Cx43 Hemichannels Research Articles

Role of Connexin 43 in an Inflammatory Model for TMJ Hyperalgesia.

Temporomandibular joint disorders (TMD) consist of a heterogeneous group of conditions that present with pain in the temporomandibular joint (TMJ) region and muscles of mastication. This project assessed the role of connexin 43 (Cx43), a gap junction protein, in the trigeminal ganglion (TG) in an animal model for persistent inflammatory TMJ hyperalgesia. Experiments were performed in male and female rats to determine if sex differences influence the expression and/or function of Cx43 in persistent TMJ hyperalgesia. Intra-TMJ injection of Complete Freund's Adjuvant (CFA) caused a significant increase in Cx43 expression in the TG at 4 days and 10 days post-injection in ovariectomized (OvX) female rats and OvX females treated with estradiol (OvXE), while TG samples in males revealed only marginal increases. Intra-TG injection of interference RNA for Cx43 (siRNA Cx43) 3 days prior to recording, markedly reduced TMJ-evoked masseter muscle electromyographic (MMemg) activity in all CFA-inflamed rats, while activity in sham animals was not affected. Western blot analysis revealed that at 3 days after intra-TG injection of siRNA Cx43 protein levels for Cx43 were significantly reduced in TG samples of all CFA-inflamed rats. Intra-TG injection of the mimetic peptide GAP19, which inhibits Cx43 hemichannel formation, greatly reduced TMJ-evoked MMemg activity in all CFA-inflamed groups, while activity in sham groups was not affected. These results revealed that TMJ inflammation caused a persistent increase in Cx43 protein in the TG in a sex-dependent manner. However, intra-TG blockade of Cx43 by siRNA or by GAP19 significantly reduced TMJ-evoked MMemg activity in both males and females following TMJ inflammation. These results indicated that Cx43 was necessary for enhanced jaw muscle activity after TMJ inflammation in males and females, a result that could not be predicted on the basis of TG expression of Cx43 alone.

Activation of Cx43 Hemichannels Induces the Generation of Ca2+ Oscillations in White Adipocytes and Stimulates Lipolysis.

The aim of the study was to investigate the mechanisms of Ca2+ oscillation generation upon activation of connexin-43 and regulation of the lipolysis/lipogenesis balance in white adipocytes through vesicular ATP release. With fluorescence microscopy it was revealed that a decrease in the concentration of extracellular calcium ([Ca2+]ex) results in two types of Ca2+ responses in white adipocytes: Ca2+ oscillations and transient Ca2+ signals. It was found that activation of the connexin half-channels is involved in the generation of Ca2+ oscillations, since the blockers of the connexin hemichannels—carbenoxolone, octanol, proadifen and Gap26—as well as Cx43 gene knockdown led to complete suppression of these signals. The activation of Cx43 in response to the reduction of [Ca2+]ex was confirmed by TIRF microscopy. It was shown that in response to the activation of Cx43, ATP-containing vesicles were released from the adipocytes. This process was suppressed by knockdown of the Cx43 gene and by bafilomycin A1, an inhibitor of vacuolar ATPase. At the level of intracellular signaling, the generation of Ca2+ oscillations in white adipocytes in response to a decrease in [Ca2+]ex occurred due to the mobilization of the Ca2+ ions from the thapsigargin-sensitive Ca2+ pool of IP3R as a result of activation of the purinergic P2Y1 receptors and phosphoinositide signaling pathway. After activation of Cx43 and generation of the Ca2+ oscillations, changes in the expression levels of key genes and their encoding proteins involved in the regulation of lipolysis were observed in white adipocytes. This effect was accompanied by a decrease in the number of adipocytes containing lipid droplets, while inhibition or knockdown of Cx43 led to inhibition of lipolysis and accumulation of lipid droplets. In this study, we investigated the mechanism of Ca2+ oscillation generation in white adipocytes in response to a decrease in the concentration of Ca2+ ions in the external environment and established an interplay between periodic Ca2+ modes and the regulation of the lipolysis/lipogenesis balance.

CO2-Sensitive Connexin Hemichannels in Neurons and Glia: Three Different Modes of Signalling?

