Reduced Gap Junction Coupling Research Articles

Ephaptic Coupling Is a Mechanism of Conduction Reserve During Reduced Gap Junction Coupling.

Many cardiac pathologies are associated with reduced gap junction (GJ) coupling, an important modulator of cardiac conduction velocity (CV). However, the relationship between phenotype and functional expression of the connexin GJ family of proteins is controversial. For example, a 50% reduction of GJ coupling has been shown to have little impact on myocardial CV due to a concept known as conduction reserve. This can be explained by the ephaptic coupling (EpC) theory whereby conduction is maintained by a combination of low GJ coupling and increased electrical fields generated in the sodium channel rich clefts between neighboring myocytes. At the same time, low GJ coupling may also increase intracellular charge accumulation within myocytes, resulting in a faster transmembrane potential rate of change during depolarization (dV/dt_max) that maintains macroscopic conduction. To provide insight into the prevalence of these two phenomena during pathological conditions, we investigated the relationship between EpC and charge accumulation within the setting of GJ remodeling using multicellular simulations and companion perfused mouse heart experiments. Conduction along a fiber of myocardial cells was simulated for a range of GJ conditions. The model incorporated intercellular variations, including GJ coupling conductance and distribution, cell-to-cell separation in the intercalated disc (perinexal width—WP), and variations in sodium channel distribution. Perfused heart studies having conditions analogous to those of the simulations were performed using wild type mice and mice heterozygous null for the connexin gene Gja1. With insight from simulations, the relative contributions of EpC and charge accumulation on action potential parameters and conduction velocities were analyzed. Both simulation and experimental results support a common conclusion that low GJ coupling decreases and narrowing WP increases the rate of the AP upstroke when sodium channels are densely expressed at the ends of myocytes, indicating that conduction reserve is more dependent on EpC than charge accumulation during GJ uncoupling.

Anti-inflammatory properties of lacosamide in an astrocyte-microglia co-culture model of inflammation

PurposeUnderstanding the effects of antiepileptic drugs on glial cells and glia-mediated inflammation is a new approach to future treatment of epilepsy. Little is known about direct effects of the antiepileptic drug lacosamide (LCM) on glial cells. Therefore, we aimed to study the LCM effects on glial viability, microglial activation, expression of gap-junctional (GJ) protein Cx43 as well as intercellular communication in an in vitro astrocyte-microglia co-culture model of inflammation. MethodsPrimary rat astrocytes co-cultures containing 5% (M5, “physiological” conditions) or 30% (M30, “pathological inflammatory” conditions) of microglia were treated with different concentrations of LCM [5, 15, 30, and 90 μg/ml] for 24 h. Glial cell viability was measured by MTT assay. Immunocytochemistry was performed to analyze the microglial activation state. Western blot analysis was used to quantify the astroglial Cx43 expression. The GJ cell communication was studied via Scrape Loading. ResultsA concentration-dependent incubation with LCM did not affect the glial cell viability both under physiological and pathological conditions. LCM induced a significant concentration-dependent decrease of activated microglia with parallel increase of ramified microglia under pathological inflammatory conditions. This correlated with an increase in astroglial Cx43 expression. Nevertheless, the functional coupling via GJs was significantly reduced after incubation with LCM. ConclusionLCM has not shown effects on the glial cell viability. The reduced GJ coupling by LCM could be related to its anti-epileptic activity. The anti-inflammatory glial features of LCM with inhibition of microglial activation under inflammatory conditions support beneficial role in epilepsy associated with neuroinflammation.

On the nature of delays allowing anatomical re-entry involving the Purkinje network: a simulation study

Clinical observations suggest that the Purkinje network can be part of anatomical re-entry circuits in monomorphic or polymorphic ventricular arrhythmias. However, significant conduction delay is needed to support anatomical re-entry given the high conduction velocity within the Purkinje network. We investigated, in computer models, whether damage rendering the Purkinje network as either an active lesion with slow conduction or a passive lesion with no excitable ionic channel, could explain clinical observations. Active lesions had compromised sodium current and a severe reduction in gap junction coupling, while passive lesions remained coupled by gap junctions, but modelled the membrane as a fixed resistance. Both types of tissue could provide significant delays of over 100 ms. Electrograms consistent with those obtained clinically were reproduced. However, passive tissue could not support re-entry as electrotonic coupling across the delay effectively increased the proximal refractory period to an extremely long interval. Active tissue, conversely, could robustly maintain re-entry. Formation of anatomical re-entry using the Purkinje network is possible through highly reduced gap junctional coupling leading to slowed conduction.

