Gap Junction Conductance Research Articles

Simulation of Conducted Responses in Microvascular Networks: Role of Current Rectification by Endothelial Cell Gap Junctions

Neuronal activity in the brain stimulates local increases in blood flow. Such flow increases require dilation of feeding arterioles, which can be initiated by upstream conducted responses in the form of electrical currents transmitted along the endothelial lining of the vessels. One potential trigger for this response is hyperpolarization of capillary endothelial cells in active regions resulting from local elevation of extracellular K+ concentration. The conducted hyperpolarization causes arteriolar dilation. For this mechanism to be effective in delivering increased flow to a specific location, the conducted response must travel in the upstream direction only, without reentry into adjacent parallel flow pathways. Such specificity in the response to activation has been observed in skeletal muscle, and may result from partial rectification of current in gap junctions connecting adjacent endothelial cells. The goal of this work is to simulate the spread of hyperpolarizing currents along vessel walls in microvascular networks of the cerebral circulation, including the effects of rectification in gap junctions. A theoretical model was developed for the spread of electrical current in the endothelial cells of a network of microvessels in response to local K+- induced hyperpolarization. Transmembrane currents are represented in the model by a KIR-channel conductance, a non-voltage-dependent K+ conductance, and a background conductance. Currents along vessel walls are represented assuming asymmetric gap junction conductance between adjacent endothelial cells, with higher conductance for downstream currents (causing upstream hyperpolarization) than for upstream currents. The model was applied to a synthetic network of 45 segments, assuming 20 nS conductance for upstream propagation, while a range of values was used for downstream propagation. The model shows effective upstream propagation of conducted responses when the gap junction conductance for downstream propagation is 1 nS or less. This result was confirmed in a simulation of a reconstructed murine cerebral cortex network with 4881 segments. In conclusion, partial current rectification by endothelial gap junctions can result in preferential upstream propagation of conducted responses, consistent with experimental observations of local flow regulation. Supported by NIH grant U01 HL133362. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Fibroblast mediated dynamics in diffusively uncoupled myocytes: a simulation study using 2-cell motifs

In healthy hearts myocytes are typically coupled to nearest neighbours through gap junctions. Under pathological conditions such as fibrosis, or in scar tissue, or across ablation lines myocytes can uncouple from their neighbours. Electrical conduction may still occur via fibroblasts that not only couple proximal myocytes but can also couple otherwise unconnected regions. We hypothesise that such coupling can alter conduction between myocytes via introduction of delays or by initiation of premature stimuli that can potentially result in reentry or conduction blocks. To test this hypothesis we have developed several 2-cell motifs and investigated the effect of fibroblast mediated electrical coupling between uncoupled myocytes. We have identified various regimes of myocyte behaviour that depend on the strength of gap-junctional conductance, connection topology, and parameters of the myocyte and fibroblast models. These motifs are useful in developing a mechanistic understanding of long-distance coupling on myocyte dynamics and enable the characterisation of interaction between different features such as myocyte and fibroblast properties, coupling strengths and pacing period. They are computationally inexpensive and allow for incorporation of spatial effects such as conduction velocity. They provide a framework for constructing scar tissue boundaries and enable linking of cellular level interactions with scar induced arrhythmia.

Gap junction modulators: Prospects in bupropion cardiotoxicity

Bupropion, a multifaceted antidepressant, is associated with serious cardiotoxicity when misused. Recently, there has been a noticeable increase in cases of intentional poisoning and current therapeutic interventions for bupropion overdose remain insufficient. The cardiotoxic effects of the drug are primarily linked to its influence on cardiac gap junctions. Small-molecule drugs enhancing gap junction conductance, such as rotigaptide and danegaptide, present potential as antidotal agents. Testing these molecules under conditions of bupropion overdose could address the urgent need for innovative approaches to mitigate bupropion-induced cardiotoxicity.

Octanol alleviates chronic constriction injury of sciatic nerve-induced peripheral neuropathy by regulating AKT/mTOR signaling.

