Myopathy with abnormal structure and function ofmuscle ... · A third case in which morphologically altered musclemitochondria andaloosely coupled state of oxidative phosphorylation

J. Neurol. Neurosurg. Psychiat., 1967, 30, 519

Myopathy with abnormal structure and functionof muscle mitochondria

W. C. HULSMANN,1 J. BETHLEM, A. E. F. H. MEIJER, P. FLEURY,AND J. P. M. SCHELLENS

From the Medical School, Rotterdam, the Departments ofNeuropathology of the Neurological Clinic, Patho-logical Anatomy, the Neurological Clinic, and the Histological Laboratory, University ofAmsterdam

When the rate of respiration of mitochrondria inthe presence of inorganic phosphate cannot be in-creased by adding adenosine diphosphate (A.D.P.)but these particles can still couple respiration to phos-phorylation of A.D.P. to adenosine triphosphate(A.T.P.), the state of respiration is called 'looselycoupled'.The first patient in whom such a loosely coupled

state of oxidative phosphorylation of the musclemitochrondria was found was described by Ernster,Ikkos, and Luft (1959). The patient suffered fromgeneralized myopathy with progressive asthenia andsevere hypermetabolism. The structure of the musclemitochondria was markedly altered as shown byelectron microscopy (Luft, Ikkos, Palmieri, Ernster,and Afzelius, 1962).The second patient with the same biochemical

disturbance of oxidative phosphorylation of themuscle mitochondria was described by Van Wijn-gaarden, Bethlem, Meijer, Hiilsmann, and Feltkamp(1967). This patient showed a proximal myopathywithout hypermetabolism. Histological examinationof a muscle biopsy revealed subsarcolemmal zonesthat contained small granules. In these zones, anincreased activity of oxidative enzymes was demon-strated. Electronmicroscopic examination showedthe presence of pathological mitochondria. Largespherical, rod-shaped or annular mitochondria wereobserved. The morphology of the pathologicalmitochondria differed in many respects from thatobserved by Luft et al. (1962), who, in addition,found peculiar inclusions in the mitochondria.Giant mitochondria with rectangular and otherinclusions were found in the muscle of a child withproximal weakness by Shy, Gonatas, and Perez(1966), in a disorder which they termed megaconialmyopathy. In this case, data on biochemical studiesof oxidative phosphorylation of the morphologicallyaltered muscle mitochondria were not given.'Temporary address: Dijkzigt Hospital, Rotterdam, the Netherlands.

A third case in which morphologically alteredmuscle mitochondria and a loosely coupled state ofoxidative phosphorylation were present will nowbe described.

CLINICAL INVESTIGATIONS

The patient's parents were both 27 years old and in goodhealth. The mother was white; the father was a Negroidfrom South Africa. An elder sister was in good health. Nomyopathies existed in the parental families and there wasno parental consanguinity. Pregnancy and parturition hadtaken a normal course.The patient started to walk when 1 year old and no

abnormal signs or symptoms were noted until the age of2, when her gait became uncertain. A few months latershe had to be carried a good deal, and she could nolonger rise from a sitting position without assistance.She was 27 months old when admitted to hospital.At examination we saw a Negroid girl who gave no

impression of illness. She displayed a staggering gait andcould not rise unassisted from a sitting or recumbentposition. The muscles of the shoulder girdle, pelvicgirdle, upper arms, and thighs were paretic, atrophic, andhypotonic. There was no hypertrophy of the calf musclesor any other muscles. Examination of the internalorgans disclosed no abnormality, and radiographs of theskull, spine, chest, and pelvis were normal. Both theE.E.G. and the E.C.G. were without abnormalities. TheE.M.G. (Dr W. J. M. Hootsmans) was derived bilaterallyfrom the gluteus medius and quadriceps femoris muscleswith the aid of concentric needle electrodes. The patient'sresistance limited the scope of this examination, whichdisclosed no unequivocal abnormalities.

LABORATORY FINDINGS The Hb concentration was 12-3g./100 ml. White cell count and E.S.R. were normal.Haematocrit value was 39 %. Blood urea was 230 mg./l.,sodium 134 mEq/l., potassium 4-3 mEq./l., calcium10-2 mEq./l., cholesterol 348 mg./100 ml.; serum lacticacid concentration 32 mg./100 ml.Serum creatine phosphokinase and aldolase activities

amounted to 5 0 mU/ml. and 4. 8mU/ml., respectively.S.G.O.T. was 33U and S.G.P.T. lOU.