Connexins can assemble into either gap junctions (between two cells) or hemichannels (from one cell to the extracellular space) and mediate cell-to-cell signalling. A subset of connexins (Cx26, Cx30, Cx32) are directly sensitive to CO2 and fluctuations in the level within a physiological range affect their open probability, and thus, change cell conductance. These connexins are primarily found on astrocytes or oligodendrocytes, where increased CO2 leads to ATP release, which acts on P2X and P2Y receptors of neighbouring neurons and changes excitability. CO2-sensitive hemichannels are also found on developing cortical neurons, where they play a role in producing spontaneous neuronal activity. It is plausible that the transient opening of hemichannels allows cation influx, leading to depolarisation. Recently, we have shown that dopaminergic neurons in the substantia nigra and GABAergic neurons in the VTA also express Cx26 hemichannels. An increase in the level of CO2 results in hemichannel opening, increasing whole-cell conductance, and decreasing neuronal excitability. We found that the expression of Cx26 in the dopaminergic neurons in the substantia nigra at P7-10 is transferred to glial cells by P17-21, displaying a shift from being inhibitory (to neuronal activity) in young mice, to potentially excitatory (via ATP release). Thus, Cx26 hemichannels could have three modes of signalling (release of ATP, excitatory flickering open and shut and inhibitory shunting) depending on where they are expressed (neurons or glia) and the stage of development.

Onjisaponin B attenuates glutamate release via inhibition of calmodulin-dependent protein kinase II and connexin 43 pathways in rat astrocytes subjected to oxygen and glucose deprivation

Background: Post-stroke depression (PSD) is one of the most common complications of stroke. Many studies have confirmed that PSD is associated with the abnormal release of glutamic acid (Glu) via the Ca2+/calmodulin (CaM)/CaM-dependent protein kinase II (CaMKII) and connexin 43 (Cx43) hemichannel pathways. Onjisaponin B, a traditional Chinese medicine, has been discovered to have neuroprotective effects. Objectives: In this study, we aimed to investigate the effects and underlying mechanisms of onjisaponin B on the release of glutamate in rat astrocytes subjected to oxygen and glucose deprivation (OGD). Materials and Methods: The ischemic and anoxic cell model was established in rat astrocytes through OGD injury. Rat astrocytes were randomly divided into control, model, 10 μM onjisaponin B, and 20 μM onjisaponin B groups. Cell viability was assessed by acridine orange/ethidium bromide bilabel assay. The intracellular Ca2+ level was measured with Fluo-3-AM as the fluorescence indicator. Western blot analysis and quantitative polymerase chain reaction were used to detect the expressions of Cx43 and CaMKII in the samples. The glutamate level of the extracellular fluid was determined using high-performance liquid chromatography-mass spectrometry. Results: The intracellular Ca2+ levels and the mRNA and protein expression of CaMKII and Cx43 in the onjisaponin B group were significantly lower than that of the model group. The glutamate level in the extracellular fluid was significantly reduced by onjisaponin B in comparison to that in the model group. Moreover, onjisaponin B attenuated the inhibitory effect of OGD on the cell viability of astrocytes. Conclusion: The results of this study suggest that onjisaponin B reduced the release of glutamate via inhibition of the Ca2+/CaMKII and Cx43 pathways in the rat astrocytes, thus inhibiting the downstream glutamic pathway and reinforcing the cell viability of astrocyte.

Cryo-EM structure of an open conformation of a gap junction hemichannel in lipid bilayer nanodiscs

To mediate cell-to-cell communication via gap junction channels (GJCs), connexins (Cx) traffic as hexameric hemichannels to the plasma membrane, which dock end-to-end between adjacent cell membranes, thereby forming a dodecameric intercellular conduit. Hemichannels also function independently to mediate the passage of contents between the cytoplasm and extracellular space. To generate hemichannels, the mutation N176Y was introduced into the second extracellular loop of Cx26. The electron cryomicroscopy structure of the hexameric hemichannel in lipid bilayer nanodiscs displays an open pore and a 4-helix bundle transmembrane design that is nearly identical to dodecameric GJCs. In contrast to the high resolution of the transmembrane α-helices, the extracellular loops are less well resolved. The conformational flexibility of the extracellular loops may be essential to facilitate surveillance of hemichannels in apposed cells to identify compatible Cx isoforms that enable intercellular docking. Our results also provide a structural foundation for previous electrophysiologic and permeation studies of Cx hemichannels.

Cx43 hemichannel microdomain signaling at the intercalated disc enhances cardiac excitability.

