Myotonia Congenita

A Histochemical and Ultrastructural Study in the Goat: ComparisonWith Abnormalities Found in Human Myotonia Dystrophica

JAMES B. ATKINSON, BA, LARRY L. SWIFT, PhD,and VIRGIL S. LEQUIRE, MD

Muscle biopsy specimens from the myotonic goat, ananimal model of heritable myotonia, were examinedhistochemically and by electron microscopy. AfterPeriodic acid-Schiff (PAS) staining with diastase diges-tion, there was increased PAS-positive material withinmyotonic goat fibers, as compared with those of nor-mal goats. Myotonic muscle stained with alizarin red S,a histochemical stain for calcium, also had an increasedstaining reaction when compared with muscle fromnormal goats. Several ultrastructural abnormalitieswere found in myotonic goat muscle using routineosmium and uranyl acetate staining. These included in-creased density ofthe t-tubules, electron-dense materialwithin t-tubules, proliferation and dilatation of sarco-

MORPHOLOGIC ABNORMALITIES of muscle invarious forms of human myotonia have been the sub-ject of numerous reports. 1-1 The most strikingchanges have been found in myotonic dystrophy, al-though histochemical and ultrastructural alterationshave been observed in myotonia congenita and para-myotonia. A great deal of electrophysiologic researchon myotonia has been done with hereditary myotoniain goats, an animal model closely resembling myo-tonia congenita in humans. However, morphologicalstudies of myotonic goats have been few and unre-vealing. 12," 3A study of muscle from myotonic goats was under-

taken to determine whether there were morphologicchanges in the goat model analogous to those reportedfor human myotonias. Due to evidence of a cytomem-brane defect in myotonia, techniques using ultrastruc-tural stains with membrane specificity were also uti-lized. The present paper describes histochemical andultrastructural abnormalities in muscle from myo-tonic goats. The relevance of these abnormalities tosimilar morphologic findings in patients is discussed.

From the Department of Pathology, Vanderbilt University, Schoolof Medicine, Nashville, Tennessee

tubular elements, and abnormal mitochondria in themyotonic biopsy specimens. To further study muscleultrastructure, ruthenium red and lanthanum were usedas electron microscopic stains with specificity for mem-branes. There was increased density of the sarcolemmaand t-tubules in myotonic muscle stained withruthenium red as compared to normal, and lanthanumproduced a darker staining reaction of the myotonicgoat sarcolemma. The histochemical and ultrastruc-tural differences between normal and myotonic goatmuscle were interpreted to be consistent with a mor-phologic basis for the abnormal contraction-relaxationproperties characteristic of myotonia. (Am J Pathol1981, 102:324-335)

Materials and Methods

Animal Model

The myotonic goats used in this study were from aherd that has been line-bred over the past 20 years.The characteristic signs of myotonia were confirmedby electromyographic measurements.'4 Normal goatswere from a separate genetic background and had noevidence of myotonia. All goats were 2-3 years old,approximately the same size, and free of other dis-eases. Muscle biopsy specimens from 7 normal and 7age- and sex-matched myotonic goats were processedin a paired manner.

Supported by grants from the Muscular Dystrophy Asso-ciation.

Accepted for publication September 3, 1980.Address reprint request to Virgil S. LeQuire, MD,

Department of Pathology, School of Medicine, VanderbiltUniversity, Nashville, TN 37232.

0002-9440/81/0313-0324$01.10 © American Association of Pathologists

324

MYOTONIA CONGENITA 325

Biopsy Procedure

Muscle biopsies were obtained after the animalswere anesthetized with intravenous injections of 150mg of ketamine hydrochloride (Ketaject, Bristol Lab-oratories, Syracuse, NY). For electron microscopystudies, the adductor longus muscle of the right hind-leg was exposed, and portions were excised after beingclamped in situ with specially designed forked clampsto prevent contraction.'lPortions of the same musclewere dissected free, excised, and either placed in anappropriate fixative or quickly frozen by immersion inisopentane cooled in liquid nitrogen for light-micro-scopic and histochemical studies. Care was taken totake biopsy specimens of identical muscles in each an-imal.

Light Microscopy

Specimens for light microscopic examination werefixed in Gendre's fixative, and paraffin-embeddedsections cut at 4 IA were stained with hematoxylin andeosin (H&E), periodic acid-Schiff (PAS) reaction be-fore and after diastase digestion, and a Gomori tri-chrome stain.'6 Some specimens were fixed in buf-fered formalin (calcium-free), and the sections werestained for five minutes with alizarin red S. '7 Frozenspecimens from two pairs of goats were sectioned at 10,u in a cryostat and stained with H&E, modified Gom-ori trichrome, PAS, phosphorylase, DPNH diaphor-ase, succinic dehydrogenase (SDH), oil red 0, andmyofibrillar adenosine triphosphatase (ATPase) atpH 9.4, 4.2, and 4.6.18 Fibers were classified as TypesI, hIa, and lIb by the scheme of Brooke and Kaiser. '9

Electron Microscopy

The clamped muscle from which biopsy specimenswere taken for ultrastructural studies was processedby several methods:

1) The muscle was fixed for 2-24 hours in coldphosphate-buffered 2%o glutaraldehyde adjusted to325 mOsM,20 then washed in phosphate-buffered7.50o sucrose. The samples were postfixed in coldphosphate-buffered 2% osmium tetroxide for 1 hour,dehydrated in a graded series of alcohol, cleared in pro-pylene oxide, preinfiltrated, and then embedded in anaraldite resin mixture. Some samples were stained enbloc with 1 Wo uranyl acetate prior to dehydration.Thick sections (1.5 ,u) stained with toluidine blue wereexamined by light microscopy, and appropriate areaswere chosen for thin (less than 1 ,) sections.

