Acta Neuropathol (1992) 83:140-148

Acta Neuropatholostca (~) Springer-Verlag 1992

The fate of dystrophin during the degeneration and regeneration of the soleus muscle of the rat* R. Vater, M. J. Cullen, L.V.B. Nicholson, and J. B. Harris Muscular Dystrophy Group Research Laboratories, Regional Neurosciences Centre, Newcastle General Hospital, Westgate Road, Newcastle-upon-TyneNE4 6BE, Great Britain Received June 25, 1991/Revised, accepted August 16, 1991

Summary. Immunocytochemistry and Western blotting were used to monitor the fate of dystrophin in the soleus muscle of the rat during a cycle of degeneration and regeneration induced by inoculation of the muscle with the venom of N o t e c h i s scutatus scutatus (the Australian tiger snake). In control muscle dystrophin was localised close to the plasma membrane. Dystrophin began to break down 3-6 h after venom inoculation, giving a characteristic discontinuous labelling pattern. At 12 h dystrophin was absent from the plasma membrane, and by 1 day the architecture of the muscle fibres had completely broken down. By 2 days post inoculation regeneration had commenced. The regenerating myofibres possessed well-organised myofibrils and the plasma membrane was intact. Dystrophin was detected by Western blot at 3 days, but was not seen in sections until regeneration of the muscle was well advanced, at 4 days post inoculation. The results suggested that although dystrophin was present in the myofibres at 3 days, it was not incorporated into the plasma membrane until 4 days post inoculation. This may be due to the influence of the functional reinnervation of the regenerating fibres, which occurs at 4-5 days, or to the growing fibres reaching a critical diameter. Key words: Dystrophin - Muscle degeneration - Muscle regeneration - Snake venom

The skeletal muscle of normal, healthy vertebrates recovers spontaneously from injury (see, e.g., [9, 15, 16, 18]). In many diseases of skeletal muscle, however, regeneration is impaired and the primary characteristic of these diseases is the progressive loss of functional muscle fibres. * Supported by the MuscularDystrophyGroup of Great Britain, the WeUcomeTrust, and the MRC Offprint requests to: R. Vater (address see above)

The best documented of the degenerative muscle diseases are the X-linked muscular dystrophies (e.g. Duchenne and Becker muscular dystrophy in man [12, 17], and the X-linked diseases of mice [5] and dogs [25]). These disorders are associated with abnormalities in the gene for the large 400-kDa cytoskeletal protein, dystrophin [20], which may be absent or produced in an abnormal form in Duchenne and Becker muscular dystrophy [2, 19, 30, 32]. Current therapeutic strategies are directed towards the introduction of a functional dystrophin gene into diseased muscle either by myoblast transfer [22, 33] or by the direct introduction of the appropriate gene [43]. If such strategies are to be successful, it is necessary for any expressed dystrophin to be correctly localised to the plasma membrane as the damaged fibres repair themselves. Little is known of the fate of dystrophin during the degeneration and regeneration of normal skeletal muscle [21].We have used a combination of biochemical and immunocytochemical techniques at both light and ultrastructural (EM) level to monitor the loss of dystrophin during experimentally induced necrosis of skeletal muscle and its re-expression during regeneration of the muscle. The aims of this study were to provide information on how and when dystrophin is lost in relation to other events during muscle degeneration, and at what stage of regeneration it can be detected again.

Materials and methods Source o f tissue

Female Wistar rats weighing90-120 g were anaesthetised (halothane/N20/O2) and a single subcutaneous injection of 15 ~tg of whole venom (0.2 ml of 75 ~g/ml in 0.9 % w/v NaC1) from the Australian tiger snake, Notechis scutatus scutatus, was made into the dorso-lateral aspect of one hindlimbat the line of demarcation between the gastrocnemiusand tibialis anterior muscles [16].The injection was made so that the venom was introduced into the vicinityof, rather than directlyinto, the underlyingsoleus muscle.

