Structural organization of the perimysium in bovine skeletal muscle: Junctional plates and associated intracellular subdomains E. Passerieux, R. Rossignol, A. Chopard, A. Carnino, J.F. Marini, T. Letellier, J.P. Delage To cite this version: E. Passerieux, R. Rossignol, A. Chopard, A. Carnino, J.F. Marini, et al.. Structural organization of the perimysium in bovine skeletal muscle: Junctional plates and associated intracellular subdomains. Journal of Structural Biology, Elsevier, 2006, 154 (2), pp.206 - 216. 10.1016/j.jsb.2006.01.002. hal- 01758589 HAL Id: hal-01758589 https://hal.umontpellier.fr/hal-01758589 Submitted on 4 Apr 2018 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. Journal of Structural Biology 154 (2006) 206–216 www.elsevier.com/locate/yjsbi Structural organization of the perimysium in bovine skeletal muscle: Junctional plates and associated intracellular subdomains E. Passerieux a, R. Rossignol a, A. Chopard b, A. Carnino b, J.F. Marini b, a a, T. Letellier , J.P. Delage ¤ a INSERM, U688 Physiopathologie Mitochondriale, Université Victor Segalen-Bordeaux 2, 146 rue Léo-Saignat, F-33076 Bordeaux Cedex, France b EA 3837, Laboratoire de Physiologie des Adaptations, Performances Motrices et Santé, Université de Nice Sophia Antipolis, Faculté des Sciences du Sport, 261 route de Grenoble, 06205 Nice Cedex, France Received 19 October 2005; received in revised form 6 January 2006; accepted 7 January 2006 Available online 8 February 2006 Abstract We analyzed the structural features of the perimysium collagen network in bovine Flexor carpi radialis muscle using various sample preparation methods and microscopy techniques. We Wrst observed by scanning electron microscopy that perimysium formed a regular network of collagen Wbers with three hierarchical levels including (i) a loose lattice of large interwoven Wbers ramiWed in (ii) numerous col- lagen plexi attaching together adjacent myoWbers at the level of (iii) speciWc structures that we call perimysial junctional plates. Second, we looked more closely at the intracellular organization underneath each plate using transmission electron microscopy, immunohisto- chemistry, and a three-dimensional reconstruction from serial sections. We observed the accumulation of myonuclei arranged in clusters surrounded by a high density of subsarcolemmal mitochondria and the proximity of capillary branches. Third, we analyzed the distribu- tion of these perimysial junctional plates, subsarcolemmal mitochondria, and myonuclei clusters along the myoWbers using a statistical analysis of the distances between these structures. This revealed a global colocalization and the existence of adhesion domains between endomysium and perimysium. Taken together, our observations give a better description of the perimysium organization in skeletal muscle, and provide evidence that perimysial junctional plates with associated intracellular subdomains may participate in the lateral transmission of contractile forces as well as mechanosensing. © 2006 Elsevier Inc. All rights reserved. Keywords: Perimysium; Myonuclear domains; Mitochondria; Muscle contraction; Skeletal muscle 1. Introduction tendinous junction (Borg and CaulWeld, 1980; Swasdison and Mayne, 1989; Trotter and Purslow, 1992). It is orga- The extracellular matrix (ECM)1 plays a fundamental nized as a regular network of thin Wbrillar collagen Wbers role, both in structural and functional aspects of skeletal composed mostly of type IV, as well as types III, VI (Listrat muscle. Its two components are the endomysium and the et al., 1999), and XII (Listrat et al., 2000) to a minor extent. perimysium. The endomysium is made of a continuous This network attaches myoWbers sarcolemma to speciWc sheath covering the full length of myoWbers until the myo- transmembrane proteins (Kovanen, 2002), thereby forming a regular mosaic pattern (Borg and CaulWeld, 1980). These * Corresponding author. Fax: +33 5 57 57 16 12. proteins, such as integrins or dystrophin–glycoprotein com- E-mail address: [email protected] (J.P. Delage). plexes (Rando, 2001), interact directly with the cytoskele- 1 Abbreviations used: COX, cytochrome c oxidase; DAPI, 4Ј,6-diamidi- ton (Berthier and Blaineau, 1997) and transmit the no-2-phenylindole, dihydrochloride; ECM, extracellular matrix; KS, contractile forces between adjacent myoWbers (Monti et al., Kolmogorov–Smirnov; PJP, perimysial junctional plate; SEM, scanning electron microscopy; TEM, transmission electron microscopy; 3D, three- 1999; Sheard et al., 2002) as far as the tendon (Purslow, dimensional. 