Received: 23 May 2019 Revised: 20 November 2019 Accepted: 30 November 2019 DOI: 10.1002/jmor.21088

RESEARCH ARTICLE

Diversity of extracellular matrix morphology in vertebrate

David A. Sleboda1 | Kristin K. Stover1 | Thomas J. Roberts1

1Department of Ecology and Evolutionary Biology, Brown University, Providence, Rhode Abstract Island Existing data suggest the extracellular matrix (ECM) of vertebrate skeletal muscle con-

Correspondence sists of several morphologically distinct layers: an , , and epi- *David A. Sleboda, Department of Ecology and mysium surrounding muscle fibers, fascicles, and whole muscles, respectively. These Evolutionary Biology, Brown University, Providence, RI. ECM layers are hypothesized to serve important functional roles within muscle, Email: [email protected] influencing passive mechanics, providing avenues for force transmission, and influenc-

Present address ing dynamic shape changes during contraction. The morphology of the skeletal muscle Kristin K. Stover, Department of Biomedical ECM is well described in mammals and birds; however, ECM morphology in other ver- Science, West Virginia School of Osteopathic Medicine, Lewisburg, WV tebrate groups including amphibians, fish, and reptiles remains largely unexamined. It remains unclear whether a multilayered ECM is a common feature of vertebrate skele- Funding information Bushnell Research Fund; Drollinger Family tal muscle, and whether functional roles attributed to the ECM should be considered in Charitable Foundation; National Institute of mechanical analyses of non-mammalian and non-avian muscle. To explore the preva- Arthritis and Musculoskeletal and Skin Diseases, Grant/Award Number: AR55295; lence of a multilayered ECM, we used a cell maceration and scanning electron micros- National Institutes of Health, Grant/Award copy technique to visualize the organization of ECM in muscle from six Number: S10 OD023461; National Science Foundation, Grant/Award Numbers: 1832795, vertebrates: bullfrogs (Lithobates catesbeianus), turkeys (Meleagris gallopavo), alligators IOS1354289 (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 was apparent in all samples taken at the muscle periphery. These findings show that a multilayered 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 collagen, ,

1 | INTRODUCTION tissues frequently subjected to tensile forces, the ECM of skeletal muscle is reinforced by fibrous collagen (Borg & Caulfield, 1980). Col- Skeletal muscle contains a highly specialized extracellular matrix lagen has a close-packed triple helix structure that grants it a high (ECM) that suits its mechanical functions. Like skin, , and other mechanical strength (Harkness, 1961), and its presence within muscle

