Urocortins Improve Dystrophic Skeletal Muscle Structure and Function through Both PKA- and Epac-Dependent Pathways
2011; Elsevier BV; Volume: 180; Issue: 2 Linguagem: Inglês
10.1016/j.ajpath.2011.10.038
ISSN1525-2191
AutoresJulie Reutenauer-Patte, François‐Xavier Boittin, O. Patthey-Vuadens, Urs T. Rüegg, O.M. Dorchies,
Tópico(s)Muscle Physiology and Disorders
ResumoIn Duchenne muscular dystrophy, the absence of dystrophin causes progressive muscle wasting and premature death. Excessive calcium influx is thought to initiate the pathogenic cascade, resulting in muscle cell death. Urocortins (Ucns) have protected muscle in several experimental paradigms. Herein, we demonstrate that daily s.c. injections of either Ucn 1 or Ucn 2 to 3-week-old dystrophic mdx5Cv mice for 2 weeks increased skeletal muscle mass and normalized plasma creatine kinase activity. Histological examination showed that Ucns remarkably reduced necrosis in the diaphragm and slow- and fast-twitch muscles. Ucns improved muscle resistance to mechanical stress provoked by repetitive tetanizations. Ucn 2 treatment resulted in faster kinetics of contraction and relaxation and a rightward shift of the force-frequency curve, suggesting improved calcium homeostasis. Ucn 2 decreased calcium influx into freshly isolated dystrophic muscles. Pharmacological manipulation demonstrated that the mechanism involved the corticotropin-releasing factor type 2 receptor, cAMP elevation, and activation of both protein kinase A and the cAMP-binding protein Epac. Moreover, both STIM1, the calcium sensor that initiates the assembly of store-operated channels, and the calcium-independent phospholipase A2 that activates these channels were reduced in dystrophic muscle by Ucn 2. Altogether, our results demonstrate the high potency of Ucns for improving dystrophic muscle structure and function, suggesting that these peptides may be considered for treatment of Duchenne muscular dystrophy. In Duchenne muscular dystrophy, the absence of dystrophin causes progressive muscle wasting and premature death. Excessive calcium influx is thought to initiate the pathogenic cascade, resulting in muscle cell death. Urocortins (Ucns) have protected muscle in several experimental paradigms. Herein, we demonstrate that daily s.c. injections of either Ucn 1 or Ucn 2 to 3-week-old dystrophic mdx5Cv mice for 2 weeks increased skeletal muscle mass and normalized plasma creatine kinase activity. Histological examination showed that Ucns remarkably reduced necrosis in the diaphragm and slow- and fast-twitch muscles. Ucns improved muscle resistance to mechanical stress provoked by repetitive tetanizations. Ucn 2 treatment resulted in faster kinetics of contraction and relaxation and a rightward shift of the force-frequency curve, suggesting improved calcium homeostasis. Ucn 2 decreased calcium influx into freshly isolated dystrophic muscles. Pharmacological manipulation demonstrated that the mechanism involved the corticotropin-releasing factor type 2 receptor, cAMP elevation, and activation of both protein kinase A and the cAMP-binding protein Epac. Moreover, both STIM1, the calcium sensor that initiates the assembly of store-operated channels, and the calcium-independent phospholipase A2 that activates these channels were reduced in dystrophic muscle by Ucn 2. Altogether, our results demonstrate the high potency of Ucns for improving dystrophic muscle structure and function, suggesting that these peptides may be considered for treatment of Duchenne muscular dystrophy. Duchenne muscular dystrophy (DMD) is a severe X-linked disorder that affects approximately 1 in 3500 male births. The disease is characterized by progressive muscle wasting starting in early childhood. Later on, respiratory complications and cardiac