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Fibrosis Rescue Improves Cardiac Function in Dystrophin-Deficient Mice and Duchenne Patient–Specific Cardiomyocytes by Immunoproteasome Modulation

2018; Elsevier BV; Volume: 189; Issue: 2 Linguagem: Inglês

10.1016/j.ajpath.2018.10.010

ISSN

1525-2191

Autores

Andrea Farini, Aoife Gowran, Pamela Bella, Clementina Sitzia, Alessandro Scopece, Elisa Castiglioni, Davide Rovina, Patrizia Nigro, Chiara Villa, Francesco Fortunato, Giacomo P. Comi, Giuseppina Milano, Giulio Pompilio, Yvan Torrente,

Tópico(s)

Cardiac Fibrosis and Remodeling

Resumo

Patients affected by Duchenne muscular dystrophy (DMD) develop a progressive dilated cardiomyopathy characterized by inflammatory cell infiltration, necrosis, and cardiac fibrosis. Standard treatments consider the use of β-blockers and angiotensin-converting enzyme inhibitors that are symptomatic and unspecific toward DMD disease. Medications that target DMD cardiac fibrosis are in the early stages of development. We found immunoproteasome dysregulation in affected hearts of mdx mice (murine animal model of DMD) and cardiomyocytes derived from induced pluripotent stem cells of patients with DMD. Interestingly, immunoproteasome inhibition ameliorated cardiomyopathy in mdx mice and reduced the development of cardiac fibrosis. Establishing the immunoproteasome inhibition–dependent cardioprotective role suggests the possibility of modulating the immunoproteasome as new and clinically relevant treatment to rescue dilated cardiomyopathy in patients with DMD. Patients affected by Duchenne muscular dystrophy (DMD) develop a progressive dilated cardiomyopathy characterized by inflammatory cell infiltration, necrosis, and cardiac fibrosis. Standard treatments consider the use of β-blockers and angiotensin-converting enzyme inhibitors that are symptomatic and unspecific toward DMD disease. Medications that target DMD cardiac fibrosis are in the early stages of development. We found immunoproteasome dysregulation in affected hearts of mdx mice (murine animal model of DMD) and cardiomyocytes derived from induced pluripotent stem cells of patients with DMD. Interestingly, immunoproteasome inhibition ameliorated cardiomyopathy in mdx mice and reduced the development of cardiac fibrosis. Establishing the immunoproteasome inhibition–dependent cardioprotective role suggests the possibility of modulating the immunoproteasome as new and clinically relevant treatment to rescue dilated cardiomyopathy in patients with DMD. Skeletal myopathy and muscular dystrophy progression are commonly associated with cardiac dysfunctions and a consequent high mortality attributable to heart failure.1Beynon R.P. Ray S.G. Cardiac involvement in muscular dystrophies.QJM. 2008; 101: 337-344Crossref PubMed Scopus (21) Google Scholar, 2Judge D.P. Kass D.A. Thompson W.R. Wagner K.R. Pathophysiology and therapy of cardiac dysfunction in Duchenne muscular dystrophy.Am J Cardiovasc Drugs. 2011; 11: 287-294Crossref PubMed Scopus (64) Google Scholar, 3Verhaert D. Richards K. Rafael-Fortney J.A. Raman S.V. Cardiac involvement in patients with muscular dystrophies: magnetic resonance imaging phenotype and genotypic considerations.Circ Cardiovasc Imaging. 2011; 4: 67-76Crossref PubMed Scopus (141) Google Scholar In particular, patients with Duchenne muscular dystrophy (DMD) present with early diastolic dysfunction and myocardial fibrosis that turn into a dilated cardiomyopathy, complicated by heart failure and arrhythmia.4Kamdar F. Garry D.J. Dystrophin-deficient cardiomyopathy.J Am Coll Cardiol. 