Activation of gap junction channels can induce neuropathic pain. Octanol can limit the conductance of gap junctions containing connexin 43 proteins. Thus, this study focused on the roles of octanol in chronic constriction injury (CCI)-induced peripheral neuropathy in mice and its mechanisms of action. Male mice were assigned into control, sham, CCI, CCI + Octanol-20mg/kg, CCI + Octanol-40mg/kg and CCI + Octanol-80mg/kg groups. CCI was performed by applying three loose ligations to mouse sciatic nerve, and the mice with CCI was administered with 20mg/kg, 40mg/kg, or 80mg/kg octanol. The neuropathic pain development was examined by assessing thermal withdrawal latency, paw withdrawal mechanical threshold, and sciatic functional index. Histopathological changes were evaluated by hematoxylin and eosin staining. The phosphorylation of protein kinase B (Akt) and mammalian target of rapamycin (mTOR) was examined by western blotting. The expression of Akt and mTOR was also evaluated by immunofluorescence staining. Octanol alleviated the CCI-induced mechanical and thermal hyperalgesia and sciatic functional loss. Additionally, octanol relieved the CCI-induced abnormal histopathological changes. Mechanistically, octanol inactivated the Akt/mTOR pathway in the mice with CCI. In conclusion, octanol can alleviate CCI-induced peripheral neuropathic by regulating the Akt/mTOR pathway and might be a novel pharmacological intervention for neuropathic pain.

Why strengthening gap junctions may hinder action potential propagation

Gap junctions are channels in cell membranes allowing ions to pass directly between cells. They are found throughout the body, including heart myocytes, neurons, and astrocytes. In cardiac tissue and throughout the nervous system, an action potential (AP) in one cell can trigger APs in neighboring cells connected by gap junctions. It is known experimentally that there is an ideal gap junction conductance for AP propagation—lower or higher conductance can lead to propagation failure. We explain this phenomenon geometrically in branching networks by analyzing an idealized model that focuses exclusively on gap junction and AP-generating currents. As expected, the gap junction conductance must be high enough for AP propagation to occur. However, if the gap junction conductance is too high, then it dominates the cell's intrinsic firing conductance and disrupts AP generation. We also identify conditions for semi-active propagation, where cells in the network are not individually excitable but still propagate action potentials.

Nernst-Planck-Gaussian modelling of electrodiffusional recovery from ephaptic excitation between mammalian cardiomyocytes.

Introduction: In addition to gap junction conduction, recent reports implicate possible ephaptic coupling contributions to action potential (AP) propagation between successive adjacent cardiomyocytes. Here, AP generation in an active cell, withdraws Na+ from, creating a negative potential within, ephaptic spaces between the participating membranes, activating the initially quiescent neighbouring cardiomyocyte. However, sustainable ephaptic transmission requires subsequent complete recovery of the ephaptic charge difference. We explore physical contributions of passive electrodiffusive ion exchange with the remaining extracellular space to this recovery for the first time. Materials and Methods: Computational, finite element, analysis examined limiting, temporal and spatial, ephaptic [Na+], [Cl-], and the consequent Gaussian charge differences and membrane potential recovery patterns following a ΔV∼130mV AP upstroke at physiological (37°C) temperatures. This incorporated Nernst-Planck formalisms into equations for the time-dependent spatial concentration gradient profiles. Results: Mammalian atrial, ventricular and purkinje cardiomyocyte ephaptic junctions were modelled by closely apposed circularly symmetric membranes, specific capacitance 1μF cm-2, experimentally reported radii a = 8,000, 12,000 and 40,000nm respectively and ephaptic axial distance w = 20nm. This enclosed an ephaptic space containing principal ions initially at normal extracellular [Na+] = 153.1mM and [Cl-] = 145.8 mM, respective diffusion coefficients D Na = 1.3 109 and D Cl = 2 109 nm2s-1. Stable, concordant computational solutions were confirmed exploring ≤1,600nm mesh sizes and Δt≤0.08ms stepsize intervals. The corresponding membrane voltage profile changes across the initially quiescent membrane were obtainable from computed, graphically represented a and w-dependent ionic concentration differences adapting Gauss's flux theorem. Further simulations explored biological variations in ephaptic dimensions, membrane anatomy, and diffusion restrictions within the ephaptic space. Atrial, ventricular and Purkinje cardiomyocytes gave 40, 180 and 2000ms 99.9% recovery times, with 720 or 360ms high limits from doubling ventricular radius or halving diffusion coefficient. Varying a, and D Na and D Cl markedly affected recovery time-courses with logarithmic and double-logarithmic relationships, Varying w exerted minimal effects. Conclusion: We thereby characterise the properties of, and through comparing atrial, ventricular and purkinje recovery times with interspecies in vivo background cardiac cycle duration data, (blue whale ∼2000, human∼90, Etruscan shrew, ∼40ms) can determine physical limits to, electrodiffusive contributions to ephaptic recovery.

