Inhibition of Activin Receptor Type IIB Increases Strength and Lifespan in Myotubularin-Deficient Mice
2011; Elsevier BV; Volume: 178; Issue: 2 Linguagem: Inglês
10.1016/j.ajpath.2010.10.035
ISSN1525-2191
AutoresMichael W. Lawlor, Benjamin Read, Rachel Edelstein, Nicole Yang, Christopher R. Pierson, Matthew J. Stein, Ariana Wermer-Colan, Anna Buj-Bello, Jennifer Lachey, Jasbir Seehra, Alan H. Beggs,
Tópico(s)Cardiomyopathy and Myosin Studies
ResumoX-linked myotubular myopathy (XLMTM) is a congenital disorder caused by deficiency of the lipid phosphatase, myotubularin. Patients with XLMTM often have severe perinatal weakness that requires mechanical ventilation to prevent death from respiratory failure. Muscle biopsy specimens from patients with XLMTM exhibit small myofibers with central nuclei and central aggregations of organelles in many cells. It was postulated that therapeutically increasing muscle fiber size would cause symptomatic improvement in myotubularin deficiency. Recent studies have elucidated an important role for the activin-receptor type IIB (ActRIIB) in regulation of muscle growth and have demonstrated that ActRIIB inhibition results in significant muscle hypertrophy. To evaluate whether promoting muscle hypertrophy can attenuate symptoms resulting from myotubularin deficiency, the effect of ActRIIB-mFC treatment was determined in myotubularin-deficient (Mtm1δ4) mice. Compared with wild-type mice, untreated Mtm1δ4 mice have decreased body weight, skeletal muscle hypotrophy, and reduced survival. Treatment of Mtm1δ4 mice with ActRIIB-mFC produced a 17% extension of lifespan, with transient increases in weight, forelimb grip strength, and myofiber size. Pathologic analysis of Mtm1δ4 mice during treatment revealed that ActRIIB-mFC produced marked hypertrophy restricted to type 2b myofibers, which suggests that oxidative fibers in Mtm1δ4 animals are incapable of a hypertrophic response in this setting. These results support ActRIIB-mFC as an effective treatment for the weakness observed in myotubularin deficiency. X-linked myotubular myopathy (XLMTM) is a congenital disorder caused by deficiency of the lipid phosphatase, myotubularin. Patients with XLMTM often have severe perinatal weakness that requires mechanical ventilation to prevent death from respiratory failure. Muscle biopsy specimens from patients with XLMTM exhibit small myofibers with central nuclei and central aggregations of organelles in many cells. It was postulated that therapeutically increasing muscle fiber size would cause symptomatic improvement in myotubularin deficiency. Recent studies have elucidated an important role for the activin-receptor type IIB (ActRIIB) in regulation of muscle growth and have demonstrated that ActRIIB inhibition results in significant muscle hypertrophy. To evaluate whether promoting muscle hypertrophy can attenuate symptoms resulting from myotubularin deficiency, the effect of ActRIIB-mFC treatment was determined in myotubularin-deficient (Mtm1δ4) mice. Compared with wild-type mice, untreated Mtm1δ4 mice have decreased body weight, skeletal muscle hypotrophy, and reduced survival. Treatment of Mtm1δ4 mice with ActRIIB-mFC produced a 17% extension of lifespan, with transient increases in weight, forelimb grip strength, and myofiber size. Pathologic analysis of Mtm1δ4 mice during treatment revealed that ActRIIB-mFC produced marked hypertrophy restricted to type 2b myofibers, which suggests that oxidative fibers in Mtm1δ4 animals are incapable of a hypertrophic response in this setting. These results support ActRIIB-mFC as an effective treatment for the weakness observed in myotubularin deficiency. X-linked myotubular myopathy (XLMTM) is a severe form of congenital myopathy with an estimated incidence of 1 in 50,000 male births, and most often manifests 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 (85) Google Scholar, 2Jungbluth H. Wallgren-Pettersson C. Laporte J. Centronuclear (myotubular) myopathy.Orphanet J Rare Dis. 2008; 3: 26Crossref PubMed Scopus (217) Google Scholar Many patients with XLMTM die of the disease within the first year of life despite use of mechanical ventilation, and there are no US Food and Drug Administration–approved treatments for this disease. XLMTM is caused by mutations in the gene encoding myotubularin (MTM1), which is a phosphoinositide phosphatase thought to be involved in endosomal trafficking and/or maintenance of the sarcoplasmic reticulum and transverse tubular (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. 