Cell. Mol. Life Sci. (2015) 72:2929–2935 DOI 10.1007/s00018-015-1968-1 Cellular and Molecular Life Sciences

MULTI-AUTHOR REVIEW

Regulation of and channels and their functional role in skeletal muscles

1,2 1,2 1,2 3,4 Juan C. Sa´ez • Bruno A. Cisterna • Anibal Vargas • Christopher P. Cardozo

Received: 4 June 2015 / Accepted: 11 June 2015 / Published online: 18 June 2015 Ó Springer Basel 2015

Abstract Myogenic precursor cells express HCs in the sarcolemma after denervation or spinal cord (Cx) and (Panx), proteins that form different injury, plus other non-selective membrane channels, membrane channels involved in cell-cell communication. including purinergic P2X7 receptors and transient receptor Cx channels connect either the cytoplasm of adjacent cells, potential type V2 channels. The absence of Cx43 and Cx45 called channels (GJC), or link the cytoplasm is sufficient to drastically reduce denervation atrophy. with the extracellular space, termed hemichannels (HC), Moreover, inflammatory cytokines also induce the while Panx channels only support the latter. In myoblasts, expression of Cxs in myofibers, suggesting the expression Panx1 HCs play a critical role in myogenic differentiation, of these Cxs as a common factor for myofiber degeneration and Cx GJCs and possibly Cx HCs coordinate metabolic under diverse pathological conditions. Inhibitors of skeletal responses during later steps of myogenesis. After inner- muscle Cx HCs could be promising tools to prevent muscle vation, myofibers do not express Cxs, but still express wasting induced by conditions associated with synaptic Panx1. In myotubes and innervated myofibers, Panx1 HCs dysfunction and inflammation. allow release of adenosine triphosphate and thus they might be involved in skeletal muscle plasticity. In addition, Keywords Pannexons Á Connexons Á Myoblasts Á Panx1 HCs present in adult myofibers mediate adenosine Myotubes Á Striate muscles Á Myogenesis Á Denervation triphosphate release and glucose uptake required for potentiation of muscle contraction. Under pathological Abbreviations conditions, such as upon denervation and spinal cord ADP Adenosine diphosphate injury, levels of Panx1 are upregulated. However, ATP Adenosine triphosphate Panx1-/- mice show similar degree of atrophy as dener- Cx Connexin vated wild-type muscles. Skeletal muscles also express Cx GJC Gap junction channel HC Hemichannel P2R Purinergic P2 receptor & Juan C. Sa´ez Panx Pannexin [email protected]

