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JOURNAL OF MORPHOLOGY 273:1246–1256 (2012)

Ring Bands in Fish : Reorienting the Myofibrils and Within a Single

Carolina Priester,* Jeremy P. Braude, Lindsay C. Morton, Stephen T. Kinsey, Wade O. Watanabe, and Richard M. Dillaman

Department of Biology and Marine Biology, University of North Carolina Wilmington, Wilmington, North Carolina 28403

ABSTRACT Skeletal muscle cells (fibers) contract by flanked on either side by layers of shortening their parallel subunits, the myofibrils. Here referred to as myosepta (Gemballa and Vogel, 2002). we show a novel pattern of myofibril orientation in white The organization of the white muscle fibers is com- muscle fibers of large black sea bass, Centropristis plex with the fibers attached to myosepta at each striata. Up to 48% of the white fibers in fish >1168 g end forming a continuous helical array relative to had peripheral myofibrils undergoing an 90o shift in orientation. The resultant ring band wrapped the middle the body’s axial skeleton (Rome et al., 1988). Gem- of the muscle fibers and was easily detected with polar- balla and Vogel (2002) refer to this arrangement as ized light microscopy. Transmission electron microscopy arch-like helical muscle fiber arrangements that showed that the reoriented myofibrils shared the cyto- span the length of several myotomes and are there- plasm with the central longitudinal myofibrils. A micro- fore intersected by multiple myosepta. In each mus- network seen throughout the fibers surrounded cle fiber the myofibrils are oriented longitudinally, nuclei but was mostly parallel to the long-axis of the connecting one myosepta to the next. Disturbances myofibrils. In the ring band portion of the fibers the of the orientation of myofibrils are rare but have microtubule cytoskeleton also shifted orientation. Sarco- been described in a variety of species (Bataillon, lemmal staining with anti-synapsin was the same in 1891; Morris, 1959; Bethlem and Wijngaarden, fibers with or without ring bands, suggesting that fibers with ring bands have normal innervation and contractile 1963; Jansen et al., 1963; Korneliussen and Nicolay- function. The ring bands appear to be related to body- sen, 1973; Mu¨ hlendyck and Ali, 1978). However, mass or age, not fiber size, and also vary along the body, they have not been reported in teleost fishes. being more frequent at the midpoint of the anteroposte- In fishes, red muscle predominantly grows by rior axis. Similar structures have been reported in dif- hyperplasia (increase in fiber number), whereas ferent taxa and appear to be associated with hypercon- white muscle undergoes hyperplasia in early post- traction of fibers not attached to a rigid structure (bone) larval stages followed by hypertrophy (increase in or with fibers with unusually weak links between the fiber size). The muscle fibers increase in size by and cytoskeleton, as in muscular dystrophy. increasing in both length and diameter (Lampilla, Fish muscle fibers are attached to myosepta, which are 1990; Kiessling et al., 1991; Johnston et al., 2011). flexible and may allow for fibers to hypercontract and thus form ring bands. The consequences of such a ring Black sea bass undergo an extreme increase in band pattern might be to restrict the further expansion body mass during postmetamorphic growth, where of the sarcolemma and protect it from further mechani- white muscle fiber diameter is positively correlated cal stress. J. Morphol. 273:1246–1256, 2012. Ó 2012 with body mass, leading to very large fibers in Wiley Periodicals, Inc. adults (Nyack et al., 2007; Priester et al., 2011). Furthermore, once white fibers become >120 lm KEY WORDS: ring band or ‘‘ringbinden’’; striated annulets; reoriented myofibrils and ; teleost white muscle; muscular dystrophy; fish white muscle Contract grant sponsor: National Science Foundation; Contract grant number: IOS-0719123.

INTRODUCTION *Correspondence to: Carolina Priester, Department of Biology and Marine Biology, University of North Carolina Wilmington, 601 South Fish skeletal muscle is composed of red and white College Road, Wilmington, NC 28403. E-mail: [email protected] muscle arranged in a highly organized fashion. In most fishes the red muscle is located laterally, in a Received 24 February 2012; Revised 16 May 2012; Accepted 26 May 2012 thin strip just under the skin along the length of the body and parallel to the lateral line (Stoiber et al., Published online 13 July 2012 in 1999). The majority of the muscle mass in fishes is Wiley Online Library (wileyonlinelibrary.com) white muscle and it is partitioned into myotomes DOI: 10.1002/jmor.20055

