Structural Determinants of Slow Conduction in the Canine Sinus Node

Structure of Sinus Node Gap Junctions. Introduction : To elucidate the role of tissue structure as a determinant of the unique conduction properties of the sinus node. we compared the spatial distribution of intercellular connections at gap junctions in the sinus node to the more rapidly conducting crista terminalis and left ventricle, which have been studied previously.
Methods and Results : Samples of four canine sinus nodes were prepared for electron microscopy. The total number and spatial orientation of neighboring myocytes connected by ultrastructurally identified intercalated disks and gap junctions to nine randomly selected index cells were determined by sequentially examining subserial sections. Sinus node cells were sparsely interconnected compared to the extent of interconnections observed previously in other tissues. A typical sinus node cell was connected to only 4.8 ± 0.7 neighbors compared with 11.3 ± 2.2 cells in the left ventricle and 6.4 ± 1.7 cells in the crista terminalis. Sinus node interconnections occurred at small intercalated disks that usually connected cells in partial side-to-side and end-to-end juxtaposition. In contrast, left ventricular myocytes are interconnected at large intercalated disks that adjoin many cells in pure side-to-side and end-to-end orientations. Crista terminalis myocytes are connected primarily in end-to-end fashion. The aggregate gap junction profile length per unit myocyte area was 26.5 times greater in the left ventricle and 5.0 times greater in the crista terminalis than in the sinus node.
Conclusion : Sinus node myocytes exhibit small, sparsely distributed gap junctions that interconnect cells in complex patterns of lateral and terminal apposition. These structural features are consistent with the unique conduction properties of the sinus node.     

Distribution of gap junctions in dog and rat ventricle studied with a double-label technique.

To assess the distribution of gap junctions in relation to the cardiac myocyte surface in paraffin sections of dog and rat ventricle, the sarcolemma was labeled with wheat germ agglutinin (WGA1) and gap junctions were labeled with antibodies to cardiac muscle gap junction protein connexin43. WGA labeled all of the myocyte sarcolemma, including that in intercalated discs and transverse tubules. Sarcolemmal WGA labeling was often interrupted at the sites of gap junctions, which were found both at the extreme ends of myocytes and along the length of adjacent myocytes. Small gap junctions predominated at plicate transverse portions of the intercalated disc; larger and sometimes ribbon-like gap junctions predominated at longitudinal portions. The longitudinal portions of the intercalated disc often extended over multiple sarcomere lengths, with ribbon-like gap junctions and linear arrays of smaller gap junctions arranged in parallel overlying successive sarcomeres. Morphometric study showed that ribbon-like gap junctions were relatively infrequent in both dog and rat left ventricular epimyocardium, and that animals with larger myocytes tended to have smaller gap junctions. In dog left ventricular epimyocardium, neither myocytes nor their larger gap junctions were randomly oriented with respect to perimysial separations; myocytes were usually somewhat flattened with their maximal diameters parallel to the separations, whereas large gap junctions were least often oriented parallel or perpendicular to the separations. Overall, the data indicate that myocyte geometry influences gap junction size and distribution; the double-label technique is ideally suited for the further exploration of that influence.     

Internalization and dephosphorylation of connexin43 in hypertrophied right ventricles of rats with pulmonary hypertension.

BACKGROUND: Altered expression and distribution of gap junctions might provide substrates for abnormal conduction and arrhythmogenesis in the heart, but little is known about the regulation of gap junctions under pathological conditions. The organization and phosphorylation state of connexin43 (Cx43) in ventricular hypertrophy will be investigated. METHODS AND RESULTS: Right ventricular (RV) hypertrophy was induced in rats by treatment with monocrotaline. Subcellular Cx43 distribution was assessed by immunoconfocal and electron microscopy. Immunolabeling of Cx43 was confined to the intercalated disks in the normal ventricular myocytes of control rats, but hypertrophied RV cells from monocrotaline-treated rats showed dispersion of Cx43 immunolabeling over the cell surface and in the cytoplasm; cytoplasmic Cx43 was increased by approximately 7-fold (n=15). The Cx43 internalization was confirmed by the double staining of monocrotaline-treated RV tissues for Cx43/wheat germ agglutinin (WGA) and Cx43/zonula occludens protein-1 (ZO-1). Electron microscopy of hypertrophied RVs showed an increase in annular gap junctions immunolabeled with Cx43. Immunoblotting revealed a significant increase in non-phosphorylated Cx43 in hypertrophied RVs (by approximately 5-fold, n=8) without changes in the total amount of Cx43. The accumulation of non-phosphorylated Cx43 in hypertrophied RVs was also recognized by immunoconfocal-microscopy with an isoform-specific antibody. CONCLUSION: Ventricular hypertrophy is associated with the dephosphorylation of Cx43 and its translocation from the intercalated disks to intracellular pools, suggesting accelerated gap junction degradation.     