dysfunctions lead to death by the age of 20 to 30 years. This disorder is caused by the absence of the structural protein dystrophin, a large protein of 427 kDa bridging the extracellular matrix to the intracellular F-actin network via a complex of transmembrane glycoproteins. Dystrophin and associated proteins are thought to play multiple roles, such as preventing mechanical damages to the sarcolemma during muscle contraction, allowing for membrane-targeted superoxide scavenging via subsarcolemmal nitric oxide production, or regulating the function of sarcolemmal components, such as cation channels.1Sabourin J. Cognard C. Constantin B. Regulation by scaffolding proteins of canonical transient receptor potential channels in striated muscle.J Muscle Res Cell Motil. 2009; 30: 289-297Crossref PubMed Scopus (23) Google Scholar Although the mechanisms leading to increased muscle cell death still remain unclear, it is generally accepted that an excessive Ca2+ influx occurring both at rest and on muscle activity is an early event in the dystrophic pathogenesis and might eventually lead to cell death.2Hopf F.W. Turner P.R. Steinhardt R.A. Calcium misregulation and the pathogenesis of muscular dystrophy.Subcell Biochem. 2007; 45: 429-464Crossref PubMed Scopus (58) Google Scholar Several hypotheses have been produced to explain the enhanced Ca2+ entry taking place in dystrophic fibers at rest and, in particular, during activity. Transient tears in the sarcolemma occurring during eccentric exercise lead to massive Ca2+ influx, allowing local proteolytic activation of cationic channels, that triggers further Ca2+ entry.3Allen D.G. Whitehead N.P. Duchenne muscular dystrophy: what causes the increased membrane permeability in skeletal muscle?.Int J Biochem Cell Biol. 2011; 43: 290-294Crossref PubMed Scopus (89) Google Scholar On the other hand, Ca2+ influxes driven by the overexpression of Ca2+ channels at the plasmalemma of otherwise normal muscles have caused a dystrophic phenotype.4Millay D.P. Goonasekera S.A. Sargent M.A. Maillet M. Aronow B.J. Molkentin J.D. Calcium influx is sufficient to induce muscular dystrophy through a TRPC-dependent mechanism.Proc Natl Acad Sci U S A. 2009; 106: 19023-19028Crossref PubMed Scopus (158) Google Scholar The lack of dystrophin may be directly responsible for enhanced Ca2+ entry in dystrophic muscle cells because of abnormal opening of store-operated channels (SOCs).1Sabourin J. Cognard C. Constantin B. Regulation by scaffolding proteins of canonical transient receptor potential channels in striated muscle.J Muscle Res Cell Motil. 2009; 30: 289-297Crossref PubMed Scopus (23) Google Scholar, 2Hopf F.W. Turner P.R. Steinhardt R.A. Calcium misregulation and the pathogenesis of muscular dystrophy.Subcell Biochem. 2007; 45: 429-464Crossref PubMed Scopus (58) Google Scholar, 5Launikonis B.S. Murphy R.M. Edwards J.N. Toward the roles of store-operated Ca2+ entry in skeletal muscle.Pflügers Arch Eur J Physiol. 2010; 460: 813-823Crossref PubMed Scopus (60) Google Scholar Recently, the proteins STIM1 and Orai1 have been identified as the Ca2+ sensor on the sarcoplasmic reticulum and the pore-forming component of SOC on the plasma membrane, respectively.5Launikonis B.S. Murphy R.M. Edwards J.N. Toward the roles of store-operated Ca2+ entry in skeletal muscle.Pflügers Arch Eur J Physiol. 2010; 460: 813-823Crossref PubMed Scopus (60) Google Scholar, 6Bolotina V.M. Orai, STIM1 and iPLA2β: a view from a different perspective.J Physiol. 2008; 586: 3035-3042Crossref PubMed Scopus (68) Google Scholar Moreover, the calcium-independent phospholipase A2 type β (iPLA2-β), an enzyme producing arachidonic acid and lysophospholipids from phospholipids at the plasma membrane, could modulate SOC activity.6Bolotina V.M. Orai, STIM1 and iPLA2β: a view from a different perspective.J Physiol. 