Author Summary Cystic fibrosis is an autosomal recessive disorder caused by mutations of the CF transmembrane conductance regula- tor (CFTR), which acts as a chloride channel and also participates in the regulation of other ions and proteins. In most CF patients, the clinical course is dominated by lung disease and recurrent pulmonary bacterial infections. Many CF patients also have significant skeletal muscle wasting and weakness, and this can affect the most essential breathing muscle, the diaphragm. Although muscle wasting in CF has generally been attributed to factors such as reduced physical activity and poor nutrition, our study reveals an intrinsic defect of skeletal muscle function caused by the lack of CFTR. Hence, we show that CFTR is normally found in skeletal muscle fibers of humans and mice. In CF muscle cells lacking CFTR, abnormal elevations of calcium and inflammatory gene expression are found. In addition, during pulmonary infections, diaphragm muscles lacking CFTR show greater weakness and induction of genes which cause muscle atrophy. These findings extend our understanding of the factors leading to exercise limitation and disability in CF and also have implications for the pathogenesis of respiratory muscle failure in CF patients during lung infections. In the present study, we sought to determine whether a specific link exists between CFTR and skeletal muscle function, with a particular emphasis on the diaphragm due to its importance for survival. Our primary objectives were two-fold: 1) to confirm the presence, localization, and functionality of CFTR in normal skeletal muscle; and 2) to determine whether CFTR deficiency predisposes to diaphragmatic muscle inflammation, atrophy, or weakness. Here we report that CFTR protein is normally found at the level of the sarcoplasmic reticulum (SR) in skeletal muscle, where it appears to modulate intracellular calcium fluxes. We also show that in the setting of lung infection, the diaphragms of Cftr2/2 mice have higher levels of pro-inflammatory gene expression, an increased induction of cachexia-inducing components of the ubiquitin-proteasome pathway, and a more severe impairment of force-generating capacity than wild-type controls. Taken together, these findings point to an intrinsic alteration of skeletal muscle function in CFTR deficiency, which is manifested by greater cytokine expression, atrophy, and force loss upon exposure to an inflammatory milieu. In view of the increased pulmonary and systemic inflammation found in CF, these phenom- ena could play a major role in promoting chronic cachexia, as well as the development of respiratory muscle failure during acute pulmonary infections in CF patients. Results Expression of CFTR in murine and human skeletal muscle We first demonstrated CFTR mRNA expression in diaphrag- matic and limb muscle tissues obtained from wild-type (Cftr+/+) mice, and this was further confirmed in primary diaphragmatic myotube cultures (see Figure 1A). As CFTR expression is classically associated with epithelial cells, we then compared CFTR expression in epithelial and skeletal muscle samples. Real-time PCR revealed that CFTR mRNA levels in both human skeletal muscle tissue and cultured muscle cells were approximately 7-fold lower than in human nasal epithelial cell cultures (Figure 1B). Furthermore, immunoprecipitation followed by western blot analysis confirmed the presence of CFTR protein in human skeletal muscle tissue and in cultured muscle cells (Figure 1C). Intrinsic Role of CFTR in Skeletal Muscle Confocal immunofluorescence labeling of CFTR together with either dystrophin or inositol triphosphate receptors (IP3Rs) – markers of the sarcolemma and sarcoplasmic reticulum (SR), respectively – were used to localize the CFTR protein. On transverse cross-sections of human skeletal muscle, only occasional surface membrane staining for CFTR could be demonstrated (Figure 2A, a–c). However, longitudinal sections showed a consistent pattern of stereotypical periodicity that co-localized to a substantial degree with IP3R immunostaining (Figure 2A, d–f), suggesting that CFTR is associated with the SR. These findings were reproduced in murine skeletal muscle (Figure 2B, d–f), and CFTR was further co-localized with another SR protein, the ryanodine receptor (RyR) (Figure 2B, g–i). As expected, no CFTR staining was observed in the muscles of Cftr2/2 mice (Figure 2B, a–c). Furthermore, in myotubes derived from Cftr+/ + mice, co-localization of CFTR with IP3Rs was also demonstrated (Figure 2C). Cftr2/2 muscle cells exhibit abnormal calcium regulation The SR in skeletal muscle is the major source of Ca2+ released via both RyRs and IP3Rs following muscle cell depolarization [18–20]. To determine whether CFTR has an influence upon this process, cultured Cftr+/+ and Cftr2/2 myotubes were loaded with the Ca2+ probe Fura-2 and exposed to high potassium (60 mM KCl) to induce depolarization in vitro. Immunostaining confirmed the absence of intracellular staining for CFTR in Cftr2/2 myotubes (Figure 3A). In Cftr+/+ myotubes, the Ca2+ response consisted of a rapid rise to a peak amplitude followed by a progressive decline to a plateau level, as shown in Figure 3B (solid line). When Cftr+/+ myotubes were pre-treated with a small molecule inhibitor of CFTR channel activity (CFTR-inh172), there was a greater initial peak as well as a higher plateau value (Figure 3B, dashed line). Quantitative analysis confirmed that the area under the Ca2+ versus time curve and the peak amplitude of Ca2+ responses were significantly greater in Cftr+/+ myotubes treated with the CFTR inhibitor (see Figure 4A). An identical approach was used to compare Ca2+ responses of Cftr+/+ and Cftr2/2 myotubes. The representative tracings in Figure 3C and group mean quantitative analyses in Figure 4B show that Cftr2/2 myotubes responded in a very similar fashion to Cftr+/+ myotubes treated with CFTR-inh172. No differences were detected between Cftr+/+ and Cftr2/2 myotubes with respect to their total SR Ca2+ content (evaluated by blocking Ca2+ re-uptake into the SR with thapsigargin), nor were the results influenced by omission of Ca2+ from the extracellular medium (data not shown). Furthermore, in Cftr2/2 muscle cells, pre-treatment with CFTR-inh172 had no effect on Ca2+ responses (Figure 3D). To better understand how CFTR could interact with RyRs and/or IP3Rs, ryanodine was used to interfere with RyR function, whereas for IP3 pathway interference two different compounds, 2- APB and U73122, were used as inhibitors of IP3Rs and phospholipase C, respectively. As shown in Figure 4C, in both Cftr+/+ and Cftr2/2 myotubes all of these treatments greatly reduced the area under curve as well as the peak amplitude of the Ca2+ response to depolarization. However, in Cftr+/+ myotubes the inhibition of RyR function had more pronounced effects than interference with the IP3 pathway. On the other hand, RyR and IP3R blockade had equivalent impacts upon intracellular Ca2+ in Cftr2/2 myotubes, providing further evidence that Ca2+ mobili- zation is dysregulated in CFTR-deficient muscle cells. Cftr2/2 muscle cells show a hyperinflammatory phenotype in vitro CFTR defects have been associated with a hyperinflammatory phenotype, with IL-8 in particular having been implicated PLoS Genetics | www.plosgenetics.org 2 July 2009 | Volume 5 | Issue 7 | e1000586