Differential Muscle Hypertrophy Is Associated with Satellite Cell Numbers and Akt Pathway Activation Following Activin Type IIB Receptor Inhibition in Mtm1 p.R69C Mice

• DOI: 10.1016/j.ajpath.2014.03.003
• Publication Date: 2014-04-13T09:02:51Z
• Authors: Michael W. Lawlor; Marissa G. Viola; Hui Meng; Rachel V. Edelstein; Fujun Liu; Ke Yan; Elizabeth J. Luna; Alexandra Lerch-G...; Raymond G. Hoffmann; Christopher R. Pi...; Anna Buj-Bello; Jennifer L. Lachey; Scott Pearsall; Lin Yang; Cecilia J. Hillard; Alan H. Beggs
• Keywords: Activin receptor

X-linked myotubular myopathy is a congenital 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 2b myofibers and modest increases strength life span in the severely myopathic Mtm1δ4 mouse model myopathy. have now performed similar study less symptomatic Mtm1 p.R69C hopes finding greater treatment efficacy. Activin animals behavioral histological evidence gastrocnemius muscles but not quadriceps or triceps. The ability respond correlated treatment-induced satellite cell number several muscle-specific abnormalities hypertrophic signaling. Treatment-responsive displayed lower levels phosphorylated ribosomal protein S6 higher eukaryotic elongation factor 2 kinase than were observed muscle wild-type littermates. Hypertrophy was associated increased S6. Our findings indicate muscle-, fiber type-, mutation-specific factors affect response therapies will be important assess future therapeutic trials. (XLMTM) form estimated incidence 1:50,000 male births most presents weakness failure.1Heckmatt J.Z. Sewry C.A. Hodes D. Dubowitz V. Congenital centronuclear (myotubular) A clinical, pathological genetic eight children.Brain. 1985; 108: 941-964Crossref PubMed Scopus (91) Google Scholar, 2Jungbluth H. Wallgren-Pettersson C. Laporte J. Centronuclear myopathy.Orphanet J Rare Dis. 2008; 3: 26Crossref (237) Scholar Many patients XLMTM die within first year despite use ventilation, no treatments approved Food Drug Administration are available. mutations gene encodes myotubularin (MTM1), which phosphoinositide phosphatase thought involved endosomal trafficking, cytoskeletal organization, apoptosis, and/or maintenance sarcoplasmic reticulum/T-tubular system myofibers.3Buj-Bello A. Fougerousse F. Schwab Y. Messaddeq N. Spehner Pierson C.R. Durand M. 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Wermer-Colan Lachey Seehra J.S. Inhibition IIb lifespan myotubularin-deficient mice.Am 178: 784-793Abstract (51) Scholar) moderately mice,13Pierson Dulin-Smith A.N. Durban Marshall M.L. J.T. Snyder A.D. Naiyer Gladman Chandler D.S. Lawlor Dowling Modeling exon 4 skipping phenotype.Hum 21: 811-825Crossref (47) both myofiber pathology seen XLMTM. Because mice, we had previously hypothesized correction would greatly improve strength. Inhibitors myostatin nonfunctional decoys its receptor, (ActRIIB), can used inhibit this negative regulator size, leading hypertrophy. Myostatin binds (and signals through) ActRIIB activate transforming factor-β pathway, prevents progression through cycle down-regulates key processes related hypertrophy.14McCroskery S. Thomas Maxwell Sharma Kambadur negatively regulates activation self-renewal.J Cell Biol. 2003; 162: 1135-1147Crossref (600) 15Joulia-Ekaza Cabello G. regulation development: molecular basis, natural mutations, physiopathological aspects.Exp Res. 2006; 312: 2401-2414Crossref (4) trial ActRIIB-mFC 17% extension span, transient weight, forelimb grip strength, restricted animals.12Lawlor Interestingly, ActRIIB-mFc produces all types (WT) mice,12Lawlor 16Cadena Tomkinson K.N. Monnell T.E. Spaits Kumar Underwood K.W. Pearsall R.S. soluble promotes independent type.J Appl Physiol. 