Intercellular Coupling Research Articles

Dynamics of neuroeffector coupling at cardiac sympathetic synapses.

The present study demonstrates, by in vitro and in vivo analyses, the novel concept that signal transmission between sympathetic neurons and the heart, underlying the physiological regulation of cardiac function, operates in a quasi-synaptic fashion. This is a result of the direct coupling between neurotransmitter releasing sites and effector cardiomyocyte membranes. Cardiac sympathetic neurons (SNs) finely tune the rate and strength of heart contractions to match blood demand, both at rest and during acute stress, through the release of noradrenaline (NE). Junctional sites at the interface between the two cell types have been observed, although whether direct neurocardiac coupling has a role in heart physiology has not been clearly demonstrated to date. We investigated the dynamics of SN/cardiomyocyte intercellular signalling, both by fluorescence resonance energy transfer-based imaging of cAMP in co-cultures, as a readout of cardiac β-adrenergic receptor activation, and in vivo, using optogenetics in transgenic mice with SN-specific expression of Channelrhodopsin-2. We demonstrate that SNs and cardiomyocytes interact at specific sites in the human and rodent heart, as well as in co-cultures. Accordingly, neuronal activation elicited intracellular cAMP increases only in directly contacted myocytes and cell-cell coupling utilized a junctional extracellular signalling domain with an elevated NE concentration. In the living mouse, optogenetic activation of cardiac SNs innervating the sino-atrial node resulted in an instantaneous chronotropic effect, which shortened the heartbeat interval with single beat precision. Remarkably, inhibition of the optogenetically elicited chronotropic responses required a high dose of propranolol (20-50mgkg-1 ), suggesting that sympathetic neurotransmission in the heart occurs at a locally elevated NE concentration. Our in vitro and in vivo data suggest that the control of cardiac function by SNs occurs via direct intercellular coupling as a result of the establishment of a specific junctional site.

Electrical dynamics of isolated cerebral and skeletal muscle endothelial tubes: Differential roles of G-protein-coupled receptors and K+ channels.

Electrical dynamics of freshly isolated cerebral endothelium have not been determined independently of perivascular nerves and smooth muscle. We tested the hypothesis that endothelium of cerebral and skeletal muscle arteries differentially utilizes purinergic and muscarinic signaling pathways to activate endothelium‐derived hyperpolarization. Changes in membrane potential (V m) were recorded in intact endothelial tubes freshly isolated from posterior cerebral and superior epigastric arteries of male and female C57BL/6 mice (age: 3‐8 months). V m was measured in response to activation of purinergic (P2Y) and muscarinic (M3) receptors in addition to small‐ and intermediate‐conductance Ca2+‐activated K+ (SKC a/IKC a) and inward rectifying K+ (KIR) channels using ATP (100 μmol·L−1), acetylcholine (ACh; 10 μmol·L−1), NS309 (0.01‐10 μmol·L−1), and 15 mmol·L−1 KCl, respectively. Intercellular coupling was demonstrated via transfer of propidium iodide dye and electrical current (±0.5‐3 nA) through gap junctions. With similarities observed across gender, peak hyperpolarization to ATP and ACh in skeletal muscle endothelial tubes was ~twofold and ~sevenfold higher, respectively, vs cerebral endothelial tubes, whereas responses to NS309 were similar (from resting V m ~−30 mV to maximum ~−80 mV). Hyperpolarization (~8 mV) occurred during 15 mmol·L−1 KCl treatment in cerebral but not skeletal muscle endothelial tubes. Despite weaker hyperpolarization during endothelial GPCR stimulation in cerebral vs skeletal muscle endothelium, the capability for robust SKC a/IKC a activity is preserved across brain and skeletal muscle. As vascular reactivity decreases with aging and cardiovascular disease, endothelial K+ channel activity may be calibrated to restore blood flow to respective organs regardless of gender.

