As in other multicellular organisms, the nematode Caenorhabditis elegans uses gap junctions to provide direct cell-to-cell contact. The nematode gap junctions are formed by innexins (invertebrate analogs of the connexins); a family of proteins that surprisingly share no primary sequence homology, but do share structural and functional similarity with connexins. The model organism C. elegans contains 25 innexin genes and innexins are found in virtually all cell types and tissues. Additionally, many innexins have dynamic expression patterns during development, and several innexins are essential genes in the nematode. C. elegans is a popular invertebrate model due to several features including a simple anatomy, a complete cell lineage, sequenced genome and an array of genetic resources. Thus, the worm has potential to offer valuable insights into the various functions of gap junction mediated intercellular communication.

In type 1 diabetes (T1D), islet dysfunction occurs prior to diabetes onset. Pro-inflammatory cytokines can disrupt insulin secretion and Ca2+ homeostasis. Connexin36 (Cx36) gap junctions electrically couple β-cells to coordinate glucose-stimulated Ca2+ and insulin secretion. Cx36 gap junction coupling can also protect against cytokine-induced apoptosis. Our goal was to determine how islet gap junction coupling and Ca2+ dynamics are altered in mouse models of T1D prior to diabetes. Glucose tolerance was assessed in NOD and immunodeficient NOD-RAG1KO mice at 6-12 weeks age. Glucose-stimulated insulin secretion, Ca2+ dynamics, and gap junction coupling were measured in islets isolated at each age. Gap junction coupling was also measured in islets from mice that underwent transfer of diabetogenic splenocytes and from chromograninA knockout NOD mice. Cell death was measured in islets isolated from wild-type, Cx36 knockout or Cx36 over-expression mice, each treated with a cocktail of pro-inflammatory cytokines and KATP or SERCA activators/inhibitors. NOD mice over-expressing Cx36 were also monitored for diabetes development, and islets assessed for insulitis and apoptosis. NOD and NOD-RAG1KO controls showed similar glucose tolerance at all ages. Ca2+ dynamics and gap junction coupling were disrupted in islets of NOD mice at 9 weeks, compared to controls. Transfer of diabetogenic splenocytes also decreased gap junction coupling. Islets from chromograninA knockout mice displayed normal coupling. Overexpression of Cx36 protected islets from cytokine-induced apoptosis. A knockout of Cx36 amplified cytokine-induced apoptosis, which was reversed by KATP activation or SERCA activation. Cx36 overexpression in NOD mice delayed diabetes development compared to NOD controls. However, apoptosis and insulitis were not improved. Decreases in islet gap junction coupling occur prior to T1D onset. Such decreases alter islet susceptibility to apoptosis due to altered Ca2+. Future studies will determine if increasing Cx36 gap junction coupling in combination with restoring Ca2+ homeostasis protects against islet decline in T1D.

Gap junctions provide pathways for intercellular communication between adjacent cells, allowing exchange of ions and small molecules. Based on the constituent protein subunits, gap junctions are classified into different subtypes varying in their properties such as unitary conductances, sensitivity to transjunctional voltage, and gating kinetics. Gap junctions couple cells electrically, and therefore the electrical activity originating in one cell can affect and modulate the electrical activity in adjacent cells. Action potentials can propagate through networks of such electrically coupled cells, and this spread is influenced by the nature of gap junctional coupling. Our study aims to computationally explore the effect of differences in gap junctional properties on oscillating action potentials in electrically coupled tissues. Further, we also explore variations in the biophysical environment by altering the size of the syncytium, the location of the pacemaking cell, as well as the occurrence of multiple pacemaking cells within the same syncytium. Our simulation results suggest that the frequency of oscillations is governed by the extent of coupling between cells and the gating kinetics of different gap junction subtypes. The location of pacemaking cells is found to alter the syncytial behavior, and when multiple oscillators are present, there exists an interplay between the oscillator frequency and their relative location within the syncytium. Such variations in the frequency of oscillations can have important implications for the physiological functioning of syncytial tissues.

