Immunocytochemical studies of cytochrome oxidase subunits in skeletal muscle of patients with partial cytochrome oxidase deficiencies

Journal of the Neurological Sciences, 1988, 87:75-90

75

Elsevier JNS 03048

Immunocytochemical studies of cytochrome oxidase subunits in skeletal muscle of patients with partial cytochrome oxidase deficiencies M . A . Johnson 1, B. Kadenbach 2, M. Droste 2, S.L. Old ~ and D . M . Turnbull 1 IDepartment of Neurology, Universityof Newcastle upon Tyne (U.K.), and 2Fachbereich Chemie (Biochemie), University of Marburg, Marburg (F.R.G.)

(Received 8 December, 1987) (Revised,received29 May, 1988) (Accepted 30 May, 1988)

SUMMARY Muscle biopsies from 17 patients with partial cytochrome oxidase deficiencies were investigated using immunocytochemical techniques for the localisation of cytochrome oxidase subunits. Antisera to subunits II/III (mitochondrially coded) and subunits IV, Vab, Vlbc, VIIa, VIIbc and VIII (nuclear coded) showed clear particulate immunoreactivity in the muscle fibres of normal control biopsies. In the patients studied, muscle fibres with absent or decreased cytochrome oxidase activity also showed decreased immunoreactivity affecting all enzyme subunits. Particularly close correlation was seen between percentages of fibres showing absent enzyme activity and those showing decreased immunoreactivity for subunits II/III which are catalytic in function. The regulatory subunits IV-VIII were affected to varying degrees with different patterns of subunit loss occurring in individual muscle fibres.

Key words: Cytochrome oxidase; Mitochondrial myopathy; Enzyme subunits; Immunolabelling INTRODUCTION Partial deficiencies of cytochrome c oxidase activity have been found in many patients with chronic progressive external ophthalmoplegia (CPEO) and myopathy, and Correspondence to: Dr. M.A. Johnson,MuscularDystrophyGroupResearchLaboratories,Regional Neurological Centre, NewcastleGeneral Hospital, Newcastleupon Tyne, NE4 6BE, U.K.

0022-510X/88/$03.50 © 1988Elsevier SciencePublishers B.V.(BiomedicalDivision)

76 in other oculocraniosomatic syndromes (Johnson et al. 1983; Maller-H0cker et al. 1983). Not all muscle fibres are equally affected; some fibres contain virtually no cytochemically demonstrable enzyme activity whereas others have relatively normal activity. This partial defect of cytochrome oxidase is thus different from that which occurs in the muscle of patients with Leigh's disease (subacute necrotising encephalomyelopathy) in which a general decrease in catalytic activity is found throughout the muscle. The fatal infantile form of total cytochrome oxidase deficiency and the benign infantile condition, characterised by a gradual resolution of the enzyme defect, also appear to affect the muscle fibre population in a relatively uniform manner. (For a review of mitochondrial myopathies associated with cytochrome oxidase deficiency see Di Mauro et al. (1985).) In the myopathy associated with CPEO many cytochrome oxidase-deficient fibres show morphological evidence of mitochondrial abnormality, such as the presence of peripheral accumulations of structurally abnormal mitochondria ("ragged-red" fibres). It is likely that the enzyme defect precedes the development of gross structural abnormalities and in occasional patients quite severe impairment of enzyme activity may occur in the absence of the usual morphological abnormalities (Turnbull et al. 1985). In some patients with oculocraniosomatic syndromes, the cytochrome oxidase deficiency may occur in conjunction with defects at other sites in the respiratory chain, notably complex I deficiency (Morgan-Hughes and Landon 1983; Sherratt et al. 1984) and complex III deficiency (Hayes et al. 1984); considerable heterogeneity exists therefore among these patients. In the present study we have examined muscle biopsies from patients with partial cytochrome oxidase deficiency using immunocytochemical techniques in order to investigate possible abnormalities affecting the protein subunits of the enzyme. Cytochrome c oxidase is a complex membrane-integrated protein consisting of 13 different subunits (Kadenbach et al. 1983; Takamiya et al. 1987) of which subunits I, II and III are mitochondrially-coded (Schatz and Mason 1974; Slonimski and Tzagoloff 1976) whereas the remainder: subunits IV, Va&b, VIa,b&c, VIIa,b&c and VIII are nuclearcoded. The main objective of this study was to determine whether muscle fibres showing little or no cytochemically-detectable cytochrome oxidase activity also showed deficiencies of anti-cytochrome oxidase immunoreactivity. We also wished to investigate whether all enzyme subunits were equally affected or whether there was evidence that one or more subunits were selectively decreased.