Connexin 43 Phosphorylation: Implications in Multiple Diseases.

Connexin 43 (Cx43) is most widely distributed in mammals, especially in the cardiovascular and nervous systems. Its phosphorylation state has been found to be regulated by the action of more than ten kinases and phosphatases, including mitogen-activated protein kinase/extracellular signaling and regulating kinase signaling. In addition, the phosphorylation status of different phosphorylation sites affects its own synthesis and assembly and the function of the gap junctions (GJs) to varying degrees. The phosphorylation of Cx43 can affect the permeability, electrical conductivity, and gating properties of GJs, thereby having various effects on intercellular communication and affecting physiological or pathological processes in vitro and in vivo. Therefore, clarifying the relationship between Cx43 phosphorylation and specific disease processes will help us better understand the disease. Based on the above clinical and preclinical findings, we present in this review the functional significance of Cx43 phosphorylation in multiple diseases and discuss the potential of Cx43 as a drug target in Cx43-related disease pathophysiology, with an emphasis on the importance of connexin 43 as an emerging therapeutic target in cardiac and neuroprotection.

Calcineurin-dependent regulation of gap junction conductance and connexin phosphorylation in guinea pig left atrium

Atrial fibrillation (AF) occurs from disordered atrial action potential conduction and is associated with reduced gap junction electrical conductance (Gj). The Ca2+ and calmodulin-dependent phosphatase, calcineurin, reduces Gj in ventricular myocardium via a protein phosphatase-1 (PP1)-dependent pathway culminating in phosphorylation of serine368 on connexin43 (pSer368-Cx43). However, characterisation of corresponding pathways in left atrial myocardium, which have a more complex connexin subtype profile, is undefined and was the aim of this study. Gj was measured in guinea-pig left atrium from the frequency-dependent variation of intracellular impedance; intracellular [Ca2+], ([Ca2+]i) in low-Na solution was measured by Fura-2 fluorescence. Phosphorylation of guinea-pig Ser368-Cx43 residues was measured by Western blot; Cx40 was immunoprecipitated and probed for serine/threonine residue phosphorylation. Low-Na solution reversibly reduced Gj, in turn attenuated or prevented by calcineurin inhibitors cyclosporin-A or CAIP, respectively. Moreover, Ser368-Cx43 phosphorylation in low-Na solution was also prevented by CAIP. Changes were partially prevented by fostreicin (FST), a protein phosphatase-2A (PP2A) inhibitor; but not by tautomycin, a PP1 inhibitor. Serine/threonine residues on Cx40 were also phosphorylated in low-Na solution; prevented by CAIP and attenuated by FST. Reduced Gj with raised [Ca2+]i is paralleled by a changed Cx43/Cx40 phosphorylation status; changes mediated by calcineurin and PP2A-dependent pathways, but not PP1. The pharmacological profile underlying changes to guinea-pig atrial gap junction electrical conductance with raised intracellular [Ca2+]i is fundamentally different from that in ventricular myocardium. This provides a targeted drug model whereby atrial and ventricular myocardium can be selectively targeted to correct conduction defects.

Diminishing neuronal acidification by channelrhodopsins with low proton conduction

Many channelrhodopsins are permeable to protons. We show that in electrically polarized cells, activation of a high-current channelrhodopsin, CheRiff, led to significant acidification. From an initial intracellular pH of 7.3, cells acidified to pH 6.9 ± 0.24 (mean ± s.d., n = 34 cells) after 60 seconds of illumination. To determine whether this acidification arose via proton transport through the opsin vs through other voltage-dependent proton channels, we performed experiments with patterned optogenetic stimulation in monolayers of HEK293 cells expressing CheRiff, an inward-rectifier potassium channel, Kir2.1, and a red-shifted pH indicator, pHoran4. Illumination with stripes of blue light led to electrical depolarization that extended beyond the illuminated spots, due to gap junctional conduction; but acidification was localized to the illuminated spots. These observations established that the acidification required proton transport through the opsin itself. We then identified and characterized two opsins which showed large photocurrents, but small proton permeability, PsCatCh2.0 and ChR2 3M. These opsins were effective at evoking neural activity, but neither induced detectable acidification. PsCatCh2.0 was also spectrally compatible with simultaneous voltage imaging with QuasAr6a. Stimulation-evoked acidification is a possible source of artifacts and disruptions to cell health in scientific and prospective therapeutic applications of optogenetics. Channelrhodopsins with low proton permeability are a promising strategy for avoiding these problems.