Journal of Morphology. 2019;1–10. wileyonlinelibrary.com/journal/jmor © 2019 Wiley Periodicals, Inc. 1 2 SLEBODA ET AL. influences many mechanical behaviors of the tissue. ECM collagen as the most likely vector through which forces are transmitted both influences the mechanical response of muscle to deformation, deter- laterally and serially between neighboring muscle fibers within the mining in part the passive tension developed by stretched muscle muscle belly (Purslow & Trotter, 1994). The perimysium contains rela- (Gindre, Takaza, Moerman, & Simms, 2013; Meyer & Lieber, 2011, tively larger collagen fibers (each composed of multiple collagen fibrils) 2018; Prado et al., 2005). The collagenous ECM also provides avenues which form fibrous septa that are shared by and delineate adjacent for lateral and serial transmission of force between adjacent muscle muscle fascicles (Borg & Caulfield, 1980; Purslow, 1989). Mathemati- fibers within the muscle belly (Huijing, Baan, & Rebel, 1998; Purslow & cal modeling suggests that the thickness of the perimysium renders it Trotter, 1994; Street, 1983), a function that allows coordination of an unlikely vector for transmission of force within muscle in vivo force from multiple muscle fibers within a fascicle, and which prevents (Purslow, 2010); however, an alternative role for the perimysium may interruptions to muscle function during tissue growth or repair be to provide slip planes within the muscle belly that accommodate (Purslow, 2010). The collagenous ECM also likely influences dynamic dynamic shape changes during contraction (Purslow, 1999; Purslow, changes in muscle shape during contraction, allowing the ECM to have 2002). The epimysium surrounds the muscle periphery and, like the a direct effect on the force and speed of contraction (Azizi, Brainerd, & perimysium, is composed of collagen fibers. Anatomical continuity Roberts, 2008; Eng, Azizi, & Roberts, 2018) as well as the useful between the epimysium, endomysium, perimysium, and in-series ten- mechanical work produced by a shortening muscle (Azizi, Deslauriers, dons suggest that the epimysium acts as a surface tendon, aiding in Holt, & Eaton, 2017). In , ECM connections to vascula- the transmission of force from muscle fibers to the skeletal system in ture help maintain patency and prevent longitudinal slippage of vessels some muscles (Passerieux, Rossignol, Letellier, & Delage, 2007). during contraction (Caulfield & Borg, 1979) and the ECM may play a Predominant among morphological and functional studies of skel- similar protective role for vessels and in skeletal muscle. etal muscle ECM are studies of mammalian and avian muscle. Mam- Existing data suggest the ECM of vertebrate skeletal muscle con- mals including cows, sheep, rats, rabbits, cats, pigs, and mice have sists of several morphologically distinct, hierarchically arranged layers been shown to display distinct endomysial, perimysial, and epimysial (Figure 1). These layers are the endomysium, perimysium, and epimy- ECM layers (see Table S1 for a list of studies of vertebrate skeletal sium, and they surround muscle fibers, fascicles, and whole muscles, muscle ECM morphology organized by species). Comparisons of ECM respectively (Borg & Caulfield, 1980; Gillies & Lieber, 2011; Rowe, morphology both across mammalian species (e.g., Rowe, 1981), and 1981). The endomysium consists of a network of criss-crossing colla- across anatomically distinct muscles within a single mammalian spe- gen fibrils that ensheathe and anchor on the cell membranes (sarco- cies (e.g., Borg & Caulfield, 1980), reveal that a collagenous, trilayered lemma) of muscle fibers. Endomysial collagen fibrils wrap the long ECM is typical of mammalian skeletal muscle. A similar morphological axes of individual muscle fibers, but are also shared between adjacent arrangement of the ECM has been observed in birds. Broiler chicken muscle fibers within a fascicle (Trotter & Purslow, 1992). They serve muscles display a collagenous ECM like that of mammals, with distinct endomysial, perimysial, and epimysial layers (e.g., Nakamura, Iwamoto, Tabata, & Ono, 2003; Roy et al., 2006). Although 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 rein- force 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 verte- brate group, it cannot be stated with confidence that a multilayered ECM is a general feature of vertebrate skeletal muscle, or that func- tional roles attributed to the endomysium, perimysium, and epimy- sium 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 FIGURE 1 Spatial organization of muscle fibers and extracellular lateral force transmission (Street, 1983) and passive tension devel- matrix as observed in mammalian and avian skeletal muscle. The extracellular matrix is a hierarchical, interlinked network composed of oped during stretch (Sleboda & Roberts, 2017; Sleboda, Wold, & Rob- distinct layers that surround muscle fibers (endomysium), fascicles erts, 2019). Knowledge of ECM morphology in these animals is (perimysium), and the muscle periphery (epimysium) needed to ensure the applicability of findings from these studies SLEBODA ET AL. 3 across vertebrates. Given the functional roles attributed to the samples were obtained opportunistically from animals used in other endomysium, perimysium, and epimysium, we hypothesized that a studies (n=1 individual per species). All muscles were sampled from multilayered collagenous ECM is a fundamental mechanical compo- fully grown animals with the exception of alligator triceps brachii, nent of skeletal muscle. which was sampled from a juvenile. All animals were obtained from commercial breeders and were healthy and free of apparent neuro- muscular disorders. Depending on availability, samples were taken 2 | MATERIALS AND METHODS either from the center of the muscle belly or from the muscle periph- ery and subjected to the cell maceration technique (Ohtani et al., To explore the prevalence of a multilayered ECM in vertebrate mus- 1988; Trotter & Purslow, 1992) which uses a 10% aqueous solution cle, we used a cell-maceration and scanning electron microscopy tech- of sodium hydroxide (NaOH) to selectively dissolve cellular material nique (Ohtani, Ushiki, Taguchi, & Kikuta, 1988) to visualize the away from chemically fixed tissue, allowing visualization of the collag- morphology of ECM collagen in the following species and muscles: enous component of the ECM. This technique has been used previ- (a) American bullfrog (Lithobates catesbeianus), semimembranosus; ously to visualize ECM morphology in mammals (e.g., Purslow & (b) turkey (Meleagris gallopavo), lateral gastrocnemius; (c) American alli- Trotter, 1994) and birds (e.g., Nakamura et al., 2003). That this tech- gator (Alligator mississippiensis), triceps brachii; (d) cane toad (Rhinella nique exposes collagen fibrils preserved in natural arrangements has marina), plantaris; (e) laboratory mouse (Mus musculus), lateral gastroc- been verified via comparison of NaOH digested and undigested mus- nemius; and (f) common carp (Cyprinus carpio), epaxial muscle. All ani- cle samples using transmission electron microscopy (Ohtani et al., mal use was approved by the Brown University Institutional Animal 1988; Trotter & Purslow, 1992). Higher concentration NaOH solu- Care and Use Committee (IACUC) protocol 1602000189. Muscle tions have been used to selectively degrade endomysial collagen in

