The area composita of adhering junctions connecting heart muscle cells of vertebrates - IV: coalescence and amalgamation of desmosomal and adhaerens junction components - late processes in mammalian heart development

In the adult mammalian heart, the cardiomyocytes and thus their terminally anchored myofibrillar bundles are connected by large arrays of closely spaced or even fused adhering junctions (AJs), termed "intercalated disks" (IDs). In recent years, the ID complex has attracted special attention as it has become clear that several human hereditary cardiomyopathies are caused by mutations of genes encoding ID marker proteins, in particular some that are also known as constituents of epithelial desmosomes. Previously, we have shown that in the mature myocardial ID the compositional differences between desmosome-like and adhaerens junctions are, by and large, lost and a composite hybrid structure, the area composita, is formed. We now report results from immunofluorescence and (immuno-)electron microscopic studies of heart formation during mouse embryogenesis and postnatal growth and show that the formation of the IDs with extended area composita structures is a late, primarily postnatal process. While up to birth small distinct desmosomes and AJs are resolved as predominant ID structures, areae compositae of increasing sizes and merged marker protein patterns occupy most of the IDs in the mature heart. Differences in the patterns of ID formation and amalgamation of the two ensembles of junction proteins in time and space are also demonstrated. Together with corresponding observations during rat and human heart development our results indicate that ID topogenesis and area composita formation are also late developmental processes in other mammals. We discuss the importance of the ID and the areae compositae in cardiac functions and, consequently, in cardiomyopathies and possible myocardial regeneration processes.     

The area composita of adhering junctions connecting heart muscle cells of vertebrates. I. Molecular definition in intercalated disks of cardiomyocytes by immunoelectron microscopy of desmosomal proteins

Among sarcomeric muscles the cardiac muscle cells are unique by, inter alia, a systemic and extended cell-cell contact structure, the intercalated disk (ID), comprising frequent and closely spaced arrays of plaque-coated cell-cell adhering junctions (AJs). As some of these junctions may look somewhat like desmosomes and others like fasciae adhaerentes, the dogma has emerged in the literature that IDs contain - like epithelial cells - both kinds of AJs formed by - for the most - mutually exclusive molecular ensembles. This, however, is not the case. In comprehensive immunoelectron microscopic studies of mammalian (human, bovine, rat, mouse) and non-mammalian (chicken, amphibia, fishes) heart muscle tissues, we have localized major constituents of the desmosomal plaques of polar epithelia, desmoplakin, plakophilin-2 and plakoglobin, as well as the desmosomal cadherins, desmoglein Dsg2 and desmocollin Dsc2, in both kinds of ID AJs, independent of the specific morphological appearance. The desmosomal molecules are not restricted to the desmosome-like-looking junctions but can also be detected in junctions appearing similar to the zonula or fascia adhaerens structures. These AJs of cardiac ID are therefore subsumed under the collective term area composita. We discuss our results with respect to the importance of ID junction molecules for the formation, maintenance and function of the heart, particularly in relation to recent findings that deletions of - or mutations in - genes encoding such proteins can cause severe, sometimes lethal damages.     

Assembly of the Cardiac Intercalated Disk during Pre- and Postnatal Development of the Human Heart

Background

In cardiac muscle, the intercalated disk (ID) at the longitudinal cell-edges of cardiomyocytes provides as a macromolecular infrastructure that integrates mechanical and electrical coupling within the heart. Pathophysiological disturbance in composition of this complex is well known to trigger cardiac arrhythmias and pump failure. The mechanisms underlying assembly of this important cellular domain in human heart is currently unknown.

Methods

We collected 18 specimens from individuals that died from non-cardiovascular causes. Age of the specimens ranged from a gestational age of 15 weeks through 11 years postnatal. Immunohistochemical labeling was performed against proteins comprising desmosomes, adherens junctions, the cardiac sodium channel and gap junctions to visualize spatiotemporal alterations in subcellular location of the proteins.

Results

Changes in spatiotemporal localization of the adherens junction proteins (N-cadherin and ZO-1) and desmosomal proteins (plakoglobin, desmoplakin and plakophilin-2) were identical in all subsequent ages studied. After an initial period of diffuse and lateral labelling, all proteins were fully localized in the ID at approximately 1 year after birth. Na_v1.5 that composes the cardiac sodium channel and the gap junction protein Cx43 follow a similar pattern but their arrival in the ID is detected at (much) later stages (two years for Na_v1.5 and seven years for Cx43, respectively).

