Characterisation of re-entrant circuit (or rotational activity) in vitro using the HL1-6 myocyte cell line.

Fibrillation is the most common arrhythmia observed in clinical practice. Understanding of the mechanisms underlying its initiation and maintenance remains incomplete. Functional re-entries are potential drivers of the arrhythmia. Two main concepts are still debated, the "leading circle" and the "spiral wave or rotor" theories. The homogeneous subclone of the HL1 atrial-derived cardiomyocyte cell line, HL1-6, spontaneously exhibits re-entry on a microscopic scale due to its slow conduction velocity and the presence of triggers, making it possible to examine re-entry at the cellular level. We therefore investigated the re-entry cores in cell monolayers through the use of fluorescence optical mapping at high spatiotemporal resolution in order to obtain insights into the mechanisms of re-entry. Re-entries in HL1-6 myocytes required at least two triggers and a minimum colony area to initiate (3.5 to 6.4 mm2). After electrical activity was completely stopped and re-started by varying the extracellular K+ concentration, re-entries never returned to the same location while 35% of triggers re-appeared at the same position. A conduction delay algorithm also allows visualisation of the core of the re-entries. This work has revealed that the core of re-entries is conduction blocks constituted by lines and/or groups of cells rather than the round area assumed by the other concepts of functional re-entry. This highlights the importance of experimentation at the microscopic level in the study of re-entry mechanisms.

Functional consequences of co-expressing connexin40 or connexin45 with connexin43 on intercellular electrical coupling.

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.

Impact of interface roughness distributions on the operation of quantum cascade lasers.

We study the impact of interface roughness on the operation of mid-IR and THz quantum cascade lasers. Particular emphasis is given towards the differences between the Gaussian and exponential roughness distribution functions, for which we present results from simulation packages based on nonequilibrium Green's functions and density matrices. The Gaussian distribution suppresses scattering at high momentum transfer which enhances the lifetime of the upper laser level in mid-IR lasers. For THz lasers, a broader range of scattering transitions is of relevance, which is sensitive to the entire profile of the interface fluctuations. Furthermore we discuss the implementation of interface roughness within a two band model.

Electronic temperatures of terahertz quantum cascade active regions with phonon scattering assisted injection and extraction scheme.

We measured the lattice and subband electronic temperatures of terahertz quantum cascade devices based on the optical phonon-scattering assisted active region scheme. While the electronic temperature of the injector state (j = 4) significantly increases by ΔT = T(e)(4) - T(L) ~40 K, in analogy with the reported values in resonant phonon scheme (ΔT ~70-110 K), both the laser levels (j = 2,3) remain much colder with respect to the latter (by a factor of 3-5) and share the same electronic temperature of the ground level (j = 1). The electronic population ratio n(2)/n(1) shows that the optical phonon scattering efficiently depopulates the lower laser level (j = 2) up to an electronic temperature T(e) ~180 K.

Cytoplasm resistivity of mammalian atrial myocardium determined by dielectrophoresis and impedance methods.

Many cardiac arrhythmias are caused by slowed conduction of action potentials, which in turn can be due to an abnormal increase of intracellular myocardial resistance. Intracellular resistivity is a linear sum of that offered by gap junctions between contiguous cells and the cytoplasm of the myocytes themselves. However, the relative contribution of the two components is unclear, especially in atrial myocardium, as there are no precise measurements of cytoplasmic resistivity, R(c). In this study, R(c) was measured in atrial tissue using several methods: a dielectrophoresis technique with isolated cells and impedance measurements with both isolated cells and multicellular preparations. All methods yielded similar values for R(c), with a mean of 138 ± 5 Ω·cm at 23°C, and a Q(10) value of 1.20. This value is about half that of total intracellular resistivity and thus will be a significant determinant of the actual value of action potential conduction velocity. The dielectrophoresis experiments demonstrated the importance of including divalent cations (Ca(2+) and Mg(2+)) in the suspension medium, as their omission reduced cell integrity by lowering membrane resistivity and increasing cytoplasm resistivity. Accurate measurement of R(c) is essential to develop quantitative computational models that determine the key factors contributing to the development of cardiac arrhythmias.