2) To study membrane-associated acid mucopoly-saccharides, muscle was processed with the use of ru-thenium red.21 The muscle was fixed in 3.6% glutar-

aldehyde, 0.1 M cacodylate, pH 7.3, 0.5% rutheniumred for 24 hours. The specimens were cut into blocks0.5-1.0 mm in diameter and 3 mm long and replacedin the fixative for 24 hours. They were soaked in 0.15M cacodylate, pH 7.3, for at least 12 hours, postfixedin 2% osmium tetroxide, 0.1 M cacodylate, pH 7.3,0.5% ruthenium red for 3 hours, dehydrated in alco-hol, and embedded in araldite. Controls were pro-cessed without osmium. Thick sections were examinedby light microscopy to obtain sections from compar-ably stained areas for electron microscopy.

3) For demonstration of calcium binding sites,muscle was fixed in 3 % glutaraldehyde, 0.175 M caco-dylate, pH 7.3, 20o polyvinylpyrrolidine, 0.5% Alcianblue for 24 hours and processed with the use of lan-thanum by the method of Waugh et al.22 Tissue wasprocessed with and without uranyl acetate en blocstaining.The thin sections (less than 1 i) were examined un-

stained and stained with lead citrate in a Philips 300electron microscope.

For comparative studies, muscle biopsies from twopatients (a 15-year-old male and a 37-year-old female)with the diagnosis of myotonic dystrophy were exam-ined. Specimens were obtained from the tibialis ante-rior muscle with the forked biopsy clamps. They werefixed in glutaraldehyde and processed for electron-mi-croscopic examination with osmium and uranyl ace-tate en bloc staining.

Analytic Methods

Mitochondria for calcium measurements were iso-lated from normal and myotonic goat muscle by dif-ferential ultracentrifugation. Flexor and extensormuscles from both hindlimbs were removed, finelyminced with scalpels, and homogenized in 3 x volumeof 0.1 M KCl, 5 mM histidine, pH 7.4, in a WaringBlendor. The homogenate was centrifuged at lOOOgfor 15 minutes in a Beckman 19 rotor, and the super-natant was decanted and centrifuged again in the 19rotor at 9500g for 15 minutes. The pellets were washedby resuspension in 0.1 M KCl, 5mM histidine, pH 7.4,and pelleting at 9500g. The washed pellets were sus-pended with a Teflon homogenizer in 0.1 M KCl, 5mM histidine, pH 7.4. Protein was estimated by themethod of Lowry et al. 23 The pellets were examined byelectron microscopy, and succinate cytochrome c re-ductase and monoamine oxidase activity were assayedas markers for inner and outer mitochondrial mem-branes, respectively.24,25

For measurements of calcium in whole muscle,samples were frozen, lyophilized, and weighed. Cal-cium content was measured with a Perkin-Elmer 290

Vol. 102 * No. 3

326 ATKINSON ET AL

atomic absorption spectrophotometer after sampleswere digested in a 5% LaCl3 and concentrated HNO3solution.

Reagents

Glutaraldehyde and osmium tetroxide were ob-tained from Polysciences, Inc. (Warrington, Pa);araldite 502 resin, dodecenyl succinic anhydride andtri(dimethylaminomethyl)phenol from Ladd Re-search Industries, Inc. (Burlington, Vt); alizarin red Sfrom Fisher Scientific Co. (Fair Lawn, NJ); and ru-

thenium red from Chroma-Gesellschaft (Stuttgart,West Germany). Glutaraldehyde was either fresh (lessthan 6 months old) or, in some cases, distilled. Allother chemicals were reagent grade.

Results

The histochemical reactions in normal and myo-tonic goat muscle produced the usual checkerboardpattern of Types I and II fibers seen in mammalianskeletal muscle. Oxidative enzyme reactions did notreveal any abnormality of the intermyofibrillar net-work pattern, and no striking differences were notedbetween normal and myotonic for the phosphorylase,DPNH diaphorase, SDH, myofibrillar ATPase, or oilred 0 staining reactions. Sections staining with H&Eand Gomori trichrome of myotonic muscle were alsounremarkable. Types I, Ila, and Ilb fibers constitutedapproximately 35.0%o, 39.5% and 25.5%7, respective-ly, of the fibers in both normal and myotonic muscle.There was moderate hypertrophy of myotonic fibers(Table 1). The mean fiber size in normal muscle was 40p, while that for myotonic was 44 ,u. Types I, Ila, andlIb fibers measured an average of 31.7, 47.6, and 40.2,u, respectively, in normal muscle as compared with33.8, 54.8, and 43.7 in myotonic muscle. Occasionaldegenerative changes and central nuclei were seen insome myotonic fibers, but these findings were not pro-nounced.The PAS reaction was identical in both normal and

myotonic goat muscle. However, following diastasetreatment, it was observed that some of the PAS-posi-tive material in the myotonic biopsies was more resis-tant to diastase digestion than in the normal muscle.