1 Departamento de Fisiologı´a, Pontificia Universidad Cato´lica Introduction de Chile, Santiago, Chile 2 Instituto Milenio, Centro Interdisciplinario de Neurociencias Satellite cells or their precursors, pluripotent mesenchymal de Valparaı´so, Universidad de Valparaı´so, Valparaı´so, Chile stem cells, become committed to myogenic differentiation 3 Center of Excellence for the Medical Consequences of Spinal and fuse to form skeletal muscle fibers after muscle injury Cord Injury, James J. Peters Veterans Affairs Medical Center, Bronx, NY 10468, USA during either regeneration after muscle injury or in devel- opment of skeletal muscles. This process requires the 4 Departments of Medicine, Rehabilitation Medicine and Pharmacology and Systems Therapeutics, Icahn School of expression of myogenic transcriptional factors, including Medicine at Mount Sinai, New York, NY 10039, USA myogenic differentiation 1 protein, myogenic factor 5 and 123 2930 J. C. Sa´ez et al. myogenin, which transform these cells into proliferative YO-PRO-1, in different cell types, including myoblasts [3, myoblasts and, subsequently, drive their continued differ- 10]. In macrophages, the increased permeability of the cell entiation [1]. A critical step that triggers the expression of membrane to dyes induced by P2X7 receptor activation is these transcription factors is the increase in intracellular abrogated by knockdown of Panx1 expression [11], indi- free calcium concentration that promotes activation of a cating that Panx1 HCs mediate the dye uptake induced by great variety of protein kinases and phosphatases, including P2X7 receptor activation. Nevertheless, recent arguments the calcium-dependent protein phosphatase calcineurin, have been presented that inhibition of dye uptake by pro- which promotes the expression of myogenic factor 5 [2]. benecid is independent of Panx HCs, suggesting that dye Therefore, identification of the mechanisms that lead to uptake induced by extracellular ATP acting on P2X7 increases in intracellular free calcium concentration is a receptor is independent of Panx1 HCs [12]. Thus, further relevant issue to be solved and will be discussed in the studies are required to clarify this apparent controversy. Of present review. In this respect, the discovery of pannexin1 interest, activation of Panx1 HCs has also been associated hemichannels (Panx1 HCs) in myotubes and adult myofi- with activation of P2YRs by ATP [13]. bers opens new avenues to further understand several Panxs are expressed by cells and are ortho- physiologic responses. In addition, new reports have logues of innexins found in [14]. Panxs are demonstrated that denervated fast skeletal muscles express membrane glycoproteins that form nonselective membrane several nonselective membrane channels that might explain HCs [15] and are encoded by three gene members, namely, a crucial step between the lack of nerve continuity and Panx1, Panx2 and Panx3. Panx1 is the most ubiquitously muscle atrophy. These two findings are also described and expressed member of this family. Current available infor- discussed in this review. There are several other review mation indicates that Panx2 expression is rather restricted papers describing the roles of Panx1 and connexin (Cx) to few cell types and tissues, including brain [16] and proteins in skeletal muscle biology and pathology [3–6]. T-cells [17]. Panx3 is preferentially expressed in bone and Here, we focus on the role of these channels in the com- skin [16]. Several investigators have proposed that Panx1 mitment of myogenic precursors to the myogenic lineage, does not form gap junction channels (GJC) because the to their differentiation, fusion into myotubes and ultimate glycosylation moiety would hinder stabilization of GJCs formation of mature skeletal muscle fibers. The function of [18]. A few reports have demonstrated the formation of these channels in muscle physiology and pathology, Panx1 GJCs in exogenous expression systems [19, 20]. including recent advances linking such channels to calcium Further studies are required to determine whether such release through dihydropyridine and ryanodine receptors, is GJCs participate in physiological cell-cell communication. also discussed. Finally, advances regarding the participa- Here, Panx1 channels will be referred to as Panx1 HCs. tion of these channels in the rapid atrophy that follows HCs connect the intracellular and extracellular com- paralysis by nerve transection will be reviewed. partments, allowing the diffusional passage of ions and small molecules, including ATP and glucose [21, 22]. Although numerous studies indicate that Panx1 HCs are Pannexin hemichannels in striate muscle ontogeny permeable to ATP, these findings have been challenged by Ramanov and group, who described that Panx1 transfected The increase in intracellular free calcium concentration in three different cell lines yields outward rectifying anion required for initiating the expression of transcription fac- channels with negligible permeability to anions exceeding tors involved in muscle differentiation could result from 250 Da, thus excluding ATP. Again, this apparent contro- calcium diffusion from the extracellular milieu as well as versy could be clarified with more studies on permeability from calcium release from intracellular stores. The former properties of Panx1 HCs [23]. could be triggered by activation of store operated calcium Several lines of evidence indicate that Panx1 HCs entry channels, including calcium sensor stromal interac- function to switch myogenic progenitors from proliferation tion molecule 1 and 2 channels and calcium release- to myogenic differentiation as well as in subsequent fusion activated protein 1 channels [7, 8], and/or of such progenitors to form myotubes and, eventually, activation of purinergic P2 receptors (P2R) by adenosine muscle fibers. Early studies in L6 cells, a cell line derived diphosphate (ADP) and adenosine triphosphate (ATP) from rat myoblasts, treated with b-glycyrrhetinic acid, a (Fig. 1). Calcium release from intracellular stores is mainly blocker of Cx HCs, GJCs and Panx1 HCs [24], showed induced by inositol triphosphate, since ryanodine receptors inhibition of myogenin and myogenic regulatory factor 4 are either inactive or absent [9]. expression as well as inhibition of the cell fusion process Activation of P2XR2, P2XR4 and P2XR7 has also been that leads to myotube formation [25]. In contrast, treatment shown to increase cellular membrane permeability to small with octanol, a blocker of Cx HCs, GJCs, but not of Panx1 molecules, such as Lucifer yellow, ethidium bromide and HCs [11, 19], did not alter myogenesis [25]. These findings 123 Regulation of pannexin and connexin channels and their functional role in skeletal muscles 2931

