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The function of the heart is to provide the tissues of the body with sufficient oxygenated blood and metabolites to meet the moment-to-moment needs as dictated by physical activity and postural and emotional changes. Cardiac myocytes are the contractile cells of the heart and constitute the bulk of heart mass. There are differences between the myocytes of the ventricles, the atria, and the conduction system: ventricular myocytes are elongated cells, packed with myofibrils (the contractile apparatus) and mitochondria (for ATP production). Myofibrils are repeating units (sarcomeres) made up of thin actin filaments anchored at the Z-discs at either end of the sarcomere, and thick myosin filaments which interdigitate and interact with the thin filaments. Contraction results from sarcomere shortening produced by the ATP-dependent movement of the thin and thick filaments relative to one another. Transverse (T-) tubules facilitate extracellular Ca...

The functional characteristics of the co-expression of connexin43, connexin40, and connexin45 proteins in human myocardium are thought to play an important role in governing normal propagation of the cardiac electrical impulse and in generating the myocardial substrate for some arrhythmias and conduction disturbances. A rat liver epithelial cell line, that endogenously expresses connexin43, was used to induce also expression of connexin40 or connexin45 after stable transfection using an inducible ecdysone system. Electrical coupling was estimated from measurement of the input resistance of transfected cells using an intracellular microelectrode to inject current and record changes to membrane potential. However, varied expression of the transfected connexin40 or connexin45 did not change electrical coupling, although connexin43/40 co-expression led to better coupling than connexin43/45 co-expression. Quantification of endogenous connexin43 expression, at both mRNA and protein levels, showed that it was altered in a manner dependent on the transfected connexin isotype. The data using rat liver epithelial cells indicate an increased electrical coupling upon expression of connexin40 and connexin43 but decreased coupling with connexin45 and connexin43 co-expression.

Cardiac myocytes are the contractile cells that enable the heart to function as a pump. They are, thus, responsible for ensuring that sufficient oxygenated blood and metabolites reach the tissues to meet the body’s needs, whether at rest or during exercise. Each myocyte beats over three billion times in a typical human lifespan; to work as an effective pump, the activities of over three billion myocytes must be synchronized. To fulfill these functions, the cardiac myocyte is equipped with highly specialized subcellular machinery. With the aid of a range of complementary high resolution imaging techniques, this chapter illustrates and explains how the key structural components of the myocyte are organized to enable the coupling of electrical excitation to contraction of the myofibrils, and how the contractile activities of individual cells are coordinated to achieve organ function.

Objective : The scavenger receptor SR-PSOX/CXCL16, which is identical to the chemokine CXCL16, is thought to be involved in atherogenesis. However, the presence and function of SR-PSOX/CXCL16 in the endothelium of atherosclerotic arteries has not been substantiated. Methods and results : In rabbit aorta immunocytochemistry revealed SR-PSOX/CXCL16 primarily in the endothelium at sites predisposed to lesion formation, in the endothelium of early atherosclerotic lesions, and mainly in intimal macrophages of more developed lesions, indicating that SR-PSOX/CXCL16-expression shifts during atherogenesis. In addition to its function as scavenger receptor and chemokine, SR-PSOX mediated the adhesion of THP-1 monocytes to endothelial cells in vitro. Both THP-1 monocytes and endothelial cells express SR-PSOX/CXCL16, and THP-1 monocytes express CXCR6, the specific receptor for SR-PSOX/CXCL16. Anti-SR-PSOX/CXCL16 and anti-CXCR6 antibody block monocyte adhesion, showing that SR-PSOX/CXCL16–CXCR6 interaction mediates monocyte-endothelial cell adhesion. SR-PSOX/CXCL16 expression of endothelial cells is upregulated by pro-inflammatory cytokines, and is reversed by incubation with ciglitazone and lovastatin. Conclusions : We suggest that SR-PSOX/CXCL16 may promote the adhesion of monocytes to the endothelium during early atherogenesis and that accumulating cytokines enhance SR-PSOX/CXCL16-mediated adhesion by upregulating SR-PSOX/CXCL16 expression. Manipulation of SR-PSOX/CXCL16 expression with anti-inflammatory agents may be of therapeutic value.

Bidirectional transport of early endosomes (EEs) involves microtubules (MTs) and associated motors. In fungi, the dynein/dynactin motor complex concentrates in a comet-like accumulation at MT plus-ends to receive kinesin-3-delivered EEs for retrograde transport. Here, we analyse the loading of endosomes onto dynein by combining live imaging of photoactivated endosomes and fluorescent dynein with mathematical modelling. Using nuclear pores as an internal calibration standard, we show that the dynein comet consists of ∼55 dynein motors. About half of the motors are slowly turned over (T1/2: ∼98 s) and they are kept at the plus-ends by an active retention mechanism involving an interaction between dynactin and EB1. The other half is more dynamic (T1/2: ∼10 s) and mathematical modelling suggests that they concentrate at MT ends because of stochastic motor behaviour. When the active retention is impaired by inhibitory peptides, dynein numbers in the comet are reduced to half and ∼10% of the EEs fall off the MT plus-ends. Thus, a combination of stochastic accumulation and active retention forms the dynein comet to ensure capturing of arriving organelles by retrograde motors.

We have shown that the gene SCN10A encoding the sodium channel Na(v)1.8 is a susceptibility factor for heart block and serious ventricular arrhythmia. Since Na(v)1.8 is known to be present in nerve fibres that mediate pain, it may be related to both cardiac pain and dysrhythmia. The localisation of Na(v)1.8 and other key nociceptive ion channels, including Na(v)1.7, Na(v)1.9, capsaicin receptor TRPV1, and purinergic receptor P2X(3), have not been reported in human heart. The aim of this study was to determine the distribution of Na(v)1.8, related sodium and other sensory channels in human cardiac tissue, and correlate their density with sympathetic nerves, regenerating nerves (GAP-43), and vascularity. Human heart atrial appendage tissues (n = 13) were collected during surgery for valve disease. Tissues were investigated by immunohistology using specific antibodies to Na(v)1.8 and other markers. Na(v)1.8 immunoreactivity was detected in nerve fibres and fascicles in the myocardium, often closely associated with small capillaries. Na(v)1.8 nerve fibres per mm(2) correlated significantly with vascular markers. Na(v)1.8-immunoreactivity was present also in cardiomyocytes with a similar distribution pattern to that seen with connexins, the specialised gap junction proteins of myocardial intercalated discs. Na(v)1.5-immunoreactivity was detected in cardiomyocytes but not in nerve fibres. Na(v)1.7, Na(v)1.9, TRPV1, P2X(3)/P2X(2), and GAP43 positive nerve fibres were relatively sparse, whereas sympathetic innervation and connexin43 were abundant. We conclude that sodium channel Na(v)1.8 is present in sensory nerves and cardiomyocytes of human heart. Na(v)1.8 and other pain channels provide new targets for the understanding and treatment of cardiac pain and dysrhythmia.