Gap junction remodeling in hypertrophied left ventricles of aortic-banded rats: prevention by angiotensin II type 1 receptor blockade

Remodeling of gap-junctional organization in hypertrophied left ventricle (LV) in response to pressure overload in rats induced by abdominal aorta banding was investigated by immunoconfocal and electron microscopy. Eight to 12 weeks after banding, rats developed significant LV hypertrophy. In contrast to control LV myocytes, which showed connexin43 (Cx43) labeling largely confined to the intercalated disks, LV myocytes from aortic-banded rats showed dispersion of punctate Cx43 labeling over the entire cell surface. In LV tissues sectioned longitudinally, the proportion of Cx43 label at the intercalated disk decreased significantly (control, 0.87 v aortic-banded, 0.62). En-face views of intercalated disks of hypertrophied myocardium revealed a reduction of Cx43 gap junctions in the disk center, giving rise to a significant decrease in the proportion of the disk occupied by gap-junctional membrane (control, 0.32 v aortic-banded, 0.24). Electron microscopy of hypertrophied LV tissue revealed that Cx43-containing gap junctions were frequently displaced from their usual locations to form side-to-side contacts distant from the disk, and also appeared as annular profiles. In aortic-banded rats treated with the angiotensin II (AII) type 1 receptor (AT1) antagonist, losartan (10 mg/kg/day, 11 weeks) not only LV hypertrophy, but also the gap junction disorganization was markedly reduced. These results suggest that LV hypertrophy induced by pressure overload is associated with Cx43 gap junction disorganization and that AII may play an important role either directly or indirectly in gap-junctional remodeling.     

Quantitative analysis of intercellular connections by immunohistochemistry of the cardiac gap junction protein connexin43

Polyclonal antisera directed against epitopes in the cytoplasmic domain of rat connexin43, the predominant cardiac gap junction protein, were used to delineate immunohistochemically the distribution of gap junctions in sections of canine left ventricle. Antigen-antibody binding and tissue structure were preserved after paraformaldehyde fixation and paraffin embedment of canine myocardium. Specific binding of antibody to the cytoplasmic surfaces of ultrastructurally identified gap junctions was confirmed with electron microscopy. Light microscopic morphometric analysis of immunostained sections in five separate experiments revealed a mean gap junction surface density of 0.0052 micron2/micron3 myocyte volume, which is consistent with previously reported values determined by use of quantitative electron microscopy. This new method permits quantitative determinations of gap junction surface density and distribution in relatively large heterogeneous areas of myocardium in which ultrastructural morphometry would be impractical. This approach should facilitate analysis of the relation between potential alterations in electrical coupling of myocytes and abnormalities of myocardial conduction occurring at the macroscopic scale in regions such as structurally heterogeneous infarct border zones.     

Remodeling of myocyte gap junctions in arrhythmogenic right ventricular cardiomyopathy due to a deletion in plakoglobin (Naxos disease).

OBJECTIVES: We tested the hypothesis that defective interactions between adhesion junctions and the cytoskeleton caused by the plakoglobin mutation in Naxos disease lead to remodeling of gap junctions and altered expression of the major gap junction protein, connexin43. BACKGROUND: Naxos disease, a recessive form of arrhythmogenic right ventricular cardiomyopathy, is associated with a high incidence of arrhythmias and sudden cardiac death. Naxos disease is caused by a mutation in plakoglobin, a protein that links cell-cell adhesion molecules to the cytoskeleton. METHODS: Myocardial expression of connexin43 and other intercellular junction proteins was characterized in 4 patients with Naxos disease. Immunohistochemistry was performed in all 4 patients, and immunoblotting and electron microscopy were performed in 1 patient who died in childhood before overt arrhythmogenic right ventricular cardiomyopathy had developed. RESULTS: Connexin43 expression at intercellular junctions was reduced significantly in both right and left ventricles in all patients with Naxos disease. Electron microscopy revealed smaller and fewer gap junctions interconnecting ventricular myocytes. Mutant plakoglobin was expressed but failed to localize normally at intercellular junctions. Localization of N-cadherin, alpha- and beta-catenins, plakophilin-2, desmoplakin-1, and desmocollin-2 at intercalated disks appeared normal. CONCLUSIONS: Remodeling of gap junctions occurs early in Naxos disease, presumably because of abnormal linkage between mechanical junctions and the cytoskeleton. Gap junction remodeling may produce a coupling defect which, combined with the subsequent development of pathologic changes in myocardium, could contribute to a highly arrhythmogenic substrate and enhance the risk of sudden death in Naxos disease.     