Physiologic regulation of heart rate and blood pressure involves connexin 36-containing gap junctions.

Chronically elevated sympathetic nervous activity underlies many cardiovascular diseases. Elucidating the mechanisms contributing to sympathetic nervous system output may reveal new avenues of treatment. The contribution of the gap junctional protein connexin 36 (Cx36) to the regulation of sympathetic activity and thus blood pressure and heart rate was determined using a mouse with specific genetic deletion of Cx36. Ablation of the Cx36 protein was confirmed in sympathetic preganglionic neurons of Cx36-knockout (KO) mice. Telemetric analysis from conscious Cx36 KO mice revealed higher variance in heart rate and blood pressure during rest and activity compared to wild-type (WT) mice, and smaller responses to chemoreceptor activation when anesthetized. In the working heart–brain stem preparation of the Cx36-KO mouse, respiratory-coupled sympathetic nerve discharge was attenuated and responses to chemoreceptor stimulation and noxious stimulation were blunted compared to WT mice. Using whole cell patch recordings, sympathetic preganglionic neurons in spinal cord slices of Cx36-KO mice displayed lower levels of spikelet activity compared to WT mice, indicating reduced gap junction coupling between neurons. Cx36 deletion therefore disrupts normal regulation of sympathetic outflow with effects on cardiovascular parameters.—Lall, V. K., Bruce, G., Voytenko, L., Drinkhill, M., Wellershaus, K., Willecke, K., Deuchars, J., Deuchars, S. A. Physiologic regulation of heart rate and blood pressure involves connexin 36–containing gap junctions.

Ephaptic coupling rescues conduction failure in weakly coupled cardiac tissue with voltage-gated gap junctions.

Electrical conduction in cardiac tissue is usually considered to be primarily facilitated by gap junctions, providing a pathway between the intracellular spaces of neighboring cells. However, recent studies have highlighted the role of coupling via extracellular electric fields, also known as ephaptic coupling, particularly in the setting of reduced gap junction expression. Further, in the setting of reduced gap junctional coupling, voltage-dependent gating of gap junctions, an oft-neglected biophysical property in computational studies, produces a positive feedback that promotes conduction failure. We hypothesized that ephaptic coupling can break the positive feedback loop and rescue conduction failure in weakly coupled cardiac tissue. In a computational tissue model incorporating voltage-gated gap junctions and ephaptic coupling, we demonstrate that ephaptic coupling can rescue conduction failure in weakly coupled tissue. Further, ephaptic coupling increased conduction velocity in weakly coupled tissue, and importantly, reduced the minimum gap junctional coupling necessary for conduction, most prominently at fast pacing rates. Finally, we find that, although neglecting gap junction voltage-gating results in negligible differences in well coupled tissue, more significant differences occur in weakly coupled tissue, greatly underestimating the minimal gap junctional coupling that can maintain conduction. Our study suggests that ephaptic coupling plays a conduction-preserving role, particularly at rapid heart rates.