This difference was appreciated by visual inspectionand comparison of the slides prior to observation withthe microscope. The difference in staining intensitybetween normal and myotonic muscle was observedby light-microscopic examination to be due to a PAS-positive, diastase-resistant material in a reticulatedform within myotonic fibers (Figures 1 and 2).An additional difference between normal and myo-

tonic goat muscle was found by light-microscopic ex-

amination with alizarin red S as a histochemical stainfor calcium. Myotonic fibers had a diffusely increasedstaining reaction with this technique, with focal accu-

mulation of stain at the surface of certain fibers (Fig-ures 3 and 4). The presence of an increased calciumcontent in myotonic muscle was confirmed biochem-ically. Muscle from normal goats contained 1.54 +

0.08 ,.moles calcium/g dry weight of muscle whereasmyotonic had 1.87 ± 0.16 imoles calcium/g dryweight of muscle (P < 0.05, Table 2). Part of this in-creased calcium was found in the mitochondria, as mi-tochondria isolated from normal goats contained 2.40± 0.33 nmoles calcium/mg protein, while that frommyotonic had 7.40 ± 2.59 nmoles calcium/mg protein(P< 0.05).

Electron-microscopic studies revealed several ab-normalities in the muscle from myotonic goats. Myo-tonic muscle stained with osmium and uranyl acetatecontained an electron-dense material within the t-tu-bules (Figure 6), unlike normal fibers (Figure 5). Thewalls of the t-tubules were also excessively dense, as

was the sarcolemma in some cases. These findingswere common to all the myotonic goats studied, al-though all the fibers within a given biopsy did notshow this t-tubule abnormality. Electron-dense,amorphous material was also distributed as focalaccumulation in the terminal cisternae of the sarco-

plasmic reticulum (SR) in both normal and myotonicfibers. An additional feature in some myotonic fiberswas an apparent proliferation of tubular elements(Figure 7). In other myotonic fibers, we observeddilatation of terminal cisternae of the SR (Figures8 and 9). There was no evidence of myotonic mus-cle of any alteration of the myofibrils, I bands, Z lines,or nuclei, or in the distribution and amount of glyco-gen in biopsies not stained with uranyl acetate.

Table 1-Values of Muscle Fiber Diameters in Normal and Myotonic Goats*

Type I Type Ila Type llb

Diameter (A Range Diameter (M) Range Diameter (,u) Range

Normal 31.7 22-50 47.6 28-72 40.2 22-50Myotonic 33.8 22-50 54.8 28-80 43.7 28-66

* Measurements are based on 200 fibers from each of 2 normal and 2 myotonic goats. The fibers were measured across their smallest diam-eter with a micrometer slide graduated at 10-M intervals.

AJP * March 1981

Vol. 102 * No. 3 MYUUTNIA LtNUEtNITA JL/

..j.....;.

.....

M.' ~~~ ~ ~ ~ ~ ~ ~ ~ ~ ~ 4

F r ( ntFg ss

ns4anpcsiteamnrshctlpinnir1P-si.iteesnmeaiiaeca

pater wthn hefier._hee ibrsapea°lage tanth nrml onro fbes,an sveal enra ncli reprset.(PS tanig it

diastae dIgsinx52)Fur3-Normal goat.PSsannihdats (Aizartion,rd x 160) Figure2-Myotonic goat. This section was cut at the sametik

thickness (4 $A) and stained with the control specimen in Figure 3. The overall intensity of staining is greater than for the normal biopsyspecimen, and there is focal accumulation of stain at the surface of individual myotonic fibers (arrows). (Alizarin red 5, x 160)

lkfflt7d'%rlMr%lkTTA d"d'%lPLTt41UlLTTqrA V)7

328 ATKINSON ET AL

Table 2-Calcium Content of Muscle and IsolatedMitochondria From Normal and Myotonic Goats*

Normal Myotonic

Muscle calciumt 1.54 + 0.08 1.87 + 0.16(Mmoles calcium/g dry weight)

Mitochondrial calciumt 2.40 ± 0.33 7.40 + 2.59(nmoles calcium/mg protein)* Data are significant at p < 0.05 as analyzed by the Student t test.t n = 3 normal and 3 myotonic goats.n = 4 normal and 4 myotonic goats.

Electron-microscopic studies revealed abnormal mi-tochondria in many myotonic goat muscle fibers (Fig-ures 7-9). These mitochondria were pleomorphic,swollen, and in various stages of disruption. Theyoften contained inclusions of large osmiophilic bodiesand myelin figures that were either loosely coiled orlamellar. Some mitochondria contained inclusionsthat were less osmiophilic, resembling lipid. In manycases the mitochondria were closely apposed to lipiddroplets. Similar mitochondrial changes were alsofound after the muscle was fixed in 2% osmium te-troxide or when 5 Ne formaldehyde was substituted forglutaraldehyde (Figures 10-12). These findings werenot present in all fibers within a given biopsy. Theywere, however, observed in all myotonic goats biop-sied and were never found in normal muscle which hadbeen simultaneously fixed and processed. The ultra-structural changes observed could not be correlatedwith any degenerative changes of corresponding fibersin the toluidine-blue-stained, araldite-embedded thicksections.