Desmoglein Binding Properties are Regulated by Plakophilins

Desmosomes are highly organized protein complexes required for strong intercellular adhesion of keratinocytes. To maintain desmosome integrity, desmosomal cadherins provide intercellular mechanical coupling with their extracellular domains whereas plaque proteins such as plakophilins (Pkp), plakoglobin and desmoplakin mediate the anchorage to the intermediate filament cytoskeleton. In addition, Pkp isoforms are involved in the regulation of desmosomal turnover and several signaling pathways and are thereby crucial for desmosomal adhesion. However, their contribution to binding properties of desmosomal cadherins has not been elucidated yet. Here, we used murine keratinocytes lacking either Pkp1 or Pkp3 (Pkp1 k.o. / Pkp3 k.o.) and characterized Dsg1 and 3 binding properties in comparison to wildtype cells (Pkp wt). Both, Pkp1 and 3 k.o. cells showed compromised intercellular adhesion and reduced membrane levels of Dsg1 and Dsg3. Consistent with these findings, the amount of Dsg3 oligomers detected by membrane‐impermeable extracellular crosslinking was drastically reduced in both, Pkp1 and 3 k.o. cell lines. Along the same line, binding frequencies of both, Dsg1 and Dsg3‐dependent binding events as well as binding strength and the step position of Dsg3 interaction, the latter of which represents a measure for cytoskeletal anchorage, were drastically reduced in Pkp1 and 3 k.o. cell lines. Further, Dsg1 binding frequency was higher at cell borders compared to cell surface in wt but not in Pkp1 and Pkp3 k.o. cells, indicating a Pkp‐dependent control of Dsg1 membrane positioning. Moreover, alteration of p38MAPK signaling, a central signaling pathway for the regulation of desmosomal adhesion, correlated with intercellular adhesion in all cell lines. However, Pkp3 k.o. cells showed reduced activity of p38MAPK under untreated conditions as well as a diminished response to the p38MAPK activator anisomycin, indicating an important role of Pkp3 for p38MAPK signaling. Taken together, these data suggest (1) that Pkps are important for localization and binding of desmosomal cadherins and (2) that Pkp3 may participate in the regulation of desmosomal adhesion via p38MAPK signaling.Support or Funding InformationFunding Information: Supported by Else‐Kröner‐Fresenius‐Stiftung (2016_A157).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Induction of Gap Junctional Coupling by Secreted Factor(s) from Mesothelial Cells is Required for the Invasiveness of Endometriosis

Intercellular interactions, including gap junction intercellular coupling (GJIC), have been implicated in many invasive processes (e.g. extravasation, metastasis and oocyte implantation). Yet the role of gap junctions in endometriosis, a major invasive medical problem that affects 6–10% of women worldwide, has not been investigated. Comparisons of the expression profile of endometrial cells (ECs) from endometriosis patients and normal subjects revealed a consistent reduction in the expression levels of connexins (the protein components of gap junctions), including the most abundant isoform, Cx43. This was most notable in stromal cells as the disease progressed, but was also evident in epithelial cells. GJIC of ECs from endometriosis patients was also reduced compared to normal subjects, again most notably in stromal cells. In mimicking the endometriosis process, heterotypic interactions with mesothelial cells induced an increase in GJIC, with stromal cells from endometriosis patients showing the largest response. This enhanced coupling correlated with enhanced trafficking of Cx43 to the cell surface, and enhanced invasiveness through a mesothelial monolayer. Blocking GJIC between endometrial and mesothelial cells using either siRNA or a Cx43 specific peptide blocker dramatically reduced this enhanced invasiveness of endometriosis derived ECs, whether epithelial or stromal in origin. Finally, GJIC induction by mesothelial cells was shown to be mediated by a secreted factor, which is heat resistant and approximately 5–10K is size. This presents a unique opportunity for developing novel therapy for treating endometriosis that would prevent the formation of lesions that cause the pain and incidence of infertility associated with this widespread disease. Currently the only therapies are based on hormonal treatments that only further compromise fertility problems associated with endometriosis (representing 35% of the cases on infertility in the nation).Support or Funding InformationWomen's Health Initiative at UTHSA School of Medicine and CPRIT grants RP150600 and RP160844, and NIH grant UL1 TR001120 for CoresThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Connexin 30 controls astroglial polarization during postnatal brain development.