To identify genetic factors influencing cardiac conduction, we carried out a genome-wide association study of electrocardiographic time intervals in 6,543 Indian Asians. We identified association of a nonsynonymous SNP, rs6795970, in SCN10A (P = 2.8 x 10(-15)) with PR interval, a marker of cardiac atrioventricular conduction. Replication testing among 6,243 Indian Asians and 5,370 Europeans confirmed that rs6795970 (G>A) is associated with prolonged cardiac conduction (longer P-wave duration, PR interval and QRS duration, P = 10(-5) to 10(-20)). SCN10A encodes Na(V)1.8, a sodium channel. We show that SCN10A is expressed in mouse and human heart tissue and that PR interval is shorter in Scn10a(-/-) mice than in wild-type mice. We also find that rs6795970 is associated with a higher risk of heart block (P < 0.05) and a lower risk of ventricular fibrillation (P = 0.01). Our findings provide new insight into the pathogenesis of cardiac conduction, heart block and ventricular fibrillation.

An understanding of how lipid droplets grow in the cell is important to current human health issues. Homotypic fusion of small lipid droplets to create larger ones is one proposed mechanism though the evidence for this process continues to be debated. By applying the technique of freeze-fracture electron microscopy to cells that have been stimulated to accumulate lipid droplets, we here present images which suggest that at least some large lipid droplets may indeed result from amalgamation of multiple smaller ones. These visual data add significantly to the notion that fusion contributes to lipid droplet growth.

GFP-tagging is widely used as a molecular tool to localize and visualize the trafficking of proteins in cells but interpretation is frequently limited by the low resolution afforded by fluorescence light microscopy. Although complementary thin-section immunogold electron microscopic techniques go some way in aiding interpretation, major limitations, such as relatively poor structural preservation of membrane systems, low labelling efficiency and the two-dimensional nature of the images, remain. Here we demonstrate that the electron microscopic technique freeze-fracture replica immunogold labelling overcomes these disadvantages and can be used to define, at high resolution, the precise location of GFP-tagged proteins in specific membrane systems and organelles of the cell. Moreover, this technique provides information on the location of the protein within the phospholipid bilayer, potentially providing insight into mis-orientation of tagged proteins compared to their untagged counterparts. Complementary application of the freeze-fracture replica immunogold labelling technique alongside conventional fluorescence microscopy is seen as a novel and valuable approach to verification, clarification and extension of the data obtained using fluorescent-tagged proteins. The application of this approach is illustrated by new findings on PAT-family proteins tagged with GFP transfected into fibroblasts from patients with Niemann-Pick type C disease.

Objective— Uptake of lipids by macrophages (MΦ) leads to lipid droplet accumulation and foam cell formation. The PAT family proteins are implicated in lipid droplet formation, but the precise function of the 47-kDa tail interacting protein (TIP47), a member of this family, is poorly defined. The present study was performed to determine the function of TIP47 in MΦ lipid metabolism. Methods and Results— Freeze-fracture cytochemistry demonstrates that TIP47 is present in the plasma membrane of MΦ and is aggregated into clusters when the cells are incubated with oleate. Suppression of adipophilin levels using siRNA knockdown leads to migration of TIP47 from a cytoplasmic pool to the lipid droplet. Further, reduction of TIP47 decreases triglyceride levels, whereas raising TIP47 levels by expression of EGFP-TIP47 shows the opposite effect. Conclusion— Our results show that the TIP47 protein levels directly correlate with triglyceride levels. We propose that TIP47 may act as a carrier protein for free fatty acids and in this way participates in conversion of MΦ into foam cells.

In the human heart connexin(Cx)40, Cx43 and Cx45-containing gap junctional channels electrically couple cardiomyocytes, forming a functional syncytium. In the mouse heart, additionally, Cx30.2-containing gap junctions have been detected in the atrioventricular node where they are implicated, together with Cx45, in impulse delay. However, whether the human ortholog of Cx30.2, Cx31.9, is expressed in the human heart has not previously been investigated. We therefore generated Cx31.9 specific antibodies to test for the expression of Cx31.9 in the human heart. These antibodies recognized the Cx31.9 protein in HeLaCx31.9 transfectants by immunofluorescence and immunoblot analyses. However, we did not find punctate Cx31.9 specific immunofluorescence signals in the working myocardium or in the impulse generation and conduction system of adult or fetal human heart. Complementary immunoblot analyses did not reveal Cx31.9 protein in the adult atrial or ventricular myocardium. We conclude that the Cx31.9 protein, unlike its counterpart in the mouse, is not expressed in detectable quantities and is thus unlikely to contribute to the impulse generation and conduction system or the working myocardium of the human heart.

Gap junctions form the cell-to-cell pathways for propagation of the precisely orchestrated patterns of current flow that govern the regular rhythm of the healthy heart. As in most tissues and organs, multiple connexin types are expressed in the heart: connexin43 (Cx43), Cx40 and Cx45 are found in distinctive combinations and relative quantities in different, functionally-specialized subsets of cardiac myocyte. Mutations in genes that encode connexins have only rarely been identified as being a cause of human cardiac disease, but remodelling of connexin expression and gap junction organization are well documented in acquired adult heart disease, notably ischaemic heart disease and heart failure. Remodelling may take the form of alterations in (i) the distribution of gap junctions and (ii) the amount and type of connexins expressed. Heterogeneous reduction in Cx43 expression and disordering in gap junction distribution feature in human ventricular disease and correlate with electrophysiologically identified arrhythmic changes and contractile dysfunction in animal models. Disease-related alterations in Cx45 and Cx40 expression have also been reported, and some of the functional implications of these are beginning to emerge. Apart from ventricular disease, various features of gap junction organization and connexin expression have been implicated in the initiation and persistence of the most common form of atrial arrhythmia, atrial fibrillation, though the disparate findings in this area remain to be clarified. Other major tasks ahead focus on the Purkinje/working ventricular myocyte interface and its role in normal and abnormal impulse propagation, connexin-interacting proteins and their regulatory functions, and on defining the precise functional properties conferred by the distinctive connexin co-expression patterns of different myocyte types in health and disease.

The freeze-fracture technique consists of physically breaking apart (fracturing) a frozen biological sample; structural detail exposed by the fracture plane is then visualized by vacuum-deposition of platinum-carbon to make a replica for examination in the transmission electron microscope. The four key steps in making a freeze-fracture replica are (i) rapid freezing, (ii) fracturing, (iii) replication and (iv) replica cleaning. In routine protocols, a pretreatment step is carried out before freezing, typically comprising fixation in glutaraldehyde followed by cryoprotection with glycerol. An optional etching step, involving vacuum sublimation of ice, may be carried out after fracturing. Freeze fracture is unique among electron microscopic techniques in providing planar views of the internal organization of membranes. Deep etching of ultrarapidly frozen samples permits visualization of the surface structure of cells and their components. Images provided by freeze fracture and related techniques have profoundly shaped our understanding of the functional morphology of the cell.