Fig. 1 Scheme of early myogenic event where Panx-based and Cx- which induce calcium from intracellular stores located in the based channels play a relevant role. In cells without acquisition of endoplasmic reticulum (ER). Then, GJCs and Cx HCs located at cell muscle commitment, Panx1 HCs and ionotropic and metabotropic interfaces and cells surface, respectively, participate in coordinating purinergic P2X and P2Y receptors play a critical role controlling the different metabolic responses that trigger differentiation. Since GJCs increase in intracellular free calcium levels required for activation of are permeable to IP3, they coordinate diverse metabolic responses intracellular signaling pathways that regulate the expression of among coupled cells. P2X7 receptor and Cx HCs are permeable to transcription factors of the muscle differentiation process, such as calcium and when activated allow calcium influx. The permeability to myogenic differentiation 1 protein (MyoD). Activation of P2YRs by calcium of Cx39-based HCs and Cx45-based HCs as well as Panx1 extracellular ATP released via Panx1 HCs leads to activation of HCs remains unknown phospholipase C d (PLC d) that generate inositol triphosphate (IP3), strongly suggest that Cx HCs and GJCs do not participate critical role in differentiation of striated muscles, undif- in the process of myoblast commitment and fusion during ferentiated C2C12 cells that are not yet committed to the myogenesis. In line with this conclusion, Panx1 HCs myogenic lineage and that are negative for myogenic dif- activated by extracellular ATP were detected in C2C12 ferentiation 1 protein, called reserve cells, were treated cells before commitment of acquisition to myogenesis [26]. with Panx1 HC blockers or transfected with small inter- Moreover, Panx1 HCs play a critical role in skeletal fering RNA directed against Panx1 mRNA, which muscles progenitor differentiation (Fig. 1), since inhibition completely prevented subsequent differentiation of these or knockdown of Panx1HCs drastically reduces the dif- cells [26]. Results of these 2 experimental approaches and ferentiation process, whereas overexpression of Panx1 the fact that reserve cells do not express activatable Cx enhances myogenic differentiation and inhibits prolifera- HCs when exposed to divalent cation-free solution, known tion [26, 27]. However, it remains puzzling that embryonic to increase the open probability of Cx HCs [26], support stem cell deletions of Panx1 exhibit no defects in myoge- the interpretation that functional Panx1 HCs and not Cx nesis. Possibly, they express other channels that take over HCs are required for commitment of early myogenic pre- the role of Panx1 HC in the early steps of muscle cursor cells to the myogenic fate. differentiation. At late stages of myogenesis, myotubes also express Myogenic differentiation is a multistep process that Panx1 HCs that allow ATP release induced by repetitive starts with commitment of cells of the myogenic lineage to electrical stimulation [28]. In these cells, Panx1 seems to myogenic fate characterized by expression of myogenin be closely associated to dihidropyridine and P2Y2 recep- and myogenic differentiation 1 protein, and ends with cell tors, since they coprecipitate and this molecular triad seems fusion and innervation-induced changes. In order to to play an important role in modulating the intracellular specifically dissect the step in which Panx1 HCs play a calcium homeostasis [28].

123 2932 J. C. Sa´ez et al.

