Ring bands in fish skeletal muscle: Reorienting the ...

Ring Bands in Fish Skeletal Muscle: Reorienting theMyofibrils and Microtubule Cytoskeleton Withina 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 byshortening their parallel subunits, the myofibrils. Herewe show a novel pattern of myofibril orientation in whitemuscle fibers of large black sea bass, Centropristisstriata. Up to 48% of the white fibers in fish >1168 ghad peripheral myofibrils undergoing an !90o shift inorientation. The resultant ring band wrapped the middleof the muscle fibers and was easily detected with polar-ized light microscopy. Transmission electron microscopyshowed that the reoriented myofibrils shared the cyto-plasm with the central longitudinal myofibrils. A micro-tubule network seen throughout the fibers surroundednuclei but was mostly parallel to the long-axis of themyofibrils. In the ring band portion of the fibers themicrotubule cytoskeleton also shifted orientation. Sarco-lemmal staining with anti-synapsin was the same infibers with or without ring bands, suggesting that fiberswith ring bands have normal innervation and contractilefunction. The ring bands appear to be related to body-mass or age, not fiber size, and also vary along the body,being more frequent at the midpoint of the anteroposte-rior axis. Similar structures have been reported in dif-ferent taxa and appear to be associated with hypercon-traction of fibers not attached to a rigid structure (bone)or with fibers with unusually weak links between thesarcolemma and cytoskeleton, as in muscular dystrophy.Fish muscle fibers are attached to myosepta, which areflexible and may allow for fibers to hypercontract andthus form ring bands. The consequences of such a ringband pattern might be to restrict the further expansionof the sarcolemma and protect it from further mechani-cal stress. J. Morphol. 273:1246–1256, 2012. ! 2012

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KEY WORDS: ring band or ‘‘ringbinden’’; striatedannulets; reoriented myofibrils and microtubules; teleostwhite muscle; muscular dystrophy; fish white muscle

INTRODUCTION

Fish skeletal muscle is composed of red and whitemuscle arranged in a highly organized fashion. Inmost fishes the red muscle is located laterally, in athin strip just under the skin along the length of thebody and parallel to the lateral line (Stoiber et al.,1999). The majority of the muscle mass in fishes iswhite muscle and it is partitioned into myotomes

flanked on either side by layers of connective tissuereferred to as myosepta (Gemballa and Vogel, 2002).The organization of the white muscle fibers is com-plex with the fibers attached to myosepta at eachend forming a continuous helical array relative tothe body’s axial skeleton (Rome et al., 1988). Gem-balla and Vogel (2002) refer to this arrangement asarch-like helical muscle fiber arrangements thatspan the length of several myotomes and are there-fore intersected by multiple myosepta. In each mus-cle fiber the myofibrils are oriented longitudinally,connecting one myosepta to the next. Disturbancesof the orientation of myofibrils are rare but havebeen described in a variety of species (Bataillon,1891; Morris, 1959; Bethlem and Wijngaarden,1963; Jansen et al., 1963; Korneliussen and Nicolay-sen, 1973; Muhlendyck and Ali, 1978). However,they have not been reported in teleost fishes.

In fishes, red muscle predominantly grows byhyperplasia (increase in fiber number), whereaswhite muscle undergoes hyperplasia in early post-larval stages followed by hypertrophy (increase infiber size). The muscle fibers increase in size byincreasing in both length and diameter (Lampilla,1990; Kiessling et al., 1991; Johnston et al., 2011).Black sea bass undergo an extreme increase inbody mass during postmetamorphic growth, wherewhite muscle fiber diameter is positively correlatedwith body mass, leading to very large fibers inadults (Nyack et al., 2007; Priester et al., 2011).Furthermore, once white fibers become >120 lm

Contract grant sponsor: National Science Foundation; Contractgrant number: IOS-0719123.