Ó 2012 WILEY PERIODICALS, INC. RING BANDS IN FISH SKELETAL MUSCLE 1247 in diameter, a redistribution of mitochondria and organisms (Bataillon, 1891; Morris, 1959; Bethlem nuclei is observed, presumably due to increasing and Wijngaarden, 1963; Jansen et al., 1963; Kor- diffusion constraints (Kinsey et al., 2007, 2011; neliussen and Nicolaysen, 1973; Mu¨ hlendyck and Locke and Kinsey, 2008). Nuclei have been found Ali, 1978) prompted us to further investigate the to be strictly subsarcolemmal (SS) in fibers <120 ultrastructure of those fibers using additional mi- lm but become distributed throughout the sarco- croscopic techniques. In this study we describe the plasm (intermyofibrillar or IM) in fibers >120 lm, myofibril arrangement in white muscle fibers hav- which is presumably a response to avoid increased ing this circular array of nuclei. Additionally, diffusion distances for nuclear products such as because MTs have been shown to organize the mRNA and (Hardy et al., 2009, 2010; position of nuclei and myofibrils in skeletal muscle Kinsey et al., 2011; Priester et al., 2011). (Bugnard et al., 2005; Pizon et al., 2005; Bruus- Because nuclei are long lived , this gaard et al., 2006), we also investigated the fibers’ reported shift in distribution raises the question of MT cytoskeleton. Finally, we examined the inner- what controls the spacing and location of nuclei vation pattern in white muscle cells with and within skeletal muscle cells. Nuclei have been without the ring arrangement of nuclei to investi- reported to be anchored in a nonrandom fashion to gate whether the presence or absence of ring band the sarcolemma through varying complexes of pattern correlated with innervation or whether the anchoring proteins and cytoskeletal elements, innervation pattern or the appearance of the syn- including and microtubules (MTs; Apel et al., apses on the sarcolemma differed. We also wanted 2000; Starr and Han, 2003; Grady et al., 2005; to determine if the outer band of reoriented myofi- Bruusgaard et al., 2006; Starr, 2007, Star, 2009). brils had a separate pattern of innervation from Myofibrils are also organized and aligned by the core of the fiber because the presence of sepa- anchoring proteins. and dystrophin- rately innervated functional myofibrils with oppos- associated proteins form a complex that links the ing orientations might act as a muscular hydrostat myofibrils and cytoskeleton to the extracellular such as those described in Kier and Smith (1985). matrix (Campbell, 1995; Starr and Han, 2003; Starr, 2007; Han et al., 2009). A lack of dystrophin leads to a derangement of the MT network in mus- MATERIALS AND METHODS cle cells (Prins et al., 2009) and the appearance of C. striata (0.4 g to 4840 g; n 5 18) were obtained from the IM nuclei in several species, including fish (Berger University of North Carolina Wilmington (UNCW) Aquaculture Facility (Wrightsville Beach, NC) and wild fish were provided et al., 2010). Disturbances or absence of anchoring by Dr. F. S. Scharf (UNCW). Fish were maintained and proc- proteins have also been shown to lead to muscular essed according to the UNCW Institutional Animal Care and dystrophies (Weller et al., 1990; Banks et al., 2008; Use Committee standards. Han et al., 2009; Goldstein and McNally, 2010). Small, rectangular pieces of epaxial white muscle were Additionally, MTs are involved with the alignment excised parallel to the fibers’ long axis orientation at a site im- mediately posterior to the operculum. In large fish (>2 Kg) six and organization of filaments in developing different regions (cranial to caudal) were sampled at equal myotubes (Pizon et al., 2002), as well as trafficking intervals, each 17% (1/6th) of the distance from the operculum nuclear products and proteins within the fiber to the caudal peduncle. All tissue was fixed for 24 hr in 4% (Ralston et al., 1999, 2001; Scholz et al., 2008), paraformaldehyde in So¨rensen’s PBS pH 7.4 or Bouin’s fixative (Presnell and Schreibman, 1997) and subsequently processed therefore playing a crucial role in organizing the respectively for cryomicrotomy or paraffin . For trans- ultrastructural features of muscle cells. mission electron microscopy (TEM) teased muscle fibers were are sarcolemmal protein assemblies fixed in 2.5% glutaraldehyde and 1.5% paraformaldehyde in also involved in organizing muscle components, PBS and processed using the protocol described in Nyack et al. keeping , and therefore the myofibrils, (2007). Cryosections (30 lm) were stained with 40,6-diamidino- 2-phenylindole (DAPI) and acridine orange (AO) and examined aligned longitudinally and in registration during with an Olympus FV1000 laser scanning confocal microscope. contraction and relaxation cycles. , plectin, Stacks of 30 fluorescence and DIC images were collected at 1 dystrophin and the complex are part lm intervals. Companion sections were stained overnight at of this costameric network that has been proposed 48C with a primary antibody against b-tubulin (E-7; Develop- mental Studies Hybridoma Bank; diluted 1:400) or for 4 hours to surround and attach to myofibrils at the Z-line at room temperature with a primary antibody against synapsin so as to provide support and prevent mechanical (SV2; Developmental Studies Hybridoma Bank, diluted 1:500). stress (Kierzenbaum, 2007; Goldstein and McNally, These were followed by incubation for one hour at room temper- 2010; Garcı´a-Pelagio et al., 2011). ature with a 1:200 dilution of a 2 mg/mL secondary goat anti- The IM nuclei described in our previous study mouse antibody conjugated with Alexa 594 fluorochrome (A- 11032; Invitrogen). Negative controls included incubation with on black sea bass (Priester et al., 2011) were some- secondary antibody only for cross reactivity. Control for auto- times randomly distributed, but were often fluorescence was done using sections exposed to blocking serum arranged in a circular or ring pattern which can only, with no primary or secondary antibody. None of the con- be seen in Figure 1A. This curious arrangement of trols showed any fluorescence at 594 nm. Serial sections (10 lm) of paraffin embedded tissue were col- central nuclei, together with the fact that a reor- lected and every 10th section was stained with hematoxylin ientation of myofibrils in skeletal muscle fibers has and eosin (H & E; Presnell and Schreibman, 1997) and exam- been previously described in a wide range of ined with both bright field and polarized light microscopy. The

Journal of Morphology 1248 C. PRIESTER ET AL.

