Physical continuity of the perimysium from myofibers to ...

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Physical continuity of the perimysium from myofibers totendons: Involvement in lateral force transmission in

skeletal muscleE. Passerieux, R Rossignol, T. Letellier, J P Delage

To cite this version:E. Passerieux, R Rossignol, T. Letellier, J P Delage. Physical continuity of the perimysium from my-ofibers to tendons: Involvement in lateral force transmission in skeletal muscle. Journal of StructuralBiology, Elsevier, 2007, 159 (1), pp.19 - 28. �10.1016/j.jsb.2007.01.022�. �hal-01785064�

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

E. Passerieux a,b, R. Rossignol b, T. Letellier b, J.P. Delage a,b,*

a UFR STAPS, Universite Victor Segalen-Bordeaux 2, 12 Avenue Camille Jullian, F-33607 Pessac Cedex, Franceb INSERM, U688 Physiopathologie mitochondriale, Universite Victor Segalen-Bordeaux 2, 146 rue Leo-Saignat, F-33076 Bordeaux Cedex, France

Received 25 July 2006; received in revised form 9 January 2007; accepted 30 January 2007Available online 3 March 2007

Abstract

Advances in muscle physiology suggest that the perimysium plays a role in the transmission of lateral contractile forces. This hypoth-esis is strongly supported by our recent demonstration of the existence of ‘‘Perimysial Junctional Plates’’ in bovine Flexor carpi radialismuscle [Passerieux, E., Rossignol, R., Chopard, A., Carnino, A., Marini, J.F., Letellier, T., Delage, J.P. 2006. Structural organization ofthe 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 beendescribed in detail throughout the entire muscle. We used an extension of the standard NaOH digestion technique and scanning electronmicroscopy to analyze perimysium architecture in bovine Flexor carpi radialis muscle. First, we observed that the perimysium is made ofa 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 reachthe myofiber surface at the level of the previously described ‘‘Perimysial Junctional Plates’’. At a higher level of organization, these cablesstick together to form the walls of numerous tubes arranged in a overlapping honeycomb pattern around the myofibers. At the ends ofthese tubes, the straight portions of the collagen cables ramify in large bundles that merge with the tendons. Taken together, these obser-vations identify four levels of organization in the perimysium: (i) Perimysial Junctional Plates that constitute the focal attachmentbetween 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 oflateral force transmission from myofibers to tendons and adjacent muscles." 2007 Elsevier Inc. All rights reserved.

Keywords: Perimysium; Myofibers; Tendons; NaOH cell maceration; Lateral muscular forces

1. Introduction

Skeletal myofibers are embedded in a complex networkof connective tissue consisting of endomysium and perimy-sium (Borg and Caulfield, 1980; Rowe, 1981). The formercan be considered the main component of the extracellular

matrix involved in muscle flexibility, while the latter is gen-erally described as simple packing tissue. However, there isa great deal of evidence that the perimysium plays a role inthe lateral transmission of contractile forces (Tidball andChan, 1989; Huijing et al., 1998; Monti et al., 1999). Thishypothesis is strongly supported by the recent demonstra-tion of the existence of ‘‘Perimysial Junctional Plates’’(PJP) (Passerieux et al., 2006).

These adhesive regions connect the myofiber surfacewith a specific intracellular subdomain. However, ourobservations were restricted to the vicinity of myofibersand there was no global description of the perimysium

1047-8477/$ - see front matter " 2007 Elsevier Inc. All rights reserved.doi:10.1016/j.jsb.2007.01.022

Abbreviations: ECM; extracellular matrix; FCR; Flexor carpi radialis;PJP; Perimysial Junctional Plate; SEM; scanning electron microscopy.

* Corresponding author. Address: UFR STAPS, Universite VictorSegalen-Bordeaux 2, 12 Avenue Camille Jullian, F-33607 Pessac Cedex,France. Fax: +33 5 57 57 16 12.

E-mail address: [email protected] (J.P. Delage).

www.elsevier.com/locate/yjsbi

Journal of Structural Biology 159 (2007) 19–28

Journal of

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from tendon to tendon. In particular, the overall organiza-tion of the perimysium collagen network, as well as itscontinuity and heterogeneity throughout the entire musclehad never been investigated.