Hyperglycaemia-induced impairment of the autorhythmicity and gap junction activity of mouse embryonic stem cell-derived cardiomyocyte-like cells.

Diabetes mellitus with hyperglycaemia is a major risk factor for malignant cardiac dysrhythmias. However, the underlying mechanisms remain unclear, especially during the embryonic developmental phase of the heart. This study investigated the effect of hyperglycaemia on the pulsatile activity of stem cell-derived cardiomyocytes. Mouse embryonic stem cells (mESCs) were differentiated into cardiac-like cells through embryoid body (EB) formation, in either baseline glucose or high glucose conditions. Action potentials (APs) were recorded using a voltage-sensitive fluorescent dye and gap junction activity was evaluated using scrape-loading lucifer yellow dye transfer assay. Molecular components were detected using immunocytochemistry and immunoblot analyses. High glucose decreased the spontaneous beating rate of EBs and shortened the duration of onset of quinidine-induced asystole. Furthermore, it altered AP amplitude, but not AP duration, and had no impact on neither the expression of the hyperpolarisation-activated cyclic nucleotide-gated isoform 4 (HCN4) channel nor on the EB beating rate response to ivabradine nor isoprenaline. High glucose also decreased both the intercellular spread of lucifer yellow within an EB and the expression of the cardiac gap junction protein connexin43 as well as upregulated the expression of transforming growth factor beta1 (TGF-β1) and phosphorylated Smad3. High glucose suppressed the autorhythmicity and gap junction conduction of mESC-derived cardiomyocytes, via mechanisms probably involving TGF-β1/Smad3 signalling. The results allude to glucotoxicity related proarrhythmic effects, with potential clinical implications in foetal diabetic cardiac disease.

TNNI3K delays atrioventricular conduction and reduces connexin-45 gap junctional coupling

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): The Dutch Research Council (NWO Talent Scheme) Introduction Cardiac conduction delay is the main substrate for triggering arrhythmias. Hence, prolongation of the PR interval on the electrocardiogram (ECG) is a strong predictor of atrial fibrillation, the most common cardiac arrhythmia. In previous research, cardiac troponin I-interacting kinase (TNNI3K) has been identified as a regulator of the PR interval. Various inbred mouse strains showed a correlation between Tnni3k expression levels and PR interval durations. Additionally, transgenic mice overexpressing hTNNI3K presented an extreme PR interval prolongation. Objective This study aims to unravel the mechanism underlying TNNI3K-driven PR interval prolongation. Methods ECG parameters were recorded in mice expressing physiological levels of Tnni3k (congenic), overexpressing hTNNI3K (TNNI3Ktg), or overexpressing kinase-dead hTNNI3K (TNNI3K-KDtg) and were compared to low-Tnni3k expressing DBA/2J control mice. Atrioventricular (AV) conduction was measured in Langendorff-perfused isolated hearts by electrical mapping. Cellular electrophysiology and conductance were measured using the (dual) patch-clamp technique. AV nodal collagen levels were identified by Pico Sirius Red staining. Candidate interactors were identified by immunoprecipitation of transfected HEK293A cell lysates. In stable HeLa-Connexin 45 (Cx45) cell lines expressing TNNI3K, Cx45 protein expression and phosphorylation levels were investigated by Western blot. Localisation of Cx45 was characterised by immunofluorescence. Results At six weeks of age, congenic and TNNI3Ktg mice show a 17% and 35% prolonged PR interval duration, respectively. Nonetheless, the PR interval of TNNI3K-KDtg mice did not change. Electrical mapping experiments on TNNI3Ktg mouse hearts show a corresponding AV conduction delay, which was neither driven by changes in cellular atrial cardiomyocyte electrophysiology nor driven by AV-nodal fibrosis. We next identified Cx45, a highly expressed connexin in the AV node, as a potential TNNI3K interactor. HeLa-Cx45 cells expressing TNNI3K demonstrated a reduced Cx45 conductance compared to controls without changes in kinetics. Moreover, we observed relatively decreased Cx45 phosphorylation and increased Cx45 intracellular accumulation. Conclusion We here present TNNI3K as a kinase-driven modulator of PR interval prolongation and AV conduction delay, which is independent of atrial electrophysiology and AV nodal fibrosis. We further identified Cx45 as a novel interactor of TNNI3K. The presence of TNNI3K reduces Cx45 gap junctional conductance and promotes intracellular Cx45 protein accumulation, which could explain the PR interval prolongation in vivo. Altogether, this study implies a crucial role for TNNI3K in AV nodal conduction.

