Differential Muscle Hypertrophy Is Associated with Satellite Cell Numbers and Akt Pathway Activation Following Activin Type IIB Receptor Inhibition in Mtm1 p.R69C Mice
2014; Elsevier BV; Volume: 184; Issue: 6 Linguagem: Inglês
10.1016/j.ajpath.2014.03.003
ISSN1525-2191
AutoresMichael W. Lawlor, Marissa G. Viola, Hui Meng, Rachel V. Edelstein, Fujun Liu, Ke Yan, Elizabeth J. Luna, Alexandra Lerch-Gaggl, Raymond G. Hoffmann, Christopher R. Pierson, Anna Buj‐Bello, Jennifer Lachey, Scott Pearsall, Lin Yang, Cecilia J. Hillard, Alan H. Beggs,
Tópico(s)Genetics and Physical Performance
ResumoX-linked myotubular myopathy is a congenital myopathy caused by deficiency of myotubularin. Patients often present with severe perinatal weakness, requiring mechanical ventilation to prevent death from respiratory failure. We recently reported that an activin receptor type IIB inhibitor produced hypertrophy of type 2b myofibers and modest increases of strength and life span in the severely myopathic Mtm1δ4 mouse model of X-linked myotubular myopathy. We have now performed a similar study in the less severely symptomatic Mtm1 p.R69C mouse in hopes of finding greater treatment efficacy. Activin receptor type IIB inhibitor treatment of Mtm1 p.R69C animals produced behavioral and histological evidence of hypertrophy in gastrocnemius muscles but not in quadriceps or triceps. The ability of the muscles to respond to activin receptor type IIB inhibitor treatment correlated with treatment-induced increases in satellite cell number and several muscle-specific abnormalities of hypertrophic signaling. Treatment-responsive Mtm1 p.R69C gastrocnemius muscles displayed lower levels of phosphorylated ribosomal protein S6 and higher levels of phosphorylated eukaryotic elongation factor 2 kinase than were observed in Mtm1 p.R69C quadriceps muscle or in muscles from wild-type littermates. Hypertrophy in the Mtm1 p.R69C gastrocnemius muscle was associated with increased levels of phosphorylated ribosomal protein S6. Our findings indicate that muscle-, fiber type-, and mutation-specific factors affect the response to hypertrophic therapies that will be important to assess in future therapeutic trials. X-linked myotubular myopathy is a congenital myopathy caused by deficiency of myotubularin. Patients often present with severe perinatal weakness, requiring mechanical ventilation to prevent death from respiratory failure. We recently reported that an activin receptor type IIB inhibitor produced hypertrophy of type 2b myofibers and modest increases of strength and life span in the severely myopathic Mtm1δ4 mouse model of X-linked myotubular myopathy. We have now performed a similar study in the less severely symptomatic Mtm1 p.R69C mouse in hopes of finding greater treatment efficacy. Activin receptor type IIB inhibitor treatment of Mtm1 p.R69C animals produced behavioral and histological evidence of hypertrophy in gastrocnemius muscles but not in quadriceps or triceps. The ability of the muscles to respond to activin receptor type IIB inhibitor treatment correlated with treatment-induced increases in satellite cell number and several muscle-specific abnormalities of hypertrophic signaling. Treatment-responsive Mtm1 p.R69C gastrocnemius muscles displayed lower levels of phosphorylated ribosomal protein S6 and higher levels of phosphorylated eukaryotic elongation factor 2 kinase than were observed in Mtm1 p.R69C quadriceps muscle or in muscles from wild-type littermates. Hypertrophy in the Mtm1 p.R69C gastrocnemius muscle was associated with increased levels of phosphorylated ribosomal protein S6. Our findings indicate that muscle-, fiber type-, and mutation-specific factors affect the response to hypertrophic therapies that will be important to assess in future therapeutic trials. X-linked myotubular myopathy (XLMTM) is a severe form of congenital myopathy with an estimated incidence of 1:50,000 male births that most often presents with severe perinatal weakness and respiratory failure.1Heckmatt J.Z. Sewry C.A. Hodes D. Dubowitz V. Congenital centronuclear (myotubular) myopathy. A clinical, pathological and genetic study in eight children.Brain. 