2010; 109: 635-642Crossref (116) interferes pathways oxidative fibers. transience effects treated mice may been disease, so repeated affected mouse.13Pierson Surprisingly, did produce significant animal weight only muscles. these main difference treatment-responsive (gastrocnemius) treatment-resistant (quadriceps) low 6 (p-rpS6) high (eEF2K) other WT mice. rpS6 eEF2K terminal signaling molecules insulinlike factor-1/Akt extracellular signal-related (ERK) fine-tuning global synthesis, role determination remains unclear (reviewed Meyuhas17Meyuhas Physiological roles S6: one kind.Int Rev 268: 1-37Crossref (173) Scholar). agents does always correlate activities known pathways, such Akt unexpectedly varies nature mutation. These highlight much still do understand about control emphasize importance evaluating multiple trials therapies. All studies approval Institutional Animal Care Use Committee Boston Children's Hospital. Genotyping MTM1/C57BL6 (Mtm1δ4) p.R69C/C57BL6 (Mtm1 p.R69C) described.12Lawlor 13Pierson 18Buj-Bello Laugel Zahreddine Pellissier essential for myogenesis mice.Proc 2002; 99: 15060-15065Crossref Male given intraperitoneal injections twice per week (alias RAP-031; Acceleron Pharma, Cambridge, MA) dose 20 mg/kg equivalent volume Tris-buffered saline (the vehicle ActRIIB-mFc), described12Lawlor 14 days until months (MOL). Animals sacrificed MOL because plateau observable effects. weighed during period. Forelimb measured weekly Chatillon force meter (Columbus Instruments, Columbus, OH) placing on horizontal grid allowing it pull away experimenter using fore limbs. maximum three measurements, 1-minute recovery period subsequent statistical analysis. To evaluate antigravity hanging performance, tested rigid mesh surface, inverting surface height approximately 40 cm above their cage, recording amount time necessary fall back into cage. 60 seconds lowered cages. euthanized carbon dioxide followed cervical dislocation, regulations Hospital Boston. photographed intact after removal skin torso quadriceps, gastrocnemius, triceps, soleus, extensor digitorum longus, diaphragm removed, weighed, frozen liquid nitrogen-cooled isopentane. Cross sections (8 μm) isopentane-frozen triceps taken midway down length stained H&E evaluation Olympus BX53 microscope DP72 camera cellSens Standard software version 1.5 (Olympus, Center Valley, PA). Fiber determined measurements MinFeret diameter, used,12Lawlor measurement offers relatively orientation.19Brooke M.H. Engel W.K. histographic analysis regard types. 4. biopsies.Neurology. 1969; 591-605Crossref Overall internally manually micrographs two ×100 magnification fields (using 10× objective lens) H&E-stained slides (range, 259 1113 fibers; n = 5 quantified group). type-specific responses, 8 μm transverse double-stained rabbit anti-dystrophin antibodies (ab15277; Abcam, monoclonal against myosin heavy chain 1 (Skeletal, Slow, clone NOQ7.5.4D; Sigma-Aldrich, St. Louis, MO), 2a (clone SC-71; Developmental Studies Hybridoma Bank, Iowa City, IA), BF-F3; Bank). Secondary included fluorescein isothiocyanate-conjugated anti-mouse IgG (Sigma-Aldrich) IgM (dilution 1:100; Sigma-Aldrich) Alexa Fluor-conjugated anti-rabbit (Molecular Probes, Carlsbad, CA). variation (type fibers) muscle, quantification myosin-positive (glycolytic) myosin-negative (oxidative) populations whole-slide scan four vehicle-treated ActRIIB-mFc–treated six diameter evaluated novel automated