Mitochondrial DNA and TLR9 drive muscle inflammation upon Opa1 deficiency
2018; Springer Nature; Volume: 37; Issue: 10 Linguagem: Inglês
10.15252/embj.201796553
ISSN1460-2075
AutoresAida Rodríguez‐Nuevo, Angels Díaz‐Ramos, Manuel Noguera, Francisco Díaz‐Sáez, Xavier Durán, Juan Pablo Muñoz, Montserrat Romero, Natàlia Plana, David Sebastián, Caterina Tezze, Vanina Romanello, Francesc Ribas‐Aulinas, Jordi Seco, Evarist Planet, Susan R. Doctrow, J. González, Miquel Borràs, Marc Liesa, Manuel Palacı́n, Joan Vendrell, Francesc Villarroya, Marco Sandri, Orian S. Shirihai, António Zorzano,
Tópico(s)Inflammasome and immune disorders
ResumoArticle6 April 2018free access Transparent process Mitochondrial DNA and TLR9 drive muscle inflammation upon Opa1 deficiency Aida Rodríguez-Nuevo Institute for Research in Biomedicine (IRB Barcelona), Barcelona, Spain Departament de Bioquímica i Biomedicina Molecular, Facultat de Biologia, Universitat de Barcelona, Barcelona, Spain CIBER de Diabetes y Enfermedades Metabólicas Asociadas (CIBERDEM), Instituto de Salud Carlos III, Madrid, Spain Search for more papers by this author Angels Díaz-Ramos Institute for Research in Biomedicine (IRB Barcelona), Barcelona, Spain Departament de Bioquímica i Biomedicina Molecular, Facultat de Biologia, Universitat de Barcelona, Barcelona, Spain CIBER de Diabetes y Enfermedades Metabólicas Asociadas (CIBERDEM), Instituto de Salud Carlos III, Madrid, Spain Search for more papers by this author Eduard Noguera Institute for Research in Biomedicine (IRB Barcelona), Barcelona, Spain Departament de Bioquímica i Biomedicina Molecular, Facultat de Biologia, Universitat de Barcelona, Barcelona, Spain CIBER de Diabetes y Enfermedades Metabólicas Asociadas (CIBERDEM), Instituto de Salud Carlos III, Madrid, Spain Search for more papers by this author Francisco Díaz-Sáez Departament de Bioquímica i Biomedicina Molecular, Facultat de Biologia, Universitat de Barcelona, Barcelona, Spain Search for more papers by this author Xavier Duran CIBER de Diabetes y Enfermedades Metabólicas Asociadas (CIBERDEM), Instituto de Salud Carlos III, Madrid, Spain Hospital Universitari de Tarragona Joan XXIII-IISPV, Facultat de Medicina, Universitat Rovira i Virgili, Tarragona, Spain Search for more papers by this author Juan Pablo Muñoz Institute for Research in Biomedicine (IRB Barcelona), Barcelona, Spain Departament de Bioquímica i Biomedicina Molecular, Facultat de Biologia, Universitat de Barcelona, Barcelona, Spain CIBER de Diabetes y Enfermedades Metabólicas Asociadas (CIBERDEM), Instituto de Salud Carlos III, Madrid, Spain Search for more papers by this author Montserrat Romero Institute for Research in Biomedicine (IRB Barcelona), Barcelona, Spain Departament de Bioquímica i Biomedicina Molecular, Facultat de Biologia, Universitat de Barcelona, Barcelona, Spain CIBER de Diabetes y Enfermedades Metabólicas Asociadas (CIBERDEM), Instituto de Salud Carlos III, Madrid, Spain Search for more papers by this author Natàlia Plana Institute for Research in Biomedicine (IRB Barcelona), Barcelona, Spain Departament de Bioquímica i Biomedicina Molecular, Facultat de Biologia, Universitat de Barcelona, Barcelona, Spain CIBER de Diabetes y Enfermedades Metabólicas Asociadas (CIBERDEM), Instituto de Salud Carlos III, Madrid, Spain Search for more papers by this author David Sebastián Institute for Research in Biomedicine (IRB Barcelona), Barcelona, Spain Departament de Bioquímica i Biomedicina Molecular, Facultat de Biologia, Universitat de Barcelona, Barcelona, Spain CIBER de Diabetes y Enfermedades Metabólicas Asociadas (CIBERDEM), Instituto de Salud Carlos III, Madrid, Spain Search for more papers by this author Caterina Tezze Venetian Institute of Molecular Medicine, Padova, Italy