Based on the appearance of myotonic goat muscleafter PAS staining with diastase digestion and the ul-trastructural appearance of the t-tubules and sarco-lemma, biopsies from normal and myotonic animalswere stained with ruthenium red and processed forelectron microscopy (Figures 13 and 14). This stain,specific for acid mucopolysaccharides,2I produced anincreased electron density of the t-tubules in myotonicmuscle. The increased staining of the t-tubules was ob-served at the periphery as well as in the center of themyotonic fibers. Myotonic sarcolemma was also moreelectron-dense, as compared with normal, with stainlocalized to the outer leaf of the unit membrane andexternal lamina. There was variability in the stainingintensity of the SR, and no consistent differences be-tween normal and myotonic muscle could be found.Ruthenium red was localized within the terminal cis-ternae of the SR and appeared to stain the material vis-ualized in osmium and uranyl acetate stained sections,as has been noted previously.26 Ruthenium red had anaffinity for pinocytotic vesicles, but no staining of themitochondria was detected. The pattern and intensity

of staining by ruthenium red in control samples werenot affected by the omission of osmium.Lanthanum was used as a stain for the ultrastruc-

tural demonstration of the muscle cytomembranesand as a marker for membrane-associated calciumbinding sites. Our results confirmed those from otherstudies in which similarities were noted between thestaining pattern of cell membranes by ruthenium redand by lanthanum.2' Lanthanum had an increased af-finity for the sarcolemma and external lamina in myo-tonic goat muscle as compared with normal, resultingin a denser staining reaction (Figures 15 and 16). Intra-cellular localization of lanthanum in normal and myo-tonic muscle was inconsistent. This was interpreted asbeing due to incomplete penetration of the stain.When penetration into the fibers did occur, no differ-ences were noted. Lanthanum had an equal affinityfor both the longitudinal and terminal portions ofnormal and myotonic SR, but staining of the t-tubuleswas variable.

Electron-microscopic examination of muscle bi-opsy specimens from two patients with myotonic dys-trophy revealed abnormalities similar to those pre-viously reported.2-810 II We observed central nuclei,accumulation of lysosomes, myelinlike structures,and destruction of myofilaments. Specific featuressimilar to those found in the myotonic goat were alsoseen. These included proliferation and dilatation ofterminal components of the SR, abnormal mitochon-dria, and increased electron density of the t-tubules(Figures 17 and 18). Many mitochondria had unusualshapes, with increased density of the matrix, and theywere closely associated with lipid.

Discussion

Histologic studies of myotonia congenita in hu-mans have generally shown only hypertrophy or mini-mal changes in muscle.27 However, in a recent report,the absence of Type Ilb fibers was demonstrated,9 andhistochemical evidence of "mitochondrial aggregates"was found in muscle biopsy specimens from a patientwith features of myotonia congenita.28 Ultrastruc-tural abnormalities in muscle from patients with myo-tonia congenita and myotonic dystrophy have been re-ported by several investigators. Schroder and Beckernoted a proliferation of certain components of the SRin myotonic dystrophy, myotonia congenita, andparamyotonia.7 In some cases they observed electron-dense precipitate on the sarcolemma, t-tubules, andpinocytotic vesicles. Other nonspecific ultrastructuralchanges have been seen in myotonic dystrophy, in-cluding swelling and proliferation of sarcotubularmembranes, myelin figures, and abnormal mitochon-

AJP * March 1981

MYOTONIA CONGENITA 329

65

7

Figure 5-Normal goat. In this electron micrograph, normal appearing triads (arrow) are seen at the A-I band junction. (Lead citrate, x 23,000)Figure 6-Myotonic goat. The t-tubules of the triads (arrows) are stained excessively dense as compared with those in the normal goat biopsy inFigure 5. (Lead citrate, x 23,000) Figure 7-Myotonic goat. The subsarcolemmal mitochondria in this section shows loss of cristae and con-tains myelin figures. There are vesicular structures, which may represent disrupted mitochondria (v). The sarcotubular elements appear ex-cessively abundant, and some are distended (arrow). Note the convoluted tubular element which is continuous with the sarcolemma (arrow-head). (Uranyl acetate, lead citrate, x31,000) Figure 8-Myotonic goat. Mitochondria are of variable size and shape, and the twovacuolated structures in the center appear to be mitochondrial remnants. Dilatation of t-tubules and SR can be seen (arrows). (Uranyl acetate,lead citrate, x 19,000)

8

Vol. 102 * No. 3

330 ATKINSON ET AL

9

11

10

12

Figure 9-Myotonic goat. The two mitochondria in this section contain disrupted cristae and myelin-type inclusions. Dilatation of sarcotubu-lar elements can also be noted (arrow). (Uranyl acetate, lead citrate, x 31,000) Figure 10-Normal goat. The mitochondria in this formalde-hyde-fixed section appear normal, with well-defined cristae in the usual configuration. (Lead citrate, x 19,000) Figure 11-Myotonic goat.In this formaldehyde-fixed myotonic biopsy specimen, mitochondria are pleomorphic, with variation in their matrix density. An osmiophilic in-clusion can be seen (arrow). There are numerous vesicles that represent distended sarcotubular elements. (Lead citrate, x 19,000) Figure12-Myotonic goat. In this section there are mitochondria with abnormal shapes and disruption of cristae organization. Some mitochondriacontain inclusions, including lipid (arrow). Distended and proliferated sarcotubular membranes can again be seen. (Lead citrate, x 19,000)

