Contractile properties, structure and fiber phenotype of intact and regenerating slow-twitch muscles of mice treated with cyclosporin A

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CONTRACTILE PROPERTIES, STRUCTURE AND PHENOTYPE

OF INTACT AND REGENERATING SLOW-TWITCH MUSCLES

OF MICE TREATED WITH CYCLOSPORIN A

A. Irintchev, M. Zweyer, R. N. Cooper*, G. S. Butler-Browne* and A. Wernig

Department of Physiology, Neurophysiology, University of Bonn,

Wilhelmstraße 31, D-53111 Bonn, Germany

and

* CNRS UMR 7000, Cytosquelette et Développement,

Hôpital Pitié-Salpêtrière, 105, Blvd. de µhôpital, 75013 Paris, France

Running title: Cyclosporine effects on muscle

Key words: Skeletal muscle, Cyclosporin A, Regeneration

Correspondence and proofs to:

Prof. Dr. Anton Wernig,

Physiologisches Institut der Universität Bonn,

Wilhelmstraße 31, D-53111 Bonn, FRG

Tel.: +49 228 287 2274

Fax: +49 228 287 2208

E-mail: [email protected]

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SUMMARY

1. We have studied the contractile properties, structure, fibre type composition and myosin heavy

chain (MyHC) expression pattern of regenerating and intact soleus muscles of adult CBA/J mice

treated with Cyclosporin A (CsA, 50 mg kg-1 day-1 s.c.) or vehicle solutions (Cremophor, saline).

2. After 4-7 weeks of drug application, as compared to vehicle treatment, the isometric contractile

force of intact muscles was reduced (tetanus, -21%, twitch, -34%) despite normal mass and

muscle cross-sectional area. The frequency of fast-twitch fibres was increased whereas innervation

deficits, histopathological alterations or changes in fibre numbers were not observed.

3. Regeneration after cryolesion of the contralateral soleus appeared to proceed more slowly, as

estimated from histological sections, in CsA- than in vehicle-treated animals. Despite this, when

muscle properties reached mature levels (4-7 weeks) muscle mass recovery was better (30%

higher weight, 50% more fibre profiles in cross-sections). Deficient was force production per unit

cross-sectional area but not maximum tension. Twitch time to peak and half-relaxation time were

shorter than control correlating with a predominancy of fast-twitch fibres (98% type II fibres

versus 16-18% in control muscles) and fast MyHC isoforms.

4. Partial reversal of this fast phenotype, as well as an increase in muscle force, were observed when

the animals were left to recover without treatment for 5-8 weeks after CsA application over 7

weeks. The high numbers of fibre profiles in CsA-treated regenerated muscles and increased mass

remained unchanged after withdrawal.

5. The results show that CsA treatment has a hyperplastic effect on regenerating muscles. The

increase in the proportions of fast fibres, much more prominent in regenerated muscles, is most

likely due to direct drug influence on signal transduction mechanisms. The reason for impairment

of force production during drug application have to be elucidated.

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INTRODUCTION

During the last two decades Cyclosporin A (CsA) has been one of the most important

immunosupressive drugs used for clinical prevention of transplant rejection as well as therapy of

various diseases. Despite the rapid progress which has been made in the discovery of new

immunosuppressive strategies and drugs in the last years, its clinical significance has not apparently

diminished (Weinblatt, 1999; Linden & Weinstein, 1999; Ponticelli & Tarantino, 1999; Billaud,

2000). Major mechanisms of action of CsA have been identified, which have helped explain both

desired (immunosuppressive) and undesired (adverse) tissue effects (Ruhlmann & Nordheim,

1997; Resch & Szamel, 1997; Olyaei et al., 1999; Matsuda & Koyasu, 2000). Research on the

action of this drug on skeletal muscle has however remained in the background for a considerable

period of time most likely because the side effects of CsA administration have rarely been

associated with skeletal muscle. In a limited number of cases myopathic complications were

attributed to CsA, however this assumption was difficult to prove clinically because of the usual

multi-drug management of the patients (Breil & Chariot, 1999). A more common clinical

observation was the decreased exercise performance of transplant patients (Savin et al., 1980;

Kavanagh et al., 1988; Jensen et al., 1991). This deficit was thought to be related to the skeletal

muscles response to the immunosuppressive treatment since mitochondrial respiration is impaired,

and muscle capillary density and endurance are reduced in CsA-treated individuals (Hokanson et

al., 1995; Mercier et al., 1995; Lampert et al., 1996; Biring et al., 1998). Another interesting

clinical observation is that, for unknown reasons, muscle force improves transiently in Duchenne

muscular dystrophy patients during CsA administration (Sharma et al., 1993; Miller et al., 1997).

Also in cases of inflammatory myopathies (polymyositis or dermatomyositis) CsA, in combination

with corticosteroids, appears to be beneficial (Vencovsky et al., 2000).

Apart from its clinical value, CsA is also useful as a pharmacological agent in studies on

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signal transduction pathways. It inhibits transcriptional activation mediated through the calcineurin

/ nuclear factor of activated T lymphocytes (NFAT) pathway (Guerini, 1997; Rao et al., 1997;

Matsuda & Koyasu, 2000). Recently, it was found that NFAT is present and functional in skeletal

muscle (Abbott et al., 1998). In addition, inhibition of its nuclear translocation by CsA leads to

an impairment of myogenic differentiation (Abbott et al., 1998). Other investigations have

identified a role of the calcineurin / NFAT-dependent pathway in controlling MyHC expression

(Chin et al., 1998). CsA application in vivo induces a slow-to-fast transformation of muscle fibres

indicating that calcineurin activation is required for slow MyHC expression. Control of the slow

muscle phenotype through the calcineurin pathway is not restricted to MyHC but involves

phenotype-specific expression of other proteins such as sarcoplasmic reticulum ATPase and

cytosolic creatine kinase (Bigard et al., 2000). Furthermore, CsA has been found to inhibit skeletal

muscle hypertrophy caused by overload through calcineurin inhibition (Musaro et al., 1999; Dunn

et al., 1999; Semsarian et al., 1999). However, muscles of transgenic mice which express activated

calcineurin are not hypertrophic even though the number of slow muscle fibres is increased

(Musaro et al., 1999; Naya et al., 2000). This finding indicates that activation of additional

pathways is required for hypertrophy but not for phenotype control.

These previous studies indicate the skeletal muscle-specific effects of CsA in vivo. This

warrants, both from a clinical and cell biological point of view, studies designed to evaluate and

correlate the consequences of CsA treatment on skeletal muscle at the level of function, structure

and fibre diversity, aims of the work reported in this article. We have restricted our observations

to one muscle, the slow-twitch soleus, but have extended our study to both intact and regenerating

adult muscles. The effects of chronic CsA administration were studied during regeneration /

treatment periods ranging from 1 to 7 weeks. The results presented here show significant CsA

effects on muscle contractility and MyHC expression, as well as time course and efficiency of

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muscle regeneration. A preliminary report of this work has been publshed as an abstract (Irintchev

et al., 1999).

