Effects of torbafylline on muscle atrophy: prevention and recovery SOUADABOUDWAR AND DOMINIQUE DESPLANGHES~ U M 1341 CARS, kboratodre de phj~siobogie,Facult&Be mkdecm'ne, Lysn Grangc-Bbanc4~e,69373 Lyosr , cede.x 58,France
FwA~zrGRABER-VON BERGEN Can. J. Physiol. Pharmacol. Downloaded from www.nrcresearchpress.com by Simon Fraser University on 11/10/14 For personal use only.
Depastment of Anatomy, Unt~~ersikg! of Bern, Bern, Switzerland
ROLANDFAVIER U M 1341 CNRS, kboraeoire de ghj~siologde,f i e ~ l t iBe mkdecinc, &yon Grange-Blarlckae, 693 73 Lyun , c e h 88, Francs
AND
HANSHOPPELER Department- sf Anatomy, Uuiversn'ty of Bern, Bern, Switzerland Received September 24, 1991 ABOUDRAW, S., DESPLANCHES, B.,GRABER-$78~ BERGEN,E, FAVIER, R . , OKYAYUZ-BAK~UTI, I., and HOPPELBW, W. 1992. Effects of torbafylline on muscle atrophy: prevention and recovery. Can. 5. Physiol. Pharmacol. 70: 814-820. The effects of torbafylline on the prevention of and the recovery from 5 weeks of hindlimb suspension induced atrophy were analyzed in rat soleus and extensor digitomm longus muscles. Muscle alterations were investigated by determining a suite of electrophysiologisal, histochemical, and muscle ultrastmctural characteristics. Administration of torbafylline during the suspension period was ineffective in preventing any sf the observed muscle atrophic changes. Application of torbafylline during the recovery period resulted in a faster recovery of some soleus muscle structural and functional properties. Mitochondrial volume densities and capillary to fiber ratios returned towards baseline values earlier in the recovery process with tsrbafylline. Furthemore, the drug significantly improved soleus muscle fatigue resistance 4 weeks after cessation of hindlimb suspension. Key w~urds:xanthine, rat muscle contraction, histocytochernistry, mitochondria, capillaries. ABOUBRAR, S., BESPLANCHES, B., GRABER-VON BERGEN, E, FAVIER, R., OKYAYWZ-BAK~UTI, I., et HOPPELEW, H. 1992. Effects of torbafylline on muscle atrophy: prevention md recovery. Can. 5. Physiol. Phamacol, 70 : 814-820. Les effets d'une substance phamcologique (la torbafyIline) sur la prevention du dkveloppement d'une atrophie musculaire ou sur sa r6cupCration, onat tt6 6tudiCes, chez Be rat. Cette arnyotrophie a kt6 induite, au niveau des muscles solCaire et extenseur commun des doigts, par 5 sernaines de suspension Qu train postkrieur de 19animal.Les alterations musculaires ont Ct6 quamtifit5es par la mesure de certain$ paramktres Clectrophgrsiologiques, histoehimiques et ultra,atmctrmraux. L'administration de torbafylline, lors de la pkriode de suspension, s'est rkvklCe inefficace pour prkvenir Bes modifications likes B l'amyotrsphie. En revanche, lors de la ptriode Be rCcupCration, un retour plus rapide 2 la normale de certaines propri6tCs fonctionnelles (densitt volumttrique mitschondriaTe, nombre de capillaires par fibre) a kt6 obsemke, dam le muscle soldaire, aprks administration de torbafylline. De plus, cete substance augmente de facon significative la r6sismnce B la fatigue du muscle soltaire, 4 semaines aprks 19arrCtde la suspension. Mots el& : xanthine, propriktks csntractiles du muscle de rat, histocytochlmie, rnitochondries, capillaires.