2016; 67: 2533-2546Crossref PubMed Scopus (188) Google Scholar Even though recent improvements in the management of respiratory insufficiency have improved the lifespan and overall prognosis of patients with DMD, sudden deaths attributable to heart failure negatively affect their quality of life. Prompt treatment and early detection of cardiomyopathy represent the requirements for successful cardioprotective therapies that block or at least slow the processes of cardiac remodeling and heart failure.3Verhaert D. Richards K. Rafael-Fortney J.A. Raman S.V. Cardiac involvement in patients with muscular dystrophies: magnetic resonance imaging phenotype and genotypic considerations.Circ Cardiovasc Imaging. 2011; 4: 67-76Crossref PubMed Scopus (141) Google Scholar Unfortunately, the current treatments for dilated cardiomyopathy are still inadequate because a deep understanding of the specific mechanisms underlying DMD-attributable heart failure is lacking. Common approaches are standard and rely on the use of angiotensin-converting enzyme inhibitors and β-adrenoceptor antagonists.3Verhaert D. Richards K. Rafael-Fortney J.A. Raman S.V. Cardiac involvement in patients with muscular dystrophies: magnetic resonance imaging phenotype and genotypic considerations.Circ Cardiovasc Imaging. 2011; 4: 67-76Crossref PubMed Scopus (141) Google Scholar Most patients with DMD develop cardiomyopathic features between 10 and 15 years of age.4Kamdar F. Garry D.J. Dystrophin-deficient cardiomyopathy.J Am Coll Cardiol. 2016; 67: 2533-2546Crossref PubMed Scopus (188) Google Scholar Because of this tight timeline during which heart dysfunction appears, DMD offers a unique opportunity to assess strategies to limit cardiomyopathy progression. In the heart of the DMD murine animal model (the mdx mouse), from 8 weeks of age, the loss of dystrophin and membrane integrity affects Ca2+ handling and nitric oxide signaling5Williams I.A. Allen D.G. Intracellular calcium handling in ventricular myocytes from mdx mice.Am J Physiol Heart Circ Physiol. 2007; 292: H846-H855Crossref PubMed Scopus (139) Google Scholar so that mdx cardiomyocytes are susceptible to mechanical stress–induced contractile failure and necrosis.6Danialou G. Comtois A.S. Dudley R. Karpati G. Vincent G. Des Rosiers C. Petrof B.J. Dystrophin-deficient cardiomyocytes are abnormally vulnerable to mechanical stress-induced contractile failure and injury.FASEB J. 2001; 15: 1655-1657Crossref PubMed Scopus (140) Google Scholar Similarly, in skeletal muscle, the lack of dystrophin determines the pathological infiltration of immune cells, such as T lymphocytes and macrophages, and the release of inflammatory cytokines, activating NF-κB–dependent pathways.7Nakamura A. Yoshida K. Takeda S. Dohi N. Ikeda S. Progression of dystrophic features and activation of mitogen-activated protein kinases and calcineurin by physical exercise, in hearts of mdx mice.FEBS Lett. 2002; 520: 18-24Crossref PubMed Scopus (78) Google Scholar Dilated cardiomyopathy and contractile deviances are evident in 36- to 40-week–old mdx mice.8Quinlan J.G. Hahn H.S. Wong B.L. Lorenz J.N. Wenisch A.S. Levin L.S. Evolution of the mdx mouse cardiomyopathy: physiological and morphological findings.Neuromuscul Disord. 2004; 14: 491-496Abstract Full Text Full Text PDF PubMed Scopus (215) Google Scholar Cardiac stress has been recognized for its main role in the up-regulation of inflammatory cytokines and growth factors and the generation of reactive oxygen species, which modulate different signaling cascades, whose dysfunctions cause altered cytokine secretion and fibrosis that commonly affects dystrophic hearts. In particular, the Janus kinase (JAK) STAT can transduce the signal of IL-6, IL-10, and interferon-γ by means of the selective phosphorylation of STAT1/3.9Barry S.P. Townsend P.A. Latchman D.S. Stephanou A. Role of the JAK-STAT pathway in myocardial injury.Trends Mol Med. 2007; 13: 82-89Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar Similarly, STAT