JOURNAL OF ULTRASTRUCTURE RESEARCH

51,404-408 (1975)

Electron Cytochemistry of Crystalline Inclusions in Human Skeletal Muscle Mitochondria 1 EDUARDO BONILLA, DONALD

L. SCHOTLAND,SALVATORE DIMAURO,

AND BELEN ALDOVER

Department o/Neurology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104 Received October 22, 1974; accepted December 17, 1974 Limb muscle biopsies from a patient with idiopathic progressive ophthalmoplegia and a patient with a slowly progressive neuromuscular disorder since infancy revealed by conventional electron microscopy the presence of crystalline inclusions in the mitochondria as the most prominent morphological finding. Electron cytochemical studies on fresh tissue blocks showed no cytochrome C oxidase or carnitine acetyl transferase activity within the crystalline inclusions. The study of respiration linked accumulation of electron dense strontium supported by either NAD or flavoprotein linked substrates on freshly isolated mitochondrial fractions showed no electron dense strontium within the crystalline inclusions. These electron cytochemical findings suggest that the crystalline inclusions are space occupying structures that do not have an active functional role within the mitochondrion. INTRODUCTION C r y s t a l l i n e i n c l u s i o n s in t h e m i t o c h o n d r i a of h u m a n s k e l e t a l m u s c l e h a v e b e e n o b s e r v e d in a v a r i e t y of d i s o r d e r s (2, 3, 4, 8,

12, 14, 15). In electron micrographs these inclusions are a r r a n g e d p a r a l l e l to t h e l o n g i t u d i n a l axis of t h e m i t o c h o n d r i a . T h e y u s u a l l y a p p e a r as g r o u p s of p a r a l l e l f i l a m e n t s . Their dimensions may vary considerably f r o m one m i t o c h o n d r i o n to a n o t h e r a n d t h e r e is a r a t h e r b r o a d r a n g e of m e a s u r e m e n t s o b t a i n e d for t h e s e s t r u c t u r e s b y different investigators. T h e p r e s e n t s t u d y was u n d e r t a k e n to f i n d o u t w h e t h e r or n o t t h e s e i n c l u s i o n s are a f u n c t i o n a l p a r t of t h e m i t o c h o n d r i a . F o r t h i s p u r p o s e two m i t o c h o n d r i a l e n z y m e s , cytochrome C oxidase a n d carnitine acetyl t r a n s f e r a s e were s t u d i e d in fresh t i s s u e b l o c k s (7, 13). T h e r e s p i r a t i o n l i n k e d a c c u m u l a t i o n of s t r o n t i u m g i v i n g a n e l e c t r o n d e n s e r e a c t i o n p r o d u c t was also s t u d i e d u s i n g one N A D l i n k e d a n d two flavop r o t e i n l i n k e d s u b s t r a t e s o n freshly isol a t e d m i t o c h o n d r i a l f r a c t i o n s (1, 5, 6, 9). 1Supported by PHS Grant NS-08045 and by a grant from The Muscular Dystrophy Associations of America, Inc.

T h e s p e c i m e n s were o b t a i n e d f r o m one p a t i e n t w i t h i d i o p a t h i c progressive o p h t h a l m o p l e g i a a n d one p a t i e n t w i t h a slowly progressive p r o x i m a l l i m b w e a k n e s s s i n c e i n f a n c y in w h i c h t h e m o s t s t r i k i n g m o r p h o logical f i n d i n g was t h e p r e s e n c e of c r y s t a l line i n c l u s i o n s w i t h i n t h e m i t o c h o n d r i a . MATERIALS AND METHODS Under local anesthesia portions of the right quadriceps were obtained. (1) Conventional electron microscopy. The specimens were removed at rest length in a U-shaped muscle clamp and fixed immediately in 5% glutaraldehyde in cacodylate buffer, PH 7.4, for 3 hr, washed in cold buffer, postfixed in 1% OsO, for 2 hr, dehydrated in serial alcohols, and embedded in Araldite. (2) Electron cytochemistry. Small tissue blocks 1 2 mm 3 were excised as quickly as possible. Cytochrome C oxidase was demonstrated by the method of Seligman (13). Control samples were incubated in a medium containing 2 mM potassium cyanide. Carnitine acetyl transferase was demonstrated by the uranyl-£errocyanide method of Higgins and Barnett (7). Control samples were incubated in the absence of acetyl coA and carnitine. All samples were fixed for 1 hr in 2% Os04 in distilled water, dehydrated in serial alcohols and embedded in Epon. For the study of strontium accumulation, mitochondria were isolated by the method of Makinen and Lee (11): 0.1 ml of mitochondria in 0.25 M sucrose was incubated in a fixed volume of 2 ml of the reaction media described by Kerpel-Fronius and Hajos at 37°C for 20 min (6, 9). Pyruvate, succinate and a-glycero-