*Correspondence to: Carolina Priester, Department of Biology andMarine Biology, University of North Carolina Wilmington, 601 SouthCollege Road, Wilmington, NC 28403. E-mail: [email protected]

Received 24 February 2012; Revised 16 May 2012;Accepted 26 May 2012

Published online 13 July 2012 inWiley Online Library (wileyonlinelibrary.com)DOI: 10.1002/jmor.20055

JOURNAL OF MORPHOLOGY 273:1246–1256 (2012)

! 2012 WILEY PERIODICALS, INC.

in diameter, a redistribution of mitochondria andnuclei is observed, presumably due to increasingdiffusion constraints (Kinsey et al., 2007, 2011;Locke and Kinsey, 2008). Nuclei have been foundto be strictly subsarcolemmal (SS) in fibers <120lm but become distributed throughout the sarco-plasm (intermyofibrillar or IM) in fibers >120 lm,which is presumably a response to avoid increaseddiffusion distances for nuclear products such asmRNA and ribosomes (Hardy et al., 2009, 2010;Kinsey et al., 2011; Priester et al., 2011).

Because nuclei are long lived organelles, thisreported shift in distribution raises the question ofwhat controls the spacing and location of nucleiwithin skeletal muscle cells. Nuclei have beenreported to be anchored in a nonrandom fashion tothe sarcolemma through varying complexes ofanchoring proteins and cytoskeletal elements,including actin and microtubules (MTs; Apel et al.,2000; Starr and Han, 2003; Grady et al., 2005;Bruusgaard et al., 2006; Starr, 2007, Star, 2009).Myofibrils are also organized and aligned byanchoring proteins. Dystrophin and dystrophin-associated proteins form a complex that links themyofibrils and cytoskeleton to the extracellularmatrix (Campbell, 1995; Starr and Han, 2003;Starr, 2007; Han et al., 2009). A lack of dystrophinleads to a derangement of the MT network in mus-cle cells (Prins et al., 2009) and the appearance ofIM nuclei in several species, including fish (Bergeret al., 2010). Disturbances or absence of anchoringproteins have also been shown to lead to musculardystrophies (Weller et al., 1990; Banks et al., 2008;Han et al., 2009; Goldstein and McNally, 2010).Additionally, MTs are involved with the alignmentand organization of myosin filaments in developingmyotubes (Pizon et al., 2002), as well as traffickingnuclear products and proteins within the fiber(Ralston et al., 1999, 2001; Scholz et al., 2008),therefore playing a crucial role in organizing theultrastructural features of muscle cells.

Costameres are sarcolemmal protein assembliesalso involved in organizing muscle components,keeping sarcomeres, and therefore the myofibrils,aligned longitudinally and in registration duringcontraction and relaxation cycles. Desmin, plectin,dystrophin and the dystroglycan complex are partof this costameric network that has been proposedto surround and attach to myofibrils at the Z-lineso as to provide support and prevent mechanicalstress (Kierzenbaum, 2007; Goldstein and McNally,2010; Garcıa-Pelagio et al., 2011).

The IM nuclei described in our previous studyon black sea bass (Priester et al., 2011) were some-times randomly distributed, but were oftenarranged in a circular or ring pattern which canbe seen in Figure 1A. This curious arrangement ofcentral nuclei, together with the fact that a reor-ientation of myofibrils in skeletal muscle fibers hasbeen previously described in a wide range of

organisms (Bataillon, 1891; Morris, 1959; Bethlemand Wijngaarden, 1963; Jansen et al., 1963; Kor-neliussen and Nicolaysen, 1973; Muhlendyck andAli, 1978) prompted us to further investigate theultrastructure of those fibers using additional mi-croscopic techniques. In this study we describe themyofibril arrangement in white muscle fibers hav-ing this circular array of nuclei. Additionally,because MTs have been shown to organize theposition of nuclei and myofibrils in skeletal muscle(Bugnard et al., 2005; Pizon et al., 2005; Bruus-gaard et al., 2006), we also investigated the fibers’MT cytoskeleton. Finally, we examined the inner-vation pattern in white muscle cells with andwithout the ring arrangement of nuclei to investi-gate whether the presence or absence of ring bandpattern correlated with innervation or whether theinnervation pattern or the appearance of the syn-apses on the sarcolemma differed. We also wantedto determine if the outer band of reoriented myofi-brils had a separate pattern of innervation fromthe core of the fiber because the presence of sepa-rately innervated functional myofibrils with oppos-ing orientations might act as a muscular hydrostatsuch as those described in Kier and Smith (1985).