phosphorylation can be driven by different serine kinases, such as p38 mitogen–activated protein kinase and extracellular-regulated kinase (ERK).9Barry S.P. Townsend P.A. Latchman D.S. Stephanou A. Role of the JAK-STAT pathway in myocardial injury.Trends Mol Med. 2007; 13: 82-89Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar In addition, STATs together with NF-κB synergistically up-regulate inducible nitric oxide synthase in DMD muscles, causing the inhibition of the soluble guanylate cyclase 1α,10Xuan Y.T. Guo Y. Han H. Zhu Y. Bolli R. An essential role of the JAK-STAT pathway in ischemic preconditioning.Proc Natl Acad Sci U S A. 2001; 98: 9050-9055Crossref PubMed Scopus (246) Google Scholar whereas STAT3 interacts with osteopontin in extracellular matrix development.11Hilfiker-Kleiner D. Hilfiker A. Fuchs M. Kaminski K. Schaefer A. Schieffer B. Hillmer A. Schmiedl A. Ding Z. Podewski E. Podewski E. Poli V. Schneider M.D. Schulz R. Park J.K. Wollert K.C. Drexler H. Signal transducer and activator of transcription 3 is required for myocardial capillary growth, control of interstitial matrix deposition, and heart protection from ischemic injury.Circ Res. 2004; 95: 187-195Crossref PubMed Scopus (315) Google Scholar A murine model of hyperglycemia found that the proliferative capacity of cardiac fibroblasts and their ability to express collagen was regulated by STAT1/3 possibly through phosphorylated ERK1/2 blockade of collagen expression.12Dai B. Cui M. Zhu M. Su W.L. Qiu M.C. Zhang H. STAT1/3 and ERK1/2 synergistically regulate cardiac fibrosis induced by high glucose.Cell Physiol Biochem. 2013; 32: 960-971Crossref PubMed Scopus (48) Google Scholar Intriguingly, most of these proteins and cytokines are deregulated in patients with DMD and their functions are commonly influenced by the immunoproteasome. The immunoproteasome is formed by the replacement of the catalytic subunits of the constitutive proteasome with other subunits, termed PSMB8, PSMB9, and PSMB10, that are induced by inflammatory stimuli, such as tumor necrosis factor α (TNF-α) and interferon-γ. Moreover, it is a critical regulator of NF-κB signaling driven by selective phosphorylation of STAT1/3.9Barry S.P. Townsend P.A. Latchman D.S. Stephanou A. Role of the JAK-STAT pathway in myocardial injury.Trends Mol Med. 2007; 13: 82-89Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar In the cells of the immune system, the highly expressed immunoproteasome plays a role in generating peptide ligands for major histocompatibility complex class I antigen presentation.13Ebstein F. Kloetzel P.M. Kruger E. Seifert U. Emerging roles of immunoproteasomes beyond MHC class I antigen processing.Cell Mol Life Sci. 2012; 69: 2543-2558Crossref PubMed Scopus (89) Google Scholar The immunoproteasome is also present in the heart and is overexpressed in dystrophic muscles.14Chen C.N. Graber T.G. Bratten W.M. Ferrington D.A. Thompson L.V. Immunoproteasome in animal models of Duchenne muscular dystrophy.J Muscle Res Cell Motil. 2014; 35: 191-201Crossref PubMed Scopus (18) Google Scholar Immunoproteasome up-regulation was observed with concomitant loss of cardiac muscle mass,15Zu L. Bedja D. Fox-Talbot K. Gabrielson K.L. Van Kaer L. Becker L.C. Cai Z.P. Evidence for a role of immunoproteasomes in regulating cardiac muscle mass in diabetic mice.J Mol Cell Cardiol. 2010; 49: 5-15Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar inflammation,16Opitz E. Koch A. Klingel K. Schmidt F. Prokop S. Rahnefeld A. Sauter M. Heppner F.L. Volker U. Kandolf R. Kuckelkorn U. Stangl K. Kruger E. Kloetzel P.M. Voigt A. Impairment of immunoproteasome function by beta5i/LMP7 subunit deficiency results in severe enterovirus myocarditis.PLoS Pathog. 