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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 (513) Google Scholar Muscle biopsy specimens from patients with XLMTM exhibit excessively small fibers with increased numbers of central nuclei and aggregation of organelles within the central regions of many cells.7Pierson 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 (83) Google Scholar While the number of centrally nucleated fibers bears little relationship to prognosis, there is a clear correlation between the degree of fiber smallness and severity of the disease.8Pierson 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 A murine model of myotubularin deficiency, the Mtm1δ4 mouse (also referred to as Mtm1 KO in previous studies),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 (97) Google Scholar, 9Al-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. 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The lipid phosphatase myotubularin is essential for skeletal muscle maintenance but not for myogenesis in mice.Proc Natl Acad Sci USA. 2002; 99: 15060-15065Crossref PubMed Scopus (172) Google Scholar The relationship between myofiber size and symptomatic severity in patients with XLMTM and Mtm1δ4 mice suggests that therapies that increase fiber size may lead to symptomatic improvement. Myostatin (formerly termed “growth differentiation factor 8”) is a protein of the transforming growth factor beta (TGF-β) superfamily that is selectively expressed in skeletal muscle, cardiac muscle, and adipose tissue during late embryogenesis and adulthood and is an important negative regulator of myofiber size.11McPherron A.C. Lawler A.M. Lee S.J. Regulation of skeletal muscle mass in mice by a new TGF-beta superfamily member.Nature. 1997; 387: 83-90Crossref PubMed Scopus (3168) Google Scholar Myostatin binds to and signals through the activin type IIB receptor (ActRIIB) to activate the TGF-β pathway, which prevents progression through the cell cycle and down-regulates several key processes related to myofiber hypertrophy.12McCroskery 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 (579) Google Scholar, 13Joulia-Ekaza D. Cabello G. Myostatin regulation of muscle development: molecular basis, natural mutations, physiopathological aspects.Exp Cell Res. 2006; 312: 2401-2414Crossref PubMed Scopus (3) Google Scholar Examples of myostatin deficiency found in sheep,14Clop A. Marcq F. Takeda H. Pirottin D. Tordoir X. Bibe B. Bouix J. Caiment F. Elsen J.M. Eychenne F. Larzul C. Laville E. Meish F. Milenkovic D. Tobin J. Charlier C. Georges M. A mutation creating a potential illegitimate microRNA target site in the myostatin gene affects muscularity in sheep.Nat Genet. 2006; 38: 813-818Crossref PubMed Scopus (1021) Google Scholar mice,11McPherron A.C. Lawler A.M. Lee S.J. Regulation of skeletal muscle mass in mice by a new TGF-beta superfamily member.Nature. 1997; 387: 83-90Crossref PubMed Scopus (3168) Google Scholar cattle, dogs, and one human child have all exhibited generalized muscular hypertrophy and increased strength,13Joulia-Ekaza D. Cabello G. Myostatin regulation of muscle development: molecular basis, natural mutations, physiopathological aspects.Exp Cell Res. 2006; 312: 2401-2414Crossref PubMed Scopus (3) Google Scholar and compared with mice with intact myostatin expression, null mice for myostatin or ActRIIB demonstrate myofiber hypertrophy and hyperplasia.15Lee S.J. McPherron A.C. Regulation of myostatin activity and muscle growth.Proc Natl Acad Sci USA. 2001; 98: 9306-9311Crossref PubMed Scopus (1269) Google Scholar, 16Lee S.J. Reed L.A. Davies M.V. Girgenrath S. Goad M.E. Tomkinson K.N. Wright J.F. Barker C. Ehrmantraut G. Holmstrom J. Trowell B. Gertz B. Jiang M.S. Sebald S.M. Matzuk M. Li E. Liang L.F. Quattlebaum E. Stotish R.L. Wolfman N.M. Regulation of muscle growth by multiple ligands signaling through activin type II receptors.Proc Natl Acad Sci USA. 2005; 102: 18117-18122Crossref PubMed Scopus (413) Google Scholar There seems to be myostatin-independent suppression of muscle growth that is mediated by ActRIIB. Treatment with a soluble ActRIIB-mFC increases muscle mass in MTm1δ4 mice,16Lee S.J. Reed L.A. Davies M.V. Girgenrath S. Goad M.E. Tomkinson K.N. Wright J.F. Barker C. Ehrmantraut G. Holmstrom J. Trowell