MATERIALS AND METHODS

C. striata (0.4 g to 4840 g; n 5 18) were obtained from theUniversity of North Carolina Wilmington (UNCW) AquacultureFacility (Wrightsville Beach, NC) and wild fish were providedby Dr. F. S. Scharf (UNCW). Fish were maintained and proc-essed according to the UNCW Institutional Animal Care andUse Committee standards.

Small, rectangular pieces of epaxial white muscle wereexcised parallel to the fibers’ long axis orientation at a site im-mediately posterior to the operculum. In large fish (>2 Kg) sixdifferent regions (cranial to caudal) were sampled at equalintervals, each !17% (1/6th) of the distance from the operculumto the caudal peduncle. All tissue was fixed for 24 hr in 4%paraformaldehyde in Sorensen’s PBS pH 7.4 or Bouin’s fixative(Presnell and Schreibman, 1997) and subsequently processedrespectively for cryomicrotomy or paraffin histology. For trans-mission electron microscopy (TEM) teased muscle fibers werefixed in 2.5% glutaraldehyde and 1.5% paraformaldehyde inPBS and processed using the protocol described in Nyack et al.(2007). Cryosections (30 lm) were stained with 40,6-diamidino-2-phenylindole (DAPI) and acridine orange (AO) and examinedwith an Olympus FV1000 laser scanning confocal microscope.Stacks of !30 fluorescence and DIC images were collected at 1lm intervals. Companion sections were stained overnight at48C with a primary antibody against b-tubulin (E-7; Develop-mental Studies Hybridoma Bank; diluted 1:400) or for 4 hoursat room temperature with a primary antibody against synapsin(SV2; Developmental Studies Hybridoma Bank, diluted 1:500).These were followed by incubation for one hour at room temper-ature with a 1:200 dilution of a 2 mg/mL secondary goat anti-mouse antibody conjugated with Alexa 594 fluorochrome (A-11032; Invitrogen). Negative controls included incubation withsecondary antibody only for cross reactivity. Control for auto-fluorescence was done using sections exposed to blocking serumonly, with no primary or secondary antibody. None of the con-trols showed any fluorescence at 594 nm.

Serial sections (10 lm) of paraffin embedded tissue were col-lected and every 10th section was stained with hematoxylinand eosin (H & E; Presnell and Schreibman, 1997) and exam-ined with both bright field and polarized light microscopy. The

RING BANDS IN FISH SKELETAL MUSCLE 1247

Journal of Morphology

Fig. 1. Ring band pattern observed with laser confocal microscopy (A and D) polarized light microscopy (B and C) and in serialhistological 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 ofreoriented myofibrils (arrow in B) and revealing striations on these reoriented myofibrils (arrow in C). (D) Laser confocal and DICexamination 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 toa nucleus lined up with the spiraling myofibrils. (E) Section from the middle of the fiber with a visible ring band (rb) around thecore of the fiber (c). (F) Section from near the end of the fiber where only the core of the fiber is present (c).