2002). Taking into account this particular anatomy and 1047-8477/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.jsb.2006.01.002 E. Passerieux et al. / Journal of Structural Biology 154 (2006) 206–216 207 associated roles, the endomysium can be considered the easily visualize the perimysium between adjacent myoWbers, main ECM component involved in muscle Xexibility. or (ii) a fracture technique that does not alter the chemical In contrast, the second component of ECM, i.e., the peri- structure of connective tissue, and makes it possible to mysium, is formed by an areolar network of crimped colla- remove the endomysium and visualize perimysium connec- gen Wbers varying in diameter and composition, including tions at the surface of myoWbers. The digestion technique of essentially type I collagen in conjunction with types III, VI NaOH cell maceration was adapted from Ohtani et al. (Listrat et al., 1999), and XII (Listrat et al., 2000). However, (1991). For this, muscle samples were immersed in NaOH few studies have analyzed the perimysium’s Wne organization 6N and maintained at a temperature of 18°C for 5 days, so perimysium continues to be considered a simple packing before being rinsed for 3 days in water at 18 °C. Afterward, tissue, even though some early observations have suggested a the samples were Wxed in 2% tannin, freeze-dried, and frac- connection with endomysium (Borg and CaulWeld, 1980; tured. The sections were coated with gold and examined on Moore, 1983) and a possible role in the lateral transmission a Philips 515 scanning electron microscope. The fracture of contractile forces (Huijing et al., 1998). In particular, there technique consisted in the Wxation of muscle samples in 2% is a lack of detail concerning contact sites between perimy- osmic acid, followed by freeze-drying and fracture. For sium and endomysium, their organization, distribution along each analysis, a series of at least Wve diVerent samples were myoWbers, and underlying intracellular environment. We taken from three diVerent muscles. analyzed these features of the perimysium collagen network with a particular emphasis on endomysium contact sites, as 2.2. Transmission electron microscopy well as the associated intracellular subdomains in bovine Flexor carpi radialis muscle using scanning electron micros- The Flexor carpi radialis was stretched by a 300-g weight copy (SEM), transmission electron microscopy (TEM), to preserve muscle Wber straightness and immediately Wxed three-dimensional reconstruction from serial sections, and by the injection of solution A (0.5% glutaraldehyde, 2% immunohistochemistry techniques. paraformaldehyde, 7% saccharose, and 4% polyvinylpyroli- Our results show that the perimysium forms a network done in 0.1 M cacodylate buVer). This made it possible to of collagen Wbers with three hierarchical levels including (i) increase intramuscular pressure and separate muscle Wbers a regular lattice of interwoven Wbers with (ii) collagen plexi for better visualization of the connective tissue. The muscle at each angle that attach adjacent myoWbers at (iii) particu- was sectioned in 40 mm3 blocks, immersed for 1 h in Wxative lar domains that we call perimysial junctional plates (PJPs). solution A, and rinsed with water. These blocks were recut A three-dimensional reconstruction from serial sections into smaller samples of about 12 mm3 and immersed in a showed that, underneath each plate, there was an accumu- second Wxative solution B (2% osmic acid in 0.1 M cacodyl- lation of myonuclei arranged in clusters near capillary ate buVer) for 1 h. After dehydration, these blocks were branches. Surrounding these clusters, we observed a high embedded in epoxy (epon) resin and cut into longitudinal or density of subsarcolemmal mitochondria by immunohisto- transversal sections 0.1 !m thick. These sections were chemistry and TEM. Comparison of the data obtained by stained with a solution of uranyl acetate and lead citrate. these various techniques enabled us to propose a model of They were observed on a Philips CM10 microscope to ana- perimysium organization that may underline its role in lat- lyse the contact sites between perimysium and myoWbers as eral transmission of contractile forces and thus in various well as the intracellular subdomains. aspects of muscle Xexibility. 2.3. Three-dimensional reconstruction 2. Materials and methods Samples of embedded material (see above) were cut by All procedures were