Search for more papers by this author Vanina Romanello Venetian Institute of Molecular Medicine, Padova, Italy Search for more papers by this author Francesc Ribas Departament de Bioquímica i Biomedicina Molecular, Facultat de Biologia, Universitat de Barcelona, Barcelona, Spain CIBER Fisiopatologia de la Obesidad y Nutricion, Instituto de Salud Carlos III, Barcelona, Spain Search for more papers by this author Jordi Seco Institute for Research in Biomedicine (IRB Barcelona), Barcelona, Spain Departament de Bioquímica i Biomedicina Molecular, Facultat de Biologia, Universitat de Barcelona, Barcelona, Spain CIBER de Diabetes y Enfermedades Metabólicas Asociadas (CIBERDEM), Instituto de Salud Carlos III, Madrid, Spain Search for more papers by this author Evarist Planet Institute for Research in Biomedicine (IRB Barcelona), Barcelona, Spain Search for more papers by this author Susan R Doctrow Department of Medicine, Boston University School of Medicine, Boston, MA, USA Search for more papers by this author Javier González Experimental Toxicology and Ecotoxicology Unit (CERETOX), Barcelona Science Park, Barcelona, Spain Search for more papers by this author Miquel Borràs Experimental Toxicology and Ecotoxicology Unit (CERETOX), Barcelona Science Park, Barcelona, Spain Search for more papers by this author Marc Liesa orcid.org/0000-0002-5909-8570 Division of Endocrinology, Diabetes and Hypertension, Department of Medicine, UCLA David Geffen School of Medicine, Los Angeles, CA, USA Search for more papers by this author Manuel Palacín Institute for Research in Biomedicine (IRB Barcelona), Barcelona, Spain Departament de Bioquímica i Biomedicina Molecular, Facultat de Biologia, Universitat de Barcelona, Barcelona, Spain CIBER de Enfermedades Raras (CIBERER), Instituto de Salud Carlos III, Barcelona, Spain Search for more papers by this author Joan Vendrell CIBER de Diabetes y Enfermedades Metabólicas Asociadas (CIBERDEM), Instituto de Salud Carlos III, Madrid, Spain Hospital Universitari de Tarragona Joan XXIII-IISPV, Facultat de Medicina, Universitat Rovira i Virgili, Tarragona, Spain Search for more papers by this author Francesc Villarroya Departament de Bioquímica i Biomedicina Molecular, Facultat de Biologia, Universitat de Barcelona, Barcelona, Spain CIBER Fisiopatologia de la Obesidad y Nutricion, Instituto de Salud Carlos III, Barcelona, Spain Search for more papers by this author Marco Sandri Venetian Institute of Molecular Medicine, Padova, Italy Search for more papers by this author Orian Shirihai orcid.org/0000-0001-8466-3431 Department of Medicine, Boston University School of Medicine, Boston, MA, USA Division of Endocrinology, Diabetes and Hypertension, Department of Medicine, UCLA David Geffen School of Medicine, Los Angeles, CA, USA Search for more papers by this author Antonio Zorzano Corresponding Author [email protected] orcid.org/0000-0002-1638-0306 Institute for Research in Biomedicine (IRB Barcelona), Barcelona, Spain Departament de Bioquímica i Biomedicina Molecular, Facultat de Biologia, Universitat de Barcelona, Barcelona, Spain CIBER de Diabetes y Enfermedades Metabólicas Asociadas (CIBERDEM), Instituto de Salud Carlos III, Madrid, Spain Search for more papers by this author Aida Rodríguez-Nuevo Institute for Research in Biomedicine (IRB Barcelona), Barcelona, Spain Departament de Bioquímica i Biomedicina Molecular, Facultat de Biologia, Universitat de Barcelona, Barcelona, Spain CIBER de Diabetes y Enfermedades Metabólicas Asociadas (CIBERDEM), Instituto de Salud Carlos III, Madrid, Spain Search for more papers by this author Angels Díaz-Ramos Institute for Research in Biomedicine (IRB Barcelona), Barcelona, Spain Departament de Bioquímica i Biomedicina Molecular, Facultat