1248 C. PRIESTER ET AL.


frequency of cells with the circular pattern was determinedusing polarized light microscopy, which allowed identification offibers with and without the circular pattern at low magnifica-tion with a large field of view.Images of muscle fibers and ring bands were outlined with an

Intuos 3 tablet (Wacom, Vancouver, WA) in Adobe Photoshop(version 7.0; Adobe Systems, San Jose, CA) and resulting poly-gons were analyzed with ImagePro Plus (v. 6.1.0.346; Media Cy-bernetics, Bethesda, MD) to determine their cross sectionalarea (CSA), perimeter and mean diameter. The mean diameterwas determined by taking the mean of the distances across thecell through the centroid in 28 increments around the perimeterof the cell. For ring band cross sectional areas, the core areawas subtracted from the total area. For sarcomere and I-bandlengths 100 measurements were taken from each fiber region(ring band and core). Measurements were made on digitizedTEM images with ImagePro Plus (v. 6.1.0.346; Media Cybernet-ics, Bethesda, MD)

RESULTSRing Band Pattern of Peripheral Myofibrils

The use of differential interference contrast(DIC) and especially polarized light microscopyrevealed that in cross sections of those fibers thatcontained IM nuclei arranged in a circular pat-tern (Fig. 1A) the myofibrils were oriented in aring-like pattern at the periphery. The peripheralband of myofibrils had an altered orientation, spi-raling around the central core of the fiber (Fig.1B–D). This peripheral ring band of myofibrilswas close to, but not perfectly perpendicular tothe core myofibrils, which was indicated byregions of extinction at !908 intervals in the pe-ripheral, reoriented myofibrils (Fig. 1B). Further-more, examination of DIC images at higher mag-nification revealed striations on these reorientedmyofibrils corresponding to the highly organizedsarcomeres of skeletal muscle (Fig. 1C). Laserscanning confocal microscope examination ofDAPI-stained whole fibers from similar sized fishshowed that the outer myofibrils were wrappedaround the center core of myofibrils in a spiral orring band pattern (Fig. 1D). The DAPI-stainednuclei were elongate and appeared to be alignedwith the long axis of the fiber within the coremyofibrils whereas in the spiraling myofibrils thenuclei appeared to change their orientation so asto line up with the ring band myofibrils (Fig. 1D).The ring band of reoriented myofibrils was notseen throughout the length of the fibers, rather itwas usually found in the center of the fibers, asrevealed by observations of both whole isolatedfibers (Fig. 1D) and serial histological sections(Fig. 1E,F).

Ring Band Frequency as a Function of BodyMass and Cell Diameter

The smallest fish to display this ring band pat-tern weighed 1168 g (Fig. 2A,B). The smallest fiberdisplaying the ring band pattern was !100 lm indiameter and was found in a fish weighing 2258 g.

Fish with body mass less than 1168 g did not havecells with the ring band pattern, even though theyhad fibers with a mean diameter >100 lm. In fishwith a body mass greater than 1168 g, 0.6–36% ofthe white fibers had the peripheral ring band pat-tern. Muscle sampled from six different sites alongthe length of the large fish (N 5 4 fish) indicatedthat this ring band pattern was more prevalent inthe midpoint of the anteroposterior axis, where upto 48% of the fibers had this pattern of reorientedmyofibrils (Fig. 2C).

Ultrastructure, Cytoskeleton, andInnervation of the Ring Band

Ultrastructural observations verified that the pe-ripheral myofibrils were truly reoriented, with sar-comeres being almost perpendicular to those of thecentral myofibrils and with no sarcolemma at theirinterface, indicating that both were within thesame fiber (Fig. 3A,B) and thus shared the samesarcoplasm. The ring band sarcomeres appeared tobe complete, suggesting that the reoriented myofi-brils were functional.

When white muscle fibers of C. striata werestained with antibodies against b-tubulin, MTswere seen in all white muscle cells, includingthose with the peripheral ring band pattern (Fig.4A), and these MTs were present throughout thefiber, in both the SS and IM regions. In fiberswithout the ring band the MTs were predomi-nately oriented with the long axis of the cell, par-allel to the myofibrils, with numerous branchesprojecting radially from those MT bundles(Fig. 4B). The MTs also appeared to wrap thenuclei. In the fibers containing the ring band, theMTs in the core of the fiber were also mostly lon-gitudinally oriented. However, at the peripherythe MTs were reoriented in the same manner asthe myofibrils. That is, they had changed orienta-tion and were roughly perpendicular to the MTsand myofibrils present at the core of the fiber(Fig. 4A).