56-02: Spatially-discordant alternans phase islands promote arrhythmogenesis

Purpose: Two key features of ventricular remodeling are fibrosis and reduced gap junctional coupling. These are associated with abnormalities in electrical impulse generation and propagation, which predispose to reentrant tachyarrhythmias. In the absence of structural remodeling, high-frequency impulse generation can also induce such arrhythmias, via the development of dynamic electrical instabilities. However, owing to the complex and stochastic nature of arrhythmias, the combined mechanistic influences of structural and dynamical perturbations on arrhythmogenesis remain incompletely understood. Methods: Arrhythmogenesis was studied using in vitro and in silico monolayer models of neonatal rat ventricular tissue with 30% randomly distributed cardiac myofibroblasts and systematically lowered intercellular coupling achieved in vitro through graded knockdown of connexin43 expression. Summary of Results: Arrhythmia incidence and complexity (i.e., number of phase singularities) increased with decreasing intercellular coupling efficiency, at pacing frequencies > 3Hz. This coincided with the onset of a special type of spatially discordant action potential duration (APD) alternans characterized by island-like areas of opposite alternans phase (APIs). The degree of electrotonic dyssynchrony (number of APIs) correlated positively with the degree of Cx43 knockdown and arrhythmia complexity. At higher myofibroblast densities, more APIs were formed and reentrant arrhythmias were more easily induced. Conclusion: Downregulation of intercellular coupling and presence of myofibroblasts give rise to spatially-discordant APD alternans in simulated and cultured monolayers of rat ventricular cells. This study reveals islands of oppositely phased APD alternans to be important determinants of reentrant tachyarrhythmia initiation and complexity.

Delayed afterdepolarizations generate both triggers and a vulnerable substrate promoting reentry in cardiac tissue

Delayed afterdepolarizations (DADs) have been well characterized as arrhythmia triggers, but their role in generating a tissue substrate vulnerable to reentry is not well understood. The purpose of this study was to test the hypothesis that random DADs can self-organize to generate both an arrhythmia trigger and a vulnerable substrate simultaneously in cardiac tissue as a result of gap junction coupling. Computer simulations in 1-dimensional cable and 2-dimensional tissue models were performed. The cellular DAD amplitude was varied by changing the strength of sarcoplasmic reticulum calcium release. Random DAD latency and amplitude in different cells were simulated using gaussian distributions. Depending on the strength of spontaneous sarcoplasmic reticulum calcium release and other conditions, random DADs in cardiac tissue resulted in the following behaviors: (1) triggered activity (TA); (2) a vulnerable tissue substrate causing unidirectional conduction block and reentry by inactivating sodium channels; (3) both triggers and a vulnerable substrate simultaneously by generating TA in regions next to regions with subthreshold DADs susceptible to unidirectional conduction block and reentry. The probability of the latter 2 behaviors was enhanced by reduced sodium channel availability, reduced gap junction coupling, increased tissue heterogeneity, and less synchronous DAD latency. DADs can self-organize in tissue to generate arrhythmia triggers, a vulnerable tissue substrate, and both simultaneously. Reduced sodium channel availability and gap junction coupling potentiate this mechanism of arrhythmias, which are relevant to a variety of heart disease conditions.

Evidence of decreased dopamine receptor‐mediated control of gap junction coupling in the inner nuclear layer of the diabetic Ins2Akita mouse retina (1129.1)

Dopamine (DA) is a light‐adaptive factor that plays an important role in daytime vision. DA controls the gain of retinal signal transmission partly by controlling gap junction coupling of retinal neurons. Because preliminary data suggests that DA levels are lower in the diabetic Ins2Akita mouse retina, we hypothesized that gap junction coupling would be altered in diabetic retina. We assayed coupling using a “cut‐loading” technique, in which whole‐mounted retinas are cut with a razor dipped in gap junction‐permeable tracer Neurobiotin (NB) and quantified NB‐labeled cells in the inner nuclear layer (INL) using fluorescence microscopy. Experiments were performed under dark‐adapted conditions. The extent of coupling in control (C57Bl6) retinas was tested in the presence of the D2‐like receptor (D2R) agonist quinpirole, the D2R antagonist sulpiride, or the gap junction antagonist beta‐glycyrrhizic acid (BGA). As expected, D2R activation and BGA reduced gap junction coupling of the NB‐labeled cells in the INL, while D2R antagonism increased tracer coupling. In the diabetic Ins2Akita mice, we found that neurons in the INL showed increased tracer coupling compared to non‐diabetic littermates. We propose that this increase in gap junction coupling between retinal neurons reflects a decrease in DA levels. Decreased DA levels and an altered coupling state of retinal neurons may be hallmarks of diabetic retinasGrant Funding Source: Supported by American Heart Association (12BGIA12040362)

Alterations in Ionic Currents and Gap Junctional Coupling by Pan-Histone Deacetylase Inhibition