Connexin-based channels in myogenesis whether both Cx-based channels or just one of them par- ticipate in myogenesis. Relevant to this issue, Cx39 does In addition to Panxs, myoblast express Cxs, which, at least not form GJCs in exogenous expression systems [39], after commitment to myogenic differentiation, have been suggesting that for this channel, it is less likely that GJCs shown to form GJCs that allow communication between participate in the influence of Cx39 on myogenesis. It still myoblasts during differentiation [3]. Myoblasts also appear remains to be determined if Cx39 forms functional HCs. In to express Cx HCs as has been found in most vertebrate addition, the role of each protein might be independent of cells studied to date [29]. The importance of Cx HCs in their capacity to form membrane channels, as has been myogenesis has not been dissected yet. However, it is shown in other systems [42]. likely that GJCs are not required for acquisition of myo- genic commitment, since it can occur in pluripotent cells without physical contact between them [26] (Fig. 1). Pannexin-based and connexin-based channels Over the past four decades, the possible role of GJCs in in adult skeletal muscles myogenesis has been studied. Ultrastructural analyses revealed the presence of GJCs between myoblasts and Cx expression and GJC formation is progressively lost between myoblasts and myotubes [30–32]. In addition, during the late steps of myogenesis. After innervation, functional gap junctions have been found in developing myofibers of most skeletal muscles do not contain GJCs myoblasts [31, 33]. More recently, Cx43 mRNA, but not [5]. Similarly, Cx HCs are also absent in adult innervated Cx32 mRNA, was detected at high levels in skeletal muscles of rodents [43]. However, the demon- cycling L6 myoblasts and Cx43 expression was shown to stration of Cx39.9 expression in slow twitch muscles of drastically decrease in these cells following fusion [25, 34]. teleosts [44] suggests that lack of skeletal muscle Cx Moreover, Cx43 has also been detected in prefusional expression is not a general feature of all . C2C12 myoblasts [35] and transient expression of Cx40 Normal adult muscles express Panx1, but not Panx2 or was found in axial skeletal muscles of mouse embryos Panx3 [5, 22, 45]. Panx1 has been localized in transverse during myoblast fusion [36]. In addition, the application of T-tubules, where it resides next to dihidropyridine recep- 18b-glycyrrhetinic acid or octanol to L6 cells myoblasts tors [5] (Fig. 2). Repetitive electrical stimulation induces undergoing differentiation blocked the expression of Panx1 HC opening and subsequent ATP release. ATP myogenin and myotube formation [25]. However, these released in this manner is vital to potentiation of the compounds are not selective inhibitors of GJCs or HCs and skeletal muscle contraction response [22]. It has been 18b-glycyrrhetinic acid blocks Cx and Panx1 HCs. Thus, suggested that another function of ATP released via Panx1 these findings should thus be interpreted with caution. HCs could be in skeletal muscle plasticity [45]. In support Several recent studies have demonstrated that during to these proposed roles of Panx1 HC-mediated ATP myogenesis in development or regeneration, myoblasts or release, it has been shown that Panx1-/- muscles or satellite cells express at least 3 other Cx species in addition muscles treated with Panx1 HC blockers do not show to Cx40, including Cx39, Cx43 and Cx45 [36–41]. It has potentiation of the contraction response promoted by been possible to study the consequences of a lack of each repetitive electrical stimulation [22]. In addition, the of these Cxs during myogenesis. The absence of either application of exogenous ATP to Panx1-/- muscles pro- Cx43 or Cx45 was found to delay the differentiation pro- motes potentiation of the contraction response [46]. cess in vitro or in vivo [37, 38]. However, Cx39 is Another important function of Panx1 HCs is that they are expressed in murine myotubes, but not in myoblast cells or permeable to glucose and account for about 50 % of glu- adult myofibers [41], suggesting that this Cx is involved in cose uptake induced by repetitive electrical stimulation, skeletal muscle differentiation, but not in the process of while the other 50 % is taken over by glucose transporter commitment to myogenic differentiation. In addition, the type 4 [22]. lack of Cx39 accelerates skeletal muscle differentiation Opening of Panx1 HCs can be induced by extracellular [40]. Moreover, the reduced expression of myogenin, cre- ATP or repetitive electrical stimulation, but the molecular atine kinase activity and the decrease in cell fusion found mechanisms involved remain to be identified. One inter- in myoblasts after induced deletion of Cx43 suggests a role esting finding is that Panx1 has several putative of Cx43 in myoblast terminal differentiation [37]. Collec- phosphorylation sites for different protein kinases, includ- tively, these studies provide relevant information on the ing protein kinase A, protein kinase C and calmodulin- role of each protein, but it is important to keep in mind that dependent protein kinase type II, in its carboxyl-terminal the absence of a particular Cx protein abrogates the for- tail. Accordingly, Panx1 was found to be a phosphoprotein mation of both GJCs and HCs, leaving open the question of and phosphorylation of serine and threonine residues is