1985; 108: 941-964Crossref PubMed Scopus (91) Google Scholar, 2Jungbluth H. Wallgren-Pettersson C. Laporte J. Centronuclear (myotubular) myopathy.Orphanet J Rare Dis. 2008; 3: 26Crossref PubMed Scopus (237) Google Scholar Many patients with XLMTM die within the first year of life despite the use of mechanical ventilation, and no treatments approved by the Food and Drug Administration are available. XLMTM is caused by mutations in the gene that encodes myotubularin (MTM1), which is a phosphoinositide phosphatase thought to be involved in endosomal trafficking, cytoskeletal organization, apoptosis, and/or maintenance of the sarcoplasmic reticulum/T-tubular system within myofibers.3Buj-Bello A. Fougerousse F. Schwab Y. Messaddeq N. Spehner D. Pierson C.R. Durand M. Kretz C. Danos O. Douar A.M. Beggs A.H. Schultz P. Montus M. Denefle P. Mandel J.L. AAV-mediated intramuscular delivery of myotubularin corrects the myotubular myopathy phenotype in targeted murine muscle and suggests a function in plasma membrane homeostasis.Hum Mol Genet. 2008; 17: 2132-2143Crossref PubMed Scopus (101) Google Scholar, 4Cao C. Backer J.M. Laporte J. Bedrick E.J. Wandinger-Ness A. Sequential actions of myotubularin lipid phosphatases regulate endosomal PI(3)P and growth factor receptor trafficking.Mol Biol Cell. 2008; 19: 3334-3346Crossref PubMed Scopus (108) Google Scholar, 5Dowling J.J. Vreede A.P. Low S.E. Gibbs E.M. Kuwada J.Y. Bonnemann C.G. Feldman E.L. Loss of myotubularin function results in T-tubule disorganization in zebrafish and human myotubular myopathy.PLoS Genet. 2009; 5: e1000372Crossref PubMed Scopus (177) Google Scholar, 6Laporte J. Hu L.J. Kretz C. Mandel J.L. Kioschis P. Coy J.F. Klauck S.M. Poustka A. Dahl N. A gene mutated in X-linked myotubular myopathy defines a new putative tyrosine phosphatase family conserved in yeast.Nat Genet. 1996; 13: 175-182Crossref PubMed Scopus (529) Google Scholar, 7Hnia K. Tronchere H. Tomczak K.K. Amoasii L. Schultz P. Beggs A.H. Payrastre B. Mandel J.L. Laporte J. Myotubularin controls desmin intermediate filament architecture and mitochondrial dynamics in human and mouse skeletal muscle.J Clin Invest. 2011; 121: 70-85Crossref PubMed Scopus (105) Google Scholar, 8Lawlor M.W. Alexander M.S. Viola M.G. Meng H. Joubert R. Gupta V. Motohashi N. Manfready R.A. Hsu C.P. Huang P. Buj-Bello A. Kunkel L.M. Beggs A.H. Gussoni E. Myotubularin-deficient myoblasts display increased apoptosis, delayed proliferation, and poor cell engraftment.Am J Pathol. 2012; 181: 961-968Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar Muscle biopsies from patients with XLMTM display excessively small fibers with increased numbers of fibers that contain central nuclei and central aggregation of organelles.9Pierson C.R. Tomczak K. Agrawal P. Moghadaszadeh B. Beggs A.H. X-linked myotubular and centronuclear myopathies.J Neuropathol Exp Neurol. 2005; 64: 555-564Crossref PubMed Scopus (84) Google Scholar Although the number of centrally nucleated fibers bears little relationship to a patient's prognosis, there is a clear correlation between the degree of fiber smallness at birth and the severity of the patients' disease.10Pierson C.R. Agrawal P.B. Blasko J. Beggs A.H. Myofiber size correlates with MTM1 mutation type and outcome in X-linked myotubular myopathy.Neuromuscul Disord. 