Cardiac connexin 43 (Cx43), Cx40 and Cx45 are co-expressed at distinct ratios in myocytes. This pattern is considered a key factor in regulating the gap junction channels composition, properties and function and remains poorly understood. This work aims to correlate gap junction function with the connexin composition of the channels at accurate ratios Cx43:Cx40 and Cx43:Cx45. Rat liver epithelial cells that endogenously express Cx43 were stably transfected to induce expression of accurate levels of Cx40 or Cx45 that may be present in various areas of the heart (e.g. atria and ventricular conduction system). Induction of Cx40 does not increase the amounts of junctional connexins (Cx43 and Cx40), whereas induction of Cx45 increases the amounts of junctional connexins (Cx43 and Cx45). Interestingly, the non-junctional fraction of Cx43 remains unaffected upon induction of Cx40 and Cx45. Co-immunoprecipitation studies show low level of Cx40/Cx43 heteromerisation and undetectable Cx45/Cx43 heteromerisation. Functional characterisation shows that induction of Cx40 and Cx45 decreases Lucifer Yellow transfer. Electrical coupling is decreased by Cx45 induction, whereas it is decreased at low induction of Cx40 and increased at high induction. These data indicate a fine regulation of the gap junction channel make-up in function of the type and the ratio of co-expressed Cxs that specifically regulates chemical and electrical coupling. This reflects specific gap junction function in regulating impulse propagation in the healthy heart, and a pro-arrhythmic potential of connexin remodelling in the diseased heart.

The prevailing hypothesis of lipid droplet biogenesis proposes that neutral lipids accumulate within the lipid bilayer of the ER membrane from where they are budded off, enclosed by a protein-bearing phospholipid monolayer originating from the cytoplasmic leaflet of the ER membrane. We have used a variety of methods to investigate the nature of the sites of ER–lipid-droplet association in order to gain new insights into the mechanism of lipid droplet formation and growth. The three-dimensional perspectives provided by freeze-fracture electron microscopy demonstrate unequivocally that at sites of close association, the lipid droplet is not situated within the ER membrane; rather, both ER membranes lie external to and follow the contour of the lipid droplet, enclosing it in a manner akin to an egg cup (the ER) holding an egg (the lipid droplet). Freeze-fracture cytochemistry demonstrates that the PAT family protein adipophilin is concentrated in prominent clusters in the cytoplasmic leaflet of the ER membrane closely apposed to the lipid droplet envelope. We identify these structures as sites at which lipids and adipophilin are transferred from ER membranes to lipid droplets. These findings call for a re-evaluation of the prevailing hypothesis of lipid droplet biogenesis.

The molecular mechanism underlying milk fat globule secretion in mammary epithelial cells ostensibly involves the formation of complexes between plasma membrane butyrophilin and cytosolic xanthine oxidoreductase. These complexes bind adipophilin in the phospholipid monolayer of milk secretory granules, the precursors of milk fat globules, enveloping the nascent fat globules in a layer of plasma membrane and pinching them off the cell. However, using freeze-fracture immunocytochemistry, we find these proteins in locations other than those previously inferred. Significantly, butyrophilin in the residual plasma membrane of the fat globule envelope is concentrated in a network of ridges that are tightly apposed to the monolayer derived from the secretory granule, and the ridges coincide with butyrophilin labeling in the globule monolayer. Therefore, we propose that milk fat globule secretion is controlled by interactions between plasma membrane butyrophilin and butyrophilin in the secretory granule phospholipid monolayer rather than binding of butyrophilin–xanthine oxidoreductase complexes to secretory granule adipophilin.

Gap junction expression is considered to influence myocardial conduction and arrhythmogenesis, but studies in patients with atrial fibrillation (AF) have reported inconsistent results. We used human atrial conduction and arrhythmogenicity to provide clinical parameters with which to correlate quantification of connexin40 (Cx40) by different techniques to address the hypothesis that antibody-epitope binding properties may influence quantification methods.Atrial conduction properties were studied in patients undergoing coronary artery bypass grafting (N = 27) using multi-electrode array mapping. Patients were defined as having a vulnerable atrial substrate if pacing-induced AF lasted for more than 30 s. Using antibodies targeted at two different epitopes of the cytoplasmic carboxyl tail, Cx40 signal was quantified by immunoconfocal microscopy in biopsies taken from the mapped atria. Only patients with a vulnerable substrate subsequently developed post-operative AF (P0.02). Immunoconfocal Cx40 signal measured by one antibody, designated S15C(R85), was lower in patients with sustained induced AF (0.013 +/- 0.009 microm(2)/microm(2) vs. 0.024 +/- 0.010 microm(2)/microm(2), P = 0.005) and was negatively correlated with atrial conduction velocity during sinus rhythm (P0.05). However, these relationships did not exist when the Cx40 signal was measured using a different antibody (Y21Y(R968)). Further studies suggested that quantification technique was reproducible against the same epitope (P0.001) but not against different epitopes indicating variable exposure of Cx40 epitopes.Quantification of Cx40 by immunoconfocal microscopy appears to be epitope dependent and evidence of epitope masking raises the possibility of more than one conformational form of the Cx40 carboxyl tail, suggesting functionally important conformations of the protein.

Up-regulation of the gap-junctional protein connexin43 (Cx43) in arterial smooth muscle cells (SMCs) features in response to injury and in atherosclerosis, in parallel with phenotypic transition to the synthetic state. TGF-beta1 is known to have a role in SMC differentiation and extracellular matrix (ECM) synthesis, key characteristics of phenotypic state. Here, we set out to examine the effects of TGF-beta1 on Cx43-gap junction expression in relation to SMC differentiation, ECM synthesis and growth. Cx43 expression was analysed by immunoconfocal microscopy and Western blotting in primary human aortic SMCs treated with TGF-beta1 over a 48-h period, with assessment of gap-junctional communication by cell-to-cell transfer of microinjected ethidium bromide. In parallel, synthetic activity was analysed by Northern blotting for ECM components alpha-1(I) and alpha1(III) procollagen transcripts, contractile differentiation was assessed by immunoconfocal microscopy and Western blotting of the markers smooth muscle alpha-actin, calponin and smooth muscle heavy chain isoform 1 (SM1), and growth was measured by BrdU incorporation. Our results demonstrate that TGF-beta1 significantly up-regulates Cx43 expression and intercellular communication, in concert with increased expression of alpha-actin, calponin and SM1. Concomitant with contractile protein expression, ECM synthesis was increased rather than decreased, TGF-beta1 inducing a significant up-regulation of both procollagen transcripts. These effects were independent of growth. We conclude that in human aortic SMCs, TGF-beta1 treatment leads to up-regulation of Cx43-mediated gap-junctional communication and increased synthetic activity yet, somewhat paradoxically, also enhanced contractile differentiation.

