Distinct moieties underlie biphasic H+ gating of connexin43 channels, producing a pH optimum for intercellular communication.

Most mammalian cells can intercommunicate via connexin-assembled, gap-junctional channels. To regulate signal transmission, connexin (Cx) channel permeability must respond dynamically to physiological and pathophysiological stimuli. One key stimulus is intracellular pH (pHi), which is modulated by a tissue's metabolic and perfusion status. Our understanding of the molecular mechanism of H+ gating of Cx43 channels-the major isoform in the heart and brain-is incomplete. To interrogate the effects of acidic and alkaline pHi on Cx43 channels, we combined voltage-clamp electrophysiology with pHi imaging and photolytic H+ uncaging, performed over a range of pHi values. We demonstrate that Cx43 channels expressed in HeLa or N2a cell pairs are gated biphasically by pHi via a process that consists of activation by H+ ions at alkaline pHi and inhibition at more acidic pHi. For Cx43 channel-mediated solute/ion transmission, the ensemble of these effects produces a pHi optimum, near resting pHi. By using Cx43 mutants, we demonstrate that alkaline gating involves cysteine residues of the C terminus and is independent of motifs previously implicated in acidic gating. Thus, we present a molecular mechanism by which cytoplasmic acid-base chemistry fine tunes intercellular communication and establishes conditions for the optimal transmission of solutes and signals in tissues, such as the heart and brain.-Garciarena, C. D., Malik, A., Swietach, P., Moreno, A. P., Vaughan-Jones, R. D. Distinct moieties underlie biphasic H+ gating of connexin43 channels, producing a pH optimum for intercellular communication.

Computational simulations of asymmetric fluxes of large molecules through gap junction channel pores.

Gap junction channels are formed out of connexin isoforms, which enable molecule and ion selective diffusion amongst neighboring cells. HeLa cells expressing distinct connexins (Cx) allow the formation of heterotypic channels, where we observed a molecular charge-independent preferential flux of large fluorescent molecules in the Cx45 to Cx43 direction. We hypothesize that the pore's shape is a significant factor along-side charge and transjunctional voltages for this asymmetric flux. To test this hypothesis, we developed a 3D computational model simulating Brownian diffusion of large molecules in a gap junction channel pore. The basic pore contour was derived from x-ray crystallographic structures of Cx43 and Cx26 and approximated using basic geometric shapes. Lucifer yellow dye molecules and cesium counter-ions were modeled as spheres using their respective Stokes radii. Our simulation results from simple diffusion and constant concentration gradient experiments showed that only charged particles yield asymmetric fluxes in heterotypic pores. While increasing the inner mouth size resulted in a near-quadratic rise in flux, the rise was asymptotic for outer mouth radii increase. Probability maps and average force per particle per pore section explain the asymmetric flux with variation in pore shape. Furthermore, the simulation results are in agreement with our in vitro experimental results with HeLa cells in Cx43-Cx45 heterotypic configurations. The presence of asymmetric fluxes can help us to understand effects of the molecular structure of the pore and predict potential differences in vivo.

Novel xeno-free human heart matrix-derived three-dimensional scaffolds.

RATIONALE: Myocardial infarction (MI) results in damaged heart tissue which can progress to severely reduce cardiac function, leading to death. Recent studies have injected dissociated, suspended cardiac cells into coronary arteries to restore function with limited results attributed to poor cell retention and cell death. Extracellular matrix (ECM) injected into damaged cardiac tissue sites show some promising effects. However, combined use of human cardiac ECM and cardiac cells may produce superior benefits to restore cardiac function. OBJECTIVE: This study was designed to assess use of new three-dimensional human heart ECM-derived scaffolds to serve as vehicles to deliver cardiac-derived cells directly to damaged heart tissue and improve cell retention at these sites while also providing biomechanical support and attracting host cell recruitment. METHODS AND RESULTS: ECM-derived porous protein scaffolds were fabricated from human heart tissues. These scaffolds were designed to carry, actively promote and preserve cardiac cell phenotype, viability and functional retention in tissue sites. ECM scaffolds were optimized and were seeded with human cardiomyocytes, cultured and subsequently implanted ex vivo onto infarcted murine epicardium. Seeded human cardiomyocytes readily adhered to human cardiac-derived ECM scaffolds and maintained representative phenotypes including expression of cardiomyocyte-specific markers, and remained electrically synchronous within the scaffold in vitro. Ex vivo, cardiomyocyte-seeded ECM scaffolds spontaneously adhered and incorporated into murine ventricle. CONCLUSIONS: Decellularized human cardiac tissue-derived 3D ECM scaffolds are effective delivery vehicles for human cardiac cells to directly target ischemic heart tissue and warrant further studies to assess their therapeutic potential in restoring essential cardiac functions.