2007; 17: 562-568Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar Two murine models of myotubularin deficiency are used, the severely symptomatic Mtm1δ4 (also referred to as Mtm1 knockout in prior studies3Buj-Bello A. Fougerousse F. Schwab Y. Messaddeq N. Spehner D. Pierson C.R. Durand M. Kretz C. Danos O. Douar A.M. Beggs A.H. Schultz P. Montus M. Denefle P. Mandel J.L. AAV-mediated intramuscular delivery of myotubularin corrects the myotubular myopathy phenotype in targeted murine muscle and suggests a function in plasma membrane homeostasis.Hum Mol Genet. 2008; 17: 2132-2143Crossref PubMed Scopus (101) Google Scholar, 11Al-Qusairi L. Weiss N. Toussaint A. Berbey C. Messaddeq N. Kretz C. Sanoudou D. Beggs A.H. Allard B. Mandel J.L. Laporte J. Jacquemond V. Buj-Bello A. T-tubule disorganization and defective excitation-contraction coupling in muscle fibers lacking myotubularin lipid phosphatase.Proc Natl Acad Sci U S A. 2009; 106: 18763-18768Crossref PubMed Scopus (147) Google Scholar, 12Lawlor M.W. Read B.P. Edelstein R. Yang N. Pierson C.R. Stein M.J. Wermer-Colan A. Buj-Bello A. Lachey J.L. Seehra J.S. Beggs A.H. Inhibition of activin receptor type IIb increases strength and lifespan in myotubularin-deficient mice.Am J Pathol. 2011; 178: 784-793Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar) and the moderately symptomatic Mtm1 p.R69C mice,13Pierson C.R. Dulin-Smith A.N. Durban A.N. Marshall M.L. Marshall J.T. Snyder A.D. Naiyer N. Gladman J.T. Chandler D.S. Lawlor M.W. Buj-Bello A. Dowling J.J. Beggs A.H. Modeling the human MTM1 p.R69C mutation in murine Mtm1 results in exon 4 skipping and a less severe myotubular myopathy phenotype.Hum Mol Genet. 2012; 21: 811-825Crossref PubMed Scopus (47) Google Scholar both of which display weakness and myofiber smallness and similar pathology to that seen in XLMTM. Because of the relationship between myofiber size and symptomatic severity in patients with XLMTM and in Mtm1δ4 mice, we had previously hypothesized that correction of myofiber smallness in myotubularin deficiency would greatly improve strength. Inhibitors of myostatin or nonfunctional decoys of its receptor, the activin type IIB receptor (ActRIIB), can be used to inhibit this negative regulator of myofiber size, leading to myofiber hypertrophy. Myostatin binds to (and signals through) the ActRIIB to activate the transforming growth factor-β pathway, which prevents progression through the cell cycle and down-regulates several key processes related to myofiber hypertrophy.14McCroskery S. Thomas M. Maxwell L. Sharma M. Kambadur R. Myostatin negatively regulates satellite cell activation and self-renewal.J Cell Biol. 2003; 162: 1135-1147Crossref PubMed Scopus (600) Google Scholar, 15Joulia-Ekaza D. Cabello G. Myostatin regulation of muscle development: molecular basis, natural mutations, physiopathological aspects.Exp Cell Res. 2006; 312: 2401-2414Crossref PubMed Scopus (4) Google Scholar We recently reported a trial of ActRIIB-mFC in Mtm1δ4 mice, which produced 17% extension of life span, with transient increases in weight, forelimb grip strength, myofiber size, and myofiber hypertrophy restricted to type 2b myofibers in Mtm1δ4 animals.12Lawlor M.W. Read B.P. Edelstein R. Yang N. Pierson C.R. Stein M.J. Wermer-Colan A. Buj-Bello A. Lachey J.L. Seehra J.S. Beggs A.H. Inhibition of activin receptor type IIb increases strength and lifespan in myotubularin-deficient mice.Am J Pathol. 2011; 178: 784-793Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar Interestingly, ActRIIB-mFc produces hypertrophy in all muscle fiber types in wild-type (WT) mice,12Lawlor M.W. Read B.P. Edelstein R. Yang N. Pierson C.R. Stein M.J. Wermer-Colan A. Buj-Bello A. Lachey J.L. Seehra J.S. Beggs A.H. Inhibition of activin receptor type IIb increases strength and lifespan in myotubularin-deficient mice.Am J Pathol. 