Physical continuity of the perimysium from myofibers to tendons: Involvement in lateral force transmission in skeletal muscle

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Abstract

Advances in muscle physiology suggest that the perimysium plays a role in the transmission of lateral contractile forces. This hypothesis is strongly supported by our recent demonstration of the existence of “Perimysial Junctional Plates” in bovine Flexor carpi radialis muscle [Passerieux, E., Rossignol, R., Chopard, A., Carnino, A., Marini, J.F., Letellier, T., Delage, J.P. 2006. Structural organization of the perimysium in bovine skeletal muscle: junctional plates and associated intracellular subdomains. J. Struct. Biol. 154 (2), 206--216] However, the overall organization of the perimysium collagen network, as well as its continuity and heterogeneity, have still not been described in detail throughout the entire muscle. We used an extension of the standard NaOH digestion technique and scanning electron microscopy to analyze perimysium architecture in bovine Flexor carpi radialis muscle. First, we observed that the perimysium is made of a highly ordered network of collagen fibers, binding the myofibers from tendon to tendon. We identified basic collagen cable structures, characterized by a straight portion (3 cm long) in the direction of the myofibers and a curved terminal portion at 60°. These cables reach the myofiber surface at the level of the previously described “Perimysial Junctional Plates”. At a higher level of organization, these cables stick together to form the walls of numerous tubes arranged in a overlapping honeycomb pattern around the myofibers. At the ends of these tubes, the straight portions of the collagen cables ramify in large bundles that merge with the tendons. Taken together, these observations identify four levels of organization in the perimysium: (i) Perimysial Junctional Plates that constitute the focal attachment between the perimysium and the myofibers, (ii) collagen plexi attaching adjacent myofibers, (iii) a loose lattice of large interwoven fibers, and (iv) honeycomb tubes connecting two tendons. This spatial arrangement of the perimysium supports the view of a complex pattern of lateral force transmission from myofibers to tendons and adjacent muscles.

Introduction

Skeletal myofibers are embedded in a complex network of connective tissue consisting of endomysium and perimysium (Borg and Caulfield, 1980, Rowe, 1981). The former can be considered the main component of the extracellular matrix involved in muscle flexibility, while the latter is generally described as simple packing tissue. However, there is a great deal of evidence that the perimysium plays a role in the lateral transmission of contractile forces (Tidball and Chan, 1989, Huijing et al., 1998, Monti et al., 1999). This hypothesis is strongly supported by the recent demonstration of the existence of “Perimysial Junctional Plates” (PJP) (Passerieux et al., 2006).

These adhesive regions connect the myofiber surface with a specific intracellular subdomain. However, our observations were restricted to the vicinity of myofibers and there was no global description of the perimysium from tendon to tendon. In particular, the overall organization of the perimysium collagen network, as well as its continuity and heterogeneity throughout the entire muscle had never been investigated.

In fact, perimysium organization is essentially known from the pioneering observations of Rowe (1974), using optical microscopy, describing crimped collagen fibers running through the muscle, some in the direction of myofibers and others at 60°. The first observations using scanning electron microscopy (SEM) on formolized muscle samples were presented by Borg and Caulfield (1980) and Rowe (1981), who demonstrated the presence of collagen fibers in various muscles from different animal species. The existence of these collagen fibers was more recently confirmed by Nishimura et al., 1996, Jarvinen et al., 2002 and Nakamura et al. (2003) using the 2 N NaOH cell-maceration digestion technique, originally introduced by Ohtani et al. (1991). Unfortunately, the contaminating presence of endomysium made it impossible to observe perimysium continuity with the tendons by this method, nor was it possible with 5 N NaOH cell maceration (Passerieux et al., 2006).

In this study, we adapted this technique to digest the myofibers and endomysium of bovine Flexor carpi radialis (FCR) muscle selectively, thus making it possible to visualize the perimysium collagen network. Myofibers and endomysium were digested in different concentrations of NaOH (pH) at varying incubation temperatures, and then examined by SEM. This revealed the entire perimysium architecture: a highly ordered network of collagen fibers binding myofibers to the tendon. In particular, we identified perimysium cables that stuck together to form the walls of tubes in a honeycomb arrangement. The ends of these tubes formed the tendons.

Section snippets

Materials and methods

All procedures were compliant with institutional guidelines for animal care. All FCR samples were taken just after slaughter.

Anatomy of the FCR muscle and associated connective tissue

The belly of the FCR muscle is about 20 cm long (Fig. 1A). Myofibers are about 6–8 cm long and join the two tendinous sheaths at an angle ranging in 15–30°: the proximal tendinous sheath (pts) on the back of figure is directly attached to the humerus and the distal tendinous sheath (dts) ends in a long tendon joining the carpe. The pennated architecture of the FCR is summarized in Fig. 1B, to facilitate understanding of the indications of the main directions shown on the following figures.

The

Discussion

The objective of this work was to clarify the perimysium organization of FCR bovine muscle, with a particular emphasis on its continuity from tendon to tendon. To achieve this, we adapted the standard NaOH cell-maceration digestion technique to eliminate specific elements: (i) myofibers and endomysium, to observe the arrangement of cables of collagen-fiber bundles through the muscle, (ii) the endomysium alone, to observe the direction of the perimysial tubes and their attachment to tendons and

Acknowledgments

The authors wish to thank INSERM, Université Victor Segalen Bordeaux 2, Association Française contre les Myopathies (AFM), Association contre les Maladies Mitochondriales (Ammi), and Région Aquitaine for financial support, Service Commun de Microscopie (SERCOMI), Université Victor Segalen Bordeaux 2, for technical assistance, and service vétérinaire de Bordeaux for bovine muscle supply.

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