Absence of glucose transporter 4 diminishes electrical activity of mouse hearts during hypoxia.

Insulin resistance, which characterizes type 2 diabetes, is associated with reduced translocation of glucose transporter 4 (GLUT4) to the plasma membrane following insulin stimulation, and diabetic patients with insulin resistance show a higher incidence of ischaemia, arrhythmias and sudden cardiac death. The aim of this study was to examine whether GLUT4 deficiency leads to more severe alterations in cardiac electrical activity during cardiac stress due to hypoxia. To fulfil this aim, we compared cardiac electrical activity from cardiac-selective GLUT4-ablated (G4H-/-) mouse hearts and corresponding control (CTL) littermates. A custom-made cylindrical 'cage' electrode array measured potentials (Ves) from the epicardium of isolated, perfused mouse hearts. The normalized average of the maximal downstroke of Ves ( (|d Ves/dt(min)|na), which we previously introduced as an index of electrical activity in normal, ischaemic and hypoxic hearts, was used to assess the effects of GLUT4 deficiency on electrical activity. The |d Ves/dt(min)|na of G4H −/− and CTL hearts decreased by 75 and 47%, respectively (P < 0.05), 30 min after the onset of hypoxia. Administration of insulin attenuated decreases in values of |d Ves/dt(min)|na in G4H −/− hearts as well as in CTL hearts, during hypoxia. In general, however, G4H −/− hearts showed a severe alteration of the propagation sequence and a prolonged total activation time. Results of this study demonstrate that reduced glucose availability associated with insulin resistance and a reduction in GLUT4-mediated glucose transport impairs electrical activity during hypoxia, and may contribute to cardiac vulnerability to arrhythmias in diabetic patients.

Modulating OPG and TGF-ß1 mRNA expression via bioelectrical stimulation.

BACKGROUND: Bone remodeling is a lifelong process that ranges from orthodontic tooth movement/alignment to bone damage/healing, to overall bone health. Osteoprotegerin (OPG) and transforming growth factor ß1 (TGF-ß1) are secreted by osteoblasts and participate in bone remodeling. OPG promotes bone remineralization and stabilization prominent in post-mechanical repositioning of the teeth in the dental alveolus. TGF-ß1 participates in regulatory processes to promote osteoblast and osteoclast equilibrium. In the context of orthodontic tooth movement, post-treatment fixation requires additional, exogenous, stabilization support. Recent research showcases supplementary solutions, in conjunction to standard tooth fixation techniques, such as OPG injections into gum and periodontal tissues to accelerate tooth anchorage; however, injections are prone to post-procedure complications and discomfort. This study utilizes noninvasive bioelectric stimulation (BES) to modulate OPG and TGF-ß1 as a novel solution to regulate bone remineralization specifically in the context of post-orthodontic tooth movement. PURPOSE: The aim of this study was to investigate a spectrum of BES parameters that would modulate OPG and TGF-ß1 expression in osteoblasts. METHODS: Osteoblasts were cultured and stimulated using frequencies from 25 Hz to 3 MHz. RT-qPCR was used to quantify changes in OPG and TGFb-1 mRNA expression. RESULTS: OPG mRNA expression was significantly increased at frequencies above 10,000 Hz with a maximum expression increase of 332 ± 8% at 100 kHz. Conversely, OPG mRNA expression was downregulated at frequencies lower than 1000 Hz. TGF-ß1 mRNA expression increased throughout all stimulation frequencies with a peak of 332 ± 72% at 250 kHz. Alizarin Red tests for calcium, indicated that mineralization of stimulated osteoblasts in vitro increased 28% after 6 weeks in culture. DISCUSSION: Results support the working hypothesis that OPG and TGF-ß1 mRNA expression can be modulated through BES. Noninvasive BES approaches have the potential to accelerate bone remineralization by providing a novel tool to supplement the anchorage process, reduce complications, and promote patient compliance and reduce post-treatment relapse. Noninvasive BES may be applicable to other clinical applications as a novel therapeutic tool to modulate bone remodeling.