2011; 178: 784-793Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar, 16Cadena S.M. Tomkinson K.N. Monnell T.E. Spaits M.S. Kumar R. Underwood K.W. Pearsall R.S. Lachey J.L. Administration of a soluble activin type IIB receptor promotes skeletal muscle growth independent of fiber type.J Appl Physiol. 2010; 109: 635-642Crossref PubMed Scopus (116) Google Scholar which suggests that myotubularin deficiency interferes with the activation of hypertrophic pathways in oxidative fibers. We hypothesized that the transience of the therapeutic effects observed in treated Mtm1δ4 mice may have been related to the severity of the disease, so we have now repeated this study in the less severely affected Mtm1 p.R69C mouse.13Pierson C.R. Dulin-Smith A.N. Durban A.N. Marshall M.L. Marshall J.T. Snyder A.D. Naiyer N. Gladman J.T. Chandler D.S. Lawlor M.W. Buj-Bello A. Dowling J.J. Beggs A.H. Modeling the human MTM1 p.R69C mutation in murine Mtm1 results in exon 4 skipping and a less severe myotubular myopathy phenotype.Hum Mol Genet. 2012; 21: 811-825Crossref PubMed Scopus (47) Google Scholar Surprisingly, treatment of Mtm1 p.R69C mice did not produce significant increases in animal weight or grip strength, and treatment-induced myofiber hypertrophy was only observed in the Mtm1 p.R69C gastrocnemius muscles. The ability of these muscles to respond to ActRIIB-mFC treatment correlated with treatment-induced increases in satellite cell number and several muscle-specific abnormalities of hypertrophic signaling. The main difference between treatment-responsive (gastrocnemius) and treatment-resistant (quadriceps) muscles in Mtm1 p.R69C mice was related to low levels of phosphorylated ribosomal protein 6 (p-rpS6) and high levels of eukaryotic elongation factor 2 kinase (eEF2K) in the treatment-responsive gastrocnemius muscle that were not observed in other Mtm1 p.R69C muscles or in WT mice. rpS6 and eEF2K are terminal signaling molecules of the insulinlike growth factor-1/Akt and extracellular signal-related kinase (ERK) pathways that are involved in the fine-tuning of global protein synthesis, with a role in the determination of cell size that remains unclear (reviewed in Meyuhas17Meyuhas O. Physiological roles of ribosomal protein S6: one of its kind.Int Rev Cell Mol Biol. 2008; 268: 1-37Crossref PubMed Scopus (173) Google Scholar). Our findings indicate that the response to hypertrophic agents does not always correlate with activities of known hypertrophic pathways, such as the Akt pathway, but unexpectedly varies both by muscle type and fiber type and in XLMTM is affected by the nature of the Mtm1 mutation. These results highlight that there is much we still do not understand about the control of muscle size and emphasize the importance of evaluating multiple muscle and fiber types in future trials of hypertrophic therapies. All studies were performed with approval from the Institutional Animal Care and Use Committee at Boston Children's Hospital. Genotyping of MTM1/C57BL6 (Mtm1δ4) and Mtm1 p.R69C/C57BL6 (Mtm1 p.R69C) mice was performed as previously described.12Lawlor M.W. Read B.P. Edelstein R. Yang N. Pierson C.R. Stein M.J. Wermer-Colan A. Buj-Bello A. Lachey J.L. Seehra J.S. Beggs A.H. Inhibition of activin receptor type IIb increases strength and lifespan in myotubularin-deficient mice.Am J Pathol. 2011; 178: 784-793Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar, 13Pierson C.R. Dulin-Smith A.N. Durban A.N. Marshall M.L. Marshall J.T. Snyder A.D. Naiyer N. Gladman J.T. Chandler D.S. Lawlor M.W. Buj-Bello A. Dowling J.J. Beggs A.H. Modeling the human MTM1 p.R69C mutation in murine Mtm1 results in exon 4 skipping and a less severe myotubular myopathy phenotype.Hum Mol Genet. 