The connexin43 carboxyl terminus and cardiac gap junction organization
Biochimica et Biophysica Acta (BBA) - Biomembranes, 2012
The precise spatial order of gap junctions at intercalated disks in adult ventricular myocardium is thought vital for maintaining cardiac synchrony. Breakdown or remodeling of this order is a hallmark of arrhythmic disease of the heart. The principal component of gap junction channels between ventricular cardiomyocytes is connexin43 (Cx43). Protein-protein interactions and modifications of the carboxyl-terminus of Cx43 are key determinants of gap junction function, size, distribution and organization during normal development and in disease processes. Here, we review data on the role of proteins interacting with the Cx43 carboxyl-terminus in the regulation of cardiac gap junction organization, with particular emphasis on Zonula Occludens-1. The rapid progress in this area suggests that in coming years we are likely to develop a fuller understanding of the molecular mechanisms causing pathologic remodeling of gap junctions. With these advances come the promise of novel approach to the treatment of arrhythmia and the prevention of sudden cardiac death. This article is part of a Special Issue entitled: The Communicating junctions, composition, structure and characteristics.
Connexin defects underlie arrhythmogenic right ventricular cardiomyopathy in a novel mouse model
Human Molecular Genetics, 2014
Arrhythmogenic right ventricular cardiomyopathy (ARVC) termed a 'disease of the desmosome' is an inherited cardiomyopathy that recently underwent reclassification owing to the identification of left-dominant and biventricular disease forms. Homozygous loss-of-function mutations in the desmosomal component, desmoplakin, are found in patients exhibiting a biventricular form of ARVC; however, no models recapitulate the postnatal hallmarks of the disease as seen in these patients. To gain insights into the homozygous loss-of-function effects of desmoplakin in the heart, we generated cardiomyocyte-specific desmoplakin-deficient mice (DSP-cKO) using ventricular myosin light chain-2-Cre mice. Homozygous DSP-cKO mice are viable but display early ultrastructural defects in desmosomal integrity leading to a cardiomyopathy reminiscent of a biventricular form of ARVC, which includes cell death and fibro-fatty replacement within the ventricle leading to biventricular dysfunction, failure and premature death. DSP-cKO mice also exhibited ventricular arrhythmias that are exacerbated with exercise and catecholamine stimulation. Furthermore, DSP-cKO hearts exhibited right ventricular conduction defects associated with loss of connexin 40 expression and electrical wavefront propagation defects associated with loss of connexin 43 expression. Dose-dependent assessment of the effects of loss of desmoplakin in neonatal ventricular cardiomyocytes revealed primary loss of connexin 43 levels, phosphorylation and function independent of the molecular dissociation of the mechanical junction complex and fibro-fatty manifestation associated with ARVC, suggesting a role for desmoplakin as a primary stabilizer of connexin integrity. In summary, we provide evidence for a novel mouse model, which is reminiscent of the postnatal onset of ARVC while highlighting mechanisms underlying a biventricular form of human ARVC.
Cardiomyocyte-restricted deletion of connexin43 during mouse development
Journal of Molecular and Cellular Cardiology, 2006
Although the gap junction protein Connexin43 (Cx43) is expressed in various cell types during embryonic development, mice with a global inactivation of Cx43 survive until birth but die perinatally due to an obstruction of the right ventricular outflow tract of the heart. To analyze the functional role of Cx43 gap junction channels in cardiomyocytes of the developing and early postnatal heart, we used αMyHC-Cre mice to ablate Cx43 expression selectively in cardiomyocytes during development. We found efficient ablation of Cx43 in cardiomyocytes during embryonic development starting at embryonic day (ED) 9.5 in the ventricular wall. Analyses of cardiac Cx43 protein at birth indicated complete loss of Cx43 expression in cardiomyocytes. All mice homozygously deficient for Cx43 in cardiomyocytes died until postnatal day (PD) 16. Heterozygous inactivation of Cx43 in cardiomyocytes neither altered atrial nor ventricular activation, but homozygous ablation led to changes in ventricular activation, i.e. significant decrease of the QRS-amplitude and prolonged QRS-duration already at PD 4. Cardiac morphology was similar to controls until PD 1, but subtle morphological changes were found in a subgroup of mutant mice at later stages. Besides narrowing of the ventricular outlet region at PD 6, hypertrophy of ventricular myocardium was found at PD 12. Our data indicate that complete inactivation of cardiac Cx43 during development predisposes hearts to develop postnatal morphological alterations, which differ from outflow tract obstructions described for Cx43 null mice. In addition, complete loss of cardiac Cx43 protein during development correlates with slowed ventricular activation at PD 4, impairs viability during development, and leads to death of all mutant mice until PD 16.