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First published online 10 July 2006
doi: 10.1242/dev.02479

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The calcineurin pathway links hyperpolarization (Kir2.1)-induced Ca²⁺ signals to human myoblast differentiation and fusion

¹ Department of Clinical Neurosciences, University Hospital, Geneva, Switzerland.
² Department of Basic Neurosciences, University Medical Center, Rue Michel Servet 1, 1211 Geneva 4, Switzerland.

View larger version (44K): [in a new window]   Fig. 1. Calcineurin, CaMK, p38-MAPK and PI3K regulatory pathways are involved in human myoblast differentiation. (A) Induction of myogenic differentiation requires a membrane hyperpolarization. Neither myogenin nor MEF2 were detected in myoblast maintained in growing medium (GM). Myoblasts were induced to differentiate for 3 days either in differentiation medium (DM) or in DM in which 116 mM Na+ was replaced by equivalent concentration of K+ to prevent the hyperpolarization (KCl). After 3 days in high K+ differentiation medium, myoblasts were replaced for 3 more days in control differentiation medium (wash KCl) in which they differentiated nicely. Myogenin was revealed with Alexa488, MEF2 with Alexa546, and nuclei with DAPI. Scale bar: 20 µm. (B) Differentiation is expressed as a percentage of myogenin- and MEF2-positive nuclei. Myoblast were induced to differentiate for 3-4 days in differentiation medium (DM). Myoblast differentiation is partially inhibited by 10 mM Cs+ (Cs) to block the hyperpolarization, by 10 µM SB202190 (SB) to block p38-MAPK, by 30 µM KN93 (KN) to block CaMK, by 50 µM LY294002 (LY) to block PI3K, and by a combination of 7 µM cyclosporin A (CsA) and 5 µM FK506 (FK) to block calcineurin. Results are expressed as mean±s.e.m.

View larger version (22K): [in a new window]   Fig. 2. Hyperpolarization controls the early activation of calcineurin pathway during human myoblast differentiation. Human primary myoblasts were transiently transfected with either 9NFAT-luc or 4RE-luc or 3MEF2-luc plasmids. Luciferase activity is in arbitrary units. Increased level of NFAT transcription factor activity reflects calcineurin activity. (A) Kinetics of activation of NFAT, myogenic bHLH and MEF2 transcription factors. Luciferase extracts were prepared from proliferating myoblasts (GM) and from myoblasts maintained for 1 to 4 days in differentiation medium (DM). (B) Depolarization inhibits calcineurin activation. Calcineurin activity was assessed using NFAT-luc plasmid. Human myoblasts were induced to differentiate for 4 days either in control conditions (DM) or in presence of 10 mM Cs+ (Cs), 116 mM high extracellular K+ (KCl), or a combination of 7 µM cyclosporin A (CsA) and 5 µM FK506 (FK). (C) Inhibition of calcineurin pathway strongly decreases myogenic bHLH and MEF2 transcription factors activation. Myogenic bHLH and MEF2 activity was assessed using 4RE-luc and 3MEF2-luc plasmid, respectively. Same conditions as in B. Results are expressed as mean±s.e.m.

View larger version (23K): [in a new window]   Fig. 3. CaMKII activation is unrelated to the membrane hyperpolarization. (A) CaMKII activity was assessed by measuring the ability of myoblast extracts to phosphorylate a specific peptide substrate in presence of 32P-ATP. Total cell extracts containing phosphatase inhibitors were prepared from proliferating myoblast (GM) or proliferating myoblast with 1.8 mM Ca2+ (GM1.8), and from myoblast induced to differentiate for 1 or 24 hours in differentiation medium (DM), or in DM containing 10 mM Cs+ (Cs), 116 mM KCl (KCl), 15 µM mM Ca2+ (DMlow) or 0.7 mM Ca2+ (DM0.7). Endogenous CaMKII activity (activity in cultured) was assessed in absence of added Ca2+ or calmodulin; total CaMKII activity (maximum activity of the myoblast sample) was assessed after addition of 5 mM Ca2+ and 5 µM calmodulin. Specific activation of CaMKII was calculated as the ratio between the endogenous and the total activity. For each experiment, the ratio obtained with myoblasts maintained in differentiation medium containing 15 µM BAPTA-AM was set to 1. Results are expressed as mean±s.e.m. (B) CaMKII activation in high Ca2+ proliferating medium does not induce myoblast differentiation. Differentiation is expressed as a percentage of myogenin- and MEF2-positive nuclei. Results are expressed as mean±s.e.m.

View larger version (16K): [in a new window]   Fig. 4. p38-MAPK and PI3K are activated in proliferation and are not controlled by the differentiation-linked hyperpolarization. (A) Activation of p38-MAPK pathway was detected by western blot using an antibody directed against phospho-p38-MAPK (Thr180/Tyr182). Phospho-p38-MAPK is present in proliferating myoblasts (GM), is maintained in differentiating myoblasts (DM) and depolarization induced by 10 mM Cs+ (Cs10 mM) does not affect the level of phosphorylation. By contrast, myogenin expression associated with differentiation is reduced in presence of Cs+. Protein extracts were prepared at the indicated time. (B) Phosphorylation of recombinant ATF2 by immunoprecipitated phospho-p38-MAPK was used to assess p38-MAPK activity. ATF2 phosphorylation has been detected with an antibody specific for phospho-ATF2 (Thr71). Phospho-p38-MAPK was immunoprecipitated from proliferating myoblasts (GM), and from myoblasts differentiated for 4 and 24 hours in presence or absence of 10 mM Cs+. (C) Detection of phospho-AKT by western blot. Myoblasts were grown in media without exogenous insulin. Phospho-AKT (Ser473/Thr308) is present in proliferating myoblasts (GM) and myoblasts differentiated for 24 hours (DM). Phospho-AKT expression is not affected by 10 mM Cs+ (Cs10) or 15 µM BAPTA-AM (BA). Myogenic differentiation is confirmed by myogenin expression.

View larger version (14K): [in a new window]   Fig. 5. Schematic representation of the role of membrane hyperpolarization during human myoblast differentiation. Chronological illustration of the mechanisms of myoblast differentiation, that begins with calcineurin activation as a consequence of Kir2.1-induced membrane hyperpolarization. Calcineurin activation is strictly associated with Kir2.1 activity and the onset of the differentiation process. Activation of Ca2+ influx through T-type Ca2+ channels (CaT) or store-operated channels (SOCs), or release of Ca2+ for endoplasmic reticulum (stores), could provide the increase of intracellular Ca2+ concentration responsible for myoblast differentiation and calcineurin activation (Arnaudeau et al., 2006). However, p38-MAPK, PI3K and CaMK are (or can be) activated during myoblast proliferation without inducing myoblast differentiation. CaMK can be activated by an increase in extracellular Ca2+ concentration not linked to the differentiation process. Myogenin and MEF2 expression is observed after calcineurin activation.