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MATERIAL AND METHODS

Animals

Female CBA/J mice aged 2 months were purchased from Charles River Deutschland

(Sultzfeld, Germany) and kept in a conventional animal unit. Upon arrival, the animals were

allowed to recover from transportation and adapt to the conditions in the unit for 2-3 days. After

that they were divided at random into three groups, one receiving CsA and two - vehicle solutions

(see below). A total of 90 animals was used in these experiments.

Drug treatment

In one group of animals, CsA (SandimmunR, Sandoz, Nürnberg, Germany, diluted 1:9 with

physiologic saline) was administered once a day subcutaneously at a dose of 50 mg kg-1 body

weight (10 µl diluted SandimmunR per g body weight). This dosage is immunosuppressive in the

mouse and we have used it successfully in previous experiments (Irintchev et al., 1995; Wernig

et al., 1995). Another experimental group received injections of saline solution containing the

vehicles in SandimmunR, polyoxyethylated castor oil (Cremophor EL, Sigma, Deisenhofen,

Germany) and ethanol. The concentrations of Cremophor and ethanol were similar to those

present in the diluted SandimmunR solution (650 and 278 mg ml-1, respectively). The mice

received 10 µl of this solution per gram body weight. The third group of animals were injected

with physiologic saline (10 µl g-1). All animals were weighed prior to each injection.

Treatment with CsA, but not with vehicle solutions, had visible side effects. There was loss

of body mass (see Results). The excrements were often, especially in the first days of treatment,

yellowish and had soft consistency. The pelt gradually acquired a shaggy appearance and pelt

colour changed from agouty to white in circumscribed areas. Although most animals tolerated the

immune suppression without special care some died (mortality rate about 10%). The reason(s) for

the lethality were not investigated.

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Experimental design

In one set of experiments, the aim was to evaluate the effects of continuous CsA treatment

over several weeks on the physiological properties and morphological features of intact and

regenerating muscles. For this purpose, left (intact) and right soleus muscles (regenerated after

cryolesion) were studied 4 - 7 weeks after injury, a time period sufficient for muscle recovery and

manifestation of drug effects. CsA application was initiated 8 days prior to surgery to achieve

intracellular drug accumulation and presumably steady-state levels during the recovery period

(Irintchev et al., 1995; Wernig et al., 1995). To dissociate CsA effects, other animals were treated

with a saline solution containing the vehicles of the CsA preparation used (see above). Finally, to

control for vehicle effects, some of the animals were treated with physiologic saline alone. In view

of initial results, and in order to the study the dynamics of the induced effects, this experimental

set was later supplemented with animals studied after recovery period of 1 - 4 weeks.

The goal in the second experimental set was to evaluate the reversibility of the drug-

induced functional and structural alterations after discontinuation of treatment. In this case, the

animals were systemically treated as above for 1 week prior to and 7 weeks following surgery after

which application of CsA, vehicles or saline was discontinued. Muscles were studied 12 - 15

weeks after muscle injury, i.e. 5 - 8 weeks after withdrawal.

Surgery for induction of muscle regeneration

The animals were anaesthetised by intraperitoneal injections of 0.4 mg kg-1 fentanyl

(Fentanyl-Janssen, Janssen, Neuss, Germany), 10 mg kg-1 droperidol (Dehydrobenzperidol,

Janssen) and 5 mg kg-1 diazepam (Valium 10 Roche, Roche, Grenzach-Wyhlen, Germany) and the

right soleus muscles were surgically exposed. The injury consisted of a single freeze-thaw cycle

of the muscle in situ. Freezing was performed by applying the flat end (3 x 0.7 mm) of a copper

rod precooled in liquid nitrogen to the surface of the mid-portion of the muscle and maintaning

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it in this position for 10 s. After thawing of the muscles, the wounds were closed with polyamide

threads (7-0 Ethilon, Ethicon, Norderstedt, Germany) and thereafter the animals were kept for

several hours on a warm plate (37°C) to prevent hypothermia. Previous work has shown that

spontaneous recovery of muscle structure and function after this injury is good (Wernig &

Irintchev, 1995; Wernig et al. 1995; , Irintchev et al. 1995, 1997a).

Isometric force measurements

The isometric contractile properties of the regenerated and intact contralateral soleus

muscles were studied in vitro according to an established protocol (for a recent description see

Wernig et al., 2000). Briefly, the muscles with the intact nerve supply were surgically removed

from anaesthesised animals (see above) after which the mice were killed by cervical dislocation.

The nerve-muscle preparations were mounted in a horizontal LuciteR chamber containing Tyrode’s

solution (composition in mM: NaCl, 125; MgCl2, 1; CaCl2, 1.8; KCl, 5.4; NaHCO3, 24; and

glucose, 10). The chamber was continuously perfused with the aerated (95% v/v O2, 5% v/v CO2)

and heated (25°C) physiologic solution. Tension was recorded with a transducer carrying two

strain gauges (DMS LY41 1,5/350, Hottinger Baldwin Messtechnik, Darmstadt, Germany).

Force measurements were performed at optimal muscle length. Muscles were stimulated

directly with suprathreshold electrical pulses via silver electrodes in the bath (direct stimulation,

pulses of 0.5 ms duration, typically 20-25 V) or via nerve with a suction electrode (indirect

stimulation, 0.1 ms pulse duration, 3-6 V). The variables measured were isometric twitch and

tetanic (20, 50 and 100 Hz for 2 seconds) force, twitch time to peak (contraction time) and twitch

half-relaxation time. For each stimulation frequency, muscles were stimulated first directly and

then indirectly. Between two stimulations, muscles were allowed to recover for three minutes.

After these recordings, acetylcholine (ACh) sensitivity was tested by rapid exchange of the normal

perfusion solution with Tyrode’s solution containing 0.2 mM ACh perchlorate. The amplitude of

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the ACh-evoked contracture was expressed as a fraction of the amplitude of the preceding direct

100-Hz tetanus.

After the force recordings, the muscles were weighed and frozen at resting length on pieces

of liver as described previously (Irintchev et al., 1997a). Twitch-tetanus ratio was calculated from

the twitch and maximum tetanic force. Tetanic force was normalised per unit wet muscle weight

(P0/muscle weight) or unit muscle cross-sectional area. The mean rate of rise of the twitch was

calculated from twitch force and twitch contraction time.

