Impaired Adaptive Response to Mechanical Overloading in Dystrophic Skeletal Muscle Pierre Joanne 1 , Christophe Hourde ´ 2 , Julien Ochala 3 , Yvain Caude ´ ran 2 , Fadia Medja 2 , Alban Vignaud 2 , Etienne Mouisel 2 , Wahiba Hadj-Said 2 , Ludovic Arandel 2 , Luis Garcia 2 , Aure ´ lie Goyenvalle 2 , Re ´ mi Mounier 4 , Daria Zibroba 5 , Kei Sakamato 5 , Gillian Butler-Browne 2 , Onnik Agbulut 1 , Arnaud Ferry 2,6 * 1 Universite ´ Paris Diderot, Sorbonne Paris Cite ´, CNRS EAC4413, Unit of Functional and Adaptive Biology, Laboratory of Stress and Pathologies of the Cytoskeleton, Paris, France, 2 Universite ´ Pierre et Marie Curie-Paris6, Sorbonne Universite ´s, UMR S794, INSERM U974, CNRS UMR7215, Institut de Myologie, Paris, France, 3 Department of Neuroscience, Uppsala University, Uppsala, Sweden, 4 Universite ´ Paris Descartes, Sorbonne Paris Cite ´ , INSERM U1016, CNRS UMR8104, Institut Cochin, Paris, France, 5 MRC Protein Phosphorylation Unit, College of Life Sciences, University of Dundee, Dundee, United Kingdom, 6 Universite ´ Paris Descartes, Sorbonne Paris Cite ´, Paris, France Abstract Dystrophin contributes to force transmission and has a protein-scaffolding role for a variety of signaling complexes in skeletal muscle. In the present study, we tested the hypothesis that the muscle adaptive response following mechanical overloading (ML) would be decreased in MDX dystrophic muscle lacking dystrophin. We found that the gains in muscle maximal force production and fatigue resistance in response to ML were both reduced in MDX mice as compared to healthy mice. MDX muscle also exhibited decreased cellular and molecular muscle remodeling (hypertrophy and promotion of slower/oxidative fiber type) in response to ML, and altered intracellular signalings involved in muscle growth and maintenance (mTOR, myostatin, follistatin, AMPKa1, REDD1, atrogin-1, Bnip3). Moreover, dystrophin rescue via exon skipping restored the adaptive response to ML. Therefore our results demonstrate that the adaptive response in response to ML is impaired in dystrophic MDX muscle, most likely because of the dystrophin crucial role. Citation: Joanne P, Hourde ´ C, Ochala J, Caude ´ran Y, Medja F, et al. (2012) Impaired Adaptive Response to Mechanical Overloading in Dystrophic Skeletal Muscle. PLoS ONE 7(4): e35346. doi:10.1371/journal.pone.0035346 Editor: Daniel Tome ´, Paris Institute of Technology for Life, Food and Environmental Sciences, France Received December 6, 2011; Accepted March 14, 2012; Published April 12, 2012 Copyright: ß 2012 Joanne et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: Financial support has been provided by Universite ´ Pierre et Marie Curie (UPMC), CNRS, INSERM, Universite ´ Paris Descartes, ANR-Genopath In-A-Fib, ANR-Blanc Androgluco, the Association Franc ¸aise contre les Myopathies (AFM), MyoAge (EC 7th FP, contract 223576), and Agence Franc ¸aise de Lutte contre le Dopage. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: arnaud.ferry@upmc.fr Introduction Skeletal muscle exhibits a crucial capacity to adapt to changing use. In response to repeated high-force contractions, i.e., mechanical overloading (ML), both absolute maximal force production and fatigue resistance of the muscle markedly increase. Tension, stretch and deformation generated during ML, together with other activity- related signals (e.g. muscle action potential, metabolites…), are sensed and in turn activate different intracellular signaling pathways that converge to induce muscle cellular and molecular adaptations, such as hypertrophy and fiber-type conversion, resulting in muscle performance improvement [1,2,3,4,5]. The gain in absolute maximal force induced by ML is, at least partly, attributed to muscle hypertrophy, since absolute maximal force is roughly proportional to muscle cross-sectional area. Muscle hypertrophy in response to ML results from increased protein synthesis via activation of the mammalian target of rapamycin (mTOR) signaling pathway [6,7]. More precisely, it is the mTOR complex-1 (mTORC1) that contains mTOR and the rapamycin-sensitive raptor subunit that promotes protein synthesis via the phosphorylation of the initiation factor-4E binding protein- 1 and the S6 kinase (S6K) that in turn phosphorylates at the S240/ 244 site the ribosomal S6 protein (rS6)[1,2]. Akt is also activated by ML [6] and it is known that genetic activation of Akt promotes muscle hypertrophy [8,9]. Recently, we demonstrated that the alpha-1 isoform of the AMP-activated protein kinase (AMPK-a 1) plays an important role in limiting muscle growth during ML, via the reduced activation of mTOR signaling [10]. In addition, it has been shown that myostatin is down-regulated in response to ML [11]. Since myostatin deactivates mTOR [12,13,14], these results suggest that together with AMPK, myostatin also limits muscle hypertrophy induced by ML. In addition, the myostatin inhibitor follistatin [15] and the stress response genes REDD1 and REDD2 (regulated in development and DNA damage response)[1,16] are possibly involved in the control of mTOR signaling in response to ML. Of note, the activation of the mitogen-activated protein kinase pathway (MAPK) may be not sufficient to induce muscle growth [17,18] and a recent study reported that satellite cells are not necessary for a robust ML-induced hypertrophy [19]. The fast/glycolytic -to slow/oxidative fiber conversion is likely the cellular and molecular adaptation responsible for the increase in fatigue resistance induced by ML. Indeed, it is well established that slow/oxidative fibers expressing type-1 and -2a myosin heavy chain (MHC-1 and MHC-2a) are more fatigue-resistant than fast/ glycolytic fibers expressing MHC-2x and MHC-2b [20]. Calci- neurin/NFAT signaling is a key player in the promotion of the slow/oxidative fiber phenotype [3,21]. Pharmacological inhibition or genetic loss of calcineurin blocks the fast/glycolytic -to slow/ oxidative fiber-type conversion induced by ML [22,23,24]. The peroxisome proliferator-activated receptor c coactivator-1 (PGC- PLoS ONE | www.plosone.org 1 April 2012 | Volume 7 | Issue 4 | e35346