Remodeling of gap junctional coupling in hypertrophied right ventricles of rats with monocrotaline-induced pulmonary hypertension

The present study investigates the remodeling of gap junctional organization in relation to changes in anisotropic conduction properties in hypertrophied right ventricles (RVs) of rats with monocrotaline (MCT)-induced pulmonary hypertension. In contrast to controls that showed immunolocalization of connexin43 (Cx43) labeling largely confined to the intercalated disks, RV myocytes from MCT-treated rats showed dispersion of Cx43 labeling over the entire cell surface. The disorganization of Cx43 labeling became more pronounced with the progression of hypertrophy. Desmoplakin remained localized to the intercalated disks, as in controls. In RV tissues, the proportion of Cx43 label at the intercalated disk progressively decreased. Quantitative analysis of en face views of intercalated disks revealed a significant decrease in the disk gap junctional density in RV tissues of MCT-treated rats (control, 0.18 versus MCT-treated, 0.14 at 2 weeks; control, 0.16 versus MCT-treated, 0.11 at 4 weeks). Conduction velocity in RVs parallel to the fiber orientation was significantly lower (30.2% [n=9]) in MCT-treated rats at 4 weeks than in control rats, whereas there was no significant difference observed in the conduction velocity across the fiber orientation between control and MCT-treated rats. The anisotropic ratio of MCT-treated rats (1.38+/-0.10) was significantly lower than that of control rats (1.98+/-0.12). These results suggest that RV hypertrophy induced by pressure overload is associated with both disorganization of gap junction distribution and alteration of anisotropic conduction properties.     

Structural differences in the cytoarchitecture and intercalated discs between the working myocardium and conduction system in the human heart

Working and specialized cardiac myocytes and their intercalated discs in adult human hearts without history of cardiac disease were examined by scanning electron microscopy. The NaOH/ultrasonication treatment of cardiac tissues resulted in the digestion of connective tissue and separation of intercellular junctions. Auricular and ventricular working cardiac myocytes were quasi-cylindrical in shape, bifurcated, and connected end-to-end at the intercalated discs. The intercalated discs in the working cardiac myocytes showed a stair-like profile, consisting of steps (plicate segments) and corresponding risers (interplicate segments). The ventricular myocytes, in particular, had many steps and risers. The plicate segments were filled with numerous finger-like microprojections. The strands of the myocytes in the sinoatrial node were oriented linearly, while those in the atrioventricular node formed a reticular network. The intercalated discs in both nodal cells were underdeveloped, having few microprojections. Myocytes in the atrioventricular bundle (His) and the right limb were arranged in parallel, and were characterized by the presence of slender branches. Purkinje cell strands formed reticular networks. The intercalated discs in the His-Purkinje system were irregular in appearance, and the microprojections were larger in size and smaller in number than those of working myocytes. The myocytes in the crista terminalis and surrounding the fossa ovalis resembled cells in the His-Purkinje system rather than auricular working myocytes in morphology, and may act as the internodal pathway. It is concluded that morphological differences in both the cytoarchitecture and intercalated discs were closely related with contraction and impulse propagation in the various regions of the human heart. Received: December 13, 2001 / Accepted: May 18, 2002 Present address: The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, Japan Tel. +81-3-5800-8654; Fax +81-3-5684-3989 e-mail: miyamotot-sur@h.u.-tokyo.ac.jp Acknowledgments The authors thank Professor Y. Uchida, Oita Medical University, for his continuous support. We also thank S. Akizuki for excellent technical help. Correspondence to T. Miyamoto     

The Wolff-Parkinson-White syndrome: the cellular substrate for conduction in the accessory atrioventricular pathway