Dynamic balance of histone acetyl transferase and histone deacetylases (HDACs)is maintained in multiple signaling pathways determining expression and repression of genes. Pan-HDAC inhibitors like Vorinostat(VOR, ZolinzaTM) and Romidepsin (Istodax(r)) have been approved for treatment of peripheral and cutaneous T-cell lymphomas. On rare occasions during clinical trials,HDACi has been associated with symptomatic ECG alterations, cardiac arrhythmias, and even sudden cardiac death for unknown reasons. Rationale of this study is to identify electrophysiological and molecular determinants involved in asymptomatic cardiac abnormalities in patients treated with HDACi during clinical trials. We investigated the effects of VOR on ionic currents and gap junctions responsible for cardiac excitability and propagation. Cultured neonatal mouse ventricular myocytes (NMVM) were treated with 1-5μM VOR overnight and patch clamp experiments were performed to measure the transient (Ito) and steady state (IK) outward K+, peak inward Na+ (INa), and peak inward L-type Ca2+ (ICa,L) current densities. Dose-dependent decreases in INa density and NaV1.5 protein levels occurred along with +10mV shift in INa activation in myocytes treated with 1-5μM VOR. Conversely, ICa,L density increased significantly without concomitant change in CaV1.2 protein expression levels. Moreover, augmentation of ICa,L byVOR was independent of stimulation with 5μM isoproterenol, suggesting discrete acetylation and phosphorylation dependent mechanisms for modulation of cardiac ICa,L However, VOR did not alter any IK or Ito current densities. Also, VOR dose dependently reduced gap junction coupling and protein expression of connexin43. Pan-HDACi treatment significantly reduces Cx43 protein expression and coupling along with salient alterations in INa and ICa,L densities. These ion channel changes present possible etiologies for cardiac abnormalities in patients. VOR being a pan-HDAC inhibitor, we plan to determine the effects of isoform-selective HDACi on ionic currents and APD in human cardiomyocytes derived from induced pluripotent stem cells.

Cerebral ischemic injury is enhanced in a model of oculodentodigital dysplasia

Oculodentodigital dysplasia (ODDD) is a rare autosomal dominant disease that results in visible developmental anomalies of the limbs, face, eyes and teeth. Recently analysis of human connexin43 (Cx43) DNA sequences has revealed a number of different missense, duplication and frame shift mutations resulting in this phenotype. A mouse model of this disorder has been created with a missense point mutation of the glycine amino acid at position 60 to serine (G60S). Heterozygote +/G60S mice exhibit a similar ODDD phenotype as observed in humans. In addition to the malformations listed above, ODDD patients often have neurological findings. In the brain, Cx43 is highly expressed in astrocytes and has been shown to play a role in neuroprotection. We were interested in determining the effect of the +/G60S mutation following stroke. Four days after middle cerebral artery occlusion the volume of infarct was larger in mice with the +/G60S mutation. In astrocyte-neuron co-cultures, exposure to glutamate also resulted in greater cellular death in the +/G60S mutants. Protein levels of Cx43 in the mutant mouse were found to be reduced when compared to the normal tissue. Cx43 protein was observed as a continual line of small punctate aggregates in the plasma membrane with increased intracellular localization, which is distinct from the larger plaques seen in the normal mouse astrocytes. Functionally, primary +/G60S astrocytes exhibited reduced gap junctional coupling and increased hemichannel activity, which may underlie the mechanism of increased damage during stroke.This article is part of the Special Issue Section entitled ‘Current Pharmacology of Gap Junction Channels and Hemichannels’.

Ischemic treatment hyperpolarizes cochlear arteriolar smooth muscle and endothelial cells via different channel mechanisms