Fig. 1. Ring band pattern observed with laser confocal microscopy (A and D) polarized light microscopy (B and C) and in serial histological sections (E and F). (A) DAPI-stained nuclei (arrows) arranged in a circular array inside some of the acridine orange- stained white muscle fibers in a large (>1168 g) fish. (B and C) Polarized light microscopy showing fibers with the ring band of reoriented myofibrils (arrow in B) and revealing striations on these reoriented myofibrils (arrow in C). (D) Laser confocal and DIC examination of DAPI-stained whole mounts of fibers showing the ring band myofibrils (rb) wrapped around the core myofibrils (c). The arrowhead in D points to a nucleus aligned with the long axis of the fiber within the core myofibrils. The arrow in D points to a nucleus lined up with the spiraling myofibrils. (E) Section from the middle of the fiber with a visible ring band (rb) around the core of the fiber (c). (F) Section from near the end of the fiber where only the core of the fiber is present (c).

Journal of Morphology RING BANDS IN FISH SKELETAL MUSCLE 1249 frequency of cells with the circular pattern was determined Fish with body mass less than 1168 g did not have using polarized light microscopy, which allowed identification of cells with the ring band pattern, even though they fibers with and without the circular pattern at low magnifica- tion with a large field of view. had fibers with a mean diameter >100 lm. In fish Images of muscle fibers and ring bands were outlined with an with a body mass greater than 1168 g, 0.6–36% of Intuos 3 tablet (Wacom, Vancouver, WA) in Adobe Photoshop the white fibers had the peripheral ring band pat- (version 7.0; Adobe Systems, San Jose, CA) and resulting poly- tern. Muscle sampled from six different sites along gons were analyzed with ImagePro Plus (v. 6.1.0.346; Media Cy- the length of the large fish (N 4 fish) indicated bernetics, Bethesda, MD) to determine their cross sectional 5 area (CSA), perimeter and mean diameter. The mean diameter that this ring band pattern was more prevalent in was determined by taking the mean of the distances across the the midpoint of the anteroposterior axis, where up cell through the centroid in 28 increments around the perimeter to 48% of the fibers had this pattern of reoriented of the cell. For ring band cross sectional areas, the core area myofibrils (Fig. 2C). was subtracted from the total area. For and I-band lengths 100 measurements were taken from each fiber region (ring band and core). Measurements were made on digitized Ultrastructure, Cytoskeleton, and TEM images with ImagePro Plus (v. 6.1.0.346; Media Cybernet- ics, Bethesda, MD) Innervation of the Ring Band Ultrastructural observations verified that the pe- RESULTS ripheral myofibrils were truly reoriented, with sar- Ring Band Pattern of Peripheral Myofibrils comeres being almost perpendicular to those of the central myofibrils and with no sarcolemma at their The use of differential interference contrast interface, indicating that both were within the (DIC) and especially polarized light microscopy same fiber (Fig. 3A,B) and thus shared the same revealed that in cross sections of those fibers that . The ring band sarcomeres appeared to contained IM nuclei arranged in a circular pat- be complete, suggesting that the reoriented myofi- tern (Fig. 1A) the myofibrils were oriented in a brils were functional. ring-like pattern at the periphery. The peripheral When white muscle fibers of C. striata were band of myofibrils had an altered orientation, spi- stained with antibodies against b-tubulin, MTs raling around the central core of the fiber (Fig. were seen in all white muscle cells, including 1B–D). This peripheral ring band of myofibrils those with the peripheral ring band pattern (Fig. was close to, but not perfectly perpendicular to 4A), and these MTs were present throughout the the core myofibrils, which was indicated by fiber, in both the SS and IM regions. In fibers regions of extinction at 908 intervals in the pe- without the ring band the MTs were predomi-  ripheral, reoriented myofibrils (Fig. 1B). Further- nately oriented with the long axis of the cell, par- more, examination of DIC images at higher mag- allel to the myofibrils, with numerous branches nification revealed striations on these reoriented projecting radially from those MT bundles myofibrils corresponding to the highly organized (Fig. 4B). The MTs also appeared to wrap the sarcomeres of skeletal muscle (Fig. 1C). Laser nuclei. In the fibers containing the ring band, the scanning confocal microscope examination of MTs in the core of the fiber were also mostly lon- DAPI-stained whole fibers from similar sized fish gitudinally oriented. However, at the periphery showed that the outer myofibrils were wrapped the MTs were reoriented in the same manner as around the center core of myofibrils in a spiral or the myofibrils. That is, they had changed orienta- ring band pattern (Fig. 1D). The DAPI-stained tion and were roughly perpendicular to the MTs nuclei were elongate and appeared to be aligned and myofibrils present at the core of the fiber with the long axis of the fiber within the core (Fig. 4A). myofibrils whereas in the spiraling myofibrils the After confirming that the myofibrils and MTs of nuclei appeared to change their orientation so as the ring band shared with the core of to line up with the ring band myofibrils (Fig. 1D). the fiber we wished to determine if the altered The ring band of reoriented myofibrils was not fibers also had a similar pattern of innervation. seen throughout the length of the fibers, rather it Staining for synapsin revealed that the cells with was usually found in the center of the fibers, as the ring band pattern were innervated at the pe- revealed by observations of both whole isolated riphery of the fiber, in the same manner (i.e. rele- fibers (Fig. 1D) and serial histological sections gated to the sarcolemma and having a stippled (Fig. 1E,F). appearance) as the cells without the pattern (Fig. 4C–F). In summary, our observations suggest that in Ring Band Frequency as a Function of Body fibers with the ring band pattern all of the fiber’s Mass and Cell Diameter ultrastructural elements seem to change orienta- The smallest fish to display this ring band pat- tion forming a ring around the core of the muscle tern weighed 1168 g (Fig. 2A,B). The smallest fiber fiber. Myofilaments, nuclei and MTs all become displaying the ring band pattern was 100 lm in spirally wound around the core of the fiber, which diameter and was found in a fish weighing 2258 g. remains unchanged.