Cx43, a major cardiac connexin, forms precursor hemichannels that accrue at the intercalated disc to assemble as gap junctions. While gap junctions are crucial for electrical conduction in the heart, little is known about the potential roles of hemichannels. Recent evidence suggests that inhibiting Cx43 hemichannel opening with Gap19 has antiarrhythmic effects. Here, we used multiple electrophysiology, imaging, and super-resolution techniques to understand and define the conditions underlying Cx43 hemichannel activation in ventricular cardiomyocytes, their contribution to diastolic Ca2+ release from the sarcoplasmic reticulum, and their impact on electrical stability. We showed that Cx43 hemichannels were activated during diastolic Ca2+ release in single ventricular cardiomyocytes and cardiomyocyte cell pairs from mice and pigs. This activation involved Cx43 hemichannel Ca2+ entry and coupling to Ca2+ release microdomains at the intercalated disc, resulting in enhanced Ca2+ dynamics. Hemichannel opening furthermore contributed to delayed afterdepolarizations and triggered action potentials. In single cardiomyocytes, cardiomyocyte cell pairs, and arterially perfused tissue wedges from failing human hearts, increased hemichannel activity contributed to electrical instability compared with nonfailing rejected donor hearts. We conclude that microdomain coupling between Cx43 hemichannels and Ca2+ release is a potentially novel, targetable mechanism of cardiac arrhythmogenesis in heart failure.

Basic science: Beta cells, islets and complications

Introduction: Inflammation has been linked to hemichannel-mediated release of adenosine triphosphate (ATP) and downstream fibrosis in various tissue type-specific complications of diabetes. In the current study we examined how inflammatory cytokines alter markers of renal tubular injury ahead of evaluating the efficacy of the connexin-43 (Cx43) hemichannel blocker Tonabersat in negating these effects. Methods: Human Kidney (HK-2) proximal tubule epithelial cells were cultured in low (5mM) or high (25mM) glucose ± interleukin (IL)-1β (10ng) and tumour necrosis factor alpha (TNFα; 10ng) for 48 hours. Immunoblotting determined expression of key proteins, whilst carboxyfluorescein (200µM) dye uptake examined hemichannel activity in treated cells +/- Tonabersat (10µM). Results: Under high glucose conditions, IL-1β and TNFα significantly increased hemichannel activity to 161±16%, (P<0.05, n=5) an effect which was negated when co-incubated with hemichannel blocker Tonabersat (123±8.5%, n=5). Moreover, stimulation with IL-1β and TNFα increased expression of the inflammatory protein IL-6 (206±42%, P<0.05, n=5) and the extracellular matrix proteins, fibronectin (365±81%, P<0.05, n=4), collagen-I (516±36%, P<0.05, n=4) and collagen-IV (830±217%, P<0.01, n=5) as compared to low glucose control. The effect was augmented at high (25mM) glucose for IL-6 (491±164%, P<0.05, n=5), fibronectin (455±112%, n=6), collagen-I (725±85%, P<0.05, n=4) and colIagen-IV (868±77%, P<0.001, n=6). Conclusion: We demonstrate that IL1β and TNFα increase expression of inflammatory and profibrotic proteins in glucose treated HK-2 cells. Paralleled by a Tonabersat-sensitive increase in Cx43 hemichannel activity, we believe that blocking hemichannels has therapeutic potential and protects against changes associated with tubular injury in the diabetic kidney.

Inhibition of connexin 43 induces hearing loss in postnatal mice.

Connexin 43 (Cx43) is the most ubiquitously expressed member of the family of connexins, constituting gap junctions and mediating cell communication, still its role in hearing loss has been little studied. Immunohistochemistry was used to detect the expression pattern of Cx43. Spiral ganglia neurons (SGNs) and Corti co-culture were utilized to assay the re-innervation of hair cells by newborn SGNs. Gap19 was utilized to inhibit Cx43 hemichannels. Auditory brainstem responses (ABR) and endocochlear potential (E.P.) were measured to confirm the hearing loss. The expression of Cx43 in P14 mice was higher than in P0 and P28 (adult) mice, the earlier time point coinciding with the early inner ear development. Additionally, the growth and synapse generation of fibers were inhibited after Gap 19 treatment of the co-cultures of the Corti and SGNs from newborn mice. Furthermore, the inhibition of Cx43 could increase the ABR threshold and decrease E.P. level in postnatal mice, whereas such an effect was not observed in adult mice. The function of Cx43 is critical during the early development of mouse cochlea but is dispensable in adult mice.

Inhibition of astrocyte hemichannel improves recovery from spinal cord injury.