de Biologia, Universitat de Barcelona, Barcelona, Spain CIBER de Diabetes y Enfermedades Metabólicas Asociadas (CIBERDEM), Instituto de Salud Carlos III, Madrid, Spain Search for more papers by this author Eduard Noguera Institute for Research in Biomedicine (IRB Barcelona), Barcelona, Spain Departament de Bioquímica i Biomedicina Molecular, Facultat de Biologia, Universitat de Barcelona, Barcelona, Spain CIBER de Diabetes y Enfermedades Metabólicas Asociadas (CIBERDEM), Instituto de Salud Carlos III, Madrid, Spain Search for more papers by this author Francisco Díaz-Sáez Departament de Bioquímica i Biomedicina Molecular, Facultat de Biologia, Universitat de Barcelona, Barcelona, Spain Search for more papers by this author Xavier Duran CIBER de Diabetes y Enfermedades Metabólicas Asociadas (CIBERDEM), Instituto de Salud Carlos III, Madrid, Spain Hospital Universitari de Tarragona Joan XXIII-IISPV, Facultat de Medicina, Universitat Rovira i Virgili, Tarragona, Spain Search for more papers by this author Juan Pablo Muñoz Institute for Research in Biomedicine (IRB Barcelona), Barcelona, Spain Departament de Bioquímica i Biomedicina Molecular, Facultat de Biologia, Universitat de Barcelona, Barcelona, Spain CIBER de Diabetes y Enfermedades Metabólicas Asociadas (CIBERDEM), Instituto de Salud Carlos III, Madrid, Spain Search for more papers by this author Montserrat Romero Institute for Research in Biomedicine (IRB Barcelona), Barcelona, Spain Departament de Bioquímica i Biomedicina Molecular, Facultat de Biologia, Universitat de Barcelona, Barcelona, Spain CIBER de Diabetes y Enfermedades Metabólicas Asociadas (CIBERDEM), Instituto de Salud Carlos III, Madrid, Spain Search for more papers by this author Natàlia Plana Institute for Research in Biomedicine (IRB Barcelona), Barcelona, Spain Departament de Bioquímica i Biomedicina Molecular, Facultat de Biologia, Universitat de Barcelona, Barcelona, Spain CIBER de Diabetes y Enfermedades Metabólicas Asociadas (CIBERDEM), Instituto de Salud Carlos III, Madrid, Spain Search for more papers by this author David Sebastián Institute for Research in Biomedicine (IRB Barcelona), Barcelona, Spain Departament de Bioquímica i Biomedicina Molecular, Facultat de Biologia, Universitat de Barcelona, Barcelona, Spain CIBER de Diabetes y Enfermedades Metabólicas Asociadas (CIBERDEM), Instituto de Salud Carlos III, Madrid, Spain Search for more papers by this author Caterina Tezze Venetian Institute of Molecular Medicine, Padova, Italy Search for more papers by this author Vanina Romanello Venetian Institute of Molecular Medicine, Padova, Italy Search for more papers by this author Francesc Ribas Departament de Bioquímica i Biomedicina Molecular, Facultat de Biologia, Universitat de Barcelona, Barcelona, Spain CIBER Fisiopatologia de la Obesidad y Nutricion, Instituto de Salud Carlos III, Barcelona, Spain Search for more papers by this author Jordi Seco Institute for Research in Biomedicine (IRB Barcelona), Barcelona, Spain Departament de Bioquímica i Biomedicina Molecular, Facultat de Biologia, Universitat de Barcelona, Barcelona, Spain CIBER de Diabetes y Enfermedades Metabólicas Asociadas (CIBERDEM), Instituto de Salud Carlos III, Madrid, Spain Search for more papers by this author Evarist Planet Institute for Research in Biomedicine (IRB Barcelona), Barcelona, Spain Search for more papers by this author Susan R Doctrow Department of Medicine, Boston University School of Medicine, Boston, MA, USA Search for more papers by this author Javier González Experimental Toxicology and Ecotoxicology Unit (CERETOX), Barcelona Science Park, Barcelona, Spain Search for more papers by this author Miquel Borràs Experimental Toxicology and Ecotoxicology Unit (CERETOX), Barcelona