After confirming that the myofibrils and MTs ofthe ring band shared cytoplasm with the core ofthe fiber we wished to determine if the alteredfibers also had a similar pattern of innervation.Staining for synapsin revealed that the cells withthe ring band pattern were innervated at the pe-riphery of the fiber, in the same manner (i.e. rele-gated to the sarcolemma and having a stippledappearance) as the cells without the pattern (Fig.4C–F).

In summary, our observations suggest that infibers with the ring band pattern all of the fiber’sultrastructural elements seem to change orienta-tion forming a ring around the core of the musclefiber. Myofilaments, nuclei and MTs all becomespirally wound around the core of the fiber, whichremains unchanged.



Cross Sectional Areas and SarcomereLengths

These measurements indicated that while ringbands were only seen in specimens greater than1168 g, there was no correlation (r2 5 0.003)between fiber diameter and the percentage of eachfiber’s cross-sectional area devoted to the ringband (Fig. 5A), and all specimens with bodymasses greater than 2258 g varied widely in theextent of the fiber area represented by the ringband pattern (Fig. 5B). Sarcomere and I-bandlengths were also examined in the core and ringband regions of fibers from multiple sections fromtwo fish. All fibers were processed for TEM in thesame manner and mean sarcomere lengths (Z-lineto Z-line) from myofibrils forming the ring bandwere significantly longer than the mean sarcomerelength of the core myofibrils (ANOVA: DF 5 1, F5 144.8, P < 0.0001; Fig. 5C). The mean lengthfor ring band sarcomeres was 1.74 6 0.0152 lm (N

5 2), whereas the mean length for the core sarco-meres was 1.50 6 0.0131 lm (N 5 2). Mean I-bandlength was significantly shorter for myofibrilsforming the ring band. Mean I-band length was0.31 6 4.823e-3lm in the ring band region (N 5 2),and 0.54 6 0.0180 lm in the core region of thefibers (N 5 2; ANOVA: DF 5 1, F 5 167.4, P <0.0001; Fig. 5D).

DISCUSSION

The ring band pattern observed in white fibersof large black sea bass in this study is virtuallyidentical to previously described microscopic struc-tures variously called ‘‘ringbinden,’’ ‘‘striated annu-lets,’’ ‘‘rings bands,’’ or ‘‘ring fibers.’’ These ringbands were observed as early as 1891 and havebeen seen in a variety of muscles from a variety ofdifferent organisms, including hagfish, Xenopuslarva, rabbit, mouse and humans (Bataillon, 1891;

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 ringband versus body mass (N 5 100 fibers per fish). (B) Plot of mean fiber diameters versus body mass (N 5 100 fibers per fish). Greycircles represent the mean diameter of fibers without the ring band, whereas white circles depict the mean diameter of fibers withthe ring band pattern. (C) Plot of % of fibers with the ring band versus six regions along the body length (cranial to caudal) of fourof the largest fish (N 5 100 fibers per site, per fish). The bars represent the average of the percentages of fibers with the ring band6 S.E.M. in four large fish weighing 2258, 2691, 3921, and 4840 g.



Morris, 1959; Bethlem and Wijngaarden, 1963;Jansen et al., 1963; Korneliussen and Nicolaysen,1973; Muhlendyck and Ali, 1978). However, the or-igin and function of ring bands has remainedunclear.

In hagfish, the ring bands were seen in normalswimming muscle composed of white fibers(Jansen et al., 1963; Korneliussen and Nicolaysen,1973). Ring bands in humans have been observedin older, but healthy individuals and particularlyin muscles that do not have an attachment to bonesuch as the extra-ocular motor muscles, dia-phragm, tongue, vocallis, uvulla, and others(Bethlem and Wijngaarden, 1963; Muhlendyck andAli, 1978). Additionally, ring bands have beenexperimentally induced in skeletal muscle ofhealthy rats and rabbits by severing their attach-ment to a rigid structure (e.g., bone) by either cut-ting the tendon or the muscle itself (Bethlem andWijngaarden, 1963).