Simulation of atrial fibrillation in a non-ohmic propagation model with dynamic gap junctions.

Gap junctions exhibit nonlinear electrical properties that have been hypothesized to be relevant to arrhythmogenicity in a structurally remodeled tissue. Large-scale implementation of gap junction dynamics in 3D propagation models remains challenging. We aim to quantify the impact of nonlinear diffusion during episodes of arrhythmias simulated in a left atrial model. Homogenization of conduction properties in the presence of nonlinear gap junctions was performed by generalizing a previously developed mathematical framework. A monodomain model was solved in which conductivities were time-varying and depended on transjunctional potentials. Gap junction conductances were derived from a simplified Vogel-Weingart model with first-order gating and adjustable time constant. A bilayer interconnected cable model of the left atrium with 100 μm resolution was used. The diffusion matrix was recomputed at each time step according to the state of the gap junctions. Sinus rhythm and atrial fibrillation episodes were simulated in remodeled tissue substrates. Slow conduction was induced by reduced coupling and by diffuse or stringy fibrosis. Simulations starting from the same initial conditions were repeated with linear and nonlinear gap junctions. The discrepancy in activation times between the linear and nonlinear diffusion models was quantified. The results largely validated the linear approximation for conduction velocities >20 cm/s. In very slow conduction substrates, the discrepancy accumulated over time during atrial fibrillation, eventually leading to qualitative differences in propagation patterns, while keeping the descriptive statistics, such as cycle lengths, unchanged. The discrepancy growth rate was increased by impaired conduction, fibrosis, conduction heterogeneity, lateral uncoupling, fast gap junction time constant, and steeper action potential duration restitution.

Intrinsic Sources and Functional Impacts of Asymmetry at Electrical Synapses

Electrical synapses couple inhibitory neurons across the brain, underlying a variety of functions that are modifiable by activity. Despite recent advances, many functions and contributions of electrical synapses within neural circuitry remain underappreciated. Among these are the sources and impacts of electrical synapse asymmetry. Using multi-compartmental models of neurons coupled through dendritic electrical synapses, we investigated intrinsic factors that contribute to effective synaptic asymmetry and that result in modulation of spike timing and synchrony between coupled cells. We show that electrical synapse location along a dendrite, input resistance, internal dendritic resistance, or directional conduction of the electrical synapse itself each alter asymmetry as measured by coupling between cell somas. Conversely, we note that asymmetrical gap junction (GJ) conductance can be masked by each of these properties. Furthermore, we show that asymmetry modulates spike timing and latency of coupled cells by up to tens of milliseconds, depending on direction of conduction or dendritic location of the electrical synapse. Coordination of rhythmic activity between two cells also depends on asymmetry. These simulations illustrate that causes of asymmetry are diverse, may not be apparent in somatic measurements of electrical coupling, influence dendritic processing, and produce a variety of outcomes on spiking and synchrony of coupled cells. Our findings highlight aspects of electrical synapses that should always be included in experimental demonstrations of coupling, and when assembling simulated networks containing electrical synapses.

Abstract 14217: Myofibroblasts Enhance the Intrinsic Sinus Rhythm at Low Cell-Cell Coupling