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520 W. C. Hulsmann, J. Bethlem, A. E. F. H. Meijer, P. Fleury, and J. P. M. Schellens

Urinary creatinine excretion was 190-230 ml./l. andcreatine excretion 300-500 mg./l.

It was impossible to collect 24-hour urines. The a-amino-nitrogen excretion was 22 mg./100 ml. Thecerebrospinal fluid was normal; serological reactions inserum and cerebrospinal fluid were negative. The B.M.R.,as estimated on the basis of oxygen consumption (Dr J.Muller), was- 19 %.

MORPHOLOGICAL STUDIES

MATERIAL AND METHODS For light microscopy speci-mens from the right and the left vastus lateralis werefixed in Heidenhain's fixation fluid and then embedded inparaffin for cutting longitudinal and transverse sectionswhich were stained with haematoxylin-eosin and Gomori'strichrome stain.For histochemical investigation other parts of the

specimens were rapidly frozen by immersing smallblocks of tissue in isopentane cooled to - 150° C. withliquid nitrogen. Longitudinal and transverse serial sec-tions were cut in a cryostat. Fixed or unfixed sectionswere stained for the activities of the following oxidativeenzymes: N.A.D.H.-tetrazolium reductase, N.A.D.P.H.-tetrazolium reductase, lactate dehydrogenase, malatedehydrogenase (N.A.D.+), malate dehydrogenase(N.A.D.P.+) decarboxylating, isocitrate dehydrogenase(N.A.D.+), isocitrate dehydrogenase (N.A.D.P.+), gly-ceraldehyde-3-phosphate dehydrogenase, glycerol-3-phos-phate dehydrogenase (N.A.D.+), glycerol-3-phosphateoxidase (menadione), glucose-6-phosphate dehydro-genase, glutamate dehydrogenase, 3-hydroxybutyratedehydrogenase, succinate dehydrogenase, and mono-amine oxidase. Other enzyme activities tested were:alpha-glucan phosphorylase a and b, adenosine tri-phosphatase (Mg-activated), myosin-adenosine triphos-phatase, 5'-nucleotidase, fructose-1,6-diphosphate ald-olase, peroxidase, cytochrome c oxidase and acid phos-phatase. Glycogen was estimated by the periodic-acid-Schiff reaction before and after digestion by diastase.The histochemical methods used are described in aprevious paper. (Van Wijngaarden et al., 1967).For electron microscopy, biopsy specimens in the

order of 1 c.mm were fixed for three hours at 0-4°C. in6% glutardialdehyde dissolved in 0-1 M cacodylatebuffer pH 7-2 with 0-22 M sucrose added. The fragmentswere then kept for a few hours in the same fluid withoutglutardialdehyde and were postfixed in buffered aqueousOsO4accordingto Millonig(1961): 1 %OS04,0 l MNaH2P04,0-1MNaOH,0-03Mglucose. After dehydration in analcohol series, the tissue was embedded in Epon. Sectionswere cut on an LKB ultramicrotome and, after 'staining'with lead tartrate, studied in a Siemens Elmiskop lawith a 400.t condensor aperture and a 50,u objectiveaperture.

RESULTS Histopathological studies disclosed twotypes of muscle fibre: those with a normal diameterand structure, and those with a very small diameterand frequently of more polygonal shape in transversesection (Fig. 1). These fibres were found among thenormal fibres in no recognizable pattern and

FIG. 1. Transverse section showing fibres with normaldiameter and fibres with a very small diameter. Gomori'strichrome x 250.

amounted to about 40% of the total number offibres. The structure of the fibres of small diametershowed pronounced changes, sometimes throughouttheir entire length (Fig. 2) and in others only throughpart of their length. Transverse sections showedthat the entire cut surface of the fibres was affected;occasional fibres showed a preponderance ofsubsarcolemmal changes. The affected fibres con-tained very finely granulated material, in whichsometimes only a few myofibrils were observed.These areas showed marked proliferation of vesi-cular nuclei with prominent nucleoli. The affectedmuscle fibres also showed a conspicuous number ofcapillaries. There were no signs of phagocytosis.Endomysial fat tissue and connective tissue wereslightly increased. Blood vessels and intramuscularnerves showed no abnormalities. Histochemicalexamination revealed that the histologically normalmuscle fibres showed medium enzyme activity withall techniques used. The fibres of small diametershowed a very high activity of the oxidative enzymes(Fig. 3). The aldolase activity was likewise high. Theactivity of ot-glucan phosphorylase a and b was lowin some and high in other fibres. The periodic-acid-



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Myopathy with abnormal structure andfunction of muscle mitochondria 521

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8w,L4\FIG. 2. Longitudinal section showing two small fibres inwhich very finely granulated material and vesicular nucleiare seen. Gomori's trichrome x 250.