ABSTRACTAstrocytes undergo intense morphological maturation during development, changing from individual sparsely branched cells to polarized and tremendously ramified cells. Connexin 30, an astroglial gap-junction channel-forming protein expressed postnatally, regulates in situ the extension and ramification of astroglial processes. However, the involvement of connexin 30 in astroglial polarization, which is known to control cell morphology, remains unexplored. We found that connexin 30, independently of gap-junction-mediated intercellular biochemical coupling, alters the orientation of astrocyte protrusion, centrosome and Golgi apparatus during polarized migration in an in vitro wound-healing assay. Connexin 30 sets the orientation of astroglial motile protrusions via modulation of the laminin/β1 integrin/Cdc42 polarity pathway. Connexin 30 indeed reduces laminin levels, inhibits the redistribution of the β1-integrin extracellular matrix receptors, and inhibits the recruitment and activation of the small Rho GTPase Cdc42 at the leading edge of migrating astrocytes. In vivo, connexin 30, the expression of which is developmentally regulated, also contributes to the establishment of hippocampal astrocyte polarity during postnatal maturation. This study thus reveals that connexin 30 controls astroglial polarity during development.

A multiscale active structural model of the arterial wall accounting for smooth muscle dynamics.

Arterial wall dynamics arise from the synergy of passive mechano-elastic properties of the vascular tissue and the active contractile behaviour of smooth muscle cells (SMCs) that form the media layer of vessels. We have developed a computational framework that incorporates both these components to account for vascular responses to mechanical and pharmacological stimuli. To validate the proposed framework and demonstrate its potential for testing hypotheses on the pathogenesis of vascular disease, we have employed a number of pharmacological probes that modulate the arterial wall contractile machinery by selectively inhibiting a range of intracellular signalling pathways. Experimental probes used on ring segments from the rabbit central ear artery are: phenylephrine, a selective α1-adrenergic receptor agonist that induces vasoconstriction; cyclopiazonic acid (CPA), a specific inhibitor of sarcoplasmic/endoplasmic reticulum Ca2+-ATPase; and ryanodine, a diterpenoid that modulates Ca2+ release from the sarcoplasmic reticulum. These interventions were able to delineate the role of membrane versus intracellular signalling, previously identified as main factors in smooth muscle contraction and the generation of vessel tone. Each SMC was modelled by a system of nonlinear differential equations that account for intracellular ionic signalling, and in particular Ca2+ dynamics. Cytosolic Ca2+ concentrations formed the catalytic input to a cross-bridge kinetics model. Contractile output from these cellular components forms the input to the finite-element model of the arterial rings under isometric conditions that reproduces the experimental conditions. The model does not account for the role of the endothelium, as the nitric oxide production was suppressed by the action of L-NAME, and also due to the absence of shear stress on the arterial ring, as the experimental set-up did not involve flow. Simulations generated by the integrated model closely matched experimental observations qualitatively, as well as quantitatively within a range of physiological parametric values. The model also illustrated how increased intercellular coupling led to smooth muscle coordination and the genesis of vascular tone.

Photoperiodic Programming of the SCN and Its Role in Photoperiodic Output.

Though the seasonal response of organisms to changing day lengths is a phenomenon that has been scientifically reported for nearly a century, significant questions remain about how photoperiod is encoded and effected neurobiologically. In mammals, early work identified the master circadian clock, the suprachiasmatic nuclei (SCN), as a tentative encoder of photoperiodic information. Here, we provide an overview of research on the SCN as a coordinator of photoperiodic responses, the intercellular coupling changes that accompany that coordination, as well as the SCN's role in a putative brain network controlling photoperiodic input and output. Lastly, we discuss the importance of photoperiodic research in the context of tangible benefits to human health that have been realized through this research as well as challenges that remain.

WNT Takes Two to Tango: Molecular Links between the Circadian Clock and the Cell Cycle in Adult Stem Cells.