Introduction A new xanhine derivative, torbaQllline, 7-ethoxymethy1- 1(5-hydroxy-%-me~yhexy1)-3-me~yhmthine, has k e n recently developed. This dmg has shown promise for the treatment of peripheral vvasculiar disease. After acute occlusion sf the femoral artery, torbafylline ameliorated the decreased skeletal muscle blood flow, red cell flux, and surface Po2. It reduced fatigability and increased the release of lactate from the ischemic muscle during and after nerve stimulation (OkyayuzBzddouti 1989). Moreover, Hudicka and Price (1990) showed that ttsrbafylline significantly decreased the proportion of glycolytic fibers in ligated fast-twitch skeletal muscles such as extensor digitomm longus and tibidis anterior (glycolytic cortex, white part). Five weeks after unilateral ligation of the common iliac artery in rat, administration sf torbafylline slightly diminished the relative loss of weight in soleus of the ligated leg compared with the contralateral control leg 'Author for correspondence. Printed in Canada ! lmprimt au Canada
(Hudlich and Torres 1990). These findings were confirmed by results of Comte et al. (1990). Loss of muscle mass and fiber type shifts are prominent events occurring during imm&~bilizaticsn-induced muscle atrophy. It therefore appeared promising to further explore the therapeutic potential of torbafylline in muscle atrophy. This was done using the headdown suspension model, developed by Morey (1979). In this model, rats are allowed to move freely, while their hindlimb muscles experience a decreased mechanical loading (hypodynamia). In general, it was found that after hindlimb suspension, slow-twitch muscles such as the soleus (SOL), containing predominantly slow-twitch, oxidative fibers (type 1) develop a greater atrophy (up to 50%) than fast-twitch muscles such as the extensor digitorurn lsngus (EDE) containing mainly fast-twitch, oxidative-glycolytic (IIa) and fasttwitch, oxidative-glycolytk (Ib)fibers (Thomason and Booth 1998). In addition, an increased expression sf fast-type myosin in slow-type fibers (Fitts et d. 1986; Reiser et d. 1987; Templeton ei HI. 1988) and changes in the major componems
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ABOUDWAW BT AL.
of the respiratory system (decrease in capillary per fiber ratio and increase in mitochondrial volume density and oxidative enzyme activities in single muscle fibers) were d s o reported (Desplanches et d.1990; Fitts et d. 1989). The purpose of the study was to explore possible effects of torbafjlline on the prevention of and the recovery from hindimb suspension induced muscle atrophy. Specificdly , we tested the hypothesis that torbafylline is capable of preventing some or all of the structural and hnctiond changes induced by 5 weeks sf hindlimb suspension when administered during the suspension period. The second hypothesis tested was that torbafylline given during the recovery period would accelerate the recovery of muscle tissue from the atrophic state. This was done by monitoring muscle fatigability, contractile properties, and the major stmcturd components of the muscle's respiratory system (capillarity and mitochondria~volume) in the SOL and the EDL muscles s f rats.
Materials and methods Thirty-five female pathogen-free Wistar rats, weighing 150 g, were housed in a temperature-controlled room (22 -24°C) with a 12-h dark-light cycle. They were provided b r i n a laboratory chow and water ad libitum. Hive rats were used as baseline controls and no manipulations were perfomed on these animals (baseline controls; C). Afier 1 week of acclimatization to the new environment, 30 rats were suspended (S) for a period of 5 weeks in individual cages using Morey's tail-suspension model (Marey 1979). These animals were assigned randomly to one of three experimental conditions. In each condition five rats were given torbafylline (25 mglkg, 1 % aqueous solution twice per day, 6 days per week) by gavage (%f T) while the remaining five hats were given comparable volumes of water (%).The first group of animals received torbafylline during the 5-week suspension period to evaluate whether the drug would prevent muscle atrophy (prevention group, S +T) . These animals were sacrificed immediately after the 5-week suspension period. The second and third groups of animals were suspended for 5 weeks without drug intervention. Thereafter, they were allowed to recover either with ar without the drug (SR),Ten animals were sacrificed after 2 weeks (treatment 2 weeks; S U ) and the remaining 10 animals after 4 weeks of recovery (treatment 4 weeks; SR4). During the 5 weeks s f suspension, the growth rate was similar in all rats. The baseline control animals were sacrificed after 5 weeks ( i s . , at the time when the animals of the prevention group were sacrificed).