Gap junction channel gating modulated through protein phosphorylation.

As a ubiquitous post-translation modification process, protein phosphorylation has proven to be a key mechanism in regulating the function of several membrane proteins, including transporters and channels. Connexins, pannexins, and innexins are protein families that form gap junction channels essential for intercellular communication. Connexins have been intensely studied, and most of their isoforms are known to be phosphorylated by protein kinases that lead to modifications in tyrosine, serine, and threonine residues, which have been reported to affect, in one way or another, intercellular communication. Despite the abundant reports on changes in intercellular communication due to the activation or inactivation of numerous kinases, the molecular mechanisms by which phosphorylation alters channel gating properties have not been elucidated completely. Hence, this chapter will cover some of the current, relevant research that attempt to explain how phosphorylation triggers and/or modulates gap junction channel gating.

Modulation of Asymmetric Flux in Heterotypic Gap Junctions by Pore Shape, Particle Size and Charge.

Gap junction channels play a vital role in intercellular communication by connecting cytoplasm of adjoined cells through arrays of channel-pores formed at the common membrane junction. Their structure and properties vary depending on the connexin isoform(s) involved in forming the full gap junction channel. Lack of information on the molecular structure of gap junction channels has limited the development of computational tools for single channel studies. Currently, we rely on cumbersome experimental techniques that have limited capabilities. We have earlier reported a simplified Brownian dynamics gap junction pore model and demonstrated that variations in pore shape at the single channel level can explain some of the differences in permeability of heterotypic channels observed in in vitro experiments. Based on this computational model, we designed simulations to study the influence of pore shape, particle size and charge in homotypic and heterotypic pores. We simulated dye diffusion under whole cell voltage clamping. Our simulation studies with pore shape variations revealed a pore shape with maximal flux asymmetry in a heterotypic pore. We identified pore shape profiles that match the in silico flux asymmetry results to the in vitro results of homotypic and heterotypic gap junction formed out of Cx43 and Cx45. Our simulation results indicate that the channel's pore-shape established flux asymmetry and that flux asymmetry is primarily regulated by the sizes of the conical and/or cylindrical mouths at each end of the pore. Within the set range of particle size and charge, flux asymmetry was found to be independent of particle size and directly proportional to charge magnitude. While particle charge was vital to creating flux asymmetry, charge magnitude only scaled the observed flux asymmetry. Our studies identified the key factors that help predict asymmetry. Finally, we suggest the role of such flux asymmetry in creating concentration imbalances of messenger molecules in cardiomyocytes. We also assess the potency of fibroblasts in aggravating such imbalances through Cx43-Cx45 heterotypic channels in fibrotic heart tissue.

Mono-Heteromeric Configurations of Gap Junction Channels Formed by Connexin43 and Connexin45 Reduce Unitary Conductance and Determine both Voltage Gating and Metabolic Flux Asymmetry.

In cardiac tissues, the expression of multiple connexins (Cx40, Cx43, Cx45, and Cx30.2) is a requirement for proper development and function. Gap junctions formed by these connexins have distinct permeability and gating mechanisms. Since a single cell can express more than one connexin isoform, the formation of hetero-multimeric gap junction channels provides a tissue with an enormous repertoire of combinations to modulate intercellular communication. To study further the perm-selectivity and gating properties of channels containing Cx43 and Cx45, we studied two monoheteromeric combinations in which a HeLa cell co-transfected with Cx43 and Cx45 was paired with a cell expressing only one of these connexins. Macroscopic measurements of total conductance between cell pairs indicated a drastic reduction in total conductance for mono-heteromeric channels. In terms of Vj dependent gating, Cx43 homomeric connexons facing heteromeric connexons only responded weakly to voltage negativity. Cx45 homomeric connexons exhibited no change in Vj gating when facing heteromeric connexons. The distributions of unitary conductances (γj) for both mono-heteromeric channels were smaller than predicted, and both showed low permeability to the fluorescent dyes Lucifer yellow and Rhodamine123. For both mono-heteromeric channels, we observed flux asymmetry regardless of dye charge: flux was higher in the direction of the heteromeric connexon for MhetCx45 and in the direction of the homomeric Cx43 connexon for MhetCx43. Thus, our data suggest that co-expression of Cx45 and Cx43 induces the formation of heteromeric connexons with greatly reduced permeability and unitary conductance. Furthermore, it increases the asymmetry for voltage gating for opposing connexons, and it favors asymmetric flux of molecules across the junction that depends primarily on the size (not the charge) of the crossing molecules.

