THE ULTRASTRUCTURE OF CARDIAC DESMOSOMES IN THE TOAD AND THEIR RELATIONSHIP TO THE INTERCALATED DISC

The fine structure of desmosomes and intercalated discs in the toad heart is discussed. A definite relationship between the dense components of these structures and the dense region of the Z band is demonstrated. The dense region of the Z band characteristically widens at its approach to the plasma membrane, and often terminates beneath it in a distinct discoidal plaque. Cardiac desmosomes appear to be structures which result from the intimate apposition of plaques of Z band material. These desmosomes retain the Z band function as sites of attachment for myofilaments. The suggestion is made that rotation of a desmosome through 90° and splitting of filaments from the adjacent sarcomere could result in the formation of a simple step-like intercalated disc. Intermediate stages in this process are illustrated. Complex discs present in the toad probably represent the alignment of groups of simple discs produced by contractile forces. Possible physiologic functions of the disc and desmosome are discussed. Other morphologic features of toad cardiac cells include a distinct amorphous outer coat to the sarcolemma, a prominent N band, and a granular sarcoplasm with poorly developed reticulum.     

Some observations on the intercalated disc of a rat cardiac muscle.

During the experimental investigations special attention was paid to the orthomorphology of the ultrastructure of cardiac muscle intercalated disc in a rat. The contact junction between the cellular membranes of adjacent cells and intercellular spaces are typical for the cardiac muscle intercalated disc. Attention was paid to the three zone system of the junctions, namely: 1. nexus (zonula occludens), 2. fascia adherens (zonula adherens) and 3. macula adherens (similar to the desmosome). Apart from this the cell membranes adjacent to the cells may form digital indentations. A single injection of adrenaline in a dose of 2.4 mg/kg causes sinuate widenings of the intercellular space, but only in the zone of the fascia adherens. The structure of the nexuses and maculae adherentes is unchanged during this time.     

Partial purification of an intercalated disc-containing cardiac plasma membrane fraction

The sarcolemma of cardiac muscle cells contains a specialised junctional region, the intercalated disc which includes three types of intercellular junction, the macula and fascia adherens and the nexus or gap junction. To facilitate the isolation of these junctions a procedure for the partial purification from mouse hearts of a subcellular fraction containing the intercalated disc region of the sarcolemma was developed. This involved investigating methods of tissue disruption that preserve the integrity of the intercalated disc and minimise myofibrillar entrapment of organelles. Examination of the distribution of marker enzymes showed that relative to the homogenate the intercalated disc fraction prepared by sucrose density centrifugation was only enriched 1.5- to 3-fold in 5'-nucleotidase and (Na+ + K+)-ATPase activities, whereas mitochondrial and sarcoplasmic reticulum marker enzymes were low. The properties of the intercalated disc-containing fraction were compared with the vesicular sarcolemmal fractions devoid of junctional complexes prepared by other methods.     

桥粒蛋白与离子转运相关通道

桥粒为细胞与细胞之间的一种连接结构,参与细胞间机械应力传导. 在心肌组织中,桥粒与粘着连接及缝隙连接共同构成闰盘,对于维护心肌闰盘结构和功能的完整性具有重要作用. 近年来,越来越多的研究表明,桥粒蛋白基因突变、表达的缺失或功能异常,可引起心肌细胞钠、钾离子通道、缝隙连接蛋白等心肌电活动相关结构的重塑,增加心肌电学异质性,进而促发心律失常. 本文将就桥粒蛋白与离子转运相关通道关系的最新研究进展进行综述.     

Isoforms of protein 4.1 are differentially distributed in heart muscle cells: relation of 4.1R and 4.1G to components of the Ca2+ homeostasis system