In fact, perimysium organization is essentially knownfrom the pioneering observations of Rowe (1974), usingoptical microscopy, describing crimped collagen fibers run-ning through the muscle, some in the direction of myofibersand others at 60!. The first observations using scanning elec-tron microscopy (SEM) on formolized muscle samples werepresented by Borg and Caulfield (1980) and Rowe (1981),who demonstrated the presence of collagen fibers in variousmuscles from different animal species. The existence of thesecollagen fibers was more recently confirmed by Nishimuraet al. (1996), Jarvinen et al. (2002) and Nakamura et al.(2003) using the 2 N NaOH cell-maceration digestion tech-nique, originally introduced by Ohtani et al. (1991). Unfor-tunately, the contaminating presence of endomysium madeit impossible to observe perimysium continuity with thetendons by this method, nor was it possible with 5 N NaOHcell maceration (Passerieux et al., 2006).

In this study, we adapted this technique to digest themyofibers and endomysium of bovine Flexor carpi radialis(FCR) muscle selectively, thus making it possible to visual-ize the perimysium collagen network. Myofibers andendomysium were digested in different concentrations ofNaOH (pH) at varying incubation temperatures, and thenexamined by SEM. This revealed the entire perimysiumarchitecture: a highly ordered network of collagen fibersbinding myofibers to the tendon. In particular, we identi-fied perimysium cables that stuck together to form thewalls of tubes in a honeycomb arrangement. The ends ofthese tubes formed the tendons.

2. Materials and methods

All procedures were compliant with institutional guide-lines for animal care. All FCR samples were taken justafter slaughter.

2.1. NaOH cell-maceration digestion technique and SEM

The usual NaOH digestion technique consists of macer-ating muscle samples previously incubated in paraformal-dehyde in 2 N NaOH solution at room temperature. Thiseliminates myofiber components, making it possible toobserve connective tissue components. In contrast, macer-ating non-formolized muscle samples results in the elimina-tion of all the muscle components. This indicates thatformaldehyde causes intermolecular crosslinking betweensome muscular proteins, as shown by Metz et al. (2004).The strength and stability of these crosslinks are differentaccording to pH and temperature (Jaenicke, 1998; Fathimaet al., 2004). Indeed, after 2 N NaOH cell maceration atroom temperature, only the endomysium and perimysiumremain (Trotter and Purslow, 1992; Nakamura et al.,2003), while cell maceration in 5 N NaOH at temperatures

below 20 !C eliminates the endomysium, whereas the myof-ibers and perimysium remain (Passerieux et al., 2006).

We extended this cell maceration technique to a widerrange of NaOH concentrations and temperatures, usingmuscle samples of different sizes (from mm to cm). For eachcondition, the procedure was as follows: fixation in 10%paraformaldehyde for 8 days and NaOH maceration at aspecific concentration at 15 !C for 5 days. The macerationwas rapidly heated to the chosen temperature and rinsedin water at this temperature for 2 days. Muscle samples werethen treated with 2% tannin and freeze-dried for easy dissec-tion and observed under a binocular microscope.

The results are summarized in Table 1, with NaOH con-centrations from 2 to 6 N and temperatures from 5 to70 !C. The results show that perimysium, epimysium, andtendons resisted all NaOH concentrations up to 37 !Cand gradually disappeared at the same pH when tempera-tures exceeded 56 !C. Myofibers remained at all NaOHconcentrations, but only below 15 !C. The endomysiumwas resistant under a set of conditions from 2 to 4 NNaOH at temperatures from 15 to 25 !C.

In this study, we applied the three cell macerationconditions:

!5 N NaOH maceration and a rinsing temperature of15 !C, noted 5–15 for myofiber, perimysium, epimy-sium, and tendon observations.!5 N NaOH maceration and a rinsing temperature of

37 !C, noted 5–37 for perimysium, epimysium, and ten-don observations.

Table 1Results of NaOH cell-maceration digestion technique after variousconditions of pH and temperature

NaOH concentrations from 2 to 6 N and temperatures from 5 to 70 !C onpreservation of muscular components (myofibers in blue, endomysium ingreen and perimysium, epimysium, and tendon in brown).

20 E. Passerieux et al. / Journal of Structural Biology 159 (2007) 19–28

!2 N NaOH maceration and a rinsing temperature of25 !C, noted 2–25 for perimysium, epimysium, tendonand fine endomysium observations.

Each sample for the three conditions was dissected, thengold-coated and examined using a Philips 515 scanningelectron microscope.