Science Park, Barcelona, Spain Search for more papers by this author Marc Liesa orcid.org/0000-0002-5909-8570 Division of Endocrinology, Diabetes and Hypertension, Department of Medicine, UCLA David Geffen School of Medicine, Los Angeles, CA, USA Search for more papers by this author Manuel Palacín Institute for Research in Biomedicine (IRB Barcelona), Barcelona, Spain Departament de Bioquímica i Biomedicina Molecular, Facultat de Biologia, Universitat de Barcelona, Barcelona, Spain CIBER de Enfermedades Raras (CIBERER), Instituto de Salud Carlos III, Barcelona, Spain Search for more papers by this author Joan Vendrell CIBER de Diabetes y Enfermedades Metabólicas Asociadas (CIBERDEM), Instituto de Salud Carlos III, Madrid, Spain Hospital Universitari de Tarragona Joan XXIII-IISPV, Facultat de Medicina, Universitat Rovira i Virgili, Tarragona, Spain Search for more papers by this author Francesc Villarroya Departament de Bioquímica i Biomedicina Molecular, Facultat de Biologia, Universitat de Barcelona, Barcelona, Spain CIBER Fisiopatologia de la Obesidad y Nutricion, Instituto de Salud Carlos III, Barcelona, Spain Search for more papers by this author Marco Sandri Venetian Institute of Molecular Medicine, Padova, Italy Search for more papers by this author Orian Shirihai orcid.org/0000-0001-8466-3431 Department of Medicine, Boston University School of Medicine, Boston, MA, USA Division of Endocrinology, Diabetes and Hypertension, Department of Medicine, UCLA David Geffen School of Medicine, Los Angeles, CA, USA Search for more papers by this author Antonio Zorzano Corresponding Author [email protected] orcid.org/0000-0002-1638-0306 Institute for Research in Biomedicine (IRB Barcelona), Barcelona, Spain Departament de Bioquímica i Biomedicina Molecular, Facultat de Biologia, Universitat de Barcelona, Barcelona, Spain CIBER de Diabetes y Enfermedades Metabólicas Asociadas (CIBERDEM), Instituto de Salud Carlos III, Madrid, Spain Search for more papers by this author Author Information Aida Rodríguez-Nuevo1,2,3, Angels Díaz-Ramos1,2,3, Eduard Noguera1,2,3, Francisco Díaz-Sáez2, Xavier Duran3,4, Juan Pablo Muñoz1,2,3, Montserrat Romero1,2,3, Natàlia Plana1,2,3, David Sebastián1,2,3, Caterina Tezze5, Vanina Romanello5, Francesc Ribas2,6, Jordi Seco1,2,3, Evarist Planet1, Susan R Doctrow7, Javier González8, Miquel Borràs8, Marc Liesa9, Manuel Palacín1,2,10, Joan Vendrell3,4, Francesc Villarroya2,6, Marco Sandri5, Orian Shirihai7,9 and Antonio Zorzano *,1,2,3 1Institute for Research in Biomedicine (IRB Barcelona), Barcelona, Spain 2Departament de Bioquímica i Biomedicina Molecular, Facultat de Biologia, Universitat de Barcelona, Barcelona, Spain 3CIBER de Diabetes y Enfermedades Metabólicas Asociadas (CIBERDEM), Instituto de Salud Carlos III, Madrid, Spain 4Hospital Universitari de Tarragona Joan XXIII-IISPV, Facultat de Medicina, Universitat Rovira i Virgili, Tarragona, Spain 5Venetian Institute of Molecular Medicine, Padova, Italy 6CIBER Fisiopatologia de la Obesidad y Nutricion, Instituto de Salud Carlos III, Barcelona, Spain 7Department of Medicine, Boston University School of Medicine, Boston, MA, USA 8Experimental Toxicology and Ecotoxicology Unit (CERETOX), Barcelona Science Park, Barcelona, Spain 9Division of Endocrinology, Diabetes and Hypertension, Department of Medicine, UCLA David Geffen School of Medicine, Los Angeles, CA, USA 10CIBER de Enfermedades Raras (CIBERER), Instituto de Salud Carlos III, Barcelona, Spain *Corresponding author. Tel: +34 934037197; Fax: +34 934034717; E-mail: [email protected] EMBO J (2018)37:e96553https://doi.org/10.15252/embj.201796553 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Opa1 participates in inner mitochondrial membrane fusion and cristae morphogenesis. Here, we