The 4.1 proteins are cytoskeletal adaptor proteins that are linked to the control of mechanical stability of certain membranes and to the cellular accumulation and cell surface display of diverse transmembrane proteins. One of the four mammalian 4.1 proteins, 4.1R (80 kDa/120 kDa isoforms), has recently been shown to be required for the normal operation of several ion transporters in the heart (Stagg MA et al. Circ Res, 2008; 103: 855-863). The other three (4.1G, 4.1N and 4.1B) are largely uncharacterised in the heart. Here, we use specific antibodies to characterise their expression, distribution and novel activities in the left ventricle. We detected 4.1R, 4.1G and 4.1N by immunofluorescence and immunoblotting, but not 4.1B. Only one splice variant of 4.1N and 4.1G was seen whereas there are several forms of 4.1R. 4.1N, like 4.1R, was present in intercalated discs, but unlike 4.1R, it was not localised at the lateral plasma membrane. Both 4.1R and 4.1N were in internal structures that, at the level of resolution of the light microscope, were close to the Z-disc (possibly T-tubules). 4.1G was also in intracellular structures, some of which were coincident with sarcoplasmic reticulum. 4.1G existed in an immunoprecipitable complex with spectrin and SERCA2. 80 kDa 4.1R was present in subcellular fractions enriched in intercalated discs, in a complex resistant to solubilization under non-denaturing conditions. At the intercalated disc 4.1R does not colocalise with the adherens junction protein, β-catenin, but does overlap with the other plasma membrane signalling proteins, the Na/K-ATPase and the Na/Ca exchanger NCX1. We conclude that isoforms of 4.1 proteins are differentially compartmentalised in the heart, and that they form specific complexes with proteins central to cardiomyocyte Ca(2+) metabolism.     

Cardiac-myocyte-specific excision of the vinculin gene disrupts cellular junctions, causing sudden death or dilated cardiomyopathy

Vinculin is a ubiquitously expressed multiliganded protein that links the actin cytoskeleton to the cell membrane. In myocytes, it is localized in protein complexes which anchor the contractile apparatus to the sarcolemma. Its function in the myocardium remains poorly understood. Therefore, we developed a mouse model with cardiac-myocyte-specific inactivation of the vinculin (Vcl) gene by using Cre-loxP technology. Sudden death was found in 49% of the knockout (cVclKO) mice younger than 3 months of age despite preservation of contractile function. Conscious telemetry documented ventricular tachycardia as the cause of sudden death, while defective myocardial conduction was detected by optical mapping. cVclKO mice that survived through the vulnerable period of sudden death developed dilated cardiomyopathy and died before 6 months of age. Prior to the onset of cardiac dysfunction, ultrastructural analysis of cVclKO heart tissue showed abnormal adherens junctions with dissolution of the intercalated disc structure, expression of the junctional proteins cadherin and beta1D integrin were reduced, and the gap junction protein connexin 43 was mislocalized to the lateral myocyte border. This is the first report of tissue-specific inactivation of the Vcl gene and shows that it is required for preservation of normal cell-cell and cell-matrix adhesive structures.     

The subcellular distribution of dystrophin in mouse skeletal, cardiac, and smooth muscle

We use a highly specific and sensitive antibody to further characterize the distribution of dystrophin in skeletal, cardiac, and smooth muscle. No evidence for localization other than at the cell surface is apparent in skeletal muscle and no 427-kD dystrophin labeling was detected in sciatic nerve. An elevated concentration of dystrophin appears at the myotendinous junction and the neuromuscular junction, labeling in the latter being more intense specifically in the troughs of the synaptic folds. In cardiac muscle the distribution of dystrophin is limited to the surface plasma membrane but is notably absent from the membrane that overlays adherens junctions of the intercalated disks. In smooth muscle, the plasma membrane labeling is considerably less abundant than in cardiac or skeletal muscle and is found in areas of membrane underlain by membranous vesicles. As in cardiac muscle, smooth muscle dystrophin seems to be excluded from membrane above densities that mark adherens junctions. Dystrophin appears as a doublet on Western blots of skeletal and cardiac muscle, and as a single band of lower abundance in smooth muscle that corresponds most closely in molecular weight to the upper band of the striated muscle doublet. The lower band of the doublet in striated muscle appears to lack a portion of the carboxyl terminus and may represent a dystrophin isoform. Isoform differences and the presence of dystrophin on different specialized membrane surfaces imply multiple functional roles for the dystrophin protein.     

Myosin filament sliding through the Z-disc relates striated muscle fibre structure to function

Striated muscle contraction requires intricate interactions of microstructures. The classic textbook assumption that myosin filaments are compressed at the meshed Z-disc during striated muscle fibre contraction conflicts with experimental evidence. For example, myosin filaments are too stiff to be compressed sufficiently by the muscular force, and, unlike compressed springs, the muscle fibres do not restore their resting length after contractions to short lengths. Further, the dependence of a fibre''s maximum contraction velocity on sarcomere length is unexplained to date. In this paper, we present a structurally consistent model of sarcomere contraction that reconciles these findings with the well-accepted sliding filament and crossbridge theories. The few required model parameters are taken from the literature or obtained from reasoning based on structural arguments. In our model, the transition from hexagonal to tetragonal actin filament arrangement near the Z-disc together with a thoughtful titin arrangement enables myosin filament sliding through the Z-disc. This sliding leads to swivelled crossbridges in the adjacent half-sarcomere that dampen contraction. With no fitting of parameters required, the model predicts straightforwardly the fibre''s entire force–length behaviour and the dependence of the maximum contraction velocity on sarcomere length. Our model enables a structurally and functionally consistent view of the contractile machinery of the striated fibre with possible implications for muscle diseases and evolution.     