B. Gertz B. Jiang M.S. Sebald S.M. Matzuk M. Li E. Liang L.F. Quattlebaum E. Stotish R.L. Wolfman N.M. Regulation of muscle growth by multiple ligands signaling through activin type II receptors.Proc Natl Acad Sci USA. 2005; 102: 18117-18122Crossref PubMed Scopus (413) Google Scholar which supports the role of multiple ligands that control muscle growth postnatally and suggests that targeting ActRIIB rather than myostatin alone may provide additional therapeutic benefit. The potential for myostatin inhibition to promote muscle growth has led to development of a new class of myostatin and ActRIIB inhibitors as prospective therapeutic agents for myopathic, dystrophic, and neurologic disorders. A soluble activin-receptor type IIB fusion protein (ActRIIB-mFC) has been developed that potently binds to TGF-β family members to produce muscle fiber growth in vitro and in vivo17Cadena 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 [published online ahead of print May 13, 2010].J Appl Physiol. 2010; 109: 635-642Crossref PubMed Scopus (113) Google Scholar. Recent studies using ActRIIB-mFC in murine models of neurologic disorders including amyotrophic lateral sclerosis and spinal muscular atrophy have demonstrated variable effects on muscle strength and no effects on animal survival.18Morrison B.M. Lachey J.L. Warsing L.C. Ting B.L. Pullen A.E. Underwood K.W. Kumar R. Sako D. Grinberg A. Wong V. Colantuoni E. Seehra J.S. Wagner K.R. A soluble activin type IIB receptor improves function in a mouse model of amyotrophic lateral sclerosis.Exp Neurol. 2009; 217: 258-268Crossref PubMed Scopus (65) Google Scholar, 19Sumner C.J. Wee C.D. Warsing L.C. Choe D.W. Ng A.S. Lutz C. Wagner K.R. Inhibition of myostatin does not ameliorate disease features of severe SMA mice.Hum Mol Genet. 2009; 18: 3145-3152Crossref PubMed Scopus (64) Google Scholar The effectiveness of this agent for treatment of primary disorders of skeletal muscle is being investigated. Myostatin and ActRIIB inhibitors are currently being evaluated for treatment of several neuromuscular disorders, but have not yet been considered for treatment of any congenital myopathies. It is believed that they hold considerable promise for treatment of XLMTM because of the direct relationship between fiber size and prognosis in affected patients. To evaluate the effectiveness of ActRIIB inhibition on myotubularin-deficient myofibers, wild-type and Mtm1δ4 mice were treated with ActRIIB-mFC, and disease progression was evaluated both behaviorally and pathologically. Treatment in Mtm1δ4 mice caused a significant extension of lifespan in this severely affected model of the disease, with transient increases in weight and forelimb grip strength. Pathologic analysis of muscles harvested while therapy was most effective revealed marked increases in muscle bulk and muscle weight, and dramatic hypertrophy of type 2b myofibers. All studies were performed with approval from the institutional animal care and utilization committee at Children's Hospital Boston (Boston, MA). MTM1/HSA mice20Buj-Bello A. Furling D. Tronchere H. Laporte J. Lerouge T. Butler-Browne G.S. Mandel J.L. Muscle-specific alternative splicing of myotubularin-related 1 gene is impaired in DM1 muscle cells.Hum Mol Genet. 2002; 11: 2297-2307Crossref PubMed Scopus (108) Google Scholar were a gift from Anna Buj-Bello and colleagues at the Université de Strasbourg, Collège de France (Illkirch, France), and were subsequently back-crossed onto a C57BL6 background. Genotyping of these MTM1/C57BL6 mice (Mtm1δ4) was performed as previously described.20Buj-Bello A. Furling D. Tronchere H. Laporte J. Lerouge T. Butler-Browne G.S. Mandel J.L. Muscle-specific alternative splicing of myotubularin-related 1 gene is impaired in DM1 muscle cells.Hum Mol Genet. 