2008; 586: 3035-3042Crossref PubMed Scopus (68) Google Scholar More important, (i) iPLA2-β expression is increased in muscles from dystrophin-deficient mice,7Boittin F.-X. Petermann O. Hirn C. Mittaud P. Dorchies O.M. Roulet E. Ruegg U.T. Ca2+-independent phospholipase A2 enhances store-operated Ca2+ entry in dystrophic skeletal muscle fibers.J Cell Sci. 2006; 119: 3733-3742Crossref PubMed Scopus (101) Google Scholar and PLA2 activity is elevated severalfold in muscles from patients with DMD8Lindahl M. Backman E. Henriksson K.G. Gorospe J.R. Hoffman E.P. Phospholipase A2 activity in dystrophinopathies.Neuromuscul Disord. 1995; 5: 193-199Abstract Full Text PDF PubMed Scopus (38) Google Scholar; (ii) store-operated Ca2+ entry (SOCE) in dystrophic fibers is corrected to normal values by selective inhibitors of iPLA27Boittin F.-X. Petermann O. Hirn C. Mittaud P. Dorchies O.M. Roulet E. Ruegg U.T. Ca2+-independent phospholipase A2 enhances store-operated Ca2+ entry in dystrophic skeletal muscle fibers.J Cell Sci. 2006; 119: 3733-3742Crossref PubMed Scopus (101) Google Scholar; and (iii) hydrolysis products of iPLA2 stimulate SOCE.9Smani T. Dominguez-Rodriguez A. Hmadcha A. Calderon-Sanchez E. Horrillo-Ledesma A. Ordonez A. Role of Ca2+-independent phospholipase A2 and store-operated pathway in urocortin-induced vasodilatation of rat coronary artery.Circ Res. 2007; 101: 1194-1203Crossref PubMed Scopus (49) Google Scholar, 10Boittin F.-X. Shapovalov G. Hirn C. Ruegg U.T. Phospholipase A2-derived lysophosphatidylcholine triggers Ca2+ entry in dystrophic skeletal muscle fibers.Biochem Biophys Res Commun. 2010; 391: 401-406Crossref PubMed Scopus (17) Google Scholar Figure 1 provides an overview of proteins involved in urocortin (Ucn) signaling and calcium influx pathways in skeletal muscle. Therefore, interventions decreasing iPLA2-β expression and/or activity are likely to improve Ca2+ homeostasis and to be beneficial to dystrophic muscle.11Wilkins 3rd, W.P. Barbour S.E. Group VI phospholipases A2: homeostatic phospholipases with significant potential as targets for novel therapeutics.Curr Drug Targets. 2008; 9: 683-697Crossref PubMed Scopus (40) Google Scholar Currently, the only drugs proposed to patients with DMD are the glucocorticoids prednisolone and deflazacort.12Manzur A. Kuntzer T. Pike M. Swan A. Glucocorticoid Corticosteroids for Duchenne Muscular Dystrophy (Review).The Cochrane Collaboration/John Wiley & Sons, Ltd. 2008; : 1-93Google Scholar Their action on deregulated Ca2+ homeostasis13Metzinger L. Passaquin A.C. Leijendekker W.J. Poindron P. Ruegg U.T. Modulation by prednisolone of calcium handling in skeletal muscle cells.Br J Pharmacol. 1995; 116: 2811-2816Crossref PubMed Scopus (42) Google Scholar might partly explain their therapeutic effects. Ucns are neuropeptides related to the hypothalamic corticotropin-releasing factor (CRF). They comprise approximately 40 amino acids, are expressed in the brain and in peripheral tissues, and are involved in anxiety, cardiovascular and renal functions, and inflammatory responses.14Fekete E.M. Zorrilla E.P. Physiology, pharmacology, and therapeutic relevance of urocortins in mammals: ancient CRF paralogs.Front Neuroendocrinol. 2007; 28: 1-27Crossref PubMed Scopus (203) Google Scholar Two mammalian CRF receptor (CRFR) subtypes have been identified15Dautzenberg F.M. Hauger R.L. The CRF peptide family and their receptors: yet more partners discovered.Trends Pharmacol Sci. 2002; 23: 71-77Abstract Full Text Full Text PDF PubMed Scopus (459) Google Scholar: CRF1R and CRF2R. Skeletal muscles express high levels of the CRF2R mRNA and protein.16Kishimoto T. Pearse R.V. Lin C.R. Rosenfeld M.G. A sauvagine/corticotropin-releasing factor receptor expressed in heart and skeletal muscle.Proc Natl Acad Sci U S A. 1995; 92: 1108-1112Crossref PubMed Scopus (374) Google Scholar, 17Samuelsson S. Lange J.S. Hinkle R.T. Tarnopolsky M. Isfort R.J. Corticotropin-releasing factor 2 receptor localization in skeletal muscle.J Histochem Cytochem. 