Histology

Stainings were performed on transverse sections (6 µm) from the mid-portion (end-plate

region) of the muscles. Unfixed sections were stained with aqueous Toluidine blue-borax solution

(both at 1% w/v) to reveal the general morphology. Calcium accumulations were visualised using

glyoxal-bis-(hydroxyanyl) and Alizarine Red S stains. For muscle fibre typing (see quantitative

analysis below), histochemical stainings for myofibrillar ATPase activity were performed after acid

(pH 4.3 or 4.65) or alkaline preincubation (pH 10.3) of the sections (modified after Guth &

Samaha, 1970; Irintchev et al., 1990; Wernig et al., 1990). Macrophages were visualised by

histochemical detection of lysosomal acid phosphatase activity according to the modified

azocoupling method of Lojda using naphthol-AS-TR-phosphate (Sigma) as substrate (Lojda et

al., 1976). Existence of immature muscle cells (myoblasts, myotubes, immature fibres) in

regenerated muscles beyond 4 weeks post-injury was monitored by immunostainings of two

developmentally regulated molecules, the neural cell adhesion molecule (NCAM, mouse

monoclonal antibody, clone H-28, Hirn et al., 1981) and the embryonic form of myosin heavy

chain molecule (MyHC, mouse monoclonal antibody F1.652, CRL-2039, American Type Culture

Collection, ATCC, Manassas, Virginia, USA), in conjunction with morphological criteria like cell

diameter and basophilia (Toluidine blue staining). Expression of mature isoforms of MyHC was

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studied by monoclonal mouse antibodies against slow (clone A 4.951, CRL-2046, ATCC), fast

IIA (clone A4.74, CRL-2041, ATCC) and fast/neonatal isoforms (N 3-36, CRL 2042, ATCC).

For immunofluorescence stainings, methanol-fixed sections were overlaid with normal goat

serum (20 % v/v in PBS) for 30 min and then incubated overnight at 4°C with primary antibodies

diluted in PBS containing 0.7% w/v lambda-carrageenan (Sigma) and 0.02% w/v sodium azide.

After washing in PBS, the first antibody was visualised with 5-((dichlorotriazin-2-yl)amino)-

fluorescein (DTAF)-IgG raised against the appropriate species, or with biotinylated IgG and

DTAF- or rhodamine-conjugated streptavidin. Secondary antibodies and conjugates were

purchased from Jackson ImmunoResearch Laboratories (Dianova, Hamburg, Germany).

Electrophoretic analysis of myosin isoform expression

Proteins were extracted from muscle samples using four volumes of 0.3 M NaCl, 0.1M

NaH2PO4, 0.01 M sodium pyrophosphate, 1 mM MgCl2, 10 mM EDTA and 1.4 mM 2-

mercaptoethanol, adjusted to pH 6.5. Separation of MyHC isoforms was performed in 8%

polyacrylamide gels containing 30% glycerol as previously described (Agbulut et al., 1996).

Muscle extracts (20µl per lane) were loaded into minigels fixed in Mini-PROTEAN II dual slab

cell (Bio-Rad, München, Germany). The electrophoresis buffer was composed of 20 mM sodium

pyrophosphate (pH 8.5), 1 mM EDTA and 1.4 mM 2-mercaptoethanol. During electrophoresis

the temperature of the buffer was maintained at 5°C in a refrigerator. For protein separation,

current (72 V) was applied for 30-31 hours. The gels were then stained with silver nitrate solution

(0.2% w/v).

Quantitative procedures

Total numbers of muscle fibre profiles, numbers of different fibre type, muscle cross-

sectional area and muscle fibre diameters were evaluated on videoprint reconstructions of

complete muscle cross-sections stained for myofibrillar ATPase activity after acid (pH 4.3) pre-

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incubation of the sections (final magnification 244x for total cross-sectional area, 610x for the

other parameters) as described previously (Irintchev et al., 1990; Wernig et al., 1990). For fibre

type analysis, the staining pattern in the print reconstruction (Type I fibres dark, Type II fibres

pale) was compared with the reversed staining pattern (Type I pale, Type II dark) in a section

stained for myofibrillar ATPase after alkaline preiincubation (pH 10.3). In addition to Type I and

II fibres, Type IIc fibres were identified by their intermediate staining intensity after both pre-

incubations. Mean orthogonal diameters (mean of the longest axis and a short one passing through

the middle of the longest at right angle) were measured using a digitizing tablet. All fibres in a

muscle section were evaluated, except for diameter measurements of occasional mechanically

damaged muscle fibres.

Statistical analysis

Multiple comparisons of group mean values were performed with ONEWAY analysis of

variance and a subsequent Tukey's test to compare mean values of more than two experimental

groups using SigmaStat 2.0 statistical software package (SPSS Europe, Erkrath, Germany). Mean

values of two groups were compared with Student´s t test. The accepted level of significance was

0.05 or less. Unless otherwise indicated, mean group values are given with standard deviations.

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RESULTS

Body weight

CsA- but not saline- or Cremophor-treated animals lost body weight during the first two

and a half weeks of treatment (Fig. 1, drug or vehicle treatment initiated 8 days before surgery at

day 0). Following this initial loss, body weight increased at a rate similar to control mice (Fig. 1,

left half < 30 days). The daily fluctuations in mean body weight and coefficients of variation were

larger in the immunosuppressed than in the control groups (Fig. 1). At the time when muscle

contractile properties were evaluated (4 - 7 weeks, Fig. 1), the mean body weight of CsA-treated

animals was about 10% lower than in the control groups (P < 0.05, Student´s t test). Reduced

body weight gain has been previously observed in rats treated with a high dose of CsA (25 mg kg-1

day-1 per os, Richter-Reichhelm & Schulte, 1998). This effect of CsA on body weight can be

attributed to a systemic toxicity (see Materials and Methods).

In another group of mice studied after CsA treatment (7 weeks) and withdrawal period of

5 - 8 weeks (Fig. 1, “after withdrawal”) mean body weight was, in contrast to the previous group,

10% higher than in control animals (P < 0.05, Student´s t test). The reason for this difference is

not known.

Isometric contractile properties during drug application

Intact muscles

Soleus muscle mass was not affected by the CsA treatment (Table 1, upper half). The mean

ratio of muscle weight / body weight (0.49 ± 0.12 mg g-1, n = 8) was similar to that of the control

groups (overall mean 0.42 ± 0.03, n = 11, n.s., Mann-Whitney rank sum test). Despite normal

muscle mass, the maximum force produced following twitch or tetanic stimulation were

significantly lower (-21%) than in control muscles (Table 1). Force generation per unit cross-

sectional area (Specific force in Table 1) or wet muscle weight (P0 / muscle weight in Table 1)

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were, on average, 25% lower than control values (P < 0.05 for both parameters, t test, CsA-

treated versus pooled control group). The twitch time-to-peak was shortened proportionally to

the reduction in twitch amplitude and the mean rate of rise of the twitch was not different from

that in control animals (Table 1). Twitch half-relaxation time was also unchanged. Tetanic and

twitch forces developed upon nerve stimulation (not shown) were similar in amplitude to those

upon direct stimulation (Table 1) in all three animal groups. This was further supplemented by the

finding of weak muscle contractures upon ACh application (Table 1), an indication for low

numbers of extrasynaptic ACh receptors characteristic for normally innervated muscle.