Connexin phosphorylation as a regulatory event linked to channel gating.

The main proteins required for functional gap junction channels are known as connexins and most of their isoforms indicate that they can become phosphorylated. Connexin phosphorylation has been reported to participate in modifying junctional communication and the mechanisms involved apparently depend on which kinase becomes involved. Although multiple reports have suggested a strong influence of phosphorylation on channel gating, not enough physiological studies have been performed to determine precisely the gating mechanisms implicated. Moreover, gap junction channels follow other various gating mechanisms, including voltage gating and chemical gating, where phosphorylation could act as a modulator. The quest for this chapter has been to discriminate those instances where phosphorylation acts directly as a gating trigger and where it acts indirectly or only as a modulator. Despite recent efforts, the mechanisms involved in all these cases are barely understood.

Connexin43 and connexin45 form heteromeric gap junction channels in which individual components determine permeability and regulation.

Two gap junction proteins, connexin43 (Cx43) and connexin45 (Cx45), are coexpressed in many cardiac and other cells. Homomeric channels formed by these proteins differ in unitary conductance, permeability, and regulation. We sought to determine the ability of Cx43 and Cx45 to oligomerize with each other to form heteromeric gap junction channels and to determine the functional and regulatory properties of these heteromeric channels. HeLa cells were transfected with Cx45 or (His)(6)-tagged Cx43 or sequentially transfected with both connexins. Immunoblots verified production of the transfected connexins, and immunofluorescence demonstrated that they were colocalized in the HeLa-Cx43(His)(6)/Cx45 cells. Connexons were solubilized from HeLa-Cx43(His)(6)/Cx45 cells by using Triton X-100 and were applied to a Ni(2+)-NTA column, which binds the His(6) sequence. Cx45 was coeluted from the column with Cx43(His)(6), demonstrating that some hemichannels contain both connexins. Single-channel recordings showed that the HeLa-Cx43(His)(6)/Cx45 cells exhibited single-channel conductances that were not observed in cells expressing either connexin alone. Dye-coupling experiments showed that HeLa-Cx43(His)(6) cells readily passed Lucifer yellow and N-(2-aminoethyl)biotinamide hydrochloride (neurobiotin); in contrast, HeLa-Cx45 and HeLa-Cx43(His)(6)/Cx45 cells showed extensive intercellular passage of neurobiotin but little coupling with Lucifer yellow. Treatment with the protein kinase C activator 12-O-tetradecanoylphorbol 13-acetate reduced junctional conductance in cells expressing Cx43, Cx45, or both connexins, but it reduced the extent of neurobiotin transfer only in HeLa-Cx43(His)(6) and HeLa-Cx43(His)(6)/Cx45 cells but not in the HeLa-Cx45 cells. Thus, biochemical and electrophysiological evidence suggests that Cx43 and Cx45 extensively mix to form heteromeric channels; however, individual connexin components dominate aspects of the physiological behavior of these channels.

Role of the carboxyl terminal of connexin43 in transjunctional fast voltage gating.

Previous studies show that chemical regulation of connexin43 (Cx43) gap junction channels depends on the integrity of the carboxyl terminal (CT) domain. Experiments using Xenopus oocytes show that truncation of the CT domain alters the time course for current inactivation; however, correlation with the behavior of single Cx43 channels has been lacking. Furthermore, whereas chemical gating is associated with a "ball-and-chain" mechanism, there is no evidence whether transjunctional voltage regulation for Cx43 follows a similar model. We provide data on the properties of transjunctional currents from voltage-clamped pairs of mammalian tumor cells expressing either wild-type Cx43 or a mutant of Cx43 lacking the carboxyl terminal domain (Cx43M257). Cx43 transjunctional currents showed bi-exponential decay and a residual steady-state conductance of approximately 35% maximum. Transjunctional currents recorded from Cx43M257 channels displayed a single, slower exponential decay. Long transjunctional voltage pulses caused virtual disappearance of the residual current at steady state. Single channel data revealed disappearance of the residual state, increase in the mean open time, and slowing of the transition times between open and closed states. Coexpression of CxM257 with Cx43CT in a separate fragment restored the lower conductance state. We propose that Cx43CT is an effector of fast voltage gating. Truncation of Cx43CT limits channel transitions to those occurring across the higher energy barrier that separates open and closed states. We further propose that a ball-and-chain interaction provides the fast component of voltage-dependent gating between CT domain and a receptor affiliated with the pore.