MATERIALS AND METHODS

Muscle biopsies were obtained for diagnostic purposes from 17 patients with oculocraniosomatic syndromes. All biopsies were taken from m. quadriceps except for 1 biceps brachii and 1 pectoralis major. All patients showed evidence of chronic progressive external ophthalmoplegia and/or ptosis and had muscle weakness of varying degrees, usually proximal in distribution. Patients at time of biopsy were 14-70 years old (mean 37) with approximately equal incidence of males (8) and females (9).

77 Control biopsies were obtained from m. quadriceps of 4 female and 2 male subjects between the ages of 18 and 38, in whom there was no evidence of a neuromuscular disorder. Subunit specific antisera were raised in rabbits as documented previously (Merle et al. 1981) using purified human heart cytochrome oxidase subunits as immunogens (Kuhn-Nentwig and Kadenbach 1986). Western blot analysis of antisera was performed as described (Kuhn-Nentwig and Kadenbach 1985). The blotting buffer consisted of 150 mM glycine, 20 mM Tris, 20~ methanol, 0.1~o SDS, pH 8.3. Determination of antisera titer by ELISA was done in fiat-bottom micro-ELISA plates (Immulon from Dynatech). Each well was incubated overnight at 4 °C with 0.3 #g human heart cytochrome ¢ oxidase, dissolved in 0.1 m150 mM sodium borate, 8 M urea, pH 9.5. All other incubations were done at room temperature. After washing with 0.2 ml PBS buffer 1~o BSA for 1 h, and with 0.2 ml PBS for 10 min, each well was incubated with 0.1 ml of one of the various antisera, diluted with PBS, 1~o BSA as indicated in Fig. 1 for 2 h at room temperature. After 3 washings with 0.2 ml PBS, 0.1 ~o Triton X-100 for 5, 10 and 15 min respectively, 0.1 ml swine anti-rabbit peroxidase conjugated antibody (Dakopatts, Hamburg) (diluted 1 : 1000 with PBS, 1~ BSA) was added and incubated for 1 h at room temperature. After washing for 10 and 15 min with 0.2 ml PBS, 0.1Fo Triton X- 100 and for 10 min with 0. I M sodium-acetate, 0.05 M NaH2PO4, pH 4.2, the wells were incubated for 40 min at room temperature with 0.1 ml 2 mM 2,2'-azino-di(3-ethylbenzthiazoline)sulfonic acid (ABTS) in 0.1 M sodium acetate, 0.05 M NaH2PO 4, pH 4.2 and 2.5 mM H202. The absorbance of the stain in the wells was read automatically by a Titertek Multiskan photometer. Muscle biopsies were frozen in dichlorodifiuoromethane (Arcton 12, ICI) cooled to - 150 °C in liquid nitrogen. For cytochemical studies 10-#m thick sections were cut using a motorised cryostat microtome (Reichert Frigocut) and used to demonstrate activities ofmyofibrillar ATPase at pH 9.5 after preincubation at pH 4.3 or 4.6 (Brooke and Kaiser 1970), succinate dehydrogenase, NADH-tetrazolium reductase (Pearse 1972) and cytochrome c oxidase (Seligman et al. 1968). For immunocytochemical studies serial 6-#m thick sections were cut, mounted on chrome-gelatin coated slides and air-dried for 2 h. The slides were then wrapped in cling-film and stored at - 40 °C for periods up to 3 weeks. Prior to immunostaining, sections were fixed for 1 h at 20 °C in formol-calcium (4~ aqueous formaldehyde containing 0.1 M calcium chloride, pH 7.0), and were then transferred through a graded ethanol series to 100~o ethanol for 15 min before being rehydrated and rinsed in 0.0125 M Tris-buffered saline (TBS) pH 7.6 for 5 min. Subunit-specific antisera were optimally diluted (see Results) in TB S, pH 7.6, and 50 #1 diluted antiserum was applied to each section for 1 h at 20 ° C. Dehydration was prevented by coveting sections with a coverglass, which also ensured even distribution of antiserum. Sections were then washed 4 × in TBS during 30 min. Swine anti-rabbit immunoglobulins (Dako, Z196) were diluted 1 : 100 in TBS and applied to sections for 1 h followed by washing 4 × in TBS as above. Rabbit peroxidase-antiperoxidase complex (PAP, Dako, Z113) diluted 1 : 100 was used to detect bound immunoglobulins (Sternberger et al. 1970). A medium containing 0.05~o 3,3'-diaminobenzidine hydro-