2.2. Embedding of the capillary network andimmunohistochemistry

2.2.1. Capillary bedOne of the FCR arteries was perfused using successive

2 ml injections of colored ink (black and white). When amyofiber bundle was dissected, it was possible to distin-guish between arteries (white) and veins (black).

2.2.2. ImmunohistochemistryFCR muscle samples were rapidly frozen in liquid

nitrogen-cooled isopentane and stored at !80 !C. Longitu-dinal sections (10 lm thick) were obtained using a cryostat(Frigocut 2800, Reichert Jung) at !22 !C. The sections werefixed in a solution of 4% paraformaldehyde (SigmaAldrich) at 25 !C for 30 min. After two washes with PBS(5 min each), the sections were dehydrated in successivebaths of 70%, 90%, and 100% ethanol (2 min each), fol-lowed by rehydration in successive baths of 90% and 70%ethanol, then washed twice in PBS (2 min each). Sectionswere then incubated in a blocking solution [10% fetal calfserum (Hyclone) diluted in PBS] at 25 !C for 2 h. Theywere then incubated overnight with primary antibody(rabbit polyclonal anti-collagen III, Rockland). After twowashes in PBS (10 min each), the sections were incubatedin 10% normal goat serum (Sigma) in PBS at 25 !C for1 h. Subsequently, sections were incubated with the second-ary antibody (Alexa Fluor 546 goat anti-rabbit IgG,Molecular Probes) at 25 !C for 90 min, and then washedthree times with PBS (15 min each). Finally, sections weremounted with Mowiol 4–88 (Calbiochem). Sections wereobserved on an epifluorescence microscope (Nikon EclipseE600), photographed by a digital camera (COHU HighPerformance CCD Camera), and acquired using Visiolabsoftware (Biocom).

3. Results

3.1. Anatomy of the FCR muscle and associated connectivetissue

The belly of the FCR muscle is about 20 cm long(Fig. 1A). Myofibers are about 6–8 cm long and join thetwo tendinous sheaths at an angle ranging in 15!–30!: theproximal tendinous sheath (pts) on the back of figure isdirectly attached to the humerus and the distal tendinoussheath (dts) ends in a long tendon joining the carpe. Thepennated architecture of the FCR is summarized in

Fig. 1B, to facilitate understanding of the indications ofthe main directions shown on the following figures.

The general architecture of perimysium was observed inlarge sections of FCR, cut along the myofibers, and treatedat 5–37 (Fig. 2A and B). Parallel perimysial structures(arrow) are visible on both figures. In Fig. 2A, these struc-tures form sections across large tubes (tu) joining the prox-imal tendon (t) at a 30! angle. These tubes are apparentlyfilled with a thin network of collagen fibers. Observationof a cross-section of a muscle sample treated at 5–37(Fig. 2C) confirmed that the perimysium consisted of tubesin a honeycomb arrangement, with a maximum diameter of3 mm. This is consistent with the sections observed inFig. 2A and B. These tubes show a large amount of thinlateral strips (ts) from their walls scattered through theinternal space, but also large lateral collagen strips (ls)from their walls that resemble the ends of tubes or junc-tions with adjacent tubes. Examination of samples of2–25 treated muscle revealed the arrangement of walls(w) and strips (s), as well as thin honeycomb structuresof endomysium (en) embedding myofibers (Fig. 2D). Thetube walls consisted of large bands of thick accumulationsof collagen fibers embedding honeycomb structures ofendomysium, while the strips were thinner bands of colla-gen fibers between the thin honeycomb structures of theendomysium.

3.2. Perimysium tube arrangement and wall structure

Muscle sample sections along the myofibers treated at5–15 showed the direction of the tubes (tu), myofibers(mf), and tendon (t) (Fig. 3). Fig. 3A shows that the tubewalls are parallel with the myofibers and in continuity withtendon collagen bundles. Fig. 3B shows that the tube wallsconsist of parallel bundles of collagen fibers (cf) crossingthe myofibers at a 60! angle (arrow).

Tubes from muscle samples treated at 5–37 were dis-sected to investigate the arrangement of their walls

Fig. 1. (A) Photograph of the bovine Flexor carpi radialis (FCR) muscle.The belly (dark portion) consists of 6–8 cm long myofibers connecting thedistal tendinous sheath (dts) on the upper face with the proximal tendinoussheath (pts) on the lower face. The belly is about 20 cm long. (B) Schematicrepresentation of FCR architecture. mf, myofibers; dts, distal tendinoussheath; pts, proximal tendinous sheath; a, pennation angle.