Contmcile properties All the rats were anesthetized by inhalation of halothane. Halothane (2.5 % , Fluothane, Pitman-Moore, NJ) in oxygen was introduced through an inlet in the rats' Plexiglas container at a rate of 2.5 Llmin. After 3 -4 rnin of exposure to 2.5 % halothane, anesthesia was maintained with a 1.5 % hdothane -oxygen mixture introduced by face m s k (funnel in glass), throughout the recording of the electrophysiological parameters and all the muscular sampling procedure. The SOL of the right leg was exposed amad dissected free of surrounding tissue, leaving its nerve and blood supply intact. The rat was then placed ventrally in a chamber maintained at 97'6. For muscle stimulation, the leg was fixed in a cradle with steel pins through the ankle and h e h e e joints. After section of the plantaris and gastrocnemius tendons, a small steel hook was tied to the distal tendon of the SOL with a silk suture for connection to a Grass FT03 force transducer. The sciatic nerve was severed in the gluteal region, and the distal end of the nerve was drawn in a suction electrode. To prevent the muscles from drying out, the preparation was humidified with a Krebs solution a d covered with a small piece of parafilm. Supramaximd square-wave pulses of 0.5 ms duration were administered with a Grass 548 stimulator. The resting tension of the muscles was adjusted so that twitch tension was maximal. After a 5 4 1 1 rest period to dlow the muscle to recover from the adjustment to optimal length, the
8 15
muscle was stimulated via the nerve. An isometric twitch contraction was produced with supramaximal 0.5-ms square-wave pulse and peak tetanic contraction with a 338-ms supramaximal pulse at 100 Hz. Isometric fatigue properties were determined by 330-rns trains of 40 Hz (SOL) or 50 Hz (EDL), delivered 11s for 4 min. Twitch contraction time (TPT), half-relaxation time (1I2 RT), peak twitch tension during a twitch, peak tetanic tension, half rise time of maxim1 tetanus (112 TPT), and the tetanus 112 RT were recorded on a high speed galvansmetric recorder (Eastman Ksdak, Rochester, NY). The electrostimulation protocol led to some edema, as previously described by Herbert et al. (1988). It induced an acute gain in muscle mass of the order of 15 (for baseline control rats) to 40% (for suspended rats) with regard to the contralateral (unstimulated) muscles. For the calculations of weight-specific values of the hnctiond parameters, we used the muscle mass of the contralateral muscles for normalization. The weight gain was probably due to an increased fluid volun~e,with no change in the contractile protein mass, which might bias some of the electrophysiological results. At the end of the physiological measurements, the stimulated SOL and EDL, muscles were rapidly removed from the hindlimbs, and were weighed and processed for histochemical analysis as described below. The same muscles from the contralateral leg were processed for morphometric analysis as described below.
Histochemistry A 5-mm thick block from the midportion of each muscle was mounted in an embedding medium (TEK ACT compound) and stored at -80°C. Serial transverse sections (18 pm) were cut on a microtome at --3Q°C and were stained by the myosin - adenosine triphosphatase (ATPase) method (Grandmontagne et al. 1982). After preincubation at pH 4.4 in acid buffer (50 n M acetic acid) with 25 mM CaCl, for 4 min at 25'6, the ATPase reaction was carried out in buffer (pH 9.4) with 18 mM CaC1, and 2.7 mM ATP at 37°C for 28 rnin. Muscle fibers were classified into three major types (I, IIa, and I&) and intermediate type fibers (Int 1 or IIc, Int I%or IIab) (Hemansen et d. 1983; Pierobon-Bomioli et d o 1981). The histochemical method used in this report was based on the observed difference in pH lability of the myosin A T P a e activity of the isomyosins in the different fibers. A comparative imunohistochemical and enzyme histochemical analysis had been previously performed (Grandmantagme et al. 1982; Pierobon-%rmioli et aI. 1981) to validate the histochemical method used. Fiber type distribution was expressed as number of fibers of each type relative to the total number of fibers. Measurements were made on some 200 fibers from each muscle. Moiphsrnetry Samples for EM morphometry were processed as previously described in detail (Woppeler et al. 1981). Carefully the total muscle was removed, weighed, and cut longitudinally into thin strips. The strips were aligned in linear sequence and a total of six relatively large blocks were taken at random intervals. They were immersion fixed in a 6.25 % solution of glutaraldehyde in 0.1 M sodium cacodylate buffer adjusted to 438 mosmol with NaCl, total osmolarity 1100 mosmol, pH 7.4. After 1 h of fixation, samples were cut into small blocks suitable for electron microscopy and fixed for mother 5 h. After rinsing in sodium cacodylate buffer the tissue was postfmed in a solution of 1% osmic acid in 0.06 M veronal acetate buffer for 2 h. After block staining in 8.5% uranyl acetate in 0.05 M maleate buffer and dehydration in increasing concentrations of ethanol, the samples were finally embedded in Epon. For the stereological amlysis two tissue blocks per muscle and animal were transversely sectioned. Capillary to fiber ratio, capillary density, and mean fiber cross-sectional area were estimated at a final magnification of x 85W.Six pictures per block and, hence, 12 pictures per animal were taken in consecgltive frames of slotted grids (type R 100A; Veco Company, Netherlands) yielding in each animal some 150 fiber profiles for analysis. Point counting was done on a grid A 100 (1W test points). A final magnification of x 24 OOCB was used for the estimation of the volume of mitochondria, myofibrils, and residual sarcoplasmic
CAN. J. PHYSIOL. PHARMACOL. VOL. 70, 1992
TABLEI. Prevention: muscle weight and isometric contractile characteristics of soleus muscle in control rats, 5-week hindlimb-suspended rats treated with or without torbsQlline
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Control group
Suspended group
Suspended group torbafylline
+
Muscle weight, rng Twitch TPT, rns 112 RT, m Peak tension, g Specific tension, g/g Tetanus 1/2TPT, ms 112 RT, ms Peak tension, g Specific tension, g/g NOTE:Values are means f SE (n = 5). Abbreviations: TPT, twitch contraction time; 112 RT, half-relaxation time; 112 TPT,half rise time of maximal tetanus. aSignificantly different between control and suspended rats. '~i~nificantlydifferent between control and suspended rats treated with torbafylline. "Significantly different between suspended rats untreated or treated with torbafjlline.