2002; 11: 2297-2307Crossref PubMed Scopus (108) Google Scholar Male wild-type and Mtm1δ4 mice were injected semiweekly beginning at 14 days of life with ActRIIB-mFC (also called RAP-031; Acceleron Pharma Inc., Cambridge, MA) at a dose of 20 mg/kg. This dosage was chosen because it was above the level at which muscle hypertrophy plateaued in wild-type mice (data not shown). Vehicle-treated animals were injected with an equivalent volume of Tris-buffered saline solution. Treatment was continued until Mtm1δ4 animals were judged to be at end stage, and wild-type animals were sacrificed at equivalent time points. Behavior of wild-type and Mtm1δ4 animals was evaluated throughout the treatment period, and they were considered at end stage when they had either lost 20% of their highest body mass measurement or demonstrated complete inability to use their hindlimbs. The age at which Mtm1δ4 animals reached end stage was closely tracked to enable construction of Kaplan-Meier survival curves using commercially available software (Prism 4; GraphPad Software, Inc., San Diego, CA). Animals were weighed five times per week during the treatment period. For statistical analysis, running averages of the animals' weight over 3 days were calculated to provide daily measurements of animal weight. Forelimb grip strength was measured weekly using a Chatillon grip force meter (Columbus Instruments International, Columbus, OH) by placing the animal on a horizontal grid and allowing it to pull away from the experimenter by using only its forelimbs. The average of three independent measurements, with a 1-minute recovery period between measurements, was used for subsequent statistical analysis. Antigravity hanging performance was tested three times weekly by placing the animals on a rigid mesh surface, inverting the surface at 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. Footprint analysis was performed weekly by immersion of an animal's feet in nontoxic ink and allowing the animal to walk across an 8.5 × 11-inch piece of paper contained in an acrylic safety glass (Plexiglas) walkway. Footsteps and foot drags were counted, and the ratio of foot drags to foot steps was calculated for subsequent statistical analysis.21Crawley J.N. What's Wrong with My Mouse? Behavioral Phenotyping of Transgenic and Knockout Mice.in: 2nd ed. Wiley-Liss, Hoboken, NJ2007: 368Google Scholar Statistical analysis was performed using commercially available software (Prism 4; GraphPad, Inc.). For statistical analysis of animal weight, forelimb grip strength, antigravity hanging performance, and foot drag, analysis of variance was performed, with Bonferroni posttests. For measurement of muscle weight and mean myofiber diameter, one-way analysis of variance was performed, with Bonferroni posttests. For survival data, statistical significance was evaluated using a log-rank test. Tissues from the quadriceps, gastrocnemius, and triceps muscles and from elsewhere in the forelimbs and hindlimbs were frozen at necropsy and stored at −80°C until analysis. Protein isolation and Western blot procedures were performed as previously described.22Wattanasirichaigoon 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 (73) Google Scholar Transferred proteins were probed with antibodies against myostatin (MAB788, 1:250 dilution; R&D Systems, Inc., Minneapolis, MN) and GAPDH [glyceraldehyde-3-phosphate dehydrogenase (6C5), 1:10,000 dilution; Abcam PLC, Cambridge, MA] and visualized using enhanced chemiluminescence. Adequacy of transfer was determined using Ponceau S staining. Quantification of protein levels normalized to GAPDH was performed using the program QuantityOne, version 4.2.1 (Bio-Rad Laboratories, Inc., Hercules, CA) on an Image Station 440 (Kodak DS; Eastman Kodak Co., Rochester, NY). Animals were euthanized using CO2 followed by cervical dislocation, per the regulations of the institutional animal care and utilization committee at Children's Hospital Boston. Animals were photographed externally and after removal of the skin from the torso and limbs. The quadriceps, gastrocnemius, triceps, soleus, extensor digitorum longus, tibialis anterior, and diaphragm muscles were removed and weighed. For subsequent ultrastructural studies, a small portion of the quadriceps muscle was fixed in 5% glutaraldehyde, 2.5% paraformaldehyde, and 0.06% picric acid in 0.2 mmol/L of cacodylate buffer, pH 7.4. Eight-micrometer cross sections of isopentane-frozen quadriceps muscle were obtained midway down the length of the muscle and stained with H&E for evaluation using an Eclipse 50i microscope (Nikon Instruments Inc., Melville, NY). Light microscopic images were captured using a SPOT Insight 4 Meg FW Color Mosaic camera and SPOT 4.5.9.1 software (Diagnostic Instruments Inc., Sterling Heights, MI). For immunofluorescence studies, 8-μm frozen transverse sections of quadriceps muscle were double stained with rabbit antidystrophin antibodies (ab15277, 1:100; Abcam PLC) and mouse monoclonal antibodies against myosin heavy chain type 1 (Skeletal, Slow, clone