Like two dancers, the circadian clock and cell cycle are biological oscillators engaged in bidirectional communication, resulting in circadian clock-gated cell division cycles in species ranging from cyanobacteria to mammals. The identified mechanisms for this phenomenon have expanded beyond intracellular molecular coupling components to include intercellular connections. However, detailed molecular mechanisms, dynamics, and physiological functions of the circadian clock and cell cycle as coupled oscillators remain largely unknown. In this review, we discuss current understanding of this connection in light of recent findings that have uncovered intercellular coupling between the circadian clock in Paneth cells and the cell cycle in intestinal stem cells via WNT signaling. This extends the impact of circadian rhythms regulating the timing of cell divisions beyond the intracellular domain of homogenous cell populations into dynamic, multicellular systems. In-depth understanding of the molecular links and dynamics of these two oscillators will identify potential targets and temporal regimens for effective chronotherapy.

Critical and Supercritical Spatiotemporal Calcium Dynamics in Beta Cells.

A coordinated functioning of beta cells within pancreatic islets is mediated by oscillatory membrane depolarization and subsequent changes in cytoplasmic calcium concentration. While gap junctions allow for intraislet information exchange, beta cells within islets form complex syncytia that are intrinsically nonlinear and highly heterogeneous. To study spatiotemporal calcium dynamics within these syncytia, we make use of computational modeling and confocal high-speed functional multicellular imaging. We show that model predictions are in good agreement with experimental data, especially if a high degree of heterogeneity in the intercellular coupling term is assumed. In particular, during the first few minutes after stimulation, the probability distribution of calcium wave sizes is characterized by a power law, thus indicating critical behavior. After this period, the dynamics changes qualitatively such that the number of global intercellular calcium events increases to the point where the behavior becomes supercritical. To better mimic normal in vivo conditions, we compare the described behavior during supraphysiological non-oscillatory stimulation with the behavior during exposure to a slightly lower and oscillatory glucose challenge. In the case of this protocol, we observe only critical behavior in both experiment and model. Our results indicate that the loss of oscillatory changes, along with the rise in plasma glucose observed in diabetes, could be associated with a switch to supercritical calcium dynamics and loss of beta cell functionality.

The stress kinase JNK regulates gap junction Cx43 gene expression and promotes atrial fibrillation in the aged heart

BackgroundThe stress kinase c-jun N-terminal kinase (JNK) is critical in the pathogenesis of cardiac diseases associated with an increased incidence of atrial fibrillation (AF), the most common arrhythmia in the elderly. We recently discovered that JNK activation is linked to the loss of gap junction connexin43 (Cx43) and enhanced atrial arrhythmogenicity. However, direct evidence for JNK-mediated impairment of intercellular coupling (cell-cell communication) in the intact aged atrium is lacking, as is evidence for whether and how JNK suppresses Cx43 in the aged human atrium. Methods and resultsJNK activity in human atrial samples is correlated with both reduced Cx43 expression and increasing age. Using a unique technique of optical mapping space constant measurement, we found that impaired intercellular coupling and reduced Cx43 were linked to enhanced activation of JNK in intact aged rabbit atria. These JNK-associated alterations were further confirmed in naturally JNK activated aged mice and in cardiac-specific inducible MKK7D (JNK upstream activator) young mice. Moreover, JNK inhibition, using either JNK specific inhibitors in aged wild-type (WT) mice and JNK activator anisomycin-treated young WT mice or JNK1/2 dominant-negative mice with genetically inhibited cardiac JNK activity, completely eliminated these functional abnormalities. Furthermore, we discovered for the first time that long-term JNK activation downregulates Cx43 expression via c-jun suppressed transcriptional activity of the Cx43 gene promoter. ConclusionOur results demonstrate that JNK is a critical regulator of Cx43 expression, and that augmented JNK activation in aged atria downregulates Cx43 to impair cell-cell communication and promote the development of AF. JNK inhibition may represent a promising therapeutic approach to prevent or treat AF in the elderly.

A Method for the Analysis of AP Foot Convexity: Insights into Smooth Muscle Biophysics.