The longstanding quest for the anatomical basis of the Wolff-Parkinson-White syndrome has left many unanswered questions. The ultrastructuralmorphology of the myocytes comprising accessory atrioventricularpathways, which are capable of rapid and variable conduction,is central to understanding the development and behaviour ofthis congenital anomaly, but remains unknown. Examination ofthree surgically resected pathways was performed to determinetheir underlying cellular morphology and the pattern of intercellularcoupling, by correlative light microscopy, electron microscopyand confocal scanning laser microscopy combined with immunohistochemicallocalization of the cardiac gap-junctional protein, connexin43. Two left-sided pathways were composed of myocardium of ‘normalworking ventricular’ type. The right-sided pathway wascomposed almost entirely of highly abnormal myocytes characterizedby aberrant myofibril organisation, with a lack of A-band materialand abnormal mitochondria, but normal intact intercalated disksno different from those seen in left-sided pathways. The gapjunctions of all pathways were composed of connexin43 distributedas in ventricular myocardium, and not as found in atrial oratrioventricular nodal tissues. While myocytes of abnormal structure were present in one ofthe accessory atrioventricular pathways examined, all pathwayshad morphologically normal gap junctions, the structures responsiblefor efficient intercellular coupling, with a pattern of distributionsuggestive of working ventricular myocardium.     

Microtubules and desmin filaments during onset of heart hypertrophy in rat: a double immunoelectron microscope study

The distribution of tubulin and desmin, the constituent proteins of microtubules and intermediate filaments, respectively, were studied in normal and hypertrophied rat myocardium by high-resolution immunofluorescence and immunoelectron microscopy. Cardiac hypertrophy was induced in 25-day-old rats by aortic stenosis. In the normal heart, double immunolabelling of ultrathin frozen sections of papillary muscle using gold-labelled probes for tubulin and desmin showed that microtubules ran primarily in a longitudinal direction through the intermyofibrillar spaces, perpendicularly to the desmin filaments. Microtubules were present near nuclei, mitochondria, and plasma membranes, while desmin filaments formed transverse connections between adjacent Z disks. No tubulin was observed near the intercalated disks, which were rich in desmin filaments. In hypertrophied hearts, myocytes exhibited the typical morphological features of developing hypertrophy. While there was little difference in the distribution of the microtubules around mitochondria and at the plasma membrane, considerable increases were seen near the nuclei and along the myofibrils. Desmin labelling was distributed transversely as in the controls; however, sometimes it was longitudinally oriented either in the intermyofibrillar space linking 2 Z disks out of register or along digitations of the intercalated disks connecting neighboring desmosomes. The unique rearrangement of desmin and tubulin filaments in hypertrophied cardiac myocytes emphasizes their distinct role in myocyte organization. We suggest that, during the development of cardiac hypertrophy, desmin filaments are mainly involved in maintaining the myofibrils in register, whereas the degree of assembly of microtubules is correlated with the rate of protein synthesis and with myofibrillogenesis.     

Role of gap junctions in the propagation of the cardiac action potential

Gap junctions play a pivotal role for the velocity and the safety of impulse propagation in cardiac tissue. Under physiologic conditions, the specific subcellular distribution of gap junctions together with the tight packaging of the rod-shaped cardiomyocytes underlies anisotropic conduction, which is continuous at the macroscopic scale. However, when breaking down the three-dimensional network of cells into linear single cell chains, gap junctions can be shown to limit axial current flow and to induce 'saltatory' conduction at unchanged overall conduction velocities. In two- and three-dimensional tissue, these discontinuities disappear due to lateral averaging of depolarizing current flow at the activation wavefront. During gap junctional uncoupling, discontinuities reappear and are accompanied by slowed and meandering conduction. Critical gap junctional uncoupling reduces conduction velocities to a much larger extent than does a reduction of excitability, which suggests that the safety for conduction is higher at any given conduction velocity for gap junctional uncoupling. In uniformly structured tissue, gap junctional uncoupling is accompanied by a parallel decrease in conduction velocity. However, this is not necessarily the case for non-uniform structures like tissue expansion where partial uncoupling paradoxically increases conduction velocity and has the capacity to remove unidirectional conduction blocks. Whereas the impact of gap junctions on impulse conduction is generally assessed from the point of view of cell coupling among cardiomyocytes, it is possible that other cell types within the myocardium might be coupled to cardiomyocytes as well. In this context, it has been shown that fibroblasts establish successful conduction between sheets of cardiomyocytes over distances as long as 300 microm. This might not only explain electrical synchronization of heart transplants but might be of importance for cardiac diseases involving fibrosis. Finally, the intriguing fact that sodium channels are clustered at the intercalated disc recently rekindled the provocative question of whether gap junctions alone are responsible for impulse propagation or whether electric field mechanisms might account for conduction as well. Whereas computer simulations show the feasibility of conduction in the absence of gap junctional coupling, a definite answer to this question must await further investigations into the biophysical properties of the intercalated disc.     