Calsarcin-3, a novel skeletal muscle-specific member of the calsarcin family, interacts with multiple Z-disc proteins

The Z-disc is a highly specialized multiprotein complex of striated muscles that serves as the interface of the sarcomere and the cytoskeleton. In addition to its role in muscle contraction, its juxtaposition to the plasma membrane suggests additional functions of the Z-disc in sensing and transmitting external and internal signals. Recently, we described two novel striated muscle-specific proteins, calsarcin-1 and calsarcin-2, that bind alpha-actinin on the Z-disc and serve as intracellular binding proteins for calcineurin, a calcium/calmodulin-dependent phosphatase shown to be integral in cardiac hypertrophy as well as skeletal muscle differentiation and fiber-type specification. Here, we describe an additional member of the calsarcin family, calsarcin-3, which is expressed specifically in skeletal muscle and is enriched in fast-twitch muscle fibers. Like calsarcin-1 and calsarcin-2, calsarcin-3 interacts with calcineurin, and the Z-disc proteins alpha-actinin, gamma-filamin, and telethonin. In addition, we show that calsarcins interact with the PDZ-LIM domain protein ZASP/Cypher/Oracle, which also localizes to the Z-disc. Calsarcins represent a novel family of sarcomeric proteins that serve as focal points for the interactions of an array of proteins involved in Z-disc structure and signal transduction in striated muscle.     

Ultrastructural and cytochemical localization of calcium in a rat cardiac muscle in normal and experimental conditions.

The experiment carried out by us on the, stimulated by adrenaline, cardiac muscle allowed the activation of calcium localized mainly in the mitochondria, MA and FA of the intercalated discs and SR to be translocated in the direction of the sarcomere myofilaments and this especially to the thin actin filaments. The authors' experimental proves, that during the contraction--relaxation function of the cardiac muscle there exists a circulation rythm or a oscillatory functional flow of calcium ions between the mitochondria, intercalated discs and SR and the contractile fibrillae of the sarcomere.     

Orientation of adhering junctions between bovine pulmonary fibroblasts

The ultrastructure and orientation of microfilament-attaching junctions between pulmonary fibroblasts (also known as myofibroblasts or contractile interstitial cells) of bovine lung septa were investigated by transmission electron microscopy. Adhering junctions similar to the fascia adherens of the intercalated disc, but of a macular design, link microfilament bundles of adjacent fibroblasts. The bundles of microfilaments joined by junctions were usually aligned perpendicularly to the axis of the alveolar wall. Gap junctions were located in close proximity to the adhering junctions, presumably to co-ordinate the contraction of the cells. The data indicate that fibroblasts are able to form multicellular contractile units within healthy, mature lung parenchyma.     

Epithelial cell-cell junctions and plasma membrane domains

Epithelial cells form a barrier against the environment, but are also required for the regulated exchange of molecules between an organism and its surroundings. Epithelial cells are characterised by a remarkable polarization of their plasma membrane, evidenced by the appearance of structurally, compositionally, and functionally distinct surface domains. Here we consider the (in)dependence of epithelial cell polarisation and the function of smaller plasma membrane domains (e.g. adherens junctions, gap junctions, tight junctions, apical lipid rafts, caveolae, and clathrin-coated pits) in the development and maintenance of cell surface polarity. Recent evidence of cross-talk and/or overlap between the different cell-cell junction components and alternate functions of junction components, including gene expression regulation, are discussed in the context of cell surface polarity.     

Ultrastructural and biochemical localization of N-RAP at the interface between myofibrils and intercalated disks in the mouse heart.

