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BACKGROUND: We tested the hypothesis that vardenafil, a common drug used for improving erectile dysfunction and able to partially normalize transepithelial chloride transport in cystic fibrosis (CF), modulates CF lung inflammation. METHODS: Inflammatory markers in lungs of F508del-CF and wild-type mice were monitored in response to lipopolysaccharide from Pseudomonas aeruginosa (LPS). The effect of pretreatment with vardenafil (0.14 mg/kg) was evaluated. RESULTS: A latent inflammatory status, characterized by neutrophil infiltrate, mouse macrophage inflammatory protein (MIP)-2 and tumor necrosis factor (TNF)-α, was found in baseline conditions in F508del-CF mice. Inflammatory markers were increased after LPS with higher responses in CF. Vardenafil globally attenuated inflammatory responses in both genotypes however reduction of macrophage infiltration, macrophage chemoattractant chemokine and interleukin-1β was observed in the CF group only. CONCLUSION: Vardenafil reduces lung inflammation with a more pronounced effect in F508del-CF mice, particularly on macrophage cell markers.

Skeletal muscle contraction is reputed not to depend on extracellular Ca2+. Indeed, stricto sensu , excitation-contraction coupling does not necessitate entry of Ca2+. However, we previously observed that, during sustained activity (repeated contractions), entry of Ca2+is needed to maintain force production. In the present study, we evaluated the possible involvement of the canonical transient receptor potential (TRPC)1 ion channel in this entry of Ca2+and investigated its possible role in muscle function. Patch-clamp experiments reveal the presence of a small-conductance channel (13 pS) that is completely lost in adult fibers from TRPC1−/−mice. The influx of Ca2+through TRPC1 channels represents a minor part of the entry of Ca2+into muscle fibers at rest, and the activity of the channel is not store dependent. The lack of TRPC1 does not affect intracellular Ca2+concentration ([Ca2+]i) transients reached during a single isometric contraction. However, the involvement of TRPC1-related Ca2+entry is clearly emphasized in muscle fatigue. Indeed, muscles from TRPC1−/−mice stimulated repeatedly progressively display lower [Ca2+]itransients than those observed in TRPC1+/+fibers, and they also present an accentuated progressive loss of force. Interestingly, muscles from TRPC1−/−mice display a smaller fiber cross-sectional area, generate less force per cross-sectional area, and contain less myofibrillar proteins than their controls. They do not present other signs of myopathy. In agreement with in vitro experiments, TRPC1−/−mice present an important decrease of endurance of physical activity. We conclude that TRPC1 ion channels modulate the entry of Ca2+during repeated contractions and help muscles to maintain their force during sustained repeated contractions.

An imbalance in (n-6)/(n-3) PUFA has been reported in cystic fibrosis (CF) patients. Glycerophospholipids enriched in docosahexaenoic acid (GPL-DHA) have been shown to regulate the (n-6)/(n-3) fatty acid ratio in the elderly. Here, we tested the effect of GPL-DHA supplementation on PUFA status in F508del homozygous CF mice. GPL-DHA liposomes were administrated by gavage (60 mg DHA/kg daily, i.e. at maximum 1.4 mg DHA/d) to 1.5-mo-old CF mice (CF+DHA) and their corresponding wild-type (WT) homozygous littermates (WT+DHA) for 6 wk. The PUFA status of different tissues was determined by GC and compared with control groups (CF and WT). There was an alteration in the (n-6) PUFA pathway in several CF-target organs in CF compared with WT mice, as evidenced by a higher level of arachidonic acid (AA) in membrane phospholipids or whole tissue (21 and 39% in duodenum-jejunum, 32 and 38% in ileum, and 19 and 43% in pancreas). Elevated AA levels were associated with lower linoleic acid (LA) and higher dihomo-gamma-linolenic acid levels. No DHA deficiency was observed. GPL-DHA treatment resulted in different PUFA composition changes depending on the tissue (increase in LA, decrease in elevated AA, DHA increase, increase in (n-6)/(n-3) fatty acid ratio). However, the DHA/AA ratio consistently increased in all tissues in CF+DHA and WT+DHA mice. Our study demonstrates the effectiveness of an original oral DHA formulation in counter-balancing the abnormal (n-6) fatty acid metabolism in organs of CF mice when administrated at a low dose and highlights the potential of the use of GPL-DHA as nutritherapy for CF patients.

