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Treatment with ActRIIB-mFc Produces Myofiber Growth and Improves Lifespan in the Acta1 H40Y Murine Model of Nemaline Myopathy

2016; Elsevier BV; Volume: 186; Issue: 6 Linguagem: Inglês

10.1016/j.ajpath.2016.02.008

1525-2191

Jennifer Tinklenberg, Hui Meng, Lin Yang, Fujun Liu, Raymond G. Hoffmann, Mahua Dasgupta, Kenneth P. Allen, Alan H. Beggs, Edna C. Hardeman, R. Scott Pearsall, R. H. Fitts, Michael W. Lawlor,

Neurogenetic and Muscular Disorders Research

Nemaline myopathies (NMs) are a group of congenital muscle diseases caused by mutations in at least 10 genes and associated with a range of clinical symptoms. NM is defined on muscle biopsy by the presence of cytoplasmic rod-like structures (nemaline rods) composed of cytoskeletal material. Myofiber smallness is also found in many cases of NM and may represent a cause of weakness that can be counteracted by treatment. We have used i.p. injection of activin type IIB receptor (ActRIIB)–mFc (an inhibitor of myostatin signaling) to promote hypertrophy and increase strength in our prior murine work; we therefore tested whether ActRIIB-mFc could improve weakness in NM mice through myofiber hypertrophy. We report a study of ActRIIB-mFc treatment in the Acta1 H40Y mouse model of NM. Treatment of Acta1 H40Y mice produced significant increases in body mass, muscle mass, quadriceps myofiber size, and survival, but other measurements of strength (forelimb grip strength, ex vivo measurements of contractile function) did not improve. Our studies also identified that the complications of urethral obstruction are associated with mortality in male hemizygote Acta1 H40Y mice. The incidence of urethral obstruction and histologic evidence of chronic obstruction (inflammation) were significantly lower in Acta1 H40Y mice that had been treated with ActRIIB-mFc. ActRIIB-mFc treatment produces a mild benefit to the disease phenotype in Acta1 H40Y mice. Nemaline myopathies (NMs) are a group of congenital muscle diseases caused by mutations in at least 10 genes and associated with a range of clinical symptoms. NM is defined on muscle biopsy by the presence of cytoplasmic rod-like structures (nemaline rods) composed of cytoskeletal material. Myofiber smallness is also found in many cases of NM and may represent a cause of weakness that can be counteracted by treatment. We have used i.p. injection of activin type IIB receptor (ActRIIB)–mFc (an inhibitor of myostatin signaling) to promote hypertrophy and increase strength in our prior murine work; we therefore tested whether ActRIIB-mFc could improve weakness in NM mice through myofiber hypertrophy. We report a study of ActRIIB-mFc treatment in the Acta1 H40Y mouse model of NM. Treatment of Acta1 H40Y mice produced significant increases in body mass, muscle mass, quadriceps myofiber size, and survival, but other measurements of strength (forelimb grip strength, ex vivo measurements of contractile function) did not improve. Our studies also identified that the complications of urethral obstruction are associated with mortality in male hemizygote Acta1 H40Y mice. The incidence of urethral obstruction and histologic evidence of chronic obstruction (inflammation) were significantly lower in Acta1 H40Y mice that had been treated with ActRIIB-mFc. ActRIIB-mFc treatment produces a mild benefit to the disease phenotype in Acta1 H40Y mice. The nemaline myopathies (NMs) are a clinically and genetically heterogeneous group of diseases, with a wide range of clinical phenotypes1North K.N. Laing N.G. Wallgren-Pettersson C. Nemaline myopathy: current concepts. The ENMC International Consortium and Nemaline Myopathy.J Med Genet. 1997; 34: 705-713Crossref PubMed Scopus (155) Google Scholar associated with mutations in at least 10 genes (ACTA1, NEB, TPM3, TPM2, TNNT1, CFL2, KBTBD13, KLHL40, KLHL41, and LMOD3).2Wallgren-Pettersson C. Sewry C.A. Nowak K.J. Laing N.G. Nemaline myopathies.Semin Pediatr Neurol. 