78 chloride (DAB) and 0.01Y/o H 2 0 2 in 0.1 M Tris, pH 7.6, was used to visualise peroxidase activity. Incubation was for 10 rain at 20 °C; sections were then washed, dehydrated and mounted in DPX.

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Fig. 1. Western blot of human heart cytochrome c oxidase with various antisera to isolated subunits. Cytochrome c oxidase from human heart (lane 1-10) or rat liver (lane 11) was separated by SDS-polyacrylamide gel electrophoresis (Kadenhach et al. 1983), blotted on nitroccUulose (Schleicher & Schilll, Dassel) and stained with amido black (lanes 10 and 11) or incubated with antisera either to the total human heart enzyme, lane 1; or to isolated subunit VIII, lane 2; VIIbc, lane 3; VIIa, lane 4; VIbc, lane 5; Via, lane 6; Va,b, lane 7; IV, lane 8; II, III, lane 9. The antisera were diluted 1 : 50 (lanes 2,6); 1 : 200 (lanes 1,9); 1 : 400 (lanes 3,4,5,7); 1 : 800 (lane 4).

79 RESULTS

Normal skeletal muscle Monospecific antisera to subunits II/III, IV, Yah, Via, VIbc, VIIa, VIIbc and VIII were characterised by Western blot (Fig. 1) and ELISA (Fig. 2). The antiserum to subunit VIIa also reacts strongly with subunits VIIbc. Strong particulate (mitochondrial) immunoreactivity was noted using sections of all six normal control muscles with antisera to subunits II/III, Vab, VIbc, VIIa and VIIbc at dilutions of 1 : 100 and with antiserum to subunit IV at 1:200 dilution. The antiserum to subunit Via gave consistently faint immunostaining of normal muscle sections even at dilutions of 1 : 50 and below. Antiserum to subunit VIII gave particulate immunostaining at 1 : 50 dilution (Fig. 3a-i). Immunostaining of sites outside mitochondria was rare. Antiserum to subunit VIIa gave a nuclear reaction which was more pronounced in some normal control muscle samples than in others. Antiserum to subunit VIII gave a marked capillary endothelial reaction in one normal control subject but not in any of the other five. Control reactions using non-immune rabbit serum as the In'st layer antiserum gave consistently negative results as did subunit-specific antisera preabsorbed using an excess of purified human heart cytochrome c oxidase. Preabsorption was done using antigen-coated microtitre plates and diluted antisera incubated at 4 °C overnight.

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oooo 1:100(20 1:2000 1:500 I:100 Antiserum dilution Fig. 2. Characterisation of antisera to subunits of human heart cytochrome c oxidase. The ELISA was performed as described under Methods. The symbols refer to the following antisera: I I - - - - - - - ~ , II/III; © O, IV; • V Vab; A A Via; A ~ & , VIbc; • Q, VIIa; V V, VIIbc; l ill, VIII.