404 Copyright © 1975 by AcademicPress, Inc. All rights of reproduction in any form reserved.

CRYSTALLINE INCLUSIONS IN MITOCHONDRIA phosphate were tested as respiratory substrates. Control specimens contained 2-4 dinitrophenol at a final concentration of 1 mM. The reactions were stopped by the addition of 2 volumes of 1% glutaraldehyde. The fractions were centrifuged in a Sorvall RC-2-B Centrifuge at 40 000 g for 10 min. The supernatants were discarded and the pellets were post fixed in 1% OsO4 at 4°C for 30 min, dehydrated in graded ethanols, and embedded in Araldite. The embedded material was sectioned with a diamond knife mounted on an LKB Ultramicrotome. Thin sections were picked up on coated 400-mesh grids. For conventional electron microscopy the sections were stained with uranyl acetate and lead citrate. For electron cytochemistry the sections were stained with lead citrate for 60 see. Sections were examined and micrographed in an AEI-6B Electron Microscope operating at 60 kv. RESULTS

Conventional electron microscopy. Numerous m i t o c h o n d r i a contained crystalline inclusions. Occasionally a m i t o c h o n d r i o n contained more t h a n one inclusion. In some instances they were located within the cristae or between the inner and outer m e m b r a n e s of the mitochondria. Their internal structure consisted of groups of parallel filaments surrounded by a thicker limiting m e m b r a n e . These filaments, on occasion, a p p e a r e d to be composed of rect a n g u l a r repeating subunits measuring 80-150 A in length (Fig. 1). All sections examined contained m i t o c h o n d r i a with inclusions. Electron cytochemistry. The preparations for the d e m o n s t r a t i o n of c y t o c h r o m e C oxidase showed t h a t the inclusions were devoid of reaction product. The reaction p r o d u c t could be seen in the adjacent cristae and a moderate reaction was associated with the limiting m e m b r a n e of the inclusions (Fig. 2). In preparations for the d e m o n s t r a t i o n of carnitine acetyl transferase activity the inclusions were devoid of reaction product. Again a moderate a m o u n t of reaction was associated with the limiting m e m b r a n e of the inclusions (Fig. 3). The reaction p r o d u c t of each cytochemical procedure was confined to expected sites of e n z y m a t i c activity and were not seen in sections of control tissue.

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Mitochondrial fractions. No electron dense strontium was seen within the inclusions when the respiration was supported by p y r u v a t e (Fig. 4a), succinate (Fig. 4b), and a - g l y c e r o p h o s p h a t e (Fig. 4c). Electron dense needles indicating active a c c u m u l a tion of s t r o n t i u m were seen within the cristae and matrix in the unaffected parts of the mitochondria. Control m i t o c h o n d r i a i n c u b a t e d with succinate, strontium, and 1 m M 2-4 dinitrophenol did not show electron dense needles (Fig. 4d). DISCUSSION Although crystalline inclusions are commonly found in skeletal muscle mitochondria in a n u m b e r of n e u r o m u s c u l a r disorders, their role in mitochondrial m e t a b o lism has not been clearly elucidated. Because of their spacial relationship with the mitochondrial cristae the possibility was considered t h a t they might contain some of the e n z y m a t i c c o m p o n e n t s of the inner m e m b r a n e and therefore be involved in mitochondrial metabolism. The principal functions of the mitochondria are oxidative phosphorylation and ion translocation (I0). It has been demonstrated t h a t isolated and "in situ" mitochondria a c c u m u l a t e s t r o n t i u m in an energy d e p e n d e n t process linked to electron transport. This reaction is d e p e n d e n t on the integrity of the respiratory chain and on energy coupling, as has been shown by the inhibitory effects of potassium cyanide and 2-4 dinitrophenol. S t r o n t i u m is accum u l a t e d by respiring mitochondria in the matrix and cristae in the form of electron dense needles and large granules. This a c c u m u l a t i o n parallels the oxidation of a given substrate and as a result is an indicator of mitochondrial metabolic activity (1,