Conclusion

Our data on developmental maturation of the ID in human heart indicate that generation of the mechanical junctions at the ID precedes that of the electrical junctions with a significant difference in time. In addition arrival of the electrical junctions (Nav1.5 and Cx43) is not uniform since sodium channels localize much earlier than gap junction channels.     

The Spatiotemporal Development of Intercalated Disk in Three-Dimensional Engineered Heart Tissues Based on Collagen/Matrigel Matrix

Intercalated disk (ID), which electromechanically couples cardiomyocytes into a functional syncitium, is closely related to normal morphology and function of engineered heart tissues (EHTs), but the development mode of ID in the three-dimensional (3D) EHTs is still unclear. In this study, we focused on the spatiotemporal development of the ID in the EHTs constructed by mixing neonatal rat cardiomyocytes with collagen/Matrigel, and investigated the effect of 3D microenvironment provided by collagen/Matrigel matrix on the formation of ID. By histological and immmunofluorescent staining, the spatiotemporal distribution of ID-related junctions was detected. Furthermore, the ultra-structures of the ID in different developmental stages were observed under transmission electron microscope. In addition, the expression of the related proteins was quantitatively analyzed. The results indicate that accompanying the re-organization of cardiomyocytes in collagen/Matrigel matrix, the proteins of adherens junctions, desmosomes and gap junctions redistributed from diffused distribution to intercellular regions to form an integrated ID. The adherens junction and desmosome which are related with mechanical connection appeared earlier than gap junction which is essential for electrochemical coupling. These findings suggest that the 3D microenvironment based on collagen/Matrigel matrix could support the ordered assembly of the ID in EHTs and have implications for comprehending the ordered and coordinated development of ID during the functional organization of EHTs.     

Mutations with pathogenic potential in proteins located in or at the composite junctions of the intercalated disk connecting mammalian cardiomyocytes: a reference thesaurus for arrhythmogenic cardiomyopathies and for Naxos and Carvajal diseases

In the past decade, an avalanche of findings and reports has correlated arrhythmogenic ventricular cardiomyopathies (ARVC) and Naxos and Carvajal diseases with certain mutations in protein constituents of the special junctions connecting the polar regions (intercalated disks) of mature mammalian cardiomyocytes. These molecules, apparently together with some specific cytoskeletal proteins, are components of (or interact with) composite junctions. Composite junctions contain the amalgamated fusion products of the molecules that, in other cell types and tissues, occur in distinct separate junctions, i.e. desmosomes and adherens junctions. As the pertinent literature is still in an expanding phase and is obviously becoming important for various groups of researchers in basic cell and molecular biology, developmental biology, histology, physiology, cardiology, pathology and genetics, the relevant references so far recognized have been collected and are presented here in the following order: desmocollin-2 (Dsc2, DSC2), desmoglein-2 (Dsg2, DSG2), desmoplakin (DP, DSP), plakoglobin (PG, JUP), plakophilin-2 (Pkp2, PKP2) and some non-desmosomal proteins such as transmembrane protein 43 (TMEM43), ryanodine receptor 2 (RYR2), desmin, lamins A and C, striatin, titin and transforming growth factor-β3 (TGFβ3), followed by a collection of animal models and of reviews, commentaries, collections and comparative studies.     

The area composita of adhering junctions connecting heart muscle cells of vertebrates - III: assembly and disintegration of intercalated disks in rat cardiomyocytes growing in culture

For cell and molecular biological studies of heart formation and function cell cultures of embryonal, neonatal or adult hearts of various vertebrates, notably rat and chicken, have been widely used. As the myocardium-specific cell-cell junctions, the intercalated disks (ID), have recently been found to be particularly sensitive to losses of - or mutations in - certain cytoskeletal proteins, resulting in cardiac damages, we have examined the ID organization in primary cultures of cardiomyocytes obtained from neonatal rats. Using immunofluorescence and immunoelectron microscopy, we have studied the major ID components for up to 2 weeks in culture, paying special attention to spontaneously beating, individual cardiomyocytes and myocardial cell colonies. While our results demonstrate the formation of some ID-like cardiomyocyte-connecting junction arrays, they also reveal a variety of structural disorders such as rather extended, junction-free ID regions, sac-like invaginations and endocytotic blebs as well as accumulations of intracytoplasmic structures suggestive of endocytosed forms of junction-derived vesicles or of junction fragments resembling fascia adhaerens elements. Moreover, we have noticed a novel type of small, obviously plaque-free cytoplasmic vesicles containing one or both of the desmosomal cadherins, desmocollin Dsc2 and desmoglein Dsg2. We conclude that cardiomyocyte cultures are useful model systems for studies of certain aspects of myocardiac differentiation and functions but, on the other hand, show progressive disintegration and deterioration. The potential value of molecular markers and reagents in studies of myocardial pathology as well as in the monitoring of myocardial differentiation of so-called stem cells is discussed.     