N-RAP is a recently discovered muscle-specific protein found at cardiac intercalated disks. Double immunogold labeling of mouse cardiac muscle reveals that vinculin is located immediately adjacent to the fascia adherens region of the intercalated disk membrane, while N-RAP extends approximately 100 nm further toward the interior of the cell. We partially purified cardiac intercalated disks using low- and high-salt extractions followed by density gradient centrifugation. Immunoblots show that this preparation is highly enriched in desmin and junctional proteins, including N-RAP, talin, vinculin, beta1-integrin, N-cadherin, and connexin 43. Electron microscopy and immunolabeling demonstrate that N-RAP and vinculin are associated with the large fragments of intercalated disks that are present in this preparation, which also contains numerous membrane vesicles. Detergent treatment of the partially purified intercalated disks removed the membrane vesicles and extracted vinculin and beta1-integrin. Further separation on a sucrose gradient removed residual actin and myosin and yielded a fraction morphologically similar to fasciae adherentes that was highly enriched in N-RAP, N-cadherin, connexin 43, talin, desmin, and alpha-actinin. The finding that N-RAP copurifies with detergent-extracted intercalated disk fragments even though beta-integrin and vinculin have been completely removed suggests that N-RAP association with the adherens junction region is mediated by the cadherin system. Consistent with this hypothesis, we found that recombinant N-RAP fragments bind alpha-actinin in a gel overlay assay. In addition, immunofluorescence shows that N-RAP remains bound at the ends of isolated, detergent-treated cardiac myofibrils. These results demonstrate that N-RAP remains tightly bound to myofibrils and fasciae adherentes during biochemical purification and may be a key constituent in the mechanical link between these two structures.     

alpha-cardiac actin (ACTC) binds to the band 3 (AE1) cardiac isoform

The band 3 protein is the major integral protein present in the erythrocyte membrane. Two tissue-specific isoforms are also expressed in kidney alpha intercalated cells and in cardiomyocytes. It has been suggested that the cardiac isoform predominantly mediates the anion exchange in cardiomyocytes, but the role of the cytoplasmic domain of the band 3 (CDB3) protein in the cardiac tissue is unknown. In order to characterize novel associations of the CDB3 in the cardiac tissue, we performed the two-hybrid assay, using a bait comprising the region from leu 258 to leu 311 of the erythrocyte band 3, which must also be present in the cardiac isoform. The assay revealed two clones containing the C-terminal region of the alpha-cardiac actin. Immunoprecipitation of whole rat heart using an anti-actin antibody, immunoblotted with anti-human band 3, showed that actin binds to band 3 which was confirmed in the reverse assay. The confocal microscopy showed band 3 in the intercalated discs. Thus, besides the in vivo physical interaction in the Saccharomyces cerevisiae cell, we demonstrated using immunopreciptation that there is a physical association of band 3 with alpha-cardiac actin in cardiomyocyte, and we suggest that the binding occur "in situ," in the intercalated disc, a site of cell-cell contact and attachment of the sarcomere to the plasma membrane.     

Simulation of Electrical Interaction of Cardiac Cells

A model of the electrical activity of excitable membrane was used to simulate action potential propagation in cardiac cells. Using an implicit method for solving finite difference equations, propagation through the intercalated disc region between two abutting cells was studied. A model of interaction was constructed and parameters of the cellular junction determined. Estimates of the intercalated disc resistance were then made from these junction parameters using a field analysis of the junction. Values of approximately 4 Ω-cm² were found and correlate well with experimentally measured values.     

Cell confluence-dependent remodeling of endothelial membranes mediated by cholesterol

The plasma membranes of endothelial cells reaching confluence undergo profound structural and functional modifications, including the formation of adherens junctions, crucial for the regulation of vascular permeability and angiogenesis. Adherens junction formation is accompanied by the tyrosine dephosphorylation of adherens junctions proteins, which has been correlated with the strength and stability of adherens junctions. Here we show that cholesterol is a critical determinant of plasma membrane remodeling in cultures of growing cow pulmonary aortic endothelial cells. Membrane cholesterol increased dramatically at an early stage in the formation of confluent cow pulmonary aortic endothelial cell monolayers, prior to formation of intercellular junctions. This increase was accompanied by the redistribution of caveolin from a high density to a low density membrane compartment, previously shown to require cholesterol, and increased binding of the annexin II-p11 complex to membranes, consistent with other studies indicating cholesterol-dependent binding of annexin II to membranes. Furthermore, partial depletion of cholesterol from confluent cells with methyl-beta-cyclodextrin both induced tyrosine phosphorylation of multiple membrane proteins, including adherens junctions proteins, and disrupted adherens junctions. Both effects were dramatically reduced by prior complexing of methyl-beta-cyclodextrin with cholesterol. Our results reveal a novel physiological role for cholesterol regulating the formation of adherens junctions and other plasma membrane remodeling events as endothelial cells reach confluence.     

