Journal o f the Neurological Sciences, 1977, 33:323-334
323
((h Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
E L E C T R O N - M I C R O S C O P I C X - R A Y M I C R O A N A L Y S I S OF N O R M A L A N D DISEASED H U M A N MUSCLE
C. A. MAUNDER, R. YAROM and V. DUBOWITZ Department o f Paediatrics attd Neonatal Medicine and the Jerry Lewis Muscle Research Centre, Hammersmith Hospital, Dtt Cane Road, London WI2 0 H S (Great BritaiH)
(Received 23 February, 1977)
SUMMARY Electron-microscopic X-ray microanalysis has been used to compare elemental concentrations in specific organelles in normal and diseased human muscle. An elevated calcium to phosphorus ratio has been found in both myonuclei and interstitial cell nuclei in diseased muscle compared with controls. Preliminary observations also suggest that differences in elemental concentrations may be associated with structural abnormalities such as internal nuclei, and loss of myofibrils.
INTRODUCTION Electron-microscopic X-ray microanalysis (EMMA) is a sensitive method for correlating element content with ultrastructure. X-rays produced by excitation of the specimen with an electron beam have characteristic energies and wavelengths which are used to identify, quantitate and localise an element to specific cellular regions. Chemical information is therefore obtainable without destroying cellular structure. Ionic fluxes are known to be involved in several cellular processes (Caldwell 1968; Carafoli 1974) and alterations in ionic distributions may have pathological significance (Wrogeman n and Pena 1976). Structural abnormalities may show elemental differences (Busch, Stromer, Goll and Suzuki 1972; Cullen and Fulthorpe 1975) and similarly ionic differences in specific organelles may indicate possible biochemical defects. Our previous EMMA studies have shown that phosphorus, chlorine, sulphur, calcium and zinc can be detected in ultrathin sections of skeletal muscle of experimental
The authors are grateful to the Muscular Dystrophy Group of Great Britain for their generous support and to the Medical Research Council for a Visiting Senior Scientist award to Dr. Yarom. Correspondence to: C. A. Maunder, Jerry Lewis Muscle Research Centre, Hammersmitb Hospital, Du Cane Road, London WI20HS, Great Britain.
324 animals (Yarom,
Maunder,
Scripps, Hall and Dubowitz
1975). In t h e p r e l i m i n a r }
i n v e s t i g a t i o n s r e p o r t e d h e r e we h a v e a p p l i e d s i m i l a r m e t h o d s t o a c o m p a r i s o n
ot
n o r m a l a n d d i s e a s e d h u m a n m u s c l e t o i d e n t i f y p o s s i b l e d i f f e r e n c e s in e l e m e n t a l c o n c e n t r a t i o n s in s u b c e l l u l a r o r g a n e l l e s . M A T E R I A L A N D METHODS O p e n o r n e e d l e b i o p s i e s f r o m t h e q u a d r i c e p s o f 17 p a t i e n t s w i t h v a r i o u s n e u r o muscular disorders were compared
with samples from 1 female and 6 male control
s u b j e c t s ( T a b l e t). C o n t r o l s u b j e c t s w e r e e i t h e r h e a l t h y v o l u n t e e r s o r p a t i e n t s u n d e r going orthopaedic
o p e r a t i o n s . A l l s a m p l e s w e r e fixed i m m e d i a t e l y in 3 o/,, g l u t a r a l -
d e h y d e i n 0.1 M c a c o d y l a t e b u f f e r a t p H 7.2. S u b s e q u e n t p r o c e s s i n g , u s i n g i n c r e a s i n g concentrations
of glutaraldehyde
prior to araldite embedding,
has been described
TABLE 1 RELATIVE MASS FRACTIONS OF C A L C I U M A N D PHOSPHORUS (7:103) IN M Y O N U C L E I OF S T R U C T U R A L L Y WELL PRESERVED FIBRES ( i SEM) Case
Sex
Age
Diagnosis
1 2 3 4 5 6 7
M M M M ~M aM ~F
17 6 7 16 32 34 42
d yr yr yr yr yr yr
normal normal normal normal normal normal normal
7 5 6 7 8 7 8
8 9 10 11 12 13
M M M M M M
3too 4yr 5 yr 6 yr 8 yr 9yr
DMD DMD DMD DMD DMD DMD
6 6 4 13 4 9
14 15 16 17
F F F F
19 40 20 34
18 19 20 21 22 23
F F M M M M
9 yr 15 yr 55 yr 10 yr 4 yr 10 yr
24
M
13 yr
yr yr yr yr
?Carrier ?Carrier ?Carrier Carrier FSH FSH LGD Becker D. SMA D'myositis (on steroids) D'myositis
No. of analyses
Ca
± ± ± z_ ± :~ ±
22 62 10 88 47 20 49
4.7 3.6 4. l 2.5 2.6 4.3 5.9
146± 8 90± 6 587 ~ 102 296 ± 31 115 ± 20 153 ± 16
301 101 511 284 208 243
± ± _~:: ± ±_ ±
17 t5 69 24 41 22
5.0 8.8 11.5 10.4 5.6 6.5
4 8 6 6
211 222 149 329
14 29 13 19
636 746 334 297
:L i ± +
26 85 25 44
3.3 3.0 5.5 12.1
10 6 4 15 7 4
644 653 145 178 200 1912
± 72 ~ 43 ± 42 ± 26 ± 25 £ 369
419 671 280 304 262 582
± 37 ± 10l ± 63 i 44 ± 27 i : 57
15.4 9.7 5.2 5.9 7.6 32.9
± ± ± ~
6 14 17 14 l0 8 11
Ca ( x 10) p
190 ± 509 ~276 ± 686 i 352 ± 222 ± 339:4
7
90 182 114 172 90 96 201
P
140 5- 19
124 t
25
ll.3
Needle biopsy. D M D ~ Duchenne muscular dystrophy; Carrier : : carrier of Duchenne muscular dystrophy; FSH ~ facioscapulohumeral dystrophy; L G D -- limb-girdle dystrophy; SMA = spinal muscular atrophy; D'myositis = dermatomyositis.