80

Fig. 3. Normal control skeletal muscle showing immunoreactivity with antisera against cytochrome oxidase subunits (peroxidase-anti-peroxidase technique) in serial sections. (a) II/III, (b) IV, (c) Vab, (d) Via, (e) VIbc, (f) VIIa, (g) VIIhc, (h) VIII, (i) non-immune control. There is clear particulate immunostaining with all antisera except anti-Via and the non-immune control (calibration bar = 50 #m).

81

Optimal immunostaining of cytochrome oxidase subunit antigens was not possible using untreated frozen sections. Sections which were simply air-dried for 2 h before immunostaining gave a patchy localisation of reactive sites. It is likely that this was caused by inadequate penetration of immunoglobulins through mitochondrial membranes. Probably only mitochondria which were cut through during sectioning or otherwise damaged by freezing and thawing were accessible to antisera in the untreated sections. However, immunoreactivity of cytochrome oxidase subunits is retained in formalin-fixed, paraffin-embedded material (Mttller-HOcker et al. 1986) which indicates that a combination of formaldehyde and ethanol treatment during processing does not adversely affect immunolocalisation. In the present study it was necessary to use frozen sections for immunolocalisation in order to be able to correlate directly cytochemical findings (e.g., cytochrome oxidase-negative fibres and fibres showing peripheral accumulations of mitochondria in succinate dehydrogenase preparations) with any abnormal immunostaining patterns. It was found that brief fixation of cryostat sections in formol-calcium, a commonly used membrane-stabilising fixative, followed by treatment of the sections in a graded ethanol series resulted in much more effective access of immunoglobulins and hence much improved localisation of subunit antigens.

CytochemicalJ'mdings in muscle biopsiesfrom patients with oculocraniosomatic syndromes The incidence of muscle fibres in which cytochrome c oxidase activity was absent or severely decreased was determined using samples of ca. 150 fibres per muscle biopsy. Serial sections were used to determine the incidence of "ragged-red" fibres (those showing abnormal peripheral accumulations of mitochondria detectable in succinate dehydrogenase (SDH) preparations). In biopsies from the 17 patients studied, proportions of cytochrome oxidase-deficient fibres ranged from 12 to 67% (mean 26%). The percentage of fibres showing "ragged-red" type changes was considerably lower (mean 9%). Affected fibres of all metabolic fibre types, as defined using myofibriUar ATPase reactions, occurred but type 1 (slow oxidative) fibres were frequently more severely affected than fibres of type 2A (fast oxidative-giycolytic) or type 2B (fast giycolytic). An abnormal predominance of type 1 fibres was common in these patients with 10/17 showing this feature. In previous studies we have shown that fibres with abnormal peripheral accumulations of mitochondria ("ragged-red" fibres) are almost always cytochrome oxidasenegative (Johnson et al. 1983). However, exceptions to this do occur, especially in patients in whom a further abnormality of the respiratory chain has been demonstrated in addition to cytochrome oxidase (complex IV) deficiency (Sherratt et al. 1986). The morphological mitochondrial abnormalities in fibres with normal cytochrome oxidase activity may therefore be associated with the deficiency of the other respiratory chain component known to be abnormal, e.g. complex I or complex III. In the current study of 9 patients investigated biochemically, complex I deficiency has been identified in 5 patients and complex III deficiency in 1 (data not shown) in addition to the defect of complex IV which was common to all 17 patients. Abnormalities of mitochondrial ATPase similar to those described by Mflller-Htcker et al. (1985) were seen with varying degrees of severity in 12/17 patients.

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~i!ili Fig. 4. Skeletal muscle from patient with partial cytochrome oxidase deficiency. Serial sections show: (a) cytochrome oxidase enzyme reactivity, a cluster of 6 negative fibres is marked with asterisks. - - - ,