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

Connexin (Cx) 43 forms gap junctions and hemichannels that mediate communication between osteocytes and adjacent cells or the extracellular environment in bone, respectively. To investigate the role of each channel type in response to mechanical unloading, two transgenic mouse models overexpressing dominant-negative Cx43 predominantly in osteocytes driven by a 10 kb dentin matrix protein 1 (Dmp1) promoter were generated. The R76W mutation resulted in gap junction inhibition and enhancement of hemichannels, whereas the Δ130-136 mutation inhibited both gap junctions and hemichannels. Both mutations led to cortical bone loss with increased endocortical osteoclast activity during unloading. Increased periosteal osteoclasts with decreased apoptotic osteocytes were observed only in R76W mice. These findings indicated that inhibiting osteocytic Cx43 channels promotes bone loss induced by unloading, mainly in the cortical area; moreover, hemichannels protect osteocytes against apoptosis and promote periosteal bone remodeling, whereas gap junctions modulate endocortical osteoclast activity in response to unloading.

Increasing connexin43 (Cx43) gap junctional conductance as a means to improve cardiac conduction has been proposed as a novel antiarrhythmic modality. Yet, transmission of molecules via gap junctions may be associated with increased infarct size. To determine whether maintaining open gap junction channels impacts on infarct size and induction of ventricular tachycardia (VT) following coronary occlusion, we expressed the pH- and voltage-independent connexin isoform connexin32 (Cx32) in ventricle and confirmed Cx32 expression. Wild-type (WT) mice injected with adenovirus-Cx32 (Cx32inj) were examined following coronary occlusion to determine infarct size and inducibility of VT. There was an increased infarct size in Cx32inj hearts as compared to WT (WT 22.9 ± 4%; Cx32inj 44.3 ± 5%; p < 0.05). Programmed electrical stimulation showed no difference in VT inducibility in WT and Cx32inj mice (VT was reproducibly inducible in 55% of shams and 50% of Cx32inj mice (p > 0.05). Following coronary occlusion, improving cell-cell communication increased infarct size, and conferred no antiarrhythmic benefit.

Micromotions are phasic contractions of the bladder wall. During urine storage, such phasic activity has little effect on intravesical pressure, however, changed motile activity may underlie urodynamic observations such as detrusor overactivity. The potential for bladder motility to affect pressure reflects a summation of the overall movements, comprising the initiation, propagation, and dissipation components of micromotions. In this study, the influence of initiation of micromotions was investigated using calcium activated chloride channel blocker niflumic acid, and the effect of propagation using blockers of gap junctions. The overall bladder tone was modulated using isoprenaline. Isolated tissue strips and whole bladder preparations from juvenile rats were used. 18β-glycyrrhetinic acid was used to block gap junctions, reducing the amplitude and frequency of micromotions in in vitro and ex vivo preparations. Niflumic acid reduced the frequency of micromotions but had no effect on the amplitude of pressure fluctuations. Isoprenaline resulted in a reduction in pressure fluctuations and a decrease in pressure baseline. Using visual video data analysis, bladder movement was visible, irrespective of lack of pressure changes, which persisted during bladder relaxation. However, micromotions propagated over shorter distances and the overall bladder tone was reduced. All these results suggest that phasic activity of the bladder can be characterised by a combination of initiation and propagation of movement, and overall bladder tone. At any given moment, intravesical pressure recordings are an integration of these parameters. This synthesis gives insight into the limitations of clinical urodynamics, where intravesical pressure is the key indicator of detrusor activity.