show that muscle-specific Opa1 ablation causes reduced muscle fiber size, dysfunctional mitochondria, enhanced Fgf21, and muscle inflammation characterized by NF-κB activation, and enhanced expression of pro-inflammatory genes. Chronic sodium salicylate treatment ameliorated muscle alterations and reduced the muscle expression of Fgf21. Muscle inflammation was an early event during the progression of the disease and occurred before macrophage infiltration, indicating that it is a primary response to Opa1 deficiency. Moreover, Opa1 repression in muscle cells also resulted in NF-κB activation and inflammation in the absence of necrosis and/or apoptosis, thereby revealing that the activation is a cell-autonomous process and independent of cell death. The effects of Opa1 deficiency on the expression NF-κB target genes and inflammation were absent upon mitochondrial DNA depletion. Under Opa1 deficiency, blockage or repression of TLR9 prevented NF-κB activation and inflammation. Taken together, our results reveal that Opa1 deficiency in muscle causes initial mitochondrial alterations that lead to TLR9 activation, and inflammation, which contributes to enhanced Fgf21 expression and to growth impairment. Synopsis Mitochondrial DNA stress caused by Opa1 deficiency in skeletal muscle leads to inflammation via TLR9 activation, which contributes to enhanced Fgf21 expression and systemic growth impairment. Skeletal muscle-specific Opa1 ablation causes mitochondrial inflammatory myopathy. Opa1 deficiency results in reduced muscle mass, mitochondrial dysfunction and enhanced Fgf21 expression. Opa1 deficiency-dependent inflammation results in severe growth defects. Inflammation is a primary cell-autonomous response of muscle cells to Opa1 deficiency, leading to NF-κB activation. Opa1 deficiency activates TLR9 by a mechanism that requires mitochondrial DNA. Introduction Mitochondrial metabolism, quality control, response to apoptotic stimuli, and hormone activity are partly controlled through the balance between mitochondrial fusion and fission (Twig et al, 2008; Liesa et al, 2009; Sebastian et al, 2012; Liesa & Shirihai, 2013; Munoz et al, 2013). Mitochondrial fusion in mammalian cells is regulated by the proteins mitofusin 1 and mitofusin 2 (Mfn1 and Mfn2) and optic atrophy 1 (Opa1). Various Opa1 isoforms are located in the mitochondrial intermembrane space or inserted within the inner mitochondrial membrane (Olichon et al, 2003; Cipolat et al, 2004; Griparic et al, 2004; Ishihara et al, 2006). The existence of multiple Opa1 isoforms and cleavage mechanisms may explain the role of this protein beyond mitochondrial inner membrane fusion, such as in cristae remodeling, and supercomplex formation (Frezza et al, 2006; Cogliati et al, 2013). In humans, OPA1 mutations have been reported in patients affected by autosomal dominant optic atrophy or ADOA (Alexander et al, 2000; Delettre et al, 2000). Overall, data support the view that the pathogenesis of ADOA occurs as a result of haploinsufficiency (Pesch et al, 2001). Human fibroblasts carrying OPA1 mutations show impaired oxidative phosphorylation and mitochondrial fusion (Amati-Bonneau et al, 2008). Some of these OPA1 missense mutations are associated with altered mitophagy and parkinsonism (Carelli et al, 2015). In addition to dominant optic atrophy, OPA1 mutations also cause a multi-systemic disorder called ADOA plus syndrome, which results in severe myopathy (Amati-Bonneau et al, 2008; Hudson et al, 2008; Zeviani, 2008). Interestingly, these patients show multiple mitochondrial DNA (mtDNA) deletions in muscles, which suggests a role of OPA1 on mtDNA stability. The appropriate homeostasis of mitochondria is essential in the maintenance of cellular health. Mitochondria