Journal of Morphology 1250 C. PRIESTER ET AL.

Fig. 2. Presence of the ring band as a function of fish body mass and location on the body. (A) Plot of % of fibers with the ring band versus body mass (N 5 100 fibers per fish). (B) Plot of mean fiber diameters versus body mass (N 5 100 fibers per fish). Grey circles represent the mean diameter of fibers without the ring band, whereas white circles depict the mean diameter of fibers with the ring band pattern. (C) Plot of % of fibers with the ring band versus six regions along the body length (cranial to caudal) of four of the largest fish (N 5 100 fibers per site, per fish). The bars represent the average of the percentages of fibers with the ring band 6 S.E.M. in four large fish weighing 2258, 2691, 3921, and 4840 g.

Cross Sectional Areas and Sarcomere 5 2), whereas the mean length for the core sarco- Lengths meres was 1.50 6 0.0131 lm(N 5 2). Mean I-band length was significantly shorter for myofibrils These measurements indicated that while ring forming the ring band. Mean I-band length was bands were only seen in specimens greater than 0.31 6 4.823e-3 m in the ring band region (N 5 2), 1168 g, there was no correlation (r2 5 0.003) l and 0.54 6 0.0180 m in the core region of the between fiber diameter and the percentage of each l fibers (N 5 2; ANOVA: DF 5 1, F 5 167.4, P < fiber’s cross-sectional area devoted to the ring 0.0001; Fig. 5D). band (Fig. 5A), and all specimens with body masses greater than 2258 g varied widely in the extent of the fiber area represented by the ring DISCUSSION band pattern (Fig. 5B). Sarcomere and I-band lengths were also examined in the core and ring The ring band pattern observed in white fibers band regions of fibers from multiple sections from of large black sea bass in this study is virtually two fish. All fibers were processed for TEM in the identical to previously described microscopic struc- same manner and mean sarcomere lengths (Z-line tures variously called ‘‘ringbinden,’’ ‘‘striated annu- to Z-line) from myofibrils forming the ring band lets,’’ ‘‘rings bands,’’ or ‘‘ring fibers.’’ These ring were significantly longer than the mean sarcomere bands were observed as early as 1891 and have length of the core myofibrils (ANOVA: DF 5 1, F been seen in a variety of muscles from a variety of 5 144.8, P < 0.0001; Fig. 5C). The mean length different organisms, including hagfish, Xenopus for ring band sarcomeres was 1.74 6 0.0152 lm(N larva, rabbit, mouse and humans (Bataillon, 1891;

Journal of Morphology RING BANDS IN FISH SKELETAL MUSCLE 1251

Fig. 3. Ultrastructure of fibers with the ring band pattern. (A and B) TEM at low (A) and high (B) magnification of regions con- taining the interface (arrow in A) between the peripheral ring band seen as longitudinal myofibrils (rb in A and B) and the core of the fiber seen as cross section myofibrils (c in A and B) sharing the same cytoplasm. An intermyofibrillar nucleus (N in A) with a pronounced (n in A) is seen at the interface between the differently oriented myofibrils. Arrowheads in (B) indicate Z- disks of sarcomeres belonging to the ring band myofibrils.

