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. Author manuscript; available in PMC: 2021 Feb 1.
Published in final edited form as: J Morphol. 2019 Dec 16;281(2):160–169. doi: 10.1002/jmor.21088

Diversity of extracellular matrix morphology in vertebrate skeletal muscle

David A Sleboda †, Kristin K Stover †,‡, Thomas J Roberts †
PMCID: PMC7137673  NIHMSID: NIHMS1577456  PMID: 31840868

Abstract

Existing data suggest the extracellular matrix (ECM) of vertebrate skeletal muscle consists of several morphologically distinct layers: an endomysium, perimysium, and epimysium surrounding muscle fibers, fascicles, and whole muscles, respectively. These ECM layers are hypothesized to serve important functional roles within muscle, influencing passive mechanics, providing avenues for force transmission, and influencing dynamic shape changes during contraction. The morphology of the skeletal muscle ECM is well described in mammals and birds; however, ECM morphology in other vertebrate groups including amphibians, fish, and reptiles remains largely unexamined. It remains unclear whether a multilayered ECM is a common feature of vertebrate skeletal muscle, and whether functional roles attributed to the ECM should be considered in mechanical analyses of non-mammalian and non-avian muscle. To explore the prevalence of a multi-layered ECM, we used a cell maceration and scanning electron microscopy technique to visualize the organization of ECM collagen in muscle from six vertebrates: bullfrogs (Lithobates catesbeianus), turkeys (Meleagris gallopavo), alligators (Alligator mississippiensis), cane toads (Rhinella marina), laboratory mice (Mus musculus), and carp (Cyprinus carpio). All muscles studied contained a collagen-reinforced ECM with multiple morphologically distinct layers. An endomysium surrounding muscle fibers was apparent in all samples. A perimysium surrounding groups of muscle fibers was apparent in all but carp epaxial muscle; a muscle anatomically, functionally, and phylogenetically distinct from the others studied. An epimysium was apparent in all samples taken at the muscle periphery. These findings show that a multi-layered ECM is a common feature of vertebrate muscle and suggest that a functionally relevant ECM should be considered in mechanical models of vertebrate muscle generally. It remains unclear whether cross-species variations in ECM architecture are the result of phylogenetic, anatomical, or functional differences, but understanding the influence of such variation on muscle mechanics may prove a fruitful area for future research.

Keywords: Muscle, extracellular matrix, collagen, vertebrate

1. Introduction

Skeletal muscle contains a highly specialized extracellular matrix (ECM) that suits its mechanical functions. Like skin, tendon, and other tissues frequently subjected to tensile forces, the ECM of skeletal muscle is reinforced by fibrous collagen (Borg & Caulfield, 1980). Collagen has a close-packed triple helix structure that grants it a high mechanical strength (Harkness, 1961), and its presence within muscle influences many mechanical behaviors of the tissue. ECM collagen influences the mechanical response of muscle to deformation, determining in part the passive tension developed by stretched muscle (Prado et al., 2005; Meyer & Lieber, 2011; Gindre, Takaza, Moerman, & Simms, 2013; Meyer & Lieber, 2018). The collagenous ECM also provides avenues for lateral and serial transmission of force between adjacent muscle fibers within the muscle belly (Street, 1983; Purslow & Trotter, 1994; Huijing, Baan, & Rebel, 1998), a function that allows coordination of force from multiple muscle fibers within a fascicle, and which prevents interruptions to muscle function during tissue growth or repair (Purslow, 2010). The collagenous ECM also likely influences dynamic changes in muscle shape during contraction, allowing the ECM to have a direct effect on the force and speed of contraction (Azizi, Brainerd, & Roberts, 2008; Eng, Azizi, & Roberts, 2018) as well as the useful mechanical work produced by a shortening muscle (Azizi, Deslauriers, Holt, & Eaton, 2017). In cardiac muscle, ECM connections to vasculature help maintain patency and prevent longitudinal slippage of vessels during contraction (Caulfield & Borg, 1979) and the ECM may play a similar protective role for vessels and nerves in skeletal muscle.