NOQ7.5.4D, 1:100 dilution; Sigma Aldrich, St. Louis, MO) or types 2a (clone SC-71, 1:50 dilution) or 2b (clone BF-F3, 1:50 dilution; Developmental Studies Hybridoma Bank, University of Iowa, Iowa City). Secondary antibodies included fluorescein isothiocyanate–conjugated anti-mouse IgG or IgM (both 1:100; Sigma-Aldrich) and AlexaFluor-conjugated anti-rabbit IgG (1:50; Molecular Probes, Carlsbad, CA). Staining was evaluated using a Nikon Eclipse 90i microscope using NIS-Elements AR software (Nikon Instruments Inc.). For morphometric evaluation and estimation of fiber number, nonoverlapping fields of muscle immunostained for dystrophin were photographed using a Nikon Plan Fluor 4×/0.13 objective (Nikon Instruments Inc.). Fibers that stained positive were individually selected using NIS-Elements AR software, and fibers were automatically measured with respect to their minimum Feret (MinFeret) diameter. The MinFeret diameter is the smallest diameter across an ellipse, which corresponds to the “greatest distance between the opposite sides of the narrowest aspect of the fiber,”23Brooke 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 which is a measurement commonly used in morphometric analyses. An adequate number of fibers were counted to ensure measurements representative of the overall specimen, which involved a larger number of fibers counted in Mtm1δ4 animals (mean, 755; range, 238–1294 fibers) than in wild-type animals (mean, 340; range, 230–416 fibers) because of the small fiber size and the regional variability seen in the Mtm1δ4 animals. All MinFeret diameters for a given specimen were pooled for generation of frequency histograms and estimation of total number of fibers within the quadriceps muscle. In addition, the mean fiber MinFeret diameter for each specimen was calculated for subsequent statistical analysis. For electron microscopy, fixed tissue was subjected to osmication, stained using uranyl acetate, dehydrated in alcohols, and embedded in TAAB Epon (Marivac Ltd., Halifax, Nova Scotia, Canada). Subsequently, 1-μm scout sections were stained with toluidine blue, and evaluated and photographed as described. Areas of interest were cut at 95-nm thickness using an ultracut microtome (Leica Camera AG, Solms, Germany), picked up on 100-m formvar-coated copper grids, stained with 0.2% lead citrate, and viewed and imaged using a Tecnai BioTwin Spirit Electron Microscope (FEI Co., Hillsboro, OR). The relative levels of myostatin were investigated in untreated wild-type and Mtm1δ4 animals to ensure that the dosage would be adequate to inhibit the circulating myostatin in Mtm1δ4 mice. Similar amounts of myostatin protein are produced in Mtm1δ4 and wild-type mice (Figure 1) at 43 days of life, although the levels observed in individual wild-type mice varied markedly. The presence of similar levels of myostatin in wild-type and Mtm1δ4 mice suggests that ActRIIB-mFC could induce effective myofiber hypertrophy in Mtm1δ4 animals at dosages that are effective in wild-type mice.17Cadena 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 [published online ahead of print May 13, 2010].J Appl Physiol. 2010; 109: 635-642Crossref PubMed Scopus (113) Google Scholar, 18Morrison B.M. Lachey J.L. Warsing L.C. Ting B.L. Pullen A.E. Underwood K.W. Kumar R. Sako D. Grinberg A. Wong V. Colantuoni E. Seehra J.S. Wagner K.R. A soluble activin type IIB receptor improves function in a mouse model of amyotrophic lateral sclerosis.Exp Neurol. 2009; 217: 258-268Crossref PubMed Scopus (65) Google Scholar In animals that received semiweekly injections of Tris-buffered saline solution (the ActRIIB-mFC vehicle), Mtm1δ4 animals were distinguishable from age-matched wild-type animals on the basis of weight at 20 days of life (P < 0.05) (Figure 2A). These differences increased with age because of continued weight gain in wild-type animals in comparison with the plateau observed after 34 days of life in Mtm1δ4 animals. Compared with vehicle-treated mice, wild-type animals treated with semiweekly injections of ActRIIB-mFC, 20 mg/kg beginning at 14 days of life, showed significant weight gain after 36 days of life (P < 0.05), and continued to gain weight with age. ActRIIB-mFC treated Mtm1δ4 animals initially exhibited a modest sustained weight gain, reaching a maximum of 124% at 53 days of life; however, the weight of these animals plateaued quickly and did not increase as