2004; 52: 967-977Crossref PubMed Scopus (11) Google Scholar By contrast, CRF1R mRNA is not much expressed in skeletal muscle and the CRF1R protein has never been reported in this tissue. Although Ucn 1 acts via both receptor subtypes, Ucn 2 and Ucn 3 are highly selective for the CRF2R.14Fekete E.M. Zorrilla E.P. Physiology, pharmacology, and therapeutic relevance of urocortins in mammals: ancient CRF paralogs.Front Neuroendocrinol. 2007; 28: 1-27Crossref PubMed Scopus (203) Google Scholar The CRF1R and CRF2R are positively coupled to Gsα,15Dautzenberg F.M. Hauger R.L. The CRF peptide family and their receptors: yet more partners discovered.Trends Pharmacol Sci. 2002; 23: 71-77Abstract Full Text Full Text PDF PubMed Scopus (459) Google Scholar which, on agonist activation, activates adenylyl cyclase, resulting in cAMP formation (Figure 1). cAMP exerts its effects through two intracellular receptors: protein kinase A (PKA), an enzyme that directly phosphorylates multiple proteins on cAMP binding; and a guanine nucleotide exchange protein activated by cAMP (Epac),18de Rooij J. Zwartkruis F.J.T. Verheijen M.H.G. Cool R.H. Nijman S.M.B. Wittinghofer A. Bos J.L. Epac is a Rap1 guanine-nucleotide-exchange factor directly activated by cyclic AMP.Nature. 1998; 396: 474-477Crossref PubMed Scopus (1607) Google Scholar which activates small G-protein–dependent kinases, causing a variety of effects. PKA has long been considered as the major intracellular effector of cAMP, but Epac is equally recognized as mediating cAMP actions19Roscioni S.S. Elzinga C. Schmidt M. Epac: effectors and biological functions.Naunyn Schmiedebergs Arch Pharmacol. 2008; 377: 345-357Crossref PubMed Scopus (122) Google Scholar, 20Gloerich M. Bos J.L. Epac: defining a new mechanism for cAMP action.Ann Rev Pharmacol Toxicol. 2010; 50: 355-375Crossref PubMed Scopus (381) Google Scholar (Figure 1). The effects of cAMP on muscle function, contractility, and relaxation, and Ca2+ homeostasis have been extensively studied. Specifically, activation of the CRF2R and downstream increases in cAMP levels modulate skeletal muscle mass.21Hinkle R.T. Donnelly E. Cody D.B. Samuelsson S. Lange J.S. Bauer M.B. Tarnopolsky M. Sheldon R.J. Coste S.C. Tobar E. Stenzel-Poore M.P. Isfort R.J. Activation of the CRF 2 receptor modulates skeletal muscle mass under physiological and pathological conditions.Am J Physiol Endocrinol Metab. 2003; 285: E889-E898PubMed Google Scholar, 22Hinkle R.T. Donnelly E. Cody D.B. Bauer M.B. Sheldon R.J. Isfort R.J. Corticotropin releasing factor 2 receptor agonists reduce the denervation-induced loss of rat skeletal muscle mass and force and increase non-atrophying skeletal muscle mass and force.J Muscle Res Cell Motil. 2005; 25: 539-547Crossref Scopus (19) Google Scholar Long-term administration of a synthetic CRF2R agonist to adult mdx mice prevented the degeneration of the diaphragm, attenuated the loss of its force, and reduced fibrosis and inflammation.23Hinkle R.T. Lefever F. Dolan E. Reichart D. Dietrich J. Gropp K. Thacker R. Demuth J. Stevens P. Qu X. Varbanov A. Wang F. Isfort R.J. Corticotrophin releasing factor 2 receptor agonist treatment significantly slows disease progression in mdx mice.BMC Med. 2007; 5: 18Crossref PubMed Scopus (13) Google Scholar The same compound increased muscle mass and decreased levels of creatine kinase (CK), an index of muscle membrane damage, in dystrophic mice.24Hall J.E. Kaczor J.J. Hettinga B.P. Isfort R.J. Tarnopolsky M.A. Effects of a CRF2R agonist and exercise on mdx and wildtype skeletal muscle.Muscle Nerve. 