FIG. 3. Transverse section. Medium activity of thefibres with normal diameter and a very high enzyme

wj (;F̂,s+ {} 0 activity ofthe smallfibres. N.A.D.H -tetrazolium reductase

FIG. 4. Adjacent to a normal fibre (right) a fibre con-taining numerous mitochondria is seen. No myofibrils arepresent x 12,000-

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Schiff reaction was negative in some and positivein other pathological fibres. There was mediumactivity of Mg-activated adenosine triphosphatase,but no or only very slight activity of calcium-activated adenosine triphosphatase catalysed by themyofibrils.

The unusual light microscopic aspect of somemuscle fibres was confirmed at electron microscopy.These fibres contained very few, if any, myofilaments;instead they contained numerous mitochondria,which in many respects differed from normal ones

(Fig. 4), and were situated in the sarcoplasm withoutany apparent orientation. In some areas they didnot seem to differ very greatly from the mitochondriain the more normal fibres, although their internalstructure was always tubulo-vesicular, whereas inthe apparently normal fibres many mitochondriawith classical cristae were present. Besides thesemore or less normal ones, many obviously abnormalmitochondria were seen which were much larger thannormal (giant mitochondria) and of quite irregularshape (Figs. Sa, 5b, and 6d). The most striking

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aspect of these abnormal mitochondria was theirinternal organization. In most of them there were,apart from a tubulo-vesicular membrane system,one or more of the following unusual components:vacuoles (Figs. Sb and 6d), concentric lamellae,resembling cristae (Figs. 5a, Sb, 6c, and 6d), mem-brane configurations of high electron density(Figs. 5a and Sb). In a few mitochondria there wereparticles which, as judged by their dimensions (250-300 A) and their electron density after 'lead-staining',may be considered to be P-glycogen particles

(Figs. 6a and 6c). The particles were enclosed bywhat seemed to be a normal mitochondrial doublemembrane; the most plausible explanation of theirpresence within the mitochondria seems to be thatthey were 'caught' when mitochondria developedirregular shapes. In between the mitochondriabulky accumulations of very electron dense, coarselamellae were sometimes seen (Fig. 6c). In onefibre a curious honeycomb-like structure wasobserved (Fig. 6b), the nature of which was notclear.



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BIOCHEMICAL STUDIES

MATERIAL AND METHODs A specimen (vastus lateralis muscle was finely mincgenized in ice-cold isolation medium. TIwere isolated by differential centrifugaticgenate, using in principle the method of LThe rates of oxidation of pyruvate and gisolated mitochondria were measured btechnique, employing differential manonwith small flasks with centre wells andmain compartments of the flasks containtcomponents: 25 mM glucose, 50 mM KCHC1 buffer of pH 716, 25 mM potasbuffer ofpH 7-4, 05 mM E.D.T.A., 0-25 rc, 5 mM MgC12, 05 mM A.T.P. andstrates. The centre wells contained KOwick to bind CO2, and the side arms contahexokinase. The reactions were startedof 05 mg. of mitochondrial protein topartments. The flasks were then attachemanometers, shaken at 250 C., and relregular intervals. After 25 minutes theadded from the side arms, so that A.D.Fgenerated continuously from A.T.P. Thilater the reactions were stopped by tperchloric acid. Glucose-6-phosphate wathe deproteinized, neutralized extractsphosphate dehydrogenase and N.A.D.P.

Phosphorylation accompanying oxidatand glutamate was also measured. The Iand the results obtained in six control pcribed in detail in the paper by Van Wi(1967).

Finally the isoenzyme pattern of lactiigenase was determined in a muscle spelectrophoresis.

RESULTS The rates of oxygen uptake iand absence of phosphate acceptor aphosphorylation with pyruvate oroxidizable substrates are shown in Tal

TABLE IABSENCE OF STIMULATION OF RESPIRATION B

OF PHOSPHATE ACCEPTOR

Phosphat

Absent

AO

20 mM Pyruvate (+0 3 mM L-malate) 4 020 mM Glutamate (+0-3 mM L-malate) 4-3

The AO (in ,u At/mg. protein/h) was correctenegligible amount of) oxygen taken up in the absevate or glutamate but in the presence of malate.The AP (in ,u mol glucose-6-phosphate/mg. proteifor the amount of G-6-P formed in the presence ofsubstrate.