Influence of v5/6-His tag on the properties of gap junction channels composed of connexin43, connexin40 or connexin45.

HeLa cells expressing wild-type connexin43, connexin40 or connexin45 and connexins fused with a V5/6-His tag to the carboxyl terminus (CT) domain (Cx43-tag, Cx40-tag, Cx45-tag) were used to study connexin expression and the electrical properties of gap junction channels. Immunoblots and immunolabeling indicated that tagged connexins are synthesized and targeted to gap junctions in a similar manner to their wild-type counterparts. Voltage-clamp experiments on cell pairs revealed that tagged connexins form functional channels. Comparison of multichannel and single-channel conductances indicates that tagging reduces the number of operational channels, implying interference with hemichannel trafficking, docking and/or channel opening. Tagging provoked connexin-specific effects on multichannel and single-channel properties. The Cx43-tag was most affected and the Cx45-tag, least. The modifications included (1) V(j)-sensitive gating of I(j) (V(j), gap junction voltage; I(j), gap junction current), (2) contribution and (3) kinetics of I(j) deactivation and (4) single-channel conductance. The first three reflect alterations of fast V(j) gating. Hence, they may be caused by structural and/or electrical changes on the CT that interact with domains of the amino terminus and cytoplasmic loop. The fourth reflects alterations of the ion-conducting pathway. Conceivably, mutations at sites remote from the channel pore, e.g., 6-His-tagged CT, affect protein conformation and thus modify channel properties indirectly. Hence, V5/6-His tagging of connexins is a useful tool for expression studies in vivo. However, it should not be ignored that it introduces connexin-dependent changes in both expression level and electrophysiological properties.

Human connexin31.9, unlike its orthologous protein connexin30.2 in the mouse, is not detectable in the human cardiac conduction system.

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.

Remodelling of gap junctions and connexin expression in diseased myocardium.

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.

Gap junction remodelling in human heart failure is associated with increased interaction of connexin43 with ZO-1.

AIMS: Remodelling of gap junctions, involving reduction of total gap junction quantity and down-regulation of connexin43 (Cx43), contributes to the arrhythmic substrate in congestive heart failure. However, little is known of the underlying mechanisms. Recent studies from in vitro systems suggest that the connexin-interacting protein zonula occludens-1 (ZO-1) is a potential mediator of gap junction remodelling. We therefore examined the hypothesis that ZO-1 contributes to reduced expression of Cx43 gap junctions in congestive heart failure. METHODS AND RESULTS: Left ventricular myocardium from healthy control human hearts (n = 5) was compared with that of explanted hearts from transplant patients with end-stage congestive heart failure due to idiopathic dilated cardiomyopathy (DCM; n = 5) or ischaemic cardiomyopathy (ICM; n = 5). Immunoconfocal and immunoelectron microscopy showed that ZO-1 is specifically localized to the intercalated disc of cardiomyocytes in control and failing ventricles. ZO-1 protein levels were significantly increased in both DCM and ICM (P = 0.0025), showing a significant, negative correlation to Cx43 levels (P = 0.0029). There was, however, no significant alteration of ZO-1 mRNA (P = 0.537). Double immunolabelling demonstrated that a proportion of ZO-1 label is co-localized with Cx43, and that co-localization of Cx43 with ZO-1 is significantly increased in the failing ventricle (P = 0.003). Interaction between the two proteins was confirmed by co-immunoprecipitation. The proportion of Cx43 that co-immunoprecipitates with ZO-1 was significantly increased in the failing heart. CONCLUSION: Our findings suggest that ZO-1, by interacting with Cx43, plays a role in the down-regulation and decreased size of Cx43 gap junctions in congestive heart failure.

Concurrent micro- to macro-cardiac electrophysiology in myocyte cultures and human heart slices.

