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• W2897011663 abstract "Duchenne muscular dystrophy (DMD) is an X-linked muscle-wasting disease caused by mutations in the dystrophin gene. DMD boys are wheelchair-bound around 12 years and generally survive into their twenties. There is currently no effective treatment except palliative care, although personalized treatments such as exon skipping, stop codon read-through, and viral-based gene therapies are making progress. Patients present with skeletal muscle pathology, but most also show cardiomyopathy by the age of 10. A systemic therapeutic approach is needed that treats the heart and skeletal muscle defects in all patients. The dystrophin-related protein utrophin has been shown to compensate for the lack of dystrophin in the mildly affected BL10/mdx mouse. The purpose of this investigation was to demonstrate that AAV9-mediated micro-utrophin transgene delivery can not only functionally replace dystrophin in the heart, but also attenuate the skeletal muscle phenotype in severely affected D2/mdx mice. The data presented here show that utrophin can indeed alleviate the pathology in skeletal and cardiac muscle in D2/mdx mice. These results endorse the view that utrophin modulation has the potential to increase the quality life of all DMD patients whatever their mutation. Duchenne muscular dystrophy (DMD) is an X-linked muscle-wasting disease caused by mutations in the dystrophin gene. DMD boys are wheelchair-bound around 12 years and generally survive into their twenties. There is currently no effective treatment except palliative care, although personalized treatments such as exon skipping, stop codon read-through, and viral-based gene therapies are making progress. Patients present with skeletal muscle pathology, but most also show cardiomyopathy by the age of 10. A systemic therapeutic approach is needed that treats the heart and skeletal muscle defects in all patients. The dystrophin-related protein utrophin has been shown to compensate for the lack of dystrophin in the mildly affected BL10/mdx mouse. The purpose of this investigation was to demonstrate that AAV9-mediated micro-utrophin transgene delivery can not only functionally replace dystrophin in the heart, but also attenuate the skeletal muscle phenotype in severely affected D2/mdx mice. The data presented here show that utrophin can indeed alleviate the pathology in skeletal and cardiac muscle in D2/mdx mice. These results endorse the view that utrophin modulation has the potential to increase the quality life of all DMD patients whatever their mutation. Duchenne muscular dystrophy (DMD) is a severe and progressive muscle-wasting disorder affecting 1:5,000 boys.1Mendell J.R. Shilling C. Leslie N.D. Flanigan K.M. al-Dahhak R. Gastier-Foster J. Kneile K. Dunn D.M. Duval B. Aoyagi A. et al.Evidence-based path to newborn screening for Duchenne muscular dystrophy.Ann. Neurol. 2012; 71: 304-313Crossref PubMed Scopus (550) Google Scholar, 2Guiraud S. Aartsma-Rus A. Vieira N.M. Davies K.E. van Ommen G.-J.B. Kunkel L.M. The pathogenesis and therapy of muscular dystrophies.Annu. Rev. Genomics Hum. Genet. 2015; 16: 281-308Crossref PubMed Scopus (204) Google Scholar, 3Mah J.K. Korngut L. Dykeman J. Day L. Pringsheim T. Jette N. A systematic review and meta-analysis on the epidemiology of Duchenne and Becker muscular dystrophy.Neuromuscul. Disord. 2014; 24: 482-491Abstract Full Text Full Text PDF PubMed Scopus (277) Google Scholar DMD is caused by mutations in the dystrophin gene leading to the loss of dystrophin, a large structural protein located at the sarcolemma. In its absence, the sarcolemma is highly susceptible to contraction-induced injury causing muscle degeneration and replacement of contractile material with adipose and fibrotic tissue.4Forcina L. Pelosi L. Miano C. Musarò A. Insights into the Pathogenic Secondary Symptoms Caused by the Primary Loss of Dystrophin.J. Funct. Morphol. and Kinesiol. 