2011; 7: e1002233Crossref PubMed Scopus (66) Google Scholar and myocyte atrophy17Cosper P.F. Harvey P.A. Leinwand L.A. Interferon-gamma causes cardiac myocyte atrophy via selective degradation of myosin heavy chain in a model of chronic myocarditis.Am J Pathol. 2012; 181: 2038-2046Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar and, in contrast, with overexpression of oxygen species and development of atrial fibrosis.18Li J. Wang S. Bai J. Yang X.L. Zhang Y.L. Che Y.L. Li H.H. Yang Y.Z. Novel role for the immunoproteasome subunit PSMB10 in angiotensin II-induced atrial fibrillation in mice.Hypertension. 2018; 71: 866-876Crossref PubMed Scopus (52) Google Scholar So far, there are several promising immunoproteasome inhibitors that have been developed and already assessed in phase 1/2 of clinical trials for selectively treating patients with inflammatory and autoimmune diseases.19Basler M. Mundt S. Bitzer A. Schmidt C. Groettrup M. The immunoproteasome: a novel drug target for autoimmune diseases.Clin Exp Rheumatol. 2015; 33: S74-S79PubMed Google Scholar, 20Mundt S. Basler M. Buerger S. Engler H. Groettrup M. Inhibiting the immunoproteasome exacerbates the pathogenesis of systemic Candida albicans infection in mice.Sci Rep. 2016; 6: 19434Crossref PubMed Scopus (29) Google Scholar Of note, the potent inhibitor ONX-0914, originally named PR957 and specifically targeted toward the highly active subunit LMP7 (β5i) of the immunoproteasome,21Muchamuel T. Basler M. Aujay M.A. Suzuki E. Kalim K.W. Lauer C. Sylvain C. Ring E.R. Shields J. Jiang J. Shwonek P. Parlati F. Demo S.D. Bennett M.K. Kirk C.J. Groettrup M. A selective inhibitor of the immunoproteasome subunit LMP7 blocks cytokine production and attenuates progression of experimental arthritis.Nat Med. 2009; 15: 781-787Crossref PubMed Scopus (443) Google Scholar was successfully used to treat a viral myocarditis by diminishing the expression of proinflammatory cytokines and chemokines, reducing infiltration of inflammatory cells, and leading to a general improvement of the cardiac output.22Althof N. Goetzke C.C. Kespohl M. Voss K. Heuser A. Pinkert S. Kaya Z. Klingel K. Beling A. The immunoproteasome-specific inhibitor ONX 0914 reverses susceptibility to acute viral myocarditis.EMBO Mol Med. 2018; 10: 200-218Crossref PubMed Scopus (36) Google Scholar In line with this result, our previous work found that ONX-0914 treatment modulates dystrophic features in mdx mice by reducing the amount of infiltrating activated T cells, myofiber necrosis, and collagen deposition in skeletal muscle tissues.23Farini A. Sitzia C. Cassani B. Cassinelli L. Rigoni R. Colleoni F. Fusco N. Gatti S. Bella P. Villa C. Napolitano F. Maiavacca R. Bosari S. Villa A. Torrente Y. Therapeutic potential of immunoproteasome inhibition in Duchenne muscular dystrophy.Mol Ther. 2016; 24: 1898-1912Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar The present study confirmed the possibility of using ONX-0914 for inhibiting the immunoproteasome function, therefore countervailing inflammatory cells and fibrosis in dilated cardiomyopathy of mdx mice and improving their hemodynamic performance. ONX-0914 treatment dampened the release of proinflammatory cytokines, decreased major histocompatibility complex I expression, and increased anti-inflammatory FoxP3+ regulatory T lymphocytes. Considering that early dilated cardiomyopathy is characterized by mild left ventricular dysfunction and differs from advanced dilated cardiomyopathy for left ventricular systolic and diastolic dysfunction, the effect of ONX-0914 treatment was tested in mdx mice with early (6-week–old) and advanced (9-month–old) dilated cardiomyopathy. Interestingly, ONX-0914 could both counteract the raising of the symptoms of cardiomyopathy in younger mdx mice and alleviate the pathological findings in older mdx mice. Our study underlines