The ring band pattern has also been observed inassociation with certain pathologies, especially dif-ferent types of muscular dystrophy (Bethlem andWijngaarden, 1963; Banks et al., 2008). Dystrophicmuscle has altered or missing proteins in its costa-meres and/or dystroglycan complexes. It has beenhypothesized that individuals suffering from mus-cular dystrophy have muscles with ‘weaker’attachments between the sarcolemma and themyofibrils, cytoskeleton, and the basal lamina,which makes these linkages more prone to tearingand damage as the muscle fiber contracts (Gold-stein and McNally, 2010; Garcıa-Pelagio et al.,2011). Han et al. (2009) have shown that a mousemodel for secondary dystroglycanopathy (Largemyd)

has weaker links between the sarcolemma and ba-sal lamina due to a lack of laminin globular do-main-binding motif. Additionally, lengthening(eccentric) contraction has been shown to inducemore damage to the sarcolemma and its attach-ments to the basal lamina and intracellular struc-tures than shortening (concentric) contractionsbecause they increase membrane tension, leadingto small tears in the sarcolemma, as suggested byWeller et al. (1990) and Han et al. (2009).

As described by Kier and Smith (1985), muscleshave a constant volume and as a muscle contracts,it shortens in length and therefore it must expandradially, increasing its circumference. An increasein circumference leads to more stress on the sarco-lemma. In a muscle that is attached to bone, theextent of contraction is limited by the attachmentto a rigid structure, whereas in muscles that arenot attached to bone, such as the diaphragm andextra-ocular motor muscles, the extent of contrac-tion is less limited and can result in hypercontrac-tion and subsequent stress and strain between thesarcolemma and cytoskeleton. The same is true formuscles that have had their attachment to bonesevered. As muscle is allowed to hypercontract,there appears to be a threshold fiber length atwhich the extreme shortening of the fiber causesthe circumference to stretch to the point of break-ing the links between the sarcolemma, the myofi-brils, cytoskeleton, and basal lamina (Muhlendyckand Ali, 1978; Han et al., 2009). By extension, in adystrophic muscle fiber one would expect a signifi-cantly lower stress to result in damage to thebridge between the sarcolemma and the myofibrilsand basal lamina, even in muscle fibers that are

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 ofthe fiber seen as cross section myofibrils (c in A and B) sharing the same cytoplasm. An intermyofibrillar nucleus (N in A) with apronounced nucleolus (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.



Fig. 4. Cytoskeletal network and innervation pattern in muscle fibers with and without the ring band. (A and B) Laser confocalz-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 ringband, with MTs running the length of the fiber (arrow) with some branching perpendicular to the longitudinal axis (arrowhead). Anucleus is seen in association with the network of MTs (circle). (C–F) Paired images showing the pattern of innervation in whitemuscle fibers without (C and D) and with (E and F) the ring band as revealed by an antibody against synapsin (arrows pointing togreen 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 coreof the fiber by ‘‘c’’ in (C) and (E).



attached to bone (Banks et al, 2008; Han et al.,2009).

Therefore, it appears that the common charac-teristic seen in the muscles that display this reor-iented ring band of myofibrils is the disruption ofthe sarcolemmal attachments to intra- and extra-cellular structures. The disruptions can be causedby various events, including hypercontraction,lengthening contractions, and pathological condi-tions such as muscular dystrophies.