are a source of damage-associated molecular patterns (DAMPs), and among others, mtDNA has been shown to induce a pro-inflammatory state (Zhang et al, 2010; Oka et al, 2012; Wenceslau et al, 2014). MtDNA has been reported to activate immunity through two distinct pathways, namely Toll-like receptor 9 (TLR9; Zhang et al, 2010; Oka et al, 2012; Liu et al, 2015), and cGAS activation (White et al, 2014; West et al, 2015). Under basal conditions, TLR9 is located in the endoplasmic reticulum (ER), and upon stimulation by inducers, TLR9 translocates to the membrane of endosomes or to lysosomes, bind to ligands, and initiate cellular inflammation (Latz et al, 2004; Zhang et al, 2010; Wei et al, 2015; De Leo et al, 2016). Interaction of TLR9 with mtDNA activates the nuclear factor kappa B (NF-κB) signaling and increases the expression of other pro-inflammatory cytokines such as tumor necrosis factor-α (TNF-α), interleukin (IL)-6, IL-1β (Julian et al, 2013; Yu & Bennett, 2014; Zhang et al, 2014). Recently, cytosolic mtDNA has been reported to engage cytosolic antiviral signaling and to enhance the expression of interferon-stimulated genes (Rongvaux et al, 2014; White et al, 2014; West et al, 2015). Thus, cytosolic mtDNA activates the DNA sensor cGAS and promotes STING-IRF3-dependent signaling (Rongvaux et al, 2014; White et al, 2014; West et al, 2015). Furthermore, neutrophils extrude interferogenic mtDNA by a process dependent in part on lysosomal activity and that occurs in the presence of a constitutive defect in mitophagy (Caielli et al, 2016), Based on the observations that OPA1 mutations cause mtDNA instability (Kim et al, 2005; Amati-Bonneau et al, 2008; Hudson et al, 2008; Yu-Wai-Man et al, 2010), we reasoned that OPA1 deficiency in a non-immune cells should not only alter mitochondrial morphology but also mitochondrial stability, and in consequence trigger immune responses. Based on this, we analyzed the impact of Opa1 loss-of-function in skeletal muscle. Our data indicate that Opa1 deletion in skeletal muscle causes mitochondrial inflammatory myopathy characterized by impaired mitochondrial function, pro-inflammatory cytokine production, altered myofiber morphology, and muscle dysfunction. In addition, we found that the activation of NF-κB in Opa1-deficient muscle cells is mediated by TLR9 and by mtDNA. Results Skeletal muscle-specific Opa1 ablation causes reduced body growth and premature death In preliminary studies, we monitored skeletal muscle regeneration in a cardiotoxin (CTX) injury-induced model in gastrocnemius muscles in wild-type animals at a range of times (Fig EV1A). Two days post-injury (dpi), when satellite cells become active, muscles were treated with adenoviruses encoding for engineered miRNAs against Opa1 (miR Opa1) or with control adenoviruses encoding for miRNA with no homology in the mouse genome (miR Ctrl; Fig EV1B). Opa1 deficiency (miR Opa1) not only caused impaired muscle regeneration but also increased the presence of immune cells and reduced muscle fiber size in regenerating myofibers (Figs 1A and B, and EV1C). Immunostaining of the developmental form of MHC (dMHC) revealed a reduction in controls between 9 and 12 days post-CTX treatment, whereas Opa1-deficient muscles showed sustained high expression of dMHC (Figs 1C and EV1D), indicating impaired muscle fiber maturation. Click here to expand this figure. Figure EV1. Skeletal muscle-specific Opa1 ablation causes reduced body growth and premature death A. Scheme of the experimental design of CTX-induced injury. B. Opa1 protein levels in control C2C12 myoblasts, which were transduced with control miRNA (miR Ctrl) or in Opa1 loss-of-function C2C12 myoblasts, which were transduced with adenoviruses encoding for miRNA