gin, an inhibitor of the ER/SR Ca2+ re-uptake pump, did not point to an expansion of SR Ca2+ stores in Cftr2/2 muscle cells as an explanation for the greater Ca2+ responses [39]. Inflammatory/atrophic phenotype and force loss in CFTR–deficient muscle Many studies have reported that CF cell lines as well as airway epithelial cells from CF patients exhibit exaggerated cytokine production [7,8,11]. Several possible explanations have been proposed, including greater Ca2+ release from the endoplasmic reticulum [40,41]. Defective CFTR function has also been linked to hyperactivation of the NF-kB pathway as well as signaling molecules such as ERK1/2 MAPK [7,9–12], which were demonstrated in the present study. Amongst the pro-inflammatory genes showing enhanced upregulation within CFTR-deficient skeletal muscle in this study, all are potential targets of NF-kB, and greater binding of the NF-kB p65 subunit was observed in Cftr2/2 muscle cells. There is also an accumulating literature implicating SR-mediated Ca2+ release, particularly from the IP3R, in the regulation of transcription factors controlling the expression of pro-inflammatory and other genes in normal skeletal muscle cells [42,43]. In this regard, it was recently reported that depolarization-induced Ca2+ transients of even brief duration can activate NF-kB in myotubes [20]. Furthermore, this phenomenon has been linked to increased cytokine (IL-6) gene expression in skeletal muscle cells [44]. Therefore, although not directly examined in our study, it is plausible to speculate that the increased NF-kB activity and pro- inflammatory gene expression patterns observed in stimulated Cftr2/2 muscle cells could be linked to greater Ca2+ release from the SR. Indeed, an analogous phenomenon of exaggerated Ca2+ release from the ER leading to NF-kB activation was recently reported in CF airway epithelial cells stimulated with IL-1b [41]. In the present study, we demonstrated two potential causes for muscle weakness in CFTR-deficient skeletal muscles. Firstly, we found that Cftr2/2 mice are more prone to a loss of muscle mass when exposed to a hyperinflammatory milieu. This is likely linked at least in part to the observed upregulation of E3 ubiquitin ligases, MuRF-1 and atrogin-1, which are centrally involved in protea- somal degradation of muscle proteins atrophy [24]. Furthermore, NF-kB activation via IKK-2/b has been directly implicated in the induction of the ubiquitin ligases which cause muscle atrophy [24]. Secondly, even after correction for atrophy and reduced cross- sectional area of the muscle, diaphragmatic strength (specific force) was abnormally decreased in infected Cftr2/2 mice. This suggests an alteration in the intrinsic contractile properties of the muscle above and beyond the loss of muscle mass. Recent work has suggested that caspase- and calpain-mediated breakdown of the contractile protein apparatus precedes protein degradation by the ubiquitin-proteasome system in sepsis, and may thus be respon- sible for the loss of specific force prior to atrophy [45,46]. Indeed, the calpain system is activated by increases in intracellular Ca2+, and may therefore be particularly relevant to our findings [46]. Oxidative stress has also been strongly implicated in sepsis-induced reductions of diaphragmatic specific force [47] and it is interesting to note that Cftr2/2 hearts are abnormally vulnerable to tissue damage caused by ischemia-reperfusion, a classical inducer of free radical-mediated injury [26]. Clinical implications Patients with CF most commonly die of type II (hypercapnic) respiratory failure, where the limiting factor is not oxygen transfer but rather the presence of respiratory muscle pump failure. This occurs when the load placed on the diaphragm exceeds its capacity to deal with the increased work of breathing. In earlier phases of Intrinsic Role of CFTR in Skeletal Muscle the disease, the increased load may actually induce a beneficial training effect on the respiratory muscles [3]. However, diaphrag- matic pressure generation normalized to its muscular mass can be reduced by as much as 40% even in clinically stable CF patients [3]. Therefore, any factor which further compromises the muscular strength of the diaphragm, such as an acute pulmonary infection with P. aeruginosa, will place patients at greater risk for the development of respiratory failure. In our study, CFTR-deficient skeletal muscles demonstrated a pathological phenotype primarily during exposure to an inflammatory milieu. This is likely to be clinically relevant, since studies suggest a quasi-constant exposure to abnormally high serum levels of inflammatory mediators in many CF patients [48], as well as a greater systemic inflammatory response to exercise [49]. Taken together, we believe our findings are consistent with a scenario (see Figure 8) in which two major factors act synergistically to favor the development of skeletal muscle dysfunction in CF: 1) a higher ambient level of pulmonary/ systemic inflammation leading to greater exposure of the muscles to pro-inflammatory mediators; and 2) a greater inherent sensitivity of the muscles (related to their lack of CFTR) to the effects of pro-inflammatory mediator exposure. This can lead, in turn, to an exaggerated upregulation of atrophy pathways as well as an impairment of contractile mechanisms. On the other hand, when the level of systemic inflammation is normal or relatively low (e.g., in non-infected Cftr2/2 mice and CF patients with early or mild disease), alterations in the function of CFTR-defective skeletal are more subtle, and may only be apparent with heavy usage such as during exercise [15]. In conclusion, the identification in this study of an intrinsic alteration of skeletal muscle function in CFTR deficiency extends our understanding of the factors leading to disability in CF patients. A major implication of our study is that the excessive levels of pulmonary and systemic inflammation found in patients with advanced CF can potentially initiate a positive feedback loop, in which the abnormal vulnerability of CFTR-deficient muscle to pro-inflammatory stimulation becomes an important component of the downward spiral leading to respiratory muscle failure. Our data also suggest that the chronic cachexia found in non- respiratory skeletal muscles of many CF patients could be related to similar mechanisms, and may thus be amenable to anti- inflammatory therapies. Materials and Methods Cell culture Primary muscle cell cultures from the mouse diaphragm and limb muscle (tibialis anterior) were established using single living muscle fibers as previously described in detail [50]. Culture conditions for the E6/E7 human skeletal muscle cell line were as previously published [51]. Calu3 cells and HEK cells stably expressing CFTR were used as positive controls for western blot/ immunoprecipitation studies. Immunofluorescence for confocal microscopy Standard staining procedures were applied to myotubes grown on glass coverslips and frozen muscle tissue sections. The analysis was performed on 3 animals from the CFTR+/+ group, 2 animals from the CFTR2/2 group, and 3 human muscle samples (3 sections each). Primary antibodies consisted of the following: 1:100 dilution of anti-CFTR (clone 24–1, R&D Systems); 1:50 dilution of anti-dystrophin (clone ab15277, Abcam); 1:100 dilution of anti- IP3R (clone NR09, Calbiochem); 1:50 dilution of anti-RyR (clone sc-13942, Santa Cruz). PLoS Genetics | www.plosgenetics.org 11 July 2009 | Volume 5 | Issue 7 | e1000586