http://www.diva-portal.org This is the published version of a paper published in Investigative Ophthalmology and Visual Science. Citation for the original published paper (version of record): Rodríguez, M A., Liu, J-X., Parkkonen, K., Li, Z., Domellöf, F P. (2018) The Cytoskeleton in the Extraocular Muscles of Desmin Knockout Mice Investigative Ophthalmology and Visual Science, 59(12): 4847-4855 https://doi.org/10.1167/iovs.18-24508 Access to the published version may require subscription. N.B. When citing this work, cite the original published paper. Permanent link to this version: http://urn.kb.se/resolve?urn=urn:nbn:se:umu:diva-153128 Anatomy and Pathology/Oncology The Cytoskeleton in the Extraocular Muscles of Desmin Knockout Mice Maria Angels Rodr´ıguez,1 Jing-Xia Liu,2 Kimmo Parkkonen,1,2 Zhenlin Li,3 and Fatima Pedrosa Domell¨of1 1Department of Clinical Sciences, Ophthalmology, Ume˚a University, Ume˚a, Sweden 2Department of Integrative Medical Biology, Section for Anatomy, Ume˚a University, Ume˚a, Sweden 3Sorbonne Universite,´ Paris, France Correspondence: Fatima Pedrosa PURPOSE. To investigate the effect of absence of desmin on the extraocular muscles (EOMs) Domell¨of, Department of Clinical with focus on the structure and composition of the cytoskeleton. Sciences, Ophthalmology, Ume˚a University, Ume˚a 901 85, Sweden; METHODS. The distribution of synemin, syncoilin, plectin, nestin, and dystrophin was [email protected]. evaluated on cross and longitudinal sections of EOMs and limb muscles from 1-year-old desmin knockout mice (desminÀ/À) by immunofluorescence. General morphology was Submitted: April 5, 2018 Accepted: August 15, 2018 evaluated with hematoxylin and eosin while mitochondrial content and distribution were evaluated by succinate dehydrogenase (SDH) and modified Gomori trichrome stainings. Citation: Rodr´ıguez MA, Liu J-X, Park- À/À konen K, Li Z, Pedrosa Domell¨of F. RESULTS. The muscle fibers of the EOMs in desmin mice were remarkably well preserved in The cytoskeleton in the extraocular contrast to those in the severely affected soleus and the slightly affected gastrocnemius muscles of desmin knockout mice. muscles. There were no signs of muscular pathology in the EOMs and all cytoskeletal proteins Invest Ophthalmol Vis Sci. studied showed a correct location at sarcolemma and Z-discs. However, an increase of SDH 2018;59:4847–4855. https://doi.org/ staining and mitochondrial aggregates under the sarcolemma was detected. 10.1167/iovs.18-24508 CONCLUSIONS. The structure of the EOMs was well preserved in the absence of desmin. We suggest that desmin is not necessary for correct synemin, syncoilin, plectin, and dystrophin location on the cytoskeleton of EOMs. However, it is needed to maintain an appropriate mitochondrial distribution in both EOMs and limb muscles. Keywords: extraocular muscles, knockout, desmin, cytoskeleton, intermediate filament xtraocular muscles (EOMs) are responsible for the complex demonstrated that a subgroup of fibers from adult and fetal E voluntary and reflex movements of the eye, which involve a EOMs lack or express only trace amounts of desmin.10 Notably, high demand of activity. The EOMs are classified as a separate desmin is not required for proper muscle fiber develop- muscle allotype given their unique structural and functional ment.11,12 properties and gene expression profile.1,2 The main function of desmin is to maintain muscle fiber Several studies performed on patients and animal models of cytoarchitecture by mediating the link between peripheral muscular dystrophy and neuromuscular disorders, such as myofibrils and sarcolemma, nuclei, and mitochondria.13 Des- Duchenne muscular dystrophy,3,4 congenital muscular dystro- min also links adjacent myofibrils at the Z-disc level and is phy with absence of laminin alpha 2 chain,5 and amyotrophic enriched in myotendinous junctions (MTJs), neuromuscular lateral sclerosis,6 reveal that the EOMs are selectively spared, in junctions (NMJs), and costameres. The correct positioning and contrast to the limb muscles. However, data on the EOMs in interaction of these cell compartments and organelles are desminopathies are lacking. critical to preserve mechanical and signaling processes taking Desminopathies are a heterogeneous group of progressive place in the muscle in response to different stimuli.14,15 Desmin myopathies and cardiomyopathies caused by mutations on performs its role by binding several molecules including other desmin (DES)oraB-crystallin (CRYAB) genes and characterized IF proteins and IF linker proteins. Two IF proteins that bind to by the presence of desmin protein aggregates and functional desmin are synemin and syncoilin. Synemin can directly form and structural alterations of the