Heart rhythm is initiated by the sinoatrial node (SAN). Compared to the chamber myocardium, the healthy SAN exhibits an exceedingly high content of fibroblasts (FBs). Developmental, aging and pathophysiological cues can activate FBs to myofibroblasts (MFBs) which exhibit depolarized membrane potential and stronger electrotonic coupling compared to FBs. We hypothesized that MFBs, at a low content, can paradoxically increase the pacing frequency of pacemaker cells (PMs) by contributing to the spontaneous depolarization slope.Monolayers and scaffold-free 3D spheroids of TBX18-induced PMs (iPMs) were generated as in vitro models of SAN by somatic gene transfer of TBX18 to neonatal rat cardiomyocytes. Retrograde-perfused, adult rat heart was employed to study intact SAN ex vivo. In silico modeling was performed to simulate pacing frequencies of PMs coupled to MFBs in 2D and 3D pacemaker tissues. Upon reducing electrotonic coupling with carbenoxolone (CBX, 5μM), pacing frequency of iPM monolayers either increased from 66 ± 51 to 168 ± 92 bpm (n=4/13) or decreased from 125 ± 47 to 98 ± 28 bpm (n=9/13). GFP-transduced control NRVM monolayers (n=8) showed only occasional activity, which was not affected by CBX treatment (|ΔBR| = 57 ± 69 in iPM monolayers vs. 11± 11 bpm in control, GFP monolayers, p<0.05). In culture, FBs are known to undergo spontaneous activation to MFBs via TGFβ signaling. Treatment of iPM spheroids with a TGFβ inhibitor (SB431542, 10μM) slowed their automaticity to 124 ± 89 bpm compared to 214 ± 18 bpm in untreated iPM spheroids. Sinus rhythm of the rat heart ex vivo increased upon CBX treatment from 199 ± 64 to 242 ± 80 bpm (n=3, p=0.07). Immunostaining confirmed presence of αSMA+ MFBs in both iPMs and the SAN. Computational study demonstrated that pacing frequencies of 2D and 3D PMs rose and fell with increasing gap junction conductance when coupled to MFBs (Vrest = -25 mV, g = 0 to 4.0 nS) but not to FBs (Vrest = -50 mV, g = 0 to 0.4 nS). Dynamic range of cardiac pacing was optimal when the ratio of MFB to PM was no more than one.Our data demonstrate that MFBs positively contribute to the pacing frequency and the safety factor of the sinus rhythm. We identify fibroblast activation state as a potential target to rejuvenate heart rhythm under aging and pathological conditions.

Cell Systems Bioelectricity: How Different Intercellular Gap Junctions Could Regionalize a Multicellular Aggregate.

Simple SummaryElectric potential patterns across tissues are instructive for development, regeneration, and tumorigenesis because they can influence transcription, migration, and differentiation through biochemical and biomechanical downstream processes. Determining the origins of the spatial domains of distinct potential, which in turn decide anatomical features such as limbs, eyes, brain, and heart, is critical to a mature understanding of how bioelectric signaling drives morphogenesis. We studied theoretically how connexin proteins with different voltage-gated gap junction conductances can maintain multicellular regions at distinct membrane potentials. We analyzed a minimal model that incorporates effective conductances ultimately related to specific ion channel and junction proteins that are amenable to external regulation. We also consider a bioelectrical relationship between the connexin composition of the intercellular gap junction and different stages of cancer.Electric potential distributions can act as instructive pre-patterns for development, regeneration, and tumorigenesis in cell systems. The biophysical states influence transcription, proliferation, cell shape, migration, and differentiation through biochemical and biomechanical downstream transduction processes. A major knowledge gap is the origin of spatial patterns in vivo, and their relationship to the ion channels and the electrical synapses known as gap junctions. Understanding this is critical for basic evolutionary developmental biology as well as for regenerative medicine. We computationally show that cells may express connexin proteins with different voltage-gated gap junction conductances as a way to maintain multicellular regions at distinct membrane potentials. We show that increasing the multicellular connectivity via enhanced junction function does not always contribute to the bioelectrical normalization of abnormally depolarized multicellular patches. From a purely electrical junction view, this result suggests that the reduction rather than the increase of specific connexin levels can also be a suitable bioelectrical approach in some cases and time stages. We offer a minimum model that incorporates effective conductances ultimately related to specific ion channel and junction proteins that are amenable to external regulation. We suggest that the bioelectrical patterns and their encoded instructive information can be externally modulated by acting on the mean fields of cell systems, a complementary approach to that of acting on the molecular characteristics of individual cells. We believe that despite the limitations of a biophysically focused model, our approach can offer useful qualitative insights into the collective dynamics of cell system bioelectricity.