2012; 21: 811-825Crossref PubMed Scopus (47) Google Scholar, 18Buj-Bello A. Laugel V. Messaddeq N. Zahreddine H. Laporte J. Pellissier J.F. Mandel J.L. The lipid phosphatase myotubularin is essential for skeletal muscle maintenance but not for myogenesis in mice.Proc Natl Acad Sci U S A. 2002; 99: 15060-15065Crossref PubMed Scopus (177) Google Scholar Male WT and Mtm1 p.R69C mice were given intraperitoneal injections twice per week with a soluble ActRIIB-mFC (alias RAP-031; Acceleron Pharma, Cambridge, MA) at a dose of 20 mg/kg or an equivalent volume of Tris-buffered saline (the vehicle used with ActRIIB-mFc), as previously described12Lawlor M.W. Read B.P. Edelstein R. Yang N. Pierson C.R. Stein M.J. Wermer-Colan A. Buj-Bello A. Lachey J.L. Seehra J.S. Beggs A.H. Inhibition of activin receptor type IIb increases strength and lifespan in myotubularin-deficient mice.Am J Pathol. 2011; 178: 784-793Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar from 14 days until 6 months of life (MOL). Animals were sacrificed at 6 MOL because of a plateau in observable therapeutic effects. Animals were weighed twice per week during the treatment period. Forelimb grip strength was measured weekly with the use of a Chatillon grip force meter (Columbus Instruments, Columbus, OH) by placing the animal on a horizontal grid and allowing it to pull away from the experimenter by using only its fore limbs. The maximum of three independent measurements, with a 1-minute recovery period between measurements, was used for subsequent statistical analysis. To evaluate antigravity hanging performance, animals were tested weekly by placing the animal on a rigid mesh surface, inverting the surface at a height of approximately 40 cm above their cage, and recording the amount of time necessary for the animal to fall back into the cage. Animals that did not fall within 60 seconds were lowered back into their cages. The maximum of three independent measurements, with a 1-minute recovery period between measurements, was used for subsequent statistical analysis. Animals were euthanized with carbon dioxide followed by cervical dislocation, per the regulations of the Institutional Animal Care and Use Committee at Children's Hospital Boston. Animals were photographed intact and after removal of the skin from the torso and limbs. The quadriceps, gastrocnemius, triceps, soleus, extensor digitorum longus, and diaphragm muscles were removed, weighed, and frozen in liquid nitrogen-cooled isopentane. Cross sections (8 μm) of isopentane-frozen quadriceps, gastrocnemius, or triceps muscles were taken midway down the length of the muscle and stained with H&E for evaluation with the use of an Olympus BX53 microscope with an Olympus DP72 camera and cellSens Standard software version 1.5 (Olympus, Center Valley, PA). Fiber size was determined through measurements of MinFeret diameter, as we have previously used,12Lawlor M.W. Read B.P. Edelstein R. Yang N. Pierson C.R. Stein M.J. Wermer-Colan A. Buj-Bello A. Lachey J.L. Seehra J.S. Beggs A.H. Inhibition of activin receptor type IIb increases strength and lifespan in myotubularin-deficient mice.Am J Pathol. 2011; 178: 784-793Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar because this measurement offers a myofiber size measurement that is relatively independent of fiber orientation.19Brooke M.H. Engel W.K. The histographic analysis of human muscle biopsies with regard to fiber types. 4. Children's biopsies.Neurology. 