observed in wild-type animals. At antigravity hanging assay, in which animals are suspended from a mesh grid until they either drop into the cage or have been hanging for 60 seconds, wild-type mice were able to hang for up to 60 seconds starting from 3 weeks of life (Figure 2B). Treatment of wild-type mice with ActRIIB-mFC led to a slight decrease in antigravity hanging performance, which was statistically significant only at 26 to 27 days of life (P < 0.05). In contrast, vehicle-treated Mtm1δ4 animals exhibited impaired hanging performance at 2 weeks of life, which subsequently degenerated into nearly complete inability to remain suspended against gravity by 28 to 29 days of life. Treatment with ActRIIB-mFC did not measurably improve the antigravity hanging performance of Mtm1δ4 mice. Mtm1δ4 animals exhibited consistently lower forelimb grip strength measurements at all ages tested (P < 0.001) (Figure 2C). Forelimb grip force measurements in vehicle-treated wild-type animals showed consistent gains in grip strength as the animals aged, whereas the grip force of Mtm1δ4 animals was greatest at 5 weeks of life and then decreased as the disease progressed. Compared with their vehicle-treated counterparts, ActRIIB-mFC–treated wild-type animals also demonstrated increased grip strength as the treatment period progressed, with grip force up to 135% greater at 9 weeks of life, the latest time point tested. In ActRIIB-mFC–treated Mtm1δ4 animals, grip strength was transiently improved by 116% at 5 weeks of life (P < 0.05), after which grip strength declined in both vehicle- and ActRIIB-mFC–treated Mtm1δ4 animals. Abnormalities of gait can be used to differentiate wild-type from Mtm1δ4 animals. Mtm1δ4 mice experience hindlimb weakness with disease progression, which can be visualized and quantified as foot drags on a footprinting assay. While foot drags are extremely uncommon in wild-type mice, the number of drags observed in Mtm1δ4 mice steadily increased after 3 weeks of life (P < 0.05) (Figure 2D). Treatment with ActRIIB-mFC did not have any effect on the number of foot drags observed at footprint analysis. Similar to the first published reports using Mtm1δ4 mice on the HSA background, which stated that the survival of Mtm1δ4 animals ranged from 6 to 12 weeks (mean, 59 days), in the present study, untreated and vehicle-treated Mtm1δ4 animals demonstrated a maximum lifespan of approximately 8 to 9 weeks (mean, 56.1 days; range, 32–65 days). In comparisons of vehicle- and ActRIIB-mFC–treated Mtm1δ4 mice, treatment with ActRIIB-mFC significantly lengthened this lifespan by 17% (mean, 67.6 days; range, 61–74 days), with a shift in median survival from 58 days to 68 days (P < 0.05) (Figure 2E). This survival benefit was due to both a decrease in the number of early deaths and delayed death in the oldest treated animals. This survival benefit also seems to be dose-dependent; reducing the dose to 5 mg/kg of ActRIIB-mFC in a pilot study of six animals resulted in an increase in median survival to 65 days (data not shown), which is still a significant improvement over that in the vehicle-treated animals (P < 0.05). Mtm1δ4 mice with late-stage disease can be easily differentiated from wild-type mice at gross and histologic examination. Mtm1δ4 mice are much smaller than age-matched wild-type mice and have proportionately smaller muscles (Figure 3A). At 43 days of life, when treated and untreated mice of both genetic backgrounds are easily distinguishable from their vehicle-treated counterparts on the basis of weight, ActRIIB-mFC treatment resulted in larger muscles in both wild-type and Mtm1δ4 mice (Figure 3B). Necropsy performed in treated animals at 8 to 10 weeks of life, which corresponded to the end-stage in treated and untreated Mtm1δ4 mice, revealed dramatic increases in muscle size in ActRIIB-mFC–treated wild-type animals. In contrast, vehicle- and ActRIIB-mFC–treated Mtm1δ4 mice were grossly indistinguishable from one another at end stage; both groups of mice were severely emaciated (Figure 3A). It should be noted, however, that the survival benefit produced by ActRIIB-mFC therapy prevented age-matching of these mice for this comparison. While the end-stage appearance of treated and untreated Mtm1δ4 mice was similar, the vehicle-treated mice were approximately 10 days younger than the ActRIIB-mFC–treated mice when th
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