Vol. 102 * No. 3

MYOTONIA CONGENITA 331

dria.2-8'10"' Dilatation and proliferation of SR, di-lated t-tubules, mitochondrial changes (swelling, dis-ruption, and inclusions), and myelinlike figures havealso been found in the drug-induced forms of myo-tonia in animals.29-32The best established and oldest known animal

model for an inherited human skeletal muscle diseaseis the myotonic goat.33 Research on this animal modelhas provided most of the data leading to our presentunderstanding of the physiology of myotonia and hashelped define the role of muscle membranes in expres-sion of the disease. The results of inbreeding in ourherd of myotonic goats suggest that the goat model isinherited as an autosomal dominant and recessivetrait, as was recently determined for myotonia con-genita in humans.34 Adams and Thompson reportedincreased osmiophilic granules and alterations in thedistribution of sarcotubular membranes in myotonicgoat muscle examined by electron microscopy.'3 Ex-cept for those and early light-microscopic studies,'2detailed histochemical and ultrastructural studies ofmuscle from myotonic goats are lacking. The resultsof our present light- and electron-microscopic studiescorrelate with many of the findings described in pa-tients and further establish the analogy of this animalmodel to human myotonia.The routine histochemical stains commonly used

for muscle revealed no abnormalities in the myotonicgoat. There were, however, striking differences notedwhen specialized staining procedures were used. PASstaining with diastase digestion showed significantdifferences between normal and myotonic goat mus-cle, with increased PAS-positive material in a reticu-lated form within myotonic fibers. The relevance ofthese findings to human myotonia is suggested by aprevious report in which cultured fibroblasts from pa-tients with myotonic dystrophy had histochemical evi-dence of a larger amount of acid mucopolysaccharidesas compared with those from control subjects.35

It seems plausible that the PAS-positive, diastase-resistant substance observed in myotonic goat muscleby light microscopy represents the same materialfound on the t-tubules and sarcolemma by electronmicroscopy. To investigate this hypothesis we used ru-thenium red, a stain with specificity for acid muco-polysaccharides. Increased staining was localized tosimilar areas in myotonic fibers in which increasedelectron density had been observed in the osmium anduranyl acetate stained sections. Although the specific-ity of ruthenium red had been questioned,36 there islittle doubt that ruthenium red has an affinity forsialic acid. 3 7 Our previous studies in which frag-mented SR (FSR) from myotonic goats was found tohave an increased sialic acid content correlate well

with the presence of an increased ruthenium red stain-ing reaction on myotonic cytomembranes. Further-more, the increased PAS-positive, diastase-resistantmaterial and the increased staining by alizarin red S inthe myotonic biopsy specimens suggest a correlationbetween sialic acid and calcium, as has been shown bybiochemical studies.38,39 This hypothesis will be fur-ther studied by experiments in progress using an-timonate for calcium localization at the ultrastruc-tural level.The lanthanum technique revealed differences be-

tween normal and myotonic goat muscle similar tothose found with ruthenium red. The similarity in ap-pearance between lanthanum and ruthenium redstaining has been noted previously2' and may be ac-counted for by the fact that lanthanum is a goodmarker for membrane calcium binding sites.40 Al-though intracellular penetration by lanthanum hasbeen considered to be fortuitous,22 recent studies haveshown that the interaction of lanthanum at intracellu-lar sites prior to fixation results from a specific uptakewith subsequent binding.41

It has been documented that intracellular stainingby lanthanum and ruthenium red is unpredictable.21'22Frank et al found variability in staining of the externallamina by lanthanum in the heart,42 and it has beensuggested that there may be loss of lanthanum duringthe embedding procedure.43 We observed no variationin the affinity of lanthanum to the surface mem-branes, however, and consistently found increasedstaining on the myotonic sarcolemma. Questions havealso been raised regarding the variability of rutheniumred penetration into tissue.2' In an attempt to mini-mize these sources of variation in our studies, compar-ably stained thick sections were chosen for electron-microscopic examination. We also--inspected a largenumber of fibers as well as all areas within individualfibers. Both staining methods revealed consistent dif-ferences between normal and myotonic muscle. Onthe basis of our present knowledge of the mechanismof interaction of these stains with biologic mem-branes, these results indicate that myotonic cytomem-branes have increased sites for calcium binding as wellas increased polyanionic sites, most likely acid muco-polysaccharides.The most striking ultrastructural alterations that we

observed in myotonic goat muscle were abnormal mi-tochondria and dilatation and proliferation of the sar-cotubular elements. These findings were comparableto similar observations in myotonic dystrophy anddrug-induced myotonia described above. The mito-chondrial changes were also similar to nonspecific al-terations seen in numerous myopathies, including Du-chenne's dystrophy, ocular myopathies, and some of

AJP * March 1981

332 ATKINSON ET AL

13 14

* .~~~~~ ~ ~~~~~~ t ..-.

15 __}116

Figure 13 Normal goat. This section is stained by the ruthenium red technique and demonstrates focal accumulation of ruthenium red in ter-minal cisternae of the SR in the normal biopsy (arrowhead). There is no staining of the t-tubules by ruthenium red (arrows), although the sar-colemma does have an affinity for the stain. (Ruthenium red, uranyl acetate, lead citrate, x 23,000) Figure 14 Myotonic goat. Enhancedstaining of the t-tubules (arrows) can be seen in this section from a myotonic goat, in contrast to the normal control in Figure 13. The sarcolem-ma is also stained densely, as are micropinocytotic vesicles. Mitochondria appear unstained by ruthenium red. (Ruthenium red, lead citrate,x 23,000) Figure 15-Normal goat. This fiber was stained by the lanthanum technique and shows enhancement of the sarcolemma andexternal lamina by lanthanum. The terminal cisternae of the SR are also stained (arrow). (Lanthanum, lead citrate, x 45,000) Figure 16-Myotonic goat. In this section there is increased staining of the sarcolemma and external lamina as compared wtih the normal goat biopsyshown in Figure 15. The increased staining reaction by lanthanum is most noticeable in focal areas (arrow). (Lanthanum, lead citrate, x 45,000)