Cardiac-specific loss of N-cadherin leads to alteration in connexins with conduction slowing and arrhythmogenesis

The remodeling of ventricular gap junctions, as defined by changes in size, distribution, or function, is a prominent feature of diseased myocardium. However, the regulation of assembly and maintenance of gap junctions remains poorly understood. To investigate N-cadherin function in the adult myocardium, we used a floxed N-cadherin gene in conjunction with a cardiac-specific tamoxifen-inducible Cre transgene. The mutant animals appeared active and healthy until their sudden death approximately 2 months after deleting N-cadherin from the heart. Electrophysiologic analysis revealed abnormal conduction in the ventricles of mutant animals, including diminished QRS complex amplitude consistent with loss of electrical coupling in the myocardium. A significant decrease in the gap junction proteins, connexin-43 and connexin-40, was observed in N-cadherin-depleted myocytes. Perturbation of connexin function resulted in decreased ventricular conduction velocity, as determined by optical mapping. Our data suggest that perturbation of the N-cadherin/catenin complex in heart disease may be an underlying cause, leading to the establishment of the arrythmogenic substrate by destabilizing gap junctions at the cell surface.     

Cardiac myocyte interconnections at gap junctions Role in normal and abnormal electrical conduction

Slow conduction leading to reentrant ventricular tachycardias in patients with healed myocardial infarcts appears to depend primarily on alterations in intercellular coupling at gap junctions of myocytes bordering the infarct scar. Results of correlative morphometric and electrophysiologic studies indicate that the elongated shape of individual myocytes, their complex overlapped packing in myocardium, and the number and distribution of gap junctions that electrically couple myocytes are all important structural determinants of anisotropic patterns of current spread in normal myocardium. Alterations of these structural features likely contribute to electrophysiologic derangements critical in reentrant arrhythmogenesis. Recent observations that cardiac myocytes may be coupled by multiple gap junction channel proteins having unique electrophysiologic properties provide new insights into potential mechanisms regulating intercellular current transfer in the heart.     

Fate of gap junctions in isolated adult mammalian cardiomyocytes

The fate of gap junctions in dissociated adult myocytes, maintained for up to 22 hours in culture medium, was investigated by semiquantitative analysis of thin sections and by freeze-fracture electron microscopy. Gap junctions in the dissociated myocyte are intact bimembranous structures seen either as invaginated surface-located structures or as annular profiles in the cytoplasm. Surface-located junctions are sealed from the exterior by a sheet of nonjunctional membrane originating (together with the "outer" junctional membrane) from the formerly neighboring cell. Serial sectioning was used to establish that at least part of the annular gap junction population in the freshly isolated myocyte represents truly discrete cytoplasmic vesicles; thus, some gap junctions are rapidly endocytosed after myocyte separation. Analysis of the surface-located-to-annular gap junction ratio suggested that no further endocytosis occurred in rabbit and cat myocytes maintained for 22 and 15 hours, respectively. Guinea pig myocytes, by contrast, did appear to continue endocytosis in culture. Analysis of the distance of gap junctional structures from the cell surface suggested that little if any inward migration of gap junction vesicles occurred. Hypoxia had no detectable effect on the internalization or inward movement of gap junctions. The quantity of ultrastructurally detectable gap junction membrane appeared to remain constant over time, as did the incidence of "complex structures" (i.e., annular gap junction profiles with features previously suggested to represent degradation). New gap junction formation was negligible, and a reappraisal of the nature of "complex structures" led to the conclusion that the origin of these structures need not be related to degradation. Taken together, the findings suggest that degradation and disappearance of gap junctional membrane after isolation of the mature myocyte constitute a much slower process than previously believed, and the possibility that the cardiac gap junction protein has a longer half-life than its counterpart in liver remains open.     