Essential fatty acid deficiency has been increasingly reported in patients with cystic fibrosis. The purpose of this work is to critically summarize previous data on fatty acid status and omega3 supplementation in cystic fibrosis. Although the reported abnormalities differ from study to study, the two most consistent features appeared to be reduced circulating levels of linoleic acid and docosahexaenoic acid (DHA). On the assumption that the fatty acid composition of erythrocyte cell membranes may be similar to that of other organs, it seems appropriate to monitor the phospholipid profile from erythrocyte membranes together with circulating blood levels. Formulations containing widely variable DHA doses, ranging from 300 mg to 5 g per day, have been administered to patients with cystic fibrosis with discrepant outcomes. Randomized controlled trials are needed in order to draw firm conclusions on the therapeutic effect of omega3 fatty acid supplementation in cystic fibrosis.

In skeletal muscle, Ca(2+) is implicated in contraction, and in regulation of gene expression. An alteration of [Ca(2+)](i) homeostasis is responsible, at least partially, for the muscle degeneration that occurs after eccentric contractions in Duchenne muscular dystrophy, a disease characterized by the loss of the cytoskeletal protein dystrophin. Using patch clamp in the cell-attached configuration, we characterized the store-operated channels (SOCs) and the stretch-activated channels (SACs) present in isolated mouse skeletal muscle. SOCs were voltage independent, had a unitary conductance between 7 and 8 pS (110 mm Ca(2+) in the pipette), and their open probability increased when the sarcoplasmic reticulum was depleted by thapsigargin. These SOCs were identical to those previously described in the pathophysiology of Duchenne muscular dystrophy. Under the same experimental conditions, we detected a channel activity that was increased by applying a negative pressure to the patch electrode. The SACs responsible for this current had the same unitary conductance and current-voltage relationship as those observed for SOCs. SOCs and SACs had a similar sensitivity to pharmacological agents such as Gd(3+), SKF-96365, 2-aminoethoxydiphenyl borate and GsMTx4 toxin. Moreover, stimulation with IGF-1 increased the occurrence of the activity of both channel types. Together, these observations suggest that SOCs and SACs might belong to the same population or share common constituents. From a functional point of view, treatment of soleus muscle with SKF-96365 or GsMTx4 toxin increased its sensitivity to a fatigue protocol, suggesting that the influx of Ca(2+) that occurs through these channels during contraction is also involved in force maintaining during repeated stimulations.

We have used a new approach to study the effects of overload on skeletal muscle phenotype in mice. The method used avoids any traumatising contact with muscles and the inflammatory reaction that this may provoke. Blocks of lead embedded in silicone were inserted under the skin of the lower part of the back. After 1 month, a 17% hypertrophy was found to have occurred in the tonic soleus muscle, but no change was observed in the fast-twitch extensor digitorum longus (EDL) muscle. The main effects on the contractile properties of the soleus muscle were a decrease in the tetanic relaxation rate and a reduction in the maximal velocity of shortening. Immunohistological analysis of the soleus muscles revealed an increase in the proportion of fibres that express myosin heavy chain (MHC) 1, from 54.2% to 73.9%, with a reduction in the proportion of MHC2a-positive fibres, from 45.8% to 30.2%. These changes were accompanied by an increase in the proportion of fibres that express the slow type of sarcoplasmic reticulum calcium pump (SERCA2a), from 61.8% to 84.7%. In EDL muscles, overload induced only minor changes. Thus, this method of overload affected the soleus muscle in particular. The observed changes in the control of muscle contraction were significantly larger than the changes in typical myofibrillar properties that were observed. These results indicate that there is a temporal dissociation between the relative expression of MHCs and SERCAs.