Intercalated discs (ICDs), which connect adjacent cardiomyocytes, are composed of desmosomes, adherens junctions (AJs) and gap junctions (GJs). Previous data demonstrated that adrenergic signaling enhances cardiac myocyte cohesion, referred to as positive adhesiotropy, via PKA-mediated phosphorylation of plakoglobin (PG). However, it was unclear whether positive adhesiotropy caused ultrastructural modifications of ICDs. Therefore, we further investigated the role of PG in adrenergic signaling-mediated ultrastructural changes in the ICD of cardiomyocytes. Quantitative transmission electron microscopy (TEM) analysis of ICD demonstrated that cAMP elevation caused significant elongation of area composita and thickening of the ICD plaque, paralleled by enhanced cardiomyocyte cohesion, in WT but not PG-deficient cardiomyocytes. STED microscopy analysis supported that cAMP elevation ex vivo enhanced overlap of desmoglein-2 (Dsg2) and N-cadherin (N-cad) staining in ICDs of WT but not PG-deficient cardiomyocytes. For dynamic analyses, we utilized HL-1 cardiomyocytes, in which cAMP elevation induced translocation of Dsg2 and PG but not of N-cad to cell junctions. Nevertheless, depletion of N-cad but not of Dsg2 resulted in a decrease in basal cell cohesion whereas positive adhesiotropy was abrogated in monolayers depleted for either Dsg2 or N-cad. In the WT mice, ultrastrutural changes observed after cAMP elevation were paralleled by phosphorylation of PG at serine 665. Our data demonstrate that in murine hearts adrenergic signaling enhanced N-cad and Dsg2 in the ICD paralleled by ultrastrutural strengthening of ICDs and that effects induced by positive adhesiotropy were strictly dependent on Pg.

A wide range of arrhythmogenic phenotypes have been associated with heterogeneous mechanical dyskinesis. Pro-arrhythmic effects are often associated with dysregulated intra-cellular calcium handling, especially via the development of intra- and inter-cellular calcium waves. Experimental evidence suggests that mechanical strain can contribute to the generation and maintenance of these calcium waves via a variety of mechano-electric coupling mechanisms. Most model studies of mechano-electric coupling mechanisms have been focused on mechano-sensitive ion channels, even though experimental studies have shown that intra- and inter-cellular calcium waves triggered by mechanical perturbations are likely to be more prevalent pro-arrhythmic mechanisms in the diseased heart. A one-dimensional strongly coupled computational model of electromechanics in rabbit ventricular cardiomyocytes showed that specific myocyte stretch sequences can modulate the susceptibility threshold for delayed after-depolarizations. In simulations of mechanically-triggered calcium waves in cardiomyocytes coupled to fibroblasts, susceptibility to calcium wave propagation was reduced as the current through the gap junction caused current drain from the myocytes. In 1D multi-cellular arrays coupled via gap junctions, mechanically-induced waves may contribute to synchronizing arrhythmogenic calcium waves and after-depolarizations.

Na+:K+:2Cl- cotransporters (NKCCs) belong to the SLC12A family of cation-coupled Cl- transporters. We investigated whether enamel-producing mouse ameloblasts express NKCCs. Transcripts for Nkcc1 were identified in the mouse dental epithelium by RT-qPCR and NKCC1 protein was immunolocalized in outer enamel epithelium and in the papillary layer but not the ameloblast layer. In incisors of Nkcc1-null mice late maturation ameloblasts were disorganized, shorter and the mineral density of the enamel was reduced by 10% compared to wild-type controls. Protein levels of gap junction protein connexin 43, Na+-dependent bicarbonate cotransporter e1 (NBCe1), and the Cl--dependent bicarbonate exchangers SLC26A3 and SLC26A6 were upregulated in Nkcc1-null enamel organs while the level of NCKX4/SLC24A4, the major K+, Na+ dependent Ca2+ transporter in maturation ameloblasts, was slightly downregulated. Whole-cell voltage clamp studies on rat ameloblast-like HAT-7 cells indicated that bumetanide increased ion-channel activity conducting outward currents. Bumetanide also reduced cell volume of HAT-7 cells. We concluded that non-ameloblast dental epithelium expresses NKCC1 to regulate cell volume in enamel organ and provide ameloblasts with Na+, K+ and Cl- ions required for the transport of mineral- and bicarbonate-ions into enamel. Absence of functional Nkcc1 likely is compensated by other types of ion channels and ion transporters. The increased amount of Cx43 in enamel organ cells in Nkcc1-null mice suggests that these cells display a higher number of gap junctions to increase intercellular communication.