The contact cardiac electrogram is derived from the extracellular manifestation of cellular action potentials and cell-to-cell communication. It is used to guide catheter based clinical procedures. Theoretically, the contact electrogram and the cellular action potential are directly related, and should change in conjunction with each other during arrhythmogenesis, however there is currently no methodology by which to concurrently record both electrograms and action potentials in the same preparation for direct validation of their relationships and their direct mechanistic links. We report a novel dual modality apparatus for concurrent electrogram and cellular action potential recording at a single cell level within multicellular preparations. We further demonstrate the capabilities of this system to validate the direct link between these two modalities of voltage recordings.

Analytical approaches for myocardial fibrillation signals.

Atrial and ventricular fibrillation are complex arrhythmias, and their underlying mechanisms remain widely debated and incompletely understood. This is partly because the electrical signals recorded during myocardial fibrillation are themselves complex and difficult to interpret with simple analytical tools. There are currently a number of analytical approaches to handle fibrillation data. Some of these techniques focus on mapping putative drivers of myocardial fibrillation, such as dominant frequency, organizational index, Shannon entropy and phase mapping. Other techniques focus on mapping the underlying myocardial substrate sustaining fibrillation, such as voltage mapping and complex fractionated electrogram mapping. In this review, we discuss these techniques, their application and their limitations, with reference to our experimental and clinical data. We also describe novel tools including a new algorithm to map microreentrant circuits sustaining fibrillation.

Relationship of connexin43 expression to phenotypic modulation in cultured human aortic smooth muscle cells.

Transition of arterial smooth muscle cells from the contractile to the synthetic phenotype in vivo is associated with up-regulation of the gap-junctional protein, connexin43 (Cx43). However, the role of increased Cx43 expression in relation to the characteristic features of the synthetic phenotype - altered growth, differentiation or synthetic activity - has not previously been defined. In the present study, growth was induced in cultured human aortic smooth muscle cells by treatment with thrombin and with PDGF-bb; growth arrest was induced by serum deprivation and contact inhibition. Alterations in Cx43 expression and gap-junctional communication were analyzed in relation to expression of markers for contractile differentiation and extracellular matrix synthesis. Treatment with thrombin, but not PDGF-bb, led to up-regulation of Cx43 gap junctions, increased synthetic activity yet also enhanced contractile differentiation. Inhibition of growth by deprivation of serum growth factors in sub-confluent cultures had no effect on Cx43 expression or contractile differentiation. Growth arrest by contact inhibition led to progressive reduction in Cx43 expression, in parallel with progressive increase in expression of differentiation markers but no alteration in synthetic activity. Of a range of stimuli examined, only thrombin had the combined effect of increasing Cx43 gap-junction communication, growth and synthesis, yet it also enhanced contractile differentiation. Down-regulation of Cx43 and improved contractile differentiation occurred only when growth arrest was induced through the contact-inhibition pathway, though, in this instance, synthesis remained undiminished. We conclude that Cx43 levels, though having common correlates, are not exclusively linked to the cell phenotype or the state of growth.

Rotor Tracking Using Phase of Electrograms Recorded During Atrial Fibrillation.

Extracellular electrograms recorded during atrial fibrillation (AF) are challenging to interpret due to the inherent beat-to-beat variability in amplitude and duration. Phase mapping represents these voltage signals in terms of relative position within the cycle, and has been widely applied to action potential and unipolar electrogram data of myocardial fibrillation. To date, however, it has not been applied to bipolar recordings, which are commonly acquired clinically. The purpose of this study is to present a novel algorithm for calculating phase from both unipolar and bipolar electrograms recorded during AF. A sequence of signal filters and processing steps are used to calculate phase from simulated, experimental, and clinical, unipolar and bipolar electrograms. The algorithm is validated against action potential phase using simulated data (trajectory centre error <0.8 mm); between experimental multi-electrode array unipolar and bipolar phase; and for wavefront identification in clinical atrial tachycardia. For clinical AF, similar rotational content (R 2 = 0.79) and propagation maps (median correlation 0.73) were measured using either unipolar or bipolar recordings. The algorithm is robust, uses standard signal processing techniques, and accurately quantifies AF wavefronts and sources. Identifying critical sources, such as rotors, in AF, may allow for more accurate targeting of ablation therapy and improved patient outcomes.