We studied the response of the superior vena cava (SVC) myocardial sleeve to atrial fibrillation (AF). We examined adult male dogs without pacing (N=6) and after rapid atrial pacing (600 bpm) for 2 weeks (P2w; N=5) and 6–8 weeks (P6–8w; N=5). After pacing, the sleeve was increased in thickness (non-paced vs. either paced group, both P

The actin-binding proteins, spectrin and dystrophin, are key components of the plasma membrane-associated cytoskeleton of the cardiac muscle cell. From confocal immunofluorescence studies, the distribution of spectrin appears to overlap with that of dystrophin, but the precise functional differentiation, molecular distributions and spatial relationship of these two cytoskeletal systems remain unclear. Freeze-fracture replica immuno-electron microscopy, in parallel with immunofluorescence/confocal microscopy, were applied to examine at high resolution the spatial relationships between the spectrin and dystrophin membrane-associated cytoskeleton systems in cardiac muscle. Application of freeze-fracture replica cytochemistry, with single and double immunogold labeling, permitted simultaneous examination of the organization of spectrin and dystrophin in en-face views of the plasma membrane at high resolution. In contrast to the close spatial relationship previously demonstrated for dystrophin and beta-dystroglycan, no association between the gold label marking dystrophin and that marking spectrin was observed. Our freeze-fracture cytochemical results suggest that the two membrane skeletal networks formed by dystrophin and spectrin in cardiac muscle are independently organized, implying that whatever overlap of function (e.g., in structural support to the plasma membrane) may exist between them, the two systems may each have additional distinctive roles.

Background— The 3D structure of the atrioventricular conduction axis incorporating detailed cellular and molecular composition, especially that relating to gap-junctional proteins, is still unclear, impeding mechanistic understanding of cardiac rhythmic disorders. Methods and Results— A 3D model of the rabbit atrioventricular conduction axis was reconstructed by combining histological and immunofluorescence staining on serial sections. The exact cellular boundaries, especially those between transitional cells and atrial myocardium, were demarcated by a dense and irregular desmin-labeling pattern in conductive myocardium. The model demonstrates that the atrioventricular conduction axis is segregated into 2 connecting compartments, 1 predominantly expressing connexin45 (compact node and transitional cells) and the other predominantly coexpressing connexin43 and connexin45 (His bundle, lower nodal cells, and posterior nodal extension). The transitional zone shows unique features of spatial complexity, including a bridging bilayer structure (a deep transitional zone connecting with a superficial atrial-transitional overlay) and asymmetrical continuity (wider atrial-transitional interfaces and shorter atrial-axial distances in the hisian portion than in the ostial portion). In the latter compartment, the His bundle, lower nodal cells, and posterior nodal extension form a continual axis and longitudinal transitional-axial interface. Conclusions— Key findings of the present study are the demonstration of a distinct anatomical border between transitional and atrial cells, connection between transitional cells and both lower nodal cells and posterior nodal extension, and distinctive connexin expression patterns in different compartments of the rabbit atrioventricular conduction axis. These features, synthesized in a novel 3D model, provide a structural framework for the interpretation of nodal function.

Large uncontrollable detrusor contractions, decreased compliance that increases luminal pressures during filling, and detrusor underactivity are all examples of abnormal bladder function. Studies of the nervous control of lower urinary tract function and measurement of the cellular properties of the component tissues of the bladder wall have been performed to deepen our knowledge of these problems. The resultant data have suggested that lower urinary tract smooth muscle should not be regarded solely as a collection of independent cellular contractile units that are each activated by separate neural inputs, but also as a syncytium of cells. Although this syncytial arrangement may not be as well developed as in other tissues, it should impose a new layer of activity that will affect overall bladder function. Recent studies have addressed this issue through investigation of spontaneous contractile activity, the cellular basis of syncytial function, and their normal and abnormal functional consequences. The results suggest that individual detrusor cells possess membrane properties that may lead to spontaneous activity fluctuations, which can affect adjacent cells and, thus, produce multicellular aberrant responses. It remains unclear whether these responses manifest themselves as dysfunctional activity in the whole bladder defects because the extent of local multicellular abnormalities is not known at present. The data do imply that myogenic defects can contribute to abnormal bladder function and, thus, suggest several new targeted drug models that should be explored.

Introduction: Gap junctional connexin proteins (connexin40 [Cx40], connexin43 [Cx43]) are a determinant of myocardial conduction and are implicated in the development of atrial fibrillation (AF). We hypothesized that atrial activation pattern during AF is related to connexin expression and that this relationship is altered by AF-induced remodeling in the fibrillating atria of chronic AF. Methods and Results: Isochronal activation mapping was performed during cardiac surgery on the right atria of patients in chronic AF (n = 13) using an epicardial electrode array. The atrial activation pattern was categorized using a complexity score based on the number of propagating wavefronts of activation and by grouping atria into those capable of uniform planar activation (simple) and those that were not (complex). The activation pattern was correlated with the levels of Cx43 and Cx40 signal measured by immunoconfocal quantification of biopsies from the mapped region. We studied the impact of electrical remodeling by comparing these findings with the unremodeled atria of patients in sinus rhythm during pacing-induced sustained AF (n = 17). In chronic AF, atria with complex activation had lower Cx40 signal than atria showing simple activation (0.013 ± 0.006 μm2/μm2 vs 0.027 ± 0.009 μm2/μm2, P < 0.02), with the relative connexin signal (Cx40/Cx40+Cx43) correlating with complexity score (P = 0.01, r =−0.74). This relationship did not occur in the unremodeled atria, and increased heterogeneity of distribution of Cx40 labeling in chronic AF was the only evidence of connexin remodeling that we detected in the overall group. Conclusion: The pattern of atrial activation is related to immunoconfocal connexin signal only in the fully remodeled atria of chronic AF. This suggests that intercellular coupling and pattern of atrial activation are interrelated, but only in conjunction with the remodeling of atrial electrophysiology that occurs in chronic AF. (J Cardiovasc Electrophysiol, Vol. 15, pp. 206-213, February 2004)

Objective— We sought to clarify the response of endothelial connexins to hyperlipidemia and lipid-lowering therapy. Methods and Results— Aortic endothelial gap junctions were analyzed by en face immunoconfocal microscopy and electron microscopy in C57BL/6 mice subjected to the following regimens: (1) normal chow (NC) for 3 months (3 mo), (2) NC for 9 mo, (3) NC for 3 mo, followed by a cholesterol-enriched diet (CED) for 6 mo, (4) NC for 3 mo and CED for 6 mo, with simvastatin in the final week, and (5) (in apoprotein E [apoE]-deficient mice) NC and examined at 3 mo and 7 to 9 mo. In wild-type mice, connexin37 (Cx37) and Cx40 were markedly downregulated in the CED-fed animals compared with those fed NC (CED vs 9-mo NC, 77% reduction in Cx37 and 65% reduction in Cx40; both P P P Conclusions— Mouse aortic endothelial gap junctions and connexins are downregulated during long-term hyperlipidemia. Short-term treatment with simvastatin leads to recovery of Cx37 expression but not Cx40 expression.