2017; 2: 44Crossref Scopus (11) Google Scholar, 5Head S. Williams D. Stephenson G. Increased susceptibility of EDL muscles from mdx mice to damage induced by contraction with stretch.J. Muscle Res. Cell Motil. 1994; 15: 490-492PubMed Google Scholar These processes typically lead to loss of ambulation between 8 and 12 years, and patients succumb to respiratory and/or cardiac failure in their second or third decade of life.6Bach J.R. O’Brien J. Krotenberg R. Alba A.S. Management of end stage respiratory failure in Duchenne muscular dystrophy.Muscle Nerve. 1987; 10: 177-182Crossref PubMed Scopus (102) Google Scholar DMD is primarily recognized as a skeletal muscle disorder, and historically, respiratory muscle insufficiencies accounted for the vast majority of patient deaths.7Rideau Y. Gatin G. Bach J. Gines G. Prolongation of life in Duchenne’s muscular dystrophy.Acta Neurol. (Napoli). 1983; 5: 118-124PubMed Google Scholar However, with improved disease management, cardiomyopathy has emerged as a leading cause of death in patients receiving assisted ventilation.8Birnkrant D.J. Ararat E. Mhanna M.J. Cardiac phenotype determines survival in Duchenne muscular dystrophy.Pediatr. Pulmonol. 2016; 51: 70-76Crossref PubMed Scopus (42) Google Scholar Despite recent progress and extensive research over the last three decades, there is still no effective treatment for DMD. Thus, development of treatments that target skeletal and cardiac muscle is essential for improving both quality of life and longevity of DMD patients. We have focused on the development of utrophin modulation therapy using small molecules; this approach will potentially target the skeletal and cardiac muscle and is applicable to all patients irrespective of their mutation. Utrophin is a structural and functional autosomal paralog of dystrophin9Love D.R. Hill D.F. Dickson G. Spurr N.K. Byth B.C. Marsden R.F. Walsh F.S. Edwards Y.H. Davies K.E. An autosomal transcript in skeletal muscle with homology to dystrophin.Nature. 1989; 339: 55-58Crossref PubMed Scopus (420) Google Scholar, 10Tinsley J.M. Blake D.J. Roche A. Fairbrother U. Riss J. Byth B.C. Knight A.E. Kendrick-Jones J. Suthers G.K. Love D.R. et al.Primary structure of dystrophin-related protein.Nature. 1992; 360: 591-593Crossref PubMed Scopus (350) Google Scholar that is localized to the sarcolemma during fetal development and confined to the neuromuscular junction (NMJ) and myotendinous junctions (MTJs) in mature muscle.11Lin S. Burgunder J.-M. Utrophin may be a precursor of dystrophin during skeletal muscle development.Brain Res. Dev. Brain Res. 2000; 119: 289-295Crossref PubMed Scopus (30) Google Scholar, 12Pons F. Robert A. Fabbrizio E. Hugon G. Califano J.-C. Fehrentz J.A. Martinez J. Mornet D. Utrophin localization in normal and dystrophin-deficient heart.Circulation. 1994; 90: 369-374Crossref PubMed Scopus (60) Google Scholar Utrophin is also expressed in regenerating myofibers in adult muscle.13Galvagni F. Cantini M. Oliviero S. The utrophin gene is transcriptionally up-regulated in regenerating muscle.J. Biol. Chem. 2002; 277: 19106-19113Crossref PubMed Scopus (37) Google Scholar, 14Weir A.P. Burton E.A. Harrod G. Davies K.E. A- and B-utrophin have different expression patterns and are differentially up-regulated in mdx muscle.J. Biol. Chem. 2002; 277: 45285-45290Crossref PubMed Scopus (102) Google Scholar Over the last two decades, we have shown in pre-clinical studies that both transgenic overexpression and pharmacological modulation of utrophin prevents skeletal muscle pathology in dystrophin-deficient (mdx) mice.15Tinsley J. Deconinck N. Fisher R. Kahn D. Phelps S. Gillis J.