Existing data suggest the ECM of vertebrate skeletal muscle consists of several morphologically distinct, hierarchically arranged layers (figure 1). These layers are the endomysium, perimysium, and epimysium, and they surround muscle fibers, fascicles, and whole muscles, respectively (Borg & Caulfield, 1980; Rowe, 1981; Gillies & Lieber, 2011). The endomysium consists of a network of criss-crossing collagen fibrils that ensheathe and anchor on the cell membranes (sarcolemma) of muscle fibers. Endomysial collagen fibrils wrap the long axes of individual muscle fibers but are also shared between adjacent muscle fibers within a fascicle (Trotter & Purslow, 1992). They serve as the most likely vector through which forces are transmitted both laterally and serially between neighboring muscle fibers within the muscle belly (Purslow & Trotter, 1994). The perimysium contains relatively larger collagen fibers (each composed of multiple collagen fibrils) which form fibrous septa that are shared by and delineate adjacent muscle fascicles (Borg & Caulfield, 1980; Purslow, 1989). Mathematical modeling suggests that the thickness of the perimysium renders it an unlikely vector for transmission of force within muscle in vivo (Purslow, 2010); however, an alternative role for the perimysium may be to provide slip planes within the muscle belly that accommodate dynamic shape changes during contraction (Purslow, 1999; Purslow, 2002). The epimysium surrounds the muscle periphery and, like the perimysium, is composed of collagen fibers. Anatomical continuity between the epimysium, endomysium, perimysium, and in-series tendons suggest that the epimysium acts as a surface tendon, aiding in the transmission of force from muscle fibers to the skeletal system in some muscles (Passerieux, Rossignol, Letellier, & Delage, 2007).

Figure 1:

Figure 1:

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Spatial organization of muscle fibers and extracellular matrix as observed in mammalian and avian skeletal muscle. The extracellular matrix is a hierarchical, interlinked network composed of distinct layers that surround muscle fibers (endomysium), fascicles (perimysium), and the muscle periphery (epimysium).

Predominant among morphological and functional studies of skeletal muscle ECM are studies of mammalian and avian muscle. Mammals including cows, sheep, rats, rabbits, cats, pigs, and mice have been shown to display distinct endomysial, perimysial, and epimysial ECM layers (see supplemental table S1 for a list of studies of vertebrate skeletal muscle ECM morphology organized by species). Comparisons of ECM morphology both across mammalian species (e.g. Rowe, 1981), and across anatomically distinct muscles within a single mammalian species (e.g. Borg & Caulfield, 1980), reveal that a collagenous, tri-layered ECM is typical of mammalian skeletal muscle. A similar morphological arrangement of the ECM has been observed in birds. Broiler chicken muscles display a collagenous ECM like that of mammals, with distinct endomysial, perimysial, and epimysial layers (see for example Nakamura, Iwamoto, Tabata, & Ono, 2003; Roy et al., 2006). While these studies show that a multilayered, collagen reinforced ECM is typical of mammals and birds, the morphological arrangement of collagen in the ECMs of other vertebrate groups including fish, amphibians, and reptiles has received relatively little study. Fibrous collagen has been shown to reinforce the ECMs of certain frogs (Schmalbruch, 1974) and fish (Ando, Toyohara, & Sakaguchi, 1992; Ando, Yoshimoto, Inabu, Nakagawa, & Makinodan, 1995), but whether these animals contain distinct endomysium, perimysium, and epimysium structurally similar to those described in mammals and birds is not clear from these studies.

Without knowledge of ECM morphology in much of the vertebrate group, it cannot be stated with confidence that a multi-layered ECM is a general feature of vertebrate skeletal muscle, or that functional roles attributed to the endomysium, perimysium, and epimysium should be considered in mechanical analyses of non-mammalian and non-avian muscle. Additionally, the efficacy of using non-mammalian or non-avian muscles as model systems for the study of ECM function remains untested. Amphibian muscle, for example, has been used as a model system to explore the influence of the ECM on lateral force transmission (Street, 1983) and passive tension developed during stretch (Sleboda & Roberts, 2017; Sleboda, Wold, & Roberts, 2019). Knowledge of ECM morphology in these animals is needed to ensure the applicability of findings from these studies across vertebrates.