FIGURE 2 American bullfrog (Lithobates catesbeianus) semimembranosus muscle. (a) Cross- section of NaOH digested muscle revealing an interconnected collagenous extracellular matrix (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 4 SLEBODA ET AL.

FIGURE 3 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 with distinct endoneurial (NEn) sheaths delineating spaces for individual nerve fibers and (NEp) surrounding the nerve periphery (see Ushiki & Ide, 1990 for a full description of nerve ECM anatomy)

FIGURE 4 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 SLEBODA ET AL. 5 skeletal muscle, allowing study of the perimysium and epimysium in muscle fibers, providing cross-sectional views of the collagenous isolation (Passerieux et al., 2007). Maceration in 10% NaOH solution ECM. Fixed samples were washed in 0.1 mol l−1 sodium cacodylate has been shown to leave the endomysial network intact (Purslow & buffer, and then immersed in 10% aqueous NaOH at room tempera- Trotter, 1994; Trotter & Purslow, 1992), allowing visualization of the ture (~22C) for 4–7 days depending on the size and thickness of the collagenous ECM in its entirety. sample. Muscle samples were monitored visually during this period Muscle samples (n=3-5 per muscle) were harvested freshly from and kept in NaOH solution until they faded to a transparent white euthanized animals and preserved in 10% formalin prior to processing. color, indicating disintegration of cellular material. Decellularized sam- Disc-shaped pieces of formalin-fixed muscle roughly 2 mm thick and ples were promptly removed from NaOH, washed in distilled water 3–10 mm in diameter were isolated manually using a fine razor blade for 3 days and then prepared for scanning electron microscopy. Sam- and immersed in Karnovsky's fixative (4% glutaraldehyde, 4% parafor- ples were immersed in 1% aqueous tannic acid for 3 hr, washed with maldehyde, 0.2 mol l−1 sodium cacodylate buffer, pH 7.4) overnight. distilled water, and postfixed in 1% aqueous osmium tetroxide over- Care was taken to section muscles perpendicular to the long axis of night. Samples were dehydrated via a graded series of ethanol and

FIGURE 5 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 6 SLEBODA ET AL.