Evaluation of Multiple Biological Therapies for Ischemic Cardiac Disease.

The development of cell- and gene-based strategies for regenerative medicine offers a therapeutic option for the repair and potential regeneration of damaged cardiac tissue post-myocardial infarction (MI). Human umbilical cord subepithelial cell-derived stem cells (hUC-SECs), human bone marrow-derived mesenchymal stem cells (hBM-MSCs), and human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), all derived from human tissue, have been shown to have in vitro and in vivo therapeutic potential. Additionally, S100a1, VEGF165, and stromal-derived factor-1α (SDF-1α) genes all have the potential to improve cardiac function and/or effect adverse remodeling. In this study, we compared the therapeutic potential of hBM-MSCs, hUC-SECs, and hiPSC-CMs along with plasmid-based genes to evaluate the in vivo potential of intramyocardially injected biologics to enhance cardiac function in a mouse MI model. Human cells derived from various tissue types were expanded under hypoxic conditions and injected intramyocardially into mice that had undergone left anterior descending (LAD) artery ligation. Similarly, plasmids were also injected into three groups of mice after LAD ligation. Seven experimental groups were studied in total: (1) control (saline), (2) hBM-MSCs, (3) hiPSC-CMs, (4) hUC-SECs, (5) S100a1 plasmid, (6) VEGF165 plasmid, and (7) SDF-1α plasmid. We evaluated echocardiography, hemodynamic catheterization measurements, and histology at 4 and 12 weeks post-biologic injection. Significant improvement was observed in cardiac function and contractility in hiPSC-CM and S100a1 groups and a significant reduction in left ventricle scar within the hUC-SEC group and a slight improvement in the SDF-1α and VEGF165 groups compared to the control group. These results demonstrate the potential for new biologic therapies to reduce scar burden and improve contractile function.

Biophysical properties of homomeric and heteromultimeric channels formed by cardiac connexins.

Substantial advances have been made in characterizing the biophysical properties of channels formed exclusively by connexin isoforms expressed mainly in the heart, e.g., Cx43, Cx45 or Cx40. These properties include transjunctional and transmembrane voltage gating as well as their perm-selectivity, chemical gating and, at a single channel level, their multiple open states and changes in mode behavior. Nonetheless, these connexins are rarely expressed individually in a cell and the presence of functional channels constituted by distinct connexin isoforms is now suspected to be a norm. In fact, combinations of the connexins that form heteromeric channels have been described in some tissues, increasing the necessity to reinforce the research that leads to understanding the effects of these heteromeric interaction on the gating and conducting characteristics of the channels. Furthermore, protein-protein interaction studies will help to understand which connexin domains are involved in these interactions and how they affect the physiology of channels and their interaction with other biological and structural molecules in the cell. New information on the biophysical properties of heteromultimeric channels suggests that interaction between connexins and connexons is not as simple as once thought. Theoretically, changes in the coupling of homomeric connexons (Cx43) in the myocardium may not be significant enough to change the physiology of the heart or to incite arrhythmias, but when heteromeric channels are present, alteration in conductance, differential gating sensitivity to bio-gating molecules and changes in voltage sensitivity increase substantially the cell resources to modulate intercellular coupling, which may participate in the physiology and/or pathology of the cardiovascular tissues.

A carboxyl terminal domain of connexin43 is critical for gap junction plaque formation but not for homo- or hetero-oligomerization.

We have initiated a series of experiments to analyze the biosynthesis and oligomerization of Cx43 in cells containing other connexins through the expression of site-directed mutants and chimeric connexin polypeptides. Here we report studies concerning a mutant of Cx43 (Cx43tr) that has been truncated after amino acid 251 to remove most of the Cx43 carboxy-terminal region. In stably transfected HeLa cells, full length Cx43 localized primarily to appositional membranes while much more Cx43tr was observed in the cytoplasm. Both Cx43 and Cx43tr showed similar oligomerization profiles based on centrifugation through sucrose gradients. HeLaCx43tr cells showed limited transfer of microinjected Lucifer Yellow but did show electrical coupling. Co-expression of Cx43tr with Cx43 or Cx45 led to Cx43tr localization at appositional membranes and co-localization with the other connexins. Moreover, cells co-expressing Cx43tr with Cx43 or Cx45 showed extensive intercellular dye coupling. Thus, Cx43tr was able to oligomerize and form functional channels when expressed alone or with a compatible connexin, but it only formed plaques when co-expressed. These results suggest that the carboxyl tail of Cx43 is not important for oligomerization, but they implicate critical residues in the formation of gap junction plaques.