Given the functional roles attributed to the endomysium, perimysium, and epimysium, we hypothesized that a multi-layered collagenous extracellular matrix is a fundamental mechanical component of skeletal muscle. To explore the prevalence of a multilayered ECM in vertebrate muscle, we used a cell-maceration and scanning electron microscopy technique (Ohtani et al., 1988) to visualize the morphology of ECM collagen in the following species and muscles: 1) American bullfrog (Lithobates catesbeianus), semimembranosus; 2) turkey (Meleagris gallopavo), lateral gastrocnemius; 3) American alligator (Alligator mississippiensis), triceps brachii; 4) cane toad (Rhinella marina), plantaris; 5) laboratory mouse (Mus musculus), lateral gastrocnemius; and 6) common carp (Cyprinus carpio), epaxial muscle.

2. Materials and Methods

All animal use was approved by the Brown University Institutional Animal Care and Use Committee (IACUC). Muscle samples were obtained opportunistically from animals used in other studies. All muscles were sampled from fully grown animals with the exception of alligator triceps brachii, which was sampled from a juvenile. All animals were obtained from commercial breeders and were healthy and free of apparent neuromuscular disorders. Depending on availability, samples were taken either from the center of the muscle belly or from the muscle periphery and subjected to the cell maceration technique (Ohtani, 1988; Trotter & Purslow, 1992) which uses a 10% aqueous solution of sodium hydroxide (NaOH) to selectively dissolve cellular material away from chemically fixed tissue, allowing visualization of the collagenous component of the extracellular matrix. This technique has been used previously to visualize ECM morphology in mammals (e.g. Purslow & Trotter, 1994) and birds (e.g. Nakamura et al., 2003). That this technique exposes collagen fibrils preserved in natural arrangements has been verified via comparison of NaOH digested and undigested muscle samples via transmission electron microscopy (Ohtani et al., 1988; Trotter & Purslow, 1992). Higher concentration NaOH solutions have been used to selectively degrade endomysial collagen in skeletal muscle, allowing study of the perimysium and epimysium in isolation (Passerieux et al., 2006). Maceration in 10% NaOH solution has been shown to leave the endomysial network intact (Trotter & Purslow 1992; Purslow & Trotter, 1994), allowing visualization of the collagenous ECM in its entirety.

Muscle samples were harvested freshly from euthanized animals and preserved in 10% formalin prior to processing. Disc-shaped pieces of formalin-fixed muscle roughly 2 mm thick and 3–10 mm in diameter were isolated manually using a fine razor blade and immersed in Karnovsky’s fixative (4% glutaraldehyde, 4% paraformaldehyde, 0.2 M sodium cacodylate buffer, pH 7.4) overnight. Care was taken to section muscles perpendicular to the long axis of muscle fibers, providing cross-sectional views of the collagenous extracellular matrix. Fixed samples were washed in 0.1 M sodium cacodylate buffer, and then immersed in 10% aqueous NaOH at room temperature (~22 °C) for 4–7 days depending on the size and thickness of the sample. Muscle samples were monitored visually during this period and kept in NaOH solution until they faded to a transparent white color, indicating disintegration of cellular material. Decellularized samples were promptly removed from NaOH, washed in distilled water for 3 days and then prepared for scanning electron microscopy. Samples were immersed in 1% aqueous tannic acid for 3 hours, washed with distilled water, and post-fixed in 1% aqueous osmium tetroxide overnight. Samples were dehydrated via a graded series of ethanol and critical point-dried with liquid CO2. Dry samples were mounted on aluminum stubs with double-sided carbon tape and sputter-coated with gold. Dry, coated samples were viewed using either a Hitachi 2700 or Thermo Apreo Volume Scope scanning electron microscope (SEM) at an accelerating voltage of 5–8 kV.