141 Contralateral soleus muscles served as controls. Both soleus muscles were removed at 1, 3, 6, and 12 hours, and 1,2, 3, 4, 7, and 21 days post inoculation (p.i.) for examination.Whenthe solei were removed, part of each muscle was taken and snap-frozen in liquid nitrogen for use in polyacrylamide gel electrophoresis and Western blotting.

Preparation of muscles for resin sectioning The muscles were pinned to dental wax under slight tension, and immersed for 1 h in 5 % w/v glutaraldehyde in 0.1 M phosphate buffer (PB) (composition 0.02 M NaH2PO42H20; 0.08 M Na2HPO4; pH 7.35) at room temperature. The muscles were then trimmed, and their mid-belly portions cut into small blocks of approximately 1.0 m m • 0.5 mm • 0.5 ram. These were fixed for a further 1 h in the same solution, post-fixed in 1% osmium tetroxide in 0.1 M PB, dehydrated in graded alcohols, and embedded in Araldite. Tissue sections were cut using a Reichert OMU4 ultramicrotome. Thick (1 ~tm) sections were collected on glass slides, and stained with 1% toluidine blue for examination by light micros~ copy. Thin (70-80 nm) sections were collected on cleaned crosswire grids (Athene G300), and stained with 30 % w/v uranyl acetate in methanol, followed by 1% w/v aqueous lead citrate. Sections were viewed using a JEOL 1200 EX electron microscope.

Preparation of muscles for immunocytochemistry and gold immunolabelling The muscles were pinned to dental wax under slight tension, and immersed for 1 h in 2 % paraformaldehyde plus 0.001% glutaral dehyde in 0.1 M PB at 4 ~ The muscles were then washed in phosphate-buffered saline (PBS) (composition 0.02 M NaH2PO42H20; 0.08 M Na2HPO4; 0.15 M NaC1; pH 7.2), trimmed, and their mid-belly portions cut into small blocks of approximately 1.0 mm x 0.5 mm • 0.5 mm. The small tissue blocks were immersed overnight in 2.3 M sucrose in 0.1 M PB (as cryoprotectant) at 4 ~ The blocks were then mounted on aluminium rivets and plunge-frozen in liquid nitrogen. The frozen muscle blocks were stored in liquid nitrogen until use.

lmmunocytochem&oy. Longitudinal and

transverse 1 ~tm tissue sections were cut using a Reichert FC4D cryoultramicrotome (knife temperature -95 ~ block temperature --75~ and transferred onto gelatin-coated slides on a drop of 2.3 M sucrose. The slides were then immersed in PBS, washed in 50 mM ammonium chloride in PBS, and excess moisture removed by gentle blotting. The sections were incubated in 10 ~1 of the primary antibody (monoclonal anti-dystrophin antibody, Dy4/6D3, which recognises an epitope in the rod domain of dystrophin between amino acids 1181 and 1388 [29]; or monoclonal anti-dystrophin antibody, Dy8/6C5, which recognises an epitope within the last 17 carboxyterminal dystrophin residues [28, 35]), diluted 1 : 5 with PBS, for 1 h at room temperature. The sections were then washed twice in PBS, and further incubated in 20 ~,1 of the secondary antibody (rhodamine-conjugated rabbit anti-mouse serum, DAKO R270), diluted 1:100 in PBS containing 0.5 % bovine serum albumin (BSA), for 1 h at room temperature. The slides were washed twice in PBS, once in distilled water, dried around the sections, and mounted with Uvinert before examination using a fluorescence-optics microscope. Control sections were processed as described above, except that D10 tissue culture medium was used instead of primary antibody.

