Bruno A. Cisterna

De novo expression of connexin hemichannels in denervated fast skeletal muscles leads to atrophy

Significance In this paper two biological findings are described and explain several muscle changes induced by denervation: (i) the sarcolemma of fast myofibers are permeabilized to small molecules such as Evans blue via connexin (Cx) hemichannels and (ii) the absence of Cx43/Cx45 hemichannels greatly attenuates the inflammasome activation and muscle atrophy. The first finding explains the activation of proteolysis in denervated muscles. The second demonstrates that muscle inflammation can occur without inflammatory cell infiltration, offering an explanation how denervated muscles can alter other tissues. These findings unveil therapeutic targets to reduce atrophy in diverse clinical conditions. Because Cx hemichannels are permeable to Evans blue, the use of this dye as tracer of cell damage should be reevaluated in different systems. Denervation of skeletal muscles induces atrophy, preceded by changes in sarcolemma permeability of causes not yet completely understood. Here, we show that denervation-induced Evans blue dye uptake in vivo of fast, but not slow, myofibers was acutely inhibited by connexin (Cx) hemichannel/pannexin1 (Panx1) channel and purinergic ionotropic P2X7 receptor (P2X7R) blockers. Denervated myofibers showed up-regulation of Panx1 and de novo expression of Cx39, Cx43, and Cx45 hemichannels as well as P2X7Rs and transient receptor potential subfamily V, member 2, channels, all of which are permeable to small molecules. The sarcolemma of freshly isolated WT myofibers from denervated muscles also showed high hemichannel-mediated permeability that was slightly reduced by blockade of Panx1 channels or the lack of Panx1 expression, but was completely inhibited by Cx hemichannel or P2X7R blockers, as well as by degradation of extracellular ATP. However, inhibition of transient receptor potential subfamily V, member 2, channels had no significant effect on membrane permeability. Moreover, activation of the transcription factor NFκB and higher mRNA levels of proinflammatory cytokines (TNF-α and IL-1β) were found in denervated WT but not Cx43/Cx45-deficient muscles. The atrophy observed after 7 d of denervation was drastically reduced in Cx43/Cx45-deficient but not Panx1-deficient muscles. Therefore, expression of Cx hemichannels and P2X7R promotes a feed-forward mechanism activated by extracellular ATP, most likely released through hemichannels, that activates the inflammasome. Consequently, Cx hemichannels are potential targets for new therapeutic agents to prevent or reduce muscle atrophy induced by denervation of diverse etiologies.

Fast skeletal myofibers of mdx mouse, model of Duchenne muscular dystrophy, express connexin hemichannels that lead to apoptosis.

Skeletal muscles of patients with Duchenne muscular dystrophy (DMD) show numerous alterations including inflammation, apoptosis, and necrosis of myofibers. However, the molecular mechanism that explains these changes remains largely unknown. Here, the involvement of hemichannels formed by connexins (Cx HCs) was evaluated in skeletal muscle of mdx mouse model of DMD. Fast myofibers of mdx mice were found to express three connexins (39, 43 and 45) and high sarcolemma permeability, which was absent in myofibers of mdx Cx43fl/flCx45fl/fl:Myo-Cre mice (deficient in skeletal muscle Cx43/Cx45 expression). These myofibers did not show elevated basal intracellular free Ca2+ levels, immunoreactivity to phosphorylated p65 (active NF-κB), eNOS and annexin V/active Caspase 3 (marker of apoptosis) but presented dystrophin immunoreactivity. Moreover, muscles of mdx Cx43fl/flCx45fl/fl:Myo-Cre mice exhibited partial decrease of necrotic features (big cells and high creatine kinase levels). Accordingly, these muscles showed similar macrophage infiltration as control mdx muscles. Nonetheless, the hanging test performance of mdx Cx43fl/flCx45fl/fl:Myo-Cre mice was significantly better than that of control mdx Cx43fl/flCx45fl/fl mice. All three Cxs found in skeletal muscles of mdx mice were also detected in fast myofibers of biopsy specimens from patients with muscular dystrophy. Thus, reduction of Cx expression and/or function of Cx HCs may be potential therapeutic approaches to abrogate myofiber apoptosis in DMD.