Using SEM, decellularized muscle samples were examined for endomysial, perimysial, and epimysial extracellular matrix. Endomysial ECM was defined by the presence of criss-crossing curvilinear collagen fibrils forming honeycomb-like networks of interconnected sheaths surrounding the spaces once occupied by muscle fibers. Collagen fibrils typically range in diameter from 10–300 nm (Bancelin, 2014), and this range informed our identification of endomysial collagen. Perimysial ECM was identifiable as large fibrous septa that divided groups of muscle fibers and was distinguishable from endomysial ECM by the presence of larger, ribbon-like collagen fibers (composed of many individual collagen fibrils) ~0.6–5.0 μm wide (Borg & Caulfield, 1980; Nakamura et al., 2003). Epimysial ECM was defined by thickened regions of ECM morphologically similar to the perimysium but present at the muscle periphery. Following collection of micrographs, measurements of collagen fibril diameter, collagen fiber width and thickness, and perimysium thickness were made in ImageJ (Shindelin et al., 2012). Following the convention of Rowe (Rowe, 1974; Rowe, 1981) and Borg and Caulfield (Borg & Caulfield, 1980), we use the term collagen fiber to describe organized bundles of collagen fibrils in the perimysium and epimysium, but note that such structures have also been referred to as collagen cables (Gillies & Lieber, 2011; Gillies, Bushong, Deerinck, Ellisman, & Lieber, 2014; Gillies et al., 2017).

3. Results

In all sampled muscles, digestion in sodium hydroxide revealed an ECM reinforced by complex, interconnected networks of fibrous collagen. Bullfrog semimembranosus (figure 2), domestic turkey lateral gastrocnemius (figure 3), American alligator triceps brachii (figure 4), cane toad plantaris (figure 5), and mouse lateral gastrocnemius (figure 6) muscles displayed morphologically distinct regions of endomysial and perimysial connective tissue. Epimysial ECM was visible in all micrographs taken at the muscle periphery (figures 2a,b, 5a, 6a, 7a,b), and in turkey lateral gastrocnemius muscle, though not presented here. Samples of alligator triceps brachii (figure 4) were collected from the center of the muscle belly such that the presence or absence of a distinct epimysium at the periphery of this muscle could not be assessed. Carp epaxial muscle (figure 7) contained endomysial and epimysial ECM but was unique among the muscles sampled in that a distinct perimysium was not evident. Endomysial collagen in carp epaxial muscle was intimately associated with collagenous myoseptal tendons (figure 7b,c), which are typical of fish body musculature (Gemballa et al 2003).

Figure 2:

Figure 2:

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American bullfrog (Lithobates catesbeianus) semimembranosus muscle. A Cross-section of NaOH digested muscle revealing an interconnected collagenous ECM. Epimysium (Ep) is indicated at the muscle periphery. White rectangle shows area magnified in panel B. B Closer view of the area denoted by the white rectangle in A. Distinct endomysial (En), perimysial (P), and epimysial (Ep) regions are indicated. Continuity between perimysial and epimysial ECM is indicated by arrows. Endomysial sheaths near the muscle periphery are continuous with the epimysium. C Closer view of the area denoted by the white rectangle in B. The perimysium contains multiple collagen fibers (white arrows). The walls of endomysial sheaths (En) are continuous with the outer edge of the perimysium (black arrows). D One collagen fiber isolated by peeling a layer of perimysial ECM apart. Fiber has a flat, ribbon-like morphology and is composed of multiple collagen fibrils running in parallel. E Oblique view of one endomysial sheath with proximal wall torn open, revealing a network of criss-crossing endomysial collagen fibrils.

Figure 3:

Figure 3:

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Domestic turkey (Meleagris gallopavo) lateral gastrocnemius muscle. A Cross-section showing morphologically distinct regions of endomysial (En) and perimysial (P) ECM. Sample is from the muscle belly where epimysium is not visible. Perimysial septa divide and delineate groups of endomysial sheaths. B Closer view of the area denoted by the white rectangle in A. Endomysial sheaths (En) and perimysium (P) are indicated. C Close-up view of interconnected endomysial sheaths (En) and an adjacent region of perimysium (P). Walls of endomysial sheaths at the fascicle periphery are continuous with the outer edge of the perimysium. Ribbon-like collagen fibers are visible in the perimysium (lassos, indicated by arrows). D Cross-section of an intramuscular nerve with distinct endoneurial (NEn) sheaths delineating spaces for individual nerve fibers and epineurium (NEp) surrounding the nerve periphery (see Ushiki and Ide, 1990 for a full description of nerve ECM anatomy).