Regenerated muscles

As judged from maximum force, 4 to 7 weeks after operation the injured muscles of drug-

treated mice behaved as well as control muscles (Table 1, Fig. 2). Muscle mass at 4-7 weeks of

regeneration was on average 40% higher than control values but specific force was low (Table 1).

Regenerated muscles of immunosuppressed animals had shorter twitch time to peak than any other

group of muscles (Table 1). This was due to an increased rate of rise of the twitch and not simply

to reduced twitch force, as compared to intact muscles, which was the case in the regenerated

control muscles (Table 1). This finding correlates with the observed slow-to-fast transformation

in CsA-treated muscles (see below). Half-relaxation time of CsA-treated and control regenerated

muscles did not differ. Both ACh sensitivity (Table 1) and nerve-evoked muscle contractions (data

not shown) were normal.

Contractile properties after transient immunosuppression

Five to eight weeks after discontinuation of drug application force generation in the intact

muscles of immunosuppressed animals was largely improved, as compared to continuously treated

muscles and no difference was present between the solei of the treated and untreated animals any

more (Table 1). These findings indicate that the drug-induced changes in the function of intact

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muscles are reversible. A similar improvement in the tetanic force was observed in regenerated

muscles after CsA withdrawal (Table 1, Fig. 2). Maximum tetanic tension was now similar to

intact control muscles and exceeded by nearly 40% that of regenerated control muscles. Similar

to the period of drug application, the mass of the CsA-treated muscles was approximately one-

third higher than that of control regenerated muscles (Table 1, significant in t test). Contraction

time was prolonged when compared to the period of drug application but it was still significantly

shorter than in the other groups (Table 1). This correlates with the partial reversal of the drug-

induced fibre type transformation (see below). Thus, as estimated in vitro, the transient treatment

with CsA was beneficial in eliminating functional deficits consistently observed after cryodamage

(Table 1, regenerated versus intact control muscles, see also Wernig et al., 1995; Wernig &

Irintchev, 1995; Irintchev et al., 1997a; 1997b).

Histological features and analysis of MyHC isoforms

Muscle cross-sectional area

In the 4 control groups of animals regenerated muscles had, on average, smaller cross-

sectional areas than the intact contralateral solei (15 - 44% deficit, Cremophor and saline, during

application and after withdrawal, Table 2). Force production correlated well with these deficits

(see specific force in Table 1). In contrast, cross-sectional areas of CsA-treated muscles were

larger than that in both control regenerated and contralateral muscles (Table 2). However, this was

associated with an increased force production only after CsA withdrawal (Table 1). Increase in

muscle girth was obviously due to the formation of larger numbers of fibres and/or more extensive

branching in the regenerated fibres (see Numbers of fibre profiles) as well as persistence of dead

muscle fibre remnants (see Other histological observations). It should be noted that, intact

muscles treated continuously with CsA had cross-sectional areas similar to the control intact

muscles (Table 2, during application) but produced lower forces (Table 1).

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Numbers of fibre profiles

Muscle cross-sections from the endplate regions of intact muscles contained similar

numbers of fibre profiles irrespective of the type of treatment or the period of observation (Table

2). In accordance with previous results, the number of fibre profiles in regenerated control muscles

were higher than in intact muscles (Table 2, Wernig et al., 1990; 1991a). This is obviously due to

abnormalities in muscle fibre architecture (split and branched fibres) commonly present in

regenerated muscles (Irintchev & Wernig, 1987; Wernig et al., 1990). The number of profiles in

regenerated muscles of immunosuppressed animals by far exceeded those in all other groups

(Table 2). No distinction has been made between true and/or pseuso-hyperplasia (increase in

number of profiles as a result of new fibre formation and due to the presence of split/branched

fibres, respectively).

Histochemical analysis of fibre phenotypes

Fibre type composition of intact control muscles were similar to those previously reported

for soleus muscles of untreated CBA/J mice (Table 2, upper and lower part, Wernig et al., 1990).

In regenerated control muscles there was a significant increase in the percentage of Type I fibres

when compared to intact controls, and, initially (at 4-7 weeks post-injury), of intermediate (Type

IIc) fibres; accordingly the numbers of Type II fibres were reduced (Fig. 3C, Table 2). Such

changes in fibre type frequencies after muscle injury and regeneration have been previously

observed in both mouse (Irintchev & Wernig, 1987; Wernig et al., 1990; 1991b) and rat (Whalen

et al., 1990) soleus muscles. In regenerated muscles of immunosuppressed animals a nearly

complete transformation in the opposite direction was present at 4-7 weeks after injury with 98%

of the profiles staining histochemically as Type II fibres (Fig. 3A, Table 2, upper part). A moderate

increase in the percentage of Type II fibres was also found in the intact CsA-treated muscles

(Table 2, upper half, Fig. 3B). These changes were reversed to a large extent after drug

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withdrawal but regenerated muscles still contained many more Type II profiles than regenerated

control muscles (Fig. 3D, Table 2, lower half). The increased frequency of undifferentiated (Type

IIc) fibres in regenerated control muscles 4 - 7 weeks after injury, and in regenerated CsA-treated

muscles after drug withdrawal (Table 2) is probably due to ongoing fibre type transformation.

Sections from regenerated and intact muscles of CsA-treated animals (4-7 weeks after

injury, n = 4) and appropriate control animals (Cremophor treatment, n = 4) were also stained with

monoclonal antibodies against MyHC isoforms (Fig. 4A-D). Observations on these muscles

confirmed the results from the histochemical typing (Table 2). Sections of the regenerated muscles

from the CsA group (4 sections/muscle approximately 100 µm apart) contained exclusively fibers

reacting with an antibody against fast/neonatal MyHC isoforms (Fig. 4C). Between 0 and 15 fibre

profiles per section were stained for slow MyHC (Fig. 4A, compare with the contralateral muscle

shown in Fig. 4B) and 8 - 23 profiles were positive for the embryonic isoform (Fig. 4D).

Electrophoretic analysis of MyHC isoform expression

The results of the histological analysis of fibre type composition were verified by

electrophoretic separation of MyHCs extracted from muscles of Cremophor- or saline-treated (n

= 14, 4-7 weeks after unilateral injury) and of CsA-treated animals (n = 11). The electrophoretic

patterns obtained from extracts of intact and regenerated muscles are shown in Figure 5. The most

significant observation in these experiments was the apparent phenotypic transformation, as

compared to control muscles. In regenerated CsA-treated muscles the band corresponding to the

slow (I) myosin heavy chain was either absent or faint. This resulted in a shift to a faster phenotype

with the appearance of fast IIB myosin, not present in intact and control regenerated solei, and an

enhanced signal at the level of migration of fast IIA/IIX isoforms (Fig. 5, lanes 1r, 2r, 3r and 4r).