Morris, 1959; Bethlem and Wijngaarden, 1963; has weaker links between the sarcolemma and ba- Jansen et al., 1963; Korneliussen and Nicolaysen, sal lamina due to a lack of globular do- 1973; Mu¨ hlendyck and Ali, 1978). However, the or- main-binding motif. Additionally, lengthening igin and function of ring bands has remained (eccentric) contraction has been shown to induce unclear. more damage to the sarcolemma and its attach- In hagfish, the ring bands were seen in normal ments to the basal lamina and intracellular struc- swimming muscle composed of white fibers tures than shortening (concentric) contractions (Jansen et al., 1963; Korneliussen and Nicolaysen, because they increase membrane tension, leading 1973). Ring bands in humans have been observed to small tears in the sarcolemma, as suggested by in older, but healthy individuals and particularly Weller et al. (1990) and Han et al. (2009). in muscles that do not have an attachment to bone As described by Kier and Smith (1985), muscles such as the extra-ocular motor muscles, dia- have a constant volume and as a muscle contracts, phragm, tongue, vocallis, uvulla, and others it shortens in length and therefore it must expand (Bethlem and Wijngaarden, 1963; Mu¨ hlendyck and radially, increasing its circumference. An increase Ali, 1978). Additionally, ring bands have been in circumference leads to more stress on the sarco- experimentally induced in skeletal muscle of lemma. In a muscle that is attached to bone, the healthy rats and rabbits by severing their attach- extent of contraction is limited by the attachment ment to a rigid structure (e.g., bone) by either cut- to a rigid structure, whereas in muscles that are ting the tendon or the muscle itself (Bethlem and not attached to bone, such as the diaphragm and Wijngaarden, 1963). extra-ocular motor muscles, the extent of contrac- The ring band pattern has also been observed in tion is less limited and can result in hypercontrac- association with certain pathologies, especially dif- tion and subsequent stress and strain between the ferent types of muscular dystrophy (Bethlem and sarcolemma and cytoskeleton. The same is true for Wijngaarden, 1963; Banks et al., 2008). Dystrophic muscles that have had their attachment to bone muscle has altered or missing proteins in its costa- severed. As muscle is allowed to hypercontract, meres and/or dystroglycan complexes. It has been there appears to be a threshold fiber length at hypothesized that individuals suffering from mus- which the extreme shortening of the fiber causes cular dystrophy have muscles with ‘weaker’ the circumference to stretch to the point of break- attachments between the sarcolemma and the ing the links between the sarcolemma, the myofi- myofibrils, cytoskeleton, and the basal lamina, brils, cytoskeleton, and basal lamina (Mu¨ hlendyck which makes these linkages more prone to tearing and Ali, 1978; Han et al., 2009). By extension, in a and damage as the muscle fiber contracts (Gold- dystrophic muscle fiber one would expect a signifi- stein and McNally, 2010; Garcı´a-Pelagio et al., cantly lower stress to result in damage to the 2011). Han et al. (2009) have shown that a mouse bridge between the sarcolemma and the myofibrils model for secondary dystroglycanopathy (Largemyd) and basal lamina, even in muscle fibers that are

Journal of Morphology 1252 C. PRIESTER ET AL.

Fig. 4. Cytoskeletal network and innervation pattern in muscle fibers with and without the ring band. (A and B) Laser confocal z-stack of an oblique section stained for microtubules (anti-b-tubulin seen in green) and nuclei (DAPI seen in red for better con- trast). (A) Fiber with ring band (rb). Arrow points to MTs spiralling around the core of the fiber (c). (B) Fiber without the ring band, with MTs running the length of the fiber (arrow) with some branching perpendicular to the longitudinal axis (arrowhead). A nucleus is seen in association with the network of MTs (circle). (C–F) Paired images showing the pattern of innervation in white muscle fibers without (C and D) and with (E and F) the ring band as revealed by an antibody against synapsin (arrows pointing to green fluorescence in D and F). DAPI-stained nuclei are seen as red. (C and E) are 1 lm optical sections of fluorescence plus DIC. (D and F) are z-stacks of 30 1 lm confocal fluorescence images without DIC. The ring band is indicated by ‘‘rb’’ in (E) and the core of the fiber by ‘‘c’’ in (C) and (E).

Journal of Morphology RING BANDS IN FISH SKELETAL MUSCLE 1253

Fig. 5. (A) Plot of ring band cross sectional area (CSA) as a percentage of total fiber CSA versus mean fiber diameter. (B) Plot of ring band CSA as a percentage of total fiber CSA versus fish body mass. (C) Plot of sarcomere lengths (N 5 100 per category) ver- sus fiber category (ring band vs. core). Grey circles represent individual measurements, black circles are mean sarcomere length 6 S.E.M. (bars), and the asterisks indicate that the means are significantly different. (D) Plot of I-band lengths versus fiber category (ring band vs. core). Grey circles represent individual measurements, black circles are mean sarcomere length 6 S.E.M. (bars), and the asterisks indicate that the means are significantly different. attached to bone (Banks et al, 2008; Han et al., not known. It does seem that the ring band con- 2009). sists of newly formed myofibrils because the fibers Therefore, it appears that the common charac- are continuing to grow hypertrophically. In fact, in teristic seen in the muscles that display this reor- some fibers the area composed of the ring band iented ring band of myofibrils is the disruption of was very large, reaching over 75% of the CSA (Fig. the sarcolemmal attachments to intra- and extra- 5A,B), indicating that these fibers were still under- cellular structures. The disruptions can be caused going hypertrophy by adding myofibrils in this by various events, including hypercontraction, reoriented, ring band pattern. lengthening contractions, and pathological condi- Staining for synapsin revealed no obvious differ- tions such as muscular dystrophies. ences in innervation between fibers with and with- Once the sarcolemma is disrupted, the breaking out the ring band, which suggests that if the fiber of its linkages leads to a disconnect between intra- is stimulated at the , all of cellular structures (cytoskeleton, myofibrils and the myofibrils would contract, both the core and nuclei) and the sarcolemma and basal lamina. ring band myofibrils. Contraction of the myofibrils These intracellular components may then become of the ring band around the middle point of the free to reorient (or disorient) and form new connec- fiber would potentially restrict the expansion of tions with the sarcolemma. What dictates the ori- the fiber’s circumference. By doing this, the reor- entation of the myofibrils, cytoskeleton and nuclei iented ring band myofibrils would be reinforcing at this roughly 908 angle to the core of the fiber is the fiber’s middle portion, thereby preventing fur-