Figure 4:

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American alligator (Alligator mississippiensis) triceps brachii muscle. A Cross-section showing endomysial (En) and perimysial (P) ECM. Sample is from the muscle belly where epimysium is not visible. Perimysial septa divide and delineate groups of endomysial sheaths. B Closer view of the area denoted by the larger white rectangle in A. Perimysium (P) forms a septum that separates two groups of endomysial sheaths (En). C Closer view of the area denoted by the white rectangle in B. Perimysium is composed of layers of ribbon-like collagen fibers rectangular in cross section. D Closer view of the area denoted by the smaller white rectangle in A showing interconnected endomysial sheaths.

Figure 5:

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Cane toad (Rhinella marina) plantaris muscle. A Cross-section of an entire plantaris muscle. A thick epimysium (Ep) covers the dorsal surface of the muscle, contributing to the large surface tendon (aponeurosis) of this muscle. A large internal tendon typical of the plantaris is also visible (T). Many endomysial sheaths terminate by attaching on the internal tendon (white arrows). B Close-up view of endomysium (En) and perimysium (P). A neurovascular bundle is visible in the perimysium consisting of spaces previously occupied by blood vessels (V) and a nerve (N, white lasso). C Close up view of perimysium. Collagen fibers are less defined than in other samples, giving the perimysium an appearance similar to sectioned plywood. D Close up view of endomysial sheaths.

Figure 6:

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Mouse (Mus musculus) lateral gastrocnemius muscle. A Cross-section showing endomysium (En), perimysium (P), and epimysium (Ep). Perimysium is continuous with epimysium near the muscle periphery (arrow). Endomysial sheaths near the muscle periphery are also continuous with the epimysium. B Closer view of the area denoted by the white rectangle in A. Endomysium (En) and perimysium (P) are indicated. C Close up view of the area indicated by the white rectangle in B. A few ribbon-like collagen fibers are visible in the perimysium (lassos, indicated with arrows). D Endomysial sheaths. Pellet-like structures attached to endomysial sheath walls are likely remnants of cellular elements, e.g. cell nuclei (Ohtani et al., 1988).

Figure 7:

Figure 7:

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Common carp (Cyprinus carpio) epaxial muscle. A Cross-section near the muscle periphery showing endomysium (En) and a myoseptal tendon (MT). Epimysium (Ep) is visible in the upper left corner of the micrograph. Myoseptal tendon divides groups of endomysial sheaths. B Closer view of the area denoted by the white rectangle in A. Endomysial sheaths (En) terminate on myoseptal tendon (MT). C Closer view of the area denoted by the white rectangle in B showing connection between endomysial sheath (En) and myoseptal tendon (MT). Myoseptal tendons contain layered, ribbon-like collagen fibers (arrows). D Close-up of an endomysial sheath.

The morphologies of observed endomysial, perimysial, and epimysial ECM were similar to those described in past studies of vertebrate ECM (e.g. Borg & Caulfield, 1980; Rowe, 1981). In all samples, the endomysium was composed of collagen fibrils ranging in diameter from ~100–300 nm. Fibrils formed long tubular sheaths that surrounded the spaces once occupied by muscle fibers (e.g. figures 2e, 3c, 4d, 5d, 6d, and 7d). Individual fibrils ran at a range of angles relative to the long axes of muscle fibers. Many fibrils were shared between adjacent endomysial sheaths, such that the endomysium comprised a continuous and highly interconnected network, as has been described previously (Trotter & Purslow, 1992). Endomysial sheaths varied in diameter both within and across muscle samples, indicating variation in muscle fiber size. Endomysial sheaths at the periphery of muscle fascicles were connected to the perimysium (figures 2c, 3c, 4b, 5c, 6b) and endomysial sheaths at the muscle periphery were connected to the epimysium (figures 2b, 6a, 7a,b), such that a continuous collagenous network spanned the entire cross section of all muscle samples.