83

Immunocytochemical studies using subunit antisera Sections serial to those used for enzyme reactivities were used throughout the immunocytochemical investigations. Immunostaining of muscle from patients was always done in parallel with a sample of control muscle. The normal control muscles always showed discrete, particulate (mitochondrial) reactivity using optimally diluted antisera, immunostaining being present in all fibres in the biopsy sections. Distinction between type 1 (oxidative) and type 2 (glycolytic) fibres was considerably less clear than with cytochemical methods for the demonstration of the catalytic activity of cytochrome oxidase. This is because the immunocytochemical method gives an intensity ofimmunolabelling proportional to the amount of enzyme protein present whereas the enzyme cytochemical technique demonstrates the difference in catalytic activity which exists between the major metabolic fibre types. Even in the fibres with lowest cytochrome oxidase activity in control muscles (type 2B fibres) clear immunostaining was seen in all cases. In all CPEO biopsies studied, a proportion of the muscle fibres showed abnormal immunostaining reactions (see Fig. 4a-i). Abnormalities were of three main types: (i) virtual absence of reaction, (ii) absence of particulate (mitochondrial) reaction but diffuse immunostaining present, (iii) severely decreased particulate reaction. In each biopsy the proportion of fibres showing abnormal immunostaining was quantitated and correlated with the enzyme cytochemical f'mdings in serial sections. The location of fibres showing absent or decreased immunostaining using each of the subunit antisera was plotted on tracings of photomicrographs of the areas of muscle under examination. The analysis of the immunostaining pattern in fibres with absent or decreased cytochrome oxidase activity is shown for four patients in Fig. 5. These patients (JW, AJ, RL and DB) showed 14 ~o, 25 ~ , 37 ~ and 67 ~o cytochrome oxidasedeficient fibres, respectively. In the 17 patients studied, an average of 40 cytochrome oxidase deficient fibres were analysed (range 14-75) from samples consisting of ca. 150 fibres. As may be seen from Fig. 5 the pattern of defective immunostaining varied from fibre to fibre but there were no constantly recurring associations of two or more defective subunits in individual fibres. Thus, although some subunits were affected with greater frequency than others, the pattern of occurrence of defective subunits was essentially random. (b) lI/lIl, the 6 fibres show examplesof negativereaction(o), diffusereaction(~7), and decreasedreaction (V). (c) IV, 3 of the fibres show a decreased reaction but the others show normal (O) or near-normal (0) immunoreactivity. (d) Vab, decreased or diffuseimmunostalningin most of the cytox - ve fibres. (e) VIbc, fibres show decreased immunoreactivity. (f) VIIa, most fibres show decreased or negative reaction but note positivefibre as in (c) and (g). (g) VIIbc, note closelysimilar pattern to VIIa. (h) VIII, diffuseimmunostainingseen in type 1 fibres,rest of fibres negative; reaction in muscle capillaries is apparent. (i) MyofibrillarATPase after preincubationat pH 4.6, one fibre of the cytox - ve cluster is type 1, rest are type 2A (calibration bar = 50 #m).

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Fig. 5. Immunoreactivityusing anti-subunitsera in cytochromeoxidase-deficientfibres from 4 patients. In this figure negative or decreased immunostainingis designated by the symbol o. Fibres with negative enzyme reaction are also designated o, and those with severely decreased activity are indicated by *. A representative sample of 25 fibres from each patient is shown.

When the percentages of cytochrome oxidase deficient fibres showing abnormal immunostaining with each of the subunit antisera was examined, it was found that antiserum to subunits II/III showed the closest correlation with absent or decreased enzyme activity. With this antiserum ca. 80~o of enzyme-deficient fibres showed decreased immunostaining. The mean percentages (obtained from analyses of the 17 patients) in the case of the other subunit antisera varied from 50 to 70 ~o (see Table 1). In spite of the variability between patients in the proportions of affected fibres, within individual patients the pattern of severity of decrease in individual subunits was fairly constant and followed the progression II/III > VIIbc > Vab and VIIa > Vlbc > IV in most cases. Whereas the optimal dilution of antisera was determined by titration on normal muscle sections, a range of dilutions on either side of optimal was also used. This allowed the following distinction to be made. If"negative" immunostaining was due to

85 absence of detectable antigen, affected fibres remained negative even using higher antibody concentrations. If decreased immunostaining was detectable only using lower antibody concentrations this indicated that some antigen was still present though in decreased amounts. TABLE 1 PERCENTAGES OF CYTOCHROME O X I D A S E - D E F I C I E N T FIBRES S H O W I N G ABNORMAL IMMUNOSTAINING Data are derived from percentages of affected fibres in individual patients; n = 17.