the substantial contribution of the immunoproteasome in infiltrating myocardial immune cells, which actively participate in cardiomyocyte death and successive fibrosis of the DMD heart. Furthermore, the data were translated from mdx mice to human cardiomyocytes derived from the induced pluripotent stem cells (iPSCs) of patients with DMD, which also displayed aberrant involvement of the immunoproteasome pathway. On the basis of the present and the previous findings,23Farini A. Sitzia C. Cassani B. Cassinelli L. Rigoni R. Colleoni F. Fusco N. Gatti S. Bella P. Villa C. Napolitano F. Maiavacca R. Bosari S. Villa A. Torrente Y. Therapeutic potential of immunoproteasome inhibition in Duchenne muscular dystrophy.Mol Ther. 2016; 24: 1898-1912Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar we suggest that the immunoproteasome possesses a key role directly involved in the poor clinical outcomes observed in patients with DMD, therefore representing a promising candidate target for rescuing dystrophic dilated cardiomyopathy. All the procedures performed on living animals comply with Italian law (D.L.vo 116/92 and subsequent additions) and are approved by local ethics committees. This work was authorized by the Ministry of Health and Local University of Milan Committee (authorization number 859/2017-PR, 5247B.35, 10/07/2017). Six-week–old, male, wild-type (C57Bl) mice, 6-week–old mdx mice, and 9-month–old mdx mice were provided by Charles River Laboratories (Wilmington, MA). All animals were housed in a controlled ambient environment (12-hour light/dark cycle) at a temperature between 21°C and 23°C. The mice had free access to clean water and food. Intraperitoneal injection of the immunoproteasome inhibitor ONX-0914 (6 mg/kg; CliniSciences, Nanterre, France) was performed on 6-week–old and 9-month–old mdx mice for 5 weeks (two injections per week, n = 10). Untreated, aged-matched mdx mice were used as controls. All investigations were conducted after informed consent was provided under regulation of local ethics committee approval (Centro Cardiologico Monzino, Milan, Italy). Human fibroblasts were isolated from the skin biopsy specimens of patients with DMD. Healthy adult dermal fibroblasts were obtained from tebu-bio (Le-Perray-en-Yvelines, France) and served as controls. Fibroblasts were culture expanded in Dulbecco's modified Eagle's medium supplemented with 10% HyClone fetal bovine serum (GE Healthcare Life Sciences, Buckinghamshire, UK), 1× MEM Non-Essential Amino Acid Solution, 2 mmol/L l-glutamine (both from Stemcell Technologies, Vancouver, Canada), and basic fibroblast growth factor. To generate iPSCs, fibroblasts were transfected with four episomal vectors (pCXLE-hUL, pCXLE-hSK, pCXLE-hOCT3/4-shp53-F, and a positive control pCXLE-EGFP) by electroporation (1650 V, 10 ms, 3 pulses) with the Neon transfection system (Invitrogen, Carlsbad, CA). Transfected fibroblasts were grown on human recombinant vitronectin–coated multiwell plates and maintained in solution for 48 hours at which point the media was changed to TeSR reprogramming media (Stemcell Technologies) with daily media changes. Emergent iPSC colonies were manually isolated between posttransfection days 21 to 30 with a 25-G syringe and seeded onto vitronectin-coated multiwell plates in mTeSR1 media (Stemcell Technologies). Fresh medium was replaced daily. From postoperative day 4 onward iPSCs were passaged without the use of enzymes every 3 to 4 days with ReLeSR (Stemcell Technologies) and plated as cell aggregates onto vitronectin-coated multiwell plates. Alkaline phosphatase activity was detected in iPSCs after incubation with Alkaline Phosphatase Live Stain (Invitrogen) for 30 minutes at 37°C. After washing, fluorescent-labeled colonies were visualized with a fluorescein isothiocyanate (FITC) filter and 20× objective (ApoTome, Zeiss, Oberkochen, Germany). For the analysis of pluripotency protein expression, stage-specific embryonic antigen-4 (SSEA4) was detected using a commercially available antibody (mouse anti-SSEA4, 1:100 in 5% normal goat serum overnight at 4°C; Abcam, Cambridge, UK) and revealed by Alexa Fluor 488 anti-mouse secondary antibody (1:400). Nuclei were counterstained with Hoechst 33,342 (Invitrogen). iPSCs were analyzed with FITC filter and 20× objective (LSM710; Zeiss). For FACS analyses, iPSCs were gently dissociated using cell dissociation reagent (Stemcell Technologies) and stained with Tra-1-60 (1:100, 1 hour; Abcam). Five percent bovine serum albumin (BSA; Sigma-Aldrich, St. Louis, MO) in phosphate-buffered saline was used as blocking solution for excluding nonspecific staining (Lonza, Caravaggio, Italy). Goat anti-mouse IgM FITC (1:200, 1 hour; Life Technologies, Carlsbad, CA) was exploited as secondary antibody. Cells were analyzed using a FACSCalibur (BD Biosciences, Franklin Lakes, NJ) or Gallios (Beckman Coulter Life Sciences, Indianapolis, IN) flow cytometers. Cardiomyocyte differentiation of iPSCs was performed following the Lian et al24Lian Q. Zhang Y. Zhang J. Zhang H.K. Wu X. Zhang Y. Lam F.F. Kang S. Xia J.C. Lai W.H. Au K.W. Chow Y.Y. Siu C.W. Lee C.N. Tse H.F. Functional mesenchymal stem cells derived from human induced pluripotent stem cells attenuate limb ischemia in mice.Circulation. 2010; 121: 1113-1123Crossref PubMed Scopus (424) Google Scholar monolayer-directed cardiomyocyte differentiation protocol. Briefly, on day 0 of differentiation, iPSCs were treated with a GSK3 inhibitor [12 μmol/L CHIR99021 in RPMI 1640 medium supplemented with insulin-free B27 (Selleck Chemicals LLC, Houston, TX, and Invitrogen, respectively)]. The media was replaced with RPMI supplemented with insulin-free B27 after 24 hours. On day 3, a combined medium was prepared that contained 1 mL of conditioned media and 1 mL of 10 μmol/L IWP2 (a Wnt signaling inhibitor, final concentration of 5 μmol/L) in RPMI supplemented with insulin-free B27. On day 5, the combined medium was replaced with RPMI supplemented with insulin-free B27. On day 7, the medium was changed to RPMI supplemented with B27 containing insulin. From this point, the medium was changed every 3 days. After 16 days in culture, cardiomyocytes derived from human iPSCs (CMs-d-iPSCs) were processed for further analysis. For immunofluorescence analyses of cardiac troponin T type 2 (cTnT2), a commercial cardiomyocyte characterization kit (Life Technologies) was used following the manufacturer's instructions, with the exception of a secondary antibody that was conjugated to Alexa Fluor 633 (Life Technologies). Cell dissociation reagent (Stemcell Technologies) was used for gently dissociating CM-d-iPSCs for flow cytometry analysis. Afterward, cells underwent fixation, permeabilization (BD Biosciences), and blocking using 5% BSA (Sigma-Aldrich) in phosphate-buffered saline (Lonza). Cells were stained with an anti-cTnT2 antibody (1:100, 1 hour; Life Technologies) followed by goat anti-mouse IgG1 FITC (1:200, 1 hour; Life Technologies). Cells were analyzed using a FACS Calibur flow cytometer (BD Biosciences). After 14 days of differentiation, CMs-d-iPSCs were harvested and plated onto fibronectin-coated multielectrode array plates. Cardiac depolarization and repolarization, T waves, and field potential durations were detected with the Maestro multielectrode array system's cardiac beat detector data processor (Axion BioSystems, Atlanta, GA). Mean field potential durations were obtained with the Comprehensive in Vitro Proarrhythmia Assay analysis tool version 9.2 (Axion BioSystems) and plotted using the AxIS Metric plotting Tool (Axion BioSystems). The