Immunolabelling for electron microscopy. Longitudinal sections, 80-nm thick, were cut on a Reichert FC4D cryoultramicrotome

(knife temperature -110~ block temperature -90~ The sections were transferred in a drop of 2.3 M sucrose to formvar/carbon-coated copper grids on iced PBS. They were quenched in 80 mM ammonium chloride in PBS for 10 min, then washed in 0.1 M PBS containing 0.5 % BSA and 0.15 % glycine, followed by normal goat serum diluted 1:20 in PBS/BSA. After further washing, the sections were incubated for 1 h in 10 ~tl of the primary antibody (Dy4/6D3, diluted 1:5 in PBS/BSA), then washed again three times in PBS/BSA. The sections were then incubated for 30 min in rabbit anti-mouse serum (DAKO Z412, diluted 1:100 in PBS/BSA), and washed five times in PBS/BSA, before being incubated for a further 1 h in the tertiary antibody (10 nm gold-conjugated goat anti-rabbit IgG, BioCell, Cardiff, diluted 1:20 in PBS/BSA). The sections were then washed six times in PBS/BSA, four times in distilled water, stabilised in 2 % methyl cellulose containing 0.2 % uranyl acetate, and allowed to dry. The sections were examined using a JEOL 1200 EX electron microscope. Control sections were processed as described above, except that D10 tissue culture medium was used instead of primary antibody.

Quantitative analysis of gold immunolabelling Nearest neighbour analyses were made on a representative selection of electron micrographs from various time points in the series. The distance from the centre of one particle to the centre of the next particle in a plane parallel with the plasma membrane was measured (see [10]). Where the labelling site was marked by a cluster of more than one gold particle, the centre of the cluster was taken as the putative site. The lower and upper cut-off limits were set at 50 and 550 nm, respectively. The justification for setting such limits has been described by Cullen et al. [10]. A lower limit of 50 nm allows for the addition of an intermediate antibody step in the labelling procedure. The calibration of the magnification of the images of the muscle fibres was based on the method employed in [10], using the 43-nm axial repeat of the myosin filaments as an internal standard.

Analysis of Western blots Small blocks of muscle (20-25 rag) were used for polyacrylamide gel electrophoresis and Western blotting [29]. The blots were probed with the monoclonal antibody to dystropbin, Dy4/6D3.

Results

Normal muscle B o t h i m m u n o f l u o r e s c e n c e (Fig. l a , b) a n d i m m u n o g o l d l a b e l l i n g (Fig. lc) s h o w e d d y s t r o p h i n to b e localised to

Table 1. Nearest neighbour distances between dystrophin epitopes in cryosections from normal, 4-, 7- and 21 days post-venom inoculation (p.i.) samples from rat soleus muscle Sample

Mean nearest neighbour distance(nm) + SE

Normal (n = 309) 4 days p.i. (n = 315) 7 days p.i. (n = 293) 21 days p.i. (n = 310)

165_+6 233_+9* 178_+7 180_+6

* Significantly different at P < 0.01 when compared with norreal, 7- and 21-day data

142 By 6-12 h p.i. muscle fibre breakdown was widespread. In many fibres there was extensive disruption of sarcomeres and many were filled with phagocytes. Blood vessels were largely undamaged but some appeared oedematous. There was immunocytochemical evidence that the distribution of dystrophin in the plasma m e m b r a n e was very patchy, i.e. there were discrete gaps in the m e m b r a n e labelling (Fig. 2a, b), and many fibres exhibited delta lesions or vacuoles. Phagocytic cells also displayed fluorescence (Fig. 2a, b), possibly as a result of the ingestion of labelled dystrophin still attached to fragments of membrane. At the ultrastructural level, no myofilaments were seen in the fibres from 6 h, and the membranes of the fibres were fragmented. Gold labelling was no longer exclusively

Fig. I a-c. Transverse (a) and longitudinal (b,c) cryosections from normal rat soleus muscle labelled for dystrophin using immunofluorescence (a,b) and immunogold labelling (c). Note the location of dystrophin at the plasma membrane, shown in more detail in (c) (arrowheads). Bars a,b = 25 gin; c = 200 nm

the plasma membrane. In the ultrathin sections the gold particles labelling the sites of immunoreactivity were spaced at regular intervals of approximately 165 nm along the cytoplasmic face of the plasma m e m b r a n e (Fig. lc, and Table 1).

