Intrinsic Role of CFTR in Skeletal Muscle 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 Cftr2/2 myotubes. (C) Effects of inhibiting RyR or IP3R function in Cftr+/+ (open bars) and Cftr2/2 (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. doi:10.1371/journal.pgen.1000586.g004 shown in Figure 5B, greater binding of the NF-kB p65 subunit was observed in Cftr2/2 diaphragm muscle cells at 10 min after initiating stimulation. In addition, inhibition of the NF-kB pathway by pre-treatment with the IkB kinase (IKK)-2 inhibitor SC-514 had similar effects in downregulating MIP-1a mRNA expression in Cftr2/2 and Cftr+/+ myotubes (3–4 fold reductions for both; p,0.05, n = 6 per group), but it was only effective in doing so for MIP-2 (2.4 fold; p,0.05) and RANTES (5.8 fold; p,0.05) in Cftr2/2 myotubes, suggesting a greater reliance upon this pathway in the latter. Finally, we observed that ERK1/2 MAPK, another signal transduction molecule implicated in NF-kB activation and cytokine gene upregulation [20], also showed greater phosphorylation following cytomix/LPS stimulation in the Cftr2/2 group (Figure 5C). Exaggerated pro-inflammatory and atrophy gene responses in vivo To evaluate the potential relevance of the above findings to the in vivo context, Cftr2/2 mice were infected with P. aeruginosa. We used the Cftr2/2 strain (CFTRtm1UNC) backcrossed onto the C57Bl/6 back- ground that has been shown to develop spontaneous lung disease [21]. As has been previously reported [22,23], the pulmonary bacterial PLoS Genetics | www.plosgenetics.org 6 July 2009 | Volume 5 | Issue 7 | e1000586