AJP * March 1981

MYOTONIA CONGENITA 333

17 18

Figure 17-Myotonic dystropy (human). This biopsy from a patient with myotonic dystrophy shows several abnormalities, including mitochon-dria with abnormal configurations and increased matrix density, and vacuolization which appears to represent dilated and proliferated sarco-tubular structures (arrows). (Uranyl acetate, lead citrate, x 19,000) Figure 18-Myotonic dystrophy (human). The striking features in thissection include abnormal mitochondria with internal disorganization and increased matrix density, dilatation of some terminal cisternae ofthe SR (arrowhead), and proliferation of other sarcotubular elements, lipid droplets, and increased density of the t-tubular membranes (arrow).(Uranyl acetate, lead citrate, x 19,000)

the so-called mitochondrial myopathies. "Moth-eaten" fibers have been described in myotonic dystro-phy,44 and in muscle made ischemic these "moth-eaten" areas were shown to be comprised of areas withdisruption and ultrastructural alterations of mito-chondria.45 Myotonic discharges in association withabnormal mitochondria and myelin figures have alsobeen found in chloroquine-induced myopathy.46 Al-though myelinlike figures have been described as arti-facts of different fixations,47-49 they are also thoughtto represent focal membrane degeneration by variousstimuli (including 20,25-diazacholesterol) or degen-erative changes secondary to defects in oxidativephosphorylation.50,5' Studies are thus currently un-derway to compare oxidative phosphorylation and thequantity and availability of energy in mitochondriafrom normal and myotonic goats to characterize thesignificance of our morphologic observations.

It is not clear whether the mitochondrial changes weobserved in myotonic goat muscle represent a reactionto a primary defect or reflect a secondary reaction tosome other alteration, such as increased calcium. Mi-tochondria which sequester excess calcium at the ex-pense of ATP production subsequently undergo pro-found morphologic changes.52 We found an increasedcalcium content in mitochondria isolated from myo-

tonic goats, as compared with normal goats. Carafoliet al likewise measured increased levels of calcium instructurally abnormal mitochondria from patientswith myotonic dystrophy.5 These related findings indystrophic and nondystrophic forms of myotoniamay provide insight into the mechanism of fiber ne-crosis and degeneration in dystrophic myopathies.

Because of the fact that myotonia can be a featureof dystrophic muscle and occurs in an animal modelwith genetic expression, studies were possible thatwould compare morphologic and biochemical fea-tures of human myotonic dystrophy to the animalmodel of myotonia congenita. In myotonia congenita,whether human or in the goat model, no convincingevidence has been presented indicative of a dystrophicprocess. The present studies have explored this in theanimal model of myotonia congenita. Our findingshave demonstrated morphologic alterations compar-able to several of those seen in human myotonic dys-trophy.The most distinguishing clinical feature of myoto-

nia is the delayed relaxation following contraction ofskeletal muscle, and it is generally thought to be due torepetitive firing of action potentials that follow depo-larization of the sarcolemma. Bryant has presentedevidence that the instability of the muscle surface

Vol. 102 * No. 3

riwil

334 ATKINSON ET AL AJP * March 1981

membrane is the result of decreased chloride conduc-tance, resulting in a high resting membrane resis-tance.53 Few investigators have studied the composi-tional characteristics of the muscle cytomembranes inmyotonia in relation to a generalized membrane de-fect. Our previous studies of FSR from normal andmyotonic goats have shown increased membrane cal-cium, an increased rate of calcium uptake and efflux,and an increased content of sialic acid in the myo-tonic.'4 Our studies of erythrocyte membranes haveindicated comparable alterations in calcium and sialicacid content in the animal model as well as increasedsialic acid in erythrocyte membranes from patientswith myotonic dystrophy.54 Although membranelipid studies of the goat FSR indicated differences incomposition consistent with a less fluid membrane inthe myotonic, no differences were detected in lipidcomposition of the erythrocyte membranes, and elec-tron-paramagnetic resonance studies of goat erythro-cyte membranes using fatty acid probes revealed nodifferences in "fluidity" between normal andmyotonic. 5 5Based on biochemical and morphologic data in the

goat model, we propose that a generalized membranedefect in myotonia is responsible for an altered intra-cellular calcium homeostasis, which produces thecharacteristic prolonged contraction of the musclefollowing depolarization. Such a defect would involveincreased permeability of the sarcolemma and sarco-tubular membranes to calcium, mitochondrial abnor-malities secondary to excess calcium, and prolongedresponse of the myotonic muscle fiber to a contractionstimulus. Although mathematical models have beenconstructed to explain repetitive electrical activitybased on decreased chloride conductance,56-58 DaRonch et a159 have proposed a comparable model formyotonia based on increased calcium permeability. Itis also possible that repetitive depolarization of thesarcolemma due to decreased chloride permeability isinfluenced by calcium.60,6'The present study has described abnormalities in

skeletal muscle from myotonic goats, including histo-chemical evidence of increased mucopolysaccharideand calcium, and ultrastructural evidence of cyto-membrane alterations and mitochondrial abnormali-ties. Several of the morphologic features in musclefrom myotonic goats correlate with previously re-ported findings in patients with myotonia congenitaand myotonic dystrophy. These observations are con-sistent with our previous biochemical data of a struc-tural and functional defect of cytomembranes in myo-tonia and further support the postulated role that ab-normal calcium homeostasis plays in the expression ofmyotonia.

