JOURNAL OF MORPHOLOGY 273:1246–1256 (2012) Ring Bands in Fish Skeletal Muscle: Reorienting the Myofibrils and Microtubule Cytoskeleton Within a Single Cell 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 connective tissue 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- tubule 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 sarcolemma 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 microtubules; 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 ribosomes (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 organelles, 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 actin 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. Dystrophin 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 myosin 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 histology. For trans- ultrastructural features of muscle cells. mission electron microscopy (TEM) teased muscle fibers were Costameres 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 sarcomeres, 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. Desmin, plectin, Stacks of 30 fluorescence and DIC images were collected at 1 dystrophin and the dystroglycan 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).