-M. Davies K. Expression of full-length utrophin prevents muscular dystrophy in mdx mice.Nat. Med. 1998; 4: 1441-1444Crossref PubMed Scopus (482) Google Scholar, 16Krag T.O. Bogdanovich S. Jensen C.J. Fischer M.D. Hansen-Schwartz J. Javazon E.H. Flake A.W. Edvinsson L. Khurana T.S. Heregulin ameliorates the dystrophic phenotype in mdx mice.Proc. Natl. Acad. Sci. USA. 2004; 101: 13856-13860Crossref PubMed Scopus (104) Google Scholar, 17Amenta A.R. Yilmaz A. Bogdanovich S. McKechnie B.A. Abedi M. Khurana T.S. Fallon J.R. Biglycan recruits utrophin to the sarcolemma and counters dystrophic pathology in mdx mice.Proc. Natl. Acad. Sci. USA. 2011; 108: 762-767Crossref PubMed Scopus (110) Google Scholar, 18Tinsley J.M. Fairclough R.J. Storer R. Wilkes F.J. Potter A.C. Squire S.E. Powell D.S. Cozzoli A. Capogrosso R.F. Lambert A. et al.Daily treatment with SMTC1100, a novel small molecule utrophin upregulator, dramatically reduces the dystrophic symptoms in the mdx mouse.PLoS ONE. 2011; 6: e19189Crossref PubMed Scopus (138) Google Scholar, 19Guiraud S. Squire S.E. Edwards B. Chen H. Burns D.T. Shah N. Babbs A. Davies S.G. Wynne G.M. Russell A.J. et al.Second-generation compound for the modulation of utrophin in the therapy of DMD.Hum. Mol. Genet. 2015; 24: 4212-4224Crossref PubMed Scopus (57) Google Scholar Although we have shown the capacity of small compounds to induce utrophin expression in the hearts of mdx mice,18Tinsley J.M. Fairclough R.J. Storer R. Wilkes F.J. Potter A.C. Squire S.E. Powell D.S. Cozzoli A. Capogrosso R.F. Lambert A. et al.Daily treatment with SMTC1100, a novel small molecule utrophin upregulator, dramatically reduces the dystrophic symptoms in the mdx mouse.PLoS ONE. 2011; 6: e19189Crossref PubMed Scopus (138) Google Scholar, 19Guiraud S. Squire S.E. Edwards B. Chen H. Burns D.T. Shah N. Babbs A. Davies S.G. Wynne G.M. Russell A.J. et al.Second-generation compound for the modulation of utrophin in the therapy of DMD.Hum. Mol. Genet. 2015; 24: 4212-4224Crossref PubMed Scopus (57) Google Scholar the functional impact of utrophin on the dystrophic myocardium still needs to be fully investigated. Adeno-associated viral (AAV) vectors are well characterized as gene-therapy tools with the capacity for systemic delivery.20Lynch G.S. Rafael J.A. Hinkle R.T. Cole N.M. Chamberlain J.S. Faulkner J.A. Contractile properties of diaphragm muscle segments from old mdx and old transgenic mdx mice.Am. J. Physiol. 1997; 272: C2063-C2068Crossref PubMed Google Scholar, 21Gregorevic P. Blankinship M.J. Allen J.M. Crawford R.W. Meuse L. Miller D.G. Russell D.W. Chamberlain J.S. Systemic delivery of genes to striated muscles using adeno-associated viral vectors.Nat. Med. 2004; 10: 828-834Crossref PubMed Scopus (544) Google Scholar There are multiple serotypes of AAV that have different tissue tropisms; of these, AAV9 can target skeletal muscle and has shown the strongest transduction of the myocardium.22Bostick B. Ghosh A. Yue Y. Long C. Duan D. Systemic AAV-9 transduction in mice is influenced by animal age but not by the route of administration.Gene Ther. 2007; 14: 1605-1609Crossref PubMed Scopus (137) Google Scholar As the 14-kb full-length dystrophin gene is too large to be packaged into an AAV vector (limited to ∼4.8 kb), truncated (“mini” and “micro”) dystrophin constructs have been developed and optimized for use in DMD.23Bostick B. Yue Y. Long C. Marschalk N. Fine D.M. Chen J. Duan D. Cardiac expression of a mini-dystrophin that normalizes skeletal muscle force only partially restores heart function in aged Mdx mice.Mol. Ther. 2009; 17: 