325 previously (Yarom et al. 1975). Osmium fixation, buffer rinses, alcohols and propylene oxide were avoided. Sections (100-120 nm) were cut on a Cambridge Huxley Mark II Ultramicrotome and collected on celloidin carbon-coated nickel or titanium grids. Celloidin coating was found later to be unnecessary. Carbon-coated sections were analysed unstained in an EMMA 4 (AEI Manchester) electron microscope using a Kevex Si (Li) energy dispersive detector. Analyses were performed using an accelerating voltage of 40 kV, a probe current of 200 nA (later reduced to 80 nA), a probe diameter of 200 nm and an analysis time of 50 sec. Characteristic X-ray intensities were displayed as a spectrum on a videoscreen and X-ray counts of each element recorded. Specimen mass was estimated by simultaneously recording the continuous (Bremsstrahlung) radiation at 20 keV (Hall 1972). The relative mass fraction (R) of each element was expressed as a ratio of the characteristic X-ray counts (P) to the continuum (W), after correction for background (B) (Hall 1972), i.e. R = P - - B . W Analyses of myonuclei, interstitial nuclei and myofibrils (A-band) of each sample were made. Heterochromatic areas of nuclei were selected and initially only wellpreserved fibres were analysed. Latterly, however, we have been able to make a few analyses of abnormal structural features. RESULTS The ultrastructural appearance of human muscle prepared for X-ray analysis is shown in Fig. 1. Despite the thickness of the section and lack of stain subcellular components of fibres are easily identifiable. The elements detected in human skeletal muscle using our methods of specimen preparation are illustrated in the energy-dispersive spectrum in Fig. 2. Nuclei consistently showed prominent peaks of phosphorus, sulphur, chlorine and calcium and occasionally traces of potassium, iron and zinc. Myofibrillar regions contained lower but detectable concentrations of phosphorus, sulphur, chlorine and calcium. Age and biopsy technique did not appear to influence element concentration. The myonuclear calcium to phosphorus ratio of the female control was higher than that of 6 male controls suggesting that elemental concentrations may differ between sexes. The calcium to phosphorus ratio was used as a means of comparison because of known intracellular relationships between calcium and phosphorus (Abood, Kurahasi, Gruner and Perez del Cerro 1968; De Meis and Carvalho 1974). Phosphorus ratios have also been used in studies of human muscle electrolytes (Bergstr6m 1962; Edwards, Jones, Maunder and Batra 1975).
Myonuclei of well-preserved.fibres The most consistent differences between normal and diseased muscle were found in the calcium and phosphorus content (Fig. 3). Variation between samples and to a lesser extent between different nuclei was found (Table 1) but in 6 cases of Duchenne
326
Fig. 1. Electron micrograph of human muscle prepared for X-ray analysis without osmication, dehydration with alcohol or staining, ~ 10,500. Z-line (Z), A-band (A), l-band (I), mitochondria (M), nucleus (N), lipid droplets (L) and a blood vessel (bv) can be distinguished.
Fig. 2. Energy dispersive spectrum of a myonucleus of human muscle recorded under the conditions described, Elements labelled in white relate to the specimen and those in black to instrumentation.
327
Fig. 3. Spectra of myonuclei of (a) normal muscle, (b) Ducbenne muscular dystrophy, (c) dermatomyositis and (d) embedding resin only. Note the variation in P and Ca peaks. muscular dystrophy ( D M D ) mean values suggest that calcium content may be increased and phosphorus decreased (Table 2). Becker dystrophy and limb-girdle dystrophy ( L G D ) showed slightly lower phosphorus concentrations but less alteration in calcium. This is in contrast to 2 cases of facio-scapulohumeral dystrophy (FSH) who showed high levels of calcium. Calcium was exceptionally high ( x 15) in I child suffering from dermatomyositis. This patient showed no sign of calcinosis but was being treated with steroids. The ratio of calcium to phosphorus in diseased muscle was consistently higher than that of normal muscle (Table 1) and in 6 cases of D M D the difference was significant (P < 0.01). One carrier of D M D also showed an elevated ratio and the deviation from normal appeared to be caused by similar alterations to calcium and phosphorus as D M D itself. Three possible carriers had low ratios similar to those of male controls and below that of the normal female. hlterstitia[ nuclei In the majority of cases interstitial nuclei showed a slightly lower calcium to phosphorus ratio than myonuclei (Table 3). In diseased muscle the interstitial nuclei showed a similar elevated calcium to phosphorus ratio as did the myonuclei, and in all
328 TABLE 2 MEAN RELATIVE MASS FRACTIONS OF C A L C I U M A N D PHOSPHORUS 1N MYONUCLEI OF N O R M A L A N D D M D MUSCLE Diagnosis
No. of cases
Ca
P
Ca (~: 10) ~-
Significance (Student's t-test)
Normal (males) DMD
6 6
124 5:17 236 5 : 7 6
372 ,, 78 281 5:55
3.6 -- 0.37 8.0 5:1.07
P < 0.01
TABLE 3 RELATIVE MASS FRACTIONS (R) OF C A L C I U M A N D P H O S P H O R U S ( × I03) IN INTERSTITIAL N U C L E I (~_ SEM) Case
Sex
Age
Diagnosis
1 2 3 4 5 6 7
M M M M aM aM aF
17 d 6 yr 7 yr 16 yr 32 yr 34 yr 42 yr
normal normal normal normal normal normal normal
6 1 6 6 4 4 6
92 185 97 177 88 132 167
8 9 10 11 12 13
M M M M M M
DMD DMD DMD DMD DMD DMD
6 5 4 10 8 9
120 101 544 304 89 130
16 17
F F
20 yr 34 yr
?Carrier Carrier
3 3
19 20 21 22 23
F M M M M
15 55 10 4 10
24
M
13 yr
FSH LGD Becker D. SMA D'myositis (on steroids) D'myositis
3 mo 4yr 5 yr 6 yr 8 yr 9yr
yr yr yr yr yr
No. of analyses
Ca
P
±
15
Ca ( × 10) p-
± 10 ± 74 5: 12 5: 10 5: 10
223 539 352 713 369 222 272
± 46 5:307 _5: 88 5: 20 5: 39
3.8 3.4 2.8 2.5 2.4 3.6 6.1
5: 9 5:26 :~- 89 5: 22 5: 4 ~ 10
230 143 885 385 214 306
± 19 5:26 5:257 :! 54 5:25 % 34
5.2 5.7 6.1 7.9 4.4 5.0
163 5: 17 285 5 : 8 0
291 ± 37 436 5- 31
5.6 6.5
6 6 8 6 4
768 174 321 122 703
799 338 383 185 824
9.6 5.2 8.3 6.6 8.5
6
149 ,,
:L 67 5:23 :L: 68 ~- 13 % 283 73
~
27
5:116 ± 36 5:82 5:21 5:356
154 -[
50
9.7
Needle biopsy. For abbreviations, see Table 1. 6 c a s e s o f D M D t h e r a t i o w a s a g a i n s i g n i f i c a n t l y h i g h e r t h a n t h a t o f c o n t r o l s ( P < 0.01),
Internal nuclei Two cases of DMD
and 1 of LGD
showed differences between internal and
peripheral nuclei of the same biospy, Although phosphorus
o n l y a few a n a l y s e s w e r e p o s s i b l e
and chlorine concentrations were consistently higher in internal nuclei
( T a b l e s 4 a n d 5).