The elasticity of single kettin molecules using a two-bead laser-tweezers assay

Kettin is a high molecular mass protein of insect muscle associated with thin filaments and alpha-actinin in the Z-disc. It is thought to form a link between thin and thick filaments towards its C-terminus, contributing significantly to passive sarcomere stiffness. Here the elastic properties were characterised by mechanical stretches on an antibody-delimited region of the single molecule using two independent optical traps capable of exerting forces up to 150 pN. Step-like events were observed in the force-extension relationships consistent with the unfolding of Ig domains at moderate force and refolding of these domains at significantly higher forces than have been observed for related modular proteins.     

Heart rhythm genomic fabric in hypoxia

The molecular mechanisms by which chronic hypoxia, whether constant (CCH) or intermittent (CIH), alters the heart rhythm are still under debate. Expression level, control, maturational profile and intercoordination of 54 genes encoding heart rhythm determinants (HRDs) were analyzed in 36 mice subjected for 1, 2 or 4 weeks of their early life to normal atmospheric conditions or to CCH or CIH. Our analysis revealed a complex network of genes encoding various heart rate, inotropy and development controllers, receptors, ion channels and transporters, ankyrins, epigenetic modulators and intercalated disc components (adherens, cadherins, catenins, desmosomal, gap and tight junction proteins). The network is remodeled during maturation and substantially and differently altered by CIH and CCH. Gene Prominence Analysis that ranks the genes according to their expression stability and networking within functional gene webs, confirmed the HRD status of certain epigenetic modulators and components of the intercalated discs not yet associated with arrhythmia.     

Alterations at the intercalated disk associated with the absence of muscle LIM protein

In this study, we investigated cardiomyocyte cytoarchitecture in a mouse model for dilated cardiomyopathy (DCM), the muscle LIM protein (MLP) knockout mouse and substantiated several observations in a second DCM model, the tropomodulin-overexpressing transgenic (TOT) mouse. Freshly isolated cardiomyocytes from both strains are characterized by a more irregular shape compared with wild-type cells. Alterations are observed at the intercalated disks, the specialized areas of mechanical coupling between cardiomyocytes, whereas the subcellular organization of contractile proteins in the sarcomeres of MLP knockout mice appears unchanged. Distinct parts of the intercalated disks are affected differently. Components from the adherens junctions are upregulated, desmosomal proteins are unchanged, and gap junction proteins are downregulated. In addition, the expression of N-RAP, a LIM domain- containing protein located at the intercalated disks, is upregulated in MLP knockout as well as in TOT mice. Detailed analysis of intercalated disk composition during postnatal development reveals that an upregulation of N-RAP expression might serve as an early marker for the development of DCM. Altered expression levels of cytoskeletal proteins (either the lack of MLP or an increased expression of tropomodulin) apparently lead to impaired function of the myofibrillar apparatus and to physiological stress that ultimately results in DCM and is accompanied by an altered appearance and composition of the intercalated disks.     

Obstructive hypertrophic cardiomyopathy is associated with reduced expression of vinculin in the intercalated disc

Mutations in the cardiac-specific insert of vinculin, metavinculin, rarely cause hypertrophic cardiomyopathy (HCM) and dilated cardiomyopathy (DCM). Subsequently, a missense mutation in the ubiquitously expressed vinculin was discovered in a patient with obstructive HCM. Microscopic examination of both myectomy specimens from patients bearing genetic defects in metavinculin and vinculin showed a marked reduction of vinculin/metavinculin expression in the intercalated disc, but normal expression in the Z-disc. Given that distinct functional domains were altered by the metavinculin and vinculin mutations, we hypothesized that the intercalated disc-specific reduction of vinculin may stem from left ventricular tract obstruction in general rather than rarely observed perturbations in VCL-encoded vinculin. To test this hypothesis, we examined the localization of vinculin/metavinculin in hypertrophied human heart tissue from patients with cardiovascular conditions associated with obstruction and hemodynamic overload using an immunohistochemistry approach. Tissue specimens derived from patients with obstructive HCM and aortic stenosis (AS) showed a universal defect of vinculin/metavinculin expression in the intercalated disc but preserved expression in the cardiac Z-disc, whereas tissue specimens derived from patients with either DCM, hypertensive heart disease (HTN), or pulmonary hypertension (PHTN) exhibited normal expression of vinculin/metavinculin in both the Z- and the intercalated disc despite being associated with hypertrophy. Results of this study suggest that cardiac hypertrophy may be associated with different expression of the marker vinculin/metavinculin depending on the underlying pathophysiology; hemodynamic overload may not affect the localization whereas obstructive disease substantially reduces the expression of vinculin preferentially in the intercalated disc.