2011; 18: 230-238Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar, 3Gupta V.A. Ravenscroft G. Shaheen R. Todd E.J. Swanson L.C. Shiina M. Ogata K. Hsu C. Clarke N.F. Darras B.T. Farrar M.A. Hashem A. Manton N.D. Muntoni F. North K.N. Sandaradura S.A. Nishino I. Hayashi Y.K. Sewry C.A. Thompson E.M. Yau K.S. Brownstein C.A. Yu T.W. Allcock R.J. Davis M.R. Wallgren-Pettersson C. Matsumoto N. Alkuraya F.S. Laing N.G. Beggs A.H. Identification of KLHL41 mutations implicates BTB-Kelch-mediated ubiquitination as an alternate pathway to myofibrillar disruption in nemaline myopathy.Am J Hum Genet. 2013; 93: 1108-1117Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar, 4Ravenscroft G. Miyatake S. Lehtokari V.L. Todd E.J. Vornanen P. Yau K.S. et al.Mutations in KLHL40 are a frequent cause of severe autosomal-recessive nemaline myopathy.Am J Hum Genet. 2013; 93: 6-18Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar, 5Yuen M. Sandaradura S.A. Dowling J.J. Kostyukova A.S. Moroz N. Quinlan K.G. et al.Leiomodin-3 dysfunction results in thin filament disorganization and nemaline myopathy.J Clin Invest. 2014; 124: 4693-4708Crossref PubMed Scopus (23) Google Scholar With the exception of KBTBD13, the proteins encoded by all these genes are structural components of (or are associated with) the sarcomeric thin filament, which is an essential component of muscle contraction.6Sanoudou D. Beggs A.H. Clinical and genetic heterogeneity in nemaline myopathy–a disease of skeletal muscle thin filaments.Trends Mol Med. 2001; 7: 362-368Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar, 7Garg A. O'Rourke J. Long C. Doering J. Ravenscroft G. Bezprozvannaya S. Nelson B.R. Beetz N. Li L. Chen S. Laing N.G. Grange R.W. Bassel-Duby R. Olson E.N. KLHL40 deficiency destabilizes thin filament proteins and promotes nemaline myopathy.J Clin Invest. 2014; 124: 3529-3539Crossref Scopus (82) Google Scholar The unifying feature of all NM cases is the presence of characteristic nemaline rods on skeletal muscle biopsy, which are aggregates of α-actinin and other sarcomeric proteins.8Dubowitz V. Sewry C. Oldfors A. Congenital myopathies and related disorders.in: Dubowitz V. Sewry C. Oldfors A. Muscle Biopsy: A Practical Approach. Saunders Elsevier, Philadelphia, PA2013: 358-405Google Scholar Although nemaline rods are a critical diagnostic feature of NM, their abundance, size, and distribution do not correlate well with disease severity,2Wallgren-Pettersson C. Sewry C.A. Nowak K.J. Laing N.G. Nemaline myopathies.Semin Pediatr Neurol. 2011; 18: 230-238Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar and their direct contribution to the weakness seen in NM is questionable. Another nonspecific pathologic feature that is seen in many cases of NM is myofiber smallness (or hypotrophy9Imoto C. Nonaka I. The significance of type 1 fiber atrophy (hypotrophy) in childhood neuromuscular disorders.Brain Dev. 2001; 23: 298-302Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar), which most often involves the type 1 myofiber population. This pattern of myofiber hypotrophy is similar to that seen in other congenital myopathies, particularly congenital fiber-type disproportion, which has genetic and phenotypic overlap with some subsets of NM.10Clarke N.F. Kolski H. Dye D.E. Lim E. Smith R.L. Patel R. Fahey M.C. Bellance R. Romero N.B. Johnson E.S. Labarre-Vila A. Monnier N. Laing N.G. North K.N. Mutations in TPM3 are a common cause of congenital fiber type disproportion.Ann Neurol. 2008; 63: 329-337Crossref PubMed Scopus (124) Google Scholar, 11Lawlor M.W. Dechene E.T. Roumm E. Geggel A.S. Moghadaszadeh B. Beggs A.H. Mutations of tropomyosin 3 (TPM3) are common and associated with type 1 myofiber hypotrophy in congenital fiber type disproportion.Hum Mutat. 