Stratifin (14-3-3 σ) Limits Plakophilin-3 Exchange with the Desmosomal Plaque

Desmosomes are prominent cell-cell adhesive junctions in stratified squamous epithelia and disruption of desmosomal adhesion has been shown to have dramatic effects on the function and integrity of these tissues. During normal physiologic processes, such as tissue development and wound healing, intercellular adhesion must be modified locally to allow coordinated cell movements. The mechanisms that control junction integrity and adhesive strength under these conditions are poorly understood. We utilized a proteomics approach to identify plakophilin-3 associated proteins and identified the 14-3-3 family member stratifin. Stratifin interacts specifically with plakophilin-3 and not with other plakophilin isoforms and mutation analysis demonstrated the binding site includes serine 285 in the amino terminal head domain of plakophilin-3. Stratifin interacts with a cytoplasmic pool of plakophilin-3 and is not associated with the desmosome in cultured cells. FRAP analysis revealed that decreased stratifin expression leads to an increase in the exchange rate of cytoplasmic plakophilin-3/GFP with the pool of plakophilin-3/GFP in the desmosome resulting in decreased desmosomal adhesion and increased cell migration. We propose a model by which stratifin plays a role in regulating plakophilin-3 incorporation into the desmosomal plaque by forming a plakophilin-3 stratifin complex in the cytosol and thereby affecting desmosome dynamics in squamous epithelial cells.     

Plakophilin-2: a cell-cell adhesion plaque molecule of selective and fundamental importance in cardiac functions and tumor cell growth

Within the characteristic ensemble of desmosomal plaque proteins, the armadillo protein plakophilin-2 (Pkp2) is known as a particularly important regulatory component in the cytoplasmic plaques of various other cell-cell junctions, such as the composite junctions (areae compositae) of the myocardiac intercalated disks and in the variously-sized and -shaped complex junctions of permanent cell culture lines derived therefrom. In addition, Pkp2 has been detected in certain protein complexes in the nucleoplasm of diverse kinds of cells. Using a novel set of highly sensitive and specific antibodies, both kinds of Pkp2, the junctional plaque-bound and the nuclear ones, can also be localized to the cytoplasmic plaques of diverse non-desmosomal cell-cell junction structures. These are not only the puncta adhaerentia and the fasciae adhaerentes connecting various types of highly proliferative non-epithelial cells growing in culture but also some very proliferative states of cardiac interstitial cells and cardiac myxomata, including tumors growing in situ as well as fetal stages of heart development and cultures of valvular interstitial cells. Possible functions and assembly mechanisms of such Pkp2-positive cell-cell junctions as well as medical consequences are discussed.     

The area composita of adhering junctions connecting heart muscle cells of vertebrates. VI. Different precursor structures in non-mammalian species

Recent studies on the formation and molecular organization of the mammalian heart have emphasized the architectural and functional importance of the adhering junctions (AJs), which are densely clustered in the bipolar end regions (intercalated disks, IDs) connecting the elongated cardiomyocytes of the adult heart. Moreover, we learned from genetic studies of mutated AJ proteins that desmosomal proteins, which for the most part are integral components of ID-specific composite AJs (areae compositae, AC), are essential in heart development and function. Developmental studies have shown that the bipolar concentration of cardiomyocyte AJs in IDs is a rather late process and only completed postnatally. Here we report that in the adult hearts of diverse lower vertebrates (fishes, amphibia, birds) most AJs remain separate and distinct in molecular character, representing either fasciae adhaerentes, maculae adhaerentes (desmosomes) or--less frequently--some form of AC. In the mature hearts of the amphibian and fish species examined a large proportion of the AJs connecting cardiomyocytes is not clustered in the IDs but remains located on the lateral surfaces where they appear either as puncta adhaerentia or as desmosomes. In many places, these puncta connect parallel cardiomyocytes in spectacular ladder-like regular arrays (scalae adhaerentes) correlated with--and connected by--electron-dense plaque-like material to sarcomeric Z-bands. In the avian hearts, on the other hand, most AJs are clustered in the IDs but only a small proportion of the desmosomes appears as AC, compared to the dominance of distinct fasciae adhaerentes. We conclude that the fusion and amalgamation of AJs and desmosomes to ACs is a late process both in ontogenesis and in evolution. The significance and possible functional implications of the specific junctional structures in vertebrate evolution and the class-specific requirements of architectural and molecular assembly adaptation during regeneration processes are discussed.     