Journal of Morphology 1254 C. PRIESTER ET AL. ther expansion of the sarcolemma and subsequent Because lengthening contractions induce more damage to the sarcolemmal linkages. damage of the sarcolemma and its attachments It has been previously suggested that chronic than isometric- or shortening-contractions (Weller stretch induces an increase in sarcomere lengths et al., 1990; Han et al., 2009), this could favor the (Houlihan and Breckenridge, 1981). This might formation of ring bands in black sea bass. explain why sarcomeres of the ring band are sig- In contrast, hagfish have an anguilliform mode nificantly longer than the sarcomeres at the core of swimming, which involves undulation of the of the fiber even though their I-bands are shorter, entire body, rather than bending in the mid-por- indicating they are more contracted. Alternatively, tion of the body like the subcarangiform swimming because they are reoriented, the Z-lines of the sar- (Lindsey, 1978; Colgate and Lynch, 2004). It is comeres in the ring band might be attaching to interesting to note that while dissecting hagfish costameres on the sarcolemma that have a differ- for a related study, we have observed that their ent spacing, i.e. that are further apart and there- swimming muscles are not firmly attached to the fore could lead to formation of longer sarcomeres. skin, and therefore are not firmly anchored. This However, it is not known whether costameres dic- characteristic would make their muscle fibers tate sarcomere length or whether sarcomere more likely to hypercontract, and may give an length dictates where Z-lines are anchored at the insight into why Korneliussen and Nicolaysen sarcolemma. (1973) found ring bands in hagfish muscle. Follow- While we have shown that the occurrence of ing this logic, one would expect to find ring bands ring band fibers is widespread, it is somewhat in fish with other types of swimming modes wher- rare, raising the question of why they appear in ever there is more bending along the fish’s body the white muscle of the black sea bass. The white length. For example, fish with carangiform swim- fibers of black sea bass in our study grow hyper- ming mode (Lindsey, 1978) would be likely to pos- trophically to very large sizes, both in diameter sess ring bands more caudally than a fish with a and length. Furthermore, they are not directly subcarangiform swimming mode, and fish with a attached to bone, but rather to a contiguous series thunniform swimming mode would be expected to of connective tissue layers (myosepta). The fibers possess ring bands only at their caudal peduncle. are aligned end-to-end, in a row from myosepta to However, a comparison between black sea bass myosepta, in such a way that the fibers at the and fish with thunniform swimming mode such as extremities attach to either the skin or the axial tuna becomes complicated due to their very differ- skeleton of the fish. When the fibers in a row con- ent arrangement of fibers and fiber types. tract, they pull on the myosepta between them The fact that this ring band pattern appears and, therefore, also pull on each other. The fibers only in larger black sea bass suggests that this located near the center of this row of fibers would phenomenon is either size related or age related, have more leeway and may hypercontract more however these two factors cannot be uncoupled. than the fibers that are attached closer to the skin Muscle fibers from larger fish have greater distan- or axial-skeleton. Furthermore, because the ends ces from connection sites, so the hypercontraction of the muscle fibers are anchored to myosepta, the possibility would increase with size. However, the portions of the sarcolemma that are close to the possible effects of aging on the anchoring proteins myosepta would likely not be disrupted during cannot be ruled out. In either case, the appearance hypercontractions, whereas the portions of the sar- of this pattern seems to be a response to damage colemma in the middle of the fiber, furthest from to the sarcolemma and its links to the cytoskeleton myosepta may be disrupted and damaged. This and basal lamina. Studies on the mechanisms for may explain why we observe: 1) the ring band pat- repairing the sarcolemma have suggested that dys- tern in the middle of each fiber rather than along ferlin is one of the proteins involved in mediating the entire length and 2) more fibers with the ring the resealing of the membrane and maintaining its band pattern towards the middle of the fish’s ante- integrity (Han and Campbell, 2007; Han et al., roposterior axis, which is the position most distant 2009). Part of that repair mechanism involves acti- from attachment sites on bone or skin. vation of proteases, such as calpain, by the high In addition, fish swim by alternating contrac- levels of calcium that are allowed to enter the fiber tions of either side of the body such that when one when the membrane is damaged. The activation of side approaches the end of a contraction cycle, the proteases leads to disassembly of cortical cytoskel- other side is already starting to contract. This eton proteins in the vicinity of the damage (Han helps maintain body stiffness during burst swim- and Campbell, 2007), which could likely also ming and leads to lengthening contractions on one release anchoring proteins and thus allow for reor- side of the fish during each tail beat. For example, ientation of intracellular structures and organ- Davies et al. (1995) have showed that during sub- elles. carangiform swimming in trout, caudal muscles The ring band likely restricts the expansion of are activated while still lengthening. Subcarangi- the middle of the fiber, therefore serving as rein- form is also the swimming mode of black sea bass. forcement against such expansion. This would pro-