Anchored-PKA regulates connexin 43 gap junctions communication in the heart

In cardiomyocytes, connexin-43 (Cx43) forms gap junction (GJ) channels at the intercalated discs (ID) of cardiomyocytes and ensures electrical impulse propagation. This is essential for sequential and coordinated cardiac contractions. In physiological stress, β-adrenergic receptor (β-AR) stimulation activates protein kinase A (PKA), which increases electrical conduction velocity. In pathological condition of myocardial infarction (MI), the β-AR system is chronically activated. Furthermore, Cx43 is altered that affects GJ communication and electrical conduction leading to fatal arrhythmias. Considering the key role of Cx43 in electrical conduction, we investigated the functional regulation of GJ by PKA-anchored through A-kinase anchoring protein (AKAP) in cardiac physiology and pathophysiology, i.e. MI. Experiments in which PKA is inhibited or displaced from AKAPs point to a PKA-scaffold protein required for PKA-mediated regulation of Cx43 phosphorylation and GJ communication. By a variety of immunoprecipitation and immunolocalization experiments, we show that the AKAP named ezrin forms a molecular complex with PKA and Cx43 in rat hearts at the cellular and tissular levels. While PKA and ezrin expression levels remain constant, Cx43 show a significant reduction after a MI. Interestingly, immunolocalization studies highlight for a decrease in Cx43 vicinity with ezrin at the ID of ventricular cardiomyocytes. All of these suggest for a role of PKA-ezrin-Cx43 complex in the regulation of GJ communication in cardiac physiology and a deregulation of this complex could explain partly the altered electrical propagation observed after a MI.

Nitric Oxide Is Essential for Generating the Minute Rhythm Contraction Pattern in the Small Intestine, Likely via ICC-DMP.

Nitrergic nerves have been proposed to play a critical role in the orchestration of peristaltic activities throughout the gastrointestinal tract. In the present study, we investigated the role of nitric oxide, using spatiotemporal mapping, in peristaltic activity of the whole ex vivo mouse intestine. We identified a propulsive motor pattern in the form of propagating myogenic contractions, that are clustered by the enteric nervous system into a minute rhythm that is dependent on nitric oxide. The cluster formation was abolished by TTX, lidocaine and nitric oxide synthesis inhibition, whereas the myogenic contractions, occurring at the ICC-MP initiated slow wave frequency, remained undisturbed. Cluster formation, inhibited by block of nitric oxide synthesis, was fully restored in a highly regular rhythmic fashion by a constant level of nitric oxide generated by sodium nitroprusside; but the action of sodium nitroprusside was inhibited by lidocaine indicating that it was relying on neural activity, but not rhythmic nitrergic nerve activity. Hence, distention-induced activity of cholinergic nerves and/or a co-factor within nitrergic nerves such as ATP is also a requirement for the minute rhythm. Cluster formation was dependent on distention but was not evoked by a distention reflex. Block of gap junction conductance by carbenoxolone, dose dependently inhibited, and eventually abolished clusters and contraction waves, likely associated, not with inhibition of nitrergic innervation, but by abolishing ICC network synchronization. An intriguing feature of the clusters was the presence of bands of rhythmic inhibitions at 4–8 cycles/min; these inhibitory patches occurred in the presence of tetrodotoxin or lidocaine and hence were not dependent on nitrergic nerves. We propose that the minute rhythm is generated by nitric oxide-induced rhythmic depolarization of the musculature via ICC-DMP.

Opposing modulation of Cx26 gap junctions and hemichannels by CO2.

A moderate increase in (55mmHg) closes Cx26 gap junctions. This effect of CO2 is independent of changes in intra- or extracellular pH. The CO2 -dependent closing effect depends on the same residues (K125 and R104) that are required for the CO2 -dependent opening of Cx26 hemichannels. Pathological mutations of Cx26 abolish the CO2 -dependent closing of the gap junction. Elastic network modelling suggests that the effect of CO2 on Cx26 hemichannels and gap junctions is mediated through changes in the lowest entropy state of the protein. Cx26 hemichannels open in response to moderate elevations of CO2 ( 55mmHg) via a carbamylation reaction that depends on residues K125 and R104. Here we investigate the action of CO2 on Cx26 gap junctions. Using a dye transfer assay, we found that an elevated of 55mmHg greatly delayed the permeation of a fluorescent glucose analogue (NBDG) between HeLa cells coupled by Cx26 gap junctions. However, the mutations K125R or R104A abolished this effect of CO2 . Whole cell recordings demonstrated that elevated CO2 reduced the Cx26 gap junction conductance (median reduction 66.7%, 95% CI, 50.5-100.0%) but had no effect on Cx26K125R or Cx31 gap junctions. CO2 can cause intracellular acidification. Using 30mm propionate, we found that acidification in the absence of a change in caused a median reduction in the gap junction conductance of 41.7% (95% CI, 26.6-53.7%). This effect of propionate was unaffected by the K125R mutation (median reduction 48.1%, 95% CI, 28.0-86.3%). pH-dependent and CO2 -dependent closure of the gap junction are thus mechanistically independent. Mutations of Cx26 associated with the keratitis ichthyosis deafness syndrome (N14K, A40V and A88V), in combination with the mutation M151L, also abolished the CO2 -dependent gap junction closure. Elastic network modelling suggests that the lowest entropy state when CO2 is bound is the closed configuration for the gap junction but the open state for the hemichannel. The opposing actions of CO2 on Cx26 gap junctions and hemichannels thus depend on the same residues and presumed carbamylation reaction.