E. Passerieux et al. / Journal of Structural Biology 159 (2007) 19–28 21

(Fig. 4). Fig. 4A, where the thin lateral strips wereremoved, presents a lateral view of a longitudinal tube sec-tion (tu1) at the junction with the tendon (t) and the conti-nuity with the other two tubes (tu2 and tu3). It revealsdetails of the perimysium tube walls and large lateral strips(ls) which form the end of tube walls near the tendon or thejunction with the two adjacent tubes. This figure shows alsothat the tube walls consist of cables made up of collagen-fiber bundles, some aligned with the tubes, others oblique.A dissection of the walls at the junction between two tubes(Fig. 4B) shows two layers (M) of crimped collagen-fiberbundles (arrow), the upper layer clearly showing thechange of direction in the same collagen-fiber bundles(—/). This preparation made it possible to dissect a set ofcollagen-fiber bundles (Fig. 4C) from the tube walls. Thefigure shows that a set of stuck collagen-fiber bundlesforms large cables consisting of a 3 cm straight portion(sp) parallel with the direction of the tubes and, therefore,the myofibers and numerous 2 cm curved portions (cp) at a60! angle in different planes. The larger cables produced thelarge lateral strips (ls) on the tube walls and the thinnerones formed thin lateral strips (ts) inside the tube, as seenin Fig. 2C.

This arrangement of perimysium cables (ca) made up oftubes (tu) is present throughout the muscle and near the

myotendinous junction. The straight portions connect tothe tendon (t) (Fig. 5A) or epimysium (e) around the bellyof the muscle (Fig. 5B).

3.3. Junction between perimysium tube and myofibers

Dissection of thin lateral strips (ts) from the internalspaces of tubes from 5 to 37 treated muscle samples(Fig. 6A) shows that each parallel collagen-fiber bundleramifies into oblique branches (b) on the myofibers (mf),as seen in Fig. 6B, a dissection of 5–15 treated musclesample. At higher magnification (Fig. 6C and D), the com-position of these bundles and branches (b) becomes clearer:the bundles are crimped and produce terminal non-crimpedbranches (tb) that spreading (arrowheads) all along themyofibers (mf) (Fig. 6C). The junction with the myofibersconsists of a small plexus (p) of collagen fibers attachingthe surfaces of adjacent myofibers (Fig. 6D). This organiza-tion corresponds to the three hierarchical levels ofperimysium network described in a previous work(Passerieux et al., 2006).

An oblique view of a transverse section of non-dissected5–15 muscle samples (Fig. 7A) indicates that the distribu-tion of collagen-fiber bundles between myofibers (mf) isheterogeneous, with denser collagen regions approximately

Fig. 2. SEM views of (A) proximal and (B) distal portions of longitudinal sections, and (C) transverse section of 5–37 NaOH-macerated FCR muscleallowing the removal of myofibers and endomysium. (D) Lateral view of 2–25 NaOH-macerated FCR muscle allowing the removal of myofibers. (A) and(B) note the tendon (t), parallel structures of perimysium (arrows), and epimysium (e). The perimysium consists of a series of tubes (tu) containing anetwork of thin collagen fibers. (C) The tubular arrangement of the perimysium is clearly visible. Note the numerous large (ls) and thin (ts) lateral stripsfrom the tube walls. (D) Note the arrangement of the tube walls (w) and strips (s) inside the remaining thin honeycombs of the endomysium (en).


200–300 lm apart (Fig. 7A). This heterogeneity is con-firmed by the distribution of type III collagen, a componentof the perimysium, observed on the immunohistochemistryFCR longitudinal section (Fig. 7B). The dense regions ofcollagen bundles in Fig. 7A may correspond to the positionof thin lateral strips and less dense regions to their branches.This corresponds to the vascular bed distribution observedafter ink injection of FCR muscle (Fig. 7C): arteries (white)and veins (black) are evenly spaced, approximately200–300 lm apart. This correspondence between the distri-bution pattern of the collagen network and vascular bedsupports previous observations (Bosman and Stamenkovic,2003) highlighting the correspondence between capillarybeds and collagen networks in muscle. This led to the con-clusion that thin lateral strips ramify with great regularityfrom the tube walls, which is consistent with the apparentlyregular distribution of branches (b) in Fig. 6A.