Neuronal involvement in muscular atrophy.

The innervation of skeletal myofibers exerts a crucial influence on the maintenance of muscle tone and normal operation. Consequently, denervated myofibers manifest atrophy, which is preceded by an increase in sarcolemma permeability. Recently, de novo expression of hemichannels (HCs) formed by connexins (Cxs) and other none selective channels, including P2X7 receptors (P2X7Rs), and transient receptor potential, sub-family V, member 2 (TRPV2) channels was demonstrated in denervated fast skeletal muscles. The denervation-induced atrophy was drastically reduced in denervated muscles deficient in Cxs 43 and 45. Nonetheless, the transduction mechanism by which the nerve represses the expression of the above mentioned non-selective channels remains unknown. The paracrine action of extracellular signaling molecules including ATP, neurotrophic factors (i.e., brain-derived neurotrophic factor (BDNF)), agrin/LDL receptor-related protein 4 (Lrp4)/muscle-specific receptor kinase (MuSK) and acetylcholine (Ach) are among the possible signals for repression for connexin expression. This review discusses the possible role of relevant factors in maintaining the normal functioning of fast skeletal muscles and suppression of connexin hemichannel expression.

Linoleic acid permeabilizes gastric epithelial cells by increasing connexin 43 levels in the cell membrane via a GPR40- and Akt-dependent mechanism

Linoleic acid (LA) is known to activate G-protein coupled receptors and connexin hemichannels (Cx HCs) but possible interlinks between these two responses remain unexplored. Here, we evaluated the mechanism of action of LA on the membrane permeability mediated by Cx HCs in MKN28 cells. These cells were found to express connexins, GPR40, GPR120, and CD36 receptors. The Cx HC activity of these cells increased after 5 min of treatment with LA or GW9508, an agonist of GPR40/GPR120; or exposure to extracellular divalent cation-free solution (DCFS), known to increase the open probability of Cx HCs, yields an immediate increase in Cx HC activity of similar intensity and additive with LA-induced change. Treatment with a CD36 blocker or transfection with siRNA-GPR120 maintains the LA-induced Cx HC activity. However, cells transfected with siRNA-GPR40 did not show LA-induced Cx HC activity but activity was increased upon exposure to DCFS, confirming the presence of activatable Cx HCs in the cell membrane. Treatment with AKTi (Akt inhibitor) abrogated the LA-induced Cx HC activity. In HeLa cells transfected with Cx43 (HeLa-Cx43), LA induced phosphorylation of surface Cx43 at serine 373 (S373), site for Akt phosphorylation. HeLa-Cx43 but not HeLa-Cx43 cells with a S373A mutation showed a LA-induced Cx HC activity directly related to an increase in cell surface Cx43 levels. Thus, the increase in membrane permeability induced by LA is mediated by an intracellular signaling pathway activated by GPR40 that leads to an increase in membrane levels of Cx43 phosphorylated at serine 373 via Akt.

Dexamethasone-induced muscular atrophy is mediated by functional expression of connexin-based hemichannels.