2007; 36: 336-341Crossref PubMed Scopus (18) Google Scholar Interestingly, recent studies9Smani T. Dominguez-Rodriguez A. Hmadcha A. Calderon-Sanchez E. Horrillo-Ledesma A. Ordonez A. Role of Ca2+-independent phospholipase A2 and store-operated pathway in urocortin-induced vasodilatation of rat coronary artery.Circ Res. 2007; 101: 1194-1203Crossref PubMed Scopus (49) Google Scholar have proposed a role for cAMP/PKA in the regulation of iPLA2 and SOCE. The extensive myofiber necrosis that affects mdx mice at approximately 3 to 5 weeks of age allows studying therapeutic interventions relevant to DMD muscle.25Grounds M.D. Radley H.G. Lynch G.S. Nagaraju K. De Luca A. Towards developing standard operating procedures for pre-clinical testing in the mdx mouse model of Duchenne muscular dystrophy.Neurobiol Dis. 2008; 31: 1-19Crossref PubMed Scopus (241) Google Scholar Herein, we describe that daily administration of Ucns attenuates this massive necrotic episode, resulting in improved muscle function and sarcolemmal integrity. Moreover, our results strongly suggest that the beneficial effects of Ucn 2 on dystrophic muscle result from PKA- and Epac-dependent inhibition of Ca2+ entry, and by down-regulation of iPLA2, an enzyme responsible for enhanced store-dependent Ca2+ entry in dystrophic fibers.7Boittin F.-X. Petermann O. Hirn C. Mittaud P. Dorchies O.M. Roulet E. Ruegg U.T. Ca2+-independent phospholipase A2 enhances store-operated Ca2+ entry in dystrophic skeletal muscle fibers.J Cell Sci. 2006; 119: 3733-3742Crossref PubMed Scopus (101) Google Scholar From a therapeutic point of view, our results suggest that Ucns could be useful for symptomatic therapy of patients with DMD. During the course of this study, we contributed to an international effort for elaborating standard operating procedures for pre-clinical investigations in the dystrophic mouse.25Grounds M.D. Radley H.G. Lynch G.S. Nagaraju K. De Luca A. Towards developing standard operating procedures for pre-clinical testing in the mdx mouse model of Duchenne muscular dystrophy.Neurobiol Dis. 2008; 31: 1-19Crossref PubMed Scopus (241) Google Scholar, 26Nagaraju K. Willmann R. TREAT-NMD Network and the Wellstone Muscular Dystrophy Cooperative Research Network: Developing standard procedures for murine and canine efficacy studies of DMD therapeutics: report of two expert workshops on “Pre-clinical testing for Duchenne dystrophy”: Washington DC. October 27th-28th 2007and Zürich, June 30th-July 1st 2008.Neuromuscul Disord. 2009; 19: 502-506Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar More information about the standard operating procedures is available at the TREAT-NMD web site (, last accessed October 15, 2011). We complied with the standard operating procedures about force measurements and histological examination, which were finalized when our study was performed. The following information regarding the materials and methods is described in sufficient detail to be read without reference to the online resource. Mouse Ucn 2 was synthesized by Genecust (Evry, France). The PKA inhibitor H-89 was obtained from Merck (VWR International, Dietikon, Switzerland). Brefeldin A and chelerythrine were obtained from Biomol (Enzolife Science, Lausen, Switzerland). PD98059 was from Tocris (LucernaChem, Lucerne, Switzerland). Other chemicals were from Sigma-Aldrich (Buchs, Switzerland), unless stated otherwise. All of the procedures involving animals were conducted in accordance with the Swiss Federal Veterinary Office's guidelines, based on Swiss Federal Law on Animal Welfare. Breeding pairs of dystrophic mdx5Cv mice27Im W.B. Phelps S.F. Copen E.H. Adams E.G. Slightom J.L. Chamberlain J.S. Differential expression of dystrophin isoforms in strains of mdx mice with different mutations.Hum Mol Genet. 