An important finding was that regenerated muscles, irrespective of treatment, did not contain

detectable levels of developmental (embryonic, neonatal) isoforms of MyHC indicating high level

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of maturation. These two observations are consistent with the physiological and histological data

presented above.

Other histological observations

A characteristic feature of CsA-treated regenerated muscles was the persistence, up to 3.5

months post-injury, of dead muscle fibre fragments which had not been phagocytised after the

cryodamage (Fig. 6A-C). Similar remnants of dead fibres were not observed in control regenerated

muscles after the second week after injury. At one week, muscle fibre debris were seen in CsA-

(n = 2), as well as Cremophor- (n = 2) or saline-treated (n =2) regenerated muscles. In most cases

individual fragments could be traced in several spaced serial sections (100 - 150 µm apart). After

the second week the remnants became more compact (Fig. 6A,B) and could rarely be traced over

more than 1-2 spaced serial sections. The remnants contained calcium accumulations as revealed

by glyoxal-bis-(hydroxyanyl) and Alizarine Red S stains (not shown) and were always surrounded

by acid phosphatase-positive cells, presumably macrophages (Fig. 6C).

It is also interesting to note that a low level of regenerative activity was consistently

observed in CsA-treated cryolesioned muscles 1 - 3.5 months after injury which is in contrast to

the rapid completion of regeneration (< 3 weeks post-injury) in control damaged muscles.

Regeneration was deduced from the finding of some immature muscle fibres, both NCAM-positive

(immunofluorescence) and basophilic (Toluidine blue, not shown), as well as the presence of

embryonic MyHCs (Fig. 4D). The regenerative activity occurred mainly in the vicinity of calcified

fibre remnants.

Finally, at early periods after muscle lesion (< 4 weeks), regeneration in CsA-treated

muscles appeared, on histological grounds, delayed as compared to control muscles (Fig. 7A-C).

This impression was mainly due to the presence of areas in the muscle cross-sections devoid of

mature muscle fibres or containing loosely packed muscle fibres (Toluidine blue, ATPase

18

stainings). Such areas varied largely in individual CsA-treated solei (Fig. 7A,B) and were not

observed in control regenerated muscles which consisted of densely arranged fibres already by 2

weeks (Fig. 7A). Despite this deficit, some solei of immunosuppressed animals studied at 8 - 21

days contained large numbers of fibre profiles (949 - 1735, n = 3) comparable with the numbers

at later periods (Table 2). Sections of other CsA-treated muscles, however, contained apparently

low numbers of fibre profiles (not quantified, compare the muscle sections shown in Fig. 7 A and

B). In agreement with the variability described here is the variability in the functional performance

of the same muscles as illustrated by the maximum force measurments in Figure 2 (black symbols

to the left of the vertical dotted line at 28 days). Taken as a whole, however, regeneration during

CsA application was, in functional terms, not impared (Fig. 2, see also Discussion).

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DISCUSSION

The results of this study show that chronic CsA treatment in mice leads to considerable

alterations in muscle physiology, structure and phenotype. We cannot, and did not aim to,

dissociate whether these changes result from direct, tissue specific, drug effects or represent

epiphenomena. Although existing evidence for direct influences on transduction pathways in

skeletal muscle (see Introduction and the following Discussion) suggests direct mechanisms, the

results have to be cautiously interpreted in view of the signs of drug toxicity observed in this

study. Irrespective of this, the present findings have relevance to the clinical situation firstly since

treatment was performed at immunosuppressive dosage for the species used and secondly, CsA

toxicity is common in patients despite therapeutic drug monitoring (Shaw et al., 1999).

The results of previous investigations have indicated that Cremophor, the vehicle of CsA,

may be responsible for some of the toxic effects of the drug treatment (Windebank et al., 1994;

Tatou et al., 1996; Sanches et al., 1994). Therefore, a group of control animals have been

included in the experiment (saline treatment) to control for the possible adverse effects of the

vehicle. No differences were found in the results obtained from both groups (Cremophor versus

saline treatment) which indicates that, under our experimental conditions, Cremophor was not

toxic.

CsA effects on intact skeletal muscles

One significant effect of the treatment was a reduction in muscle force. This deficit was

neither due to a smaller muscle mass or cross-sectional area, nor to histopathological alterations

or decreased fibre number. The moderate (10%) slow-to-fast fibre transformation (see below) is

also not a plausible explanation (20-30% force deficit) even if we speculatively assume that there

is a difference in force generation per unit cross-sectional area in fast- and slow-twitch fibres of

the soleus muscle. It seems more likely that the force deficits arise from muscle mitochondrial

20

malfunctions (Mercier et al., 1995; Lampert et al., 1996; Biring et al., 1998). However, such

adverse effects, in conjunction with reduced capillary density, may provide a plausible explanation

of motor performance deficits in CsA-treated humans but not for impaired force generation of

isolated muscle preparations studied in vitro at 25°C. The possibility that CsA directly impairs

muscle contractility has also to be considered. In cardiac muscle, CsA has a direct adverse effect

on contractile function most likely due to its effect on Ca2+ handling (Janssen et al., 2000).

Tacrolimus (FK506), another drug with an immunosuppressive action similar to CsA, has a direct

effect on excitation-contraction coupling in skeletal muscle exerted via its binding to the FK

binding protein (FKBP) associated with the ryanodine receptor (Lamb & Stephenson, 1996). The

question as to whether CsA has a direct influence on force generation in skeletal muscle is of

interest, both from a physiological and a clinical point of view, and has to be further investigated.

A second major effect of CsA treatment on normal muscles was a change in fibre type

composition. The total number of muscle fibre profiles in CsA-treated solei was similar to the

numbers found in the control groups but less Type I and more Type II fibres were found (roughly

10% difference for both types). Apparently, a slow-to-fast fibre type transformation had occurred

in a proportion of the existing fibres. As indicated by the electrophoretic data, this phenotypic

transition was restricted to mature MyHC isoforms normally expressed in mouse soleus muscle

(I, IIA/X). Such a slow-to-fast transformation has been previously reported and explained by

inhibition of calcineurin activity (Chin et al., 1998; Bigard et al., 2000; Naya et al., 2000).

The alterations induced by CsA in the soleus muscles were reversible. After a prolonged

period of drug application (7 weeks) and withdrawal (5-8 weeks) all muscle properties studied

were comparable to those of the control groups. Therefore, drug treatment does not induce any

permanent functional or structural deficits in intact skeletal muscles.