against Opa1 (miR Opa1) (n = 5). C. Distribution of myofiber size in mice treated with miR Ctrl or miR Opa1 adenoviruses at dpi 12 (n = 5). D. Representative image of dMHC immunohistochemistry from gastrocnemius muscle treated with miR Ctrl or miR Opa1 adenoviruses (12 dpi). Scale bars, 100 μm. E. Partial genomic structure of the Opa1 gene showing the scission of exon 5, thus deleting all Opa1 protein isoforms. F. Opa1 mRNA levels in the gastrocnemius muscle of loxP (non-expressing Cre Opa1loxP/loxP mice) and skeletal muscle-specific KO mice (KO) (n = 10). G. Opa1 protein levels in tissue homogenates from control (loxP) and skeletal muscle-specific KO mice (KO). H, I. Relative weight of muscles (gastrocnemius (Gast) and quadriceps (Quad)) (panel H) (n = 18) and organs (liver (L), heart (H), kidney (K), spleen (S), T thymus (T), subcutaneous adipose tissue (SAT), visceral adipose tissue (VAT), and brown adipose tissue (BAT)) (panel I) (n = 32) of 9-week-old loxP and KO mice. These data are expressed as g of tissue/g of body weight (BW). J. Distribution of myofiber size of 150 myofibers in quadriceps muscle. K, L. Mean cross-sectional area (CSA) (K) and distribution of myofiber size (L) of 150 myofibers in diaphragm muscle. M. Representative images of hematoxylin and eosin-stained lung sections from loxP and KO mice. Thick arrows indicate congestion and atelectasis. Thin arrows indicate normal parenchyma. Asterisks show bronchioles. Scale bars, 100 μm. Data information: Data represent mean ± SEM. *P < 0.001 vs. control loxP mice. Data were analyzed using Student's t-test. Download figure Download PowerPoint Figure 1. Skeletal muscle-specific Opa1 ablation causes reduced body growth and premature death A. Transversal sections of gastrocnemius muscles of 3-month-old mice injected with CTX as an injury-induced model and, 2 days later, with adenoviruses encoding for non-targeting miRNA (miR Ctrl) or miRNA targeting Opa1 (miR Opa1). Samples were taken on various days after the injury (dpi, n = 5). Scale bars, 100 μm. B. Mean cross-sectional area (CSA) of 150 myofibers per gastrocnemius muscle at dpi 12. C. Quantification of positive MHC myofibers vs. total regenerating myofibers of gastrocnemius muscle treated with miR Ctrl or miR Opa1 adenoviruses at dpi 9 and 12 (n = 5). D. Opa1 protein levels in tissue homogenates of control (loxP) and skeletal muscle-specific KO mice (KO). The skeletal muscle used was gastrocnemius muscle (n = 6). E. Survival curves for loxP and Opa1 KO mice (n = 25). F. Picture of loxP and KO mice at 9 weeks of age. G. Body weight of loxP and KO male and female mice (n = 25). H. Grip strength in loxP and KO mice. loxP (n = 7) and KO (n = 4). I. Mean cross-sectional area (CSA) of 150 myofibers in quadriceps muscle. J–K. Plasma concentration of growth hormone (GH) (J) and Igf1 (K) of loxP (n = 8) and KO mice (n = 10). Data information: Data represent mean ± SEM. *P < 0.001 vs. control loxP mice (with the exception of panel B in which P < 0.01). Data were analyzed using Student's t-test (B, C, I, J, and K) or analysis of variance test (G and H). Download figure Download PowerPoint Based on these data, we analyzed the impact of Opa1 depletion on muscle homeostasis by the generation of skeletal muscle-specific knockout mice. This was performed by crossing homozygous Opa1-loxP/loxP mice with a mouse strain expressing Cre recombinase under the control of the myogenin promoter (Li et al, 2005; Figs 1D and EV1E–G). Opa1 ablation in this specific tissue induced a dramatic reduction in life span (Fig 1E) and impaired normal growth (Fig 1F and G). At 9 weeks of age, Opa1 KO mice showed a reduction in relative weight of skeletal muscle (Fig EV1H) and no change in various organs and tissues (Fig