Objective: Connexin 43 (Cx43) is a protein constituent of gap junctions (GJs) in various barrier cells, especially astrocytes and microglia of the blood-brain-barrier (BBB), where it plays an important role in intercellular communication and regulation of the barrier. Despite the importance of Cx43 in other blood barriers, not much attention has been paid to expression and function of Cx43 in the blood-labyrinth-barrier (BLB) of the stria vascularis in the cochlea. Methods: We used multiple research approaches, including immunocytochemical staining, patch-clamp dye loading technique, real-time quantitative reverse transcription (RT)-PCR, western blot, measurement of endocochlear potential (EP) with an electrode through the scala media, and auditory brainstem response to test hearing function. Results: We found Cx43 expressed in vascular endothelial cells (ECs) and perivascular resident macrophages (PVMs) in the stria vascularis of adult C57BL/6 mouse cochleae. In particular, we found Cx43 expressed in foot processes of PVMs at points of contact with the endothelium. Consistent with Cx43 expression in vivo, we also found Cx43 expressed in EC-EC and EC-PVM interfaces in a co-cultured cell line model. Using a patch-clamp dye loading technique, we demonstrated that Alexa Fluor® 568 dye injected into PVMs diffuses to connected neighboring ECs. The functional coupling between the ECs and PVMs is blocked by 18α-Glycyrrhetinic acid (18α-GA), a GJ blocker. Suppression of Cx43 with small interfering RNA (siRNA) in vivo significantly elevated hearing threshold and caused the EP to drop and the blood barrier to become more permeable. In further study, using in vitro primary EC cell line models, we demonstrated that suppression of Cx43 disrupts intercellular tight junctions (TJs) in the EC monolayer and increases endothelial monolayer permeability. Conculsion: Taken together, these findings underscore the importance of Cx43 expression in the normal ear for maintaining BLB integrity, normal EP, and hearing function.

Cardiac fibroblasts express multiple voltage-dependent ion channels. Even though fibroblasts do not generate action potentials, they may influence cardiac electrophysiology by electrical coupling via gap junctions with cardiomyocytes, and through fibrosis. Here, we investigate the electrophysiological phenotype of cultured fibroblasts from right atrial appendage tissue of patients with sinus rhythm (SR) or atrial fibrillation (AF). Using the patch-clamp technique in whole-cell mode, we observed steady-state outward currents exhibiting either no rectification or inward and/or outward rectification. The distributions of current patterns between fibroblasts from SR and AF patients were not significantly different. In response to depolarizing voltage pulses, we measured transient outward currents with fast and slow activation kinetics, an outward background current, and an inward current with a potential-dependence resembling that of L-type Ca2+ channels. In cell-attached patch-clamp mode, large amplitude, paxilline-sensitive single channel openings were found in ≈65% of SR and ∼38% of AF fibroblasts, suggesting the presence of "big conductance Ca2+-activated K+ (BK Ca )" channels. The open probability of BK Ca was significantly lower in AF than in SR fibroblasts. When cultured in the presence of paxilline, the shape of fibroblasts became wider and less spindle-like. Our data confirm previous findings on cardiac fibroblast electrophysiology and extend them by illustrating differential channel expression in human atrial fibroblasts from SR and AF tissue.