329 TABLE 4 RELATIVE MASS F R A C T I O N S ( × 103) OF PHOSPHORUS 1N P E R I P H E R A L A N D I N T E R N A L NUCLEI ( i SEM) Diagnosis
Peripheral nuclei (p)
Internal nuclei (i)
Ratio p i
Duchenne dystrophy
243 ± 22 a(9) 208 ± 41 (4) 280 ± 63 (4)
396 ± 26 (3) 217 ± 66 (5) 499 ± 164 (2)
0.61
Duchenne dystrophy Limb girdle dystrophy
0.96 0.56
,~ Number of analyses.
TABLE 5 RELATIVE MASS FRACTIONS ( × 103) OF C H L O R I N E IN PERIPHERAL A N D INTERNAL NUCLEI ( ± SEM) Diagnosis
Peripheral nuclei (p)
Internal nuclei (i)
Ratio p ]-
Duchenne dystrophy
389 ± 54 a(5) 70 :L 13 (4) 94 ± 28 (4)
588 ± 121 (3) 85 ± 6.4 (5) 134 ± 13 (2)
0.66
Duchenne dystrophy Limb-girdle dystrophy
0.82 0.70
:' Number of analyses.
TABLE 6 RELATIVE MASS FRACTIONS ( x 10:3) OF PHOSPHORUS IN NUCLEI OF N O R M A L FIBRES A N D FIBRES WITH MYOF1BR1LLAR LOSS ( ± SEM) Diagnosis
Normal (N)
Atrophic (A)
Ratio N
Significance (Student's t-test)
Duchenne dystrophy
243 •~(9) 101 (6) 208 (4) 280 (4)
472 ± 64 (6) 161 ± 18 (7) 131 ± 1.1 (1 O) 254 ± 62 (2)
0.51
P < 0.01
0.63
P < 0.05
1.59
P < 0.05
Duchenne dystrophy Duchenne dystrophy
Limb-girdle dystrophy
Number of analyses.
± 22 ± 15 _± 41
± 63
1.1
330 TABLE 7 R E L A T I V E MASS F R A C T I O N S ( :. 10 :~)OF M Y O F I B R I L L A R C A L C I U M
Diagnosis
No. of cases
Ca
N o r m a l males DMD FSH FSH D'myositis (on steroids)
6 6 1 I 1
107 135 506 438 880
(:: S E M )
i
15 ~ 13 5_- 73 ± 56 _% 206
F o r abbreviations, see Table 1.
Nuclei of fibres with loss of myofibrils (atrophic) Preliminary investigations of nuclei of fibres with reduced myofibrillar content suggested that the phosphorus content may differ from that of well-preserved fibres (Table 6). Fibres of 2 cases of DMD with only mild myofibrillar loss showed significantly higher nuclear phosphorus concentrations than those of well-preserved fibres in the same biopsy (P < 0.01 and P < 0.05). In another case of DMD and 1 of LGD the fibres analysed had severe loss of myofibrils and the nuclei had lower phosphorus concentrations than other nuclei in the same biopsy.
M yofibrils Comparison of myofibrillar regions in normal and diseased muscle showed elevated calcium concentrations in 2 cases of FSH and marked elevation in the case of dermatomyositis being treated with steroids (Fig. 4 and Table 7). Calcium concentrations in 6 cases of DMD, although elevated, were not significantly different from those of controls. DISCUSSION
Our preliminary results demonstrate that electron-microscopic X-ray microanalysis can localise differences in intrinsic elemental concentrations between normal and diseased muscle. The elements most frequently detected were phosphorus, sulphur, chlorine and calcium. The calcium to phosphorus ratio showed a consistent and significant difference between nuclei in muscle from normal and Duchenne dystrophy subjects. Variability in the relative concentrations of calcium and phosphorus was sometimes high, as shown by the SEM values, but the present data suggests that in DMD myonuclear calcium concentration is elevated whilst phosphorus is decreased. Additional, carefully controlled analyses may reduce the variation between readings and determine the specificity and relationship of these changes to clinical states and stages. Occasionally traces of potassium, iron and zinc were found. Other methods of specimen preparation and different instrumental conditions may detect alterations in the concentration of other elements. X-ray microanalysis of myocardium and extra-
331
Fig. 4. Spectra of myofibrils of (a) normal muscle, (b) Duchenne muscular dystrophy and (c) dermatomyositis. Note the variation in calcium content. ocular muscle of experimental animals has suggested that zinc may be altered in response to injury (Peters, Yarom, Dormann and Hall 1976). These authors used glutaraldehyde/osmium fixed material and a probe current twice that used in our study. The relationship of our recorded elemental concentrations to those in vivo is difficult to assess because of the effects of fixation and specimen preparation on the redistribution of elements. Some elemental differences may be secondary and relate to damage rather than to disease entities. Although our initial analyses were of struc-
332 turally well-preserved fibres ionic alterations may precede morphological damage. This has been demonstrated in mitochondria which accumulate calcium before any observed structural changes (Judah, Ahmed and McLean 1964). Localised alterations in elemental concentrations might therefore be a useful criterion for identifying damaged fibres which are ultrastructurally normal, particularly in subclinical states. In this context it is interesting to note the elevated calcium to phosphorus ratio in the carrier of DMD. More direct involvement of calcium in degenerative changes has been implicated (Busch et al. 1972). Destruction of Z-lines is a frequent ultrastructural abnormality in myofibres and may be caused by calcium activation of a degradation catalyst. Cell volume and electrical phenomena, which are known to be influenced by ionic concentrations, may be related to increased sarcoplasmic volume, dilatation of sarcoplasmic reticulum and hypercontraction of myofibrils observed in muscular dystrophy (Cullen and Fulthorpe 1975). Wrogemann and Pena (1976) have suggested that a net influx of calcium into the cell triggers events leading to necrosis. Alterations in calcium concentrations were mainly confined to myonuclei with the