2010; 31: 176-183Crossref PubMed Scopus (62) Google Scholar Myofiber hypotrophy represents a potentially reversible pathologic phenotype that has a known association with muscle strength and should thus be considered as a therapeutic target for all cases of NM. Currently, there is no effective treatment for NM. Because myofiber smallness or suboptimal numbers of sarcomeres may contribute to weakness in NM, we hypothesized that induction of myofiber hypertrophy would be of symptomatic benefit in murine models of NM. Myostatin binds to (and signals through) the activin type IIB receptor (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.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 Nonfunctional decoys of ActRIIB can be used to inhibit this negative regulator of myofiber size, leading to myofiber hypertrophy. In our prior work, we used ActRIIB-mFc, a soluble activin type IIB receptor, to produce myofiber hypertrophy in wild-type (WT) mice and in two murine models of X-linked myotubular myopathy (XLMTM).14Lawlor 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 (50) Google Scholar, 15Lawlor M.W. Viola M.G. Meng H. Edelstein R.V. Liu F. Yan K. Luna E.J. Lerch-Gaggl A. Hoffmann R.G. Pierson C.R. Buj-Bello A. Lachey J.L. Pearsall S. Yang L. Hillard C.J. Beggs A.H. Differential muscle hypertrophy is associated with satellite cell numbers and Akt pathway activation following activin type IIB receptor inhibition in Mtm1 p.R69C mice.Am J Pathol. 2014; 184: 1831-1842Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar In both these studies, the functional effect of hypertrophy on XLMTM muscle was significantly limited by the abnormalities of excitation-contraction coupling (ECC) that are also encountered in this disease. Because physiologic studies performed in NM mouse models16Chandra M. Mamidi R. Ford S. Hidalgo C. Witt C. Ottenheijm C. Labeit S. Granzier H. Nebulin alters cross-bridge cycling kinetics and increases thin filament activation: a novel mechanism for increasing tension and reducing tension cost.J Biol Chem. 2009; 284: 30889-30896Crossref PubMed Scopus (83) Google Scholar, 17de Haan A. van der Vliet M.R. Gommans I.M. Hardeman E.C. van Engelen B.G. Skeletal muscle of mice with a mutation in slow alpha-tropomyosin is weaker at lower lengths.Neuromuscul Disord. 2002; 12: 952-957Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar, 18Nguyen M.A. Joya J.E. Kee A.J. Domazetovska A. Yang N. Hook J.W. Lemckert F.A. Kettle E. Valova V.A. Robinson P.J. North K.N. Gunning P.W. Mitchell C.A. Hardeman E.C. Hypertrophy and dietary tyrosine ameliorate the phenotypes of a mouse model of severe nemaline myopathy.Brain. 2011; 134: 3516-3529Crossref PubMed Scopus (45) Google Scholar, 19Ravenscroft G. Jackaman C. Bringans S. Papadimitriou J.M. Griffiths L.M. McNamara E. Bakker A.J. Davies K.E. Laing N.G. Nowak K.J. Mouse models of dominant ACTA1 disease recapitulate human disease and provide insight into therapies.Brain. 2011; 134: 1101-1115Crossref PubMed Scopus (47) Google Scholar, 20Witt C.C. Burkart C. Labeit D. McNabb M. Wu Y. Granzier H. Labeit S. Nebulin regulates thin filament length, contractility, and Z-disk structure in vivo.EMBO J. 2006; 25: 3843-3855Crossref PubMed Scopus (192) Google Scholar and myofibers from human patients with NM21Ottenheijm C.A. Hooijman P. DeChene E.T. Stienen G.J. Beggs A.H. Granzier H. Altered myofilament function depresses force generation in patients with nebulin-based nemaline myopathy (NEM2).J Struct Biol. 2010; 170: 334-343Crossref PubMed Scopus (76) Google Scholar, 22Ottenheijm C.A. Lawlor M.W. Stienen G.J. Granzier H. Beggs A.H. Changes in cross-bridge cycling underlie muscle weakness in patients with tropomyosin 3-based myopathy.Hum Mol Genet. 2011; 20: 2015-2025Crossref PubMed Scopus (54) Google Scholar, 23Ottenheijm C.A. Witt C.C. Stienen G.J. Labeit S. Beggs A.H. Granzier H. Thin filament length dysregulation contributes to muscle weakness in nemaline myopathy patients with nebulin deficiency.Hum Mol Genet. 