253-261Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar, 24Shin J.H. Nitahara-Kasahara Y. Hayashita-Kinoh H. Ohshima-Hosoyama S. Kinoshita K. Chiyo T. Okada H. Okada T. Takeda S. Improvement of cardiac fibrosis in dystrophic mice by rAAV9-mediated microdystrophin transduction.Gene Ther. 2011; 18: 910-919Crossref PubMed Scopus (45) Google Scholar, 25Bostick B. Yue Y. Lai Y. Long C. Li D. Duan D. Adeno-associated virus serotype-9 microdystrophin gene therapy ameliorates electrocardiographic abnormalities in mdx mice.Hum. Gene Ther. 2008; 19: 851-856Crossref PubMed Scopus (81) Google Scholar, 26Townsend D. Blankinship M.J. Allen J.M. Gregorevic P. Chamberlain J.S. Metzger J.M. Systemic administration of micro-dystrophin restores cardiac geometry and prevents dobutamine-induced cardiac pump failure.Mol. Ther. 2007; 15: 1086-1092Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar, 27Bostick B. Shin J.-H. Yue Y. Duan D. AAV-microdystrophin therapy improves cardiac performance in aged female mdx mice.Mol. Ther. 2011; 19: 1826-1832Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar, 28Harper S.Q. Hauser M.A. DelloRusso C. Duan D. Crawford R.W. Phelps S.F. Harper H.A. Robinson A.S. Engelhardt J.F. Brooks S.V. Chamberlain J.S. Modular flexibility of dystrophin: implications for gene therapy of Duchenne muscular dystrophy.Nat. Med. 2002; 8: 253-261Crossref PubMed Scopus (460) Google Scholar Clinical trials investigating micro(μ)-dystrophin (Solid Biosciences; phase I and II; NCT03368742) and mini-dystrophin (Pfizer; phase I b; NTC03362502) for the use in DMD have been initiated.29Solid Biosciences (2017). A Randomized, Controlled, Open-label, Single-ascending Dose, Phase I/II Study to Investigate the Safety and Tolerability, and Efficacy of Intravenous SGT-001 in Male Adolescents and Children With Duchenne Muscular Dystrophy. https://clinicaltrials.gov/ct2/show/NCT03368742.Google Scholar, 30Pfizer (2018). A Phase 1b Multicenter, Open-label, Single Ascending Dose Study To Evaluate The Safety And Tolerability Of Pf-06939926 In Ambulatory Subjects With Duchenne Muscular Dystrophy. https://clinicaltrials.gov/ct2/show/NCT03362502.Google Scholar Similar pre-clinical studies were also conducted using a μ-utrophin construct and were found to improve the pathology of dystrophin and utrophin double knockout (dko) mice31Odom G.L. Gregorevic P. Allen J.M. Finn E. Chamberlain J.S. Microutrophin delivery through rAAV6 increases lifespan and improves muscle function in dystrophic dystrophin/utrophin-deficient mice.Mol. Ther. 2008; 16: 1539-1545Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar and adenovirus-mediated utrophin gene transfer also mitigated the more severe pathological phenotype of golden retriever dogs with canine X-linked muscular dystrophy.32Cerletti M. Negri T. Cozzi F. Colpo R. Andreetta F. Croci D. Davies K.E. Cornelio F. Pozza O. Karpati G. et al.Dystrophic phenotype of canine X-linked muscular dystrophy is mitigated by adenovirus-mediated utrophin gene transfer.Gene Ther. 2003; 10: 750-757Crossref PubMed Scopus (114) Google Scholar Given the capacity of AAV to deliver a transgene to both cardiac and skeletal muscle, we employed an AAV9 serotype carrying the utrophin μ-gene to assess its impact on cardiac function and skeletal muscle pathology. Most of the pre-clinical studies in the mouse have been carried out in C57BL/10ScSn-Dmdmdx/J (BL10/mdx) mice that show a relatively mild phenotype, particularly in the heart. In recent years, the D2.B10-Dmdmdx/J (D2/mdx) mouse model has been recognized as a potential pre-clinical model for DMD due to the lesser regenerative capacity of the D2/2J (D2/wild-type [WT]) parent strain.33Rodrigues M. Echigoya Y. Maruyama R. Lim K.R.Q. Fukada S.I. Yokota T. Impaired regenerative capacity and lower revertant fibre expansion in dystrophin-deficient mdx muscles on DBA/2 background.Sci. Rep. 2016; 6: 38371Crossref PubMed Scopus (30) Google Scholar, 34Fukada S. Morikawa D. Yamamoto Y. Yoshida T. Sumie N. Yamaguchi M. Ito T. Miyagoe-Suzuki Y. Takeda S. Tsujikawa K. Yamamoto H. Genetic background affects properties of satellite cells and mdx phenotypes.Am. J. Pathol. 