Once the sarcolemma is disrupted, the breakingof its linkages leads to a disconnect between intra-cellular structures (cytoskeleton, myofibrils andnuclei) and the sarcolemma and basal lamina.These intracellular components may then becomefree to reorient (or disorient) and form new connec-tions with the sarcolemma. What dictates the ori-entation of the myofibrils, cytoskeleton and nucleiat this roughly 908 angle to the core of the fiber is

not known. It does seem that the ring band con-sists of newly formed myofibrils because the fibersare continuing to grow hypertrophically. In fact, insome fibers the area composed of the ring bandwas very large, reaching over 75% of the CSA (Fig.5A,B), indicating that these fibers were still under-going hypertrophy by adding myofibrils in thisreoriented, ring band pattern.

Staining for synapsin revealed no obvious differ-ences in innervation between fibers with and with-out the ring band, which suggests that if the fiberis stimulated at the neuromuscular junction, all ofthe myofibrils would contract, both the core andring band myofibrils. Contraction of the myofibrilsof the ring band around the middle point of thefiber would potentially restrict the expansion ofthe fiber’s circumference. By doing this, the reor-iented ring band myofibrils would be reinforcingthe fiber’s middle portion, thereby preventing fur-

Fig. 5. (A) Plot of ring band cross sectional area (CSA) as a percentage of total fiber CSA versus mean fiber diameter. (B) Plot ofring 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 6S.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), andthe asterisks indicate that the means are significantly different.



ther expansion of the sarcolemma and subsequentdamage to the sarcolemmal linkages.

It has been previously suggested that chronicstretch induces an increase in sarcomere lengths(Houlihan and Breckenridge, 1981). This mightexplain why sarcomeres of the ring band are sig-nificantly longer than the sarcomeres at the coreof the fiber even though their I-bands are shorter,indicating they are more contracted. Alternatively,because they are reoriented, the Z-lines of the sar-comeres in the ring band might be attaching tocostameres on the sarcolemma that have a differ-ent spacing, i.e. that are further apart and there-fore could lead to formation of longer sarcomeres.However, it is not known whether costameres dic-tate sarcomere length or whether sarcomerelength dictates where Z-lines are anchored at thesarcolemma.

While we have shown that the occurrence ofring band fibers is widespread, it is somewhatrare, raising the question of why they appear inthe white muscle of the black sea bass. The whitefibers of black sea bass in our study grow hyper-trophically to very large sizes, both in diameterand length. Furthermore, they are not directlyattached to bone, but rather to a contiguous seriesof connective tissue layers (myosepta). The fibersare aligned end-to-end, in a row from myosepta tomyosepta, in such a way that the fibers at theextremities attach to either the skin or the axialskeleton of the fish. When the fibers in a row con-tract, they pull on the myosepta between themand, therefore, also pull on each other. The fiberslocated near the center of this row of fibers wouldhave more leeway and may hypercontract morethan the fibers that are attached closer to the skinor axial-skeleton. Furthermore, because the endsof the muscle fibers are anchored to myosepta, theportions of the sarcolemma that are close to themyosepta would likely not be disrupted duringhypercontractions, whereas the portions of the sar-colemma in the middle of the fiber, furthest frommyosepta may be disrupted and damaged. Thismay explain why we observe: 1) the ring band pat-tern in the middle of each fiber rather than alongthe entire length and 2) more fibers with the ringband pattern towards the middle of the fish’s ante-roposterior axis, which is the position most distantfrom attachment sites on bone or skin.

In addition, fish swim by alternating contrac-tions of either side of the body such that when oneside approaches the end of a contraction cycle, theother side is already starting to contract. Thishelps maintain body stiffness during burst swim-ming and leads to lengthening contractions on oneside of the fish during each tail beat. For example,Davies et al. (1995) have showed that during sub-carangiform swimming in trout, caudal musclesare activated while still lengthening. Subcarangi-form is also the swimming mode of black sea bass.

Because lengthening contractions induce moredamage of the sarcolemma and its attachmentsthan isometric- or shortening-contractions (Welleret al., 1990; Han et al., 2009), this could favor theformation of ring bands in black sea bass.