Duchenne myopathy is a lethal disease due to the absence of dystrophin, a cytoskeletal protein. Muscles from dystrophin-deficient mice (mdx) typically present an exaggerated susceptibility to eccentric work characterized by an important force drop and an increased membrane permeability consecutive to repeated lengthening contractions. The present study shows that mdx muscles are largely protected from eccentric work-induced damage by overexpressing a dominant negative mutant of TRPV2 ion channel. This observation points out the role of TRPV2 channel in the physiopathology of Duchenne muscular dystrophy.

PURPOSE: To study the effect of plyometric training on Ca2+ sensitivity and the influence of troponin T (TnT) isoforms on Ca2+ -activation properties in skinned human muscle fibers. METHODS: Biopsies were obtained from the vastus lateralis of eight men before and after the training period. Chemically skinned fibers were evaluated regarding their Ca2+ -activation properties and were classified according to their myosin heavy chain (MHC) contents and analyzed regarding their slow and fast TnT isoforms. RESULTS: After training, significant improvements (P < 0.05) were found for static jump, countermovement jump, 6 x 5-m shuttle-run test, and leg-press performances. An 8% increase in the proportion of type IIa fibers (P < 0.05) was observed. Single-fiber diameters increased by 11% in type I (P < 0.01), 10% in type IIa (P < 0.001), and 15% in type IIa/IIx fibers (P < 0.001). Peak fiber force increased by 35% in type I (P < 0.001), 25% in type IIa (P < 0.001), and 57% in type IIa/IIx fibers (P < 0.01). The Ca2+ -activation threshold was not altered by training, but the Ca2+ concentration required to elicit half-maximal activation showed a decreasing trend, with significant changes in type I fibers (P < 0.001). Cooperativity at low Ca2+ concentrations was increased in type I and type IIa/IIx fibers (P < 0.05). Type I fibers exclusively expressed slow TnT isoforms, and type II fibers were always associated with fast TnT isoforms, independent of training status. Therefore, changes in Ca2+ sensitivity after training could not be explained by differential fast or slow TnT isoform expression. CONCLUSION: Plyometric training increased single-fiber Ca2+ sensitivity, especially in type I fibers. These changes could not be explained by a modified TnT isoform expression pattern.

Background Inflammation plays a critical role in lung disease development and progression in cystic fibrosis. Azithromycin is used for the treatment of cystic fibrosis lung disease, although its mechanisms of action are poorly understood. We tested the hypothesis that azithromycin modulates lung inflammation in cystic fibrosis mice. Methods We monitored cellular and molecular inflammatory markers in lungs of cystic fibrosis mutant mice homozygous for the ΔF508 mutation and their littermate controls, either in baseline conditions or after induction of acute inflammation by intratracheal instillation of lipopolysaccharide from Pseudomonas aeruginosa, which would be independent of interactions of bacteria with epithelial cells. The effect of azithromycin pretreatment (10 mg/kg/day) given by oral administration for 4 weeks was evaluated. Results In naive cystic fibrosis mice, a spontaneous lung inflammation was observed, characterized by macrophage and neutrophil infiltration, and increased intra-luminal content of the pro-inflammatory cytokine macrophage inflammatory protein-2. After induced inflammation, cystic fibrosis mice combined exaggerated cellular infiltration and lower anti-inflammatory interleukin-10 production. In cystic fibrosis mice, azithromycin attenuated cellular infiltration in both baseline and induced inflammatory condition, and inhibited cytokine (tumor necrosis factor-α and macrophage inflammatory protein-2) release in lipopolysaccharide-induced inflammation. Conclusion Our findings further support the concept that inflammatory responses are upregulated in cystic fibrosis. Azithromycin reduces some lung inflammation outcome measures in cystic fibrosis mice. We postulate that some of the benefits of azithromycin treatment in cystic fibrosis patients are due to modulation of lung inflammation.