Action potential (AP) profiles vary based on the cell type, with cells of the same type typically producing APs with similar shapes. But in certain syncytial tissues, such as the smooth muscle of the urinary bladder wall, even a single cell is known to exhibit APs with diverse profiles. The origin of this diversity is not currently understood, but is often attributed to factors such as syncytial interactions and the spatial distribution of parasympathetic nerve terminals. Thus, the profile of an action potential is determined by the inherent properties of the cell and influenced by its biophysical environment. The analysis of an AP profile, therefore, holds potential for constructing a biophysical picture of the cellular environment. An important feature of any AP is its depolarization to threshold, termed the AP foot, which holds information about the origin of the AP. Currently, there exists no established technique for the quantification of the AP foot. In this study, we explore several possible approaches for this quantification, namely, exponential fitting, evaluation of the radius of curvature, triangulation altitude, and various area based methods. We have also proposed a modified area-based approach (CX,Y) which quantifies foot convexity as the area between the AP foot and a predefined line. We assess the robustness of the individual approaches over a wide variety of signals, mimicking AP diversity. The proposed (CX,Y) method is demonstrated to be superior to the other approaches, and we demonstrate its application on experimentally recorded AP profiles. The study reveals how the quantification of the AP foot could be related to the nature of the underlying synaptic activity and help shed light on biophysical features such as the density of innervation, proximity of varicosities, size of the syncytium, or the strength of intercellular coupling within the syncytium. The work presented here is directed toward exploring these aspects, with further potential toward clinical electrodiagnostics by providing a better understanding of whole-organ biophysics.

Ontogeny of Circadian Rhythms and Synchrony in the Suprachiasmatic Nucleus.

In mammals, the suprachiasmatic nucleus (SCN) of the hypothalamus coordinates daily rhythms including sleep-wake, hormone release, and gene expression. The cells of the SCN must synchronize to each other to drive these circadian rhythms in the rest of the body. The ontogeny of circadian cycling and intercellular coupling in the SCN remains poorly understood. Recent in vitro studies have recorded circadian rhythms from the whole embryonic SCN. Here, we tracked the onset and precision of rhythms in PERIOD2 (PER2), a clock protein, within the SCN isolated from embryonic and postnatal mice of undetermined sex. We found that a few SCN cells developed circadian periodicity in PER2 by 14.5 d after mating (E14.5) with no evidence for daily cycling on E13.5. On E15.5, the fraction of competent oscillators increased dramatically corresponding with stabilization of their circadian periods. The cells of the SCN harvested at E15.5 expressed sustained, synchronous daily rhythms. By postnatal day 2 (P2), SCN oscillators displayed the daily, dorsal-ventral phase wave in clock gene expression typical of the adult SCN. Strikingly, vasoactive intestinal polypeptide (VIP), a neuropeptide critical for synchrony in the adult SCN, and its receptor, VPAC2R, reached detectable levels after birth and after the onset of circadian synchrony. Antagonists of GABA or VIP signaling or action potentials did not disrupt circadian synchrony in the E15.5 SCN. We conclude that endogenous daily rhythms in the fetal SCN begin with few noisy oscillators on E14.5, followed by widespread oscillations that rapidly synchronize on E15.5 by an unknown mechanism.SIGNIFICANCE STATEMENT We recorded the onset of PER2 circadian oscillations during embryonic development in the mouse SCN. When isolated at E13.5, the anlagen of the SCN expresses high, arrhythmic PER2. In contrast, a few cells show noisy circadian rhythms in the isolated E14.5 SCN and most show reliable, self-sustained, synchronized rhythms in the E15.5 SCN. Strikingly, this synchrony at E15.5 appears before expression of VIP or its receptor and persists in the presence of blockers of VIP, GABA or neuronal firing. Finally, the dorsal-ventral phase wave of PER2 typical of the adult SCN appears ∼P2, indicating that multiple signals may mediate circadian synchrony during the ontogeny of the SCN.

Epilepsy and astrocyte energy metabolism.