1996; 5: 1149-1153Crossref PubMed Scopus (170) Google Scholar, 28Reutenauer J. Dorchies O.M. Patthey-Vuadens O. Vuagniaux G. Ruegg U.T. Investigation of Debio 025, a cyclophilin inhibitor, in the dystrophic mdx mouse, a model for Duchenne muscular dystrophy.Br J Pharmacol. 2008; 155: 574-584Crossref PubMed Scopus (86) Google Scholar were originally obtained from Transgene (Strasbourg, France), with the agreement of The Jackson Laboratory (Bar Harbor, ME), and their genetically normal counterparts, C57BL/6J mice, were obtained from Charles River (Iffa Credo, Saint Germain sur l'Arbresle, France). Animals were housed in plastic cages containing wood granule bedding, maintained with 12-hour dark/12-hour light cycles and unlimited access to food and water throughout the study. Approximately equal numbers of males and females were used in each group. Litters of 3-week-old animals were treated daily for 2 weeks and analyzed at the age of 5 weeks. Lyophilized substances were prepared as 10−4 mol/L aqueous stock solutions, and aliquots were frozen at −20°C. The working solutions were freshly prepared from thawed stock aliquots diluted with 0.9% NaCl. A volume of ≤1% of the body weight was injected s.c. in the interscapular region. Dystrophic animals received either rat Ucn 1 at 300 μg/kg or mouse Ucn 2 at 30, 100, or 300 μg/kg. Groups of untreated C57BL/6J and mdx5Cv received only saline solution. Injections were performed at 12:00 p.m. ± 2 hours. Data were collected from 8 to 12 animals per group. Mice were weighed daily. Animals were kept with the dam until weaning (postnatal day 28) and, thereafter, were isolated in separate cages until sacrifice (postnatal day 35). Food consumption during the second week of treatment was determined as the amount (in grams) of pellets consumed per gram of body weight per day. At the end of the treatment period, animals were anesthetized by i.p. injection of a mixture of urethane (1.5 g/kg) and diazepam (5 mg/kg). The Achilles' tendon of the right hind limb was exposed and linked to a force transducer coupled to a LabView interface (National Instruments, Austin, TX) for trace acquisition and analysis. The knee joint was firmly immobilized. Two thin steel electrodes were inserted into the triceps surae muscle (comprising the fast-twitch glycolytic gastrocnemius and plantaris muscles and the slow-twitch oxidative soleus muscle). Muscles were electrically stimulated with 0.5-millisecond pulses of controlled intensity and frequency. The stimulation-recording protocol was performed as follows. After manual settings of optimal muscle length and optimal current intensity, a phasic twitch was recorded at a sampling rate of 3 kHz to determine the absolute peak twitch force (Pt), the time to peak (TTP), the time for half relaxation from the peak (RT1/2), the maximum rate of tension development (Tdev), and the maximum rate of tension loss (Tloss). After a 3-minute pause, muscles were subjected to a force-frequency test, where force was recorded using 200-millisecond bursts of increasing frequencies (from 10 to 100 Hz by increments of 10 Hz), with one burst every 30 seconds. The strongest response was taken as the absolute optimal tetanic force (Po). Finally, after another 3-minute pause, muscles were submitted to a fatigue test for 5 minutes: the frequency was set at 60 Hz, and the decrease in muscle force was recorded while 60 stimulations were delivered, each consisting of a 2-second burst and a 3-second rest. The responses were expressed as the percentage of the maximal tension. Absolute phasic and tetanic forces were converted into specific forces (mN/mm2 of muscle section) after