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Targeting the Activin Type IIB Receptor to Improve Muscle Mass and Function in the mdx Mouse Model of Duchenne Muscular Dystrophy

2011; Elsevier BV; Volume: 178; Issue: 3 Linguagem: Inglês

10.1016/j.ajpath.2010.11.071

ISSN

1525-2191

Autores

Emidio E. Pistilli, Sasha Bogdanovich, Marcus D. Goncalves, Rexford S. Ahima, Jennifer Lachey, Jasbir Seehra, Tejvir S. Khurana,

Tópico(s)

Cardiomyopathy and Myosin Studies

Resumo

The activin receptor type IIB (ActRIIB) is a transmembrane receptor for transforming growth factor-β superfamily members, including myostatin, that are involved in the negative regulation of skeletal muscle mass. We tested the translational hypothesis that blocking ligand binding to ActRIIB for 12 weeks would stimulate skeletal muscle growth and improve muscle function in the mdx mouse. ActRIIB was targeted using a novel inhibitor comprised of the extracellular portion of the ActRIIB fused to the Fc portion of murine IgG (sActRIIB), at concentrations of 1.0 and 10.0 mg/kg−1 body weight. After 12 weeks of treatment, the 10.0 mg/kg−1 dose caused a 27% increase in body weight with a concomitant 33% increase in lean muscle mass. Absolute force production of the extensor digitorum longus muscle ex vivo was higher in mice after treatment with either dose of sActRIIB, and the specific force was significantly higher after the lower dose (1.0 mg/kg−1), indicating functional improvement in the muscle. Circulating creatine kinase levels were significantly lower in mice treated with sActRIIB, compared with control mice. These data show that targeting the ActRIIB improves skeletal muscle mass and functional strength in the mdx mouse model of DMD, providing a therapeutic rationale for use of this molecule in treating skeletal myopathies. The activin receptor type IIB (ActRIIB) is a transmembrane receptor for transforming growth factor-β superfamily members, including myostatin, that are involved in the negative regulation of skeletal muscle mass. We tested the translational hypothesis that blocking ligand binding to ActRIIB for 12 weeks would stimulate skeletal muscle growth and improve muscle function in the mdx mouse. ActRIIB was targeted using a novel inhibitor comprised of the extracellular portion of the ActRIIB fused to the Fc portion of murine IgG (sActRIIB), at concentrations of 1.0 and 10.0 mg/kg−1 body weight. After 12 weeks of treatment, the 10.0 mg/kg−1 dose caused a 27% increase in body weight with a concomitant 33% increase in lean muscle mass. Absolute force production of the extensor digitorum longus muscle ex vivo was higher in mice after treatment with either dose of sActRIIB, and the specific force was significantly higher after the lower dose (1.0 mg/kg−1), indicating functional improvement in the muscle. Circulating creatine kinase levels were significantly lower in mice treated with sActRIIB, compared with control mice. These data show that targeting the ActRIIB improves skeletal muscle mass and functional strength in the mdx mouse model of DMD, providing a therapeutic rationale for use of this molecule in treating skeletal myopathies. The muscular dystrophies are a group of progressive, hereditary primary myopathies that result in skeletal muscle degeneration and wasting.1Adams R.D. Victor M. Principles of Neurology.in: ed 4. McGraw-Hill, New York1989: 1117-1132Google Scholar Duchenne's muscular dystrophy (DMD) and the milder allelic Becker form are X-linked diseases resulting from a genetic mutation in the dystrophin gene (DMD), leading to a loss or severe reduction of functional dystrophin protein.2Hoffman E.P. Brown Jr, R.H. Kunkel L.M. 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Inhibition of myostatin in adult mice increases skeletal muscle mass and strength.Biochem Biophys Res Commun. 2003; 300: 965-971Crossref PubMed Scopus (427) Google Scholar and an improvement in the dystrophic phenotype in the mdx mouse model of DMD.21Bogdanovich S. Krag T.O. Barton E.R. Morris L.D. Whittemore L.A. Ahima R.S. Khurana T.S. Functional improvement of dystrophic muscle by myostatin blockade.Nature. 2002; 420: 418-421Crossref PubMed Scopus (718) Google Scholar, 22Bogdanovich S. Perkins K.J. Krag T.O. Whittemore L.A. Khurana T.S. Myostatin propeptide-mediated amelioration of dystrophic pathophysiology.FASEB J. 2005; 19: 543-549Crossref PubMed Scopus (203) Google Scholar, 23Wagner K.R. McPherron A.C. Winik N. Lee S.J. Loss of myostatin attenuates severity of muscular dystrophy in mdx mice.Ann Neurol. 