Regional acute ischemia generally induces a compensatory vasodilation in the most vascular beds including the cochlear arterioles but the underlying cellular mechanism remains controversial. Using conventional intracellular and whole‐cell recording from smooth muscle (SMC) and endothelial cells (EC) in situ of and dispersed from segments of guinea pigs cochlear spiral modiolar artery, we analyzed the membrane actions of acute ischemic treatment (AIT, hypercapinic hypoxia, pO2 =0 mmHg, pH=6.2). The AIT for 2 to 20 min caused a reversible and partially repeatable vasodilation and a hyperpolarization in the majority of cells. ACh‐ and 10 mM K+‐induced dilation was significantly reduced, lost or reversed to a constriction during and after the AIT. Holding at −20 mV, AIT induced a reversible 10–65 pA outward current within 1–2 min in the majority of SMCs and ECs. During the AIT, the whole‐cell I/V curve became stronger outwardly rectifying at voltages >; −40 mV. The AIT‐induced net current in isolated SMCs exhibited a delayed outward rectification upon depolarization steps and partial blockade by 1–10 mM TEA or 4‐AP, suggesting an activation of both voltage‐gated (KV) and big conductance Ca2+‐activated (BK) K+‐channels. In contrast, the AIT‐induced current in isolated ECs was blocked by 30 μM niflumic acid but not by TEA, 4‐AP and linopirdine; the net current showed a voltage and time dependence typical of Ca2+‐activated Cl−‐channels (CaCC): slowly activating outwardly rectifying with a reversal potential near −30 mV. AIT (>;10 min) also reduced gap junction coupling current in in situ SMCs and ECs. We conclude that the acute ischemia in the cochlear arteriole induces hyperpolarization, dilation and loss of EDHF function via activation of KV and BK in smooth muscle cells and activation of Ca2+‐activated Cl−‐channels in endothelial cells, as well as an inhibition of gap junction coupling. Supported by NIH NIDCD DC 004716, P30 05983.

Focal Energy Deprivation Underlies Arrhythmia Susceptibility in Mice With Calcium-Sensitized Myofilaments

The Ca(2+) sensitivity of the myofilaments is increased in hypertrophic cardiomyopathy and other heart diseases and may contribute to a higher risk for sudden cardiac death. Ca(2+) sensitization increases susceptibility to reentrant ventricular tachycardia in animal models, but the underlying mechanism is unknown. To investigate how myofilament Ca(2+) sensitization creates reentrant arrhythmia susceptibility. Using hypertrophic cardiomyopathy mouse models (troponinT-I79N) and a Ca(2+) sensitizing drug (EMD57033), here we identify focal energy deprivation as a direct consequence of myofilament Ca(2+) sensitization. To detect ATP depletion and thus energy deprivation, we measured accumulation of dephosphorylated Connexin 43 (Cx43) isoform P0 and AMP kinase activation by Western blotting and immunostaining. No differences were detected between groups at baseline, but regional accumulation of Connexin 43 isoform P0 occurred within minutes in all Ca(2+)-sensitized hearts, in vivo after isoproterenol challenge and in isolated hearts after rapid pacing. Lucifer yellow dye spread demonstrated reduced gap junctional coupling in areas with Connexin 43 isoform P0 accumulation. Optical mapping revealed that selectively the transverse conduction velocity was slowed and anisotropy increased. Myofilament Ca(2+) desensitization with blebbistatin prevented focal energy deprivation, transverse conduction velocity slowing, and the reentrant ventricular arrhythmias. Myofilament Ca(2+) sensitization rapidly leads to focal energy deprivation and reduced intercellular coupling during conditions that raise arrhythmia susceptibility. This is a novel proarrhythmic mechanism that can increase arrhythmia susceptibility in structurally normal hearts within minutes and may, therefore, contribute to sudden cardiac death in diseases with increased myofilament Ca(2+) sensitivity.

Gap-Junction Suppression of Electrical Activity in Normal and Diabetic Pancreatic Islets