2009; 18: 2359-2369Crossref PubMed Scopus (114) Google Scholar do not indicate problems with ECC in NM, we hypothesized that myostatin/ActRIIB inhibition would produce both hypertrophy and significant functional benefits in murine models of NM. Although a variety of murine models are available for testing, the Acta1 H40Y mouse represented a good first model for the evaluation of myostatin inhibition due to its behavioral phenotype and use in prior treatment studies.18Nguyen M.A. Joya J.E. Kee A.J. Domazetovska A. Yang N. Hook J.W. Lemckert F.A. Kettle E. Valova V.A. Robinson P.J. North K.N. Gunning P.W. Mitchell C.A. Hardeman E.C. Hypertrophy and dietary tyrosine ameliorate the phenotypes of a mouse model of severe nemaline myopathy.Brain. 2011; 134: 3516-3529Crossref PubMed Scopus (45) Google Scholar Acta1 H40Y mice develop moderate weakness, pathologic features similar to those of NM, and mortality of approximately half of hemizygote males at 1 to 3 months of age.18Nguyen M.A. Joya J.E. Kee A.J. Domazetovska A. Yang N. Hook J.W. Lemckert F.A. Kettle E. Valova V.A. Robinson P.J. North K.N. Gunning P.W. Mitchell C.A. Hardeman E.C. Hypertrophy and dietary tyrosine ameliorate the phenotypes of a mouse model of severe nemaline myopathy.Brain. 2011; 134: 3516-3529Crossref PubMed Scopus (45) Google Scholar The pathologic phenotype and clinical course of Acta1 H40Y mice was improved by crossing them with transgenic mice that produced myofiber hypertrophy through the overexpression of insulin-like growth factor-1 or FHL1 (but not c-ski),18Nguyen M.A. Joya J.E. Kee A.J. Domazetovska A. Yang N. Hook J.W. Lemckert F.A. Kettle E. Valova V.A. Robinson P.J. North K.N. Gunning P.W. Mitchell C.A. Hardeman E.C. Hypertrophy and dietary tyrosine ameliorate the phenotypes of a mouse model of severe nemaline myopathy.Brain. 2011; 134: 3516-3529Crossref PubMed Scopus (45) Google Scholar suggesting a benefit from at least some forms of hypertrophy in these mice. In this study, Acta1 H40Y mice and WT littermates were injected twice per week with 10 mg/kg of ActRIIB-mFc, starting at 2 weeks of age and lasting until death or 16 weeks of age. Treatment of Acta1 H40Y mice produced myofiber hypertrophy and improved survival without measurable increases in limb strength. We also identified an unexpected cause of death in Acta1 H40Y mice (urinary outlet obstruction) that may represent a broader-reaching cause of mortality in muscle disease models. Our studies have found an important new phenotype of weakness that may affect survival in murine models of neuromuscular disease, while also revealing that ActRIIB inhibition has the potential to improve pathologic features and disease course in NM. All studies were performed with approval from the Institutional Animal Care and Use Committee at The Medical College of Wisconsin and Marquette University. Genotyping of the Acta1 H40Y mice was performed as previously described.18Nguyen M.A. Joya J.E. Kee A.J. Domazetovska A. Yang N. Hook J.W. Lemckert F.A. Kettle E. Valova V.A. Robinson P.J. North K.N. Gunning P.W. Mitchell C.A. Hardeman E.C. Hypertrophy and dietary tyrosine ameliorate the phenotypes of a mouse model of severe nemaline myopathy.Brain. 2011; 134: 3516-3529Crossref PubMed Scopus (45) Google Scholar Male WT and Acta1 H40Y mice were given i.p. injections twice per week with ActRIIB-mFC (also termed RAP-031; Acceleron Pharma, Cambridge, MA) at a dose of 10 mg/kg or an equivalent volume of Tris-buffered saline (the vehicle used with ActRIIB-mFc) as previously described14Lawlor 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 (50) Google Scholar from 14 days until 4 months of life. Animals were euthanized at 4 months of life because of a plateau in observable therapeutic effects. Animals were weighed daily during the treatment period beginning at 2 weeks of age when the first injection was given. Once the animals were weaned at the age of 3 weeks, forelimb grip strength was measured biweekly using a grip strength meter (Columbus Instruments, Columbus, OH) by placing the animal on a horizontal grid and allowing it to pull away from the experimenter using only its forelimbs. 