technique developed Dr. Lin extremely well our manual measurements.12Lawlor image algorithm began ridge detection enhance boundaries, robust seed based concave area identification find initial seeds final boundaries automatically delineated gradient vector flow deformable model. After each accurately delineated, calculated shortest axis along segmented contour fiber. scanned antibodies. Visiomorph 3.4.3.0 (Visiopharm, Medicon Denmark) allow distinction black pixels inside white/gray (in grayscale) areas dystrophin staining. Bayesian classification segment image, postprocessing steps applied shape disregard counted correspond myofibers. count group cells immunoperoxidase staining Pax7 (Developmental Bank) biotin-SP–conjugated donkey Fab fragment, described.8Lawlor Images counting Pax7+ field dividing total 1117 3631 3 tissues elsewhere hind limbs weeks (WOL) necropsy stored −80°C Frozen crushed nitrogen homogenized lysing buffer (Cell Signaling Technology, Danvers, contained protease (Roche, Basel, Switzerland) (Roche). Western blot procedures described.20Wattanasirichaigoon Swoboda K.J. Takada Tong H.Q. Lip Iannaccone S.T. Laing N.G. Mutations slow alpha-tropomyosin gene, TPM3, rare cause nemaline myopathy.Neurology. 59: 613-617Crossref (76) Transferred proteins probed variety antigens. Antibodies recognize following antigens obtained Technologies: (4691), phospho-Akt (Thr308; 2965), (Ser473; 4060), (2217), phospho-S6 (Ser240/244; 5364), ERK (4695), phospho-ERK (ERK 1/2, Thr202/204; 4370), p70-S6 (S6K; 2708), phospho–p70-S6K (Thr421/Ser424; 9204), p90-ribosomal (RSK)1/2/3 (9355), phospho–p90-RSK (Ser380; 9335), phospho–4E-BP1(Thr37/46; 2855), phospho-eEF2K (Ser366; 3691). Other (MAB788; R&D Systems, Minneapolis, MN), (ab76940; Abcam), glyceraldehyde-3-phosphate dehydrogenase (GAPDH; G8795; visualized chemiluminescent horseradish peroxidase antibody reagent (Denville Scientific, Metuchen, NJ). Adequacy transfer Ponceau Quantification normalized GAPDH ImageJ 1.45s (NIH, Bethesda, MD). Samples additional p.R69C/vehicle p.R69C/ActRIIB-mFc added quantitative p-rpS6 (based replicate gels) further establish interanimal variability. Statistical Prism (GraphPad, Inc., San Diego, CA) statisticians Quantitative Health Sciences Section Medical College Wisconsin. For analyses variance Bonferroni posttests. average one-way posttests performed. data, t-tests compare expression different genotypes treatments, paired same genotype group. In received injections, distinguishable age-matched basis 7 WOL (P < 0.05) (Figure 1A). differences became older continued gain animals, contrast relative animals. showed gains comparison increase age. ActRIIB-mFC–treated consistent slight (averaging g) reach significance show hang up tested) 1B). Treatment performance. Vehicle-treated exhibited significantly impaired performance 0.05), which, although variable, degenerate complete inability mice.12Lawlor consistently 12 1C). also progressed 0.01); however, measurably easily distinguished gross examination.13Pierson smaller proportionately (data shown). At (ie, 5.5 treatment), mass 2A). As described,13Pierson those found mean diameters 24 ± 0.53 51 1.27 μm, respectively, (shown means SEM; P 2). nucleation major feature model, (WT/vehicle 1.43% 0.8%, R69C/vehicle 5.3% 1.0%, WT/vehicle 0.8% 0.3%, 4.9% 1.4% decrease treatment. MOL, evidenced 5-μm (10%) percentage large Immunostaining 2a) glycolytic 2b) subtypes indicated experienced treatment, rightward shift histograms 2, D E). contrast, marked subpopulation compared counterparts, 10-μm (32%) gast