EV1I). Muscle force was also lower in Opa1 KO mice (Fig 1H). In keeping with this, histological analyses of KO mice indicated a marked decrease in fiber size and cross-sectional area in quadriceps and diaphragm (Figs 1I and EV1J, K and L). Consistent with altered diaphragm morphology, we observed extensive congestion and atelectasis in the lungs of these mice (Fig EV1M). In keeping with the reduced growth, plasma levels of GH were high (Fig 1J) and IGF1 were low (Fig 1K). Characterization of the hepatic profile of Opa1 KO mice showed reduced phosphorylation of STAT5 (Fig 2A), and decreased expression of its target genes Igf1 and Fos (Fig 2B). Also, it revealed reduced expression of growth hormone receptor (Ghr) and increased expression of the STAT5 inhibitor Socs3 (Fig 2B). Figure 2. Muscle-specific Opa1 ablation induces growth hormone resistance A. Stat5 phosphorylation in livers of loxP and KO mice (n = 10). B. mRNA levels of Igf1, Fos, growth hormone receptor (Ghr), and Socs3 in liver of loxP and KO mice (n = 5). C. Fgf21 gene expression in muscle of loxP and KO mice (n = 8). D. FGF21 levels in plasma of loxP (n = 8) and KO mice (n = 10). E–G. Opa1 mRNA levels in muscle (E), and Fgf21 gene expression in muscle (F) and liver (G) of loxP, Opa1 KO, FGF21 KO, and double KO (Opa1 + FGF21 KO, DKO) (n = 8). H. Hepatic expression of genes encoding Pgc1α, respiratory complex IV subunit Cox7a1, fatty acid oxidation components (Cpta1, Mcad, and Vlcad), and gluconeogenic enzymes (Pepck and G6p) in loxP, Opa1 KO, DKO, and Fgf21 KO mice (n = 7). I, J. Relative weight of gastrocnemius and quadriceps muscles, (I) and organs (J) of loxP, Opa1 KO, DKO, and Fgf21 KO mice (n = 10). These data are expressed as g of tissue/g of body weight (BW). K. Igf1, Fos, and Ghr mRNA levels in the livers of loxP, Opa1 KO, DKO, and Fgf21 KO mice (n = 10). Data information: Data represent mean ± SEM. *P < 0.05 vs. control loxP mice. #P < 0.05 vs. DKO mice. Data were analyzed using Student's t-test (B–D) or analysis of variance with Tukey's post hoc test (E–K). Download figure Download PowerPoint Gene set enrichment analysis of genomic profiling in muscles of control and Opa1 KO mice revealed the up-regulation of ATF4 target genes in muscles of Opa1 KO mice (Dataset EV1; Kim et al, 2013) among the large number of genes with deregulated expression (Dataset EV2; 486 genes up-regulated and 175 repressed). Fgf21, an ATF4 target gene, was greatly increased in the muscles of Opa1 KO mice (Fig 2C). Circulating Fgf21 levels were fourfold greater in these animals (Fig 2D). Given the observation that Fgf21 causes resistance to GH (Inagaki et al, 2008), we explored the possibility of a rescue by generating a double knockout (DKO) mouse for Fgf21 and Opa1 (Fig 2E–G). DKO mice normalized hepatic PGC-1α expression consistent with the regulatory role of Fgf21 (Potthoff et al, 2009; Fig 2H). In keeping with these observations, Opa1 KO mice showed enhanced expression of hepatic genes relevant in gluconeogenesis, lipid metabolism, or mitochondrial respiration, and DKO mice showed a normalized expression (Fig 2H). In contrast, DKO mice showed similar muscle and tissue weights to those of Opa1 KO mice (Fig 2I and J). Furthermore, DKO failed to enhance the hepatic expression of Igf1 or Fos genes (Fig 2K). In all, these data indicate that Fgf21 mediates the metabolic alterations occurring in liver from Opa1 KO mice, but it is not involved in the reduced growth of Opa1 KO mice. Opa1 ablation causes inflammatory myopathy Histological inspection of Opa1-deficient muscle sections revealed a substantial number of necrotic myofibers, as well as regenerating fibers (centrally located nuclei; Fig 3A). Given the
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