Muscle degeneration

Muscle fibre necrosis began within 3 h of venom inoculation, and was associated with oedema, the separation of myofibres and some hypercontraction of fibres. There was also an influx of phagocytic cells into some of the muscle fibres. At this stage dystrophin labelling was still strong and apparently intact, although in those fibres invaded by phagocytes small gaps could be seen in the m e m b r a n e labelling.

Fig. 2 a-c. Transverse (a) and longitudinal (b,c) cryosections from rat soleus muscle 6 h after inoculation of venom. Note the disrupted fibres with patchy immunofluorescent dystrophin labeling of the plasma membrane (a,b; open arrows, soloid arrows: phagocyte). Intrafusal fibres of spindles did not appear to be affected by the venom (a, inset). Electron micrograph (c) shows plasma membrane breakdown and the dispersal of dystrophin in the cytoplasm. Bars a,b = 25 gm; c = 200 nm

143 located at the periphery of the fibres, but was dispersed (Fig. 2c). All dystrophin labelling had disappeared by 12 h p.i. when the inflammatory response was at its maximum, and invasion of the necrotic fibres by phagocytic cells was well advanced. Intrafusal fibres at the equatorial region of muscle spindles remained intact and dystrophin labelling of these fibres was strongly positive and continuous (Fig. 2a, inset).

Muscle regeneration Two days following the administration of venom, the early o e d e m a had subsided, the numbers of phagocytic

Fig. 3 a-d. Resin sections of rat soleus muscle 2 days after inoculation of venom, stained with toluidine blue (a,e) and uranyl acetate and lead citrate (b,d). Regenerating myotubes, containing centrally located nuclei (asterisks, ?4), and myofibrils being laid down (Mf) in bundles of thick (TkF) and thin (TnF) filaments, with dense bodies marking the formation of Z-lines (Z). Inset: Oblique longitudinal section through a spindle, showing at least two regenerating fibres (arrowheads). Bars a,c = 25 gin; b = 500 nm; d = 200 nm

cells had decreased markedly, and very immature regenerating myotubes could be identified (Fig. 3a). It was also noted that at least two intrafusal fibres of a muscle spindle (Fig. 3a, inset) had lost their striations and were in the process of regenerating. In the extrafusal myotubes, myonuclei were located centrally (Fig. 3b, c) and small bundles of myofilaments were clearly visible (Fig. 3b, d). Occasional dense bodies could be seen (Fig. 3b), which we believe represented the early stages of Z-line formation. Although the regenerating myotubes were bounded by clearly visible plasma membrane, no dystrophin labelling was observed either by immunofluorescence or immunogold labelling (Fig. 4). At 3 days the bundles of myofilaments had increased in size (Fig. 5), and M-lines and Z-lines had become

Fig. 4a-c. Dystrophin-immunolabelled cryosections from rat soleus muscle 2 days after inoculation of venom. Regenerating myotubes can be identified (a,b) (Mt). Some fibres contained myofilaments (c) (Mf), but no dystrophin labelling was visible at this stage, despite reformation of the plasma membrane (black open arrow). White open arrow: phagocyte. Bars a, b = 25 ~tm;c = 200 nm

144

Fig. 5 a-d. Resin sections of rat soleus muscle 3 days after inoculation of venom, stained with toluidine blue (a,c) and uranyl acetate and lead citrate (b,d). Regenerating myofibres were more advanced,with myofibrils (m) being clearly visible, and the bundles of thick (TkF) and thin (TnF) filaments larger and more clearly defined. The Z- (Z) and M-lines (M) were evident in the forming sarcomeres, and most fibres displayed chains of central nuclei asterisks, N). mi: Mitochondrion. Bars a,c = 25 ~tm; b,d = 200