determination of dystrophin protein expression in CMs-d-iPSCs by Western blotting was performed by standard techniques using an anti-dystrophin antibody that recognizes human dystrophin (1:500; Abcam) and an anti–glyceraldehyde-3-phosphate dehydrogenase antibody as a housekeeping protein (1:2000; Abcam). Intracellular Ca2+ was determined with a Fluo-4 Calcium Imaging Kit immunofluorescence-based assay kit (Life Technologies) and random images were captured with an ApoTome (Zeiss). Pixel intensity or percent area were calculated using ImageJ software version 1.51i (NIH, Bethesda, MD; http://imagej.nih.gov/ij). cTnI or TNF-α in CMs-d-iPSC–conditioned media was measured with commercial enzyme-linked immunosorbent assay kits (Life Technologies) and normalized to total protein. Animals were sacrificed by cervical dislocation, and cardiac muscles of ONX-0914 treated and untreated mdx mice were collected for both histologic and biochemical analyses. Muscles destined to collagen staining were frozen in liquid-nitrogen–cooled isopentane and cut on a cryostat (Leica CM1850) into 8 μm. Staining of sections was performed with rabbit anti–collagen 1 antibody (COL1A; Cell Signaling Technology, Danvers, MA). Briefly, heart tissues were blocked for 60 minutes with 3% BSA in phosphate-buffered saline at room temperature and then incubated overnight at 4°C with primary antibody diluted 1:300 in blocking solution. Monoclonal anti-rabbit 594 (Molecular Probe, Invitrogen) was added at a dilution of 1:200 in blocking solution for 1 hour. Nuclei were stained with DAPI (Sigma-Aldrich), and images were captured with a Leica TCS SP2 confocal microscope. Before sacrificed, other animals were subjected to cardiac perfusion with saline followed by a 10% formalin flush. To determine the amount of fibrosis, cardiac tissues were stained by Azan Mallory: the percentage of collagen area per section (stained in blue) was measured by ImageJ. Hearts were isolated from ONX-0914–treated and untreated mdx mice and total protein concentration obtained as previously described.25Parolini D. Meregalli M. Belicchi M. Razini P. Lopa R. Del Carlo B. Farini A. Maciotta S. Bresolin N. Porretti L. Pellegrino M. Torrente Y. CD20-related signaling pathway is differently activated in normal and dystrophic circulating CD133(+) stem cells.Cell Mol Life Sci. 2009; 66: 697-710Crossref PubMed Scopus (10) Google Scholar Samples were resolved on polyacrylamide gels (ranging from 6% to 12%) and transferred to nitrocellulose membranes (Bio-Rad Laboratories, Hercules, CA). The membranes were incubated overnight with the primary antibodies against: PSMB5 (1:500; Abcam); PSMB8 (1:500; Abcam); PSMB9 (1:500; Abcam); β tubulin III (1:400; Sigma-Aldrich); vinculin (1:1000; Santa Cruz Biotechnology, Santa Cruz, TX); osteopontin (1:500; R&D Systems, Minneapolis, MN); FoxP3 (1:500; eBioscience, San Diego, CA); TNF-α (1:500; eBioscience); transient receptor potential channel 1 (TRPC1; 1:500; Santa Cruz Biotechnology); STAT1 (1:400; Cell Signaling Technology); phosphorylated STAT1 (1:400; Cell Signaling Technology); STAT-3 (1:400; Cell Signaling Technology); and phosphorylated STAT3 (1:400; Cell Signaling Technology). After incubation, the membranes were detected with peroxidase-conjugated secondary antibodies (Agilent Technologies, Santa Clara, CA) and developed by ECL (Amersham Biosciences, Little Chalfont, UK). Cardiac biopsy specimens were collected from ONX 0914–treated and untreated mdx mice, and the samples were prepared for analysis as previously described.26Sitzia C. Farini A. Colleoni F. Fortunato F. Razini P. Erratico S. Tavelli A. Fabrizi F. Belicchi M. Meregalli M. Comi G. Torrente Y. Improvement of endurance of DMD animal model using natural polyphenols.Biomed Res Int. 2015; 2015: 680615Crossref PubMed Scopus (8) Google Scholar Mitochondrial respiratory chain enzymes and the citrate synthase activities were measured by means of a spectrophotometer as previously described.27Bresolin N. Zeviani M. Bonilla E. Miller R.H. Leech R.W. Shanske S. Nakagawa M. DiMauro S. Fatal infantile cytochrome c oxidase deficiency: decrease of immunologically detectable enzyme in muscle.Neurology. 