The area composita of adhering junctions connecting heart muscle cells of vertebrates. II. Colocalizations of desmosomal and fascia adhaerens molecules in the intercalated disk

Using immunofluorescence histochemistry and immunoelectron microscopy on sections through myocardiac tissues of diverse mammalian (human, cow, rat, mouse) and fish species we show that both desmosomal and fascia adhaerens proteins identified by gel electrophoresis and immunoblot occur in the area composita, the by far major type of plaque-bearing junctions of the intercalated disks (IDs) connecting cardiomyocytes. Specifically, we demonstrate that desmoplakin and the other desmosomal proteins occur in these junctions, together with N-cadherin, cadherin-11, alpha- and beta-catenin as well as vinculin, afadin and proteins p120(ctn), ARVCF, p0071, and ZO-1, suggestive of colocalization. We conclude that the predominant type of adhering junction present in IDs is a junction sui generis, termed area composita, that is characterized by an unusually high molecular complexity and an intimate association of molecules of both ensembles, the desmosomal one and the fascia adhaerens category. We discuss possible myocardium-specific, complex-forming interactions between members of the two ensembles and the relevance of our findings for the formation and functioning of the heart and for the understanding of hereditary and other cardiomyopathies. We further propose to use this highly characteristic area composita ensemble of molecules as cardiomyocyte markers for the monitoring of cardiomyogenesis, cardiomyocyte regeneration and possible cardiomyocyte differentiation from mesenchymal stem cells.     

Decreased plakophilin-1 expression promotes increased motility in head and neck squamous cell carcinoma cells

Desmosomes are prominent cell-cell adhesive junctions found in a variety of epithelial tissues, including the oral epithelium. The transmembrane core of the desmosome is composed of the desmosomal cadherins that interact extracellularly to mediate cell-cell adhesion. The cytoplasmic domain of desmosomal cadherins interact with plaque proteins that in turn interact with the keratin intermediate filament cytoskeleton. Plakophilin 1 is a major desmosomal plaque component that functions to recruit intermediate filaments to sites of cell-cell contact via interactions with desmoplakin. Decreased assembly of desmosomes has been reported in several epithelial cancers. We examined plakophilin-1 expression in an esophageal squamous cell carcinoma tissue microarray and found that plakophilin-1 expression inversely correlates with tumor grade. In addition, we sought to investigate the effect of plakophilin-1 expression on desmosome assembly and cell motility in oral squamous cell carcinoma cell lines. Cell lines expressing altered levels of plakophilin-1 were generated and the ability of these cells to recruit desmoplakin to sites of cell-cell contact was examined. Our results show that decreased expression of plakophilin-1 results in decreased desmosome assembly and increased cell motility and invasion. These data lead us to propose that loss of plakophilin-1 expression during head and neck squamous cell carcinoma (HNSCC) progression may contribute to an invasive phenotype.     

The Arm-Repeat Protein NPRAP (Neurojungin) Is a Constituent of the Plaques of the Outer Limiting Zone in the Retina, Defining a Novel Type of Adhering Junction

In the retina, special plaque-bearing adhering junctions are aligned to form a planar system (the "outer limiting zone," OLZ) of heterotypic connections between the photoreceptor cells and the surrounding glial cells ("Müller cells"), together with homotypic junctions. In the plaques of these junctions, which contain N-cadherin-and possibly also related cadherins-we have identified, by immunolocalization techniques, a recently discovered neural tissue-specific protein, neurojungin, a member of the plakoglobin/armadillo protein family. In these plaques we have also detected other adherens plaque proteins, such as alpha- and beta-catenin, protein p120, and vinculin, as well as proteins known as constituents of tight junction plaques, such as symplekin and protein ZO-1, and the desmosomal plaque protein plakophilin 2. This unusual combination of proteins and the demonstrated absence of plakoglobin define the OLZ junctions as a new and distinct category of adhering junction, which probably has special architectural functions.     