The pancreatic islets of Langerhans are multicellular microorgans which play a central role in maintaining blood glucose homeostasis, through secretion of hormone insulin. Interactions between cells within the islet are critical to enhance and coordinate the β-cell insulin secretion response to glucose. Gap junction channels coordinate membrane potential between β-cells in the islet. This serves to coordinate oscillations in free-calcium activity ([Ca2+]i) and insulin secretion, but also to suppress spontaneous [Ca2+]i elevations under basal glucose. Here we quantify the gap junction suppression of β-cell excitability and describe this with a percolation model of cell-cell coupling. We utilize islets from wild-type mice and 2 transgenic mouse models in which mutations to the KATP channel either increase or decrease excitability in a population of β-cells in the islet. In each set of islets, we quantify the variation of [Ca2+]i and insulin secretion with gap junction coupling, at low glucose and high glucose. In all cases reduced gap junction coupling resulted in elevated [Ca2+]i; with the variation of [Ca2+]i with gap junction conductance showing a similar non-linear relationship which can be described using the percolation model. This variation could also describes insulin release and glucose homeostasis: large elevations in [Ca2+]i predict significant reductions in blood glucose levels. Importantly, in mice expressing KATP channel mutations that reduce insulin secretion and cause severe hyperglycemia and diabetes, a reduction in gap junction coupling elevates insulin release and prevents diabetes. Thus the relationship governing gap junction coupling and the suppression of spontaneous elevations in [Ca2+]i is dependent on the distribution of excitability across β-cells of the islet and the level of coupling between β-cells, and this governs the overall islet response. Utilizing this may be a promising therapeutic treatment for specific monogenic forms of diabetes.

ATP activates P2X receptors to mediate gap junctional coupling in the cochlea

ATP is an important extracellular signaling molecule and can activate both ionotropic (P2X) and metabotropic purinergic (P2Y) receptors to influence cellular function in many aspects. Gap junction is an intercellular channel and plays a critical role in hearing. Here, we report that stimulation of ATP reduced gap junctional coupling between cochlear supporting cells. This uncoupling effect could be evoked by nanomolar physiological levels of ATP. A P2X receptor agonist benzoylbenzoyl-ATP (BzATP) but not a P2Y receptor agonist UTP stimulated this uncoupling effect. Application of P2X receptor antagonists pyridoxalphosphate-6-azophenyl-2′,4′-disulfonic acid (PPADS, 50μM) or oxidized ATP (oATP, 0.1mM) eliminated this uncoupling effect. We further found that ATP activated P2X receptors in the cochlear supporting cells allowing Ca2+ influxing, thereby increasing intracellular Ca2+ concentration to mediate gap junctions. These data suggest that ATP can mediate cochlear gap junctions at the physiological level by the activation of P2X receptors rather than P2Y receptors. This P2X receptor-mediated purinergic control on the cochlear gap junctions may play an important role in the regulation of K+-recycling for ionic homeostasis in the cochlea and the reduction of hearing sensitivity under noise stress for protection.

Arrhythmogenic Implications of Fibroblast-Myocyte Interactions

With increasing life span as afforded by nutritional, technological, and therapeutic advancements during the last century, cardiac diseases have moved to the top of morbidity and mortality statistics and progressed into a significant socioeconomic burden. Accordingly, efforts have been substantial to understand the development of heart failure as the final disease state in a sequence of events initiated by a variety of etiologies ranging from ischemic heart disease to idiopathic cardiomyopathies, hereditary diseases, and senescence. In respect to cardiac arrhythmogenesis, these studies were primarily focused on the elucidation of parenchymal cell dysfunction and included topics such as impairment of ion-channel function, reduced gap junctional coupling, and abnormal calcium handling of cardiomyocytes. By contrast, the involvement of stromal cells in the initiation and maintenance of cardiac arrhythmias has been less aggressively pursued. This is likely due to the circumstance that at first glance, stromal cells seem to be less “exciting” than their parenchymal counterparts, which exhibit electric and contractile activity. This perception has started to change considerably during the last decade, however, and it is the goal of this short review to summarize recent findings that shed new light onto potential roles played by cardiac fibroblasts, and in particular myofibroblasts, in the development of the arrhythmic heart. Editorial see p 249 ### Cardiac Fibroblasts: Structure, Function, and 3-Dimensional Organization In normal hearts, fibroblasts outnumber cardiomyocytes by a factor of 2 to 3.1 Scanning electron microscopic studies show fibroblasts to be spindle-like, slender cells that link up to each other by filopodia, whereby they form an interconnected 3-dimensional network of cells that surrounds cardiomyocytes in a regular fashion (Figure 1).2 Similar to other tissues such as the skin, fibroblasts in the heart form a functional syncytium by expressing intercellular channels (gap junctions) at sites of cell-to-cell contacts.2,3 The ability of fibroblasts to also establish …