exception of a few cases showing increased myofibrillar calcium. Improved methods of specimen preparation might enable detection of elemental changes in other locations. However, analysis of organelles known to transport calcium, such as sarcoplasmic reticulum (Hasselbach 1964) and mitochondria (Carafoli 1974), is limited by the minimum probe diameter. In our preparations these structures were often too small for accurate localisation. Alterations in nuclear phosphorus concentrations may be related to several biochemical activities. Nucleotide content and protein synthesis have been the subject of several investigations of dystrophic muscle. Watts and Reid (1968) reported that DNA and RNA content was about twice as high in dystrophic mouse muscle compared to normal muscle, whereas dystrophic chick myoblasts in culture were shown to have a markedly reduced nuclear and cytoplasmic RNA content (Ross and Jans 1968). There is also evidence suggesting alterations in cyclic nucleotides in diseased muscle (Zymans, Epstein, Saifer, Aronson and Volk 1959; Canal, Frattola and Smirne 1975) and their involvement in several cellular processes such as cell division (Sheppard and Plagemann 1975), myogenesis (Epstein, Jiminez de Asua and Rozengart 1975; Zalin and Montague 1975) and phosphorylation of phosphoproteins (Wagner 1975). Phosphorylation of non-histone proteins has been shown to be involved in chromosome structure and gene activation (Kleinsmith 1975; Thi Man, Morris and Cole 1975) suggesting that alterations in phosphorus concentrations could be specific and relate more directly to disease processes. Our initial analyses of structural abnormalities suggest that elemental differences might be detectable in internal nuclei and fibres with myofibrillar loss. Interpretation of the observed alterations in phosphorus and chlorine is difficult at this stage and our results need to be substantiated with more data. It is important to note that elemental differences in diseased muscle were observed in more than one cell type. Both interstitial and myonuclei showed significantly higher calcium to phosphorus ratios in diseased muscle compared with normal muscle. This may reflect a generalised membrane dysfunction similar to that already suggested
333 as the basic defect in muscular dystrophy (Roses, Herbstreith and Appel 1975: Mawatari, Schonberg and Olarte 1976; Sha'afi, Rodan, Hintz, Fernandez and Rodan 1975). At this stage, however, any conclusions drawn from our results are speculative. More data is required to confirm our initial findings and to establish the normal variations of elemental content. Correlative biochemical analyses are also needed to assess the validity and advantages of X-ray analysis and to determine the reasons for observed elemental differences. However, our results provide unique information on the distribution of elements and highlight some interesting differences between normal and diseased muscle. With improved methods of specimen preparation it is hoped that this new approach may clarify some of the processes related to the pathogenesis of neuromuscular disorders. ACKNOWLEDGEMENTS
We thank Dr. T. A. Hall, Department of Zoology, University of Cambridge for the use of the EMMA 4 and his helpful advice.
REFERENCES Abood, L. G., K. Kurahasi, E. Gruner and M. Perez del Cerro (1968) Nucleotide content, calcium accumulation and phosphate metabolism in subcellular fractions of rat brain, Biochim. biophys. Acta, 53 : 531-544. Bergstr6m, J. (1962) Muscle electrolytes in man. Determined by neutron activation analysis on needle biopsy specimens - - A study on normal subjects, kidney patients and patients with chronic diarrhoea. Stand. J. clin. Lab. Invest., 14: Suppl. 68. Busch, W. A., M. H. Stromer, D. E. Goll and A. Suzuki (1972) Ca 2~ specific removal of Z lines from rabbit skeletal muscle, J. Cell Biol., 52: 367-381. Caldwel[, P. C. (1968) Factors governing movements and distribution of inorganic ions in nerve and muscle, Physiol. Rev., 48: 1-64. Canal, N., L. Frattola and S. Smirne (1975) The metabolism of cyclic-3-5 adenosine monophosphate (cAMP) in diseased muscle, J. Neurol., 208: 259-265. Carafoli, E. (1974) Mitochondrial uptake of calcium ions and regulation of cell function. In: M. S. Smellier (Ed.), Biochemical Society Symposium, No. 39, p. 89. Cullen, M. J. and J. J. Fulthorpe (1975) Stages in fibre breakdown in Duchenne muscular dystrophy, J. neurol. Sei., 24: 179-200. De Meis, L. and M. G. C. Carvalho (1974) Role of the Ca 2+ concentration gradient in the adenosine-. 5-triphosphate-inorganic phosphate exchange catalyzed by sarcoplasmic reticulum, BiochemistO', 13:5032 5038. Edwards, R. H. T., D. A. Jones, C. A. Maunder and G. J. Batra (1975) Needle biopsy for muscle chemistry, Lancet, 1 : 736. Epstein, C. J., L. Jiminez de Asua and E. Rozengart (1975) The role of cyclic AMP in myogenesis, J. cell. PhysioL, 86: 83-90. Hall, T. A. (1972) X-ray microanalysis in biology - - Quantitation, Micron, 3:93 97. Hasselbach, W. (1964) Relaxing factor and the relaxation of muscle, Progr. biophys, tool. Biol., 14: 167. Judah, J. D., K. Ahmed and A. E. M. McLean (1964) Possible role of ion shifts in liver injury. In: A. V. S. de Reuch and J. Knight (Eds.), Ciba Foundation Symposium on Cell Injury, J. and A. Churchill, London, p. 187. Kleinsmith, L. J. (1975) Phosphorylation of non-histone proteins in the regulation of chromosome structure and function, J. cell. PhysioL, 85: 459-476.