2 g.) of the leftced and homo-ie mitochondria)n of the homo-,uft et al. (1962).rlutamate by they the WarburgnPt-prc nrAvirX-A

seen that the addition of phosphate acceptor doesnot alter the rate of oxidation although phosphory-lation occurs. This phenomenon indicates thatoxidative phosphorylation catalysed by the mito-chondria is loosely coupled. The L.D.H. isoenzymepattern showed a significant diminution ofL.D.H.-5.

COMMENT

1L5s,ideoarms.l At the age of 2, the patient developed a rapidlysdthe following progressive proximal myopathy involving theed, 25 mM Tris- humeroscapular and pelvic muscles. Histological;sium-phosphate examination disclosed numerous muscle fibres ofmM cytochrome small diameter, which contained fine granules.oxidizable sub- This granular material showed a very high activityH and a paper of oxidative enzymes.Lined 500 mU of Histochemical examination disclosed no myosinby the addition A.T.P.ase in these small fibres. Electron microscopydthodifferentimal revealed the presence of large and abnormal mito-adings taken at chondria in these fibres. Mitochondria isolated fromhexokinase was a homogenate of a biopsy from a vastus lateralisP. could now be muscle showed a loosely coupled state of oxidativerty-five minutes phosphorylation. We considered the possibilitythe addition of that this biochemical finding might have resultedLs determined in from errors made in the isolation of the mitochon-with glucose-6- dria and/or the determination of oxidative phos-

phorylation. However, eight control biopsies (six ofion of pyruvate which have been described by Van Wijngaardentechniques used et al.) were examined in exactly the same manner asajingaarden et a.the present case and showed, with glutamate and

pyruvate as respiratory substrates, an increase ofc acid dehydro- oxygen consunmption upon the ,addition of phos-ecimen by agar phate acceptor which varied from 2-3 to fivefold.

The patient of Luft et al. showed severe hyper-metabolism (B.M.R. ranging from + 140% to +210%) without hyperthyroidism, and is to be

in the presence expected when large numbers of mitochondriaLnd the rate of show a loosely coupled state of oxidative phosphory-glutamate as lation. The case of Van Wijngaarden et al. and theble I. It can be present case showed the same biochemical disorder,

although no hypermetabolism was found. This maybe explained by a relatively small overall number ofmuscle mitochondria being in a loosely coupled

BY THE ADDrIION state. It may well be that the changes in structureand function of the muscle mitochondria in the

te Acceptor present case are the result of a biochemical disorderoutside the mitochondria. Swelling of mitochondria

Present was seen in electron microscopic studies of muscleAO AP biopsies from 34 patients with muscular dystrophy4-0 7-6 of various clinical types, and from five other patients4 5 8-6 with neurogenic atrophy by Fisher, Cohn, and

Danowski (1966). These authors also suggest thatdf°orthe(dalpmost it is most unlikely that these ultrastructural altera--nce of added pyru-

tions represent a significant causative factorin/h) was corrected in the pathogenesis of the dystrophic disorders.malate only as the

A tubulo-vesicular membrane system and an

Respiratory Substrate(s)



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Myopathy with abnormal structure andfunction of muscle mitochondria

arrangement of the cristae in the form of concentriclamellae are described for normal mitochondria,e.g. by Zelander (1959) in mouse adrenal cortex. Asto the giant mitochondria observed in the presentcase, it must be pointed out that mitochondrialgigantism is observed in various conditions andseems to be an aspecific response to differentabnormal circumstances (Rouiller and Jezequel,1963; Svoboda, Grady, and Higginson, 1966; Volkand Scarpelli, 1966). With respect to the internalconcentric lamellae and dense membrane con-figurations, it is of interest that some authorsdescribe similar formations in degenerative mito-chondria (Bovis, Kasten, and Okigaki, 1966;Jezequel, 1959; Kisch, Cavusoglu, and Marangoni,1959; de Robertis and Sabatini, 1958; Volk andScarpelli, 1966).

It seems possible that the accumulations ofdense coarse lamellae lying free in the sarcoplasmare remnants of degenerated mitochondria, sincemyelin-like figures are often associated with cellularregression and the membrane configurations ob-served inside the mitochondria may be interpretedas indications of degeneration.