2010; 176: 2414-2424Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar Coley et al.35Coley W.D. Bogdanik L. Vila M.C. Yu Q. Van Der Meulen J.H. Rayavarapu S. Novak J.S. Nearing M. Quinn J.L. Saunders A. et al.Effect of genetic background on the dystrophic phenotype in mdx mice.Hum. Mol. Genet. 2016; 25: 130-145Crossref PubMed Scopus (120) Google Scholar and Vohra et al.36Vohra R. Batra A. Forbes S.C. Vandenborne K. Walter G.A. Magnetic Resonance Monitoring of Disease Progression in mdx Mice on Different Genetic Backgrounds.Am. J. Pathol. 2017; 187: 2060-2070Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar showed that pathology in D2/mdx mice was more severe than age-matched BL10/mdx mice. Importantly, left ventricle (LV) systolic dysfunction has been shown in 6-month-old D2/mdx mice, whereas the equivalent cardiac defects are not present in BL10/mdx mice until 12 months of age.37Stuckey D.J. Carr C.A. Camelliti P. Tyler D.J. Davies K.E. Clarke K. In vivo MRI characterization of progressive cardiac dysfunction in the mdx mouse model of muscular dystrophy.PLoS ONE. 2012; 7: e28569Crossref PubMed Scopus (56) Google Scholar D2/mdx mice therefore offer a promising and more acutely affected model for cardiac studies. We therefore employed cardiac cine-MRI as it represents the most accurate, non-invasive approach for assessing cardiac function.38Stuckey D.J. Carr C.A. Tyler D.J. Clarke K. Cine-MRI versus two-dimensional echocardiography to measure in vivo left ventricular function in rat heart.NMR Biomed. 2008; 21: 765-772Crossref PubMed Scopus (52) Google Scholar, 39Soslow J.H. Xu M. Slaughter J.C. Stanley M. Crum K. Markham L.W. Parra D.A. Evaluation of Echocardiographic Measures of Left Ventricular Function in Patients with Duchenne Muscular Dystrophy: Assessment of Reproducibility and Comparison to Cardiac Magnetic Resonance Imaging.J. Am. Soc. Echocardiogr. 2016; 29: 983-991Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar Of the parameters acquired by MRI, ejection fraction is the most accurate measure of systolic function and therefore was the primary parameter of interest.39Soslow J.H. Xu M. Slaughter J.C. Stanley M. Crum K. Markham L.W. Parra D.A. Evaluation of Echocardiographic Measures of Left Ventricular Function in Patients with Duchenne Muscular Dystrophy: Assessment of Reproducibility and Comparison to Cardiac Magnetic Resonance Imaging.J. Am. Soc. Echocardiogr. 2016; 29: 983-991Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar The aim of this work was to elucidate the functional impact of utrophin modulation on a more severe model of muscular dystrophy in mice. We explored whether μ-utrophin delivery could attenuate the pathology in skeletal muscle and tested the functional effects of utrophin expression on the dystrophin-deficient heart. Cardiac cine-MRI assessment revealed significantly elevated LV stroke volume, right ventricle (RV) end diastolic volume, and RV stroke volume in 14-week-old BL10/mdx mice compared to age-matched BL10/WT mice (Table 1). No differences were observed between cardiac function of D2/mdx and D2/WT mice at 14 weeks of age (Table 1). Surprisingly, 21-week-old D2/mdx mice showed significantly elevated LV mass, LV ejection fraction, LV stroke volume and RV ejection fraction compared to D2/WT mice (Table 1). No differences were observed in