A role for plakophilin-1 in the initiation of desmosome assembly

Plakophilins (pkp-1, -2, and -3) comprise a family of armadillo-repeat containing proteins that are found in the desmosomal plaque and in the nucleus. Plakophilin-1 is most highly expressed in the suprabasal layers of the epidermis and loss of plakophilin-1 expression results in skin fragility-ectodermal dysplasia syndrome, which is characterized by a reduction in the number and size of desmosomes in the epithelia of affected individuals. To investigate the role of plakophilin-1 during desmosome formation, we fused plakophilin-1 to the hormone-binding domain of the estrogen receptor to create a fusion protein (plakophilin-1/ER) that can be activated in cell culture by the addition of 4-hydroxytamoxifen. When plakophilin-1/ER was expressed in A431 cells it was incorporated into endogenous desmosomes and did not disrupt desmosome formation. A derivative of A431 cells (A431D) do not form desmosomes, even though they express all the components believed to be necessary for desmosome assembly. Expression and activation of plakophilin-1/ER in A431D cells resulted in punctate desmoplakin staining on the cell surface. Co-expression of a classical cadherin (N-cadherin) and plakophilin-1/ER in A431D cells resulted in punctate desmoplakin staining at cell-cell borders. These data suggest that plakophilin-1 can induce assembly of desmosomal components in A431D cells in the absence of a classical cadherin; however a classical cadherin (N-cadherin) is required to direct assembly of desmosomes between adjacent cells. The activatable plakophilin-1/ER system provides a unique culture system to study the assembly of the desmosomal plaque in culture.     

Decreased Plakophilin-1 Expression Promotes Increased Motility in Head and Neck Squamous Cell Carcinoma Cells

Desmosomes are prominent cell-cell adhesive junctions found in a variety of epithelial tissues, including the oral epithelium. The transmembrane core of the desmosome is composed of the desmosomal cadherins that interact extracellularly to mediate cell-cell adhesion. The cytoplasmic domain of desmosomal cadherins interact with plaque proteins that in turn interact with the keratin intermediate filament cytoskeleton. Plakophilin 1 is a major desmosomal plaque component that functions to recruit intermediate filaments to sites of cell-cell contact via interactions with desmoplakin. Decreased assembly of desmosomes has been reported in several epithelial cancers. We examined plakophilin-1 expression in an esophageal squamous cell carcinoma tissue microarray and found that plakophilin-1 expression inversely correlates with tumor grade. In addition, we sought to investigate the effect of plakophilin-1 expression on desmosome assembly and cell motility in oral squamous cell carcinoma cell lines. Cell lines expressing altered levels of plakophilin-1 were generated and the ability of these cells to recruit desmoplakin to sites of cell-cell contact was examined. Our results show that decreased expression of plakophilin-1 results in decreased desmosome assembly and increased cell motility and invasion. These data lead us to propose that loss of plakophilin-1 expression during head and neck squamous cell carcinoma (HNSCC) progression may contribute to an invasive phenotype.     

The connexin43 carboxyl terminus and cardiac gap junction organization

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.     

Carboxyl terminus of Plakophilin-1 recruits it to plasma membrane, whereas amino terminus recruits desmoplakin and promotes desmosome assembly

Plakophilins are armadillo repeat-containing proteins, initially identified as desmosomal plaque proteins that have subsequently been shown to also localize to the nucleus. Loss of plakophilin-1 is the underlying cause of ectodermal dysplasia/skin fragility syndrome, and skin from these patients exhibits desmosomes that are reduced in size and number. Thus, it has been suggested that plakophilin-1 plays an important role in desmosome stability and/or assembly. In this study, we used a cell culture system (A431DE cells) that expresses all of the proteins necessary to assemble a desmosome, except plakophilin-1. Using this cell line, we sought to determine the role of plakophilin-1 in de novo desmosome assembly. When exogenous plakophilin-1 was expressed in these cells, desmosomes were assembled, as assessed by electron microscopy and immunofluorescence localization of desmoplakin, into punctate structures. Deletion mutagenesis experiments revealed that amino acids 686-726 in the carboxyl terminus of plakophilin-1 are required for its localization to the plasma membrane. In addition, we showed that amino acids 1-34 in the amino terminus were necessary for subsequent recruitment of desmoplakin to the membrane and desmosome assembly.     