Spinal cord injury (SCI) causes severe disability, and the current inability to restore function to the damaged spinal cord leads to lasting detrimental consequences to patients. One strategy to reduce SCI morbidity involves limiting the spread of secondary damage after injury. Previous studies have shown that connexin 43 (Cx43), a gap junction protein richly expressed in spinal cord astrocytes, is a potential mediator of secondary damage. Here, we developed a specific inhibitory antibody, mouse-human chimeric MHC1 antibody (MHC1), that inhibited Cx43 hemichannels, but not gap junctions, and reduced secondary damage in 2 incomplete SCI mouse models. MHC1 inhibited the activation of Cx43 hemichannels in both primary spinal astrocytes and astrocytes in situ. In both SCI mouse models, administration of MHC1 after SCI significantly improved hind limb locomotion function. Remarkably, a single administration of MHC1 30 minutes after injury improved the recovery up to 8 weeks post-SCI. Moreover, MHC1 treatment decreased gliosis and lesion sizes, increased white and gray matter sparing, and improved neuronal survival. Together, these results suggest that inhibition of Cx43 hemichannel function after traumatic SCI reduces secondary damage, limits perilesional gliosis, and improves functional recovery. By targeting hemichannels specifically with an antibody, this study provides a potentially new, innovative therapeutic approach in treating SCI.

Urothelium-Specific Deletion of Connexin43 in the Mouse Urinary Bladder Alters Distension-Induced ATP Release and Voiding Behavior.

Connexin43 (Cx43), the main gap junction and hemichannel forming protein in the urinary bladder, participates in the regulation of bladder motor and sensory functions and has been reported as an important modulator of day–night variations in functional bladder capacity. However, because Cx43 is expressed throughout the bladder, the actual role played by the detrusor and the urothelial Cx43 is still unknown. For this purpose, we generated urothelium-specific Cx43 knockout (uCx43KO) mice using Cre-LoxP system. We evaluated the day–night micturition pattern and the urothelial Cx43 hemichannel function of the uCx43KO mice by measuring luminal ATP release after bladder distention. In wild-type (WT) mice, distention-induced ATP release was elevated, and functional bladder capacity was decreased in the animals’ active phase (nighttime) when Cx43 expression was also high compared to levels measured in the sleep phase (daytime). These day–night differences in urothelial ATP release and functional bladder capacity were attenuated in uCx43KO mice that, in the active phase, displayed lower ATP release and higher functional bladder capacity than WT mice. These findings indicate that urothelial Cx43 mediated ATP signaling and coordination of urothelial activity are essential for proper perception and regulation of responses to bladder distension in the animals’ awake, active phase.

Mechanotransduction via the coordinated actions of integrins, PI3K signaling and Connexin hemichannels

Mechanical loading opens connexin 43 (Cx43) hemichannels (HCs), leading to the release of bone anabolic molecules, such as prostaglandins, from mechanosensitive osteocytes, which is essential for bone formation and remodeling. However, the mechanotransduction mechanism that activates HCs remains elusive. Here, we report a unique pathway by which mechanical signals are effectively transferred between integrin molecules located in different regions of the cell, resulting in HC activation. Both integrin α5 and αV were activated upon mechanical stimulation via either fluid dropping or flow shear stress (FSS). Inhibition of integrin αV activation or ablation of integrin α5 prevented HC opening on the cell body when dendrites were mechanically stimulated, suggesting mechanical transmission from the dendritic integrin αV to α5 in the cell body during HC activation. In addition, HC function was compromised in vivo, as determined by utilizing an antibody blocking αV activation and α5-deficient osteocyte-specific knockout mice. Furthermore, inhibition of integrin αV activation, but not that of α5, attenuated activation of the phosphoinositide 3-kinase (PI3K)-protein kinase B (AKT) signaling pathway upon mechanical loading, and the inhibition of PI3K/AKT activation blocked integrin α5 activation and HC opening. Moreover, HC opening was blocked only by an anti-integrin αV antibody at low but not high FSS levels, suggesting that dendritic αV is a more sensitive mechanosensor than α5 for activating HCs. Together, these results reveal a new molecular mechanism of mechanotransduction involving the coordinated actions of integrins and PI3K/AKT in osteocytic dendritic processes and cell bodies that leads to HC opening and the release of key bone anabolic factors.