Up-regulation of connexin43 correlates with increased synthetic activity and enhanced contractile differentiation in TGF-beta-treated human aortic smooth muscle cells.

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.

Alterations in cardiac connexin expression in cardiomyopathies.

Gap junctions, assembled from connexins, form the cell-to-cell pathways for propagation of the precisely orchestrated patterns of current flow that govern the synchronized rhythm of the healthy heart. As in most tissues and organs, multiple connexin types are co-expressed in the heart; the connexins Cx43, Cx40 and Cx45 are found in distinctive combinations and relative quantities in different, functionally specialized subsets of cardiomyocytes. Alterations in connexin expression and gap junction organization, now a well-documented feature of human cardiomyopathies, potentially contribute to the pro-arrhythmic substrate. In the diseased ventricle, the most consistently reported quantitative alteration involves heterogeneous reduction in Cx43 expression and disruption of the normal ordered pattern of Cx43 gap junction distribution. Additional studies suggest that upregulation of Cx40 and Cx45 may also feature in the failing ventricle, the former restricted to ischemic cardiomyopathy and localized to the subendocardial region. By correlating data from studies on the human patient with those from animal and cell models, alterations in connexin expression and gap junction organization have emerged as important factors to be considered in understanding the pro-arrhythmic substrate found in human cardiomyopathies.

Enhancement of Gap Junction Function During Acute Myocardial Infarction Modifies Healing and Reduces Late Ventricular Arrhythmia Susceptibility.

OBJECTIVES: The purpose of this study was to investigate the effects of enhancing gap junction (GJ) coupling during acute myocardial infarction (MI) on the healed infarct scar morphology and late post-MI arrhythmia susceptibility. BACKGROUND: Increased heterogeneity of myocardial scarring after MI is associated with greater arrhythmia susceptibility. We hypothesized that short-term enhancement of GJ coupling during acute MI can produce more homogeneous infarct scars, reducing late susceptibility to post-MI arrhythmias. METHODS: Following arrhythmic characterization of a rat 4-week post-MI model (n = 24), another 27 Sprague-Dawley rats were randomized to receive rotigaptide to enhance GJ coupling (n = 13) or to saline control (n = 14) by osmotic minipump immediately prior to and for the first 7 days following surgically induced MI. At 4 weeks post-MI, hearts were explanted for ex vivo programmed electrical stimulation (PES) and optical mapping. Heterogeneity of infarct border zone (IBZ) scarring was quantified by histomorphometry. RESULTS: Despite no detectable differences in infarct size at 4 weeks post-MI, rotigaptide-treated hearts had reduced arrhythmia susceptibility during PES (inducibility score for rotigaptide: 2.4 ± 0.8; for control: 5.0 ± 0.6; p = 0.02) and less heterogeneous IBZ scarring (dispersion of IBZ complexity score: rotigaptide: 1.1 ± 0.1; control: 1.4 ± 0.1; p = 0.04), associated with an improvement in IBZ conduction velocity (rotigaptide: 43.1 ± 3.4 cm/s; control: 34.8 ± 2.0 cm/s; p = 0.04). CONCLUSIONS: Enhancement of GJ coupling for only 7 days at the time of acute MI produced more homogeneous IBZ scarring and reduced arrhythmia susceptibility at 4 weeks post-MI. Short-term GJ modulation at the time of MI may represent a novel treatment strategy to modify the healed infarct scar morphology and reduce late post-MI arrhythmic risk.

Remodelling of gap junctions and connexin expression in heart disease.