Fig. 6 a-c. Cryosections from rat soleus muscle 4 days after

inoculation of venom. Most of the regenerating fibres exhibited dystrophin labelling of the plasma membrane (a,b), although some fibres displayed continuous labelling, and others discontinuous labelling (b, open arrows). Gold labelling of dystrophin was seen along the plasma membrane at intervals generally greater than those seen in normal muscle fibres (c, arrowheads). Bars a,b = 25 ~tm; c = 200 nm

nm

clearly visible in many of the developing sarcomeres (Fig. 5b). Dystrophin was still undetectable. Dystrophin was first detected on tissue sections at 4 days p.i. (Fig. 6). In some fibres labelling around the periphery was continuous, but in others the membrane labelling showed discrete gaps (arrows, Fig. 6b). It seemed that the continuous labelling was most prominent in the larger fibres. This was tested by measuring the diameter of approximately 100 randomly chosen fibres in a single transverse section of a 4-day regenerating muscle, and classifying the dystrophin labelling as either continuous or discontinuous. The mean diameter of the fibres with continuous labelling was approximately 2.5 ~tm greater than that of the fibres displaying discrete gaps in the labelling (Fig. 7).

Examination of the 4-day regenerating muscle at ultrastructural level revealed fibres displaying stretches of labelled and stretches of unlabelled plasma membrane. These would probably correspond to the areas of discontinuous labelling described above. The lengths of decorated plasma m e m b r a n e showed that the labelling was typically irregular at this stage (Fig. 6c). T h e r e appeared to be a regular repeat of labelling sites of between 100 and 200 nm at most stages, and calculations of the mean values were very similar (approximately 175 nm), except at 4 days p.i. where the mean interval was significantly larger (233 + 9 nm) (Table 1). The larger mean interval at 4 days was due to the higher frequency of larger intervals at this point. For example, at 4 days, 17 % of all intervals exceeded 300

145 35 30 25 20 0

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11

12

13

14

15

16

17

18

19 20

21

Fibre diameter(pm) Fig. 7. Histogram to show the distribution of the diameter of randomly selected regenerating muscle fibres (n = 84) displaying either continuous (n = 50) [] or discontinuous (n = 34) 9 dystrophin labelling 4 days after inoculation of venom. The difference between the two samples was significant at P < 0.0i

Fig. 8 a-c. Cryosections from rat soleus muscle 7 days after inoculation of venom. All the regenerated fibres exhibited complete dystrophin labelling of the plasma membrane. Electron micrograph (c) shows the gold probe at intervals indistinguishable from normal (arrowheads).Bars a,b = 25 ~xm; c = 200 nm

nm, c o m p a r e d to < 8 % in 7-, 21-day and normal tissue. T h e regenerating myofibres increased in d i a m e t e r to reach approximately 20 ~m at 7 days p.i. (c.f. 30-34 Fm in control muscle). F r o m this time the myofibrils were well organised and there was strong, complete and continuous immunofluorescent labelling of dystrophin in the plasma m e m b r a n e of the r e g e n e r a t e d fibres (Fig. 8a, b). T h e examination of the muscle fibres at the ultrastructural level at this stage also confirmed that the i m m u n o g o l d labelling of dystrophin was indistinguishable f r o m that of the controls. Similar experiments were m a d e using the antibody Dy8/6C5, a monoclonal antibody specific for the carboxy-terminal region of dystrophin. In all respects the results were similar to those obtained with the Dy4/6D3 antibody (results not shown).

Analysis of Western blots T h e samples of contralateral muscles gave reproducible bands across the whole blot, with the top b a n d in each

Fig. 9a,b. Western blots of samples taken at various time points from contralateral (a) and venom-inoculated (b) rat soleus muscle probed with anti-dystrophin monoclonal antibody Dy4/6D3. The venom-inoculated muscle samples show dystrophin breakdown begins at 3-6 h post inoculation, and the full-size 400-kDa protein is expressed again in regenerating muscle at 3 days. Note the bands of lower molecular mass corresponding to the breakdown products of dystrophin at 6 h, 12 h and 1 day post inoculation (arrowheads) (b), and the disappearance and reappearance of myosin (M)

146 lane corresponding to the full-size 400-kDa protein (Fig. 9). The pattern of dystrophin bands displayed on the blot of venom-inoculated muscle samples, however, showed significant changes in the muscle that were consistent with the immunocytochemical results. It appeared that dystrophin began to break down 3 h following the inoculation of venom. Bands corresponding to breakdown products of lower molecular mass were visible in the lower region of the blots from 3 h (arrowheads, Fig. 9). Dystrophin degradation progressed steadily until no protein was detectable at 2 days p.i. There was evidence of the full-size 400-kDa protein appearing at 3 days, but the normal banding pattern for dystrophin only became apparent from 4 days onwards.