Epilepsy is a complex neurological syndrome characterized by neuronal hyperexcitability and sudden, synchronized electrical discharges that can manifest as seizures. It is now increasingly recognized that impaired astrocyte function and energy homeostasis play key roles in the pathogenesis of epilepsy. Excessive neuronal discharges can only happen, if adequate energy sources are made available to neurons. Conversely, energy depletion during seizures is an endogenous mechanism of seizure termination. Astrocytes control neuronal energy homeostasis through neurometabolic coupling. In this review, we will discuss how astrocyte dysfunction in epilepsy leads to distortion of key metabolic and biochemical mechanisms. Dysfunctional glutamate metabolism in astrocytes can directly contribute to neuronal hyperexcitability. Closure of astrocyte intercellular gap junction coupling as observed early during epileptogenesis limits activity-dependent trafficking of energy metabolites, but also impairs clearance of the extracellular space from accumulation of K+ and glutamate. Dysfunctional astrocytes also increase the metabolism of adenosine, a metabolic product of ATP degradation that broadly inhibits energy-consuming processes as an evolutionary adaptation to conserve energy. Due to the critical role of astroglial energy homeostasis in the control of neuronal excitability, metabolic therapeutic approaches that prevent the utilization of glucose might represent a potent antiepileptic strategy. In particular, high fat low carbohydrate "ketogenic diets" as well as inhibitors of glycolysis and lactate metabolism are of growing interest for the therapy of epilepsy.

Calcium-Dependent Arrhythmogenic Foci Created by Weakly Coupled Myocytes in the Failing Heart.

Intercellular uncoupling and Ca2+ (Ca) mishandling can initiate triggered ventricular arrhythmias. Spontaneous Ca release activates inward current which depolarizes membrane potential (Vm) and can trigger action potentials in isolated myocytes. However, cell-cell coupling in intact hearts limits local depolarization and may protect hearts from this arrhythmogenic mechanism. Traditional optical mapping lacks the spatial resolution to assess coupling of individual myocytes. We investigate local intercellular coupling in Ca-induced depolarization in intact hearts, using confocal microscopy to measure local Vm and intracellular [Ca] simultaneously. We used isolated Langendorff-perfused hearts from control (CTL) and heart failure (HF) mice (HF induced by transaortic constriction). In CTL hearts, 1.4% of myocytes were poorly synchronized with neighboring cells and exhibited asynchronous (AS) Ca transients. These AS myocytes were much more frequent in HF (10.8% of myocytes, P<0.05 versus CTL). Local Ca waves depolarized Vm in HF but not CTL hearts, suggesting weaker gap junction coupling in HF-AS versus CTL-AS myocytes. Cell-cell coupling was assessed by calcein fluorescence recovery after photobleach during intracellular [Ca] recording. All regions in CTL hearts exhibited faster calcein diffusion than in HF, with HF-AS myocyte being slowest. In HF-AS, enhancing gap junction conductance (with rotigaptide) increased coupling and suppressed Vm depolarization during Ca waves. Conversely, in CTL hearts, gap junction inhibition (carbenoxolone) decreased coupling and allowed Ca wave-induced depolarizations. Synchronization of Ca wave initiation and triggered action potentials were observed in HF hearts and computational models. Well-coupled CTL myocytes are effectively voltage-clamped during Ca waves, protecting the heart from triggered arrhythmias. Spontaneous Ca waves are much more common in HF myocytes and these AS myocytes are also poorly coupled, enabling local Ca-induced inward current of sufficient source strength to overcome a weakened current sink to depolarize Vm and trigger action potentials.

GJA1-20k Arranges Actin to Guide Cx43 Delivery to Cardiac Intercalated Discs.

Delivery of Cx43 (connexin 43) to the intercalated disc is a continuous and rapid process critical for intercellular coupling. By a pathway of targeted delivery involving microtubule highways, vesicles of Cx43 hemichannels are efficiently trafficked to adherens junctions at intercalated discs. It has also been identified that actin provides rest stops for Cx43 forward trafficking and that Cx43 has a 20 kDa internally translated small C terminus isoform, GJA1-20k (Gap Junction Protein Alpha 1- 20 kDa), which is required for full-length Cx43 trafficking, but by an unknown mechanism. We explored the mechanism by which the GJA1-20k isoform is required for full-length Cx43 forward trafficking to intercalated discs. Using an in vivo Adeno-associated virus serotype 9-mediated gene transfer system, we confirmed in whole animal that GJA1-20k markedly increases endogenous myocardial Cx43 gap junction plaque size at the intercalated discs. In micropatterned cell pairing systems, we found that exogenous GJA1-20k expression stabilizes filamentous actin without affecting actin protein expression and that GJA1-20k complexes with both actin and tubulin. We also found that filamentous actin regulates microtubule organization as inhibition of actin polymerization with a low dose of latrunculin A disrupts the targeting of microtubules to cell-cell junctions. GJA1-20k protects actin filament from latrunculin A disruption, preserving microtubule trajectory to the cell-cell border. For therapeutic implications, we found that prior in vivo Adeno-associated virus serotype 9-mediated gene delivery of GJA1-20k to the heart protects Cx43 localization to the intercalated discs against acute ischemic injury. The internally translated GJA1-20k isoform stabilizes actin filaments, which guides growth trajectories of the Cx43 microtubule trafficking machinery, increasing delivery of Cx43 hemichannels to cardiac intercalated discs. Exogenous GJA1-20k helps to maintain cell-cell coupling in instances of anticipated myocardial ischemia.