Journal of Morphology RING BANDS IN FISH SKELETAL MUSCLE 1255 tect the sarcolemma from further stretch and dam- Bugnard E, Zaal KJM, Ralston E. 2005. Reorganization of age during and relaxation microtubule nucleation during muscle differentiation. Cell Motil Cytoskel 60:1–13. cycles. The opposing forces of the two groups of Bruusgaard JC, Liestøl K, Gundersen K. 2006. Distribution of differently oriented myofibrils make the fiber func- myonuclei and microtubules in live muscle fibers of young, tion somewhat like a muscular hydrostat (Kier middle-aged, and old mice. J Appl Physiol 100:2024–2030. and Smith, 1985), but by interactions of bundles of Campbell KP. 1995. Three muscular dystrophies: Loss of cyto- myofibrils in a single cell rather than groups of skeleton- linkage. Cell 80:675–679. Colgate JE, Lynch KM. 2004. Mechanics and control of swim- whole muscle cells. ming: A review. IEEE J Oceanic Eng 29:660–673. Together, these observations and the previous Davies MLF, Johnston IA, van de Wal JW. 1995. Muscle fibers literature give insights into what controls muscle in rostral and caudal myotomes of the Atlantic cod (Gadus ultrastructural organization, directionality and morhua L.) have different mechanical properties. Physiol Zool how all these factors can change orientation due to 68:673–697. Garcı´a-Pelagio KP, Bloch RJ, Ortega A, Gonza´les-Serratos H. disturbances of the connections to the sarcolemma. 2011. Biomechanics of the sarcolemma and costameres in sin- We propose that disturbing the sarcolemma gle skeletal muscle fibers from normal and dystrophin-null through stretching in the circumferential direction mice. J Muscle Res Cell Motil 31:323–336. disrupts the links between extracellular anchoring Gemballa S, Vogel F. 2002. Spatial arrangement of white mus- sites (basal lamina), the dystroglycan complexes cle fibers and myoseptal tendons in fishes. Comp Biochem Physiol A: Mol Int Physiol 133:1013–1037. and intracellular structures, namely the myofi- Goldstein JA, McNally EM. 2010. Mechanisms of muscle weak- brils, cytoskeletal elements (MTs), and nuclei. This ness in muscular dystrophy. J Gen Physiol 136:29–34. overstretching can result from hypercontraction or Grady RM, Starr DA, Ackerman GL, Sanes JR, Han M. 2005. lengthening contraction of muscle fibers that are Syne proteins anchor muscle nuclei at the neuromuscular not attached to bone (normally or experimentally). junction. Proc Natl Acad Sci USA 102:4359–4364. Han R, Campbell KP. 2007. and muscle membrane Once these links are broken, the intracellular ele- repair. Curr Opinion Cell Biol 19:409–416. ments are released from the sarcolemma and free Han R, Kanagawa M, Yoshida-Moriguchi T, Rader EP, Ng RA, to reorient in a different direction. In our study, Michele DE, Muirhead DE, Kunz S, Moore SA, Iannaccone the disturbance of sarcolemma seems to also allow ST, Miyake K, McNeil PL, Mayer U, Oldstone MBA, Faulkner for the release of nuclei from the periphery of the JA, Campbell KP. 2009. Basal lamina strengthens cell mem- brane integrity via the laminin G domain-binding motif of a- fiber, which then become IM nuclei once new myo- dystroglycan. Proc Natl Acad Sci USA 106:12573–12579. fibrils are formed around them. Hardy KM, Dillaman RM, Locke BR, Kinsey ST. 2009. A skele- tal muscle model of extreme hypertrophic growth reveals the influence of diffusion on cellular design. Am J Physiol Int ACKNOWLEDGMENTS Comp Reg Physiol 296:R1855–R1867. Hardy KM, Lema SC, Kinsey ST. 2010. The metabolic demands The authors are grateful for the technical assis- of swimming behavior influence the evolution of skeletal mus- cle fiber design in the brachyuran crab family Portunidae. tance of Mark Gay, Dr. Sonja Pyott, Dr. Fred Mar Biol 157:221–236. Scharf, and Kenneth Kelly. The monoclonal anti- Houlihan DF, Breckenridge L. 1981. Stretch-induced growth of bodies E-7, developed by Michael Klymkowsky, blowfly muscle. J Insect Physiol 27:521–525. and SV2, developed by Kathleen Buckley, were Jansen J, Andersen P, Jansen JKS. 1963. On the structure and obtained from the Developmental Studies Hybrid- innervation of the parietal muscle of the hagfish (Myxine glu- oma Bank developed under the auspices of the tinosa). Acta Morph Neerl-Scand 5:329–338. Johnston IA, Bower NI, Macqueen DJ. 2011. Growth and the NICHD and maintained by The University of regulation of myotomal muscle mass in teleost fish. J Exp Iowa, Department of Biology, Iowa City, IA 52242. Biol 214:1617–1628. Kier WM, Smith KK. 1985. Tongues, tentacles and trunks: The biomechanics of movement in muscular-hydrostats. Zool J Linnean Soc 83:307–324. LITERATURE CITED Kierzenbaum AL. 2007. . In: Ozols I, Hall A, edi- Apel ED, Lewis RM, Grady RM, Sanes JR. 2000. Syne-1, a dys- tors. Histology and : An Introduction to Pathol- trophin- and klarsicht-related protein associated with synap- ogy. Philadelphia: Mosby Elsevier. pp 197–208. tic nuclei at the neuromuscular junction. J Biol Chem Kiessling A, Storebakken T, Asgard T, Kiessling KH. 1991. 275:31986–31995. Changes in the structure and function of the epaxial muscle Banks GB, Combs AC, Chamberlain JR, Chamberlain JS. 2008. of rainbow trout (Oncorhyncus mykiss) in relation to ration Molecular and cellular adaptations to chronic myotendinous and age: I. Growth dynamics. Aquaculture 93:335–356. strain injury in mdx mice expressing a truncated dystrophin. Kinsey ST, Hardy KM, Locke BR. 2007. The long and winding Hum Mol Gen 17:39–75–3986. road: Influences of intracellular metabolite diffusion on cellu- Bataillon E. 1891. Recherches anatomiques et expe´rimentales lar organization and metabolism in skeletal muscle. J Exp sur la me´tamorphose des amphibiens anoures. Ann Univ Biol 210:3503–3512. Lyon 2:1–135. Kinsey ST, Locke BR, Dillaman RM. 2011. Molecules in motion: Berger J, Berger S, Hall TE, Lieschke GJ, Currie,PD. 2010. Influences of diffusion on metabolic structure and function in Dystrophin-deficient zebrafish feature aspects of the Duch- skeletal muscle. J Exp Biol 214:263–274. enne muscular dystrophy pathology. Neuromusc Disord Korneliussen H, Nicolaysen K. 1973. Ultrastructure of four 20:826–832. types of striated muscle fibers in the Atlantic hagfish (Myxine Bethlem J, Van Wijngaarden GK. 1963. The incidence of ringed glutinosa, L.) Z Zellforsch 143:273–290. fibres and sarcoplasmic masses in normal and diseased mus- Lampilla LE. 1990. Comparative microstructure of red , cle. J Neurol Neurosurg Psychiat 26:326–332. poultry and fish muscle. J Muscle Foods 1:247–267.