Discussion

Muscle degeneration The studies described in this report were undertaken to determine the distribution of dystrophin in the soleus muscle of the rat, and to establish when, in a cycle of degeneration and regeneration, dystrophin was lost and when it was re-expressed. The strong immunofluorescent labelling of dystrophin in the vicinity of the plasma membrane in normal rat muscle fibres is in agreement with earlier immunocytochemical reports on human, rat and mouse muscle [1, 4, 40, 45]. Ultrastructural examination showed that dystrophin molecules label at intervals of approximately 165 nm along the plasma membrane in normal rat muscle, which is in close agreement with measurements of the length of the isolated dystrophin molecule made by Pons et al. [34] using rotary shadowing.The measurements obtained are of the same order of magnitude (100-175 nm) as those also reported or predicted by Cullen et al. [10], Koenig et al. [24], Murayama et al. [27], and Watkins et al. [41]. It should be noted that Sealock et al. [38] found no indication of any regular labelling pattern onTorpedo postsynaptic membranes or the skeletal muscle of Xenopus, but their data were not reported in detail and it is difficult to make any further analysis of this apparent discrepancy. The soleus muscle fibres began to degenerate within 3 h of the inoculation of venom, and between 3 and 6 h the muscle fibres exhibited lesions in the plasma membrane, accompanied by hypercontraction and the phagocytosis of the contractile material. The discontinuity of dystrophin labelling seen 6 h after the inoculation of venom suggests that this is the time when the breakdown of dystrophin begins. The venom of the Australian tiger snake contains powerful toxic A2 phospholipases, but is weak in proteolytic activity, and it therefore seems unlikely that its primary target is either dystrophin or any other membraneassociated protein or glycoprotein. If it is assumed that the hydrolysis of phospholipids of the membrane is the primary mode of action [15], the dispersal of gold particles into the cytoplasm at the periphery of the fibres

seen at 6 h p.i. could result from the release of the glycoprotein-dystrophin complex from the membrane (still decorated with gold) into the cytoplasm, from hydrolysis of the plasma membrane and subsequent intracellular proteolytic activity on the glycoproteins to release decorated dystrophin into the cytoplasm, or from the hydrolysis of the plasma membrane and proteolysis of the glycoprotein-dystrophin complex with liberation of the free gold particles from the molecule into the cytoplasm.We cannot predict which alternative is most likely, although it is clear from Western blots that dystrophin-related breakdown products appear by 3-6 h. At 12 h it was difficult to locate dystrophin, even where the plasma membrane was still intact. This may suggest that once proteolysis has started in the damaged fibres, dystrophin becomes a target for endogenous proteases and is broken down rapidly. It was interesting to note that at the equatorial region the intrafusal fibres of the muscle spindles seen at this stage did not appear to be affected by the venom, and strong dystrophin labelling of their membranes was maintained. We presume that the intrafusal fibres at the equator were largely protected from the effects of venom by the spindle capsule [15, 16, 18], although we do have evidence from 2-day post-venom material that some intrafusal fibres were damaged, had lost their striations, and were in the process of regenerating (see Fig. 3a, inset) in at least one spindle.