Primordial oscillations in life: Direct observation of glycolytic oscillations in individual HeLa cervical cancer cells.

We report the first direct observation of glycolytic oscillations in HeLa cervical cancer cells, which we regard as primordial oscillations preserved in living cells. HeLa cells starved of glucose or both glucose and serum exhibited glycolytic oscillations in nicotinamide adenine dinucleotide (NADH), exhibiting asynchronous intercellular behaviors. Also found were spatially homogeneous and inhomogeneous intracellular NADH oscillations in the individual cells. Our results demonstrate that starved HeLa cells may be induced to exhibit glycolytic oscillations by either high-uptake of glucose or the enhancement of a glycolytic pathway (Crabtree effect or the Warburg effect), or both. Their asynchronous collective behaviors in the oscillations were probably due to a weak intercellular coupling. Elucidation of the relationship between the mechanism of glycolytic dynamics in cancer cells and their pathophysiological characteristics remains a challenge in future.

Computational assessment of the functional role of sinoatrial node exit pathways in the human heart.

AimThe human right atrium and sinoatrial node (SAN) anatomy is complex. Optical mapping experiments suggest that the SAN is functionally insulated from atrial tissue except at discrete SAN-atrial electrical junctions called SAN exit pathways, SEPs. Additionally, histological imaging suggests the presence of a secondary pacemaker close to the SAN. We hypothesise that a) an insulating border-SEP anatomical configuration is related to SAN arrhythmia; and b) a secondary pacemaker, the paranodal area, is an alternate pacemaker but accentuates tachycardia. A 3D electro-anatomical computational model was used to test these hypotheses.MethodsA detailed 3D human SAN electro-anatomical mathematical model was developed based on our previous anatomical reconstruction. Electrical activity was simulated using tissue specific variants of the Fenton-Karma action potential equations. Simulation experiments were designed to deploy this complex electro-anatomical system to assess the roles of border-SEPs and paranodal area by mimicking experimentally observed SAN arrhythmia. Robust and accurate numerical algorithms were implemented for solving the mono domain reaction-diffusion equation implicitly, calculating 3D filament traces, and computing dominant frequency among other quantitative measurements.ResultsA centre to periphery gradient of increasing diffusion was sufficient to permit initiation of pacemaking at the centre of the 3D SAN. Re-entry within the SAN, micro re-entry, was possible by imposing significant SAN fibrosis in the presence of the insulating border. SEPs promoted the micro re-entry to generate more complex SAN-atrial tachycardia. Simulation of macro re-entry, i.e. re-entry around the SAN, was possible by inclusion of atrial fibrosis in the presence of the insulating border. The border shielded the SAN from atrial tachycardia. However, SAN micro-structure intercellular gap junctional coupling and the paranodal area contributed to prolonged atrial fibrillation. Finally, the micro-structure was found to be sufficient to explain shifts of leading pacemaker site location.ConclusionsThe simulations establish a relationship between anatomy and SAN electrical function. Microstructure, in the form of intercellular gap junction coupling, was found to regulate SAN function and arrhythmia.