normalization for the muscle cross-sectional area. The cross-sectional area values (in mm2) were determined by dividing the triceps surae muscle mass (in mg) by the product of optimal muscle length (Lo, in mm) and d, the density of mammalian skeletal muscle (d = 1.06 mg/mm3). All tension and time parameters, except Tdev and Tloss, were determined directly from the traces using the LabView program. For the determination of Tdev and Tloss, the coordinates of the twitch traces were imported into a Microsoft Office Excel file (Redmond, WA). The linear region of the contraction phase (contained within the first 5 milliseconds of tension development) and of the relaxation phase (contained in a time window of approximately 30 milliseconds after the twitch peak value) was used for linear regression analysis. The extreme coordinates of the selected range were repeatedly included or excluded until the highest slope value was found and recorded as either the maximum rate of tension development (Tdev, determined from the contraction phase of the twitch) or the maximum rate of tension loss (Tloss, determined from the relaxation phase of the twitch). After the measurement of the muscle contractile properties (24 ± 4 hours after the last administration of test substance), the mice received an intracardiac injection of heparin (approximately 30 U of heparin/mL of blood), the aorta was cut, and the blood was collected in the thoracic cavity in heparinized tubes and centrifuged at 3000 × g for 10 minutes at 4°C. Fresh plasma fractions were saved at 4°C for CK determination. The remaining plasma was frozen at −20°C until measurement of plasma Ucn 2 concentrations using a YK190 mouse Ucn 2 enzyme-linked immunosorbent assay kit (Cosmo Bio, Tokyo, Japan), according to the manufacturer's instructions. Plasma CK activity was determined by spectrophotometry using the Catachem diagnostic kit (Investcare Vet, Middlesex, UK), according to the manufacturer's instructions, within 48 hours of plasma preparation. Immediately after blood collection, extensor digitorum longus (EDL), gastrocnemius, plantaris, soleus, and tibialis anterior muscles were dissected bilaterally and weighed. Heart, liver, kidneys, and spleen were also collected and weighed. The EDL and soleus from the left leg and the left hemidiaphragm were embedded in Tissue Freezing Medium (Polysciences, Chemie Brunschwig, Basel, Switzerland), frozen in liquid nitrogen–cooled isopentane, and stored at −80°C for histological analysis. Transverse sections (10 μm thick) were prepared with an HM 560M cryostat (Microm, Volketswil, Switzerland) and collected on SuperFrost Plus slides (Assistent, Sondheim, Germany). Whole muscle cross sections, taken near the midpoint of the muscle, were stained with H&E, according to classic procedures. Pictures were taken with a Spot Insight QE digital camera (Visitron Systems, Puchheim, Germany), coupled to an inverted microscope (Axiovert 200 M; Zeiss, Feldbach, Switzerland). One cross section was analyzed per animal. The samples were coded and analyzed by an observer (O.P.V.) who was blinded to the details of the study. The necrotic myofibers, infiltrated immune cells, actively proliferating myoblasts, or small caliber newly formed myotubes cannot be distinguished from the others with H&E. They form areas of darkly stained, densely packed cells. These areas were demarcated and filled black using Adobe Photoshop software (Adobe, San Jose, CA). Their surfaces were then measured using Metamorph software (Visitron Systems, Puchheim, Germany) and expressed as percentage of total muscle surface. As a whole, these structures are indicative of the muscle necrosis and subsequent early steps of regeneration that were active when the histological analysis was performed. Thereafter, these