Three of this month's Scientific Discovery papers highlight the importance of collaboration in delivering high quality scientific research. As scientific technology increases in power and cost, and specific areas of interest become more specialized, it is becoming more difficult to cover all aspects of a completeresearch story. Collaborating with other experts in the field or other fields, including industry, allows strong scientific proof to be generated for the hypothesis and aims. Building strong collaborative,inter-disciplinary, multi-institutional, international groups with academic and industrial partners is the way forward for all discovery. We look forward to publishing more of these collaborative papersin future issues of the BJU International. OBJECTIVES To test the hypothesis that intercellular electrical coupling is altered in human detrusor smooth muscle from patients with unstable bladders. MATERIALS AND METHODS Human detrusor biopsy samples were obtained from patients with stable and unstable bladders. Intracellular electrical impedance was measured with alternating current (20 Hz−300 kHz) across the ends of detrusor strips in an oil-gap, after correcting for extracellular space resistance. Gap junctions were identified by localization of connexins (Cx), specifically Cx45, Cx43 and Cx40 transcripts, using immunoconfocal microscopy. RESULTS Total intracellular resistivity was greater in strips from unstable than from stable bladders (median 1246 vs 817 Ω.cm). The increase was attributed to an increase in junctional resistance; cytoplasmic resistance was unchanged. Cx43 was localized to a submucosal layer and to connective tissue; Cx40 label was confined to endothelial cells of blood vessels. Cx45 labelling was localized to detrusor bundles and appeared to be less marked in samples from unstable bladders. Semi-quantitative analysis of Northern blots showed that Cx45 expression in unstable was less than that in stable bladders. CONCLUSIONS These data suggest that intercellular coupling is reduced in detrusor from unstable bladders. Cx45 was localized to the detrusor layer, with Cx 43 more evident in the suburothelial mucosa. Cx45 labelling was less intense in detrusor samples from unstable bladders. These results are consistent with reduced gap junction coupling in detrusor from unstable bladders.

Gap junctions, clusters of transmembrane channels that link adjoining cells, mediate myocyte-to-myocyte electrical coupling and communication. The component proteins of gap junction channels are termed connexins, and gap-junctional channels composed of different connexin types exhibit different biophysical properties. In common with other tissues, the heart expresses multiple connexin isotypes. Spatially defined patterns of expression of 3 connexin isotypes-connexin43, connexin40, and connexin45-form specific cell-to-cell conduction pathways for the spread of current flow that governs the normal cardiac rhythm. Remodeling of gap junction organization and connexin expression is a conspicuous feature of human congestive heart failure and other cardiac conditions in which there is an arrhythmic tendency. This remodeling may take the form of disturbances in the distribution of gap junctions, in which the normal ordered pathways for cell-to-cell conduction are disrupted, or quantitative alterations in connexin expression, notably reduced connexin43 levels, which may contribute to slowing of conduction. Recent evidence from studies in experimental animals strengthens the case that gap junction remodeling is a key determinant of the proarrhythmic substrate in the diseased heart.

Objective To determine whether suburothelial interstitial cells of the human bladder express gap junctions, and if so, to establish their extent and composition, using immunocytochemistry, confocal microscopy and electron microscopy. Materials and methods Bladder tissue was obtained at cystectomy; the tissue was: (i) frozen for cryosectioning and Northern blot analysis; (ii) fixed and embedded for standard thin-section electron microscopy; and (iii) processed using low-denaturation conditions in Lowicryl for immunogold-label electron microscopy. Cryosections were immunofluorescently labelled using antibodies against connexins 43, 40 and 45, vimentin, desmin and c-Kit ligand, and examined by confocal microscopy. Double labelling was used to determine the spatial relationship of labelling for connexin43 with that of vimentin and desmin. Thin-section electron microscopy was used to investigate interstitial cell ultrastructure and permit unequivocal identification of gap junctions, and immunogold labelling of Lowicryl sections was applied to localize connexin43. Results Immunoconfocal microscopy showed prominent labelling for the gap junction protein, connexin43, in a suburothelial band of cells that was also strongly positive for vimentin. The connexin43/vimentin-positive cells showed only weak labelling for desmin and c-Kit ligand, and were immunonegative for connexins 40 and 45. Northern blotting showed a corresponding abundance of connexin43 transcript in the mucosal layer but not the detrusor layer of the bladder wall. Electron microscopy revealed abundant gap junctions, recognized by their pentalaminar structure, between the cell processes of interstitial cells in the suburothelial zone. That these interstitial cell gap junctions were the source of the connexin43 immunolabelling observed by immunoconfocal microscopy was confirmed by immunogold labelling in sections of Lowicryl-embedded tissue examined by electron microscopy. Conclusion A network of interstitial cells, extensively linked by connexin43-containing gap junctions, is located beneath the urothelium in human bladder. As gap junctions provide pathways for direct cell-to-cell communication, the interstitial cellular network may operate as a functional syncytium, integrating signals and responses in the bladder wall.

Objective: Acute administration of l-arginine (LA), the physiological substrate of nitric oxide, has been used as a strategy for myocardial protection during ischemia-reperfusion. The aim of this study was to assess the effects of chronic oral LA administration on vascular functions and morphology after prolonged cold cardioplegic arrest. Methods: Adult male Sprague–Dawley rats (600–650 g) were divided into control and LA groups, which received LA (4 mg/ml) for 6 weeks. Two experimental protocols were carried out. (1) Isolated rat heart perfusion was performed and hearts were subjected to ischemia for 4 h at 48C using cold crystalloid cardioplegia (n ¼ 8 in LA, n ¼ 7i n control). Endothelial and vascular smooth muscle functions were assessed through observations of pre- and post-ischemic coronary flow response to 5-hydroxytryptamine (5-HT) and glyceryl trinitrate (GTN) (%5-HT and %GTN, respectively). (2) Semi-quantitative assessment of tissue morphology was conducted after the same ischemia-reperfusion protocol (n ¼ 4 in each group). Results: The LA group showed significantly better recovery (post-/pre-ischemic value) of %5-HT (97.0 ^ 65.6 versus 21.5 ^ 25.7%, P ¼ 0:015) and %GTN (124.5 ^ 117.6 versus 47.7 ^ 16.6%, P ¼ 0:021). The histological assessment showed no significant differences between the two groups. Conclusions: Chronic oral administration of LA significantly ameliorated the postischemic recovery of endothelial and vascular smooth muscle functions after cold cardioplegic arrest in rats. q 2002 Elsevier Science B.V. All rights reserved.