Abstract 307: GJA1-20k Regulates Actin Cytoskeleton and Promotes Delivery of Connexin 43 to Cardiac Intercalated Discs

Rationale: Delivery of connexin 43 (Cx43) to the intercalated disc is a continuous and rapid process critical for intercellular coupling. By a pathway of targeted delivery involving microtubules and actin cytoskeleton rest stops, Cx43 hemichannels are efficiently trafficked to adherens junctions at intercalated discs. It has recently been shown that an internally translated isoform of Cx43, GJA1-20k, facilitates full-length Cx43 trafficking in cell lines, although the mechanism remains unknown. Objective: We explored the mechanism by which GJA1-20k regulates the trafficking of full-length Cx43 to intercalated discs. Methods and Results: In vivo overexpression of exogenous GJA1-20k, administered via AAV9-mediated gene delivery, increases the delivery of full length Cx43 to intercalated discs in mouse hearts. Using electron microscopy and fluorescence microscopy, together with biochemical co-immunoprecipitation, we found in micro-patterned HeLa cells and cardiomyocytes that GJA1-20k not only substantially increases the number and length of actin fibers, but can also rescue the effect of actin disruption. GJA1-20k complexes with actin and tubulin, improving microtubule targeting to cell-cell borders in the setting of actin disruption. Actin is also disrupted in acute ischemia-reperfusion (IR) injury. The ex vivo rescue potential of GJA1-20k was further tested in mouse hearts subjected to myocardial IR injury. As compared to control GFP and full length GAJ1-43k, only GJA1-20k gene transfer significantly improves the targeting of Cx43 to intercalated discs following IR injury. Conclusions: These results indicate that GJA1-20k positively modulates actin cytoskeleton to facilitate microtubule-based Cx43 trafficking machinery, promoting the delivery of full-length Cx43 to cardiac cell-cell junctions under normal and ischemic conditions. Therefore, up regulation of GJA1-20k is a potential therapeutic option to reverse the loss of Cx43 in IR injuries.

Connexin43, but not connexin30, contributes to adult neurogenesis in the dentate gyrus

The subgranular zone of the dentate gyrus represents a niche in which radial glia (RG)-like cells generate new neurons throughout postnatal life in the mammalian brain. Previous data showed that RG-like cells are coupled through gap junction channels, primarily formed by connexin43 (Cx43) and Cx30, and that the expression of these proteins is required for adult neurogenesis in the hippocampus. However, their individual function and underlying mechanisms remain unclear. Here we demonstrate that Cx43, but not Cx30, is crucial for adult neurogenesis. To assess whether Cx43-dependent intercellular coupling between RG-like cells or rather channel-independent interactions of the protein regulate neurogenesis, mice bearing a Cx43 point mutation (Cx43G138R) in RG-like cells and protoplasmic astrocytes cells were employed, which was expected to cause channel closure without affecting the trafficking of the protein to the membrane. We confirmed the disruption of coupling between RG-like cells and astrocytes in the hippocampus of Cx43G138R mice. Proliferative activity and neurogenesis in the DG were significantly decreased in the mutant mouse line, indicating that functional Cx43 channels are essential for proper adult neurogenesis. The fate of proliferating cells in the DG was not affected by Cx43 mutation as revealed by 5-bromo-2-deoxyuridine (BrdU) incorporation assays. Together, these findings suggest that adult neurogenesis in the hippocampus does not require Cx30 but channel-dependent functions of Cx43.

On the propagation of a low frequency excitation in a perturbed FitzHugh–Nagumo system: Simulation and experiments

In this paper, we first report the response of an elementary FitzHugh–Nagumo electronic circuit excited by a low frequency sine wave and perturbed by an additive high frequency deterministic perturbation. This preliminary study constitutes a reference to analyze the collective behavior of a chain of 45 coupled elementary cells. In particular, we focus on the propagation of a low frequency sine wave which is only applied on the first fifteen cells of the lattice. It is shown that a high frequency sine wave which perturbs the whole network can enable or disable the propagation of this low frequency signal. By reducing the strength of the intercellular coupling below a critical value, we also establish that the propagation fails whatever the amplitude of the perturbation. Finally, by adding a stochastic perturbation to the high frequency deterministic perturbation, we numerically and experimentally investigate their combined effects on the propagation of the low frequency component. Numerical and experimental results reveal that, under certain conditions, noise can assist the propagation of the low frequency excitation.