The connexin mimetic peptide Gap27 increases human dermal fibroblast migration in hyperglycemic and hyperinsulinemic conditions in vitro

Significant increases in skin wound healing rates occur by reducing connexin-mediated communication (CMC). Gap27, a connexin (Cx) mimetic peptide targeted to the second extracellular loop of Cx43, which inhibits CMC, increases migration of human keratinocytes and dermal fibroblasts. To examine the efficacy of Gap27 in a hyperglycemic and hyperinsulinemic in vitro environment, cell migration, gap junction, and Cx hemichannel functionality and cell-substrate adhesion assays were performed on human dermal fibroblasts and diabetic fibroblast and keratinocytes. To investigate fibroblast genes involved in these processes, extra-cellular matrix (ECM) and adhesion gene expression was determined with a PCR array. Gap27 increased fibroblast migration in both euglycemia/euinsulinemia and hyperglycemia/hyperinsulinemia, and influenced migration in diabetic keratinocytes. Hyperglycemia/hyperinsulinemia reduced gap junction coupling in fibroblasts and Gap27 reduced CMC and cell adhesion to substrata in fibroblasts cultured in high glucose. Migrating dermal fibroblast ECM and cell adhesion genes were found to be differentially regulated by Gap27 in euglycemia and hyperglycemia. The PCR array showed that Gap27 upregulated 34 genes and downregulated 1 gene in euglycemic migrating fibroblasts. By contrast in hyperglycemia, Gap27 upregulated 1 gene and downregulated 9 genes. In euglycemic conditions, Gap27 induced upregulation of genes associated with ECM remodeling, whereas in hyperglycemia, ECM component genes were downregulated by Gap27. Thus, Gap27 improves cell migration during scrape-wound repair in hyperglycemia/hyperinsulinemia conditions in vitro, although migration of diabetic cells is less influenced. Our results suggest that this increase in motility may occur by decreasing gap junction and hemichannel activity and altering gene expression in the adhesion and ECM pathway.

Connexin 43 mimetic peptides inhibit spontaneous epileptiform activity in organotypic hippocampal slice cultures

Gap junctions are cytoplasmic channels connecting adjacent cells and mediating their electrical and metabolic coupling. Different cell types in the CNS express various gap junction forming proteins, the connexins, in a cell-specific manner. Using the general gap junctional blocker, carbenoxolone, and two synthetic connexin mimetic peptides, corresponding to amino acid sequences of segments within the second extracellular loop of connexin 43, we studied the role of gap junctions in the generation of epileptiform activity in rat organotypic hippocampal slice cultures. While carbenoxolone inhibited both spontaneous and evoked seizure-like events, connexin mimetic peptides selectively attenuated spontaneous recurrent epileptiform activity, and only after prolonged (> 10 h) treatment. The effects were mediated through reduced gap junctional coupling as indicated by suppressed fluorescent dye transfer between the cells. Assuming a selective inhibition of a connexin 43-dependent process by the mimetic peptides and preferential localization of this connexin isoform in astrocytes, the data suggest that, in developing hippocampal networks, the generation and/or initiation of spontaneous recurrent seizure-like activity may depend in large part upon the opening of glial gap junctions. Furthermore, this study shows that the use of a synthetic peptide that mimics a short sequence of a specific connexin isoform and, hence, blocks gap junctional communication in targeted cell types in the CNS, is a viable strategy for the modulation of cerebral activity.

Reduced gap junctional coupling leads to uncorrelated motor neuron firing and precocious neuromuscular synapse elimination

During late embryonic and early postnatal life, neuromuscular junctions undergo synapse elimination that is modulated by patterns of motor neuron activity. Here, we test the hypothesis that reduced spinal neuron gap junctional coupling decreases temporally correlated motor neuron activity that, in turn, modulates neuromuscular synapse elimination, by using mutant mice lacking connexin 40 (Cx40), a developmentally regulated gap junction protein expressed in motor and other spinal neurons. In Cx40-/- mice, electrical coupling among lumbar motor neurons, measured by whole-cell recordings, was reduced, and single motor unit recordings in awake, behaving neonates showed that temporally correlated motor neuron activity was also reduced. Immunostaining and intracellular recording showed that the neuromuscular synapse elimination was accelerated in muscles from Cx40-/- mice compared with WT littermates. Our work shows that gap junctional coupling modulates neuronal activity patterns that, in turn, mediate synaptic competition, a process that shapes synaptic circuitry in the developing brain.