The effects of muscle creatine manipulation on contractile properties in oxidative and glycolytic muscles were evaluated. Whereas control mice (NMRi; n = 12) received normal chow (5 g daily), three experimental groups were created by adding creatine monohydrate (CR group; 5%, 1 week; n = 13); beta-guanidinoproprionic acid, an inhibitor of cellular creatine uptake (beta-GPA group; 1%, 2 weeks; n = 12); or CR following beta-GPA (beta-GPA+CR group; n = 11). Total creatine (TCr) and the contractile properties of incubated soleus and extensor digitorum longus (EDL) muscles were determined. For the soleus, compared with control, TCr increased in the CR group (+25%), decreased in beta-GPA group (-50%), and remained stable in the beta-GPA+CR group, whereas, for the EDL, TCr was similar in the CR, and lower in the beta-GPA (-40%) and beta-GPA+CR (-15%) groups. None of the experimental groups (CR, beta-GPA, or beta-GPA+CR) showed changes in peak tension (P(peak)), time to peak tension, or relaxation in soleus or EDL during twitch or tetanic stimulation. For the soleus, fatigue reduced P(peak) to approximately 60% of initial P(peak); 5 min of recovery restored P(peak) to values approximately 15% higher in CR than in controls. P(peak) recovery was not affected by beta-GPA or beta-GPA+CR in the soleus or any treatment in the EDL. Thus, peak tension recovery is enhanced by creatine intake in oxidative but not glycolytic muscles. This may be implicated in the beneficial action of creatine loading.

Clenbuterol induces hypertrophy and a slow-to-fast phenotype change in skeletal muscle, but the signaling mechanisms remain unclear. We hypothesized that clenbuterol could act via local expression of insulin-like growth factor I (IGF-I). Administration of clenbuterol to 3-mo-old female Wistar rats resulted in a 10 and 13% increase of soleus muscle mass after 3 and 9 days, respectively, reaching 16% after 4 wk. When associated with triiodothyronine, clenbuterol induced a dramatic slow-to-fast phenotype change. In parallel, clenbuterol administration induced in soleus muscle a fivefold increase in IGF-I mRNA levels associated with an eightfold increase in IGF-binding protein (IGFBP)-4 and a fivefold increase of IGFBP-5 mRNA levels on day 3. This increased IGF-I gene expression was associated with an increase in muscle IGF-I content, already detected on day 1 and persisting until day 5 without increase in serum IGF-I concentrations. These data show that muscle hypertrophy induced by clenbuterol is associated with a local increase in muscle IGF-I content. They suggest that clenbuterol-induced muscle hypertrophy could be mediated by local production of IGF-I.

The cardiac sarco(endo)plasmic reticulum Ca 2+ -ATPase gene ( ATP2A2 ) encodes the following two different protein isoforms: SERCA2a (muscle-specific) and SERCA2b (ubiquitous). We have investigated whether this isoform specificity is required for normal cardiac function. Gene targeting in mice successfully disrupted the splicing mechanism responsible for generating the SERCA2a isoform. Homozygous SERCA2a −/− mice displayed a complete loss of SERCA2a mRNA and protein resulting in a switch to the SERCA2b isoform. The expression of SERCA2b mRNA and protein in hearts of SERCA2a −/− mice corresponded to only 50% of wild-type SERCA2 levels. Cardiac phospholamban mRNA levels were unaltered in SERCA2a −/− mice, but total phospholamban protein levels increased 2-fold. The transgenic phenotype was characterized by a ≈20% increase in embryonic and neonatal mortality (early phenotype), with histopathologic evidence of major cardiac malformations. Adult SERCA2a −/− animals (adult phenotype) showed a reduced spontaneous nocturnal activity and developed a mild compensatory concentric cardiac hypertrophy with impaired cardiac contractility and relaxation, but preserved β-adrenergic response. Ca 2+ uptake levels in SERCA2a −/− cardiac homogenates were reduced by ≈50%. In isolated cells, relaxation and Ca 2+ removal by the SR were significantly reduced. Comparison of our data with those obtained in mice expressing similar cardiac levels of SERCA2a instead of SERCA2b indicate the importance of the muscle-specific SERCA2a isoform for normal cardiac development and for the cardiac contraction-relaxation cycle.