334 Mawatari, S., M. Schonberg and M. Olarte (1976) Biochemical abnormalities of erythrocyte membranes in Duchenne dystrophy, Arch. Neurol. (Chic.), 33:489-493. Peters, P. D., R. Yarom, A. Dormann and T. Hall (1976) X-ray microanalysis of intracellular zinc -EMMA-4 examinations of normal and injured muscle and myocardium, J. Ultrastruct. Res., 57: 121-131. Roses, A. D., M. H. Herbstreith and S. H. Appel (1975) Membrane protein kinase alterations in Duchenne muscular dystrophy, Nature (Lond.), 254: 350-351. Ross, K. F. A. and D. E. Jans (1968) The nuclear and cytoplasmic RNA in normal differentiating and dystrophic myoblasts. In : 4th Symposium on Research in Muscular Dystrophy, Pitman Medical, London, p. 240. Sha'afi, R. I., S. B. Rodan, R. L. Hintz, S. M. Fernandez and G. A. Rodan (1975) Abnormalities in membrane microviscosity and ion transport in genetic muscular dystrophy, Nature (Lond.)~ 254: 525-526. Sheppard, J. R. and P. G. W. Plagemann (1975) Cyclic AMP, membrane transport and cell division, Part 1 (Effects of various chemicals on cyclic AMP levels and rate of transport of nucleosides, hypoxanthine and deoxyglucose in several lines of cultured cells), J. cell. Physiol., 85:163-172. Thi Man, N., G. E. Morris and R. J. Cole (1975) Gene activation during muscle differentiation and the role of non-histone chromosomal protein phosphorylation, Develop. BioL, 47: 81-96. Wagner, H. K. (1975) Regulation of protein kinase and phosphoprotein phosphatase by cyclic AMP and cyclic AMP antagonist, FEBS Letters, 57 : 60-63. Watts, D. C. and J. D. Reid (1968) Comparison of protein synthesis in normal and dystrophic mouse muscle. In: 4th Symposium on Research in Muscular Dystrophy, Pitman Medical, London, p. 336. Wrogemann, K. and S. D. J. Pena (1976) Mitochondrial calcium overload - - A general mechanism for cell-necrosis in muscle diseases, Lancet, l : 672-674. Yarom, R., C. A. Maunder, M. Scripps, T. A. Hall and V. Dubowitz (1975) A simplified method of specimen preparation for X-ray microanalysis of muscle and blood cells, Histochemistry, 45: 49-59. Zalin, R. J. and W. M. Montague (1975) Changes in cyclic AMP, adenylate cyctase and protein kinase levels during the development of embryonic chick skeletal muscle, Exp. Cell Res., 93 : 55-92. Zymans, M. C., N. Epstein, A. Saifer, S. M. Aronson and B. W. Volk (1959) Distribution of acidsoluble nucleotides in hind leg muscles of mice with dystrophia muscularis, Amer. J. Physiol., 196: 1093-1097.
323
((h Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
E L E C T R O N - M I C R O S C O P I C X - R A Y M I C R O A N A L Y S I S OF N O R M A L A N D DISEASED H U M A N MUSCLE
C. A. MAUNDER, R. YAROM and V. DUBOWITZ Department o f Paediatrics attd Neonatal Medicine and the Jerry Lewis Muscle Research Centre, Hammersmith Hospital, Dtt Cane Road, London WI2 0 H S (Great BritaiH)
(Received 23 February, 1977)
SUMMARY Electron-microscopic X-ray microanalysis has been used to compare elemental concentrations in specific organelles in normal and diseased human muscle. An elevated calcium to phosphorus ratio has been found in both myonuclei and interstitial cell nuclei in diseased muscle compared with controls. Preliminary observations also suggest that differences in elemental concentrations may be associated with structural abnormalities such as internal nuclei, and loss of myofibrils.
INTRODUCTION Electron-microscopic X-ray microanalysis (EMMA) is a sensitive method for correlating element content with ultrastructure. X-rays produced by excitation of the specimen with an electron beam have characteristic energies and wavelengths which are used to identify, quantitate and localise an element to specific cellular regions. Chemical information is therefore obtainable without destroying cellular structure. Ionic fluxes are known to be involved in several cellular processes (Caldwell 1968; Carafoli 1974) and alterations in ionic distributions may have pathological significance (Wrogeman n and Pena 1976). Structural abnormalities may show elemental differences (Busch, Stromer, Goll and Suzuki 1972; Cullen and Fulthorpe 1975) and similarly ionic differences in specific organelles may indicate possible biochemical defects. Our previous EMMA studies have shown that phosphorus, chlorine, sulphur, calcium and zinc can be detected in ultrathin sections of skeletal muscle of experimental
The authors are grateful to the Muscular Dystrophy Group of Great Britain for their generous support and to the Medical Research Council for a Visiting Senior Scientist award to Dr. Yarom. Correspondence to: C. A. Maunder, Jerry Lewis Muscle Research Centre, Hammersmitb Hospital, Du Cane Road, London WI20HS, Great Britain.