ObjectivesThis study characterizes the histology of myocardium predicted to be hibernating using three different imaging techniques to explain the discordance among them.BackgroundBoth radionuclide and functional imaging techniques were used to assess myocardial hibernation. The former have high sensitivity and the latter high specificity for predicting functional recovery.MethodsNineteen patients underwent thallium-201 and 99m-technetium tetrofosmin myocardial perfusion imaging, and dobutamine magnetic resonance imaging (MRI), prior to coronary bypass grafting. Criteria for predicted hibernation for each technique were defined before operation. Postoperative criteria for scar and true hibernation were also defined. Biopsies were analyzed for myocyte volume fraction (MVF), glycogen deposition and pathologic cell features.ResultsThallium was most sensitive in predicting hibernation (88%) and MRI most specific (84%); and, although there was good agreement between thallium and tetrofosmin (85%), agreement between MRI and thallium (59%) or tetrofosmin (59%) was poor. For each technique, MVF was higher in segments predicted to be hibernating rather than scar (p < 0.05). The MVF was higher where both thallium and MRI predicted hibernation (0.77 ± 0.07) than in segments predicted by thallium alone (0.69 ± 0.13, p < 0.05). Proven hibernating segments had a higher MVF than scar (0.72 ± 0.11 vs. 0.6 ± 0.26, p < 0.05).ConclusionsPreservation of myocyte fraction is an important determinant of functional recovery after revascularization. A higher myocyte fraction is required to maintain contractile reserve than to achieve significant tracer uptake. This explains the higher sensitivity of radionuclide imaging compared with dobutamine MRI in the identification of myocardial hibernation.

The HL-1 atrial line contains cells blocked at various developmental stages. To obtain homogeneous sub-clones and correlate changes in gene expression with functional alterations, individual clones were obtained and characterised for parameters involved in conduction and excitation-contraction coupling. Northern blots for mRNAs coding for connexins 40, 43 and 45 and calcium handling proteins (sodium/calcium exchanger, L- and T-type calcium channels, ryanodine receptor 2 and sarco-endoplasmic reticulum calcium ATPase 2) were performed. Connexin expression was further characterised by western blots and immunofluorescence. Inward currents were characterised by voltage clamp and conduction velocities measured using microelectrode arrays. The HL-1 clones had similar sodium and calcium inward currents with the exception of clone 2 which had a significantly smaller calcium current density. All the clones displayed homogenous propagation of electrical activity across the monolayer correlating with the levels of connexin expression. Conduction velocities were also more sensitive to inhibition of junctional coupling by carbenoxolone (∼80%) compared to inhibition of the sodium current by lidocaine (∼20%). Electrical coupling by gap junctions was the major determinant of conduction velocities in HL-1 cell lines. In summary we have isolated homogenous and stable HL-1 clones that display characteristics distinct from the heterogeneous properties of the original cell line.

Gap junctions, clusters of transmembrane channels that link adjoining cells, mediate myocyte-to-myocyte electrical coupling and communication. The component proteins of gap junction channels are termed connexins and, in in vitro expression systems, gap-junctional channels composed of different connexin types exhibit different biophysical properties. In common with other tissues, the heart expresses multiple connexin isoforms. Spatially defined patterns of expression of three connexin isoforms - connexin43, connexin40 and connexin45 - form the cell-to-cell conduction pathways responsible for the orderly spread of current flow that governs the normal cardiac rhythm. Remodeling of gap junction organization and connexin expression is a common feature of human heart disease conditions in which there is an arrhythmic tendency. This remodeling may take the form of disturbances in the distribution of gap junctions and/or quantitative alterations in connexin expression, notably reduced ventricular connexin43 levels. The idea that such changes may contribute to the development of a pro-arrhythmic substrate in the diseased heart has gained ground over the last decade. Recent studies using transgenic mice models have raised new opportunities to explore the significance of gap junction remodeling in the diseased heart.

Background —Atrial fibrillation (AF), a cardiac arrhythmia arising from atrial re-entrant circuits, is a common complication after cardiac surgery, but the proarrhythmic substrate underlying the development of postoperative AF remains unclear. This study investigated the hypothesis that altered expression of connexins, the component proteins of gap junctions, is a determinant of a predisposition to AF. Methods and Results —The expression of the 3 atrial connexins–connexins 43, 40, and 45—was analyzed at the mRNA and protein levels by Northern and Western blotting techniques and immunoconfocal microscopy in right atrial appendages from patients with ischemic heart disease who were undergoing coronary artery bypass surgery. Twenty percent of the patients subsequently developed AF, which allowed retrospective division of the samples into 2 groups, non-AF and AF. Connexin43 and connexin45 transcript and protein levels did not differ between the groups. However, connexin40 transcript and protein were expressed at significantly higher levels in the AF group. Connexin40 protein was markedly heterogeneous in distribution. Conclusions —Atrial myocardium susceptible to AF is distinguished from its nonsusceptible counterpart by elevated connexin40 expression. The heterogeneity of connexin distribution could give rise to different resistive properties and conduction velocities in spatially adjacent regions of tissue, which become enhanced and, hence, proarrhythmic the higher the overall level of connexin40.

Congestive heart failure is associated with a high risk of life-threatening ventricular re-entrant arrhythmias. Down-regulation of the principal gap-junctional protein of the ventricular myocytes, connexin43, has previously been implicated in arrhythmia in ischaemic heart disease, but it is not known whether connexin43 is similarly reduced in heart failure due to idiopathic dilated cardiomyopathy, whether disease-related connexin43 down-regulation occurs at the level of transcription or translation, or whether the expression of other connexin isotypes is altered in congestive heart failure. We therefore investigated the expression of the four connexins expressed in the heart-connexins 43, 40, 45 and 37-at the mRNA and protein levels in explanted hearts from transplant patients with end-stage heart failure (NYHA class 4) by immunoconfocal analysis, and northern and western blotting. Connexin43 mRNA and protein were markedly downregulated in the left ventricle in end-stage heart failure due both to ischaemic cardiomyopathy and idiopathic dilated cardiomyopathy. Connexin43 content was spatially heterogeneous in the diseased ventricle. Connexin40 mRNA was increased in the ischaemic group, more so in the left ventricle than the right. This correlated with an increased depth of connexin40 protein expression in myocytes at the endocardial surface. Connexin45 mRNA and protein, present only in very low quantities, followed a similar trend to connexin43, while connexin37 (exclusively expressed in endothelium) showed no change. Our findings show that congestive heart failure is associated with significantly reduced levels of the principal gap junction protein, connexin43, in the left ventricle, potentially contributing to enhanced arrhythmogenicity and contractile dysfunction. This down-regulation is due predominantly to a reduced transcript steady-state level. Elevated connexin40 may represent a compensatory response that improves the spread of depolarization in the otherwise compromised ischaemic ventricle.