critical point-dried with liquid CO2. Dry samples were mounted on collagen. Bullfrog semimembranosus (Figure 2), domestic turkey lat- aluminum stubs with double-sided carbon tape and sputter-coated eral gastrocnemius (Figure 3), American alligator triceps brachii with gold. Dry, coated samples were viewed using either a Hitachi (Figure 4), cane toad plantaris (Figure 5), and mouse lateral gastrocne- 2700 or Thermo Apreo Volume Scope scanning electron microscope mius (Figure 6) muscles displayed morphologically distinct regions of (SEM) at an accelerating voltage of 5–8 kV. endomysial and perimysial connective tissue. Epimysial ECM was visi- Using SEM, decellularized muscle samples were examined for ble in all micrographs taken at the muscle periphery (Figures 2a,b, 5a, endomysial, perimysial, and epimysial ECM. Endomysial ECM was 6a, and 7a,b), and in turkey lateral gastrocnemius muscle, though not defined by the presence of criss-crossing curvilinear collagen fibrils for- presented here. Samples of alligator triceps brachii (Figure 4) were col- ming honeycomb-like networks of interconnected sheaths surrounding lected from the center of the muscle belly such that the presence or spaces once occupied by muscle fibers. Collagen fibrils typically range in absence of a distinct epimysium at the periphery of this muscle could diameter from 10 to 300 nm (Bancelin, 2014), and this range informed not be assessed. Carp epaxial muscle (Figure 7) contained endomysial our identification of endomysial collagen. Perimysial ECM was identifi- and epimysial ECM, but was unique among the muscles sampled in able as large fibrous septa that divided groups of muscle fibers and was that a distinct perimysium was not evident. Endomysial collagen in distinguishable from endomysial ECM by the presence of larger, ribbon- carp epaxial muscle was intimately associated with collagenous like collagen fibers (composed of many individual collagen fibrils) myoseptal (Figure 7b,c), which are typical of fish body muscu- ~0.6–5.0 μm wide (Borg & Caulfield, 1980; Nakamura et al., 2003). lature (Gemballa et al., 2003). Epimysial ECM was defined by thickened regions of ECM morphologi- The morphologies of observed endomysial, perimysial, and cally similar to the perimysium, but present at the muscle periphery. Fol- epimysial ECM were similar to those described in past studies of ver- lowing collection of micrographs, measurements of collagen fibril tebrate ECM (e.g., Borg & Caulfield, 1980; Rowe, 1981). In all samples, diameter, collagen fiber width and thickness, and perimysium thickness the endomysium was composed of collagen fibrils ranging in diameter were made in ImageJ (Schindelin et al., 2012). Following the convention from ~100 to 300 nm. Fibrils formed long tubular sheaths that sur- of Rowe (1974, 1981) and Borg and Caulfield (1980), we use the term rounded spaces once occupied by muscle fibers (Figures 2e, 3c, 4d, collagen fiber to describe organized bundles of collagen fibrils in the peri- 5d, 6d, and 7d). Individual fibrils ran at a range of angles relative to mysium and epimysium, but note that such structures have also been the long axes of muscle fibers. Many fibrils were shared between adja- referred to as collagen cables (Gillies et al., 2017; Gillies, Bushong, cent endomysial sheaths, such that the endomysium comprised a con- Deerinck, Ellisman, & Lieber, 2014; Gillies & Lieber, 2011). tinuous 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 3 | RESULTS in muscle fiber size. Endomysial sheaths at the periphery of muscle fascicles were connected to the perimysium (Figures 2c, 3c, 4b, 5c, In all sampled muscles, digestion in sodium hydroxide revealed an and 6b) and endomysial sheaths at the muscle periphery were con- ECM reinforced by complex, interconnected networks of fibrous nected to the epimysium (Figures 2b, 6a, and 7a,b), such that a

FIGURE 6 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, for example, cell nuclei (Ohtani et al., 1988) SLEBODA ET AL. 7