structures are referred to as the active necrosis/regeneration index or surface. Normal myofibers presented as cells, usually of large caliber, with nuclei located at the membrane. The regenerated myofibers were identified by the presence of centrally located nuclei. Normal and regenerated (centronucleated) myofibers were counted manually, and the regenerated myofibers were expressed as percentage of total fiber count. The amount of centronucleated fibers is an index of prior necrosis undergone by the muscle several days before the histological analysis is performed. Frozen gastrocnemius muscles were ground to a fine powder in liquid nitrogen–cooled mortars. Samples (approximately 30 mg) were rapidly weighed into liquid nitrogen–cooled microtubes. Muscle proteins were extracted by the addition of 9 volumes (v/w) of Guba-Straub buffer [300 mmol/L NaCl, 100 mmol/L NaH2PO4, 50 mmol/L Na2HPO4, 10 mmol/L Na4P2O7, 1 mmol/L MgCl2, and 10 mmol/L EDTA (pH 6.5)] containing 0.1% 2-mercaptoethanol and 0.2% protease inhibitor cocktail. After incubation with gentle shaking for 45 minutes at 4°C, the samples were sonicated for 10 seconds, Triton X-100 (Applichem, Axonlab, Le Mont-sur-Lausanne, Switzerland) was added to a final concentration of 1%, and extracts were centrifuged at 12,000 × g for 15 minutes at 4°C. The supernatants were collected, and protein content was determined using the Bradford method (Bio-Rad, Reinach, Switzerland). Extracts were diluted with reducing Laemmli buffer at a final protein concentration of 3 mg/mL before being used for immunoblotting. To determine the quality of the samples, 50 μg of protein was resolved on gradient gels (5% to 12% of acrylamide) at 100 V before being stained with 0.1% Coomassie Blue and dried. Muscle extracts (30 to 60 μg/lane) were separated on 6% to 7% SDS-PAGE gels, and proteins were transferred onto 0.45-μm nitrocellulose membranes, according to standard procedures. Transfer efficiency was verified by staining with red Ponceau S. Membranes were blocked for 1 hour in Tris-buffered saline and Tween 20 [20 mmol/L Tris-base, 150 mmol/L NaCl, and 0.1% Tween-20 (pH 7.5)] containing 5% nonfat dry milk and incubated overnight at 4°C with one of the following antibodies (diluted 1:1000 in Tris-buffered saline and Tween 20 containing 5% bovine serum albumin): goat anti-CRF1R (LifeSpan Biosciences, LabForce, Nunningen, Switzerland), rabbit anti-CRF2R (Abcam, Cambridge, UK), rabbit anti-STIM1 (Cell Signaling Technology, Millipore, Zug, Switzerland), and rabbit anti-iPLA2-β (Cayman, Alexis, Lausen, Switzerland). After extensive washing, membranes were incubated for 1 hour with a horseradish peroxidase–conjugated donkey anti-rabbit IgG (Amersham Biosciences, Otelfingen, Switzerland) or mouse anti-goat IgG (Jackson ImmunoResearch, Newmarket, UK), diluted 1:30,000 in Tris-buffered saline and Tween 20, containing 5% milk. Signals were obtained by chemiluminescence (ECL plus kit; Amersham Biosciences) and exposed to Fuji X-ray film (Fujifilm Europe, Dusseldorf, Germany). The films were scanned, and densitometry analysis was performed using Image J (NIH, Bethesda, MD). Signals were normalized to the myosin heavy chain content (as determined on Coomassie Blue–stained gels) and corrected for the value of a reference sample loaded twice on every gel for the purpose of intergel comparison. The results were expressed in arbitrary units. Frozen gastrocnemius muscles were ground to a fine powder in liquid nitrogen–cooled mortars. Samples (approximately 30 mg) were rapidly weighed into liquid nitrogen–cooled microtubes. Muscle proteins were extracted by the addition of 3 volumes (v/w) of cold lysis buffer [10 mmol/L Tris-base and 300 mmol/L sucrose (pH 7.0)] containing 0.2% protease inhib
Referência(s)