2002; 52: 832-836Crossref PubMed Scopus (325) Google Scholar Additional ligands bind to ActRIIB, including activin A, NODAL, bone morphogenetic protein 2, bone morphogenetic protein-6/7, and growth/differentiation factor 11.33de Caestecker M. The transforming growth factor-beta superfamily of receptors.Cytokine Growth Factor Rev. 2004; 15: 1-11Abstract Full Text Full Text PDF PubMed Scopus (372) Google Scholar Some of these ligands may act in concert with MSTN to inhibit muscle growth, as recently suggested.34Lee 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 In the present study, we used a soluble form of ActRIIB (sActRIIB) as a means to suppress multiple ligands that bind to ActRIIB, and we tested the translational hypothesis that sActRIIB therapy would stimulate skeletal muscle growth and improve muscle function in the mdx mouse model of DMD. Our data demonstrate that 12 weeks of sActRIIB therapy increases body mass, muscle mass, and muscle function in the mdx mouse, providing rationale for the therapeutic use of this strategy for improving muscle wasting in skeletal myopathies such as DMD. Male C57BL/10ScSn-Dmdmdx/J (mdx) and C57BL/10ScSn wild-type mice were purchased from Jackson Laboratories (Bar Harbor, ME) at 4 weeks of age. Mice were exposed to a 12-hour light/dark cycle and 22°C ambient temperature, and received rodent chow (Lab Diet 5001; PMI Nutrition International, Gray Summit, MO) and water ad libitum. Mice were acclimated at the animal facility at the University of Pennsylvania before initiation of experiments. The mdx mice were randomly assigned to receive either sActRIIB injections (n = 14) or sterile PBS injections (n = 13). Control and sActRIIB-treated mice were housed in separate cages during the 12-week preclinical trial. Age-matched C57BL/10ScSn mice (n = 6) were used as untreated wild-type controls. All experiments were approved by the Institutional Animal Care and Use Committee at the University of Pennsylvania. Inhibition of ActRIIB signaling was achieved using a fusion protein comprised of a form of the extracellular domain of ActRIIB linked to the Fc portion of murine IgG (RAP-031; Acceleron Pharma, Cambridge, MA). Two independent drug trials were performed in mdx mice, at concentrations of 1.0 and 10.0 mg/kg−1 body weight. The mdx mice in each group were compared with a separate group of age-matched control mdx mice. Experimental mice received intraperitoneal injections of either sActRIIB or sterile PBS two times per week for 12 weeks. sActRIIB activity in vivo was verified through qPCR for expression of the transgelin gene (Tagln; synonym, Sm22) and Western immunoblotting analysis (phosphorylated Akt) analysis for markers downstream of ActRIIB, using treated and control muscles35Qiu P. Feng X.H. Li L. Interaction of Smad3 and SRF-associated complex mediates TGF-beta1 signals to regulate SM22 transcription during myofibroblast differentiation.J Mol Cell Cardiol. 2003; 35: 1407-1420Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar, 36Goncalves M.D. Pistilli E.E. Balduzzi A. Birnbaum M.J. Lachey J. Khurana T.S. Ahima R.S. Akt deficiency attenuates muscle size and function but not the response to ActRIIB inhibition.PLoS One. 2010; 5: e12707Crossref PubMed Scopus (47) Google Scholar, 37Chen S. Kulik M. Lechleider R.J. Smad proteins regulate transcriptional induction of the SM22alpha gene by TGF-beta.Nucleic Acids Res. 2003; 31: 1302-1310Crossref PubMed Scopus (96) Google Scholar, 38Amirouche A. Durieux A.C. Banzet S. Koulmann N. Bonnefoy R. Mouret C. Bigard X. Peinnequin A. Freyssenet D. Down-regulation of Akt/mammalian target of rapamycin signaling pathway in response to myostatin overexpression in skeletal muscle.Endocrinology. 2009; 150: 286-294Crossref PubMed Scopus (205) Google Scholar (see Supplementary Figure S1 at http://ajp.amjpathol.org). The mdx mice were weighed twice per week to determine the correct volume of sActRIIB to be delivered and to plot growth curves. In the 10.0 mg/kg−1 group, body composition was determined during week 11 of treatment using nuclear magnetic resonance quantification (Echo Medical Systems), as described previously.39Wong T. Hildebrandt M.A. Thrasher S.M. Appleton J.A. Ahima R.S. Wu G.D. Divergent metabolic adaptations to intestinal parasitic nematode infection in mice susceptible or resistant to obesity.Gastroenterology. 