Non-Selective Cation Currents Mediated by Cx43 Hemichannel-P2X4 Receptor Signaling Pathway in Rat Atrial Myocytes under Shear Stress

Cardiac myocytes are exposed to shear stress under physiological conditions and hemodynamic disturbances. Shear stress has been thought to trigger two distinct global Ca2+ waves in atrial myocytes through autocrine activation of P2X4 receptor (P2X4R) and P2Y1-receptor (P2Y1R) via connexin 43 (Cx43)-mediated ATP release. In this study, we examined whether such shear stress-triggered Cx43-ATP-P2 receptor signaling generates depolarizing currents in atrial myocytes. Whole-cell patch clamp and dye-flux assays were used with genetic and pharmacological interventions in rat atrial myocytes and HL-1 cells. We found that shear stress activated a non-selective inward cation (Cs+) current (IS,Cs) at resting potential which was eliminated by Cx43 blockade using siRNA, antibodies, carbenoxolone or La3+. IS,Cs was enhanced by removal of external Ca2+, whereas internal Ca2+ buffering suppressed IS,Cs. Hemichannel-mediated dye (calcein red-orange) flux was induced by shear stress. This response was augmented by removing external Ca2+ but was not affected by pannexin blocker (probenecid). The shear-activated current was significantly reduced when Cs+ was replaced with NMDG+, and this was enhanced by quinine and suppressed by La3+. P2X4R antagonists (5-BDBD and PSB-12054), P2XR-blocker (iso-PPADS) and anti-P2X4R antibodies each blocked IS,Cs by approximately 50%. About 25% of IS,Cs was suppressed by P2Y1R inhibitor (MRS2179) and/or transient receptor potential melastatin 4 blocker (9-phenanthrol). P2X4R, but not P2X5R, was co-localized with Cx43 at the cell ends. P2X4R-dependent IS,Cs was augmented in atrial myocytes from hypertrophied rat hearts by short-term aortic constriction. Our data suggest that shear stress triggers Cx43 hemichannels to gate P2X4Rs in their vicinity. This shear stress signaling may play an important role in membrane depolarization at negative potentials and in atrial remodeling under increased afterload.

Osteocytic Connexin43 Channels Regulate Bone-Muscle Crosstalk.

Bone–muscle crosstalk plays an important role in skeletal biomechanical function, the progression of numerous pathological conditions, and the modulation of local and distant cellular environments. Previous work has revealed that the deletion of connexin (Cx) 43 in osteoblasts, and consequently, osteocytes, indirectly compromises skeletal muscle formation and function. However, the respective roles of Cx43-formed gap junction channels (GJs) and hemichannels (HCs) in the bone–muscle crosstalk are poorly understood. To this end, we used two Cx43 osteocyte-specific transgenic mouse models expressing dominant negative mutants, Δ130–136 (GJs and HCs functions are inhibited), and R76W (only GJs function is blocked), to determine the effect of these two types of Cx43 channels on neighboring skeletal muscle. Blockage of osteocyte Cx43 GJs and HCs in Δ130–136 mice decreased fast-twitch muscle mass with reduced muscle protein synthesis and increased muscle protein degradation. Both R76W and Δ130–136 mice exhibited decreased muscle contractile force accompanied by a fast-to-slow fiber transition in typically fast-twitch muscles. In vitro results further showed that myotube formation of C2C12 myoblasts was inhibited after treatment with the primary osteocyte conditioned media (PO CM) from R76W and Δ130–136 mice. Additionally, prostaglandin E2 (PGE2) level was significantly reduced in both the circulation and PO CM of the transgenic mice. Interestingly, the injection of PGE2 to the transgenic mice rescued fast-twitch muscle mass and function; however, this had little effect on protein synthesis and degradation. These findings indicate a channel-specific response: inhibition of osteocytic Cx43 HCs decreases fast-twitch skeletal muscle mass alongside reduced protein synthesis and increased protein degradation. In contrast, blockage of Cx43 GJs results in decreased fast-twitch skeletal muscle contractile force and myogenesis, with PGE2 partially accounting for the measured differences.

Connexin Gap Junctions and Hemichannels Link Oxidative Stress to Skeletal Physiology and Pathology.

The goal of this review is to provide an overview of the impact and underlying mechanism of oxidative stress on connexin channel function, and their roles in skeletal aging, estrogen deficiency, and glucocorticoid excess associated bone loss. Connexin hemichannel opening is increased under oxidative stress conditions, which confers a cell protective role against oxidative stress-induced cell death. Oxidative stress acts as a key contributor to aging, estrogen deficiency, and glucocorticoid excess-induced osteoporosis and impairs osteocytic network and connexin gap junction communication. This paper reviews the current knowledge for the role of oxidative stress and connexin channels in the pathogenesis of osteoporosis and physiological and pathological responses of connexin channels to oxidative stress. Oxidative stress decreases osteocyte viability and impairs the balance of anabolic and catabolic responses. Connexin 43 (Cx43) channels play a critical role in bone remodeling, mechanotransduction, and survival of osteocytes. Under oxidative stress conditions, there is a consistent reduction of Cx43 expression, while the opening of Cx43 hemichannels protects osteocytes against cell injury caused by oxidative stress.