1969; 19: 591-605Crossref PubMed Google Scholar Overall fiber size and the number of internally nucleated fibers were manually measured with micrographs of two ×100 magnification fields (using a 10× objective lens) of H&E-stained slides (range, 259 to 1113 fibers; n = 4 to 5 animals quantified per treatment group). To evaluate fiber type-specific responses, 8 μm frozen transverse sections of quadriceps muscle were double-stained with rabbit anti-dystrophin antibodies (ab15277; Abcam, Cambridge, MA) and mouse monoclonal antibodies against myosin heavy chain type 1 (Skeletal, Slow, clone NOQ7.5.4D; Sigma-Aldrich, St. Louis, MO), type 2a (clone SC-71; Developmental Studies Hybridoma Bank, Iowa City, IA), or type 2b (clone BF-F3; Developmental Studies Hybridoma Bank). Secondary antibodies included fluorescein isothiocyanate-conjugated anti-mouse IgG (Sigma-Aldrich) or IgM (dilution 1:100; Sigma-Aldrich) and Alexa Fluor-conjugated anti-rabbit IgG (Molecular Probes, Carlsbad, CA). Because of variation in the number and type of oxidative fibers (type 1 and 2a fibers) in mouse muscle, quantification was performed by evaluating the type 2b myosin-positive (glycolytic) and type 2b myosin-negative (oxidative) populations on a whole-slide scan of one quadriceps and one gastrocnemius muscle from four vehicle-treated WT mice, four ActRIIB-mFc–treated WT mice, six vehicle-treated Mtm1 p.R69C mice, and six ActRIIB-mFc–treated Mtm1 p.R69C mice. MinFeret diameter was evaluated by using a novel automated technique developed by Dr. Lin Yang that correlated extremely well with our manual measurements.12Lawlor M.W. Read B.P. Edelstein R. Yang N. Pierson C.R. Stein M.J. Wermer-Colan A. Buj-Bello A. Lachey J.L. Seehra J.S. Beggs A.H. Inhibition of activin receptor type IIb increases strength and lifespan in myotubularin-deficient mice.Am J Pathol. 2011; 178: 784-793Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar The automated image quantification algorithm began with ridge detection to enhance the muscle fiber boundaries, followed by robust seed detection that was based on concave area identification to find initial seeds for muscle fibers. The final muscle fiber boundaries were automatically delineated with a gradient vector flow deformable model. After each fiber was automatically and accurately delineated, the MinFeret diameter was calculated by finding the shortest axis along the segmented contour of each muscle fiber. The number of muscle fibers was determined with scanned slides that had been stained with anti-dystrophin antibodies. A software analysis algorithm was developed with Visiomorph software version 3.4.3.0 (Visiopharm, Medicon Valley, Denmark) to allow the distinction between black pixels inside myofibers from the white/gray pixels (in grayscale) in areas of dystrophin staining. Bayesian classification was used to segment the image, and postprocessing steps were applied related to shape and area to disregard counted areas that did not correspond to myofibers. Fiber count analysis was performed on muscles from four to six animals per treatment group for both the quadriceps and gastrocnemius muscles. The quantification of satellite cells was performed with immunoperoxidase staining for Pax7 (Developmental Studies Hybridoma Bank) and biotin-SP–conjugated donkey anti-mouse IgG Fab fragment, as previously described.8Lawlor M.W. Alexander M.S. Viola M.G. Meng H. Joubert R. Gupta V. Motohashi N. Manfready R.A. Hsu C.P. Huang P. Buj-Bello A. Kunkel L.M. Beggs A.H. Gussoni E. Myotubularin-deficient myoblasts display increased apoptosis, delayed proliferation, and poor cell engraftment.Am J Pathol. 2012; 181: 961-968Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar Images were quantified by counting the number of Pax7+ cells within a field and dividing by the total number of fibers (range, 1117 to 3631 fibers; n = 3 animals quantified per treatment group). Muscle tissues from the quadriceps, gastrocnemius, triceps, and elsewhere in the fore and hind limbs from WT and Mtm1 p.R69C mice at 6 MOL or WT and Mtm1δ4 mice at 6 weeks of life (WOL) were frozen at the time of necropsy and stored at −80°C until analysis. Frozen muscles were crushed in liquid nitrogen and homogenized with lysing buffer (Cell Signaling Technology, Danvers, MA) that contained protease inhibitor (Roche, Basel, Switzerland) and phosphatase inhibitor (Roche). Western blot procedures were performed as previously described.20Wattanasirichaigoon D. Swoboda K.J. Takada F. Tong H.Q. Lip V. Iannaccone S.T. Wallgren-Pettersson C. Laing N.G. Beggs A.H. Mutations of the slow muscle alpha-tropomyosin gene, TPM3, are a rare cause of nemaline myopathy.Neurology. 