Induced deletion of the N-cadherin gene in the heart leads to dissolution of the intercalated disc structure

The structural integrity of the heart is maintained by the end-to-end connection between the myocytes called the intercalated disc. The intercalated disc contains different junctional complexes that enable the myocardium to function as a syncytium. One of the junctional complexes, the zonula adherens or adherens junction, consists of the cell adhesion molecule, N-cadherin, which mediates strong homophilic cell-cell adhesion via linkage to the actin cytoskeleton. To determine the function of N-cadherin in the working myocardium, we generated a conditional knockout containing loxP sites flanking exon 1 of the N-cadherin (Cdh2) gene. Using a cardiac-specific tamoxifen-inducible Cre transgene, N-cadherin was deleted in the adult myocardium. Loss of N-cadherin resulted in disassembly of the intercalated disc structure, including adherens junctions and desmosomes. The mutant mice exhibited modest dilated cardiomyopathy and impaired cardiac function, with most animals dying within two months after tamoxifen administration. Decreased sarcomere length and increased Z-line thickness were observed in the mutant hearts consistent with loss of muscle tension because N-cadherin was no longer available to anchor myofibrils at the plasma membrane. Ambulatory electrocardiogram monitoring captured the abrupt onset of spontaneous ventricular tachycardia, confirming that the deaths were arrhythmic in nature. A significant decrease in the gap junction protein, connexin 43, was observed in the N-cadherin-depleted hearts. This animal model provides the first demonstration of the hierarchical relationship of the structural components of the intercalated disc in the working myocardium, thus establishing N-cadherin's paramount importance in maintaining the structural integrity of the heart.     

Chronic hemodynamic overload of the atria is an important factor for gap junction remodeling in human and rat hearts

OBJECTIVES: The expression and distribution of connexins is abnormal in a number of cardiac diseases, including atrial fibrillation, and is believed to favor conduction slowing and arrhythmia. Here, we studied the role of atrial structural remodeling in the disorganization of gap junctions and whether redistributed connexins can form new functional junction channels. METHODS: Expression of connexin-43 (Cx43) was characterized by immunoblotting and immunohistochemistry in human right atrial specimens and in rat atria after myocardial infarction (MI). Gap junctions were studied by electron and 3-D microscopy, and myocyte-myocyte coupling was determined by Lucifer yellow dye transfer. RESULTS: In both chronically hemodynamically overloaded human atria in sinus rhythm and in dilated atria from MI-rats, Cx43 were dephosphorylated and redistributed from the intercalated disc to the lateral cell membranes as observed during atrial fibrillation. In MI-rats, the gap junctions at the intercalated disc were smaller (20% decrease) and contained very little Cx43 (0 or 1 gold particle vs. 42 to 98 in sham-operated rats). In the lateral membranes of myocytes, numerous connexon aggregates comprising non-phosphorylated Cx43 were observed. These connexon aggregates were in no case assembled into gap junction plaque-like structures. However, N-cadherin was well organized in the intercalated disc. There was very little myocyte-myocyte coupling in MI-rat atria and no myocyte-fibroblast coupling. Regression of the atrial remodeling was associated with the normalization of Cx43 localization. CONCLUSION: Structural alteration of the atrial myocardium is an important factor in the disorganization of connexins and gap junction. Moreover, redistributed Cx43 do not form junction channels.     

Altered patterns of cardiac intercellular junction distribution in hypertrophic cardiomyopathy.

OBJECTIVE: To examine the distribution pattern of intercellular junctions (the mechanically coupling desmosomes and the electrically coupling gap junctions) in hypertrophic cardiomyopathy (HCM) hearts showing myofibre disarray. DESIGN: Samples from six necropsied hearts were studied, representing the interventricular septum and the free walls of the left and right ventricles. Immunohistochemical labelling of desmoplakin was used as a marker for desmosomes, and of connexin43 as a marker for gap junctions, in single and double stainings. The slides were examined by confocal laser scanning microscopy. RESULTS: Marked disorganisation of intercalated discs was observed in areas featuring myofibre disarray. Besides overall derangement, localised abnormalities in desmosome organisation were evident, which included: (1) the formation of abnormally enlarged megadiscs; (2) the presence of intersecting disc structures; and (3) aberrant side to side desmosomal connections. Gap junctional abnormalities included: (1) random distribution of gap junctions over the surface of myocytes, rather than localisation to intercalated discs; (2) abundant side to side gap junction connections between adjacent myocytes; and (3) formation of abnormally shaped gap junctions. Circles of myocytes continuously interconnected by gap junctions were also observed. Regions of the diseased hearts lacking myofibre disarray, and control hearts of normal patients and patients with other cardiac diseases, did not show these alterations. CONCLUSIONS: The disorganisation of the intercellular junctions associated with myofibre disarray in HCM may play an important role in the pathophysiological manifestations of the disease. The remodelling of gap junction distribution may underlie the formation of an arrhythmogenic substrate, thereby contributing to the generation and maintenance of cardiac arrhythmias associated with HCM.     

