Pannexin channels mediate the acquisition of myogenic commitment in C2C12 reserve cells promoted by P2 receptor activation

Manuel A Riquelme,Luis A Cea,Mario Subiabre,Anã­Bal A Vargas,Carlos Puebla,Kenji F Shoji,Juan C Sã¡Ez,Josã© L Vega

doi:10.3389/fcell.2015.00025

Abstract

The acquisition of myoblast commitment to the myogenic linage requires rises in intracellular free Ca2+ concentration ([Ca2+]i). Putative cell membrane pathways involved in these [Ca2+]i increments are P2 receptors (P2Rs) as well as connexin (Cx) and/or pannexin (Panx) hemichannels and channels (Cx HChs and Panx Chs), respectively, which are known to permeate Ca2+. Reserve cells (RCs) are uncommitted myoblasts obtained from differentiated C2C12 cell cultures, which acquire commitment upon replating. Regarding these cells, we found that extracellular ATP increases the [Ca2+]i via P2Rs. Moreover, ATP increases the plasma membrane permeability to small molecules and a non-selective membrane current, both of which were inhibited by Cx HCh/Panx1Ch blockers. However, RCs exposed to divalent cation-free saline solution, which is known to activate Cx HChs (but not Panx Chs), did not enhance membrane permeability, thus ruling out the possible involvement of Cx HChs. Moreover, ATP-induced membrane permeability was inhibited with blockers of P2Rs that activate Panx Chs. In addition, exogenous ATP induced the expression of myogenic commitment and increased MyoD levels, which was prevented by the inhibition of P2Rs or knockdown of Panx1 Chs. Similarly, increases in MyoD levels induced by ATP released by RCs were inhibited by Panx Ch/Cx HCh blockers. Myogenic commitment acquisition thus requires a feed-forward mechanism mediated by extracellular ATP, P2Rs, and Panx Chs.

Highlights

During skeletal muscle ontogeny and regeneration, pluripotential mesodermal or satellite cells acquire myogenic commitment, which involves the expression of myogenic determination factors such as MyoD, Myf-5, and myogenin, transforming these cells into proliferative myoblasts (Charge and Rudnicki, 2004).The acquisition of myogenic commitment requires increases in intracellular free Ca2+ concentration ([Ca2+]i), which promote the activation of calcineurin that, in turn, induces the expression of the Myf5 transcriptionPanx1 and P2XRs in myogenesis factor (Friday and Pavlath, 2001)
In cells pretreated with 150 μM oxidized ATP (oATP), another P2XR blocker, the Ca2+ signal elicited by 150 μM ATP was comparable to that of cells pretreated with iso-PPDAS
We observed that extracellular ATP increases the Ca2+ signal via P2 receptors (P2Rs) as well as membrane current and permeability to Etd+ mediated by Panx1 Chs and expression levels of MyoD in C2C12 Reserve cells (RCs)

Summary

Introduction

During skeletal muscle ontogeny and regeneration, pluripotential mesodermal or satellite cells acquire myogenic commitment, which involves the expression of myogenic determination factors such as MyoD, Myf-5, and myogenin, transforming these cells into proliferative myoblasts (Charge and Rudnicki, 2004).The acquisition of myogenic commitment requires increases in intracellular free Ca2+ concentration ([Ca2+]i), which promote the activation of calcineurin (a Ca2+-dependent protein phosphatase) that, in turn, induces the expression of the Myf transcriptionPanx and P2XRs in myogenesis factor (Friday and Pavlath, 2001). The activation of P2XRs 2, 4, and 7 has been shown to increase cell membrane permeability to small molecules, including Lucifer yellow, ethidium (Etd+) and YO-PRO-1 in diverse cell types such as myoblasts and macrophages (North, 2002; Araya et al, 2004; Pelegrin and Surprenant, 2006). Increase of plasma membrane permeability to dyes induced by P2X7R activation is absent in cells lacking pannexin (Panx1) expression (Pelegrin and Surprenant, 2006; Locovei et al, 2007), suggesting that channels composed of Panx mediate dye uptake induced by P2X7R activation. Diverse stimuli can increase the open probability of Cx HChs, including membrane depolarization to positive values, pro-inflammatory conditions, reduced extracellular Ca2+ concentration and rises in intracellular [Ca2+]i, among others (Sáez and Leybaert, 2014)

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