Perimysial ECM contained collagen fibers (Figures 2c, 3c, 4b, 6b,c), each composed of many collagen fibrils arranged in parallel (figure 2d). Fibers were typically rectangular in cross section, ranging between 1–5 μm in width but rarely more than 1 μm in thickness, giving them a flat, ribbon-like appearance. Exceptionally large perimysial fibers greater than 15 μm in width and greater than 1.5 μm in thickness were observed in alligator triceps muscle (figures 4b,c). Collagen fibers were not clearly defined in cane toad plantaris, giving the perimysium of this muscle an appearance similar to sectioned plywood (figure 5c). In other samples, layered sheets of perimysial collagen fibers formed fibrous septa that divided or encircled large groups of muscle fibers (e.g. figures 3a, 4a). Perimysial septa ranging in thickness from ~10–30 μm were common across all sampled muscles, with the exception of bullfrog semimembranosus, in which no perimysial septa thicker than 15 μm were observed. Exceptionally large septa greater than 50 μm in thickness were observed in two muscles: turkey gastrocnemius (figure 3a,b) and alligator triceps (figure 4a,b).

Epimysial ECM was morphologically similar to perimysial ECM, being composed of layers of ribbon-like collagen fibers. Epimysial collagen fibers were generally larger than perimysial collagen fibers, with a fiber width of ~50 μm typical across all epimysial collagen fibers observed. Perimysial septa that extended to the muscle periphery were continuous with the epimysium (figures 2b, 6a).

In addition to muscle fibers, fascicles, and the muscle periphery, the collagenous ECM encompassed intramuscular nerves and blood vessels (figures 3d, 5b). Nerves were identifiable in digested muscle samples by their distinct collagen fibrillar structure, defined by the presence of distinct epineurium and endoneurium layers (Ushiki & Ide, 1990). Tubular spaces once occupied by blood vessels were identifiable by their position within the thickness of the perimysium, where muscle fibers are not found.

Myoseptal tendons visible in carp epaxial muscle ranged in thickness from ~20–50 um and were composed of layered, ribbon-like collagen fibers (figure 7c) that formed divisions between large groups of muscle fibers (figure 7a,b). Endomysial sheaths terminated directly on myoseptal tendons (figure 7c). A large intramuscular tendon was also visible in cane toad plantaris muscle (figure 5a) and served as the ultimate attachment point for many endomysial sheaths.

4. Discussion

Our study confirms that a multilayered collagenous ECM is present in the muscles of many vertebrates, and that distinct endomysial, perimysial, and epimysial layers are not unique to mammals and birds. An endomysium surrounding and linking muscle fibers was present in all sampled muscles, and in all samples this endomysial network was accompanied by thickened layers of perimysial ECM delineating fascicles within the muscle belly, epimysial ECM surrounding the muscle periphery, or a combination of both perimysial and epimysial ECM. These findings suggest that a multi-layered ECM is typical of vertebrate muscle, and that functional roles attributed to the ECM can reasonably be assumed to influence the mechanics of non-mammalian and non-avian muscle. We have assumed previously that a mammal-like ECM is present in amphibian muscle and that this ECM influences basic mechanical properties of the tissue, such as the response of passive muscle to stretch (Sleboda & Roberts, 2017; Sleboda et al., 2019). The current findings validate this assumption and confirm that amphibian and other non-mammalian and non-avian muscles can serve as valid models for the study of ECM biomechanics in vertebrate muscle.