Conceptualizing conduction as a pliant electrical response: impact of gap junctions and ion channels.

Vasomotor responses conduct among resistance arteries to coordinate blood flow delivery pursuant to energetic demand. Conduction is set by the electrical and mechanical properties of vascular cells, the former tied to how gap junctions and ion channels distribute and dissipate charge, respectively. These membrane proteins are subject to modulation; thus, conduction could be viewed as "pliant" to the current regulatory state. This study used in silico approaches to conceptualize electrical pliancy and to illustrate how gap junctional and ion channel properties distinctly impact conduction along a single skeletal muscle artery or a branching cerebrovascular network. Initial simulations revealed how vascular cells encoded with electrotonic properties best reproduced spreading behavior; the endothelium's importance as a charge source and a longitudinal conduit was readily observed. Alterations in gap junctional conductance produced unique electrical fingerprints: 1) decreased endothelial coupling impaired longitudinal but enhanced radial spread, and 2) reduced myoendothelial coupling limited radial but enhanced longitudinal spread. Subsequent simulations illustrated how tuning ion channel activity, e.g., inward rectifying- and voltage-gated K+ channels, modified charge dissipation, resting membrane potential, and the spread of the electrical phenomenon. Restricting ion channel tuning to a network subregion then revealed how electrical spread could be locally shaped in accordance with the aggregate changes in membrane resistance. In summary, our analysis frames and reimagines electrical conduction as a pliable process, with subtle regulatory changes to membrane proteins shaping network spread and tissue perfusion.NEW & NOTEWORTHY Conducted vasomotor responses depend on initiation and spread of electrical phenomena along arterial walls and their translation into contractile responses. Using computational approaches, we show how subtle but widespread regulation of gap junctions and ion channels can modulate the range and amplitude of electrical spread. Ion channels are regulated by endocrine and mechanical signals and may differ regionally in networks. Subregional electrical changes are not spatially confined but may affect electrical conduction in neighboring regions.

Ionic Mechanisms of Impulse Propagation Failure in the FHF2-Deficient Heart.

FHFs (fibroblast growth factor homologous factors) are key regulators of sodium channel (NaV) inactivation. Mutations in these critical proteins have been implicated in human diseases including Brugada syndrome, idiopathic ventricular arrhythmias, and epileptic encephalopathy. The underlying ionic mechanisms by which reduced Nav availability in Fhf2 knockout (Fhf2KO) mice predisposes to abnormal excitability at the tissue level are not well defined. Using animal models and theoretical multicellular linear strands, we examined how FHF2 orchestrates the interdependency of sodium, calcium, and gap junctional conductances to safeguard cardiac conduction. Fhf2KO mice were challenged by reducing calcium conductance (gCaV) using verapamil or by reducing gap junctional conductance (Gj) using carbenoxolone or by backcrossing into a cardiomyocyte-specific Cx43 (connexin 43) heterozygous background. All conditions produced conduction block in Fhf2KO mice, with Fhf2 wild-type (Fhf2WT) mice showing normal impulse propagation. To explore the ionic mechanisms of block in Fhf2KO hearts, multicellular linear strand models incorporating FHF2-deficient Nav inactivation properties were constructed and faithfully recapitulated conduction abnormalities seen in mutant hearts. The mechanisms of conduction block in mutant strands with reduced gCaV or diminished Gj are very different. Enhanced Nav inactivation due to FHF2 deficiency shifts dependence onto calcium current (ICa) to sustain electrotonic driving force, axial current flow, and action potential (AP) generation from cell-to-cell. In the setting of diminished Gj, slower charging time from upstream cells conspires with accelerated Nav inactivation in mutant strands to prevent sufficient downstream cell charging for AP propagation. FHF2-dependent effects on Nav inactivation ensure adequate sodium current (INa) reserve to safeguard against numerous threats to reliable cardiac impulse propagation.