Muscle regeneration Regeneration of the soleus muscle was first observed 2 days after the inoculation of venom. This was a little earlier than is usually reported [16, 18, 26]. Early regeneration was manifested by the appearance of multinucleate myotubes with peripherally located myofibrils. Even at this early stage, dense bodies were present, indicating, we believe, the initiation of Z-line formation. The myotube plasma membranes could be clearly resolved, but no dystrophin labelling was seen by either immunofluorescence or immunogold labelling, and this would suggest that the protein had not yet been expressed or accumulated at the plasma membrane. It would seem, therefore, that the expression of dystrophin is delayed and is not concomitant with the formation of the plasma membrane in the developing myofibre. The fluorescent labelling of the regenerating fibres with anti-dystrophin antibodies was first detected at 4 days, although it tended to be patchy, especially in the smaller fibres. This may be of some relevance given the findings of other workers, who reported the heterogeneity of dystrophin expression in Duchenne and Becket muscular dystrophy, and in carriers of the disease [3, 32, 39]. At the EM level, gold labelling was irregular with a larger number of longer intervals between the probes, suggesting that in many regenerating fibres the dystrophin lattice was still incomplete, or that the glycoproteins, to which the dystrophin molecules bind [7], had not reappeared or been incorporated into the mem-

147 brane. The period between 3 and 7 days is the time of rapid growth of regenerating muscle fibres [42], when the myofibres are becoming innervated and mechanically active [13]. Little is known of the regulation of expression of dystrophin, but our results are very similar to the description by Hagiwara et al. [14] of the expression of dystrophin during normal development, and suggest that either re-innervation of regenerating fibres, or the achievement of a given diameter during regeneration, is related to regulation; the imposition of work-loads consequent upon re-innervation may also be a factor. These possibilities are currently being investigated. By 7 days the regenerating muscle fibres had formed well-organised sarcomeres, and the localisation and immunostaining of dystrophin was indistinguishable from normal.

anchoring the cytoskeleton to the membrane to create mechanical strength [6, 23, 36, 37, 45], or in the maintenance of the non-random distribution of membrane glycoproteins [7, 11, 44]. It is hoped that further work on the loss and reaccumulation of dystrophin in muscle fibres may provide some clues to its function, e.g. relating timing to a particular function. It is also anticipated that such studies, in elucidating the importance of the time scale of dystrophin expression in regenerating muscle fibres, will be of value in the assessment of therapeutic strategies applied to the Xp-21 dystrophies.

Analysis of Western blots

References

Although immunocytochemical studies provide useful information on the loss and reaccumulation of dystrophin in skeletal muscle, it is generally accepted that an integrated analysis involving the use of Western blots can be of value in the study of protein expression [31]. Biochemical analysis of the contralateral muscle samples gave the same banding pattern as normal human muscle [29]. Western blots of the venom-inoculated muscle samples confirmed the immunocytochemical results. The blots showed that dystrophin had begun to break down at 3 h p.i., demonstrated by the appearance of lower molecular mass bands, although there was a considerable amount of the full-size 400-kDa protein remaining in the tissue. By 6 h the amount of 400-kDa protein was drastically reduced, and there was a higher abundance of breakdown product bands. This is consistent with the immunocytochemical observation of discontinuous labelling by anti-dystrophin antibodies, and, at the ultrastructural level, the dispersal of gold particles at the periphery of the muscle fibres. At 12 h the amount of detectable 400-kDa protein was negligible, the small amount present possibly being due to dystrophin in the surviving intrafusal fibres of the spindles and the few extrafusal fibres that may, at the time, survive assault by the venom and its toxins [16, 18]. By 1 day only breakdown product bands could be seen. A t 2 days no proteins were visible, as was the case with immunotabelling, but some dystrophin, in the form of the 400-kDa protein, could be observed at 3 days. This suggests that dystrophin was being expressed, but that the expressed protein was not being attached to the plasma membrane at this stage. At 4 days, however, the abundance of dystrophin had increased almost to its normal level, and the blots for the 7- and 21-days samples were identical to those of normal tissue. The function of dystrophin in muscle fibre plasma membranes is still unknown, although it has been variously suggested that the molecules are involved in the stabilisation of the plasma membrane by linking the cell cytoskeleton to the extracellular matrix [7, 8, 37], in

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Acknowledgements. We thank Keith Davison and Elizabeth O'DonnelI for assistance with the gels and blots.

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