Overexpression of cardiac connexin45 increases susceptibility to ventricular tachyarrhythmias in vivo

Electrophysiological remodeling involving gap junctions has been demonstrated in failing hearts and may contribute to intercellular uncoupling, delayed conduction, enhanced arrhythmias, and vulnerability to sudden death in patients with heart failure. Recently, we showed that failing human hearts exhibit marked increases in connexin45 (Cx45) expression in addition to previously documented decreases in connexin43 (Cx43) expression. Each of these changes results in reduced gap junction coupling. The objective of the present study was to examine functional consequences of increased Cx45 in cardiac gap junctions. Transgenic mice with cardiac-selective overexpression of the developmentally downregulated cardiac connexin, connexin45 (Cx45OE mice) were subjected to in vivo electrophysiology studies in which an intracardiac catheter was used to induce ventricular arrhythmias in anesthetized mice, and in which ambulatory ECG monitoring was used to detect spontaneous arrhythmias in unanesthetized mice. Hearts were analyzed by TaqMan RT-PCR, immunostaining, immunoblotting, and echocardiography. Lucifer yellow and neurobiotin dye transfer was used to assess coupling in transgenic and control myocyte cultures. Cx45 mRNA was two orders of magnitude greater in Cx45OE mice. Cx45-immunoreactive signal at gap junctions increased twofold and total Cx45 protein by immunoblotting increased 25% in Cx45OE mice compared with nontransgenic littermate controls. Functionally, Cx45OE mice exhibited more inducible ventricular tachycardia than controls but did not exhibit any other functional or structural derangements as assessed by echocardiography. Ventricular myocytes isolated from Cx45OE mice exhibited diminished intercellular transfer of Lucifer yellow dye and increased transfer of neurobiotin, consistent with altered cell-to-cell communication. Thus increased myocardial expression of Cx45 results in remodeling of intercellular coupling and greater susceptibility to ventricular arrhythmias in vivo.

Chronic nicotine in hearts with healed ventricular myocardial infarction promotes atrial flutter that resembles typical human atrial flutter

The potential of chronic nicotine exposure for atrial fibrillation (AF) and atrial flutter (AFL) in hearts with and without chronic myocardial infarction (MI) remains poorly explored. MI was created in dogs by permanent occlusion of the left anterior descending coronary artery, and dogs were administered nicotine (5 mg.kg(-1).day(-1) sc) for 1 mo using osmotic minipumps. High-resolution epicardial (1,792 bipolar electrodes) and endocardial Halo catheters were used to map activation during induced atrial rhythms. Nicotine promoted inducible sustained AFL at a mean cycle length of 134 +/- 10 ms in all MI dogs (n = 6) requiring pacing and electrical shocks for termination. No AFL could be induced in MI dogs (n = 6), control (non-MI) dogs (n = 3) not exposed to nicotine, and dogs with no MI and exposed to nicotine (n = 3). Activation maps during AFL showed a single reentrant wavefront in the right atrium that rotated either clockwise (60%) or counterclockwise (40%) around the crista terminalis and through the isthmus. Ablation of the isthmus prevented the induction of AFL. Nicotine caused a significant (P < 0.01) but highly heterogeneous increase in atrial interstitial fibrosis (2- to 10-fold increase in left and right atria, respectively) in the MI group but only a 2-fold increase in the right atrium in the non-MI group. Nicotine also flattened (P < 0.05) the slope of the epicardial monophasic action potential duration (electrical restitution) curve of both atria in the MI but not in non-MI dogs. Two-dimensional simulation in an excitable matrix containing an isthmus and nicotine's restitutional and reduced gap junctional coupling (fibrosis) parameters replicated the experiments. Chronic nicotine in hearts with MI promotes AFL that closely resembles typical human AFL. Increased atrial interstitial fibrosis and flattened electrical restitution are important substrates for the AFL.