2002; 59: 613-617Crossref PubMed Scopus (76) Google Scholar Transferred proteins were probed with antibodies against a variety of antigens. Antibodies that recognize the following antigens were all obtained from Cell Signaling Technologies: Akt (4691), phospho-Akt (Thr308; 2965), phospho-Akt (Ser473; 4060), S6 ribosomal protein (2217), phospho-S6 ribosomal protein (Ser240/244; 5364), ERK (4695), phospho-ERK (ERK 1/2, Thr202/204; 4370), p70-S6 kinase (S6K; 2708), phospho–p70-S6K (Thr421/Ser424; 9204), p90-ribosomal S6 kinase (RSK)1/2/3 (9355), phospho–p90-RSK (Ser380; 9335), phospho–4E-BP1(Thr37/46; 2855), and phospho-eEF2K (Ser366; 3691). Other antibodies used for Western blot studies recognize myostatin (MAB788; R&D Systems, Minneapolis, MN), ActRIIB (ab76940; Abcam), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH; G8795; Sigma-Aldrich) and were visualized with chemiluminescent horseradish peroxidase antibody detection reagent (Denville Scientific, Metuchen, NJ). Adequacy of transfer was determined by Ponceau S staining. Quantification of protein levels normalized to GAPDH was performed with ImageJ software version 1.45s (NIH, Bethesda, MD). Samples from four animals per treatment group were used for analysis. Two additional Mtm1 p.R69C/vehicle and Mtm1 p.R69C/ActRIIB-mFc animals were added to the quantitative evaluation of total and p-rpS6 (based on results from replicate gels) to further establish the degree of interanimal variability. Statistical evaluation was performed with Prism software version 4 software (GraphPad, Inc., San Diego, CA) or by statisticians at the Quantitative Health Sciences Section at the Medical College of Wisconsin. For statistical analysis of animal weight, forelimb grip strength, and antigravity hanging performance, analyses of variance were performed with Bonferroni posttests. For muscle weight and average myofiber diameter measurements, one-way analyses of variance with Bonferroni posttests were performed. For statistical analysis of Western blot data, t-tests were used to compare protein expression between different genotypes or treatments, and paired t-tests were used to compare protein expression between the gastrocnemius and quadriceps muscles of the same genotype and treatment group. In animals that received vehicle injections, Mtm1 p.R69C animals were distinguishable from age-matched WT animals on the basis of weight at 7 WOL (P < 0.05) (Figure 1A). These differences increased as the animals became older because of continued weight gain in WT animals, in contrast to the relative weight plateau seen after 7 WOL in Mtm1 p.R69C animals. ActRIIB-mFc–treated WT mice showed significant weight gains in comparison with vehicle-treated animals at 6 WOL (P < 0.05) that continued to increase with age. ActRIIB-mFC–treated Mtm1δ4 animals displayed a consistent slight increase (averaging 1 to 2 g) in weight that did not reach statistical significance and did not show the continued increases observed in WT mice. WT mice displayed the ability to hang for up to 60 seconds (the maximum period tested) after 3 WOL (Figure 1B). Treatment of WT mice with ActRIIB-mFC did not affect antigravity hanging performance. Vehicle-treated Mtm1 p.R69C animals exhibited significantly impaired hanging performance at 4 WOL (P < 0.05), which, although variable, did not degenerate into the complete inability for antigravity hanging that we observed in Mtm1δ4 mice.12Lawlor M.W. Read B.P. Edelstein R. Yang N. Pierson C.R. Stein M.J. Wermer-Colan A. Buj-Bello A. Lachey J.L. Seehra J.S. Beggs A.H. Inhibition of activin receptor type IIb increases strength and lifespan in myotubularin-deficient mice.Am J Pathol. 