cardiac function between BL10/WT and BL10/mdx mice at 21 -weeks of age (Table 1). At 28 weeks of age, no statistical differences were observed between D2/mdx and D2/WT mice in either LV or RV parameters (Table 1). A significant increase in heart mass normalized to body mass was observed in the hearts of 21-week-old D2/mdx mice compared to D2/WT mice (Table S1). These differences in heart mass were resolved by 28 weeks of age (Table S1). Skeletal muscle pathology of D2/mdx mice was consistent with previous reports35Coley W.D. Bogdanik L. Vila M.C. Yu Q. Van Der Meulen J.H. Rayavarapu S. Novak J.S. Nearing M. Quinn J.L. Saunders A. et al.Effect of genetic background on the dystrophic phenotype in mdx mice.Hum. Mol. Genet. 2016; 25: 130-145Crossref PubMed Scopus (120) Google Scholar (data not shown). As 21-week-old D2/mdx mice exhibited elevated systolic function compared to WT control mice, markers of pathology were assessed in this cohort. D2/mdx mice exhibited more prominent necrosis (Figure 1A), fibrosis (Figure 1B), calcification (Figure 1C) and immunoglobulin G (IgG)-positive regions (Figure 1D) as well as loss of components of the dystrophin glycoprotein complex (DGC) from the sarcolemma (Figures 1E and 1F) compared to age-matched WT controls. Quantification of alizarin red revealed a non-significant increase of calcification in D2/mdx mice compared to D2/WT mice (Figure 1G). Quantification of Masson’s trichrome revealed a significant increase of fibrosis in both BL10/mdx and D2/mdx mice relative to their respective WT control strains (Figure 1H). Phosphorylated glycogen synthase kinase (GSK)-3β and serine/threonine kinase (AKT) are associated with cardiac hypertrophy.40Desai M.S. Shabier Z. Taylor M. Lam F. Thevananther S. Kosters A. Karpen S.J. Hypertrophic cardiomyopathy and dysregulation of cardiac energetics in a mouse model of biliary fibrosis.Hepatology. 2010; 51: 2097-2107Crossref PubMed Scopus (61) Google Scholar Assessment of these markers revealed significantly elevated levels of phosphorylated GSK-3β relative to total expression in the hearts of 21-week-old D2/mdx mice compared to all other groups (Figure 1I). However, there were no significant differences seen in AKT phosphorylation relative to total expression between groups (Figure 1J).Table 1Cardiac Cine-MRI Assessment of 14-, 21-, and 28-Week-Old BL10/WT, BL10/mdx, D2/WT, and D2/mdx Mice14 Weeks21 Weeks28 WeeksLeft VentricleEnd diastolic volumeBL10WT68.51 ± 11.6263.84 ± 5.5460.31 ± 4.43mdx66.25 ± 2.9878.71 ± 5.7777.41 ± 9.74D2WT49.24 ± 4.4946.24 ± 5.9450.25 ± 3.71mdx47.44 ± 2.4357.66 ± 1.9654.03 ± 4.38End systolic volumeBL10WT34.32 ± 10.7422.95 ± 3.4321.70 ± 2.99mdx22.62 ± 2.0326.85 ± 3.7528.32 ± 5.92D2WT12.61 ± 2.1118.13 ± 2.4022.46 ± 3.86mdx14.34 ± 2.7114.89 ± 3.1418.76 ± 2.83Stroke volumeBL10WT34.28 ± 2.8040.89 ± 4.0138.61 ± 2.97mdx43.63 ± 1.82*51.86 ± 2.9749.09 ± 6.01D2WT36.63 ± 3.2228.11 ± 3.8927.78 ± 3.62mdx34.08 ± 1.6842.77 ± 2.51*35.28 ± 4.93Ejection fractionBL10WT53.76 ± 6.0364.32 ± 4.2264.34 ± 3.40mdx66.06 ± 2.1466.34 ± 2.4465.65 ± 6.04D2WT74.77 ± 3.1161.14 ± 2.5355.36 ± 5.95mdx64.18 ± 7.1374.19 ± 4.95*64.25 ± 6.04MassBL10WT112.13 ± 17.98116.58 ± 13.22129.41 ± 5.81mdx109.56 ± 8.00124.49 ± 10.17132.59 ± 14.39D2WT106.71 ± 5.82100.27 ± 10.98107.12 ± 12.02mdx103.05 ± 12.64131.60 ± 7.65*135.05 ± 6.80Right VentricleEnd diastolic volumeBL10WT51.77 ± 4.3950.31 ± 9.2353.65 ± 4.89mdx63.05 ± 2.26*66.20 ± 7.3274.17 ± 9.75D2WT63.41 ± 8.0940.40 ± 8.4339.75 ± 5.13mdx52.13 ± 6.2343.90 ± 6.8240.12 ± 6.26End systolic volumeBL10WT22.81 ± 5.6618.28 ± 3.5317.15 ± 1.66mdx21.24 ± 1.8423.64 ± 4.2527.41 ± 3.62*D2WT14.98 ± 1.6616.63 ± 3.9718.76 ± 4.40mdx20.19 ± 5.8213.51 ± 3.2716.62 ± 3.47Stroke volumeBL10WT28.96 ± 1.4032.03 ± 5.8936.51 ± 4.97mdx41.81 ± 1.24*42.56 ± 5.4146.76 ± 7.23D2WT48.43 ± 7.2323.77 ± 4.8820.99 ± 3.90mdx31.94 ± 2.8730.39 ± 4.6523.49 ± 4.17Ejection fractionBL10WT57.97 ± 6.1364.32 ± 2.6466.97 ± 4.01mdx66.55 ± 2.0464.06 ± 4.8062.40 ± 3.08D2WT75.50 ± 2.7158.55 ± 3.2352.89 ± 7.77mdx63.49 ± 5.4869.52 ± 3.65*58.76 ± 5.42All parameters are expressed as μL with the exception of ejection fraction (%) and mass (mg). Data represented as mean ± SEM, *p < 0.05 versus age-matched WT control, n = 6/group. Open table in a new tab All parameters are expressed as μL with the exception of ejection fraction (%) and mass (mg). Data represented as mean ± SEM, *p < 0.05 versus age-matched WT control, n = 6/group. Western blot analysis revealed expression of the truncated utrophin protein in the cardiac muscle and in the fast type II tibialis anterior (TA) and extensor digitorum longus (EDL) skeletal muscles. Transgene expression was lower in the EDL muscles compared to the TA muscles (Figures 2B, 2E, and 2F ). A lower level of truncated protein expression in diaphragm and the slow type I soleus muscle, which is more complicated to transduce, was noted. Quantification of μ-utrophin protein expression supports these observations (Figure S1A). The AAV genome copy number quantification (Figure S1B) confirmed the expected bio-distribution of the vector genome (vg), and results are in correlation with previous data generated with an AAV9.41Hakim C.H. Wasala N.B. Pan X. Kodippili K. Yue Y. Zhang K. Yao G. Haffner B. Duan S.X. Ramos J. et al.A five-repeat micro-dystrophin gene ameliorated dystrophic phenotype in the severe DBA/2J-mdx model of Duchenne muscular dystrophy.Mol. Ther. Methods Clin. Dev. 2017; 6: 216-230Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar Immunofluorescence (IF) analysis revealed that utrophin was localized to the sarcolemma in cross-sections from the heart, diaphragm, EDL, and TA muscles from treated mice compared to vehicle control mice (Figures 2A–2E). Longitudinal sections from TA muscles also confirmed localization of utrophin to the sarcolemma along the myofibers (Figure 2F). Interestingly, weak or no correlation was evident between μ-utrophin protein expression or vector genome quantification and functional parameters in the EDL muscles, diaphragm muscle strips, or the heart (Figure S2). Muscle architecture was assessed by H&E and revealed improved muscle structure and reduced necrosis in EDL, TA, diaphragm, and soleus muscle cross-sections from D2/mdx mice administered AAV9/CMV-μ-utrophin (AAV-μ-Utro) compared to vehicle control mice (Figure 3A). Ex vivo muscle physiological testing of whole EDL muscles and diaphragm muscle strips was performed to determine the impact of treatment on skeletal muscle function. Despite low μ-utrophin protein expression observed, specific force of EDL muscles (Figure 3B) and diaphragm muscle strips (Figure 3D) was significantly greater in mice treated with AAV-μ-Utro compared to vehicle control mice. Percentage force drop was assessed following a series of eccentric contractions in the EDL muscles and diaphragm muscle strips from treated and vehicle control mice. Percentage force drop was significantly rescued in EDL muscles treated with AAV-μ-Utro and vehicle control mice following eccentric contraction (Figure 3C). Percentage force drop of diaphragm muscle strips following eccentric contraction was not different between groups (Figure 3E). Kyphosis score and muscle mass (TA, EDL, quadriceps, and soleus) were not altered with treatment (Table S2). Cardiac cross-sections from treated and vehicle control mice were assessed for dystrophic pathology. Fewer dystrophic lesions were observed in the hearts of treated D2/mdx mice compared to vehicle control mice (Figure 