21

Influences on muscle regeneration

Regenerating muscles were more profoundly influenced by the treatment. At 4 to 7 weeks

after injury, regenerated solei were composed exclusively of Type II fibres as demonstrated using

histochemical and immunocytochemical approaches. This phenotype was verified by the MyHC

electrophoretic patterns obtained from treated muscles. In addition, the assay showed that

treatment could lead to quantitatively new expression profiles including that of the MyHC IIB

isoform which is not detected normally in intact or regenerating mouse solei (Whalen et al.,

1990). The degree of phenotypic maturation was high as revealed by the apparent absence of

developmental MyHC isoforms. In accordance with the predominantly fast fibre phenotype was

the finding of a shorter contraction and half-relaxation time.

After one month, no obvious histological signs of regeneration deficits were found in CsA-

treated solei. On the contrary, muscle cross-sectional areas, number of profiles per section as well

as muscle mass by far exceeded those of control muscles. In contrast, structural deficits were

found in muscles studied 1-3 weeks after cryodamage (see also Abbott et al., 1998 for deficits by

10 days of regeneration). These two findings are apparently contradictory: it appears that muscle

repair during CsA application is initially delayed or defective but finally more efficient. This

paradox can be explained if we assume that CsA inhibits myoblast differentiation and

simultaneously enhances cell proliferation. In this way, the period of myoblast proliferation will

be longer than normal ("impaired regeneration") and the "gain" of myogenic cells greater before

maturation and fusion. Experimental evidence supports this notion in regard to inhibition of

differentiation (Hardiman et al., 1993; Abbott et al., 1998; Friday et al., 2000) but no significant

effects on cell proliferation in vitro have been reported (Abbott et al., 1998). Nevertherless, may

be the influences on cell replication in vivo, direct or indirect, differ from those in vitro.

Despite tissue defects, the functional performance of regenerated CsA-treated muscles

22

studied at time periods shorter than 4 weeks was comparable to control solei (see Fig. 2). This

finding indicates the limitations of any "one-methodology" approach used to evaluate muscle

recovery after injury. If histology alone had been used, the conclusion in this particular case would

have been "impaired regeneration". The functional assay alone would have indicated "normal

recovery". These considerations are important but do not explain the disparity in the findings

obtained with the two methods. The most simple explanation is that CsA-treated and control

muscles initially contain similar amounts of contractile tissue but treated and not control muscles -

considerable additional (due to enhanced myoblast proliferation, see above), volume of

differentiating, still non-contractile muscle tissue. As soon as this additional compartment matures

(> 4 weeks of regeneration), treated muscles become superior in function as compared to control

(Fig. 2). In this way, permanent functional deficits present after regeneration of cryodamaged

muscles (Wernig et al., 1991; Wernig et al., 1995; Wernig & Irintchev, 1995; Irintchev et al.,

1997a) were compensated at the organ (whole muscle) level. Similar mechanisms may account for

the observed improvement of muscle performance in CsA-treated Duchenne patients (Sharma et

al., 1993; Miller et al., 1997).

During CsA treatment force generation in regenerated muscles, in relation to muscle cross-

sectional area, was impaired to the same extent as in the contralateral intact muscles. This may be

explained by the direct effects on the contractile mechanisms which were proposed above. An

additional explanation was the presence of non-contractile tissue (calcified fibre remnants, see

Results and following Discussion). Irrespective of the exact reason(s), it is important to note that

the force production per unit cross-sectional area in regenerated muscles did not improve after

drug withdrawal as was the case in the contralateral solei. This indicates that there is a long-lasting

or permanent deficit. In addition, expression of the fast MyHC isoforms was above control level

after withdrawal. It is probable that the restoration of a "normal" pattern requires more than 2

23

months.

An interesting observation was that dead fibre remnants persisted in the regenerated solei

throughout the period of observation, even after drug withdrawal (for time periods up to 3 months

after cryodamage). We speculate that a possible reason is the inhibition of phagocytic activity in

regenerating muscles. The resulting delay in removing cell debris after injury may result in the

deposition of calcium which hampers subsequent phagocytosis. Although the evidence concerning

CsA effects on macrophages is not always consistent (for a review see Lose Garcia et al., 1996)

observations such as inhibition of macrophage activation and of cytokine production by these cells,

as well as reduced phagocytosis (see e.g. Thorat et al., 1994; Holschermann et al., 1996; Wasaki

et al., 1997; Garcia et al., 1999) support our speculation.

Concluding remarks

Effects of CsA treatment on fibre phenotype, muscle hypertrophy and muscle

differentiation have been reported previously. By combining physiological recordings and

subsequent morphological and biochemical analyses of the same muscles in this study we could

reveal new aspects. Important are the findings of impaired function of normal muscle during CsA

treatment, profound influence on fibre phenotype in regenerated muscles and the hyperplastic

effects on regenerating muscle, among others. These findings raised questions, as discussed above,

the answers to which will contribute to better understanding of drug influences, muscle function

and muscle regeneration.

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Acknowledgements

Stefanie Briel, Jennifer Heckroth and Karin Müller-Using provided technical assistance.

Gunnar Lange was helpful with fibre type quantifications and physiological recordings. The work

was supported by grants from the Deutsche Forschungsgemeinschaft (We859) and European

Commission (BMH4-97-2767 to A.W. and QLK6-1999-02034 to G.B.B. and A.W.), and by the

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Association pour la Recherche contre les Cancers (G.B.B.).

33

Table 1. In vitro contractile properties and muscle mass of intact and regenerated soleus muscles of CBA/J mice.

Group Tetanicforce(P0, mN)

Twitchforce(Pt, mN)

Wet muscleweight(mg)

P0 / muscleweight (N g-1)

Specificforce (P0 /area,kN m-2)

Twitch timeto peak (ms)

Twitch half-relaxationtime (ms)

ACh-inducedcontracture(% of P0)

Mean rateof rise ofthe twitch(N s-1)

n

During drug application (4 - 7 weeks post-injury)

CsA-treated animals

Regenerated muscles 113 ± 18 * 26 ± 3.2 * 13 ± 4.5 9.2 ± 2.7 * 13 ± 1.6 *# 24 ± 4.1 * # $ 49 ± 19 * $ 4.0 ± 1.5 1.1 ± 0.2 # $ 8

Intact muscles 119 ± 13 * 29 ± 0.7 * 10 ± 3.1 12 ± 3.6 18 ± 3.7 38 ± 2.7 * 84 ± 21 3.9 ± 1.9 0.7 ± 0.3 8

Control animals

Regenerated muscles 97 ± 15 * 22 ± 3.7 * 9.3 ± 1.3 11 ± 1.4 * 22 ± 4.5 38 ± 4.1 * 54 ± 8.6 * 5.0 ± 2.7 0.6 ± 0.1 * 6+5