References1. Wohlfart G: Dystrophia myotonica and myotonia con-

genita: Histopathologic studies with special reference tochanges in the muscles. J Neuropathol Exp Neurol 1951,10:109-124

2. Aleu FP, Afifi AK: Ultrastructure of muscle in myo-tonic dystrophy: Preliminary observations. Am J Pathol1964, 45:221-231

3. Klinkerfuss GH: An electron microscopic study of myo-tonic dystrophy. Arch Neurol 1967, 16:181-193

4. Samaha FJ, Schroeder JM, Rebeiz J, Adams RD: Stud-ies on myotonia: Biochemical and electron microscopicstudies on myotonia congenita and myotonia dystro-phica. Arch Neurol 1967, 17:22-33

5. Carafoli E, Montevecchi MT, Patriarca P, Ballotti A:Alterazioni ultrastrutturali e biochimiche nei mitocon-dri di muscoli miotonici umani. Sperimentale 1970, 120:285-299

6. Schotland DL: An electron microscopic investigation ofmyotonic dystrophy. J Neuropathol Exp Neurol 1970,29:241-253

7. Schroder JM, Becker PE: Anomalien des T-systems unddes Sarkoplasmatischen Reticulums bei der Myotonie,Paramyotonie und Adynamie. Virchows Arch [PatholAnat] 1972, 357:319-344

8. Korenyi-Both A, Lapis K, Margit Gallai, Szobor A: Finestructural alterations of muscle fibers in diseases accom-panied by myotonia. Beitr Pathol 1975, 156:241-256

9. Crews J, Kaiser KK, Brooke MH: Muscle pathology ofmyotonia congenita. J Neurol Sci 1976, 28:449-457

10. Ketelsen UP: Ultrastructure of dystrophic skeletal mus-cle. Israel J Med Sci 1977, 13:107-120

11. Casanova G, Jerusalem F: Myopathology of myotonicdystrophy: A morphometric study. Acta Neuropathol(Berl) 1979, 45:231-240

12. Kolb LC: Congenital myotonia in goats. Bull JohnsHopkins Hosp 1938, 63:221-237

13. Adams MJ, Thompson JW: Myotonia congenita ingoats: an electron microscopic, biochemical, and bio-physical study. South Med J 1965, 58:1573

14. Swift LL, Atkinson JB, LeQuire VS: The compositionand calcium transport activity of the sarcoplasmic retic-ulum from goats with and without heritable myotonia.Lab Invest 1979, 40:384-390

15. Vick NA, Olson WH: A biopsy clamp for removal andfixation of muscle in electron microscopy. Stain Tech1970, 45:42

16. Drury RAB, Wallington EA: Carleton's HistologicalTechnique. New York, Oxford University Press, 1967

17. Puchtler H, Meloan SN, Terry MS: On the history andmechanism of alizarin and alizarin red S stains for cal-cium. J Histochem Cytochem 1969, 17:110-124

18. Dubowitz V, Brooke MH: Muscle Biopsy: A ModernApproach. London, W. B. Saunders 1973, pp 20-33

19. Brooke MH, Kaiser KK: Muscle fiber types: how manyand what kind? Arch Neurol 1970, 23:369-379

20. Glauert AM: The fixation and embedding of biologicalspecimens. Techniques for Electron Microscopy. Editedby DH Kay. Philadelphia, F. A. Davis 1965, pp 166-212

21. Luft JH: Ruthenium red and violet: II. Fine structurallocalization in animal tissues. Anat Rec 1971, 171:369-416

22. Waugh RA, Spray TL, Sommer JR: Fenestrations ofsarcoplasmic reticulum. Delineation by lanthanum act-ing as a fortuitous tracer and in situ negative stain. J CellBiol 1973, 59:254-260

23. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ: Pro-tein measurement with the Folin phenol reagent. J BiolChem 1951, 193:265-275

24. Fleischer S, Fleischer B: Removal and binding of polarlipids in mitochondria and other membrane systems,

Vol. 102 * No. 3 MYOTONIA CONGENITA 335

Methods in Enzymology. Vol 10. Edited by RW Esta-brook, ME Pullman. New York, Academic Press, 1967,pp 406-433

25. Tabor CW, Tabor H, Rosenthal SM: Purification ofamine oxidase from beef plasma. J Biol Chem 1954,208:645-661

26. Goldstein MA: Anionic binding of ruthenium red in fishextraocular muscle. Z Zellforsch 1969, 102:459-465

27. Adams RD: Diseases of Muscle: A Study in Pathology.Hagerstown, Maryland, Harper and Row, 1975

28. Engel WK: Mitochondrial aggregates in muscle disease.J Histochem Cytochem 1964, 12:46-48

29. Pachter BR, Eberstein A, Breinin GM: Electromyo-graphic and electron microscopic findings in the extra-ocular muscles of the myotonic rat. Exp Neurol 1977,57:971-983

30. Dux E, T6th I, Dux L, Joo F, Kiszely G: The possiblecellular mechanism of 2,4-dichlorophenoxyacetate-in-duced myopathy. FEBS Lett 1977, 82:219-22

31. Danon JM, Karpati G, Carpenter S: Subacute skeletalmyopathy induced by 2,4-dichlorophenoxyacetate inrats and guinea pigs. Muscle Nerve 1978, 1:89-102

32. Ontell M, Paul HS, Adibi SA, Martin JL: Involvementof transverse tubules in induced myotonia. J Neuro-pathol Exp Neurol 1979, 38:596-605

33. Bryant SH: Myotonia in the goat. Ann NY Acad Sci1979, 317:314-324

34. Becker PE: Syndromes associated with myotonia: clini-cal-genetic classification. Pathogenesis of Human Mus-cular Dystrophies. Edited by LP Rowland. Amsterdam,Excerpta Medica, 1977, pp 699-703