Astrocytic Connexin43 Channels as Candidate Targets in Epilepsy Treatment.

In epilepsy research, emphasis is put on exploring non-neuronal targets such as astrocytic proteins, since many patients remain pharmacoresistant to current treatments, which almost all target neuronal mechanisms. This paper reviews available data on astrocytic connexin43 (Cx43) signaling in seizures and epilepsy. Cx43 is a widely expressed transmembrane protein and the constituent of gap junctions (GJs) and hemichannels (HCs), allowing intercellular and extracellular communication, respectively. A plethora of research papers show altered Cx43 mRNA levels, protein expression, phosphorylation state, distribution and/or functional coupling in human epileptic tissue and experimental models. Human Cx43 mutations are linked to seizures as well, as 30% of patients with oculodentodigital dysplasia (ODDD), a rare genetic condition caused by mutations in the GJA1 gene coding for Cx43 protein, exhibit neurological symptoms including seizures. Cx30/Cx43 double knock-out mice show increased susceptibility to evoked epileptiform events in brain slices due to impaired GJ-mediated redistribution of K+ and glutamate and display a higher frequency of spontaneous generalized chronic seizures in an epilepsy model. Contradictory, Cx30/Cx43 GJs can traffic nutrients to high-energy demanding neurons and initiate astrocytic Ca2+ waves and hyper synchronization, thereby supporting proconvulsant effects. The general connexin channel blocker carbenoxolone and blockers from the fenamate family diminish epileptiform activity in vitro and improve seizure outcome in vivo. In addition, interventions with more selective peptide inhibitors of HCs display anticonvulsant actions. To conclude, further studies aiming to disentangle distinct roles of HCs and GJs are necessary and tools specifically targeting Cx43 HCs may facilitate the search for novel epilepsy treatments.

Contribution of Connexin Hemichannels to the Pathogenesis of Acute Lung Injury.

Connexin (Cx) family members form hemichannels (HCs) and gap junctions (GJs). Biological functions of Cx HCs have not been adequately characterized due to the inability to selectively target HCs or GJs. Recently, we developed a 6-mer peptide mimetic (P5) of the first extracellular loop of Cx43 and showed that it can block the permeability of HCs but not GJs formed by Cx43. In this study, we further characterized the HC blocking property of P5 and investigated the role of Cx HCs in acute lung injury (ALI). We found that P5 administration decreased HC permeability, in pulmonary microvascular endothelial cells, HepG2 cells, and even Cx43-deficient astrocytes, which express different sets of Cxs, suggesting that P5 is a broad spectrum Cx HC blocker. In addition, P5 reduced HC permeability of alveolar cells in vivo. Moreover, P5 decreased endotoxin-induced release, by vascular endothelial cells in vitro, of high mobility group box protein 1 (HMGB1), a critical mediator of acute lung injury (ALI), and reduced HMGB1 accumulation in bronchoalveolar lavage fluid (BALF) of mice subjected to intratracheal endotoxin instillation. Furthermore, P5 administration resulted in a significant decrease in the concentrations of ALT, AST, and LDH in the BALF, the accumulation of leukocytes in alveoli, and the mortality rate of mice subjected to ALI. Wright-Giemsa staining showed that P5 caused similar reductions of both neutrophils and monocytes in BALF of ALI mice. Together, these results suggest that Cx HCs mediate HMGB1 release, augment leukocyte recruitment, and contribute to ALI pathology.

Abstract 13383: The Vascular Endothelial Barrier: A Novel Therapeutic Target for Preventing Atrial Fibrillation