Computational modeling is an important tool to advance our knowledge on cardiac diseases and their underlying mechanisms. Computational models of conduction in cardiac tissues require identification of parameters. Our knowledge on these parameters is limited, especially for diseased tissues. Here, we assessed and quantified parameters for computational modeling of conduction in cardiac tissues. We used a rabbit model of myocardial infarction (MI) and an imaging-based approach to derive the parameters. Left ventricular tissue samples were obtained from fixed control hearts (animals: 5) and infarcted hearts (animals: 6) within 200 μm (region 1), 250-750 μm (region 2) and 1,000-1,250 μm (region 3) of the MI border. We assessed extracellular space, fibroblasts, smooth muscle cells, nuclei and gap junctions by a multi-label staining protocol. With confocal microscopy we acquired three-dimensional (3D) image stacks with a voxel size of 200 × 200 × 200 nm. Image segmentation yielded 3D reconstructions of tissue microstructure, which were used to numerically derive extracellular conductivity tensors. Volume fractions of myocyte, extracellular, interlaminar cleft, vessel and fibroblast domains in control were (in %) 65.03 ± 3.60, 24.68 ± 3.05, 3.95 ± 4.84, 7.71 ± 2.15, and 2.48 ± 1.11, respectively. Volume fractions in regions 1 and 2 were different for myocyte, myofibroblast, vessel, and extracellular domains. Fibrosis, defined as increase in fibrotic tissue constituents, was (in %) 21.21 ± 1.73, 16.90 ± 9.86, and 3.58 ± 8.64 in MI regions 1, 2, and 3, respectively. For control tissues, image-based computation of longitudinal, transverse and normal extracellular conductivity yielded (in S/m) 0.36 ± 0.11, 0.17 ± 0.07, and 0.1 ± 0.06, respectively. Conductivities were markedly increased in regions 1 (+75, +171, and +100%), 2 (+53, +165, and +80%), and 3 (+42, +141, and +60%). Volume fractions of the extracellular space including interlaminar clefts strongly correlated with conductivities in control and MI hearts. Our study provides novel quantitative data for computational modeling of conduction in normal and MI hearts. Notably, our study introduces comprehensive statistical information on tissue composition and extracellular conductivities on a microscopic scale in the MI border zone. We suggest that the presented data fill a significant gap in modeling parameters and extend our foundation for computational modeling of cardiac conduction.

During enamel development, formation of hydroxyapatite crystals and regulation of pH in the enamel matrix require massive transport of ions. Both ameloblasts and adjacent dental epithelial cells in the stellate reticulum co-express several transmembrane cotransporters/ion-exchangers for transport of ions across plasma membranes. Gap junctions (GJs) enable intercellular exchanges of ions between neighboring cells. This suggests that the ameloblasts and other cell layers of the enamel organ, form a functional unit. During the bell stage of tooth formation, the non-ameloblast dental epithelium highly expresses the Na-K-Cl cotransporter (Nkcc1). Nkcc1-null mice are associated with enamel hypomineralization and increased expression of GJ protein connexin 43 (Cx43), suggesting that reduced ion transport in the Nkcc1-null mouse is in part compensated by increased intercellular ion transport through GJs. To understand the role of GJs in ion transport and its effect on pH regulation, we examined in a mouse strain in which Cx43 was ablated selectively in DMP1 expressing cells (Cx43flox/flox mice crossed with DMP1-8kb-Cre mice), including ameloblasts. Micro-CT analysis showed that the mineral density at late maturation stage incisal enamel of the Cx43-null mice was 10% less than in controls, whereas that in dentin was unchanged. Maturation stage ameloblasts of mice lacking the pH regulating sodium/bicarbonate transporter NBCe1 (Nbce1-null), or chloride channel Cftr (Cftr-null) were found to have increased Cx43-immunostaining. These results support the possibility that GJs in the ameloblast-papillary complex at the maturation stage contribute to ion transport by enabling passage of ions directly from cells of the papillary layer into ameloblast layer. Increasing the number of GJs may partly compensate the reduction of ion-cotransporters and ion exchangers in dental epithelium.