FIGURE 7 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

continuous collagenous network spanned the entire cross section of muscle samples by their distinct collagen fibrillar structure, defined by all muscle samples. the presence of distinct epineurium and endoneurium layers (Ushiki & Perimysial ECM contained collagen fibers (Figures 2c, 3c, 4b, and Ide, 1990). Tubular spaces once occupied by blood vessels were iden- 6b,c), each composed of many collagen fibrils arranged in parallel tifiable by their position within the thickness of the perimysium, (Figure 2d). Fibers were typically rectangular in cross section, ranging where muscle fibers are not found. between 1 and 5 μm in width, but rarely more than 1 μm in thickness, Myoseptal tendons visible in carp epaxial muscle ranged in thick- giving them a flat, ribbon-like appearance. Exceptionally large ness from ~20 to 50 μm and were composed of layered, ribbon-like perimysial fibers greater than 15 μm in width and greater than 1.5 μm collagen fibers (Figure 7c) that formed divisions between large groups in thickness were observed in alligator triceps muscle (Figure 4b,c). of muscle fibers (Figure 7a,b). Endomysial sheaths terminated directly Collagen fibers were not clearly defined in cane toad plantaris, giving on myoseptal tendons (Figure 7c). A large intramuscular tendon was the perimysium of this muscle an appearance similar to sectioned ply- also visible in cane toad plantaris muscle (Figure 5a) and served as the wood (Figure 5c). In other samples, layered sheets of perimysial colla- ultimate attachment point for many endomysial sheaths. gen fibers formed fibrous septa that divided or encircled large groups of muscle fibers (e.g., Figure 3a and 4a). Perimysial septa ranging in thickness from ~10 to 30 μm were common across all sampled mus- 4 | DISCUSSION cles, with the exception of bullfrog semimembranosus, in which no perimysial septa thicker than 15 μm were observed. Exceptionally Our study confirms that a multilayered collagenous ECM is present in large septa greater than 50 μm in thickness were observed in two the muscles of many vertebrates, and that distinct endomysial, muscles: turkey gastrocnemius (Figure 3a,b) and alligator triceps perimysial, and epimysial layers are not unique to mammals and birds. (Figure 4a,b). An endomysium surrounding and linking muscle fibers was present in Epimysial ECM was morphologically similar to perimysial ECM, all sampled muscles, and in all samples this endomysial network was being composed of layers of ribbon-like collagen fibers. Epimysial col- accompanied by thickened layers of perimysial ECM delineating fasci- lagen fibers were generally larger than perimysial collagen fibers, with cles within the muscle belly, epimysial ECM surrounding the muscle a fiber width of ~50 μm typical across all epimysial collagen fibers periphery, or a combination of both perimysial and epimysial ECM. observed. Perimysial septa that extended to the muscle periphery These findings suggest that a multilayered ECM is typical of verte- were continuous with the epimysium (Figures 2b and 6a). brate muscle, and that functional roles attributed to the ECM can rea- In addition to muscle fibers, fascicles, and the muscle periphery, sonably be assumed to influence the mechanics of non-mammalian the collagenous ECM encompassed intramuscular nerves and blood and non-avian muscle. We have assumed previously that a mammal- vessels (Figures 3d and 5b). Nerves were identifiable in digested like ECM is present in amphibian muscle and that this ECM influences 8 SLEBODA ET AL. basic mechanical properties of the tissue, such as the response of passive septum, vertebrae, and skin, which acts like an external tendon in fish muscle to stretch (Sleboda et al., 2019; Sleboda & Roberts, 2017). The (Nursall, 1956). Myoseptal tendons were structurally similar to the peri- current findings confirm this assumption and suggest that amphibian and mysium observed in other animals, being composed of layered arrange- other non-mammalian and non-avian muscles can serve as valid models ments of ribbon-like collagen fibers (Figure 7b,c); however, the presence for the study of ECM biomechanics in vertebrate muscle. of endomysial sheaths terminating directly on myoseptal tendons distin- Although we did not attempt quantitative analysis of collagen guished them as tendons. It is possible that the presence of myoseptal content, muscles imaged in the current study display variations in tendons in fish body muscle negates the need for a distinct perimysium; ECM morphology qualitatively similar to those observed in previously however, further comparative study of the skeletal muscle ECM in fish is studied vertebrates. Differences in the amount of endomysial, needed to explore this hypothesis, and to determine whether the lack of perimysial, and epimysial ECM visible in cross section were apparent a distinct perimysium is typical of fish muscle. 