Subunit antisera

% fibres (mean _+ SD)

II/III IV Vab Via Vlbc Vlla VIIbc VIII

81 51 61 * 53 59 70 *

+ 18 + 20 + 17 + 18 + 21 + 20

* Reactions with anti-Via and anti-VIII were not strong enough to permit evaluation.

There was no evidence that any particular fibre type was especially susceptible to loss of cytochrome oxidase activity or to an associated loss of specific immunoreactivity. The fact that all fibre types show loss of catalytic activity in CPEO patients has been documented previously using quantitative cytochemical techniques (Johnson et al. 1983). In the present study the percentage incidence of affected fibres was 21.5 type 1, 26.2~o type 2A and 19.1 ~o type 2B, confn'ming that no fibre type was selectively affected. Clear particulate immunoreactivity was present in normal muscle in all fibre types including type 2B fibres which contain fewest mitochondria per unit area; hence little difficulty was experienced in deciding whether any individual fibres in CPEO patients showed decreased immunoreactivity. The antiserum to subunit IV frequently revealed a lower percentage of fibres with decreased immunostaining than other antisera and only a small proportion of these fibres showed no detectable antigen. This relatively poor correlation of decreased enzyme activity and subunit IV immunoreactivity is reflected in the regression analysis of percentages of affected fibres in the 17 patients studied (Table 2). Whereas there was a strong positive correlation between percentages ofcytochrome oxidase deficient fibres and percentages of fibres showing decreased immunoreactivity with other antisera (r = 0.745 to 0.841 ; P < 0.001) the correlation with subunit IV immunoreactivity was not significant (r = 0.441; P > 0.05). Strong positive correlations were found between proportions of fibres deficient in subunits II/III, Vab, Vlbc, VIIa and VIIbc (r = 0.851 to 0.968; P < 0.001) but the correlation with decreases in subunit IV immunoreactivity was much less strong.

86 TABLE 2 R E G R E S S I O N ANALYSIS OF P E R C E N T A G E S O F FIBRES D E F I C I E N T IN C Y T O C H R O M E O X I D A S E (CYTOX) ACTIVITY A N D A N T I - S U B U N I T I M M U N O R E A C T I V I T Y

Cytox II/III IV Vab Vlbc VIIa

II/III

IV

Vab

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Vlla

VIIbc

0.841

0.441 0.498

0.773 0.910 0.479

0.745 0.888 0.640 0.939

0.836 0.928 0.4 t 7 0.922 0.851

0.833 0.934 0.489 0.968 0.895 0.946

n = 17; when r > 0.725, P < 0.001; when r > 0.482, P < 0.05.

Of fibres showing absent or severely decreased immunoreactivity with subunit antisera, the vast majority were also cytochrome oxidase-deficient and in any one patient no subunit antiserum showed more defective fibres than the total number of cytochrome oxidase deficient fibres in that particular biopsy. However, discrepancies where decreased immunoreactivity was accompanied by normal cytochrome oxidase activity did occur. Percentage incidence of this discrepancy was low: subunit II/III 9.9~o, IV 3.2~o, Vab 5.6~o, Vlbc 6.1~, VIIa 9.7~o, VIIbc 10.8~o (mean values). Some of the anomalous situations where loss of immunoreactivity was seen in spite of normal cytochrome oxidase activity may be due to inhomogeneity along the length of muscle fibres. In this study the total length of muscle fibre traversed in a set of serial sections is ca. 200/tm. Recent work (Sengers et al. 1986) has shown that "ragged-red" fibres do not necessarily display their characteristics of abnormal mitochondrial distribution throughout their whole length. We have also confn'med that this is also true of cytochrome oxidase negativity and hence some instances of discrepancy between enzyme activity and immunostalning may be due to this inlaomogeneity. We repeated our serial immunostaining in some patients with the highest incidence of this discrepancy between catalytic and immunolabdling data, using sections at the beginning and end of the series as indicators of cytochrome oxidase activity. Even when catalytic activity was absent in both cases, some instances of positive immunostaining were seen in the intermediate sections. It seems likely that fibres in a state of transition between normal and enzyme-deficient show a patchy distribution of normal and abnormal mitochondria at this stage in the evolution of the disorder. In general, lack of cytochrome oxidase activity was found in many more fibres than either decreased immunostaining or "ragged-red" type of morphological mitochondrial abnormalities. The implication here is that loss of enzyme activity appears to precede any decline in the m o u n t of subunit protein detectable by immunocytochemical techniques, the development of gross structural mitochondrial abnormalities taking place at an even later stage. In 1 patient in this study, documented in a previous paper (Turnbull et al. 1985) no "ragged-red" fibres were seen and yet over 30~o fibres in this patient were cytochrome oxidase-deficient. This patient's muscle was also unusual as