Functional and pharmacological analysis of cardiomyocytes differentiated from human peripheral blood mononuclear-derived pluripotent stem cells.

Advances in induced pluripotent stem cell (iPSC) technology have set the stage for routine derivation of patient- and disease-specific human iPSC-cardiomyocyte (CM) models for preclinical drug screening and personalized medicine approaches. Peripheral blood mononuclear cells (PBMCs) are an advantageous source of somatic cells because they are easily obtained and readily amenable to transduction. Here, we report that the electrophysiological properties and pharmacological responses of PBMC-derived iPSC CM are generally similar to those of iPSC CM derived from other somatic cells, using patch-clamp, calcium transient, and multielectrode array (MEA) analyses. Distinct iPSC lines derived from a single patient display similar electrophysiological features and pharmacological responses. Finally, we demonstrate that human iPSC CMs undergo acute changes in calcium-handling properties and gene expression in response to rapid electrical stimulation, laying the foundation for an in-vitro-tachypacing model system for the study of human tachyarrhythmias.

The voltage-sensitive dye di-4-ANEPPS slows conduction velocity in isolated guinea pig hearts.

BACKGROUND: Voltage-sensitive dyes are important tools for mapping electrical activity in the heart. However, little is known about the effects of voltage-sensitive dyes on cardiac electrophysiology. OBJECTIVE: To test the hypothesis that the voltage-sensitive dye di-4-ANEPPS modulates cardiac impulse propagation. METHODS: Electrical and optical mapping experiments were performed in isolated Langendorff perfused guinea pig hearts. The effect of di-4-ANEPPS on conduction velocity and anisotropy of propagation was quantified. HeLa cells expressing connexin 43 were used to evaluate the effect of di-4-ANEPPS on gap junctional conductance. RESULTS: In electrical mapping experiments, di-4-ANEPPS (7.5 µM) was found to decrease both longitudinal and transverse conduction velocities significantly compared with control. No change in the anisotropy of propagation was observed. Similar results were obtained in optical mapping experiments. In these experiments, the effect of di-4-ANEPPS was dose dependent. di-4-ANEPPS had no detectable effect on connexin 43-mediated gap junctional conductance in transfected HeLa cells. CONCLUSION: Our results demonstrate that the voltage-sensitive dye di-4-ANEPPS directly and dose-dependently modulates cardiac impulse propagation. The effect is not likely mediated by connexin 43 inhibition. Our results highlight an important caveat that should be taken into account when interpreting data obtained using di-4-ANEPPS in cardiac preparations.

The maximal downstroke of epicardial potentials as an index of electrical activity in mouse hearts.

The maximal upstroke of transmembrane voltage (dV(m)/dt(max)) has been used as an indirect measure of sodium current I(Na) upon activation in cardiac myocytes. However, sodium influx generates not only the upstroke of V(m), but also the downstroke of the extracellular potentials V(e) including epicardial surface potentials V(es). The purpose of this study was to evaluate the magnitude of the maximal downstroke of V(es) (|dV(es)/dt (min)|) as a global index of electrical activation, based on the relationship of dV(m)/dt(max) to I(Na). To fulfill this purpose, we examined |dV(es)/dt(min)| experimentally using isolated perfused mouse hearts and computationally using a 3-D cardiac tissue bidomain model. In experimental studies, a custom-made cylindrical "cage" array with 64 electrodes was slipped over mouse hearts to measure V(es) during hyperkalemia, ischemia, and hypoxia, which are conditions that decrease I(Na). Values of |dV(es)/dt(min)| from each electrode were normalized (|dV(es)/dt (min)|(n)) and averaged (|dV(es)/dt(min)|(na)). Results showed that |dV(es)/dt(min)|(na) decreased during hyperkalemia by 28, 59, and 79% at 8, 10, and 12 mM [K(+)](o), respectively. |dV(es)/dt(min)| also decreased by 54 and 84% 20 min after the onset of ischemia and hypoxia, respectively. In computational studies, |dV(es)/dt(min)| was compared to dV(m)/dt(max) at different levels of the maximum sodium conductance G(Na), extracellular potassium ion concentration [K(+)](o), and intracellular sodium ion concentration [Na(+)](i), which all influence levels of I(Na). Changes in |dV(es)/dt(min)|(n) were similar to dV(m)/dt (max) during alterations of G(Na), [K(+)](o), and [Na(+)](i). Our results demonstrate that |dV(es)/dt(min)|(na) is a robust global index of electrical activation for use in mouse hearts and, similar to dV(m)/dt(max), can be used to probe electrophysiological alterations reliably. The index can be readily measured and evaluated, which makes it attractive for characterization of, for instance, genetically modified mouse hearts and drug effects on cardiac tissue.