Intact muscles 151 ± 20 44 ± 12 9.4 ± 0.8 16 ± 2.0 22 ± 3.6 49 ± 5.2 89 ± 15 3.5 ± 2.2 0.9 ± 0.2 5+5

After drug withdrawal (12 - 15 weeks post-injury)

CsA-treated animals

Regenerated muscles 165 ± 27 # 32 ± 6.1* # 12 ± 2.2 14 ± 1.7 14 ± 7.3 # 33 ± 2.7 * # $ 69 ± 17 3.9 ± 1.8 1.0 ± 0.2 # 5

Intact muscles 164 ± 19 41 ± 2.4 10 ± 1.1 16 ± 1.0 22 ± 2.3 46 ± 4.2 97 ± 28 3.9 ± 1.2 0.9 ± 0.1 5

Control animals

Regenerated muscles 119 ± 6.7 *B 27 ± 1.8 * $ 8.8 ± 2.4 14 ± 4.5 23 ± 5.8 42 ± 3.9 70 ± 7.6 7.1 ± 3.7 0.6 ± 0.1 $ 4+3

Intact muscles 143 ± 5.4 41 ± 3.1 10 ± 2.6 16 ± 1.0 22 ± 3.6 52 ± 4.1 98 ± 22 4.2 ± 2.0 0.8 ± 0.1 3+3

CBA/J mice were treated with either Cyclosporin A (CsA-treated animals, 50 mg kg-1 daily), Cremophor or saline (control animals). In three groups of animals(During drug application) treatment was initiated 8 days prior to unilateral muscle injury and continued for 4 to 7 weeks. The other groups (After drug withdrawal)were similarly treated up to end of the 7th week after injury and muscles were studied 5-8 weeks after drug withdrawal. For brevity, the two control groups(Cremophor and saline) are presented with overall mean values since group means are very similar. However, statistically (see below) they were treated asindependent groups. Values are means ± standard deviations. Statistical analysis was performed with ONEWAY analysis of variance followed by the Tukey´sprocedure for multiple comparisons. Simbols in superscript indicate that the mean value is significantly different from: * - intact control, # - regenerated control, $ -intact CsA-treated, A - control animals (saline- or Cremophor-treated). B - significantly different from intact contralateral muscles (t test).

34

Table 2. Results from morphometric and fibre phenotype analyses of intact and regenerated soleusmuscles of CsA- or vehicle-treated CBA/J mice.

TreatmentGroup of muscles

Muscle cross-sectional area(mm2)

Number offibre profilesper section

Type Iprofiles(%)

Type IIprofiles(%)

Type IIcprofiles(%)

n

During drug application (4 - 7 weeks post-injury)

Cyclosporin A

Regenerated muscles 0.86 ± 0.14 § + 1289 ± 190 * 0.4 ± 0.6 * 98 ± 1.8 * 1.3 ± 1.5 § 6

Intact muscles 0.64 ± 0.12 753 ± 79 56 ± 3.9 $ 43 ± 4.3 $ 1.6 ± 1.4 8

Cremophor

Regenerated muscles 0.40 ± 0.07 # + 865 ± 164 78 ± 5.4 # 18 ± 4.0 # 4.0 ± 2.4 # 4

Intact muscles 0.71 ± 0.16 705 ± 41 69 ± 3.8 31 ± 4.0 0.4 ± 0.2 4

Saline

Regenerated muscles 0.50 ± 0.08 881 ± 61 A 80 ± 5.5 # 16 ± 4.1 # 4.4 ± 2.1 # 5

Intact muscles 0.68 ± 0.08 710 ± 14 65 ± 3.8 34 ± 3.9 0.7 ± 0.8 4

After drug withdrawal (12 - 15 weeks post-injury)

Cyclosporin A

Regenerated muscles 1.08 ± 0.33 § 1226 ± 314 # 55 ± 13 § 38 ± 12 § 6.6 ± 6.3 6

Intact muscles 0.75 ± 0.13 723 ± 55 62 ± 3.2 37 ± 3.9 1.0 ± 0.9 5

Cremophor

Regenerated muscles 0.50 ± 0.15 848 ± 87 A 85 ± 7.6 $ 14 ± 7.5 $ 1.1 ± 0.3 4

Intact muscles 0.62 ± 0.13 733 ± 19 66 ± 8.8 33 ± 8.4 1.3 ± 0.5 4

Saline

Regenerated muscles 0.62 ± 0.15 891 ± 24 A 81 ± 2.8 $ 17 ± 2.5 $ 1.2 ± 0.8 4

Intact muscles 0.73 ± 0.11 763 ± 39 67 ± 2.3 32 ± 2.3 0.4 ± 0.4 3

Evaluations were done on transverse sections from the mid-portions of the muscles studied physiologically(Table 1). Fibre typing was performed on sections stained for myofibrillar ATPase activity. For further detailson the quantitative procedures see Material and Methods. n- number of muscles studied.Mean values ± standard deviations are given. Differences between mean group values in each experimental set(During drug application and After drug withdrawal) were analysed with ONEWAY analysis of variance andTukey´s multiple comparison procedure. Symblos in superscript indicate that the mean value is significantlydifferent from the value(s) of: * - all other 5 groups; # - all three groups of intact muscles; $ - both groups ofintact muscles treated with Cremophor or saline; § - both groups of regenerated muscles treated withCremophor or saline; +- intact contralateral muscles ( A - difference significant only in a t test, see text).

35

FIGURES AND LEGENDS

Figure 1. Body weight changes during treatment with CsA or vehicle solutions, and afterwithdrawal

In the first part of the graph (below 30 days), mean body weights with SD are shownfor groups of mice treated daily with CsA (black symbols), Cremophor (open circles) or saline(open squares). Treatment was initiated several days prior to surgery (muscle lesion at day 0)and continued for 4 - 7 weeks thereafter (only values up to day 28 are shown here). The valuesfor the same groups of animals on the day of muscle dissection have been described as 4-7weeks (overall values for the two control groups, open triangle). For comparison, in the thirdpart of the graph („after withdrawal“), values from the last day of the observation period forthree other groups (CsA - filled diamond, pooled values for the two control groups - opendiamond) are given. These animals were continuously treated with CsA or vehicle solutions for7 weeks and studied after an additional recovery period ranging between 5 and 8 weeks (i.e., at12 - 15 weeks after surgery). Numbers in brackets indicate the number of animals, asterisks -significant difference between the two groups (controls pooled together, two-sided Student´s ttest, P < 0.05).

Daily injection of vehicle solution (Cremophor or saline) did not have any obviousadverse effect on body weight and the relatively young animals (2 months of age uponinitiation of treatment) increased in mass (< 30 days) and were slightly, but still statisticallysignificantly, heavier by 4 - 7 weeks of treatment than the CsA-treated mice. Note large dailyvariations in mean body weight in the CsA group initially and weight loss after surgery at day0. In contrast to these findings, body weight after CsA withdrawal is higher in the CsA than inthe control group.

