Lack of CFTR in skeletal muscle predisposes to muscle wasting and diaphragm muscle pump failure in cystic fibrosis mice

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 Cftr(-/-) 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.

Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Figure 1. Expression of CFTR in normal skeletal muscle.

(A) CFTR transcripts in lung, tibialis anterior (TA), and diaphragm (Dia) muscle tissue (in vivo) as well as in cultured myotubes from wild-type (Cftr+/+) mouse diaphragms (in vitro). (B) Relative quantification of human CFTR mRNA in skeletal muscle tissue (MT), cultured E6/E7 skeletal muscle cells (MC), and cultured epithelial cells (EC) from human nasal mucosa. (C) Western blotting for human CFTR after immunoprecipitation (IP) of CFTR protein in human skeletal muscle tissue and cultured human muscle cells (myotubes and myoblasts), as well as CFTR-overexpressing positive control cells (Calu3 or HEK). Note the presence of both mature (Band C) and immature (Band B) forms of CFTR. Top Panel: lysate amounts for IP were 1000 (lanes 3–5), 2000 (lane 6), and 200 µg (lane 7). Bottom Panel: lysate amounts for IP were 3000, 1500, and 1000 µg for lanes 2–4, respectively.

Figure 2. Confocal localization of CFTR in skeletal muscle.

(A) Double labeling for CFTR and either membrane- (dystrophin) or SR- (IP3R) associated proteins in transverse and longitudinal sections of human skeletal muscle. (B) Double labeling of CFTR and SR (IP3R or RyR) proteins in longitudinal sections of Cftr+/+ and Cftr−/− mouse skeletal muscle (scale bar = 10 µm). (C) Triple labeling of IP3R, CFTR, and myonuclei (DAPI) in cultured mouse myotubes.

Figure 3. Abnormal calcium responses in CFTR–deficient muscle cells.

(A) Negative immunostaining for CFTR in Cftr−/− myotubes. (B) Representative Ca2+ responses in Cftr+/+ myotubes, either without (control) or with (CFTR-inh) pre-treatment by CFTR-inh172. (C) Representative Ca2+ responses in Cftr+/+ versus Cftr−/− myotubes. (D) Lack of effect of CFTR-inh172 on the area under the curve or peak amplitude of Ca2+ responses in Cftr−/− myotubes.

Figure 4. Quantification and inhibition of SR–mediated calcium release.

(A) Group mean data for intracellular Ca2+ responses in Cftr+/+ myotubes, either without (control) or with (CFTRinh) pre-treatment by CFTR-inh172. (B) Group mean data for intracellular Ca2+ responses in Cftr+/+ versus Cftr−/− myotubes. (C) Effects of inhibiting RyR or IP3R function in Cftr+/+ (open bars) and Cftr−/− (filled bars) myotubes. For all experiments, N = minimum of 30 myotubes per group. * p<0.05 versus control, † p<0.05 for ryanodine versus 2-APB and U73122.

Figure 5. Hyperinflammatory phenotype of Cftr−/− skeletal muscle cells in vitro.

(A) Upper panels: Cytokine mRNA expression levels in Cftr+/+ versus Cftr−/− diaphragmatic myotubes under control conditions (CTL) and after 4 hours of stimulation (TNF-α 1 ng/ml, IL-1α 5 U/ml, IFN-γ 20 U/m, LPS 1 ng/ml). N = 5 per group; * p<0.05 for control versus stimulated groups, † p<0.05 for Cftr+/+ versus Cftr−/−. Lower panels: Representative RNase protection assays from diaphragm (left panel) and limb muscle (right panel). (B) NFκB p65 levels in nuclear extracts (normalized for protein) obtained from diaphragmatic myotubes in Cftr+/+ versus Cftr−/− groups stimulated for the indicated time periods (0–60 min). N = 4–5 per group; * p<0.05 for Cftr+/+ versus Cftr−/−. (C) Western analysis of phosphorylated (pERK1/2) and total ERK1/2 MAPK in cytoplasmic lysates from Cftr+/+ and Cftr−/− diaphragmatic myotubes following stimulation with cytokines+LPS. Results are representative of 2 experiments.

Figure 6. Hyperinflammatory phenotype of Cftr−/− skeletal muscle in vivo.

(A) Cytokine mRNA expression levels in Cftr+/+ versus Cftr−/− diaphragms for uninfected (CTL) and Pseudomonas-infected (day 2) mice. N = 4–5 mice per group; * p<0.05 for control versus infected mice, † p<0.05 for Cftr+/+ versus Cftr−/−. (B) Real-time PCR quantification of the E3 ubiquitin ligases MuRF-1 and atrogin-1 in diaphragms of control and infected mice. * p<0.05 for Cftr+/+ versus Cftr−/−.

Figure 7. Effects of CFTR status on diaphragmatic contractile function.

(A) Response of intact diaphragmatic muscle fibers to KCl-induced depolarization in Cftr+/+ versus Cftr−/− groups, under basal conditions (N = 6–7 mice per group) or with CFTR- inh172 (N = 4–5 mice per group) added to the perfusate (100 µM). (B,C): Effects of CFTR deficiency on relaxation and contraction times of the diaphragm during electrical stimulation (white bars: Cftr+/+; red bars: Cftr−/−). (D) Differential effects of Pseudomonas lung infection on diaphragmatic force capacity in Cftr+/+ and Cftr−/− mice (N = 5 mice per group). * p<0.05 for Cftr+/+ versus Cftr−/−.

Figure 8. Schematic representation of respiratory muscle failure in CF.

A greater inherent sensitivity of CF muscle cells to pro-inflammatory stimuli, when combined with the higher levels of pulmonary and systemic inflammation found in CF, leads to exaggerated NF-kB and cytokine activation within diaphragmatic muscle fibers. This in turn provokes diaphragmatic wasting and weakness in CF, thus contributing to the pathogenesis of respiratory failure.