Atrial fibrillation (AF), the most common arrhythmia, is associated with inflammation and vascular dysfunction. AF patients have elevated levels of vascular endothelial growth factor (VEGF; 90-580 pg/ml), which promotes vascular leak and edema. We have previously identified edema-induced disruption of sodium channel (Na V 1.5) -rich intercalated disk (ID) nanodomains as a novel arrhythmia mechanism. We hypothesized that (i) elevated VEGF levels promote AF by disrupting ID nanodomains, and slowing atrial conduction, and (ii) protection of the vascular barrier can prevent these arrhythmias. Clinically-relevant VEGF levels (500 pg/ml, 60 minutes) increased FITC-dextran extravasation (99.3% vs. 24.3% in vehicle controls) in WT mouse hearts, consistent with increased vascular leak. Electron microscopy revealed ID nanodomain swelling, near both gap junctions (perinexi; 64±9nm vs 17±1nm) and mechanical junctions (63±4nm vs 27±2nm) in VEGF-treated hearts relative to controls. Super-resolution STORM microscopy revealed Na V 1.5 enrichment at perinexi (9±2 fold) and N-cadherin-rich sites (7±1 fold) relative to non-junctional ID sites in control hearts. VEGF reduced Na V 1.5 enrichment at both sites (6±1 and 4±1 fold, respectively), consistent with Na V 1.5 translocation from ID nanodomains. Atrial conduction, assessed by optical mapping, was slowed by VEGF (10±0.4 cm/s vs 21.3±1.3 cm/s at baseline). VEGF increased atrial arrhythmia burden both ex vivo (80% vs 0% in vehicle controls) and in vivo (70% vs 20% in vehicle controls). Next, we tested two strategies shown to prevent vascular barrier breakdown. Blocking connexin43 hemichannels (αCT11 peptide) decreased both incidence (40%) and duration (1.45±3.42s) of VEGF-induced arrhythmias. Likewise, blocking pannexin1 channels (Panx1-IL2 peptide) shortened VEGF-induced arrhythmias (2.48±0.83s). Mefloquine and spironolactone, which are small molecules that respectively inhibit Cx43 hemichannels and pannexin channels, were also found to effectively prevent VEGF-induced atrial arrhythmias. These results highlight VEGF-induced vascular leak as a novel mechanism for AF, and suggest vascular barrier protection as an anti-arrhythmic strategy.

Mechanosensitive collaboration between integrins and connexins allows nutrient and antioxidant transport into the lens.

The delivery of glucose and antioxidants is vital to maintain homeostasis and lens transparency. Here, we report a new mechanism whereby mechanically activated connexin (Cx) hemichannels serve as a transport portal for delivering glucose and glutathione (GSH). Integrin α6β1 in outer cortical lens fiber activated by fluid flow shear stress (FFSS) induced opening of hemichannels. Inhibition of α6 activation prevented hemichannel opening as well as glucose and GSH uptake. The activation of integrin β1, a heterodimeric partner of α6 in the absence of FFSS, increased Cx50 hemichannel opening. Hemichannel activation by FFSS depended on the interaction of integrin α6 and Cx50 C-terminal domain. Moreover, hemichannels in nuclear fiber were unresponsive owing to Cx50 truncation. Taken together, these results show that mechanically activated α6β1 integrin in outer cortical lens fibers leads to opening of hemichannels, which transport glucose and GSH into cortical lens fibers. This study unveils a new transport mechanism that maintains metabolic and antioxidative function of the lens.

Mitochondrial Fate of Regenerative Hematopoietic Stem Cells Is Sequentially Controlled By Two Specific Conformations of Connexin-43