2007; 133: 1979-1988Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar Physiological properties, including isometric twitch force, contraction time of the twitch, half-relaxation time of the twitch, isometric tetanic force, and force drop after eccentric lengthening contractions, were quantified in freshly dissected extensor digitorum longus (EDL) muscles from 16-week-old mdx mice, as described previously.21Bogdanovich S. Krag T.O. Barton E.R. Morris L.D. Whittemore L.A. Ahima R.S. Khurana T.S. Functional improvement of dystrophic muscle by myostatin blockade.Nature. 2002; 420: 418-421Crossref PubMed Scopus (718) Google Scholar, 22Bogdanovich S. Perkins K.J. Krag T.O. Whittemore L.A. Khurana T.S. Myostatin propeptide-mediated amelioration of dystrophic pathophysiology.FASEB J. 2005; 19: 543-549Crossref PubMed Scopus (203) Google Scholar Muscle length was adjusted to obtain the maximal twitch response and this length was measured and recorded as optimal length. Three maximal twitch and tetanic contractions were performed, followed by a series of five eccentric lengthening contractions. The force drop from eccentric lengthening contractions was calculated as the percent difference in maximum force between the first and fifth eccentric lengthening contraction. Muscles were stimulated in a Ringer's solution composed of 100 mmol/L NaCl, 4.7 mmol/L KCl, 3.4 mmol/L CaCl2, 1.2 mmol/L KH2PO4, 1.2 mmol/L MgSO4, 25 mmol/L HEPES, and 5.5 mmol/L d-glucose. At the end of the physiological assessment, EDL muscles were incubated in an oxygenated 1% w/v Procion Orange solution for 30 minutes, flash frozen in isopentane cooled to the temperature of liquid nitrogen, and stored at −80°C until used for sectioning. Muscle cross-sectional area (CSA) was calculated by dividing the muscle mass by the product of the muscle density coefficient (1.06 g · cm−3), muscle optimal length, and the fiber length coefficient for EDL muscles, as described previously40Brooks S.V. Faulkner J.A. Contractile properties of skeletal muscles from young, adult and aged mice.J Physiol. 1988; 404: 71-82PubMed Google Scholar, 41Lynch G.S. Hinkle R.T. Chamberlain J.S. Brooks S.V. Faulkner J.A. Force and power output of fast and slow skeletal muscles from mdx mice 6–28 months old.J Physiol. 2001; 535: 591-600Crossref PubMed Scopus (249) Google Scholar: CSA = EDL mass/(Lo × fiber length coefficient × 1.06 g/cm3). Frozen muscle sections (10 μm thickness) were cut at the midbelly of the muscle and fixed in ice-cold methanol for 5 minutes. Diaphragm and tibialis anterior sections were processed for histological examination with H&E and Masson's trichrome stains. For morphometric analyses, EDL muscle sections were stained with an anti-laminin monoclonal antibody (Sigma-Aldrich, St. Louis, MO), incubated with Alexa 488 goat anti-mouse secondary antibody (Invitrogen, Carlsbad, CA), incubated with the DNA-binding dye Hoechst 33825, and visualized using epi-fluorescence illumination on an Olympus BX51 microscope at an objective magnification of 10× or 40×. Digital images were acquired using an Olympus Magnafire digital camera. Morphometric measurements were made using ImageJ image processing software version 1.43 (NIH, Bethesda, MD; http://rsb.info.nih.gov/ij/). Morphometric measurements included single-fiber CSA, total number of fibers, percentage of centrally nucleated fibers, and number of Procion Orange-positive fibers. Blood was collected from all experimental mice via cardiac puncture and was allowed to clot for 30 minutes at room temperature. Serum was collected after centrifugation for 10 minutes at 4.0 relative centrifugal force. Creatine kinase (CK) levels were quantified using a kit as per manufacturer's instructions (catalog no. 2910-430; Stanbio Laboratories, Boerne, TX). In addition to serum samples, normal and abnormal serum standards were included in all analyses to validate the assay (catalog no. G427, G428; Stanbio Laboratories). The content of the amino acid hydroxyproline has been used as a measure of the extent of fibrosis in dystrophic skeletal muscle.42Gargioli C. Coletta M. De Grandis F. Cannata S.M. Cossu G. PlGF-MMP-9-expressing cells restore microcirculation and efficacy of cell therapy in aged dystrophic muscle.Nat Med. 2008; 14: 973-978Crossref PubMed Scopus (103) Google Scholar The tibialis anterior muscle and the diaphragm were used for hydroxyproline quantification (AAA Laboratory, Mercer Island, WA). Total RNA was isolated from gastrocnemius muscles as described previously.43Pistilli E.E. Siu P.M. Alway S.E. Interleukin-15 responses to aging and unloading-induced skeletal muscle atrophy.Am J Physiol Cell Physiol. 