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 biweekly by placing the animal on a rigid mesh surface, inverting the surface at a height of approximately 40 cm above a cage that contained at least 3 cm of bedding material, 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. Beginning at 1 month of age, mice were placed in a circular chamber (49.5 cm) and allowed to freely explore the enclosure for 10 minutes once a month. The open field tests were videotaped and analyzed for the total distance traveled by ANY-maze software version 3.96 (Stoelting, Wood Dale, IL). Also beginning at 1 month of age, animals were placed on a treadmill moving at 4 to 6 m/min (Columbus Instruments) every other week and monitored for the number of falls onto shock bars that occurred during 10 minutes. Animals were euthanized with carbon dioxide followed by cervical dislocation. Animals were photographed after the removal of the skin from the torso and limbs. The quadriceps, gastrocnemius, triceps, soleus, extensor digitorum longus (EDL), and diaphragm muscles were removed, weighed, and frozen in liquid nitrogen–cooled isopentane. Mice were euthanized with carbon dioxide and the EDL and soleus muscles rapidly removed and placed into a dissecting chamber containing a room temperature (23°C) Ringer solution (124 nM NaCl, 4 nM KCl, 1 nM MgCl2, 1 nM KH2PO4, 25 nM NaHCO3, and 2 nM CaCl2 gassed with 95% O2 to 5% CO2, pH 7.40). Muscles were prepared for study by tying a small loop onto the distal and proximal tendon at the fiber tendon junction with 4-0 silk thread.24Troup J.P. Metzger J.M. Fitts R.H. Effect of high-intensity exercise training on functional capacity of limb skeletal muscle.J Appl Physiol (1985). 1986; 60: 1743-1751PubMed Google Scholar To determine the contractile properties and fatigability of the EDL and soleus, each muscle was transferred to an experimental chamber containing Ringer solution (95% O2 to 5% CO2) and suspended with the 4-0 loops between a Cambridge model 352 ergometer (Aurora Scientific, Aurora, ON) and a fixed post. Each preparation was adjusted to its optimal length at which maximal twitch and tetanic tension was elicited (resting tension = 2 g) and isometric contractile properties studied as described previously and briefly reviewed here.25Hurst J.E. Fitts R.H. Hindlimb unloading-induced muscle atrophy and loss of function: protective effect of isometric exercise.J Appl Physiol (1985). 2003; 95: 1405-1417Crossref PubMed Scopus (49) Google Scholar The muscles were stimulated along their entire length with platinum wire electrodes. Peak twitch tension was elicited with 0.5-ms supermaximal pulse, and contraction time was measured as the time from the onset of force development to peak force. Half relaxation time (½RT) was the time required for force to decay to half of the maximum twitch value. Peak tetanic tension (Po) was elicited by stimulation at 100 Hz for 500 ms for the soleus and 150 Hz for 200 ms for the EDL. For the twitch and tetanus, the maximum rate of force development and decay were determined by measuring the peak slope of the contraction and relaxation, respectively. The output from the transducer was amplified and sampled at 3.3 kHz by a Pentium computer using custom-made software. The force-velocity association was determined, as described previously.25Hurst J.E. Fitts R.H. Hindlimb unloading-induced muscle atrophy and loss of function: protective effect of isometric exercise.J Appl Physiol (1985). 