Gap junctions play essential roles in the normal function of the heart and arteries, mediating the spread of the electrical impulse that stimulates synchronized contraction of the cardiac chambers, and contributing to co-ordination of activities between cells of the arterial wall. In common with other multicellular systems, cardiovascular tissues express multiple connexin isotypes that confer distinctive channel properties. This review highlights how state-of-the-art immunocytochemical and cellular imaging techniques, as part of a multidisciplinary approach in gap junction research, have advanced our understanding of connexin diversity in cardiovascular cell function in health and disease. In the heart, spatially defined patterns of expression of three connexin isotypes-connexin43, connexin40, and connexin45-underlie the precisely orchestrated patterns of current flow governing the normal cardiac rhythm. Derangement of gap junction organization and/or reduced expression of connexin43 are associated with arrhythmic tendency in the diseased human ventricle, and high levels of connexin40 in the atrium are associated with increased risk of developing atrial fibrillation after coronary by-pass surgery. In the major arteries, endothelial gap junctions may simultaneously express three connexin isotypes, connexin40, connexin37, and connexin43; underlying medial smooth muscle, by contrast, predominantly expresses connexin43, with connexin45 additionally expressed at restricted sites. In normal arterial smooth muscle, the abundance of connexin43 gap junctions varies according to vascular site, and shows an inverse relationship with desmin expression and positive correlation with the quantity of extracellular matrix. Increased connexin43 expression between smooth muscle cells is closely linked to phenotypic transformation in early human coronary atherosclerosis and in the response of the arterial wall to injury. Current evidence thus suggests that gap junctions in both their guises, as pathways for cell-to-cell signaling in the vessel wall and as pathways for impulse conduction in the heart, contribute to the initial pathogenesis and eventual clinical manifestation of human cardiovascular disease.

The synchronous contractions of the uterus in labour depend on electrical coupling of myometrial smooth muscle cells by gap junctions. In the human myometrium, gap junctions are scarce in the non-pregnant uterus, but become abundant at term in preparation for labour. We have previously demonstrated that in the human myometrium at term, three different gap-junctional proteins are expressed, connexins 43, 45, and 40. These connexins are known to have distinctive functional capacities in in vitro expression systems but whether, in the human myometrium in vivo, they are co-assembled into the same gap junction or form different types of gap junction has previously been unclear. By applying triple immunogold labelling to sections of Lowicryl-embedded tissue for electron microscopy, together with complementary immunoconfocal microscopy, we demonstrate here that connexins 43, 45, and 40 are commonly present as mixtures within the same gap-junctional plaque. While all gap junctions contain connexin43, the relative signal for each connexin type varies between individual junctions. The presence within single gap-junctional plaques of three different connexins, each with the potential for conferring distinctive channel properties, suggests an inherent versatility for modulation of smooth muscle cell intercellular communication properties during human parturition. Microsc. Res. Tech. 54:114–122, 2001. © 2001 Wiley-Liss, Inc.

The mouse is currently widely used as a model organism in the analysis of gene function but how developmentally regulated patterns of connexin gene expression in the mouse compare with those in the human is unclear. Here we compare the patterns of connexin expression in the heart during the development of the mouse (from embryonic day 12.5 to 6 weeks postpartum) and the human (at 9 weeks gestation and adult stage). The extent of connexin43 expression in the ventricles progressively increased during development of the mouse heart. The developmental pattern of expression for connexins 40 and 45 in the mouse heart was similar, but not identical, and in the ventricles showed a progressive and preferential expression in the conduction system. In general, these dynamic changes of connexins 43, 40 and 45 during mouse cardiac development appear to be mirrored in the human.

Background —Genetic mutations of dystrophin and associated glycoproteins underlie cell degeneration in several inherited cardiomyopathies, although the precise physiological role of these proteins remains under discussion. We studied the distribution of dystrophin in relation to the force-transducing vinculin-rich costameres in left ventricular cardiomyocytes from normal and failing human hearts to further elucidate the function of this protein complex. Methods and Results —Single- and double-label immunoconfocal microscopy and parallel high-resolution immunogold fracture-label electron microscopy were used to localize dystrophin and vinculin in human left ventricular myocytes from normal (n=6) and failing hearts (idiopathic dilated cardiomyopathy, n=7, or ischemic heart disease, n=5). In control cardiomyocytes, dystrophin had a continuous distribution at the peripheral sarcolemma, with concentrated bands corresponding to the vinculin-rich costameres. Intracellular labeling extended along transverse (T) tubule membranes. Fracture-label confirmed this distribution, showing significantly greater label on plasma membrane fractures overlying I-bands (I-band 4.1±0.3 gold particles/μm; A-band 3.3±0.2 gold particles/μm: mean±SE, P =0.02). Hypertrophied myocytes from failing hearts showed maintenance of this surface distribution except in degenerating cells; there was a clear increase in intracellular dystrophin label reflecting T-tubule hypertrophy. Conclusions —Dystrophin partially colocalizes with costameric vinculin in normal and hypertrophied myocytes, a distribution lost in degenerating cells. This suggests a primarily mechanical role for dystrophin in maintenance of cell membrane integrity in normal and hypertrophied myocytes. The presence of dystrophin in the cardiac T-tubule membrane, in contrast to its known absence in skeletal muscle T-tubules, implies additional roles for dystrophin in membrane domain organization.

SUMMARY The aim of this study was to establish, using immunolabeling, whether the Kv1.5 K 1 channel is present in the pacemaker of the heart, the sinoatrial (SA) node. In the atrial muscle surrounding the SA node and in the SA node itself (from guinea pig and ferret), Western blotting analysis showed a major band of the expected molecular weight, z 64 kD. Confocal microscopy and immunofluorescence labeling showed Kv1.5 labeling clustered in atrial muscle but punctate in the SA node. In atrial muscle, Kv1.5 labeling was closely associated with labeling of Cx43 (gap junction protein) and DPI/II (desmosomal protein), whereas in SA node Kv1.5 labeling was closely associated with labeling of DPI/II but not labeling of Cx43 (absent in the SA node) or Cx45 (another gap junction protein present in the SA node). Electron microscopy and immunogold labeling showed that the Kv1.5 labeling in atrial muscle is preferentially associated with desmosomes rather than gap junctions. (J Histochem Cytochem 48:769‐780, 2000)

Abstract —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.

The cardiac myocyte is the most physically energetic cell in the body, contracting constantly, without tiring, 3 billion times or more in an average human lifespan. By coordinating its beating activity with that of its 3 billion neighbours in the main pump of the human heart, over 7,000 litres of blood are pumped per day, without conscious effort, along 100,000 miles of blood vessels. A detailed picture of the membrane organisation of the cardiac muscle cell underpins our understanding of how the electrical impulse, generated within the heart, stimulates coordinated contraction of the cardiac chambers. This article highlights, with the aid of modern cellular imaging methods, key components of the membrane machinery responsible for coupling electrical excitation and contraction in the cardiomyocyte, focusing on plasma membrane/sarcoplasmic reticulum and plasma membrane/plasma membrane junctions. BioEssays 22:188-199, 2000.