87 regards the results obtained with subunit antisera where negative or decreased immunoreactivity was never found in more than 25 ~ of the cytoehrome oxidase-deficient fibres (anti-subunit II/III serum), with even lower proportions of fibres showing decreased immunostaining with other subunit antisera. The situation regarding subunit VIII differs from the findings with other subunits. Although discrete particulate immunoreactivity was shown by all 6 control muscle samples, albeit at a lower dilution than that used for the other subunit-specific antisera, in all patients' muscle biopsies except two the immunoreactivity was so indistinct as to make evaluation of the reaction in individual fibres not feasible. One interpretation of this finding is that unlike the other subunits whose immunoreactivity is often preserved after enzyme activity is demonstrably decreased, anti-subunit VIII immunoreactivity declines in a large proportion of fibres prior to loss of catalytic activity. With antisubunit VIII serum a marked capillary reaction was seen in 6/17 patients and also in one of the 6 control biopsies; the significance of this finding is at present unclear. The antiserum to subunit Via consistently gave a negative reaction in patients' muscle biopsies and at best a very faint reaction in the biopsies from normal controls, even at dilutions less than 1 : 50. It is possible that subunit Via in skeletal muscle differs from its counterpart in cardiac muscle to which the antiserum was raised. Western blots of normal human skeletal muscle showed that the antiserum to cardiac muscle subunit Via reacted with the corresponding isolated skeletal muscle subunit, although faintly. It may be therefore that epitopes recognised by the antiserum are not detectable in the immunocytochemicai system, possibly because they are located within the hydrophobic lipid domain of the membrane and inaccessible to aqueous reagents, even after treatment designed to increase membrane permeability.

DISCUSSION The abnormal patterns of immunoreactivity observed using subunit-specific antisera on muscle biopsies from patients with partial cytochrome c oxidase deficiencies permit several interpretations. The total absence of any one enzyme subunit might indicate failure of translation and/or transcription of the corresponding gene (i.e. mutation of the gene). However this type of abnormality was not observed in the present study. Although individual muscle fibres showing total absence of immunoreactivity towards a given subunit were seen, such fibres comprised only a proportion of those lacking catalytic activity and, moreover, normal immunoreactivity was present in much of the muscle fibre population. The absence of particulate (mitochondrial) immunostaining but the presence of diffuse (cytosolic) immunoreactivity might, in the case of nuclear-coded subunits, indicate normal expression of the subunit protein but defective transport across the mitochondrial membrane and/or defective assembly of the enzyme complex within the inner membrane. Diffuse immunostaining seen with antisera to subunits IV-VIII could be due to this type of defect; however, diffuse immunostaining was also seen in some patients' biopsies using anti-subunit II/III serum. Since subunits II and III are mito-