2003; 95: 1405-1417Crossref PubMed Scopus (49) Google Scholar At the plateau of peak tetanic contraction, the computer switched the ergometer from length to force control, and the load on the muscle was rapidly stepped to maintain three loads less than peak force. Force was held at each load for 100 ms for the soleus and 40 ms for the EDL, during which time the change in muscle length was monitored. The computer calculated the velocity of shortening from the slope of the length change during the last half of each load step. This procedure was repeated four times, such that 12 different loads were studied for each muscle. Loads were expressed as a percentage of peak force. Vmax was calculated by the straight-line form of the Hill equation (P0 − P)/V = P/b + a/b by using loads ≤50% of P0, where P is force, V is velocity, and a and b are constants with the dimensions of force and velocity, respectively. A hyperbolic curve was fit to the data by using the Hill equation (P + a) (V + b) = (P0 + a) b. Muscle power was calculated from the fitted force-velocity parameters and the maximum isometric force that was developed during the experiment.25Hurst J.E. Fitts R.H. Hindlimb unloading-induced muscle atrophy and loss of function: protective effect of isometric exercise.J Appl Physiol (1985). 2003; 95: 1405-1417Crossref PubMed Scopus (49) Google Scholar Absolute power was defined as the product of force (in mN) and shortening velocity (ML/s), yielding a final value of mN·ML−1·s−1. Cross sections (8 μm) of isopentane-frozen quadriceps muscles were taken midway down the length of the muscle and stained with hematoxylin and eosin or Gomori trichrome for evaluation using an Olympus BX53 microscope with an Olympus DP72 camera and cellSens standard software (Olympus, Center Valley, PA). Fiber size was determined through measurements of MinFeret diameter14Lawlor 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 (50) Google Scholar because this measurement offers a myofiber size measurement that is relatively independent of fiber orientation.26Brooke 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 To evaluate fiber size and fiber type–specific responses, 8-μm frozen transverse sections of quadriceps muscle were double-stained with rabbit anti-dystrophin antibodies (ab15277; 1:100; Abcam, Cambridge, MA) and mouse monoclonal antibodies against myosin heavy chain type 2b (clone BF-F3; 1:50; Developmental Studies Hybridoma Bank, Iowa City, IA). Secondary antibodies included Alexa Fluor 488–conjugated anti-mouse IgM (1:400; Sigma-Aldrich, St. Louis, MO) and Alexa Fluor 488–conjugated anti-rabbit IgG (1:200; Molecular Probes, Carlsbad, CA). Because of variation in the number and type of oxidative fibers (type 1 and 2a fibers) in mouse muscle, quantitation 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 muscle from six vehicle-treated WT mice, six ActRIIB-mFc–treated WT mice, six vehicle-treated Acta1 H40Y mice, and six ActRIIB-mFc–treated Acta1 H40Y mice. MinFeret diameter was evaluated using a novel automated technique developed by Dr. Lin Yang, as we have previously reported.14Lawlor 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 (50) Google Scholar, 15Lawlor M.W. Viola M.G. Meng H. Edelstein R.V. Liu F. Yan K. Luna E.J. Lerch-Gaggl A. Hoffmann R.G. Pierson C.R. Buj-Bello A. Lachey J.L. Pearsall S. Yang L. Hillard C.J. Beggs A.H. Differential muscle hypertrophy is associated with satellite cell numbers and Akt pathway activation following activin type IIB receptor inhibition in Mtm1 p.R69C mice.Am J Pathol. 2014; 184: 1831-1842Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar Glutaraldehyde-fixed strips of quadriceps muscle were collected from some mice, and electron microscopy was performed at the Medical College of Wisconsin's Electron Microscopy Core Facility. Organs were removed and fixed in 10% neutral buffered formalin for subsequent paraffin embedding at the Children's Hospital of Wisconsin Research Institute Histology Core, as previously described.27Kheir J.N. Scharp L.A. Borden M.A. Swanson E.J. Loxley A. Reese J.H. Black K.J. Velazquez L.A. Thomson L.M. Walsh B.K. Mullen K.E. Graham D.A. Lawlor M.W. Brugnara C. Bell