Restricted mitochondrial metabolism with low mitochondrial reactive oxygen species (ROS) and membrane potential are essential properties of repopulating hematopoietic stem cells (HSC). Upon regenerative stress, as found after chemotherapy and/or radiotherapy, HSC exit quiescence, proliferate and differentiate into mature blood cells. Understanding the mechanism controlling hematopoiesis regeneration upon replicative stress is expected to provide molecular targets for amelioration of chemotherapy induced toxicity on HSC. Recent evidence demonstrates that the coordinated regulation of mitochondrial dynamics and the clearance of damaged mitochondria are the critical determinants of HSC fate decisions. Upon myeloablative stress, hematopoietic connexin 43 (H-Cx43), a major component of the gap junctions (GJ) present in the cell, lysosome and mitochondrial membranes, preserves the survival and efficient blood formation of regenerating HSC and progenitors (HSPC) by the transfer of damaging excess ROS, preventing HSPC apoptosis and lethal hematology failure. The protective role of H-Cx43 depends on the regulation of cell-contact dependent mitochondrial transfer to BM mesenchymal stromal cells. Mitochondrial homeostasis is maintained by coordinated regulation of mitochondrial fission, fusion and lysosome dependent mitophagy. We hypothesized that hematopoietic Cx43 may exert a mitochondrial autonomous activity affecting the ability of HSC to regenerate. We created HSC mitochondrial reporter mice with hematopoietic deficiency of Cx43(H-Cx43D/D) and analyzed mitochondrial dynamics and fate in quiescent and dividing HSC. While quiescent Cx43D/D HSC function normally, Cx43 deficiency results in increased mitochondrial ROS and membrane depolarization in cycling HSC. Time lapsed imaging of photo-converted mitochondria indicate that mitochondria of Cx43D/D cycling HSC split into highly-motile, smaller fragments. Interestingly, the activating phosphorylation (Ser616) of the mitochondrial fission protein, Drp1 and its accumulation within mitochondria is higher in Cx43D/D dividing HSC. The recruitment of Drp1 to mitochondria is regulated by mitochondrial membrane adaptors Mff and Fis1. Expression of Fis1, but not Mff, is significantly increased in Cx43D/D cycling HSC. In contrast, the components of mitochondrial fusion machinery Mfn2 and active Drp1 (phospho-Drp1-Ser637) are significantly attenuated in dividing Cx43D/D HSC, suggesting that HSC Cx43 promotes mitochondrial fusion and stability, and inhibits mitochondrial fragmentation. Increased mitochondrial fission in dividing Cx43D/D HSC facilitates mitophagy as indicated by increased co-localization of mitochondria with the ubiquitin kinase Pink1 which simultaneously recruits the E3 ubiquitin ligase Parkin, autophagosome p62 and Lc3, and the lysosomal membrane protein Lamp2 on the surface of dysfunctional mitochondria. Additionally, increased phosphorylation of Ampk (Tyr172) and Ulk1 (Ser555) in mitochondria of cycling Cx43D/D HSC demonstrate that H-Cx43 is a negative regulator of Ampk dependent mitophagy in diving HSC. Inhibition of the Drp1 GTPase activity by expression of the dominant negative Drp1-K38A mutant prevents mitochondrial fragmentation, motility and mitophagy of dividing Cx43D/D HSC, confirming that the inhibitory effect on mitophagy of Cx43 depends on its role on mitochondrial fission. Expression of Cx43 structure-function mutants (cys-less mutant with impaired head-to-head hemichannel docking, but not hemichannel function; and C-terminus truncated D257 mutant with impaired signaling and intramolecular interactions needed for channel gating) in H-Cx43D/D HSC demonstrated that the negative regulatory role of Cx43 on mitochondrial fission requires functional Cx43 hemichannels while the constitutive inhibitory effect of H-Cx43 on mitophagy depends on the formation of complete functional GJ channels. Our results identify for first time the sequential role of two distinct conformations of mitochondrial H-Cx43 dependent channels on the control of mitochondrial fate: fission and mitophagy, in cycling HSC. This data provides novel targets for ex-vivo intervention to preserve HSC activity by transfer of genetically manipulated mitochondria. Figure Disclosures Cancelas: TerumoBCT: Consultancy, Research Funding; Cerus Corp: Research Funding; Hemanext Inc.: Consultancy, Research Funding; Velico LLC: Consultancy, Research Funding; Cytosorbents: Research Funding; Westat Inc: Consultancy, Research Funding; US DoD: Research Funding; NIH: Consultancy, Research Funding.

Regulation of Connexin Gap Junctions and Hemichannels by Calcium and Calcium Binding Protein Calmodulin.

Connexins are the structural components of gap junctions and hemichannels that mediate the communication and exchange of small molecules between cells, and between the intracellular and extracellular environment, respectively. Connexin (Cx) 46 is predominately expressed in lens fiber cells, where they function in maintaining the homeostasis and transparency of the lens. Cx46 mutations are associated with impairment of channel function, which results in the development of congenital cataracts. Cx46 gap junctions and hemichannels are closely regulated by multiple mechanisms. Key regulators of Cx46 channel function include Ca2+ and calmodulin (CaM). Ca2+ plays an essential role in lens homeostasis, and its dysregulation causes cataracts. Ca2+ associated CaM is a well-established inhibitor of gap junction coupling. Recent studies suggest that elevated intracellular Ca2+ activates Cx hemichannels in lens fiber cells and Cx46 directly interacts with CaM. A Cx46 site mutation (Cx46-G143R), which is associated with congenital Coppock cataracts, shows an increased Cx46-CaM interaction and this interaction is insensitive to Ca2+, given that depletion of Ca2+ reduces the interaction between CaM and wild-type Cx46. Moreover, inhibition of CaM function greatly reduces the hemichannel activity in the Cx46 G143R mutant. These research findings suggest a new regulatory mechanism by which enhanced association of Cx46 with CaM leads to the increase in hemichannel activity and dysregulation may lead to cataract development. In this review, we will first discuss the involvement of Ca2+/CaM in lens homeostasis and pathology, and follow by providing a general overview of Ca2+/CaM in the regulation of Cx46 gap junctions. We discuss the most recent studies concerning the molecular mechanism of Ca2+/CaM in regulating Cx46 hemichannels. Finally, we will offer perspectives of the impacts of Ca2+/CaM and dysregulation on Cx46 channels and vice versa.