4A). The hearts from D2/mdx mice treated with AAV-μ-Utro also showed lower levels of calcification and fibrosis compared to vehicle control mice (Figures 4B and 4C). However, only the reduction in fibrosis was statistically significant (Figures 4F and 4G). Components of the DGC are lost from the sarcolemma in dystrophic muscle and undergo proteolysis; therapies aimed at restoring dystrophin and modulating utrophin have shown to maintain these components. IF assessment of β-dystroglycan (β-DG) and β-sarcoglycan (β-SG) revealed a clear increase in localization of β-SG at the sarcolemma of AAV-μ-Utro-treated mice compared to vehicle control mice (Figure 4D). A slight increase of β-DG was also evident by IF assessment (Figure 4E), however to a lesser extent than β-SG. Western blot assessment was therefore also carried out and confirmed elevated levels of β-DG in the hearts of treated mice compared to vehicle control mice (Figure 4H). To assess the impact of treatment on hypertrophic signaling, phosphorylated GSK-3β was assessed by western blot. Reduced phosphorylation of GSK-3β was clearly evident in the hearts of D2/mdx mice administered AAV-μ-Utro compared to vehicle control mice (Figure 4J). The elevated ejection fraction observed in the D2/mdx mice compared to age-matched D2/WT was reduced following AAV-μ-Utro treatment. Reduction in ejection fraction is evident in representative MRI short axis mid-papillary slices (Figure 5A). Quantitation of serial short axis slices revealed significant reductions in LV mass, LV ejection fraction, LV stroke volume, and RV ejection fraction in mice administered AAV-μ-Utro compared to vehicle control mice (Figure 5B). End diastolic volume, end systolic volumes (LV and RV), and RV stroke volume were not significantly attenuated with treatment (Table 2). Assessment of diastolic function revealed no difference between groups in peak ejection rate, peak filling rate, or time to peak ejection rate (Table 2).Table 2Cardiac Cine-MRI Analysis of 21-Week-Old D2/mdx Mice Administered AAV-μ-Utro Compared to Vehicle Control MiceSystolicUntreatedAAV-μ-UtroLeft VentricleEnd diastolic volume (μL)57.03 ± 3.2746.70 ± 4.22End systolic volume (μL)7.43 ± 1.2610.51 ± 1.76Right VentricleEnd diastolic volume (μL)53.45 ± 9.7352.60 ± 9.06End systolic volume (μL)12.14 ± 2.7717.16 ± 3.51Stroke volume (μL)41.32 ± 7.6635.44 ± 6.70DiastolicPeak ejection rate−605.50 ± 57.60−412.76 ± 105.32Peak filling rate (mL/s)474.80 ± 60.21448.72 ± 25.59Time to peak ejection rate (mL/s)8.50 ± 2.3512.33 ± 4.24Data represented as SEM, n = 8/group. Open table in a new tab Data represented as SEM, n = 8/group. The data presented here show that AAV-μ-Utro administration ameliorates the skeletal and cardiac phenotype of D2/mdx mice, which present with a more severe pathology than the BL10/mdx mouse model due to differences in genetic background.34Fukada S. Morikawa D. Yamamoto Y. Yoshida T. Sumie N. Yamaguchi M. Ito T. Miyagoe-Suzuki Y. Takeda S. Tsujikawa K. Yamamoto H. Genetic background affects properties of satellite cells and mdx phenotypes.Am. J. Pathol. 2010; 176: 2414-2424Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar, 35Coley W.D. Bogdanik L. Vila M.C. Yu Q. Van Der Meulen J.H. Rayavarapu S. Novak J.S. Nearing M. Quinn J.L. Saunders A. et al.Effect of genetic background on the dystrophic phenotype in mdx mice.Hum. Mol. Genet. 2016; 25: 130-145Crossref PubMed Scopus (120) Google Scholar The positive effects we observed with μ-utrophin gene delivery in" @default.
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• W2897011663 title "Micro-utrophin Improves Cardiac and Skeletal Muscle Function of Severely Affected D2/mdx Mice" @default.
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