Myotonia Congenita

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  • Myotonia Congenita

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


    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 MethodsAnimal 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



    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


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


    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.


    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, resp