3.4. Model of perimysium organization from myofibers totendons

From the above detailed observations, we constructed amodel of perimysium organization in FCR. This summa-rizes the perimysium architecture and the way it is builtfrom an assembly of basic structures: perimysium cables.These cables are stuck together to form the walls of tubes(Fig. 8A) that, in turn, form honeycomb structures

arranged alternately throughout the muscle, from tendonto tendon (Fig. 8B), thus constituting the fourth level ofperimysium organization (Passerieux et al., 2006).

In this manner, myofibers are embedded in finelyorganized, three-dimensional connective tissue.

4. Discussion

The objective of this work was to clarify the perimysiumorganization of FCR bovine muscle, with a particularemphasis on its continuity from tendon to tendon. Toachieve this, we adapted the standard NaOH cell-macera-tion digestion technique to eliminate specific elements: (i)

Fig. 3. SEM views of dissections of FCR after 5–15 NaOH cellmaceration allowing the removal of endomysium. (A) Lateral view, theperimysium forms a series of tubes (tu) parallel to and surroundingmyofiber (mf) bundles. Their walls are in continuity with the tendon (t).(B) Low magnification showing a front view of lateral tube walls. Note theparallel collagen-fiber (cf) bundles in the tube walls. The myofibers andtubes cross the tendons (t) at a 15! angle and the collagen-fiber bundlescross the myofibers at a 60! angle (arrow).

Fig. 4. SEM views of dissections of tube walls after 5–37 NaOH cellmaceration of FCR allowing the removal of myofibers and endomysium.(A) Portion of a tube (tu1) in the vicinity of the tendon (t) and itscontinuity with two adjacent tubes (tu2 and tu3). Large lateral strips (ls) ofcollagen fibers form the end of the tube (tu1) and provide continuity withthe walls of two adjacent tubes (tu2 and tu3). Note that tube walls consistof some collagen-fiber bundles aligned with the tubes and otherspositioned obliquely. (B) Portion of the junction of the walls of twotubes consisting of two layers (M) of crimped collagen-fiber bundles(arrow). Note the change in direction of the bundles (—/). (C) A set ofcollagen-fiber bundles from a tube wall forming cables with a straightportion (noted sp, 3 cm long) aligned with the tube and the associatedcurved portions (noted cp, 2 cm long) where collagen-fiber bundles curvedat 60! in different planes.


Fig. 5. SEM views of dissections of FCR after 5–37 NaOH cell maceration allowing the removal of myofibers and endomysium. (A) Junction of cables(ca) with the tendon (t). Note the continuity of the straight portion of the collagen bundles with the tendon. (B) The tube (tu) walls connecting to theepimysium (e) at the junction of the belly and tendon (t).

Fig. 6. SEM views of dissections after (A) 5–37 allowing the removal of myofibers and endomysium and (B, C and D) 5–15 NaOH cell macerationallowing the removal of endomysium. (A) Thin lateral strips (ts) from cables ramifying into branches (b), forming obliquely oriented collagen-fiberbundles. (B) Thin lateral strips (ts) from the tube walls (w) ramify into collagen-fiber branches (b) between myofibers (mf). (C) Crimped collagen-fiber-bundle branches (b) from strips lead into numerous terminal non-crimped branches (tb) spread (arrowheads) on a myofiber (mf). (D) Detail of the end of abranch (b) producing a plexus (p) at the surface of adjacent myofibers (mf).


myofibers and endomysium, to observe the arrangement ofcables of collagen-fiber bundles through the muscle, (ii) theendomysium alone, to observe the direction of the perimy-sial tubes and their attachment to tendons and myofibers.Morphological criteria were sufficient to identify the peri-mysium as crimped type I collagen fibers and the endomys-ium as a network of thin type IV collagen fibrils. Thisdigestion technique under 5–37 conditions also eliminatedthe proteoglycans that assemble perimysial fibrouselements, i.e., cables, making it possible to examine themspecifically. However, the results of this digestion techniquevary considerably according to muscle type, due to the greatdiversity in the ECM components of individual muscles anddifferent muscle types (for review, see Purslow, 2002).

In this article, we clarified the perimysium organization,consisting of tubes arranged in a honeycomb structure,composed of the collagen cables connecting the tendons.