35. Swift MR, Finegold MJ: Myotonic muscular dystrophy:Abnormalities in fibroblast culture. Science 1969, 165:294-296

36. Luft JH: Ruthenium red and violet: I. Chemistry, puri-fication, methods of use for electron microscopy andmechanism of action. Anat Rec 1971, 171:347-368

37. Dorrscheidt-Kafer M, Grocki K: The effect of ruthe-nium red and its interaction with membrane-bound si-alic acid on contraction threshold in frog skeletal mus-cle. J Physiol 1978, 284:52P

38. Behr J-P, Lehn J-M: Stability constants for the com-plexation of alkali and alkaline-earth cations by N-ace-tyl-neuraminic acid. FEBS Lett 1972, 22:178-180

39. Jaques LW, Brown EB, Barrett JM, Brey WS, WeltnerW: Sialic acid: A calcium-binding carbohydrate. J BiolChem 1977, 252:4533-4538

40. Hagiwara S, Henkart MP, Kidokoro Y: Excitation-con-traction coupling in amphioxus muscle cells. J Physiol1971, 219:233-251

41. Weihe E, Hartschuh W, Metz J, Bruhl U: The use ofionic lanthanum as a diffusion tracer and as a marker ofcalcium binding sites. Cell Tissue Res 1977, 178: 285-302

42. Frank JS, Langer GA, Nudd LM, Seraydarian K: Themyocardial cell surface, its histochemistry, and the ef-fect of sialic acid and calcium removal on its structureand cellular ionic exchange. Circ Res 1977, 41:702-714

43. Sommer JR, Waugh RA: The ultrastructure of themammalian cardiac muscle cell -with special emphasison the tubular membrane systems. Am J Pathol 1976,82:192-232

44. Dubowitz V: Histochemical aspects of muscle disease,Disorders of Voluntary Muscle. Edited by JN Walton.Edinburgh, Churchill Livingstone, 1974, pp 310-359

45. Heffner RR, Barron SA: The early effects of ischemiaupon skeletal muscle mitochondria. J Neurol Sci 1978,38:295-315

46. Mastaglia FL, Papadimitriou JM, Dawkins RL, Bever-idge B: Vacuolar myopathy associated with chloro-quine, lupus erythematosus and thymoma: Report of acase with unusual mitochondrial changes and lipid ac-cumulation in muscle. J Neurol Sci 1977, 34:315-328

47. Cecio A: Electron microscopic observations of youngrat liver: I. Distribution and structure of the myelinfigures (lamellar bodies). Z Zellforsch 1964, 62:717-742

48. Trump BF, Ericsson JLE: The effect of the fixative solu-tion on the ultrastructure of cells and tissues: A com-parative analysis with particular attention to the prox-imal convoluted tubule of the rat kidney. Lab Invest1965, 14:1245-1323

49. Olah I, Rohlich P: Peculiar membrane configurationsafter fixation in glutaraldehyde. Acta Biol Hung 1966,17:65-73

50. Beux YL, Hetenyi G, Phillips MJ: Mitochondrial mye-lin-like figures: A non-specific reactive process of mito-chondrial phospholipid membranes to several stimuli. ZZellforsch 1969, 99:491-506

51. Hruban Z, Slesers A, Hopkins E: Drug-induced andnaturally occurring myeloid bodies. Lab Invest 1972,27:62-70

52. Hunter DR, Haworth RA, Southard JH: Relationshipbetween configuration, function, and permeability incalcium-treated mitochondria. J Biol Chem 1976, 251:5069-5077

53. Bryant SH: The electrophysiology of myotonia, with areview of congenital myotonia of goats, New Develop-ments in Electromyography and Clinical Neurophysiol-ogy. Vol 1. Edited by JE Desmedt. Basel, S Karger,1973, pp 420-450

54. Atkinson JB, Swift LL, Lankford PG, LeQuire VS: Ageneralized membrane defect in heritable myotonia:Studies of erythrocytes in an animal model and patients.Proc Soc Exp Biol Med 1980, 163:69-75

55. Swift LL, Atkinson JB, Perkins RC, Dalton LR, Le-Quire VS: Electron paramagnetic resonance and satura-tion transfer electron paramagnetic resonance studies onerythrocytes from goats with and without heritable myo-tonia. J Membr Biol 1980, 52:165-172

56. Bretag AH: Mathematical modelling of the myotonicaction potential, New Developments in Electromyogra-phy and Clinical Neurophysiology. Vol 1. Edited by JEDesmedt. Basel, S Karger, 1973, pp 464-482

57. Barchi RL: Myotonia: An evaluation of the chloride hy-pothesis. Arch Neurol 1975, 32:175-180

58. Adrian RH, Marshall MW: Action potentials recon-structed in normal and myotonic muscle fibres. J Phys-iol 1976, 258:125-143

59. Da Ronch A, Radu H, Caldesi-Valeri V: Computer sim-ulation of calcium-induced myotonia. J Math Biol 1978,6:225-234

60. Saiter V, Zacharova D, Zachar I: Fluxes of Ca and Srions in single crustacean muscle fibres. Physiol Bohe-moslov 1968, 17:12-13

61. Furman RE, Barchi RL: The pathophysiology of myo-tonia produced by aromatic carboxylic acids. Ann Neu-rol 1978, 4:357-365

AcknowledgmentsThe authors acknowledge the excellent technical assis-

tance of Maurice H. Haugh and Fred Morris and wish to ex-press appreciation to Dr. Mary E. Gray for her valuablehelp.