In contrast, hagfish have an anguilliform modeof swimming, which involves undulation of theentire body, rather than bending in the mid-por-tion of the body like the subcarangiform swimming(Lindsey, 1978; Colgate and Lynch, 2004). It isinteresting to note that while dissecting hagfishfor a related study, we have observed that theirswimming muscles are not firmly attached to theskin, and therefore are not firmly anchored. Thischaracteristic would make their muscle fibersmore likely to hypercontract, and may give aninsight into why Korneliussen and Nicolaysen(1973) found ring bands in hagfish muscle. Follow-ing this logic, one would expect to find ring bandsin fish with other types of swimming modes wher-ever there is more bending along the fish’s bodylength. For example, fish with carangiform swim-ming mode (Lindsey, 1978) would be likely to pos-sess ring bands more caudally than a fish with asubcarangiform swimming mode, and fish with athunniform swimming mode would be expected topossess ring bands only at their caudal peduncle.However, a comparison between black sea bassand fish with thunniform swimming mode such astuna becomes complicated due to their very differ-ent arrangement of fibers and fiber types.

The fact that this ring band pattern appearsonly in larger black sea bass suggests that thisphenomenon is either size related or age related,however these two factors cannot be uncoupled.Muscle fibers from larger fish have greater distan-ces from connection sites, so the hypercontractionpossibility would increase with size. However, thepossible effects of aging on the anchoring proteinscannot be ruled out. In either case, the appearanceof this pattern seems to be a response to damageto the sarcolemma and its links to the cytoskeletonand basal lamina. Studies on the mechanisms forrepairing the sarcolemma have suggested that dys-ferlin is one of the proteins involved in mediatingthe resealing of the membrane and maintaining itsintegrity (Han and Campbell, 2007; Han et al.,2009). Part of that repair mechanism involves acti-vation of proteases, such as calpain, by the highlevels of calcium that are allowed to enter the fiberwhen the membrane is damaged. The activation ofproteases leads to disassembly of cortical cytoskel-eton proteins in the vicinity of the damage (Hanand Campbell, 2007), which could likely alsorelease anchoring proteins and thus allow for reor-ientation of intracellular structures and organ-elles.

The ring band likely restricts the expansion ofthe middle of the fiber, therefore serving as rein-forcement against such expansion. This would pro-



tect the sarcolemma from further stretch and dam-age during muscle contraction and relaxationcycles. The opposing forces of the two groups ofdifferently oriented myofibrils make the fiber func-tion somewhat like a muscular hydrostat (Kierand Smith, 1985), but by interactions of bundles ofmyofibrils in a single cell rather than groups ofwhole muscle cells.

Together, these observations and the previousliterature give insights into what controls muscleultrastructural organization, directionality andhow all these factors can change orientation due todisturbances of the connections to the sarcolemma.We propose that disturbing the sarcolemmathrough stretching in the circumferential directiondisrupts the links between extracellular anchoringsites (basal lamina), the dystroglycan complexesand intracellular structures, namely the myofi-brils, cytoskeletal elements (MTs), and nuclei. Thisoverstretching can result from hypercontraction orlengthening contraction of muscle fibers that arenot attached to bone (normally or experimentally).Once these links are broken, the intracellular ele-ments are released from the sarcolemma and freeto reorient in a different direction. In our study,the disturbance of sarcolemma seems to also allowfor the release of nuclei from the periphery of thefiber, which then become IM nuclei once new myo-fibrils are formed around them.

ACKNOWLEDGMENTS

The authors are grateful for the technical assis-tance of Mark Gay, Dr. Sonja Pyott, Dr. FredScharf, and Kenneth Kelly. The monoclonal anti-bodies E-7, developed by Michael Klymkowsky,and SV2, developed by Kathleen Buckley, wereobtained from the Developmental Studies Hybrid-oma Bank developed under the auspices of theNICHD and maintained by The University ofIowa, Department of Biology, Iowa City, IA 52242.

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