36

Figure 2. Muscle force during and after a period of drug or vehicle treatment

Maximum tetanic force of individual muscles studied after different recovery periods followingcryodamage at day 0. The animals were treated with CsA (filled circles), Cremophor (opencircles) or saline solution (open squares). The end of the treatment period (for further detailssee text and Fig. 1) is indicated by a vertical dotted line at day 49. These and other values fromthe tension recordings of the muscles shown between the two dotted lines (28 - 49 days) andafter the treatment period (> 49 days) are represented as mean group values in Table 1(„During drug application“ and „After withdrawal“, respectively). The fitted curves appear todescribe reasonably well the data as judged by visual inspection and indicated by the high r2

values which are shown in the graph. Note the overlap of the two curves describing the controldata.

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Figure 3. Changes in fibre type composition due to CsA treatment: histochemicalanalysis

Stainings for myofibrillar ATPase (acid preincubation, pH 4.3) of cross-sections from soleusmuscles of mice treated with CsA (A, B, D) or saline (C). Darkly stained muscle fibers areType I, unstained fibers - Type II, intermediately stained fibers - Type IIc (expressing bothType I and Type II myosin isoforms, one marked with asterisk in C). The regenerated right andthe intact left soleus muscle of one animal treated for 48 days with CsA are shown in A and B,respectively. In contrast to the intact muscle (B), only Type II fibers are present in theregenerated muscle (A) except for three small profiles at the arrows. Note that capillaries andlarger blood vessels (arrowheads) are also deliniated. This staining is due to high phosphataseactivity not related to skeletal muscle myosin. Unlike the CsA treated muscle shown in A,prevalance of Type I fibres is apparent in a muscle regenerated for 43 days in the presence ofphysiologic saline (C). One intermediate (IIc) fibre is marked with an asterisk (C). White spotsin the centers of dark fibers (C and D) are centrally located muscle fiber nuclei, a persistentsign of prior muscle regeneration in the mouse. CsA-induced muscle fiber type changes inregenerating muscles are reversible as seen in the transverse section from a muscle treated withCsA for 7 weeks after muscle injury followed by a 56-day recovery period without drugapplication (D). Scale bar in A indicates 50 µm and applies also to B, C and D.

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Figure 4. Changes in fibre type composition due to CsA treatment: immunocytochemicalanalysis

Transverse sections of the regenerated (A, C and D) and the intact contralateral soleus muscle(B) removed after a 7-week period of CsA treatment of one animal. The sections are stainedwith monoclonal antibodies recognising slow (A, B), fast and neonatal (C) and embryonic (D)isoforms of the MyHCs and a fluorescent labelled secondary antibody. In contrast to thesection of the contralateral intact muscle stained on the same microscope slide (B), only a faintsignal for slow myosin is seen in a single fibre profile in the regenerated muscle (A, arrow).Staining of regenerated fibres seen in C can be attributed, as indicated by the electrophoreticanalysis (see Results), to the presence of fast, rather than neonatal, myosin isoforms. Twosmall fibre profiles appear to contain the embryonic myosin isoform (D, arrows), a presumptivesign of active regeneration process. Scale bar in A indicates 100 µm and applies also to B, Cand D.

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Figure 5. Expression of MyHC isoforms as revealed by electrophoretic separation

SDS-PAGE electrophoretic patterns of myosin isoforms extracted from regenerated (lanesmarked with r) and intact contralateral muscles (lanes marked with i) of animals treated for 7weeks after surgery with CsA (lanes 1 - 4), Cremophor (lanes 5 - 8) or saline (lanes 9 and 10).For comparison, the expression pattern in intact muscles of untreated animals (Untreatedmuscle) are shown. In addition, the position of major MyHC isoforms are indicated on eachside of the figure. Schematic representation of the electrophoretic mobility of the differentmyosin heavy-chain isoforms: embryonic, fast IIA,fast IIX, fast IIB, neonatal (NN) and slow(I) is shown at the bottom.

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Figure 6. Persisting muscle fibre remnants after muscle damage/ regeneration in CsA-treated animals

Cross-sections from CsA-treated muscles studied after regeneration periods of 15 (A), 40 (B)and 21 days (C). Toluidine blue (A, B) and acid phosphatase (C) stains. By two weeks ofregeneration, muscle fibre debris (asterisks) are seen alongside the centrally nucleatedregenerated fibres (see arrow). At later periods, the condensed and amorphous remnants (twomarked with asterisks in B), are still present and, in contrast to the regenerated fibres, appearconspicuously bright when viewed with differential interference contrast (Nomarski) optics asseen in B. Acid phosphatase activity, presumably in macrophages, is present at the periphery ofthe muscle fibre remnants (C, one at arrow) as well as dispersed among the regenerated fibres(C, spot-like staining as the one at the arrowhead). Scale bar in C indicates 100 µm and appliesalso to A and B.

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Figure 7. Morphological signs of delayed muscle regeneration in CsA-treated mice

Complete cross-sections from a Cremophor- (A) and two CsA-treated muscles (B and C),after 2 weeks of regeneration. Myofibrillar ATPase activity after acid preincubation (pH 4.65)delineates all muscle fibres irrespective of fibre type. The control muscle (A) has alreadyreached an advanced stage of regeneration by 2 weeks as demonstrated by the diameters ofmost fibres and the compact arrangement of the fibres into fascicles. Still, note the smallerdiameters of the fibres arranged as a stripe of lower staining intensity at the right border of themuscle. The white spots in the center of many fibres are due to centrally located nuclei. Theappearance of the sections from the two CsA-treated muscle differ largely although both havebeen removed after two weeks of regeneration. Regeneration in the muscle, shown in B, ispoor as apparent from the relatively small muscle cross-sectional area and the small numbers offibres located in the superficial areas of the muscle (groups marked with asterisks). The centralarea contains few, mostly small and poorly stained fibres and numerous darkly stained bloodvessels. The second CsA-treated muscle shown here (C) has a larger cross-sectional areacompared with both other muscles and contains numerous fibres of large caliber. However, themuscle fibres in this muscle are generaly loosely arranged (compare with A) and areas of lessadvanced regeneration in the central part of the muscle (asterisk) are present. The darklystained structures indicated by the two arrows are dead muscle fibre remnants; this staining ispresumably due to accumulated calcium (see Results) which reacts in a non-enzymatic fashionwith constituents of the substrate and staining solutions (see Lojda et al., 1976). Sections ofthe liver upon which muscles are fixed prior to freezing in isopentane are seen in A and B (L).Scale bar in C indicates 200 µm and applies also to A and B.