Elimination of the proteoglycans from the perimysiumrevealed the fundamental shape of perimysium cables,made up of collagen-fiber bundles. They have an unusualconfiguration, characterized by a long 3 cm portion parallelto the myofibers, and a large curved portion at a 60! angle.Dissections showed that these cables were sometimes stucktogether in opposite directions (as shown in Fig. 5A), sothat they enter in opposite tendons in the same direction.

Our findings highlight the complexity of perimysiumorganization in the FCR muscle: multi-directional orienta-tion of crimped collagen bundles and their attachmentalong the full length of myofibers as well as to a large ten-dinous sheath. . . This complexity certainly reflects that ofmuscular mechanics.

The high number of collagen terminations stuck on themyofibers may transmit forces from contracted myofibersto tendons. These terminations may collect a fraction ofthe contractile forces in a system parallel to myofibers,reducing the level of force transmission at the myotendi-nous junction in the same proportions, as proposed byTidball (1991). This view is also supported by the specificmulti-directional organization of collagen-fiber bundlesfrom myofibers to tendons and epimysium shown in ourstudy. Tension from contracted myofibers may be collectedby the perimysium terminations in various loci along theentire muscle, and then distributed to the two tendinous

Fig. 7. (A) SEM view after 5–15 NaOH cell maceration allowing theremoval of endomysium. Intact spatial distribution of the collagen-fiberbundles between myofibers (mf). Note that fiber-dense regions areseparated by approximately 200–300 lm. (B) Immunohistochemistry oftype III collagen on longitudinal section of FCR muscle. Distribution ofthe perimysium through the myofibers (mf) seen as intense domains. Notethe heterogeneous distribution. (C) Photograph of FCR muscle bundleafter injecting colored inks into the vascular bed. Straight arteries (white)and veins (black) and their oblique branches are approximately200–300 lm apart.

Fig. 8. Models of perimysium architecture. (A) Arrangement of collagen-fiber bundles from cables in a perimysium tube. Cable bundles are initiallyparallel with myofibers, then the collagen fibers curve at a 60! angle indifferent planes. Some of these fibers form portions of the tube walls, whileothers constitute collagen fiber strips inside the tubes. Lateral fibers ofstrips are attached to myofibers at the PJP (Passerieux et al., 2006). (B)Alternating distribution of tubes in a honeycomb arrangement andcontinuity with tendons.


sheaths and epimysium. This may support the transmissionof forces to bone junction and contiguous muscles, as pro-posed in our model (Fig. 9). This distribution of forces atthe muscle periphery was recently confirmed by Meijeret al. (2006). It was also proposed by Young et al. (2000)in the case of the in series-myofiber junctions. Moreover,Yucesoy et al. (2002) hypothesized the existence of an elas-

tic link between myofibers. Our results indicate that thismay correspond to the numerous crimped bundles of colla-gen fiber cables. According to Cribb and Scott (1995), de-crimping collagen fibers lengthens them considerably,which is unlikely as myofibers are linked together by ashort network of endomysium type IV collagen fibers. Toinvestigate this issue, we dissected bundles of fresh FCRmuscle and added markers (indicated by circles) to two dif-ferent bundles of myofibers (noted mf1 and mf2 in theFig. 10A). When a relative displacement up to 5 mm wasimposed on one bundle under slow strain (Fig. 10B), thesecond bundle did not move. This demonstrated that themyofiber linkage via the endomysium is not constantthrough the muscle belly, so there is the possibility of largedisplacement of portions of the muscle. The displacementof the collagen-fiber bundles shown in Fig. 10 also suggeststhat there may be significant modifications in the directionof the curved portion of the cables when the length of themuscle is modified. In the case of passive stretch, Rowe(1974) showed that the angle between the collagen fibersand the myofibers decreased. However, it is difficult to pre-dict the variation in this angle in case of contraction, asmodifications in the length of collagen fibers under stressmay interact with displacement in the muscle: changes inmyofiber angle and length in contracted muscle (Huijinget al., 1989), modifications in tendinous sheath length dur-ing contraction (Rack and Westbury, 1984), and increase indiameter of contracted muscle, etc.

Fig. 10. Photographs of a dissection of two myofiber bundles (mf1 and mf2) from FCR muscle with surrounding connective tissue. (A) Note the parallelarrangement of collagen fibers at 60! (arrowhead) (B) Low stress (arrow) applied to the bundle at the top of the dissection pulled the markers (circles)5 mm apart, whereas the second bundle did not move.