Protein LUMA is a cytoplasmic plaque constituent of various epithelial adherens junctions and composite junctions of myocardial intercalated disks: a unifying finding for cell biology and cardiology

In a series of recent reports, mutations in the gene encoding a protein called LUMA (or TMEM43), widely speculated to be a tetraspan transmembrane protein of the nuclear envelope, have been associated with a specific subtype of cardiomyopathy (arrhythmogenic cardiomyopathies) and cases of sudden death. However, using antibodies of high specificity in immunolocalization experiments, we have discovered that, in mammals, LUMA is a component of zonula adhaerens and punctum adhaerens plaques of diverse epithelia and epithelial cell cultures and is also located in (or in some species associated with) the plaques of composite junctions (CJs) in myocardiac intercalated disks (IDs). In CJs, LUMA often colocalizes with several other CJ marker proteins. In all these cells, LUMA has not been detected in the nuclear envelope. Surprisingly, under certain conditions, similar CJ localizations have also been seen with some antibodies commercially available for some time. The identification of LUMA as a plaque component of myocardiac CJs leads to reconsiderations of the molecular composition and architecture, the development, the functions, and the pathogenic states of CJs and IDs. These findings now also allow the general conclusion that LUMA has to be added to the list of mutations of cardiomyocyte junction proteins that may be involved in cardiomyopathies.     

Cell–cell junction remodeling in the heart: Possible role in cardiac conduction system function and arrhythmias?

Anchoring cell-cell junctions (desmosomes, fascia adherens) play crucial roles in maintaining mechanical integrity of cardiac muscle cells and tissue. Genetic mutations and/or loss of critical components in these macromolecular structures are increasingly being associated with arrhythmogenic cardiomyopathies; however, their specific roles have been primarily attributed to effects within the working (ventricular) cardiac muscle. Growing evidence also points to a key role for anchoring cell-cell junction components in cardiac muscle cells of the cardiac conduction system. This is not only evidenced by the molecular and ultra-structural presence of anchoring cell junctions in specific compartments/structures of the cardiac conduction system (sinoatrial node, atrioventricular node, His-Purkinje system), but also because conduction system-related arrhythmias can be found in humans and mouse models of cardiomyopathies harboring defects and/or mutations in key anchoring cell-cell junction proteins. These studies emphasize the clinical need to understand the molecular and cellular role(s) for anchoring cell-cell junctions in cardiac conduction system function and arrhythmias. This review will focus on (i) experimental findings that underline an important role for anchoring cell-cell junctions in the cardiac conduction system, (ii) insights regarding involvement of these structures in age-related cardiac remodeling of the conduction system, (iii) summarizing available genetic mouse models that can target cardiac conduction system structures and (iv) implications of these findings on future therapies for arrhythmogenic heart diseases.     

Dominant-negative connexin43-EGFP inhibits calcium-transient synchronization of primary neonatal rat cardiomyocytes.

Recent studies using mice with genetically engineered gap junction protein connexin (Cx) genes have provided evidence that reduced gap-junctional coupling in ventricular cardiomyocytes predisposes to ventricular arrhythmia. However, the pathological processes of arrhythmogenesis due to abnormalities in gap junctions are poorly understood. We have postulated a hypothesis that dysfunction of gap junctions at the single-cell level may affect synchronization of calcium transients among cardiomyocytes. To examine this hypothesis, we developed a novel system in which gap-junctional intercellular communication in primary neonatal rat cardiomyocytes was inhibited by a mutated (Delta130-137) Cx43 fused with enhanced green fluorescent protein (Cx43-EGFP), and calcium transients were imaged in real time while the mutated Cx43-EGFP-expressing cardiomyocytes were identified. The mutated Cx43-EGFP inhibited dye coupling not only in the liver epithelial cell line IAR 20 but also in primary neonatal rat cardiomyocytes in a dominant-negative manner, whereas wild-type Cx43-EGFP made functional gap junctions in otherwise communication-deficient HeLa cells. The mutated Cx43-EGFP induced desynchronization of calcium transients among cardiomyocytes with significantly higher frequency than wild-type Cx43-EGFP. These results suggest that dysfunction of gap-junctional intercellular communication at the single-cell level could hamper synchronous beating among cardiomyocytes as a result of desynchronization of calcium transients.