324 animals (Yarom,
Maunder,
Scripps, Hall and Dubowitz
1975). In t h e p r e l i m i n a r }
i n v e s t i g a t i o n s r e p o r t e d h e r e we h a v e a p p l i e d s i m i l a r m e t h o d s t o a c o m p a r i s o n
ot
n o r m a l a n d d i s e a s e d h u m a n m u s c l e t o i d e n t i f y p o s s i b l e d i f f e r e n c e s in e l e m e n t a l c o n c e n t r a t i o n s in s u b c e l l u l a r o r g a n e l l e s . M A T E R I A L A N D METHODS O p e n o r n e e d l e b i o p s i e s f r o m t h e q u a d r i c e p s o f 17 p a t i e n t s w i t h v a r i o u s n e u r o muscular disorders were compared
with samples from 1 female and 6 male control
s u b j e c t s ( T a b l e t). C o n t r o l s u b j e c t s w e r e e i t h e r h e a l t h y v o l u n t e e r s o r p a t i e n t s u n d e r going orthopaedic
o p e r a t i o n s . A l l s a m p l e s w e r e fixed i m m e d i a t e l y in 3 o/,, g l u t a r a l -
d e h y d e i n 0.1 M c a c o d y l a t e b u f f e r a t p H 7.2. S u b s e q u e n t p r o c e s s i n g , u s i n g i n c r e a s i n g concentrations
of glutaraldehyde
prior to araldite embedding,
has been described
TABLE 1 RELATIVE MASS FRACTIONS OF C A L C I U M A N D PHOSPHORUS (7:103) IN M Y O N U C L E I OF S T R U C T U R A L L Y WELL PRESERVED FIBRES ( i SEM) Case
Sex
Age
Diagnosis
1 2 3 4 5 6 7
M M M M ~M aM ~F
17 6 7 16 32 34 42
d yr yr yr yr yr yr
normal normal normal normal normal normal normal
7 5 6 7 8 7 8
8 9 10 11 12 13
M M M M M M
3too 4yr 5 yr 6 yr 8 yr 9yr
DMD DMD DMD DMD DMD DMD
6 6 4 13 4 9
14 15 16 17
F F F F
19 40 20 34
18 19 20 21 22 23
F F M M M M
9 yr 15 yr 55 yr 10 yr 4 yr 10 yr
24
M
13 yr
yr yr yr yr
?Carrier ?Carrier ?Carrier Carrier FSH FSH LGD Becker D. SMA D'myositis (on steroids) D'myositis
No. of analyses
Ca
± ± ± z_ ± :~ ±
22 62 10 88 47 20 49
4.7 3.6 4. l 2.5 2.6 4.3 5.9
146± 8 90± 6 587 ~ 102 296 ± 31 115 ± 20 153 ± 16
301 101 511 284 208 243
± ± _~:: ± ±_ ±
17 t5 69 24 41 22
5.0 8.8 11.5 10.4 5.6 6.5
4 8 6 6
211 222 149 329
14 29 13 19
636 746 334 297
:L i ± +
26 85 25 44
3.3 3.0 5.5 12.1
10 6 4 15 7 4
644 653 145 178 200 1912
± 72 ~ 43 ± 42 ± 26 ± 25 £ 369
419 671 280 304 262 582
± 37 ± 10l ± 63 i 44 ± 27 i : 57
15.4 9.7 5.2 5.9 7.6 32.9
± ± ± ~
6 14 17 14 l0 8 11
Ca ( x 10) p
190 ± 509 ~276 ± 686 i 352 ± 222 ± 339:4
7
90 182 114 172 90 96 201
P
140 5- 19
124 t
25
ll.3
Needle biopsy. D M D ~ Duchenne muscular dystrophy; Carrier : : carrier of Duchenne muscular dystrophy; FSH ~ facioscapulohumeral dystrophy; L G D -- limb-girdle dystrophy; SMA = spinal muscular atrophy; D'myositis = dermatomyositis.
325 previously (Yarom et al. 1975). Osmium fixation, buffer rinses, alcohols and propylene oxide were avoided. Sections (100-120 nm) were cut on a Cambridge Huxley Mark II Ultramicrotome and collected on celloidin carbon-coated nickel or titanium grids. Celloidin coating was found later to be unnecessary. Carbon-coated sections were analysed unstained in an EMMA 4 (AEI Manchester) electron microscope using a Kevex Si (Li) energy dispersive detector. Analyses were performed using an accelerating voltage of 40 kV, a probe current of 200 nA (later reduced to 80 nA), a probe diameter of 200 nm and an analysis time of 50 sec. Characteristic X-ray intensities were displayed as a spectrum on a videoscreen and X-ray counts of each element recorded. Specimen mass was estimated by simultaneously recording the continuous (Bremsstrahlung) radiation at 20 keV (Hall 1972). The relative mass fraction (R) of each element was expressed as a ratio of the characteristic X-ray counts (P) to the continuum (W), after correction for background (B) (Hall 1972), i.e. R = P - - B . W Analyses of myonuclei, interstitial nuclei and myofibrils (A-band) of each sample were made. Heterochromatic areas of nuclei were selected and initially only wellpreserved fibres were analysed. Latterly, however, we have been able to make a few analyses of abnormal structural features. RESULTS The ultrastructural appearance of human muscle prepared for X-ray analysis is shown in Fig. 1. Despite the thickness of the section and lack of stain subcellular components of fibres are easily identifiable. The elements detected in human skeletal muscle using our methods of specimen preparation are illustrated in the energy-dispersive spectrum in Fig. 2. Nuclei consistently showed prominent peaks of phosphorus, sulphur, chlorine and calcium and occasionally traces of potassium, iron and zinc. Myofibrillar regions contained lower but detectable concentrations of phosphorus, sulphur, chlorine and calcium. Age and biopsy technique did not appear to influence element concentration. The myonuclear calcium to phosphorus ratio of the female control was higher than that of 6 male controls suggesting that elemental concentrations may differ between sexes. The calcium to phosphorus ratio was used as a means of comparison because of known intracellular relationships between calcium and phosphorus (Abood, Kurahasi, Gruner and Perez del Cerro 1968; De Meis and Carvalho 1974). Phosphorus ratios have also been used in studies of human muscle electrolytes (Bergstr6m 1962; Edwards, Jones, Maunder and Batra 1975).
Myonuclei of well-preserved.fibres The most consistent differences between normal and diseased muscle were found in the calcium and phosphorus content (Fig. 3). Variation between samples and to a lesser extent between different nuclei was found (Table 1) but in 6 cases of Duchenne
326
Fig. 1. Electron micrograph of human muscle prepared for X-ray analysis without osmication, dehydration with alcohol or staining, ~ 10,500. Z-line (Z), A-band (A), l-band (I), mitochondria (M), nucleus (N), lipid droplets (L) and a blood vessel (bv) can be distinguished.
Fig. 2. Energy dispersive spectrum of a myonucleus of human muscle recorded under the conditions described, Elements labelled in white relate to the specimen and those in black to instrumentation.
