Ordered Assembly of the Adhesive and Electrochemical Connections within Newly Formed Intercalated Disks in Primary Cultures of Adult Rat Cardiomyocytes

Division of Pediatric Cardiology, Department of Pediatrics and Communicable Diseases, University of Michigan, 1150 West Medical Center Dr., Ann Arbor, MI 48109, USA.
BioMed Research International (Impact Factor: 2.71). 05/2010; 2010:624719. DOI: 10.1155/2010/624719
Source: PubMed


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.

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    • "Thus, decreased Cx43 in these cells can influence retinal homeostasis through compromised crosstalk that ultimately contributes to the demise of the cells and breakdown of the blood–retinal barrier. Although Cx43 deficiency has been identified as a critical mediator in several diseases, such as arrhythmias [30,31], congestive heart failure [32], and oculodentodigital dysplasia [33], among others, our current findings provide the first evidence that downregulation of Cx43 expression in the retina can promote vascular lesions characteristic of DR. "
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    ABSTRACT: Purpose To determine whether downregulation of Connexin 43 (Cx43) expression promotes development of acellular capillaries (ACs), pericyte loss (PL), excess permeability, and retinal thickening in rat retinas. Methods Control rats, diabetic rats, and rats intravitreally injected with Cx43 siRNA or scrambled siRNA were used in this study to determine if acute downregulation of Cx43 expression contributes to retinal vascular cell death and excess permeability. Western blot (WB) analysis and Cx43 immunostaining were performed to assess Cx43 protein levels and distribution in the retinal vessels. Concurrently, retinal networks were subjected to terminal deoxynucleotidyl transferase-mediated uridine 5′-triphosphate-biotin nick end labeling (TUNEL) assay and counter-stained to assess the number of apoptotic cells, ACs, and PL. Assessment of fluorescein isothiocyanate-dextran (FITC-dex) extravasation from retinal capillaries and optical coherence tomography (OCT) were performed to determine retinal vascular permeability and retinal thickness, respectively. Results WB analysis indicated a significant decrease in the Cx43 protein level in the retinas of the diabetic rats and those intravitreally injected with Cx43 siRNA compared to the retinas of the control rats. Likewise, the retinal vascular cells of the diabetic rats and the Cx43 siRNA-treated rats showed a significant decrease in Cx43 immunostaining. Importantly, the number of apoptotic cells, ACs and PL, FITC-dex extravasation, and thickness increased in the retinas of the diabetic and Cx43 siRNA-treated rats compared to those of the control rats. Conclusions Results indicate that downregulation of Cx43 expression alone induces vascular cell death and promotes vascular permeability in the retina. These findings suggest that diabetes-induced downregulation of Cx43 participates in promoting retinal vascular lesions associated with diabetic retinopathy (DR).
    Full-text · Article · Jun 2014 · Molecular vision
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    • "Important work by Delmar and colleagues in recent years has shed light on the degree to which Na v 1.5 channels are integrated into the macromolecular complex at the ID. They have demonstrated that localization of mechanical junction proteins, mainly PKP2 (a desmosomal protein), precedes recruitment of Cx43 gap junctions and AnkG (a sub-membrane adapter protein involved in localizing Na v 1.5 in the membrane) to sites of cell-to-cell contact in neonatal rat ventricular myocytes (Geisler et al., 2010). Functionally, they have demonstrated that both regulation of the sodium current PKP2 and AnkG and linked mutations in both proteins to Brugada syndrome, an inherited arrhythmia syndrome resulting from loss of sodium current density (Cerrone et al., 2013; Hashemi et al., 2009). "
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    ABSTRACT: Cardiac conduction is the propagation of electrical excitation through the heart and is responsible for triggering individual myocytes to contract in synchrony. Canonically, this process has been thought to occur electrotonically, by means of direct flow of ions from cell to cell. The intercalated disk (ID), the site of contact between adjacent myocytes, has been viewed as a structure composed of mechanical junctions that stabilize the apposition of cell membranes and gap junctions which constitute low resistance pathways between cells. However, emerging evidence suggests a more active role for structures within the ID in mediating intercellular electrical communication by means of non-canonical ephaptic mechanisms. This review will discuss the role of the ID in the context of the canonical, electrotonic view of conduction and highlight new, emerging possibilities of its playing a more active role in ephaptic coupling between cardiac myocytes.
    Full-text · Article · Apr 2014 · Cell Communication & Adhesion
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    • "Dr. Mario Delmar and colleagues have provided evidence suggesting that Cx43 and Na v 1.5 participate in a macromolecular complex at the intercalated disk which includes the desmosomal protein plakophilin-2 (PKP2) as well as ankyrin-G, a submembrane adapter protein involved in localizing cardiac sodium channels (Na v 1.5) in the membrane. In primary cultures of neonatal rat ventricular myocytes, they found that Cx43 gap junctions and ankyrin-G (AnkG) are recruited to sites of cell-to-cell contact following the localization of mechanical adhesion proteins [22]. Subsequently, they demonstrated regula-tion of the sodium current by both PKP2 and AnkG [10] [53] [54] and mutations in both proteins have been associated with Brugada syndrome, an inherited arrhythmia syndrome characterized by decreased sodium current density [9] [25]. "
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    ABSTRACT: Cardiac conduction is the process by which electrical excitation is communicated from cell to cell within the heart, triggering synchronous contraction of the myocardium. The role of conduction defects in precipitating life-threatening arrhythmias in various disease states has spurred scientific interest in the phenomenon. While the understanding of conduction has evolved greatly over the last century, the process has largely been thought to occur via movement of charge between cells via gap junctions. However, it has long been hypothesized that electrical coupling between cardiac myocytes could also occur ephaptically, without direct transfer of ions between cells. This review will focus on recent insights into cardiac myocyte intercalated disk ultrastructure and their implications for conduction research, particularly the ephaptic coupling hypothesis.
    Full-text · Article · Jan 2014 · FEBS letters
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