The juxtaglomerular apparatus (JGA) is an essential structure in the regulation of renal function. The JGA embodies two major functions: tubuloglomerular feedback (TGF) and renin secretion. TGF is one of the mechanisms mediating renal autoregulation. It is initiated by an increase in tubular NaCl concentration at the macula densa cells. This induces a local afferent arteriolar vasoconstriction and a conducted response that can be measured several 100 μm upstream from the juxtaglomerular segment. This spread of the vasomotor response into the surrounding vasculature likely plays a key role in renal autoregulation, and it requires the presence of gap junctions, intercellular pores based on connexin (Cx) proteins. Several Cx isoforms are expressed in the JGA and in the arteriolar wall. Disruption of this communication pathway is associated with reduced TGF, dysregulation of renin secretion, and hypertension. We examine if the absence of Cx40 or Cx45, expressed in the endothelial and vascular smooth muscle cells respectively, attenuates afferent arteriolar local and conducted vasoconstriction. Afferent arterioles from wildtype and Cx-deficient mice (Cx40 and Cx45) were studied using the isolated perfused juxtamedullary nephron preparation. Vasoconstriction was induced via electrical pulse stimulation at the glomerular entrance. Inner afferent arteriolar diameter was measured locally and upstream to evaluate conducted vasoconstriction. Electrical stimulation induced local vasoconstriction in all groups. The local vasoconstriction was significantly smaller when Cx40 was absent. The vasoconstriction decreased in magnitude with increasing distance from the stimulation site. In both Cx40 and Cx45 deficient mice, the vasoconstriction conducted a shorter distance along the vessel compared to wild-type mice. In Cx40 deficient arterioles, this may be caused by a smaller local vasoconstriction. Collectively, these findings imply that Cx40 and Cx45 are central for normal vascular reactivity and, therefore, likely play a key role in TGF-induced regulation of afferent arteriolar resistance.

Transcriptomes associated with wooden breast (WB) were characterized in broilers at two different market ages. Breasts (Pectoralis major) were collected, 20-min postmortem, from male Ross 308 broilers slaughtered at 6 and 7 weeks of age. The breasts were classified as "non-WB" or "WB" based on palpation hardness scoring (non-WB = no abnormal hardness, WB = consistently hardened). Total RNA was isolated from 16 samples (n = 3 for 6 week non-WB, n = 3 for 6 week WB; n = 5 for 7 week non-WB, n = 5 for 7 week WB). Transcriptome was profiled using a chicken gene expression microarray with one-color hybridization technique, and compared between non-WB and WB samples of the same age. Among 6 week broilers, 910 transcripts were differentially expressed (DE) (false discovery rate, FDR < 0.05). Pathway analysis underlined metabolisms of glucose and lipids along with gap junctions, tight junction, and focal adhesion (FA) signaling as the top enriched pathways. For the 7 week broilers, 1,195 transcripts were identified (FDR < 0.05) with regulation of actin cytoskeleton, mitogen-activated protein kinase (MAPK) signaling, protein processing in endoplasmic reticulum and FA signaling highlighted as the enriched affected pathways. Absolute transcript levels of eight genes (actinin-1 - ACTN1, integrin-linked kinase - ILK, integrin subunit alpha 8 - ITGA8, integrin subunit beta 5 - ITGB5, protein tyrosine kinase 2 - PTK2, paxillin - PXN, talin 1 - TLN1, and vinculin - VCL) of FA signaling pathway were further elucidated using a droplet digital polymerase chain reaction. The results indicated that, in 6 week broilers, ITGA8 abundance in WB was greater than that of non-WB samples (p < 0.05). Concerning 7 week broilers, greater absolute levels of ACTN1, ILK, ITGA8, and TLN1, accompanied with a reduced ITGB5 were found in WB compared with non-WB (p < 0.05). Transcriptional modification of FA signaling underlined the potential of disrupted cell-cell communication that may incite aberrant molecular events in association with development of WB myopathy.