The cardiomyocyte membrane cytoskeleton consists of the costameric proteins that mediate force transduction from the cell to the extracellular matrix, and a sub-membrane network composed of dystrophin and associated proteins. Studies of the precise cellular distribution of dystrophin and of the consequences of genetic mutations leading to abnormal expression of the dystrophin molecule, as occurs in Duchenne and Becker's muscular dystrophies, highlight potential functional roles of this sub-membrane protein complex in cardiomyocytes. Detailed investigation of dystrophin distribution using the complementary cell imaging techniques of immunoconfocal microscopy and freeze-fracture cytochemistry at the electron-microscopical level show that, in contrast to rat cardiomyocytes, the dystrophin network in human cardiomyocytes is locally enriched at costameres. Thus located, the dystrophin network appears to have a mechanical role, involving stabilization of the peripheral plasma membrane during the repetitive distortion associated with cardiac contraction and, in the human myocyte, contributing to lateral force-transduction. Evidence from animal models of muscular dystrophy and from investigation of the interactions of the sub-membrane cytoskeleton with other membrane-associated proteins including ion channels, receptors and enzymes, further suggests a role for dystrophin in organization and regulation of membrane domains. The relative preservation of the membrane cytoskeleton in non-dystrophic dilated cardiomyopathy and in ischemic cardiomyopathy, conditions in which the myocyte contractile apparatus and internal desmin-based cytoskeleton are commonly disrupted, emphasizes the vital role of the membrane cytoskeleton in cell survival. Continued cardiomyocyte survival despite loss of contractile protein organization has implications in the potential for reversibility of left ventricular remodeling that can be achieved in the clinical setting.

The pacemaker of the heart, the sinoatrial (SA) node, is characterized by unique electrical coupling properties. To investigate the contribution of gap junction organization and composition to these properties, the spatial pattern of expression of three gap junctional proteins, connexin45 (Cx45), connexin40 (Cx40), and connexin43 (Cx43), was investigated by immunocytochemistry combined with confocal microscopy. The SA nodal regions of rabbits were dissected and rapidly frozen. Serial cryosections were double labeled for Cx45 and Cx43 and for Cx40 and Cx43, using pairs of antibody probes raised in different species. Dual-channel scanning confocal microscopy was applied to allow simultaneous visualization of the different connexins. Cx45 and Cx40, but not Cx43, were expressed in the central SA node. The major part of the SA nodal-crista terminalis border revealed a sharply demarcated boundary between Cx43-expressing myocytes of the crista terminalis and Cx45/Cx40-expressing myocytes of the node. On the endocardial side, however, a transitional zone between the crista terminalis and the periphery of the node was detected in which Cx43 and Cx45 expression merged. These distinct patterns of connexin compartmentation and merger identified suggest a morphological basis for minimization of contact between the tissues, thereby restricting the hyperpolarizing influence of the atrial muscle on the SA node while maintaining a communication route for directed exit of the impulse into the crista terminalis.

Abstract —Upregulation of connexin43-gap junctions is associated with transition of contractile vascular smooth muscle cells (SMCs) to the synthetic state. To determine whether phenotypically distinct subpopulations of medial SMCs differentially express connexin43, we investigated the human distal internal mammary artery, a structurally heterogeneous vessel with features ranging from elastic to elastomuscular to muscular. Immunoconfocal microscopy combined with quantitative analysis and complemented by in situ hybridization showed that SMCs in the elastic medial regions expressed high levels of connexin43 but low levels of desmin, whereas those of muscular medial regions expressed low levels of connexin43 but high levels of desmin. Ultrastructurally, SMCs of both regions were of the contractile phenotype, but the former cells were irregular in shape with relatively prominent synthetic organelles whereas the latter were spindle shaped with fewer synthetic organelles. Vimentin, smooth muscle α-actin, calponin, h-caldesmon, and myosin heavy chains (SM1 and SM2) were equally highly expressed by most cells in both subpopulations. The connexin43/desmin expression pattern of SMCs in regions of intimal thickening resembled those of elastic medial regions. These findings refine the view suggested from previous studies that high levels of connexin43 expression are associated with SMCs of a less contractile/more synthetic phenotype. In the internal mammary artery, the 2 subpopulations of SMCs with markedly different connexin43 expression levels both represent a differentiated contractile phenotype, but the subpopulation showing high levels of connexin43-gap junctions is characterized by low levels of desmin and structural features that reflect a more synthetic tendency.

Integration of vascular endothelial function relies on multiple signaling mechanisms, including direct cell-cell communication through gap junctions. Gap junction proteins expressed in the endothelium include connexin37, connexin40, and connexin43. To investigate whether individual endothelial cells in vivo express all three connexin types and, if so, whether multiple connexins are assembled into the same gap junction plaque, we used affinity-purified connexin-specific antibodies raised in three different species to permit multiple-label immunoconfocal and immunoelectron microscopy in the rat main pulmonary artery. Immunoconfocal microscopy showed a high incidence of co-localization between connexin43 and connexin40, but lower incidences of co-localization between connexin37 and connexin40 or connexin43. Immunoelectron microscopy revealed that 83% of gap junction profiles contained all three connexins, with the proportion of connexin40 labeling being significantly higher than that of connexin37 or connexin43. The presence of three different connexin types of distinct properties in vitro provides potential for complex regulation and functional differentiation of endothelial intercellular communication properties in vivo.

We previously demonstrated that α6 (Cx45), one of the three connexins of the mammalian myocardium, is preferentially expressed in the peripheral portion of the ventricular conduction system in rats and mice. Here we report that α6 is also prominently immunolocalized in the atrioventricular node and His bundle of these species. The distribution of immunolocalized α6 reveals that the node and bundle form part of an extended central conductive network circumscribing the AV and outflow junctional regions of the fetal, and less continuously, the adult heart. Of the three cardiac connexins, α6 is the isoform most continuously expressed by conduction tissues, and may thus account for the recently reported viability of the α5 (Cx40) knockout mouse. It is concluded that α6 expression is a defining feature of the heterogenous tissues comprising the atrioventricular conduction system of the rodent heart. Dev. Genet. 24:82–90, 1999. © 1999 Wiley-Liss, Inc.

Mutations in or ablation of the gene encoding caveolin-3, a protein of muscle cell caveolae, result in forms of muscular dystrophy and cardiomyopathy. Another member of the caveolin gene family, caveolin-1, is widely considered not to be expressed in myocytes, yet ablation of the gene encoding this protein in mice also results in cardiomyopathy. By applying the high-resolution electron-microscopical imaging technique of freeze-fracture replica immunolabelling, we report here evidence that caveolin-1 is expressed in human cardiac myocytes, localized to both caveolae and non-caveolar domains in the plasma membrane. Disorders of the myocyte resulting from defects in caveolin-1 may thus arise directly, at the level of the myocyte, rather than via other cell types as previously proposed.