Because pro-inflammatory cytokines have been linked to muscle wasting via the ubiquitin-proteasome system, we proceeded to measure the diaphragmatic expression levels of two key regulators of this proteolytic pathway in skeletal muscle, MuRF-1 and atrogin-1 [24]. Diaphragmatic expression levels of both MuRF-1 and atrogin- 1 were increased in Cftr2/2 mice, but not in their wild-type littermates, after pulmonary infection with P. aeruginosa (Figure 6B). Measurements of limb muscle (soleus) weights also indicated a significant difference between infected Cftr2/2 and Cftr+/+ mice (6.4860.17 mg versus 8.2060.24 mg; p,0.0001, n = 7 per group). Therefore, these findings are consistent with a greater susceptibility of infected Cftr2/2 mouse skeletal muscles not only to cytokine upregulation, but also to ubiquitin-mediated proteolysis and atrophy. Altered contractile function and excessive force loss during P. aeruginosa lung infection in diaphragms of Cftr2/2 mice We initially examined whether the different KCl-evoked responses of cultured Cftr+/+ and Cftr2/2 myotubes could be ascertained in intact perfused diaphragmatic muscle fibers. Figure 7A shows major differences in the force response profiles of Cftr+/+ (monophasic pattern) and Cftr2/2 (biphasic pattern) fibers during KCl-induced contraction. Moreover, Cftr+/+ dia- phragms incubated with CFTR-inh172 exhibited a biphasic pattern similar to the Cftr2/2 group, while CFTR-inh172 had no effect upon Cftr2/2 diaphragms. In addition, when diaphrag- matic fibers from Cftr2/2 were electrically stimulated, a significant prolongation of relaxation and contraction were observed during tetanic (but not single twitch) stimulation as compared to Cftr+/+ diaphragms (Figure 7B and 7C). Taken together, these results corroborate the abnormal responses to depolarization in Cftr2/2 myotubes and further indicate a functional alteration in intact skeletal muscle fibers lacking CFTR. To determine whether the greater increases in pro-inflammatory and atrophy gene expression in Cftr2/2 diaphragms are associated with exaggerated muscle weakness during pulmonary infection, force measurements were performed on electrically stimulated fibers. As shown in Figure 7D, lung infection had adverse effects upon diaphragmatic force production in the Cftr2/2 group only. Hence at the P. aeruginosa inoculation dose of 16105 cfu employed, force-generating capacity of Cftr+/+ diaphragms remained normal, whereas Cftr2/2 diaphragms generated maximal force values which were approximately one-third lower than in identically infected Cftr+/+ mice. Taken together, these results indicate that the pro- inflammatory and atrophy gene expression profiles found in the diaphragms of infected Cftr2/2 mice are associated with a significant impairment of contractile function, which is manifested by profound weakness of this critically important respiratory muscle. Discussion The results of our study demonstrate a previously unrecognized role for CFTR in skeletal muscle physiology, and supply new insights into a potential mechanism favoring the development of respiratory muscle failure in CF. The main findings of our investigation can be summarized as follows: 1) CFTR is normally expressed at the SR of skeletal muscle fibers and is clearly functional, as indicated by abnormal intracellular Ca2+ and contractile responses to depolarization in muscles with CFTR deficiency; and 2) diaphragm muscles lacking CFTR exhibit an exaggerated upregulation of cytokines and cachexia-inducing factors during pulmonary infection, which is associated with a major deterioration of diaphragmatic function. Intrinsic Role of CFTR in Skeletal Muscle CFTR expression and function in normal skeletal muscle CFTR expression has been reported in both cardiac [25–27] and smooth muscle [28]. To our knowledge, there is only one prior report of CFTR expression in skeletal muscle, in which a single mRNA species of 7.5 kb was demonstrated in rat skeletal muscle, as compared to the 6.3 kb transcript found in the lung and intestines [13]. Whereas the most common mutation (F508 delta) in CF patients causes CFTR to be retained in the endoplasmic reticulum of epithelial cells, our results suggest that this may be the major location of CFTR in skeletal muscle. Although we do not exclude a co-existent surface membrane localization, in our study CFTR was mostly co-localized with RyRs and IP3Rs in their characteristic cross-striated staining pattern [18]. This is reminis- cent of ClC-4, which co-localizes at the surface membrane with CFTR in intestinal epithelial cells [29] but was recently shown to be highly expressed in the SR of skeletal muscle [30]. While the specific protein motifs responsible for differential cellular localiza- tion of the epithelial and skeletal muscle isoforms of CFTR are unknown, the CFTR gene is known to exhibit complex patterns of tissue-specific expression, with different transcription start sites and alternative splicing [27,31]. In skeletal muscle, the ClC-1 Cl2 channel at the surface membrane plays the predominant role in maintaining the normal resting electrical potential of the cell [32]. The normal physiological role of SR Cl2 channels is more ill-defined. Several Cl2 channels with distinct Cl2 conductances, selectivities, and voltage-dependencies have been characterized in SR vesicles [33,34]. However, in most cases the specific molecular identities of these channels have not been determined. Both Ca2+-induced and voltage-activated Ca2+ release are enhanced by Cl2 exposure, apparently via increased activation of RyRs [33,35]. However, Cl2 was also shown to inhibit caffeine-induced Ca2+ release [36], indicating that the regulatory effects of Cl2 channels upon SR function are complex. One proposed function for SR Cl2 channels is to permit Cl2 to act as a counter ion during high rates of Ca2+ release and uptake from the SR [33,34], in order to restore the SR membrane potential, and it is possible that CFTR plays a role in this process. CFTR could also modulate SR function in a manner which is unrelated to Cl2 channel activity per se. For example, CFTR mediates glutathione flux in epithelial cells [5] and thus plays a role in controlling the redox state, which is an important modifier of SR Ca2+ release [37]. Dysregulated calcium homeostasis in CFTR–deficient muscle In the present study, we found that defective CFTR function is associated with increased Ca2+ mobilization in myotubes stimu- lated by potassium-induced depolarization. These findings are consistent with previous observations in Cftr2/2 vascular smooth muscle, where greater KCl-induced vasoconstriction and impaired relaxation are found [28], as well as increased intracellular Ca2+ and enhanced vasoconstrictor responses during exposure to CFTR-inh172 [38]. In our study, Ca2+ responses to depolarization were similarly increased irrespective of whether CFTR function was inhibited by genetic or pharmacologic methods. Our data indicate that the SR is the primary source of this increased Ca2+ influx, since the latter was not affected by the removal of extracellular calcium. The fact that inhibiting the IP3-mediated pathway with either 2-APB or U73122 (but not the RyR) led to a decrease of Ca2+ values to levels obtained in Cftr+/+ myotubes under the same conditions, suggests that the IP3R could be the most important source of dysregulated Ca2+ release from the SR in CFTR-deficient muscle cells, although this remains to be determined. In addition, experiments performed with thapsigar- PLoS Genetics | www.plosgenetics.org 9 July 2009 | Volume 5 | Issue 7 | e1000586