cytoskeleton network that lead heteropolymers with desmin,16 whereas syncoilin can associate to muscle fiber degeneration.7 Although the disease phenotype with desmin but without heteropolymer network formation.17 is very heterogeneous and the first symptoms can appear at any Both proteins colocalize with desmin at the Z-discs, costa- time during the patient’s lifetime, these are predominantly meres, and myotendinous and neuromuscular junctions.17–19,20 characterized by distal and proximal skeletal muscular weak- Nestin and vimentin are two additional IF proteins coexpressed ness that coexists with cardiac pathology in half of the with desmin during skeletal muscle development. In contrast to patients.7,8 synemin and syncoilin, nestin and vimentin are downregulated Desmin is a 53-kilodalton (kDa) intermediate filament (IF) postnatally in healthy skeletal muscle, with vimentin disap- protein encoded by a single-copy gene.9 Desmin is the most pearing completely, whereas nestin expression in adult muscle abundant cytoskeletal protein in mature muscle and for many is restricted to NMJs and MTJs21,22 and regenerating muscle years it was considered ubiquitous in smooth, cardiac, and fibers.23 We have previously shown that nestin is normally skeletal muscle. However, recent work from our laboratory present in some muscle fibers in the adult human EOMs.10 Copyright 2018 The Authors iovs.arvojournals.org j ISSN: 1552-5783 4847 This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License. Downloaded From: https://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/937555/ on 11/09/2018 Extraocular Muscles of Desmin Knockout Mice IOVS j October 2018 j Vol. 59 j No. 12 j 4848 TABLE. Antibodies Used Antigen Host Supplier (Catalog No.) Synemin H, high molecular weight Rabbit pAb Provided by Zhenlin Li, Sorbonne Universite´ Synemin H/M, high and medium molecular weight Rabbit pAb Provided by Zhenlin Li, Sorbonne Universite´ Plectin Guinea pig pAb Progen, Heidelberg, Germany (GP21) Syncoilin Rabbit pAb Proteintech, Chicago, IL, USA (25151-1-AP) Nestin Mouse mAb R&D systems, Minneapolis, MN, USA (2736) Vimentin Mouse mAb Dako Omnis, Agilent, Santa Clara, CA, USA (M0725) Dystrophin Rabbit pAb Gene Tex, Inc., Irvine, CA, USA (GTX15277) MyHC IIa Mouse mAb Developmental Studies Hybridoma Bank, Iowa City, IA, USA (SC-71) MyHCIIb Mouse mAb Developmental Studies Hybridoma Bank (BF-F3) MyCH I Mouse mAb Developmental Studies Hybridoma Bank (BA-D5) Laminin Rabbit pAb Dako Omnis (Z0097) pAb, polyclonal antibody; mAb, monoclonal antibody. In desmin knockout (desminÀ/À) mouse models, important Consecutive serial cross and longitudinal sections (5–6 lm structural modifications including muscle fiber disorganization, thick) were cut at À248C with a Reichert Jung cryostat (Leica, fibrosis, subsarcolemmal aggregation of mitochondria, and Heidelberg, Germany). coexistence of muscle fiber regeneration and degeneration are present, particularly in the highly used muscles, such as the Histochemistry soleus muscle.11,24,25 In particular, the lack of desmin produces alterations in mitochondrial distribution in soleus, gastrocne- Frozen cross sections were stained with hematoxylin and eosin mius, and cardiac muscles.24,26 The correct positioning of (H&E).32 In addition, we used a standard protocol32 to reveal mitochondria depends on desmin interaction with plectin,27 a the mitochondrial marker enzyme succinate dehydrogenase major cytoskeleton cross-linking protein not included in the IF (SDH), and the modified Gomori trichrome staining was family. Important changes in desmin-binding protein patterns carried out as described32 to demonstrate mitochondrial comprising alterations in synemin and syncoilin have been abnormalities in the EOMs. reported in the soleus muscles from desminÀ/À mice. In the absence of desmin, both proteins are lost from the muscle fiber Z-discs, whereas their expression at the subsarcolemma is Immunofluorescence 19,28 partly affected. Moreover, in patients with desminopathy, All sections used for immunofluorescence were air dried for 15 29 7 30 the proteins synemin, syncoilin, and plectin were found to minutes and fixed in 4% paraformaldehyde (PFA) for 8 minutes, colocalize with desmin aggregates. À/À 11 except for the immunodetection of myosin heavy chain In the present study we used a desmin mouse model to (MyHC) isoforms, where no fixation was performed. Thereaf- investigate the effect of absence of desmin in the structure and ter, the sections were washed in PBS and blocked with 10% composition of the cytoskeleton of the EOMs. The study normal serum (from goat or donkey depending on the host included the important desmin-binding proteins synemin, species of the secondary antibody used) in PBS containing