Novel treatment strategies for cardiac tissue regeneration are heading for the use of engineered cardiac tissue made from induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs). Despite the proven cardiogenic phenotype of these cells, a significant lack of structural and functional properties of mature myocytes prevents safe integration into the diseased heart. To date, maturation processes of cardiomyocytes remain largely unknown but may comprise biophysical cues from the immediate cell environment. Mechanosensing is one critical ability of cells to react to environmental changes. Accordingly, the surrounding substrate stiffness, comprised of extracellular matrix (ECM), cells, and growth surface, critically influences the myocyte's physiology, as known from deleterious remodeling processes in fibrotic hearts. Conversely, the mechanical properties during culture of iPSC-CMs may impact on their structural and functional maturation. Here, we tested the hypothesis that the environmental stiffness influences structural and functional properties of iPSC-CMs and investigated the effect of different substrate stiffnesses on cell contractility, excitation-contraction (EC) coupling, and intercellular coupling. Culture surfaces with defined stiffnesses ranging from rigid glass with 25GPa to PDMS of physiological softness were coated with ECM proteins and seeded with murine iPSC-CMs. Using confocal imaging, cardiac protein expression was assessed. Ca2+ handling and contractile properties were analyzed on different substrate stiffnesses. Intercellular coupling via gap junctions was investigated by fluorescence recovery after photobleaching (FRAP). Our data revealed greater organization of L-type Ca2+ channels and ryanodine receptors and increased EC-coupling gain, demonstrating structural and functional maturation in cells grown on soft surfaces. In addition, increased shortening and altered contraction dynamics revealed increased myofilament Ca2+ sensitivity in phase-plane loops. Moreover, connexin 43 expression was significantly increased in iPSC-CMs grown on soft surfaces leading to improved intercellular coupling. Taken together, our results demonstrate that soft surfaces with stiffnesses in the physiological range improve the expression pattern and interaction of cardiac proteins relevant for EC-coupling. In parallel, soft substrates influence contractile properties and improve intercellular coupling in iPSC-CMs. We conclude that the mechanical stiffness of the cell environment plays an important role in driving iPSC-CMs toward further maturation by inducing adaptive responses.

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∼130 mV 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 1 μF cm-2, experimentally reported radii a = 8,000, 12,000 and 40,000 nm respectively and ephaptic axial distance w = 20 nm. This enclosed an ephaptic space containing principal ions initially at normal extracellular [Na+] = 153.1 mM 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 ≤1,600 nm mesh sizes and Δt≤0.08 ms 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 2000 ms 99.9% recovery times, with 720 or 360 ms 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, ∼40 ms) can determine physical limits to, electrodiffusive contributions to ephaptic recovery.

Beneficial fungi in the genus Trichoderma are among the most widespread biocontrol agents of plant pathogens. Their role in triggering plant defenses against pathogens has been intensely investigated, while, in contrast, very limited information is available on induced barriers active against insects. The growing experimental evidence on this latter topic looks promising, and paves the way toward the development of Trichoderma strains and/or consortia active against multiple targets. However, the predictability and reproducibility of the effects that these beneficial fungi is still somewhat limited by the lack of an in-depth understanding of the molecular mechanisms underlying the specificity of their interaction with different crop varieties, and on how the environmental factors modulate this interaction. To fill this research gap, here we studied the transcriptome changes in tomato plants (cultivar "Dwarf San Marzano") induced by Trichoderma harzianum (strain T22) colonization and subsequent infestation by the aphid Macrosiphum euphorbiae. A wide transcriptome reprogramming, related to metabolic processes, regulation of gene expression and defense responses, was induced both by separate experimental treatments, which showed a synergistic interaction when concurrently applied. The most evident expression changes of defense genes were associated with the multitrophic interaction Trichoderma-tomato-aphid. Early and late genes involved in direct defense against insects were induced (i.e., peroxidase, GST, kinases and polyphenol oxidase, miraculin, chitinase), along with indirect defense genes, such as sesquiterpene synthase and geranylgeranyl phosphate synthase. Targeted and untargeted semi-polar metabolome analysis revealed a wide metabolome alteration showing an increased accumulation of isoprenoids in Trichoderma treated plants. The wide array of transcriptomic and metabolomics changes nicely fit with the higher mortality of aphids when feeding on Trichoderma treated plants, herein reported, and with the previously observed attractiveness of these latter toward the aphid parasitoid Aphidius ervi. Moreover, Trichoderma treated plants showed the over-expression of transcripts coding for several families of defense-related transcription factors (bZIP, MYB, NAC, AP2-ERF, WRKY), suggesting that the fungus contributes to the priming of plant responses against pest insects. Collectively, our data indicate that Trichoderma treatment of tomato plants induces transcriptomic and metabolomic changes, which underpin both direct and indirect defense responses.