123 Regulation of pannexin and connexin channels and their functional role in skeletal muscles 2933

Fig. 2 Scheme showing the putative location of Panx1 HCs in the transverse T-tubule of adult skeletal muscles. Panx1 HCs are in close association with dihidropyridine receptors (DhpR), which in an alternate way are in close contact with the (RyR). a View in the bilipid membrane and b view in a monolipid membrane. c Perpendicular view of C (90° rotation). The putative localization of Panx1 HCs needs to be confirmed experimentally

enhanced during potentiation of muscle contraction necrosis factor a and interleukin 1b [5], which might be induced by repetitive electrical stimulation [22]. Whether relevant in diverse conditions associated with inflammation the increase in the phosphorylation status is the cause of that lead to muscle atrophy. Moreover, skeletal muscles of activation of Panx1 HCs remains unknown. mice express Panx3 in response to high-fat diet, and the A recent unexpected finding was that denervated rodent palmitate-induced expression and activity of Panx3 HCS in skeletal muscles show de novo expression of several non- myotubes has been proposed to be mediated by Toll-like selective membrane channels, including Cx HCs, P2X7 receptor 4 and nuclear factor kB [47]. Accordingly, Toll- receptors and transient receptor potential type V2 channels, like receptor 4 and nuclear factor kB inhibition or Panx3 and upregulation of Panx1 [43]. Deletion of only Cx43 and knockdown prevents monocyte chemoattraction [47]. Cx45 was sufficient to drastically reduce muscle atrophy and to completely prevent activation of the transcription factor nuclear factor kB and to increase of mRNA levels of Conclusions and perspectives proinflammatory cytokines [43]. In addition, the upregu- lated expression of P2X7 receptor was almost completely The accumulated evidence strongly suggests that Panx1 blocked, suggesting that activation of its expression is HCs are essential for myogenic fate commitment and that downstream from Cx43 and Cx45 expression. Since sev- Cx-based channels play critical roles in the coordination eral of these membrane channels, including Cx43 HCs, and progression of subsequent steps required for myogenic P2X7 receptors and transient receptor potential type V2 differentiation and myogenesis. Additional work is needed channels, are permeable to calcium, it has been proposed to understand the relative importance of Cx GJCs and Cx that they serve as membrane pathways for calcium influx HCs in these processes. Also, further studies are required to that would activate intracellular metabolic pathways pre- identify the phosphorylated amino acid residues of Panx1 viously shown to participate in muscle wasting [43]. In as well as the protein kinases responsible for this post- contrast, denervation-induced muscle atrophy was not translational modification. prevented by the lack of Panx1 expression in Panx1-/- It is clear that either repetitive electrical stimulation or mice [43]. An additional interesting finding is that in spite extracellular ATP leads to rapid opening of Panx HCs in of the Cx expression in denervated muscle, these cells do the sarcolemma. However, several questions remain to be not form functional GJCs [43]. answered, among which (1) is the increase in phosphory- Other factors that promote the expression of Cx HCs in lation state of Panx1 HCs responsible for the enhanced skeletal muscles are the proinflammatory cytokines tumor activity of this channel in the sarcolemma? (2) Is the

