Lack of CFTR in Skeletal Muscle Predisposes to Muscle Wasting and Diaphragm Muscle Pump Failure in Cystic Fibrosis Mice Maziar Divangahi1., Haouaria Balghi1., Gawiyou Danialou 1 , Alain S. Comtois1, Alexandre Demoule1,2, Sheila Ernest1, Christina Haston 1 , Renaud Robert3, John W. Hanrahan3, Danuta Radzioch4, Basil J. Petrof1* 1 Meakins-Christie Laboratories and Respiratory Division, McGill University Health Centre and Research Institute, Montreal, Quebec, Canada, 2 Universite´ Paris 6 Pierre et Marie Curie, UPRES EA2397, Paris, France, 3 Department of Physiology, McGill University, Montreal, Quebec, Canada, 4 Centre for the Study of Host Resistance, Departments of Experimental Medicine and Human Genetics, McGill University Health Centre Research Institute, Montreal, Quebec, Canada Abstract Cystic fibrosis (CF) patients often have reduced mass and strength of skeletal muscles, including the diaphragm, the primary muscle of respiration. Here we show that lack of the CF transmembrane conductance regulator (CFTR) plays an intrinsic role in skeletal muscle atrophy and dysfunction. In normal murine and human skeletal muscle, CFTR is expressed and co- localized with sarcoplasmic reticulum-associated proteins. CFTR–deficient myotubes exhibit augmented levels of intracellular calcium after KCl-induced depolarization, and exposure to an inflammatory milieu induces excessive NF-kB translocation and cytokine/chemokine gene upregulation. To determine the effects of an inflammatory environment in vivo, sustained pulmonary infection with Pseudomonas aeruginosa was produced, and under these conditions diaphragmatic force-generating capacity is selectively reduced in Cftr2/2 mice. This is associated with exaggerated pro-inflammatory cytokine expression as well as upregulation of the E3 ubiquitin ligases (MuRF1 and atrogin-1) involved in muscle atrophy. We conclude that an intrinsic alteration of function is linked to the absence of CFTR from skeletal muscle, leading to dysregulated calcium homeostasis, augmented inflammatory/atrophic gene expression signatures, and increased diaphragmatic weakness during pulmonary infection. These findings reveal a previously unrecognized role for CFTR in skeletal muscle function that may have major implications for the pathogenesis of cachexia and respiratory muscle pump failure in CF patients. Citation: Divangahi M, Balghi H, Danialou G, Comtois AS, Demoule A, et al. (2009) Lack of CFTR in Skeletal Muscle Predisposes to Muscle Wasting and Diaphragm Muscle Pump Failure in Cystic Fibrosis Mice. PLoS Genet 5(7): e1000586. doi:10.1371/journal.pgen.1000586 Editor: Gregory A. Cox, The Jackson Laboratory, United States of America Received December 17, 2008; Accepted July 3, 2009; Published July 31, 2009 Copyright: 2009 Divangahi 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: Support was received from the Canadian Cystic Fibrosis Foundation, the Canadian Institutes of Health Research and BREATHE program, the Quebec Respiratory Health Network of the Fonds de la recherche en sante´ du Que´bec, and the McGill University Health Centre Research Institute. 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: basil.petrof@mcgill.ca . These authors contributed equally to this work. Introduction Cystic fibrosis (CF) is caused by defects in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR) [1]. Despite improvements in therapy, a progressive loss of lung function, associated with recurrent pulmonary infections and inflammation, still prevails. Over time, the diaphragm and other respiratory muscles become unable to sustain the increased breathing requirement, thereby leading to respiratory failure. This ultimately fatal situation is favored by the presence of generalized skeletal muscle atrophy (cachexia) and weakness in many CF patients, which has been attributed to malnutrition, mechanical disadvantage of the respiratory muscles, and other factors [2,3]. CFTR is a member of the superfamily of ATP-binding cassette (ABC) transporter ATPases, which functions as a cAMP-regulated chloride (Cl2) channel [1,4]. The actions of CFTR extend beyond Cl2channel function, with several other roles described in the secretion or absorption of different ions as well as in the transport or regulation of other macromolecules [5,6]. It is also reported that cells with defective CFTR have a hyperinflammatory phenotype, characterized by an excessive production of cytokines triggered by various stimuli [7–11]. The exaggerated inflammatory response in CFTR-defective cells has been linked to enhanced activation of the transcription factor NF-kB [7,9–12], a central regulator of inflammation. Intriguingly, CFTR transcripts have been detected in rat skeletal muscle, although no attempts were made to elucidate a functional role [13]. However, exercise performance can be impaired in CF patients at early stages of the disease despite essentially normal pulmonary function and nutritional status [14,15]. In addition, exercise limitation in CF patients appears to differ according to the type of CFTR mutation, independent of pulmonary function [16]. These and other observations have led to speculation that a specific CFTR-related functional defect could exist in the skeletal muscles of CF patients [17]. PLoS Genetics | www.plosgenetics.org 1 July 2009 | Volume 5 | Issue 7 | e1000586