Although we did not attempt quantitative analysis of collagen content, muscles imaged in the current study display variations in ECM morphology qualitatively similar to those observed in previously studied vertebrates. Differences in the amount of endomysial, perimysial, and epimysial ECM visible in cross section were apparent across muscle samples. For example, distinct perimysial ECM was more prevalent and easier to identify in alligator triceps brachii than in mouse gastrocnemius (e.g. Figure 4a versus 6a), and collagen fibrils formed endomysial sheaths with visibly denser walls in bullfrog semimembranosus than in carp epaxial muscle (e.g. Figure 2e versus 7d). Such qualitative differences are likely the result of multiple factors, of which phylogenetic differences are only one. Previous biochemical analyses of hydroxyproline content in skeletal muscle show that the prevalence of the endomysium, perimysium, and epimysium is variable, with perimysial collagen content being more variable than either endomysial or epimysial content (Light, Champion, Voyle, & Bailey, 1985; Purslow, 2002). Variations in skeletal muscle ECM morphology and content have been attributed to multiple factors, including differences in animal size (Rowe, 1981), muscle function (Borg & Caulfield, 1980), and muscle fiber type (Nakamura et al., 2003). The prevalence and morphological arrangement of the skeletal muscle ECM also varies significantly with animal age (Alnaqeeb, Alzaid, & Goldspink, 1984; Nishimura, Ojima, Liu, Hattori, & Takahashi, 1996; Fang, Nishimura, & Takahashi, 1999; Gao, Kostrominova, Faulkner, & Wineman, 2008), and in some neuromuscular disorders (Smith, Lee, Ward, Chambers, & Lieber, 2011; Lieber & Ward, 2013). It is unclear from the current study whether cross-species variations in ECM architecture observed are the result of phylogenetic, anatomical, functional, or other differences, but the impact of these and similar variations on muscle mechanics warrant further investigation. Biochemical analyses of total collagen content within muscle have been shown to be poor predictors of fundamental mechanical properties of muscle, such as passive muscle stiffness (Bensamoun et al., 2006; Lieber & Ward, 2013), but it has been suggested that subtle differences in ECM morphology may underlie specialization of muscle for particular functional tasks, such as the production or dissipation of mechanical energy (Azizi, 2014).

Among the muscles sampled, the most unique ECM morphology was observed in carp epaxial muscle. Carp epaxial muscle was distinct in the current study both as the only axial muscle (all other were limb muscles), and as the only muscle that operates in a fully aquatic environment. The drastically different loading conditions and evolutionary and developmental histories of this muscle may underlie its distinctiveness. Carp epaxial muscle displayed a typical endomysium composed of collagen fibrils surrounding muscle fibers; however, very few collagen fibers reinforced the endomysium, and no clear perimysium was evident. Carp muscle was also unique in its display of myoseptal tendons, which are typical of fish muscle (Gemballa et al., 2003). Myoseptal tendons aid in the transmission of contractile force from trunk muscles to the horizontal septum, vertebrae, and skin, which acts like an external tendon in fish (Nursall, 1956). Myoseptal tendons were structurally similar to the perimysium observed in other animals, being composed of layered arrangements of ribbon-like collagen fibers (figures 7b,c); however, the presence of endomysial sheaths terminating directly on myoseptal tendons distinguished them as tendons. It is possible that the presence of myoseptal tendons in fish body muscle negates the need for a distinct perimysium; however, further comparative study of the skeletal muscle ECM in fish is needed to explore this hypothesis, and to determine whether the lack of a distinct perimysium is typical of fish muscle.

5. Conclusions

The findings of the current study support the hypothesis that a multilayered, collagen-reinforced ECM is a general feature of vertebrate skeletal muscle. They confirm that non-mammalian and non-avian muscle can serve as valid model systems for the study of ECM function, and that past studies of ECM mechanics in frogs (e.g. Street, 1986; Sleboda & Roberts, 2017; Sleboda et al., 2019) and other vertebrates can inform our understanding of vertebrate muscle generally. It remains unclear whether cross-species variations in ECM architecture observed in the current study are the result of phylogenetic, anatomical, or functional differences; however, such variations have the potential to influence both passive and active muscle mechanics and may prove a fruitful area for future research.

Supplementary Material

supp table

Research highlights.

A multi-layered, collagenous extracellular matrix is shown to reinforce skeletal muscle in fish, amphibians, reptiles, mammals, and birds, suggesting that such matrices are a common and functionally important feature of vertebrate muscle.

Author contributions and Acknowledgements

DAS and KKS collected, prepared, and imaged muscle samples. DAS drafted the manuscript. DAS, TJR, and KKS conceived the study and revised the manuscript. All authors gave final approval for publication. The authors thank Geoff Williams for technical assistance obtaining micrographs, and Patricia Hernandez, Payam Mohassel, and Henry Tsai for providing tissue samples. Funded by NIH grants AR55295 and S10OD023461, NSF grants IOS1354289 and 1832795, the Bushnell Research Fund, and an EEB Dissertation Development grant from the Drollinger Family Charitable Foundation.

Footnotes

Conflict of interest: The authors have no competing interests to declare.

Data Availability Statement: The data that support the findings of this study are available from the corresponding author upon reasonable request.

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