Intrinsic Role of CFTR in Skeletal Muscle 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. doi:10.1371/journal.pgen.1000586.g008 Calcium imaging Intracellular free Ca2+ measurements were performed in diaphragmatic myotubes grown on coverslips using the ratiometric probe Fura-2 (5 mM), and frames were collected at 2 s intervals for a period of 100 s. A customized system was employed for KCl perfusion and pharmacologic reagents applied to myotubes as follows: 10 mM CFTR-inh172 (Calbiochem) for 30 min; 2 mM thapsigargin (Sigma) for 400 s; 100 mM ryanodine (Sigma) for 10 min; 50 mM 2-aminoethoxydiphenylborate (APB) (Calbiochem) for 30 min; and 10 mM U73122 (Sigma) for 20 min. RT–PCR Murine CFTR primers contained the following sequences (59 to 39): TCTCTGCCTTGTGGGAAATC (forward), and AGTACC CGGCATAATCCAAG (reverse); human CFTR primers consisted of the following (59 to 39): CTACATGGAACACATACCTTCG (forward), and GGTGATAATCACTGCATAGC (reverse); GAPDH primer sequences (59 to 39) were: AGCAATGCCT CCTGCACCACC (forward), and CCGGAGGGGCCATC CACAGTC (reverse). Real-time PCR of CFTR expression was performed with FAST SYBR Green, and relative quantification was PLoS Genetics | www.plosgenetics.org 12 July 2009 | Volume 5 | Issue 7 | e1000586