123 2934 J. C. Sa´ez et al. increase in Panx1 HC activity seen during potentiation of channels in normal skeletal muscles and their possible role in muscle contraction due to increase in opening probability muscle atrophy. J Membr Biol 245(8):423–436. doi:10.1007/ s00232-012-9485-8 of available channels in the or is due to 6. Cisterna BA, Cardozo C, Saez JC (2014) Neuronal involvement recruitment of more Panx1 HCs to the sarcolemma? (3) in muscular atrophy. Front Cell Neurosci 8:405. doi:10.3389/ What mechanisms restore the activity of Panx1 HCs to the fncel.2014.00405 basal state? Similarly, the atrophy observed in adult den- 7. Darbellay B, Arnaudeau S, Ceroni D, Bader CR, Konig S, Bernheim L (2010) Human muscle economy myoblast differen- ervated or inflamed skeletal muscles can be largely tiation and excitation-contraction coupling use the same prevented in muscles that do not express Cx43 and Cx45. Is molecular partners, STIM1 and STIM2. J Biol Chem this a mechanism that obeys a hierarchical expression of 285(29):22437–22447. doi:10.1074/jbc.M110.118984 calcium-permeable channels or, instead, might all of them 8. Darbellay B, Arnaudeau S, Konig S, Jousset H, Bader C, Demaurex N, Bernheim L (2009) STIM1- and Orai1-dependent be involved such that each contribute to reach a critical store-operated calcium entry regulates human myoblast differ- increase in intracellular free calcium concentration? entiation. J Biol Chem 284(8):5370–5380. doi:10.1074/jbc. In general, the field of Cx-based and Panx-based channel M806726200 research is rather limited, but offers numerous relevant 9. Antigny F, Konig S, Bernheim L, Frieden M (2014) Inositol 1,4,5 trisphosphate receptor 1 is a key player of human myoblast dif- questions to be answered to further increase our knowledge ferentiation. Cell Calcium 56(6):513–521. doi:10.1016/j.ceca. in skeletal muscle physiology and pathophysiology. 2014.10.014 Moreover, Cx HCs might be potential molecular targets to 10. Ryten M, Dunn PM, Neary JT, Burnstock G (2002) ATP regu- reduce muscle atrophy in diverse circumstances. The lates the differentiation of mammalian skeletal muscle by activation of a P2X5 receptor on satellite cells. J Cell Biol mechanisms by which expression of sarolemmal Cx HCs is 158(2):345–355. doi:10.1083/jcb.200202025 repressed in innervated and noninflamed muscles remains 11. Pelegrin P, Surprenant A (2006) Pannexin-1 mediates large pore unknown. In addition, the development of potent and formation and interleukin-1beta release by the ATP-gated P2X7 selective Cx HC blockers might be useful to reduce muscle receptor. EMBO J 25(21):5071–5082. doi:10.1038/sj.emboj. 7601378 degeneration induced by spinal cord injury and many other 12. Bhaskaracharya A, Dao-Ung P, Jalilian I, Spildrejorde M, Skar- pathological conditions in which the integrity of nerve ratt KK, Fuller SJ, Sluyter R, Stokes L (2014) Probenecid blocks muscle contact and functional regulation has been altered. human P2X7 receptor-induced dye uptake via a pannexin-1 independent mechanism. PLoS One 9(3):e93058. doi:10.1371/ Acknowledgments This work was partially funded by FONDECYT journal.pone.0093058 grants 1150291 (to Juan C. Sa´ez), ICM-Economı´a P09-022-F Centro 13. Locovei S, Wang J, Dahl G (2006) Activation of pannexin 1 Interdisciplinario de Neurociencias de Valparaı´so (to Juan C. Sa´ez) channels by ATP through P2Y receptors and by cytoplasmic and the Department of Veterans Affairs Rehabilitation Research and calcium. FEBS Lett 580(1):239–244. doi:10.1016/j.febslet.2005. Development Service (B9212C) and the James J. Peters VA Medical 12.004 Center. Bruno A. Cisterna and Anibal Vargas acknowledge the sup- 14. 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