Fig. 9. Model of diffusion of forces through the perimysium network. Acontracted myofiber (fi‹) pulls on perimysium branches at the PJP. Thiscreates tension ( ) in the curved and straight portions of the cables.Tensions diffuse through the entire perimysium network, affecting non-contracted myofiber, as well as the various portions of the tendons andepimysium.


The last property of the perimysium organization, sug-gested by our observations in FCR muscle, concerns thepossible increase in tube-wall tension during muscle elon-gation or contraction. During stretching, the elasticity ofthe curved portion of the cables creates tension in the tubewalls, compressing the myofibers inside. This may explainthe increase in intramuscular pressure demonstrated byDavis et al. (2003). However, the behavior of the perimy-sium network during contractions is still unknown, so noconclusions can be drawn on this aspect.

One interesting question is whether other muscles haveequivalent structures. We performed first investigationson the rat Peroneus digiti quarti muscle. An SEM view ofa transverse section of 5–56 NaOH-macerated muscleshows a similar tubular structure (tu) in the perimysium(Fig. 11A). In addition, a muscle fracture proceduredescribed by Passerieux et al. (2006) shows that collagen-fiber bundles of perimysium (p) connect myofibers (mf)to form a similar PJP to that described in FCR muscle(Passerieux et al., 2006). These results indicate that the lat-eral force transmission may be a general mechanical prop-erty of muscles. This view is also supported by the indirectevidence of lateral force transmission in the rat Extensordigitorum longus muscle (Huijing et al., 1998). However,the consequences of lateral force transmission by theperimysium may be expected to vary among musclesdepending on the content of perimysial connective tissue,

the density of PJPs, and tube direction, which differ accord-ing to the muscles’ general architecture (pennation angle,etc.).

To conclude, these observations from FCR, togetherwith our previous work (Passerieux et al., 2006), distin-guish four levels of organization within the perimysium:(i) Perimysial Junctional Plates that constitute the focalattachment of the perimysium to the myofibers, (ii) numer-ous collagen plexi attaching adjacent myofibers, (iii) a looselattice of large interwoven fibers, and (iv) a honeycombarrangement of tubes connecting the two tendons. Hence,the perimysium can be considered a complex elasticstructure may transmit contraction forces and producetension at the PJP, with their underlying myonuclei andsubsarcolemmal mitochondria (Passerieux et al., 2006).Thus it constitutes a good candidate for participating inmechanosensing and associated control of the skeletalmuscle metabolism.

Acknowledgments

The authors wish to thank INSERM, Universite VictorSegalen Bordeaux 2, Association Francaise contre lesMyopathies (AFM), Association contre les MaladiesMitochondriales (Ammi), and Region Aquitaine forfinancial support, Service Commun de Microscopie (SER-COMI), Universite Victor Segalen Bordeaux 2, for techni-cal assistance, and service veterinaire de Bordeaux forbovine muscle supply.

References

Borg, T.K., Caulfield, J.B., 1980. Morphology of connective tissue inskeletal muscle. Tissue Cell. 12, 197–207.

Bosman, F.T., Stamenkovic, I., 2003. Functional structure and compo-sition of the extracellular matrix. J. Pathol. 200, 423–428.

Cribb, A.M., Scott, J.E., 1995. Tendon response to tensile stress: anultrastructural investigation of collagen: proteoglycan interactions instressed tendon. J Anat. 187 (Pt. 2), 423–428.

Davis, J., Kaufman, K.R., Lieber, R.L., 2003. Correlation between activeand passive isometric force and intramuscular pressure in the isolatedrabbit tibialis anterior muscle. J. Biomech. 36, 505–512.

Fathima, N.N., Madhan, B., Rao, J.R., Nair, B.U., Ramasami, T.,2004. Interaction of aldehydes with collagen: effect on thermal,enzymatic and conformational stability. Int. J. Biol. Macromol. 34,241–247.

Huijing, P.A., Baan, G.C., Rebel, G.T., 1998. Non-myotendinous forcetransmission in rat extensor digitorum longus muscle. J. Exp. Biol. 201(Pt. 5), 683–691.

Huijing, P.A., van Lookeren Campagne, A.A., Koper, J.F., 1989. Musclearchitecture and fibre characteristics of rat gastrocnemius and semi-membranosus muscles during isometric contractions. Acta Anat.(Basel) 135, 46–52.