2007; 292: C1298-C1304Crossref PubMed Scopus (60) Google Scholar RNA quantity and integrity was accessed using a NanoDrop spectrophotometer (Thermo Scientific, Wilmington, DE); the 260/280 ratio for all samples used was between 1.9 and 2.1. Two micrograms of total RNA was reverse transcribed to make cDNA using a high-capacity cDNA reverse transcription kit according to the manufacturer's instructions (Applied Biosystems, Foster City, CA). The wells of a 96-well optical reaction plate were loaded with a 20-μL volume consisting of 10× PCR master mix (Applied Biosystems, Foster City, CA), cDNA diluted in sterile H2O, and either a primer mix for the gene of interest or the housekeeping gene 18S (Rn18s).The relative quantification of each gene was calculated using the ΔΔCt method, with one control B6129 cDNA sample used as the assay calibrator. Quadriceps tissue was homogenized in lysis buffer containing 50 mmol/L Tris · HCl (pH 7.4), 250 mmol/L mannitol, 50 mmol/L NaF, 1 mmol/L sodium pyrophosphate, 1 mmol/L benzamidine, and 1 mmol/L phenylmethylsulfonyl fluoride with 0.5% (w/v) Triton X-100, supplemented with complete protein inhibition cocktail tablet from Roche (Penzberg, Germany), as described previously.36Goncalves M.D. Pistilli E.E. Balduzzi A. Birnbaum M.J. Lachey J. Khurana T.S. Ahima R.S. Akt deficiency attenuates muscle size and function but not the response to ActRIIB inhibition.PLoS One. 2010; 5: e12707Crossref PubMed Scopus (47) Google Scholar Protein extracts (50 μg) were separated using 4% to 12% NuPAGE Bis-Tris gels (Invitrogen) and transferred to nitrocellulose membranes with wet transfer cells (Bio-Rad Laboratories, Hercules, CA). After 1 hour of blocking with Tris-buffered saline with 0.1% (v/v) Tween 20 containing 10% (w/v) nonfat dried milk, membranes were incubated with a polyclonal antibody against phosphorylated (Ser473) Akt, Akt1/2/3 (Santa Cruz), and GAPDH (Cell Signaling Technology, Danvers, MA). Blots were washed with the Tris-buffered saline with Tween 20 and were incubated with anti-rabbit IgG conjugated to horseradish peroxidase (Santa Cruz Biotechnology, Santa Cruz, CA). The signals were detected with enhanced enzymatic chemiluminescence (Amersham-GE Healthcare, Chalfont St Giles, UK). Student's t-test was used to analyze the data from the two independent trials, using independent control groups for comparisons with the two doses of sActRIIB used. Data were analyzed using the GraphPad Prism 4 statistical software package (GraphPad Software, La Jolla, CA), with statistical significance set at P < 0.05. Data are reported as means ± SEM. To test the effects of sActRIIB therapy, two independent preclinical drug trials were performed in which 4-week-old mdx mice received intraperitoneal injections twice weekly for 12 weeks at a dose of 1.0 mg/kg−1 or 10.0 mg/kg−1 body weight and were compared with trial-matched control mdx mice that received equal volumes of sterile PBS. Mice treated with the 10.0 mg/kg−1 dose had a significant increase in body weight, which began after only 3.5 weeks of treatment (ie, seven injections; Figure 1A). This resulted in a 27% increase in body weight at the termination of the trial (Figure 1B). In contrast, the body weight of mdx mice that received the 1.0 mg/kg−1 dose was not significantly different from control (Figure 1, A and B). Nuclear magnetic resonance quantification revealed that treatment with the 10.0 mg/kg−1 dose produced a selective increase in muscle mass. Skeletal muscle mass was 33% greater in treated mdx mice, compared with control mdx mice, with no changes in the amount of adipose tissue mass (Figure 1C). The increases in lean mass were clearly visible in the gross muscle anatomy of mice treated with the 10.0 mg/kg−1 dose, compared with control. The upper and lower limb skeletal muscles of treated mice, as well as the muscles along the caudal region, were all visibly larg

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