Long-term treatment with high glucocorticoid doses induces skeletal muscle atrophy. However, the molecular mechanism of such atrophy remains unclear. We evaluated the possible involvement of connexin-based hemichannels (Cx HCs) in muscle atrophy induced by dexamethasone (DEX), a synthetic glucocorticoid, on control (Cx43(fl/fl)Cx45(fl/fl)) and Cx43/Cx45 expression-deficient (Cx43(fl/fl)Cx45(fl/fl):Myo-Cre) skeletal myofibers. Myofibers of Cx43(fl/fl)Cx45(fl/fl) mice treated with DEX (5h) expressed several proteins that form non-selective membrane channels (Cx39, Cx43, Cx45, Panx1, P2X7 receptor and TRPV2). After 5h DEX treatment in vivo, myofibers of Cx43(fl/fl)Cx45(fl/fl) mice showed Evans blue uptake, which was absent in myofibers of Cx43(fl/fl)Cx45(fl/fl):Myo-Cre mice. Similar results were obtained in vitro using ethidium as an HC permeability probe, and DEX-induced dye uptake in control myofibers was blocked by P2X7 receptor inhibitors. DEX also induced a significant increase in basal intracellular Ca(2+) signal and a reduction in resting membrane potential in Cx43(fl/fl)Cx45(fl/fl) myofibers, changes that were not elicited by myofibers deficient in Cx43/Cx45 expression. Moreover, treatment with DEX induced NFκB activation and increased mRNA levels of TNF-α in control but not in Cx43/Cx45 expression-deficient myofibers. Finally, a prolonged DEX treatment (7days) increased atrogin-1 and Murf-1 and reduced the cross sectional area of Cx43(fl/fl)Cx45(fl/fl) myofibers, but these parameters remained unaffected in Cx43(fl/fl)Cx45(fl/fl):Myo-Cre myofibers. Therefore, DEX-induced expression of Cx43 and Cx45 plays a critical role in early sarcolemma changes that lead to atrophy. Consequently, this side effect of chronic glucocorticoid treatment might be avoided by co-administration with a Cx HC blocker.

Regulation of pannexin and connexin channels and their functional role in skeletal muscles

Myogenic precursor cells express connexins (Cx) and pannexins (Panx), proteins that form different membrane channels involved in cell-cell communication. Cx channels connect either the cytoplasm of adjacent cells, called gap junction channels (GJC), or link the cytoplasm with the extracellular space, termed hemichannels (HC), while Panx channels only support the latter. In myoblasts, Panx1 HCs play a critical role in myogenic differentiation, and Cx GJCs and possibly Cx HCs coordinate metabolic responses during later steps of myogenesis. After innervation, myofibers do not express Cxs, but still express Panx1. In myotubes and innervated myofibers, Panx1 HCs allow release of adenosine triphosphate and thus they might be involved in skeletal muscle plasticity. In addition, Panx1 HCs present in adult myofibers mediate adenosine triphosphate release and glucose uptake required for potentiation of muscle contraction. Under pathological conditions, such as upon denervation and spinal cord injury, levels of Panx1 are upregulated. However, Panx1−/− mice show similar degree of atrophy as denervated wild-type muscles. Skeletal muscles also express Cx HCs in the sarcolemma after denervation or spinal cord injury, plus other non-selective membrane channels, including purinergic P2X7 receptors and transient receptor potential type V2 channels. The absence of Cx43 and Cx45 is sufficient to drastically reduce denervation atrophy. Moreover, inflammatory cytokines also induce the expression of Cxs in myofibers, suggesting the expression of these Cxs as a common factor for myofiber degeneration under diverse pathological conditions. Inhibitors of skeletal muscle Cx HCs could be promising tools to prevent muscle wasting induced by conditions associated with synaptic dysfunction and inflammation.

On Biophysical Properties and Sensitivity to Gap Junction Blockers of Connexin 39 Hemichannels Expressed in HeLa Cells