327
Fig. 3. Spectra of myonuclei of (a) normal muscle, (b) Ducbenne muscular dystrophy, (c) dermatomyositis and (d) embedding resin only. Note the variation in P and Ca peaks. muscular dystrophy ( D M D ) mean values suggest that calcium content may be increased and phosphorus decreased (Table 2). Becker dystrophy and limb-girdle dystrophy ( L G D ) showed slightly lower phosphorus concentrations but less alteration in calcium. This is in contrast to 2 cases of facio-scapulohumeral dystrophy (FSH) who showed high levels of calcium. Calcium was exceptionally high ( x 15) in I child suffering from dermatomyositis. This patient showed no sign of calcinosis but was being treated with steroids. The ratio of calcium to phosphorus in diseased muscle was consistently higher than that of normal muscle (Table 1) and in 6 cases of D M D the difference was significant (P < 0.01). One carrier of D M D also showed an elevated ratio and the deviation from normal appeared to be caused by similar alterations to calcium and phosphorus as D M D itself. Three possible carriers had low ratios similar to those of male controls and below that of the normal female. hlterstitia[ nuclei In the majority of cases interstitial nuclei showed a slightly lower calcium to phosphorus ratio than myonuclei (Table 3). In diseased muscle the interstitial nuclei showed a similar elevated calcium to phosphorus ratio as did the myonuclei, and in all
328 TABLE 2 MEAN RELATIVE MASS FRACTIONS OF C A L C I U M A N D PHOSPHORUS 1N MYONUCLEI OF N O R M A L A N D D M D MUSCLE Diagnosis
No. of cases
Ca
P
Ca (~: 10) ~-
Significance (Student's t-test)
Normal (males) DMD
6 6
124 5:17 236 5 : 7 6
372 ,, 78 281 5:55
3.6 -- 0.37 8.0 5:1.07
P < 0.01
TABLE 3 RELATIVE MASS FRACTIONS (R) OF C A L C I U M A N D P H O S P H O R U S ( × I03) IN INTERSTITIAL N U C L E I (~_ SEM) Case
Sex
Age
Diagnosis
1 2 3 4 5 6 7
M M M M aM aM aF
17 d 6 yr 7 yr 16 yr 32 yr 34 yr 42 yr
normal normal normal normal normal normal normal
6 1 6 6 4 4 6
92 185 97 177 88 132 167
8 9 10 11 12 13
M M M M M M
DMD DMD DMD DMD DMD DMD
6 5 4 10 8 9
120 101 544 304 89 130
16 17
F F
20 yr 34 yr
?Carrier Carrier
3 3
19 20 21 22 23
F M M M M
15 55 10 4 10
24
M
13 yr
FSH LGD Becker D. SMA D'myositis (on steroids) D'myositis
3 mo 4yr 5 yr 6 yr 8 yr 9yr
yr yr yr yr yr
No. of analyses
Ca
P
±
15
Ca ( × 10) p-
± 10 ± 74 5: 12 5: 10 5: 10
223 539 352 713 369 222 272
± 46 5:307 _5: 88 5: 20 5: 39
3.8 3.4 2.8 2.5 2.4 3.6 6.1
5: 9 5:26 :~- 89 5: 22 5: 4 ~ 10
230 143 885 385 214 306
± 19 5:26 5:257 :! 54 5:25 % 34
5.2 5.7 6.1 7.9 4.4 5.0
163 5: 17 285 5 : 8 0
291 ± 37 436 5- 31
5.6 6.5
6 6 8 6 4
768 174 321 122 703
799 338 383 185 824
9.6 5.2 8.3 6.6 8.5
6
149 ,,
:L 67 5:23 :L: 68 ~- 13 % 283 73
~
27
5:116 ± 36 5:82 5:21 5:356
154 -[
50
9.7
Needle biopsy. For abbreviations, see Table 1. 6 c a s e s o f D M D t h e r a t i o w a s a g a i n s i g n i f i c a n t l y h i g h e r t h a n t h a t o f c o n t r o l s ( P < 0.01),
Internal nuclei Two cases of DMD
and 1 of LGD
showed differences between internal and
peripheral nuclei of the same biospy, Although phosphorus
o n l y a few a n a l y s e s w e r e p o s s i b l e
and chlorine concentrations were consistently higher in internal nuclei
( T a b l e s 4 a n d 5).
329 TABLE 4 RELATIVE MASS F R A C T I O N S ( × 103) OF PHOSPHORUS 1N P E R I P H E R A L A N D I N T E R N A L NUCLEI ( i SEM) Diagnosis
Peripheral nuclei (p)
Internal nuclei (i)
Ratio p i
Duchenne dystrophy
243 ± 22 a(9) 208 ± 41 (4) 280 ± 63 (4)
396 ± 26 (3) 217 ± 66 (5) 499 ± 164 (2)
0.61
Duchenne dystrophy Limb girdle dystrophy
0.96 0.56
,~ Number of analyses.
TABLE 5 RELATIVE MASS FRACTIONS ( × 103) OF C H L O R I N E IN PERIPHERAL A N D INTERNAL NUCLEI ( ± SEM) Diagnosis
Peripheral nuclei (p)
Internal nuclei (i)
Ratio p ]-
Duchenne dystrophy
389 ± 54 a(5) 70 :L 13 (4) 94 ± 28 (4)
588 ± 121 (3) 85 ± 6.4 (5) 134 ± 13 (2)
0.66
Duchenne dystrophy Limb-girdle dystrophy
0.82 0.70
:' Number of analyses.
TABLE 6 RELATIVE MASS FRACTIONS ( x 10:3) OF PHOSPHORUS IN NUCLEI OF N O R M A L FIBRES A N D FIBRES WITH MYOF1BR1LLAR LOSS ( ± SEM) Diagnosis
Normal (N)
Atrophic (A)
Ratio N
Significance (Student's t-test)
Duchenne dystrophy
243 •~(9) 101 (6) 208 (4) 280 (4)
472 ± 64 (6) 161 ± 18 (7) 131 ± 1.1 (1 O) 254 ± 62 (2)
0.51
P < 0.01
0.63
P < 0.05
1.59
P < 0.05
Duchenne dystrophy Duchenne dystrophy
Limb-girdle dystrophy
Number of analyses.
± 22 ± 15 _± 41
± 63
1.1
330 TABLE 7 R E L A T I V E MASS F R A C T I O N S ( :. 10 :~)OF M Y O F I B R I L L A R C A L C I U M
Diagnosis
No. of cases
Ca
N o r m a l males DMD FSH FSH D'myositis (on steroids)
6 6 1 I 1
107 135 506 438 880
(:: S E M )
i
15 ~ 13 5_- 73 ± 56 _% 206
F o r abbreviations, see Table 1.