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 vs. 6a), and collagen fibrils 5 | CONCLUSIONS formed endomysial sheaths with visibly denser walls in bullfrog semi- membranosus than in carp epaxial muscle (e.g., Figure 2e vs. 7d). Such The findings of the current study support the hypothesis that a multilay- qualitative differences are likely the result of multiple factors, of ered, collagen-reinforced ECM is a general feature of vertebrate skeletal which phylogenetic differences are only one. Previous biochemical muscle. They confirm that non-mammalian and non-avian muscle can analyses of hydroxyproline content in skeletal muscle show that the serve as valid model systems for the study of ECM function, and that past prevalence of the endomysium, perimysium, and epimysium is vari- studies of ECM mechanics in frogs (e.g., Sleboda et al., 2019; Sleboda & able, with perimysial collagen content being more variable than either Roberts, 2017; Street, 1983) and other vertebrates can inform our under- endomysial or epimysial content (Light, Champion, Voyle, & Bailey, standing of vertebrate muscle generally. It remains unclear whether cross- 1985; Purslow, 2002). Variations in skeletal muscle ECM morphology species variations in ECM architecture observed in the current study are and content have been attributed to multiple factors, including differ- the result of phylogenetic, anatomical, or functional differences; however, ences in animal size (Rowe, 1981), muscle function (Borg & Caulfield, such variations have the potential to influence both passive and active 1980), and muscle fiber type (Nakamura et al., 2003). The prevalence muscle mechanics and may prove a fruitful area for future research. and morphological arrangement of the skeletal muscle ECM also var- ies significantly with animal age (Alnaqeeb, Alzaid, & Goldspink, 1984; ACKNOWLEDGMENTS Fang, Nishimura, & Takahashi, 1999; Gao, Kostrominova, Faulkner, & The authors thank Geoff Williams for technical assistance obtaining Wineman, 2008; Nishimura, Ojima, Liu, Hattori, & Takahashi, 1996), micrographs, and Patricia Hernandez, Payam Mohassel, and Henry and in some neuromuscular disorders (Lieber & Ward, 2013; Smith, Tsai for providing tissue samples. Funded by NIH grants AR55295 Lee, Ward, Chambers, & Lieber, 2011). It is unclear from the current and S10OD023461, NSF grants IOS1354289 and 1832795, the study whether cross-species variations in ECM architecture observed Bushnell Research Fund, and an EEB Dissertation Development grant are the result of phylogenetic, anatomical, functional, or other differ- from the Drollinger Family Charitable Foundation. ences, but the impact of these and similar variations on muscle mechanics warrant further investigation. Biochemical analyses of total CONFLICT OF INTEREST collagen content within muscle have been shown to be poor predic- The authors have no competing interests to declare. tors of fundamental mechanical properties of muscle, such as passive muscle stiffness (Bensamoun et al., 2006; Lieber & Ward, 2013), but it AUTHOR CONTRIBUTIONS has been suggested that subtle differences in ECM morphology may D.A.S. and K.K.S. collected, prepared, and imaged muscle samples. underlie specialization of muscle for particular functional tasks, such D.A.S. drafted the manuscript. D.A.S., T.J.R., and K.K.S. conceived the study as the production or dissipation of mechanical energy (Azizi, 2014). and revised the manuscript. All authors gave final approval for publication. Among the muscles sampled, the most unique ECM morphology was observed in carp epaxial muscle. Carp epaxial muscle was distinct DATA AVAILABILITY STATEMENT in the current study both as the only axial muscle (all other were limb The data that support the findings of this study are available from the muscles), and as the only muscle that operates in a fully aquatic envi- corresponding author upon reasonable request. ronment. The different loading conditions and evolutionary and devel- opmental histories of this muscle may underlie its distinctiveness. ORCID Carp epaxial muscle displayed a typical endomysium composed of colla- David A. Sleboda https://orcid.org/0000-0003-0170-9540 gen fibrils surrounding muscle fibers; however, few collagen fibers reinforced the endomysium, and no clear perimysium was evident. Carp REFERENCES muscle was also unique in its display of myoseptal tendons, which are Alnaqeeb, M. A., Alzaid, N. S., & Goldspink, G. (1984). Connective tissue typical of fish muscle (Gemballa et al., 2003). 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