Effects of angiotensin II on expression of the gap junction channel protein connexin43 in neonatal rat ventricular myocytes

Objectives. To elucidate signal transduction pathways regulating expression of myocardial gap junction channel proteins (connexins) and to determine whether mediators of cardiac hypertrophy might promote remodeling of gap junctions, we characterized the effects of angiotensin II on expression of the major cardiac gap junction protein connexin43 (Cx43) in cultured neonatal rat ventricular myocytes.Background. Remodeling of the distribution of myocardial gap junctions appears to be an important feature of anatomic substrates of ventricular arrhythmias in patients with heart disease. Remodeling of intercellular connections may be initiated by changes in connexin expression caused by chemical mediators of the hypertrophic response.Methods. Cultures were exposed to 0.1 μmol/liter angiotensin II for 6 or 24 h, and Cx43 expression was characterized by immunoblotting, confocal microscopy and electron microscopy.Results. Immunoblot analysis revealed a twofold increase in Cx43 content in cells treated for 24 h with angiotensin II (n = 4, p < 0.05). This response was inhibited by the presence of 1.0 μmol/liter losartan, an AT1-receptor blocker. Confocal and electron microscopy demonstrated enhanced Cx43 immunoreactivity and increases in the number and size of gap junction profiles in cells exposed to angiotensin II for 24 h. These effects were also blocked by losartan. Immunoprecipitation of Cx43 from cells metabolically labeled with [³⁵S]methionine demonstrated 2.4- and 2.9-fold increases in Cx43 radioactivity after 6 and 24 h exposure to angiotensin II, respectively (p < 0.03 at each time point).Conclusions. Angiotensin II up-regulates gap junctions in cultured neonatal rat ventricular myocytes by increasing Cx43 synthesis. Signal transduction pathways activated by angiotensin II under pathophysiologic conditions could initiate remodeling of conduction pathways, leading to the development of anatomic substrates of arrhythmias.     

Designer gap junctions that prevent cardiac arrhythmias

Cardiac gap junctions are specialized membrane structures comprised of arrays of intercellular channels responsible for propagation of the cardiac impulse. These channels are formed by oligomerization of individual protein subunits known as connexins. In response to a broad array of pathologic stressors, gap junction expression is disturbed, resulting in aberrant cardiac conduction and increased propensity for rhythm disturbances. In this article, we review some of the recently identified molecular regulators of connexin assembly, membrane targeting, and degradation, focusing on the role of post-translational phosphorylation of connexin 43, the major gap junctional protein expressed in ventricular myocardium. We also describe efforts to engineer “designer” gap junctions that are resistant to pathologic remodeling.     

Voltage-gated K(+)Channel, Kv4.2, localizes predominantly to the transverse-axial tubular system of the rat myocyte

Kv4.2 subunit, a member of K(+)channel gene family, is considered to play a major role in the formation of depolarization-activated transient outward K(+)current channels in the mammalian heart. We investigated the subcellular localization of Kv4.2 subunit in the rat heart by immunofluorescence and immunoelectron microscopy. In atrial cells, Kv4.2 immunofluorescent staining was intensely observed in the peripheral sarcolemma and the intercalated disks, but seldom found in transverse tubules, which are rare or absent in atrial cells. In ventricular cells, the labeling of Kv4.2 immunofluorescent staining was found throughout the entire cell membrane, and the staining was stronger in the transverse-axial tubular system than in the peripheral sarcolemma. Correlative immunoconfocal and immunoelectron microscopy using FluoroNanogold confirmed that Kv4.2 distributed in the transverse-axial tubular system including the longitudinally oriented axial tubules. Immunogold electron microscopy of ultrathin cryosections revealed that Kv4.2 was distributed on the plasma membranes of the T-tubules. The extensive distribution of Kv4.2 on the entire cell membrane of myocytes would provide rat myocardial cells with a large capability for the transport of K(+)ions through the channels in the repolarization phase.