Jaenicke, R., 1998. What ultrastable globular proteins teach us aboutprotein stabilization. Biochemistry (Mosc) 63, 312–321.

Jarvinen, T.A., Jozsa, L., Kannus, P., Jarvinen, T.L., Jarvinen, M., 2002.Organization and distribution of intramuscular connective tissue innormal and immobilized skeletal muscles. An immunohistochemical,polarization and scanning electron microscopic study. J. Muscle Res.Cell Motil. 23, 245–254.

Fig. 11. (A) SEM view of dissections of rat Peroneus digiti quarti muscleafter 5–56 NaOH cell maceration to remove myofibers and endomysium.Note the tubular arrangement (tu) of the perimysium. (B) SEM view ofPeroneus digiti quarti muscle prepared according to the fracture technique.Note the collagen-fiber bundles of the perimysium (p) connecting themyofibers (mf).


Meijer, H.J., Baan, G.C., Huijing, P.A., 2006. Myofascial force transmis-sion is increasingly important at lower forces: firing frequency-relatedlength-force characteristics of rat extensor digitorum longus. ActaPhysiol. (Oxf). 186, 185–195.

Metz, B., Kersten, G.F., Hoogerhout, P., Brugghe, H.F., Timmermans,H.A., de Jong, A., Meiring, H., ten Hove, J., Hennink, W.E.,Crommelin, D.J., Jiskoot, W., 2004. Identification of formaldehyde-induced modifications in proteins: reactions with model peptides. J.Biol. Chem. 279, 6235–6243.

Monti, R.J., Roy, R.R., Hodgson, J.A., Edgerton, V.R., 1999. Transmis-sion of forces within mammalian skeletal muscles. J. Biomech. 32,371–380.

Nakamura, Y.N., Iwamoto, H., Tabata, S., Ono, Y., 2003. Comparison ofcollagen fibre architecture between slow-twitch cranial and fast-twitchcaudal parts of broiler M. latissimus dorsi. Br. Poult. Sci. 44, 374–379.

Nishimura, T., Ojima, K., Liu, A., Hattori, A., Takahashi, K., 1996.Structural changes in the intramuscular connective tissue duringdevelopment of bovine semitendinosus muscle. Tissue Cell. 28,527–536.

Ohtani, O., Kikuta, A., Ohtsuka, A., Murakami, T., 1991. Organization ofthe reticular network of rabbit Peyer’s patches. Anat. Rec. 229,251–258.

Passerieux, E., Rossignol, R., Chopard, A., Carnino, A., Marini, J.F.,Letellier, T., Delage, J.P., 2006. Structural organization of theperimysium in bovine skeletal muscle: Junctional plates and associatedintracellular subdomains. J. Struct. Biol. 154 (2), 206–216.

Purslow, P.P., 2002. The structure and functional significance of variationsin the connective tissue within muscle. Comp. Biochem. Physiol. AMol. Integr. Physiol. 133, 947–966.

Rack, P.M., Westbury, D.R., 1984. Elastic properties of the cat soleustendon and their functional importance. J. Physiol. 347, 479–495.

Rowe, R.W.D., 1974. Collagen fibre arrangement in intramuscularconnective tissue. Changes associated with muscle shorteningand their possible relevance to raw meat toughness measurements.J. Fd. Technol. 9, 501–508.

Rowe, R.W.D., 1981. Morphology of perimysial and endomysial connec-tive tissue in skeletal muscle. Tissue Cell. 13, 681–690.

Tidball, J.G., 1991. Force transmission across muscle cell membranes.J. Biomech. 24 (Suppl. 1), 43–52.

Tidball, J.G., Chan, M., 1989. Adhesive strength of single muscle cells tobasement membrane at myotendinous junctions. J. Appl. Physiol. 67,1063–1069.

Trotter, J.A., Purslow, P.P., 1992. Functional morphology of theendomysium in series fibered muscles. J. Morphol. 212, 109–122.

Young, M., Paul, A., Rodda, J., Duxson, M., Sheard, P., 2000.Examination of intrafascicular muscle fiber terminations: implicationsfor tension delivery in series-fibered muscles. J. Morphol. 245,130–145.

Yucesoy, C.A., Koopman, B.H., Huijing, P.A., Grootenboer, H.J., 2002.Three-dimensional finite element modeling of skeletal muscle using atwo-domain approach: linked fiber-matrix mesh model. J. Biomech.35, 1253–1262.


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