1985; 35: 802-812Crossref PubMed Google Scholar The value of citrate synthase was used to normalize the values of the other complexes. Total RNA was extracted from cardiac biopsies obtained from ONX-0914–treated and untreated mdx mice. cDNA was generated using the Reverse Transcriptase Kit (ThermoFisher Scientific) followed by the SYBR-Green method to quantify the expression of the genes listed in Table 1. All the cDNA samples were tested in duplicate, and the threshold cycle (CT) of each target gene was normalized against GAPDH, which was considered a housekeeping gene. Relative transcript levels were calculated from the CT values as X = 2−Δct, where X is the fold difference in amount of target gene versus GAPDH and ΔCT = CTtarget − CTGAPDH.Table 1List of Primers for Quantitative RT-PCRPrimer namePrimer sequencem-TNF-α Forward5′-CTACCTTGTTGCCTCCTCTTT-3′ Reverse5′-GAGCAGAGGTTCAGTGATGTAG-3′m-IL-1β Forward5′-TCTGATGGGCAACCACTTAC-3′ Reverse5′-GTTGACAGCTAGGTTCTGTTCT-3′m-CCL2 Forward5′-TTTCTTAAATGCAAGGTGTGGA-3′ Reverse5′-CCTTGGAATCTCAAACACAAAGT-3′m-ICAM-1 Forward5′-AGTAGATCAGTGAGGAGGTGAA-3′ Reverse5′-GCATCCTGACCAGTAGAGAAAC-3′m-ROR-γt Forward5′-GACTGACAATCAGCAGGGATAA-3′ Reverse5′-GGGAAATACAATGAGGTATTGAAAGG-3′COL3A Forward5′-GCCTTCTACACCTGCTCCTG-3′ Reverse5′-GATCCAGGATGTCCAGAGG-3′CCL2, chemokine (C-C motif) ligand 2; COL3A, collagen 3A; ICAM-1, intercellular adhesion molecule 1; m-, mouse-specific; ROR-γt, retinoic acid receptor–related orphan receptor γ; TNF-α, tumor necrosis factor α. Open table in a new tab CCL2, chemokine (C-C motif) ligand 2; COL3A, collagen 3A; ICAM-1, intercellular adhesion molecule 1; m-, mouse-specific; ROR-γt, retinoic acid receptor–related orphan receptor γ; TNF-α, tumor necrosis factor α. Histochemical staining was imaged with an Axioskop II microscope using a digital camera (AxioCamColor) and Axiovision software version 4.7 (all by Zeiss), a Leica TCS SP2 confocal system, or a Zeiss 710 multiphoton confocal mircroscope with ZEN software version 2010D. Densitometric analyses and manual or automatic counting (Threshold Color Plug-in) were performed using ImageJ software version 2.0.0-rc-2 in 20 sections per muscle. Data were analyzed by GraphPad Prism software version 6 (GraphPad Software, San Diego, CA) and expressed as means ± SD or means ± SEM. To compare multiple group means, one-way and two-way analysis of variance was used, and multiplicity adjusted P values for each comparison were reported for each analysis. For the comparison of two groups, a t-test was applied assuming equal variances. The difference among groups was considered significant at P < 0.05. The number of animals per experiment was calculated with the help of the dedicated G POWER online software version 3.1,28Colatsky T. Fermini B. Gintant G. Pierson J.B. Sager P. Sekino Y. Strauss D.G. Stockbridge N. The comprehensive in vitro proarrhythmia assay (CiPA) initiative — update on progress.J Pharmacol Toxicol Methods. 2016; 81: 15-20Crossref PubMed Scopus (258) Google Scholar considering a power of 80% and a significance level of P < 0.05. Based on literature data, the minimum number of animals per experimental group required to observe statistically significant differences is 6 mice per group imaging (ultrasonographic and hemodynamic) and molecular techniques, whereas 4 mice per group are required for immunohistochemical analyses for ea

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