Gene expression of IGF-I, IGFBP-4 and IGFBP-5 was studied in hindhimb skeletal muscle of mice, which were either overloaded or unloaded for 8 days. Overloading induced a 15% hypertrophy in soleus muscle associated with a 60% increase of IGF-I transcript levels and a doubling of IGFBP-4 mRNA levels. IGFBP-5 mRNA levels were decreased to one third of the control value. Changes in IGFBPs mRNA always preceded changes in IGF-I gene expression. Unloading by hindlimb suspension resulted in atrophy of soleus muscle (20%) and phenotype change towards the fast type associated with a transient decrease of IGF-I mRNA (30%) and a sustained increase (×2) of IGFBP-5 transcript. These alterations in IGFBPs expression, in unloaded or overloaded soleus, suggest that they may play a role in skeletal muscle adaptation to changes in loading.

Summary Expression levels of fast-twitch (SERCA1), slow-twitch (SERCA2a) and “housekeeping” (SERCA2 b) isoforms of the sarcoplasmic reticulum Ca 2+ -transport ATPase were monitored during regeneration of rat soleus muscles following necrosis induced by the toxin notexin at the tissue level by Western blot analysis and at the cellular level by immunocytochemical analysis. Due to necrosis, levels of muscle-specific SERCA1 and SERCA2 a isoforms dropped to low levels on the third day after injection of the toxin. Subsequently, during regeneration both isoforms recovered but with a different time course. Expression of the fast type SERCA1 increased first. This type showed its most pronounced increase between day 3 and 10. Expression of the slow type SERCA2 a was biphasic. After an increase to approximately one third of the control value on days 5-10, it showed its main increase up to the control level between day 10 and 21. Expression levels of the housekeeping SERCA2 b isoform remained relatively constant throughout the 4 weeks of regeneration. Between day 10 and 28, when new innervation is established, SERCA2 a expression spread gradually over almost all fibers whereas the number of SERCA1-expressing fibers decreased and only a limited number of fibers co-expressed SERCA1 and SERCA2 a. At 4 weeks of regeneration, expression of the fast isoform was found only in 12% of the fibers, whereas the slow form was found in 98% of the fibers. In the contralateral untreated soleus muscles, 26% SERCA1-positive and 81% SERCA2 a-positive fibers were observed. Immunocytochemical analysis showed that SERCA1 and SERCA2 a were co-expressed with fast and slow myosin isoforms in fibers of normal muscles but in regenerated muscle only slow myosin and slow SERCA isoforms correlated. The results show that during regeneration levels of fast and slow SERCA proteins change in a similar way as their mRNAs do. However, in regenerated soleus, unlike in normal muscle, expression of slow SERCA is coregulated only with the slow myosin isoform. This finding is in agreement with the fact that the number of slow type fibers is increased in regenerated soleus.