Although connexins (Cxs) are broadly expressed by cells of mammalian organisms, Cx39 has a very restricted pattern of expression and the biophysical properties of Cx39-based channels [hemichannels (HCs) and gap junction channels (GJCs)] remain largely unknown. Here, we used HeLa cells transfected with Cx39 (HeLa-Cx39 cells) in which intercellular electrical coupling was not detected, indicating the absence of GJCs. However, functional HCs were found on the surface of cells exposed to conditions known to increase the open probability of other Cx HCs (e.g., extracellular divalent cationic-free solution (DCFS), extracellular alkaline pH, mechanical stimulus and depolarization to positive membrane potentials). Cx39 HCs were blocked by some traditional Cx HC blockers, but not by others or a pannexin1 channel blocker. HeLa-Cx39 cells showed similar resting membrane potentials (RMPs) to those of parental cells, and exposure to DCFS reduced RMPs in Cx39 transfectants, but not in parental cells. Under these conditions, unitary events of ~75 pS were frequent in HeLa-Cx39 cells and absent in parental cells. Real-time cellular uptake experiments of dyes with different physicochemical features, as well as the application of a machine-learning approach revealed that Cx39 HCs are preferentially permeable to molecules characterized by six categories of descriptors, namely: (1) electronegativity, (2) ionization potential, (3) polarizability, (4) size and geometry, (5) topological flexibility and (6) valence. However, Cx39 HCs opened by mechanical stimulation or alkaline pH were impermeable to Ca2+. Molecular modeling of Cx39-based channels suggest that a constriction present at the intracellular portion of the para helix region co-localizes with an electronegative patch, imposing an energetic and steric barrier, which in the case of GJCs may hinder channel function. Results reported here demonstrate that Cx39 form HCs and add to our understanding of the functional roles of Cx39 HCs under physiological and pathological conditions in cells that express them.

Connexins and Pannexins in Bone and Skeletal Muscle

Purpose of reviewTo discuss current knowledge on the role of connexins and pannexins in the musculoskeletal system.Recent findingsConnexins and pannexins are crucial for the development and maintenance of both bone and skeletal muscle. In bone, the presence of connexin and more recently of pannexin channels in osteoblasts, osteoclasts, and osteocytes has been described and shown to be essential for normal skeletal development and bone adaptation. In skeletal muscles, connexins and pannexins play important roles during development and regeneration through coordinated regulation of metabolic functions via cell-to-cell communication. Further, under pathological conditions, altered expression of these proteins can promote muscle atrophy and degeneration by stimulating inflammasome activity.SummaryIn this review, we highlight the important roles of connexins and pannexins in the development, maintenance, and regeneration of musculoskeletal tissues, with emphasis on the mechanisms by which these molecules mediate chemical (e.g., ATP and prostaglandin E2) and physical (e.g., mechanical stimulation) stimuli that target the musculoskeletal system and their involvement in the pathophysiological changes in both genetic and acquired diseases.

Connexin hemichannels explain the ionic imbalance and lead to atrophy in denervated skeletal muscles.

Denervated fast skeletal muscles undergo atrophy, which is associated with an increase in sarcolemma permeability and protein imbalance. However, the mechanisms responsible for these alterations remain largely unknown. Recently, a close association between de novo expression of hemichannels formed by connexins 43 and 45 and increase in sarcolemma permeability of denervated fast skeletal myofibers was demonstrated. However, it remains unknown whether these connexins cause the ionic imbalance of denervates fast myofibers. To elucidate the latter and the role of hemichannels formed by connexins (Cx HCs) in denervation-induced atrophy, skeletal myofibers deficient in Cx43 and Cx45 expression (Cx43fl/flCx45fl/fl:Myo-Cre mice) and control (Cx43fl/flCx45fl/fl mice) were denervated and several muscle features were systematically analyzed at different post-denervation (PD) times (1, 3, 5, 7 and 14days). The following sequence of events was found in denervated myofibers of Cx43fl/flCx45fl/fl mice: 1) from day 3 PD, increase in sarcolemmal permeability, 2) from day 5 PD, increases of intracellular Ca2+ and Na+ signals as well as a significant increase in protein synthesis and degradation, yielding a negative protein balance and 3) from day 7 PD, a fall in myofibers cross-section area. All the above alterations were either absent or drastically reduced in denervated myofibers of Cx43fl/flCx45fl/fl:Myo-Cre mice. Thus, the denervation-induced Cx HCs expression is an early event that precedes the electrochemical gradient dysregulation across the sarcolemma and critically contributes to the progression of skeletal muscle atrophy. Consequently, Cx HCs could be a therapeutic target to drastically prevent the denervation-induced atrophy of fast skeletal muscles.