It should be stressed that the irregular myelin-likefigures may be fixation artifacts. According toOlah and Rohlich (1966), glutaraldehyde fixationcan cause this type of formation.The pictures of glycogen in mitochondria agree

with those obtained by Fain-Maurel (1966) from a

mollusc gland; both in the material described bythis author and in the present case, glycogenappears to be enclosed by the double mitochondrialmembrane (Fig. 6a).The present report is the second from our group

to describe a loose coupling of the isolated skeletalmuscle mitochondria. The fact that these two cases

were seen within a period of six months may mean

that this kind of phenomenon is not extremely rare.It seems probable that more of these cases will befound when morphological studies are correlatedwith biochemical investigations, even in the absenceof hypermetabolism.

SUMMARY

The case is described of a 27-month-old girl suf-

fering from a myopathy involving the proximalmuscles. Histopathological studies revealed thepresence of muscle fibres of very small diameter,which contained finely granulated material. A veryhigh activity of the oxidative enzymes was seen inthese small fibres. Electron microscopy showednumerous mitochondria, many of which were ofabnormal size and shape. Biochemical studiesshowed that the isolated mitochondria were looselycoupled.

REFERENCES

Bovis, R., Kasten, F. H., and Okigaki, T. (1966). Electron micro-scopic study of the toxic effect of sodium fluoroacetate on ratmyocardial cultures. Exp. Cell Res., 43, 611-621.

Ernster, L., Ikkos, D., and Luft, R. (1959). Enzymic activities ofhuman skeletal muscle mitochondria: a tool in clinical metabolicresearch. Nature (Lond.), 184, 1851-1854.

Fain-Maurel, M. A. (1966). Localisations intramitochondriale etintracisternale de glycogsne monoparticulaire. C. R. Acad.Sci. Paris serie D., 263, 1107-1110.

Fisher, E. R., Cohn, R. E., and Danowski, T. S. (1966). Ultrastructuralobservations of skeletal muscle in myopathy and neuropathywith special reference to muscular dystrophy. Lab. Invest.,15, 778-793.

J&z6quel, A. M. (1959). D16gn6rescence my6linique des mitochondriesde foie humain dans un 6pith6lioma du chol6doque et unict6re viral. J. Ultrastruct. Res.,3, 210-215.

Kisch, B., Cavusoglu, M., and Marangoni, B. A. (1959). Electronmicroscopic changes in human heart in cardiac failure. Exp.Med. Surg., 17, 85-100.

Luft, R., Ikkos, D., Palmieri, G., Ernster, L., and Afzelius, B. (1962).A case of severe hypermetabolism of nonthyroid origin with adefect in the maintenance of mitochondrial respiratory control:a correlated clinical, biochemical and morpholcgical study.J. clin. Invest., 41, 1776-1804.

Millonig, G. (1961). A modified procedure for lead staining of thinsections. J. biophys. biochem. Cytol., 11, 736-739.

Olah, I., and Rohlich, P. (1966). Peculiar membrane configurationsafter fixation in glutaraldehyde. Acta biol. Acad. Sci. Hung.,17, 65-74.

De Robertis, E., and Sabatini, D. (1958). Mitochondrial changes inthe adrenocortex of normal hamsters. J. biophys. biochem.Cytol., 4,667-678.

Rouiller, Ch., and J6zEquel, A. M. (1963). Electron microscopy of theliver. In The Liver vol. I, edited by Ch. Rouiller, pp. 195-264.Academic Press, New York.

Shy, G. M., Gonatas, N. K., and Perez, M. (1966). Two childhoodmyopathies with abnormal mitochondria. I. Megaconialmyopathy. IT. Pleoconial myopathy. Brain, 89, 133-158.

Svoboda, D., Grady, H., and Higginson, J. (1966). The effects ofchronic protein deficiency in rats. It. Biochemical and ultra-structural changes. Lab. Invest., 15, 731-749.

Volk, T. L., and Scarpelli, D. G. (1966). Mit)chondrial g-gantism inthe adrenal cortex following hypophysectomy. Ibid., 15,707-715.

Van Wijngaarden, G. K., Bethlem, J., Meijer, A. E. F. H., Hulsmann,W. Ch., and Feltkamp, C. A. (1967). Skeletal muscle diseusewith abnormal mitochondria. Brain, 90, 577-592.

Zelander, T. (1959). Ultrastructure of the mouse adrenal cortex. Anelectron microscopical study in intact and hydrocortisone-treated male adults. J. Ultrastruct. Res., suppl. 2.

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Myopathy with abnormal structure and function ofmuscle ... · A third case in which morphologically altered musclemitochondria andaloosely coupled state of oxidative phosphorylation