D.C. McGowan Jr., F.X. Oxygen gas-filled microparticles provide intravenous oxygen delivery.Sci Transl Med. 2012; 4: 140ra88Crossref PubMed Scopus (84) Google Scholar, 28Moghadaszadeh B. Rider B.E. Lawlor M.W. Childers M.K. Grange R.W. Gupta K. Boukedes S.S. Owen C.A. Beggs A.H. Selenoprotein N deficiency in mice is associated with abnormal lung development.FASEB J. 2013; 27: 1585-1599Crossref Scopus (29) Google Scholar The histopathologic findings of all thoracic and abdominal organs were assessed in a pilot group of four to six animals per treatment group using hematoxylin and eosin–stained slides. After recognizing the distribution of genitourinary disease in Acta1 H40Y males, the pelvic contents of subsequent cohorts of mice were dissected and histologically evaluated in the sagittal and axial planes. Because the axial view of the membranous urethra offered the most consistent view of skeletal muscle features between animals, blocks revealing axial sections of the membranous urethra at a comparable site (including the urethra, prostate, striated muscle, and rectum) were further evaluated by staining for dystrophin (ab15277; Abcam) or skeletal muscle actin (ab52218; Abcam) to determine the degree of NM-associated disease in urethral striated muscles and visualized using biotinylated horse anti-rabbit IgG (BA-1100; Vector Laboratories, Burlingame, CA). These findings were quantified by manually counting the number of fibers with actin-positive aggregates and the total number of fibers in a single medium-powered (magnification, ×100) field from each of the two urethral striated muscle fascicles that were reproducibly found along the lateral aspects of the urethra in axial sections. The skeletal muscle actin antibody that was used had cross-reactivity with cardiac actin and smooth muscle actin on testing of this antibody in cardiac and aortic tissue, respectively. Muscle tissues from the quadriceps muscle was 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) containing protease inhibitor (Roche, Basel, Switzerland) and phosphatase inhibitor (Roche). Western blot procedures were performed as previously described.15Lawlor M.W. Viola M.G. Meng H. Edelstein R.V. Liu F. Yan K. Luna E.J. Lerch-Gaggl A. Hoffmann R.G. Pierson C.R. Buj-Bello A. Lachey J.L. Pearsall S. Yang L. Hillard C.J. Beggs A.H. Differential muscle hypertrophy is associated with satellite cell numbers and Akt pathway activation following activin type IIB receptor inhibition in Mtm1 p.R69C mice.Am J Pathol. 2014; 184: 1831-1842Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar, 29Wattanasirichaigoon 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 a variety of antigens. Antibodies recognizing the following antigens were all obtained from Cell Signaling Technologies (Danvers, MA): Akt (4691), phospho-Akt (Ser473; 4060), phospho-Akt (Thr308; 2965), p70-S6K (2708), phospho-p70-S6K (Thr421/Ser424; 9204), S6 Ribosomal Protein (2217), phospho-S6 Ribosomal Protein (Ser240/244; 5364), phospho-eEF2k (Ser366; 3691), and phospho-4E-BP1 (Thr37/46; 2855). Other antibodies used for Western blot studies recognize: myostatin (MAB788; R&D Systems, Minneapolis, MN), ActRIIB (ab76940; Abcam), and glyceraldehyde-3-phosphate dehydrogenase (G8795; Sigma-Aldrich) and were visualized using 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 glyceraldehyde-3-phosphate dehydrogenase was performed with ImageJ software version 1.48 (NIH, Bethesda, MD; http://imagej.nih.gov/ij).30Rasband W.S. ImageJ. National Institutes of Health, Bethesda, Maryland1997-2012Google Scholar Samples from four animals per treatment group were used for analysis. Statistical evaluation was performed using Prism software version 6.0 (GraphPad Software, Inc., La Jolla, CA) by SAS software version 9.2 (SAS Institute Inc., Cary, NC) or STATA software version 13.1 (StataCorp, Chicago, IL). For statistical analysis of animal weight, forelimb grip strength, op