Nuclei of fibres with loss of myofibrils (atrophic) Preliminary investigations of nuclei of fibres with reduced myofibrillar content suggested that the phosphorus content may differ from that of well-preserved fibres (Table 6). Fibres of 2 cases of DMD with only mild myofibrillar loss showed significantly higher nuclear phosphorus concentrations than those of well-preserved fibres in the same biopsy (P < 0.01 and P < 0.05). In another case of DMD and 1 of LGD the fibres analysed had severe loss of myofibrils and the nuclei had lower phosphorus concentrations than other nuclei in the same biopsy.
M yofibrils Comparison of myofibrillar regions in normal and diseased muscle showed elevated calcium concentrations in 2 cases of FSH and marked elevation in the case of dermatomyositis being treated with steroids (Fig. 4 and Table 7). Calcium concentrations in 6 cases of DMD, although elevated, were not significantly different from those of controls. DISCUSSION
Our preliminary results demonstrate that electron-microscopic X-ray microanalysis can localise differences in intrinsic elemental concentrations between normal and diseased muscle. The elements most frequently detected were phosphorus, sulphur, chlorine and calcium. The calcium to phosphorus ratio showed a consistent and significant difference between nuclei in muscle from normal and Duchenne dystrophy subjects. Variability in the relative concentrations of calcium and phosphorus was sometimes high, as shown by the SEM values, but the present data suggests that in DMD myonuclear calcium concentration is elevated whilst phosphorus is decreased. Additional, carefully controlled analyses may reduce the variation between readings and determine the specificity and relationship of these changes to clinical states and stages. Occasionally traces of potassium, iron and zinc were found. Other methods of specimen preparation and different instrumental conditions may detect alterations in the concentration of other elements. X-ray microanalysis of myocardium and extra-
331
Fig. 4. Spectra of myofibrils of (a) normal muscle, (b) Duchenne muscular dystrophy and (c) dermatomyositis. Note the variation in calcium content. ocular muscle of experimental animals has suggested that zinc may be altered in response to injury (Peters, Yarom, Dormann and Hall 1976). These authors used glutaraldehyde/osmium fixed material and a probe current twice that used in our study. The relationship of our recorded elemental concentrations to those in vivo is difficult to assess because of the effects of fixation and specimen preparation on the redistribution of elements. Some elemental differences may be secondary and relate to damage rather than to disease entities. Although our initial analyses were of struc-
332 turally well-preserved fibres ionic alterations may precede morphological damage. This has been demonstrated in mitochondria which accumulate calcium before any observed structural changes (Judah, Ahmed and McLean 1964). Localised alterations in elemental concentrations might therefore be a useful criterion for identifying damaged fibres which are ultrastructurally normal, particularly in subclinical states. In this context it is interesting to note the elevated calcium to phosphorus ratio in the carrier of DMD. More direct involvement of calcium in degenerative changes has been implicated (Busch et al. 1972). Destruction of Z-lines is a frequent ultrastructural abnormality in myofibres and may be caused by calcium activation of a degradation catalyst. Cell volume and electrical phenomena, which are known to be influenced by ionic concentrations, may be related to increased sarcoplasmic volume, dilatation of sarcoplasmic reticulum and hypercontraction of myofibrils observed in muscular dystrophy (Cullen and Fulthorpe 1975). Wrogemann and Pena (1976) have suggested that a net influx of calcium into the cell triggers events leading to necrosis. Alterations in calcium concentrations were mainly confined to myonuclei with the exception of a few cases showing increased myofibrillar calcium. Improved methods of specimen preparation might enable detection of elemental changes in other locations. However, analysis of organelles known to transport calcium, such as sarcoplasmic reticulum (Hasselbach 1964) and mitochondria (Carafoli 1974), is limited by the minimum probe diameter. In our preparations these structures were often too small for accurate localisation. Alterations in nuclear phosphorus concentrations may be related to several biochemical activities. Nucleotide content and protein synthesis have been the subject of several investigations of dystrophic muscle. Watts and Reid (1968) reported that DNA and RNA content was about twice as high in dystrophic mouse muscle compared to normal muscle, whereas dystrophic chick myoblasts in culture were shown to have a markedly reduced nuclear and cytoplasmic RNA content (Ross and Jans 1968). There is also evidence suggesting alterations in cyclic nucleotides in diseased muscle (Zymans, Epstein, Saifer, Aronson and Volk 1959; Canal, Frattola and Smirne 1975) and their involvement in several cellular processes such as cell division (Sheppard and Plagemann 1975), myogenesis (Epstein, Jiminez de Asua and Rozengart 1975; Zalin and Montague 1975) and phosphorylation of phosphoproteins (Wagner 1975). Phosphorylation of non-histone proteins has been shown to be involved in chromosome structure and gene activation (Kleinsmith 1975; Thi Man, Morris and Cole 1975) suggesting that alterations in phosphorus concentrations could be specific and relate more directly to disease processes. Our initial analyses of structural abnormalities suggest that elemental differences might be detectable in internal nuclei and fibres with myofibrillar loss. Interpretation of the observed alterations in phosphorus and chlorine is difficult at this stage and our results need to be substantiated with more data. It is important to note that elemental differences in diseased muscle were observed in more than one cell type. Both interstitial and myonuclei showed significantly higher calcium to phosphorus ratios in diseased muscle compared with normal muscle. This may reflect a generalised membrane dysfunction similar to that already suggested
333 as the basic defect in muscular dystrophy (Roses, Herbstreith and Appel 1975: Mawatari, Schonberg and Olarte 1976; Sha'afi, Rodan, Hintz, Fernandez and Rodan 1975). At this stage, however, any conclusions drawn from our results are speculative. More data is required to confirm our initial findings and to establish the normal variations of elemental content. Correlative biochemical analyses are also needed to assess the validity and advantages of X-ray analysis and to determine the reasons for observed elemental differences. However, our results provide unique information on the distribution of elements and highlight some interesting differences between normal and diseased muscle. With improved methods of specimen preparation it is hoped that this new approach may clarify some of the processes related to the pathogenesis of neuromuscular disorders. ACKNOWLEDGEMENTS
We thank Dr. T. A. Hall, Department of Zoology, University of Cambridge for the use of the EMMA 4 and his helpful advice.
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