Different combinations and relative quantities of three connexins-connexin43, connexin40 and connexin45-are expressed in different subsets of cardiomyocyte. In the healthy heart, gap junctions assembled from these different connexin combinations form the cell-to-cell pathways for the precisely orchestrated patterns of current flow that govern the normal heart rhythm. Remodelling of gap junction organization and connexin expression is a conspicuous feature of human heart disease in which there is an arrhythmic tendency. This remodelling may take the form of structural remodelling, involving disturbances in the distribution of gap junctions (i.e., disruption of the normal ordered pathways for cell-to-cell conduction), and remodelling of connexin expression, involving alteration in the amount or type of connexin(s) present. Most notable among quantitative alterations in connexin expression is a reduction in ventricular connexin43 levels in human congestive heart failure. By correlating data from studies in experimental animal models, gap junction and connexin remodelling emerges as a factor to be considered in understanding the pro-arrhythmic substrate characteristic of many forms of heart disease. However, our knowledge of the functional correlates of the specific patterns of multiple connexin expression found in different regions of the heart in health and disease remains rudimentary, and the development of new experimental cell models heralds advances in this area over the next few years.

Gap junction alterations in human cardiac disease.

Gap junctions, assembled from connexins, 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, connexin40 and connexin45 are found in distinctive combinations and relative quantities in different, functionally specialized subsets of cardiomyocyte. Alterations of gap junction organization and connexin expression are now well established as a consistent feature of human heart disease in which there is an arrhythmic tendency. These alterations may take the form of structural remodelling, involving disturbances in the distribution of gap junctions and/or alteration of the amount or type of connexin(s) expressed. In the diseased ventricles, the most consistent quantitative alteration involves heterogeneous reduction in connexin43 expression. In the atria, features of gap organization and connexin expression have been implicated in the initiation of atrial fibrillation and, once the condition becomes chronic, gap junction alterations associated with remodelling may contribute to persistence of the condition. By correlating data from studies on the human patient with those from animal and cell models, alterations in gap junctions and connexins have emerged as important factors to be considered in understanding the pro-arrhythmic substrate found in a variety of forms of heart disease.

Development of a cell model for functional and structural analysis of connexin co-expression: achieving homogeneous and inducible expression of multiple connexins in stable transfectants.

We set out to develop an in vitro cell model in which connexins 43, 40 and 45 are co-expressed in the same combinations as found in different sub-types of cardiomyocyte in vivo, using inducible promoters of the Tet-Off and Ecdysone systems. In initial studies, a heterogeneous pattern of gene expression was observed. To achieve homogeneous expression, an Internal Ribosome Entry Site (IRES) sequence was employed, ensuring that a single mRNA coded for connexin and antibiotic resistance. We then constructed plasmids that combine the inducibility of the Tet-Off and Ecdysone systems with the homogeneous expression given by the IRES constructs. These were demonstrated to give inducible and homogeneous expression. By using the reporter gene, Enhanced Green Fluorescent Protein (EGFP), it was further shown in the Tet-Off system that expression of the transfected gene was modulated homogeneously in all cells when induction was repressed. The cell model is now at a suitable stage of development for investigation of the functional correlates of the distinctive connexin co-expression found in different regions of the heart.

Alteration of gap junctions and connexins in the right atrial appendage during cardiopulmonary bypass.

OBJECTIVES: We investigated the influence of cardiopulmonary bypass on cardiomyocyte gap junctions and connexins. METHODS: Samples were collected at intervals during operation from the right atrial appendage in 21 patients (mean [+/- SD] age 55 +/- 21 years). Immunodetection of connexins was conducted by Western blotting and confocal microscopy with parallel electron microscopic examination of gap junctions. RESULTS: Downregulation of connexin 43 during the course of operation occurred in more than half of the patients. The mean densitometric value of connexin 43 decreased by 23%, with samples from patients with coronary artery disease showing a greater reduction than seen in those from patients with other diseases (31% +/- 22% vs 10% +/- 24%, P =.04). Such alterations were confirmed by confocal microscopy, which also demonstrated reduced connexin 45 immunolabeling in most patients. Electron microscopy revealed a reduction in the dimensions of cell membrane-located gap junctions and more frequent intracytoplasmic gap junctional membrane in samples from later time points (P =.04). CONCLUSIONS: Downregulation of connexins accompanied by a reduction in gap junctions is common in the cardiomyocytes of the right atrial appendage during cardiopulmonary bypass. The association of a marked reduction in connexin 43 with coronary artery disease may imply inadequate intraoperative cardiac protection in patients with this disease.