2011; 178: 784-793Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar Treatment with ActRIIB-mFC did not significantly improve the antigravity hanging performance of Mtm1 p.R69C mice. Forelimb grip strength measurements were consistently lower in Mtm1 p.R69C mice than in WT mice after 12 WOL (P < 0.05) (Figure 1C). ActRIIB-mFC–treated WT animals also showed an increase in grip strength as the treatment period progressed (P < 0.01); however, treatment with ActRIIB-mFC did not measurably improve forelimb grip strength performance of Mtm1 p.R69C mice. Mtm1 p.R69C mice can be easily distinguished from WT mice by gross and histological examination.13Pierson C.R. Dulin-Smith A.N. Durban A.N. Marshall M.L. Marshall J.T. Snyder A.D. Naiyer N. Gladman J.T. Chandler D.S. Lawlor M.W. Buj-Bello A. Dowling J.J. Beggs A.H. Modeling the human MTM1 p.R69C mutation in murine Mtm1 results in exon 4 skipping and a less severe myotubular myopathy phenotype.Hum Mol Genet. 2012; 21: 811-825Crossref PubMed Scopus (47) Google Scholar Mtm1 p.R69C mice are smaller than age-matched WT mice and have proportionately smaller muscles (data not shown). At 6 MOL (ie, after 5.5 months of treatment), ActRIIB-mFC treatment of WT animals produced significant increases in mass of the quadriceps, triceps, and gastrocnemius muscles (P < 0.05), but only the gastrocnemius muscle of treated Mtm1 p.R69C mice showed significant treatment-induced increases in mass (P < 0.05) (Figure 2A). As previously described,13Pierson C.R. Dulin-Smith A.N. Durban A.N. Marshall M.L. Marshall J.T. Snyder A.D. Naiyer N. Gladman J.T. Chandler D.S. Lawlor M.W. Buj-Bello A. Dowling J.J. Beggs A.H. Modeling the human MTM1 p.R69C mutation in murine Mtm1 results in exon 4 skipping and a less severe myotubular myopathy phenotype.Hum Mol Genet. 2012; 21: 811-825Crossref PubMed Scopus (47) Google Scholar quadriceps muscle fibers of vehicle-treated Mtm1 p.R69C mice are smaller than those found in age-matched WT mice, with mean fiber diameters of 24 ± 0.53 and 51 ± 1.27 μm, respectively, by 6 MOL (shown as means ± SEM; P < 0.05) (Figure 2). Although central nucleation is not a major pathological feature in this animal model, muscles of Mtm1 p.R69C mice showed increased numbers of centrally nucleated fibers in comparison with WT muscles (WT/vehicle quadriceps = 1.43% ± 0.8%, R69C/vehicle quadriceps = 5.3% ± 1.0%, WT/vehicle gastrocnemius = 0.8% ± 0.3%, and R69C/vehicle gastrocnemius = 4.9% ± 1.4% internally nucleated fibers; P < 0.05), and the number of internally nucleated fibers did not significantly increase or decrease after ActRIIB-mFc treatment. Treatment of WT animals with ActRIIB-mFC increased myofiber size by 6 MOL, as evidenced by a 5-μm (10%) increase in mean fiber diameter and an increased percentage of large fibers within the gastrocnemius and quadriceps muscles (Figure 2). Immunostaining for oxidative (type 1 or 2a) or glycolytic (type 2b) myosin subtypes within these muscles indicated that all types of WT muscle fibers experienced hypertrophy in response to ActRIIB-mFC treatment, as evidenced by a rightward shift in histograms of fiber size (Figure 2, D and E). In contrast, the gastrocnemius, but not quadriceps, muscles from ActRIIB-mFC–treated Mtm1 p.R69C animals showed marked hypertrophy in only a subpopulation of muscle fibers compared with their vehicle-treated counterparts, with a 10-μm (32%) increase in mean fiber diameter in the gast
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