5,6,9). The s t r o n t i u m studies indicate a lack of utilization of both N A D and flavoprotein linked substrates within the inclusions. This could be explained by either a block in the respiratory chain or an absence of mitochondrial function. The lack of cyto-

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FIG. 1. Conventional electron microscopy. Several mitochondria contain crystalline inclusions (single arrows). Uranyl acetate and lead citrate. × 45 000. FIG. 2. Cytochrome C oxidase. No activity is noted within crystalline inclusion (Inc). Reaction product is seen in adjacent cristae and in the membrane surrounding the inclusion (single arrows). × 60 000. FIG. 3. Carnitine acetyl transferase. No activity is noted within crystalline inclusion (Inc). Reaction product is seen in the membrane surrounding the inclusion and in adjacent mitochondria. × 45 000.

CRYSTALLINE INCLUSIONS IN MITOCHONDRIA

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Fro. 4. Strontium accumulation supported by succinate (a), alpha glycerophosphate (b), and pyruvate (c). No electron dense needles are present within the inclusions (Inc). Reaction product is present within adjacent portions of the mitochondrion and within other mitochondria (single arrows). Control mitochondria (d) demonstrating the inhibitory effect of 1 m M dinitrophenol on succinate supported accumulation of strontium. × 30 000.

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chrome C oxidase within the inclusions does not resolve this question. However, t h e c o n c o m i t a n t l a c k of c a r n i t i n e a c e t y l transferase supports the second hypothesis. I t is h i g h l y u n l i k e l y t h a t t w o i n d e p e n d ent mitochondrial enzymes would be absent from an otherwise normal mitochondrial structure. I n c o n c l u s i o n , t h e s u m of t h e s e r e s u l t s suggests that the observed mitochondrial inclusions are space occupying structures t h a t do n o t p l a y a n a c t i v e f u n c t i o n a l role within the mitochondrion. REFERENCES 1. CARAFOL1, E., WEILAND, S., AND LEHNINGER, A., Biochem. Biophys. Acta 97, 88 (1965). 2. CHOU, S. M., Aeta Neuropath. 12, 68 (1969). 3. Dx MAURO, S., SCHOTLAND, D. L., BONILLA,E., LEE, C. P., GAMBETTI,P. L., AND ROWLAND,L. P., Arch. Neurol. 29, 170 (1973).

4. GONATAS,N. K., EVANGELISTA,I., ANDMARTIN,J., Am. J. Med. 42, 169 (1967). 5. GREENWALT,J. W., ANDCARAFOLI,E., J. Cell Biol.

29, 37 (1966). 6. HAJOS, F., AND KERPEL-FRONIUS,S., J. Cell Biol.

51, 216 (197l). 7. HIGGINS,J. A., ANDBARNETT,R. J., J. Cell Sci. 6, 29 (1970). 8. HUDGSON, P., BRADLEY, W. G., AND JENKINSON, M., J. NeuroI. Sci. 16, 343 (1972). 9. KERPEL-FRoNIUS,S., ANDHAJOS,F., J. Histochem. and Cytochem. 18,740 (1970). 10. LEHNINGER, A., Biochemistry, pp. 384 and 401,

Worth, New York, 1970. 11. MAK1NEN,M. W., ANDLEE, C. P., Arch. Biochem. Biophys. 126, 75 (1968). 12. OLSON, W., ENGEL, W. K., WALSH, G. 0., AND EINAUGLER,R., Arch. Neurol. 26, 193 (1972). 13. SELIGMAN, A. M., KARNOVSKY, M. J., WASSERKRUG, H. L., AND HANKER, J. S., J. Cell Biol.

38, 1 (1968). 14. SHAFIQ,S. A., MILHORAT,A. T., AND GORYCKI,M. A., Arch. Neurol. 17, 666 (1967). 15. SHY, G. M., GONATAS, N. K., AND PEREZ, M., Brain 89, 133 (1966).

Electron cytochemistry of crystalline inclusions in human skeletal muscle mitochondria.

JOURNAL OF ULTRASTRUCTURE RESEARCH 51,404-408 (1975) Electron Cytochemistry of Crystalline Inclusions in Human Skeletal Muscle Mitochondria 1 EDUARD...
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