The connexin43 C-terminal region mediates neuroprotection during stroke.

Connexin43 plays an important role in neuroprotection in experimental stroke models; reducing the expression of this gap junction protein in astrocytes enhances injury upon middle cerebral artery occlusion (MCAO). Because the C-terminal region of connexin43 isimportant for channel activity, we carried out MCAO stroke experiments in mice expressing a truncated form of connexin43 (Cx43DeltaCT mice). Brain sections were analyzed for infarct volume, astrogliosis, and inflammatory cell invasion 4 days after MCAO. Adult cortices and astrocyte cultures were examined for connexin43 (Cx43) expression by immunohistochemistry and Western blot. Cultured astrocytes were also examined for dye coupling, channel conductance, hemichannel activity, and Ca wave propagation. The Cx43DeltaCT mice exhibit enhanced cerebral injury after stroke. Astrogliosis was reduced and inflammatory cell invasion was increased inthe peri-infarct region in these mice compared with controls; Cx43 expression was also altered. Lastly, cultured astrocytes from Cx43DeltaCT mice were less coupled and displayed alterations in channel gating, hemichannel activity, and Ca wave properties. These results suggest that astrocytic Cx43 contributed to the regulation of cell death after stroke and support the view that the Cx43 C-terminal region is important in protection in cerebral ischemia.

Electrophysiological modeling of fibroblasts and their interaction with myocytes.

Experimental studies have shown that cardiac fibroblasts are electrically inexcitable, but can contribute to electrophysiology of myocardium in various manners. The aim of this computational study was to give insights in the electrophysiological role of fibroblasts and their interaction with myocytes. We developed a mathematical model of fibroblasts based on data from whole-cell patch clamp and polymerase chain reaction (PCR) studies. The fibroblast model was applied together with models of ventricular myocytes to assess effects of heterogeneous intercellular electrical coupling. We investigated the modulation of action potentials of a single myocyte varying the number of coupled fibroblasts and intercellular resistance. Coupling to fibroblasts had only a minor impact on the myocyte's resting and peak transmembrane voltage, but led to significant changes of action potential duration and upstroke velocity. We examined the impact of fibroblasts on conduction in one-dimensional strands of myocytes. Coupled fibroblasts reduced conduction and upstroke velocity. We studied electrical bridging between ventricular myocytes via fibroblast insets for various coupling resistors. The simulations showed significant conduction delays up to 20.3 ms. In summary, the simulations support strongly the hypothesis that coupling of fibroblasts to myocytes modulates electrophysiology of cardiac cells and tissues.

SRC utilizes Cas to block gap junctional communication mediated by connexin43.

The Src tyrosine kinase phosphorylates Cas (Crk-associated substrate) to confer anchorage independence and invasive growth potential to transformed cells. Gap junctional communication is often lower between aggressive tumor cells compared with normal or benign precursors. The gap junction protein connexin43 (Cx43) is a tumor suppressor that can inhibit tumor cell growth. Src can phosphorylate Cx43 to block gap junctional communication between transformed cells. However, mechanisms by which this event actually closes intercellular channels have not been clearly defined. Here, we report that Src and Cas associate with each other at intercellular junctions. In addition, Cas is required for Src to reduce dye transfer and electrical coupling between cells expressing Cx43. Thus, Src utilizes Cas to inhibit gap junctional communication mediated by Cx43. This finding introduces a novel role of the Cas focal adhesion linker protein in the gap junction complex. This observation may help explain how gap junctional communication can be suppressed between malignant and metastatic tumor cells.