88 chondrially-coded and synthesised, defective transport cannot be invoked to explain this finding. It is more likely that abnormal mitochondria may liberate subunit proteins or breakdown products thereof and that these may be the cause of the observed diffuse cytosolic reaction. In normal control muscles diffuse cytoplasmic immunoreactivity was not observed. The most commonly observed abnormality was that of decreased particulate immunoreactivity; this probably indicates defective regulation of gene expression, the corresponding gene itself not being primarily involved. Such a mechanism would seem most likely to operate in situations such as the partial enzyme deficiency described here, where a large proportion of muscle fibres retain both normal catalytic activity and immunoreactivity. All the patients studied showed a fairly constant pattern of severely and less severely affected subunits. Antisera to subunits II/III gave the greatest degree of correlation with decreased enzyme activity; this is not surprising since these two subunits are involved in the catalytic activity of the enzyme complex. Using anti-subunit II/III sera, 11/17 patients showed decreased immunostaining in 80-100~o fibres where catalytic activity was impaired. Strong positive correlation of decreased enzyme activity with decreased immunoreactivity was also seen with subunits Vab, Vlbc, VIIa and VIIbc, all these subunits being regulatory rather than catalytic in function. Subunit IV showed somewhat less decrease in immunoreactivity with considerable variation between the biopsies from the 17 patients. The reaction of subunits in situ with their corresponding antisera may differ profoundly from what occurs with isolated subunits. Investigations using chemical labelling (Ludwig et al. 1979) and the reactions of subunit-specific antisera with isolated mitochondria or mitoplasts (Chan and Tracey 1978; Kulm-Nentwig and Kadenbach 1985) have suggested that some subunits may be orientated to the cytosolic face of the membrane. Individual subunits appear to be buried to different extents in the hydrophobic lipid domain of the membrane (for review see Kadenbach et al. 1987). It is possible, therefore, that epitopes against which the immune response is directed may be inaccessible, because of steric hindrance or because they are located deep in the hydrophobic phase of the membrane. The results reported in the present study, involving significant decreases in immunoreactivity affecting a majority of subunits, may be explicable by a defective assembly of the enzyme complex. Since it appears that the cytochrome oxidase defect in these patients is progressive, affecting an increasing proportion of muscle fibres during the course of the disease (Bresolin et al. 1987), loss of cytochrome oxidase activity may be due to an increasing failure of subunits to be properly incorporated into the membrane. Jarausch and Kadenbach (1985) have analysed the nearest neighbour relationships of cytochrome oxidase subunits using cleavable cross-linking agents. Subunit II links directly with subunits IV, Va, VIa,b,c, and VIIc; subunit III links to VIb and subunit I links to Va and b, VIIb and VIII. There are thus direct links between at least one of the "catalytic" subunits I, II and III and each of the "regulatory" subunits with the exception of VIIa which appears not to form cross-links with any other subunit. If we assume that the partial cytochrome oxidase defect described in the present paper

89 is due to defective regulation of the synthesis of the mitochondrially-coded catalytic subunits II/III, then the associated decrease in immunoreactivity of nuclear-coded subunits may be due to the impossibility of incorporating these subunits correctly when subunits I I / I I I are absent, and their consequent proteolytic degradation when not correctly assembled. A mechanism involving defective incorporation is compatible with the varying involvement of individual subunits seen from fibre to fibre. Whereas the cytochemical properties of normal muscle fibres are uniform along the length of the fibres, the defect of cytochrome oxidase and the presence of morphological mitochondrial abnormalities may be confined initially to segments of the fibres. Since the defect is progressive it is likely that it not only affects a greater proportion of muscle fibres as it advances, but also affects individual muscle fibres more extensively. The nature of the primary defect in chronic progressive external ophthalmoplegia (CPEO) is not known. In addition to the deficiency of complex IV (cytochrome c oxidase), defects involving complexes I and III and mitochondrial ATPase may occur. Frequently more than one of these defects are present in the muscle of individual patients. The enzyme proteins involved are all coded in part by the mitochondrial genome (Anderson et al. 1981). The defects involving cytochrome oxidase and mitochondrial ATPase are known to affect subpopulations of muscle fibres; this may also be true of defects of complex I and complex III but remains to be proven. Although the decreases in cytochrome oxidase activity recorded in muscle homogenates from C P E O patients may be relatively modest, due to the continued presence of fibres with normal enzyme activity, effects within individual cytochrome oxidase-deficient fibres may be severe and it is possible that the viability of such fibres will be impaired. A more complete understanding of this disorder requires continued investigation of individual respiratory complexes at the cellular level.

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