Berbey et al. (1) recently reported that overexpressed TRPC1-YFP fusion protein is targeted to the sarcoplasmic reticulum of adult skeletal muscle fibers and operates as a calcium leak channel. This observation is in accordance with previous reports showing that the movement of TRPC1 to the plasma membrane is dependent on co-expression with TRPC4 or TRPC5 ((2); for review see Refs. 3 and 4) and that, when expressed alone, TRPC1 forms functional endoplasmic reticulum (ER) homotetrameric channels (5). In this paper, the authors also show by immunocytochemistry with a Chemicon antibody that endogenous TRPC1 is localized exclusively in the sarcoplasmic reticulum. This observation contradicts previous data reporting its electrophysiological detection in the plasma membrane (6) and its co-localization and co-immunoprecipitation with dystrophin, α1-syntrophin, and caveolin (7,–9). To clarify this apparent discrepancy, we performed immunocytochemistry with the same anti-TRPC1 antibody as Berbey et al. (1) on FDB muscle fibers isolated from wild-type and TRPC1-deficient mice (10). As shown in Fig. 1, immunolabeling with this antibody gave similar results in WT and TRPC1-deficient mice (upper and lower panel, respectively). We observed a striated pattern in both groups (large staining in A bands and faint staining in M bands, as shown on the profile curves along the red line). Similar results were obtained on longitudinal sections of paraffin-embedded EDL muscles (not shown). This staining seems very similar to that presented by Berbey et al. (1). Our data strongly suggest that anti-TRPC1 Chemicon antibody is not specific of TRPC1 when used in these conditions. Fig. 1. In conclusion, we believe that the results presented by Berbey and colleagues (1) do not exclude the possible localization of endogenous TRPC1 in the plasma membrane and that the possible localization of endogenous TRPC1 in the sarcoplasmic reticulum (SR) needs further investigation.

1. Parvalbumin content, heat rate and rate of relaxation were measured in two mouse muscles: the slow-twitch soleus and the fast-twitch extensor digitorum longus (EDL). 2. No trace of parvalbumin was found in the soleus; EDL contained a mean of 4.86 mg of this protein per gram of fresh muscle (S.D. = 1.25). 3. Heat rate during 7 s isometric tetani in isolated soleus muscle at 20 degrees C can be described by the sum of an exponentially decaying term and a constant term. The exponential term is reduced by 67% in a second tetanus performed 1 s after a first one; its repriming is complete after a resting period of about 1 min. The exponential term has therefore the properties of labile heat. 4. Relaxation rate measured during 15 s of isometric interrupted tetani at 20 degrees C is nearly constant in the soleus, but decreases continuously with increasing tetanus duration in the EDL. In the latter, isometric tension also decreases continuously. 5. Therefore, parvalbumin can account neither for the labile heat production in mouse soleus nor for the slowing of relaxation associated with muscle fatigue observed after a few seconds of tetanus in EDL. The role of parvalbumin in striated muscles is thus reassessed, and other possible causes of labile heat production and slowing of relaxation are discussed.

The 27-amino-acid gastrin-releasing peptide (GRP1-27) is a neuropeptide and growth factor that is synthesized by various neural and neuroendocrine cells. The major pro-GRP hormone (isoform I) contains both GRP1-27 and a novel C-terminal extension peptide termed pro-GRP31-125. In order to define potentially active neuropeptides that could be generated from this novel protein domain, we analyzed the posttranslational processing of endogenous human pro-GRP1-125 in a small-cell lung cancer cell line. Because such studies are much easier in an overexpression system, we investigated at the same time the posttranslational processing of baculovirus-expressed human pro-GRP1-125 in an insect ovary cell line. In the small-cell lung cancer cell line, GRP1-27 was cleaved as expected from the endogenous prohormone at a pair of basic amino acids (29 and 30) and alpha-amidated at its C-terminal methionine; however, a number of novel peptides were generated by additional cleavages in the pro-GRP31-125 domain. In the insect ovary cell line, GRP1-27 was cleaved from the expressed prohormone by a different mechanism, as were a number of other peptides that appeared to be similar in size to those produced by the human neuroendocrine tumor cell line. These data show for the first time that an insect ovary cell line that is widely used to overexpress proteins can process a human neuropeptide precursor. They also reveal the existence of novel pro-GRP-derived peptides that are candidates for biologically active ligands.