Journal of Morphology 1256 C. PRIESTER ET AL.

Lindsey,CC. 1978. Form, function, and locomotory habits in Prins KW, Humston JL, Mehta A, Tate V, Ralston E, Ervasti fish. In: Hoar, WS, Randall, DJ, editors. Fish Physiology, Vol. JM. 2009. Dystrophin is a microtubule-associated protein. J 7. Locomotion. New York: Academic Press. pp 1–100. Cell Biol 186:363–369. Locke BR, Kinsey ST. 2008. Diffusional constraints on energy Ralston E, Lu Z, Ploug T. 1999. The organization of the Golgi metabolism in skeletal muscle. J Theor Biol 254:417–429. complex and microtubules in skeletal muscle is fiber type-de- Morris WR. 1959. Striated annulets (Ringbinden), their experi- pendent. J Neurosci 19:10694–10705. mental production in mammalian muscle. Arch Path 68:438– Ralston E, Ploug T, Kalhovde J, Lømo T. 2001. Golgi complex, 444. exit sites, and microtubules in skeletal Mu¨ hlendyck H, Ali SS. 1978. Histological and ultrastructural muscle fibers are organized by pattern activity. J Neurosci studies on the ringbands in human extraocular muscles. 21:875–883. Albrecht Von Graefes Arch Kiln Exp Ophthalmol 208:177– 191. Rome LC, Funke RP, Alexander RM, Lutz G, Aldridge H., Scott Nyack AC, Locke BR, Valencia A, Dillaman RM, Kinsey ST. F, Freadman M. 1988. Why animals have different muscle 2007. Scaling of post-contractile PCr recovery in fish white fibre types. Nature 355:824–827. muscle: Effect of intracellular diffusion. Am J Physiol Scholz D, Baicu CF, Tuxworth WJ, Xu L, Kasiganesam H, 292:R2077–R2088. Menick DR, Cooper IVG. 2008. Microtubule-dependent distri- Pizon V, Iakovenko A, van der Ven PFM, Kelly R Fatu C, Fu¨rst bution of mRNA in adult cardiocytes. Am J Physiol DO, Karsenti E, Gautel M. 2002. Transient association of Circ Physiol 294:H1135–H1144. and myosin with microtubules in nascent myofibrils Starr DA, Han M. 2003. Anchors away: An actin based mecha- directed by the MURF2 RING-finger protein. J Cell Sci nism of nuclear positioning. J Cell Sci 116:211–216. 115:4469–4482. Starr DA. 2007. Communication between the cytoskeleton and Pizon V, Gerbal F, Diaz CC, Karsenti E. 2005. Microtubule-de- the nuclear envelope to position the nucleus. Mol Biosyst pendent transport and organization of sarcomeric myosin dur- 3:583–589. ing skeletal muscle differentiation. Europ Mol Biol Org Star DA. 2009. A nuclear-envelope bridge positions nuclei and 24:3781–3792. moves chromosomes. J Cell Sci 122:577–586. Presnell JK, Schreibman MP. 1997. Solution preparation. In: Humason’s Animal Tissue Techniques, Fifth Edition. Balti- Stoiber W, Haslett JR, Sa¨nger AM. 1999. Myogenic patterns in more: John Hopkins University Press. pp 473–474. teleosts: What does the present evidence really suggest? J Priester C, Morton LC, Kinsey ST, Watanabe WO, Dillaman Fish Biol 55:84–99. RM. 2011. Growth patterns and nuclear distribution in white Weller B, Karpati G, Carpenter,S. 1990. Dystrophin-deficient muscle fibers from black sea bass, Centropristis striata: Evi- mdx muscle fibers are preferentially vulnerable to necrosis dence for the influence of diffusion. J Exp Biol 214:1230– induced by experimental lengthening contractions. J Neurol 1239. Sci 100:9–13.

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