TABLE2. Treatment: muscle weight and isometric contractile characteristics of soleus muscle in rats recavering 2 or 4 weeks, with or without torbafylline, after a 5-week period of hindlimb suspension 2 weeks recovery
2 weeks recovery torbafylline
+
4 weeks recovery torbafylline
+
4 weeks recovery
Muscle weight, rang Twitch TPT, rns 112 RT, ms Peak tension, g Specific tension, g/g Tetanus 112 TPT, ms 112 RT, rns Peak tension, g Specific tension, g/g NOTE:Values are means f SB (n = 5). Abbreviations: TPT,twitch csntraction time; 112 RT,half-relaxationtime; 112 TPT, hdf rise time s f maximal tetanus. d~ignificantlydifferent between control rats and rats recovering afger hindlimb suspension with torbafylline for 2 weeks, "Significantly different between control rats and rats recovering after kindlimb suspension for 4 weeks. f~i~nificantly different between control rats and rats recovering after hindlimb suspension with torba%ylline for 4 weeks. gSignificantly different between rats recovering after hindlimb suspension with or without torbafjlline for 4 weeks.
components per unit volume of muscle fiber. Systematic sampling with a random stating point was done in consecutive frames of 200-square mesh grids. Twenty micrographs per block and hence 40 micrographs per were taken and analys& by p i n t counting with a grid C 16 (144 test points). All stereological variables were calculated by applying standard procedures (Weibel 1979) previously established (Hoppeler et al. 1981). Morphological parameters are expressed in relative and absolute terns. For example, the absolute volume of mitochondria (Vmt in mL) was calculated from the mass of the soleus (Ms in g) and its fractional volume of mitochondria (Vv(mt,f), unitless) as V(mt) = Vv(mt,fl x Ms/l .M, whereby the interstitial space is neglected and the muscle is assumed to consist entirely of muscle fibers and 1.06 g/mL is the specific density of muscle tissue (see Csdey et al. 1987). statisfics
For dl data, intergroup differences were analysed with a two-way analysis of variance and an appropriate (Sheffk) post-hoe test for muZtiple comparison. Data are expressed as means SE; statistical significance was accepted at p < 0.05.
+
Results Baseline control rats and rats after 5 weeks of tail suspension with and without torbafyl'ine had weights f After weeks of 6 , Ig7 f and 217 recovery the body weights for unveated and treated rats were 224 5 and 216 3 g, respectively; after 4 weeks of recovery- they- were 233 _+ 8 and 248 f 5 g. There were no diferences in the growth rates of animals with or without the drug. 99
+
g9
+
Muscle mass and finetional memurements Muscle mass and bnctional measurements of soleus muscles are reported in Table I . There was a similar, close to 60% decrease in SOL muscle mass for both treated and untreated hindlimb suspended animals. Afer 2 weeks of recovery, muscle mass had dready returned to control values but not for the treated animals (Table 2). Ht has to be comsidered that for this study, we only had age-matched baseline controls (i.e., animals that were not hindlimb suspended) for the
ABQUDRWR ET AL.
Fatigability
% Fibers
index(g/g)
PREVENTION
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PREVENTION
I
"
a
.
I
.
1
.
I
TREATMENT ( 2 Weeks ) TREATMENT (2 Weeks)
IOOr
[LIZ.
TREATMENT (4 Weeks)
I
TREATMENT (4 Weeks)
FIG. 1. Fatigability i d e x (g/g) In SOL muscle. Abbreviations: PREVENTION, control group (G), 5-week hindlimbsuspended rats untreated ( S ) or treated with torbafylline ( S f T). TREATMENT, rats recovering 2 or 4 weeks, after a 5-week period of hindlimb suspension, with (SW +T, SR4 +T, respectively) or without torbafylline (SW, SR4). Values are means f SE (n = 5). Values are significantly different between rats recovering 4 weeks with or without torbafylline.
11-week-old animals (immediately after suspension). However, data from the literature show that this recovery was at least close to complete because SOL muscle mass in female Wistar control rats of age 19 weeks has been reported to be 1 12 f 5 rng (Besplmches et al. 1987b). As a consequence of muscle atrophy, weight-specific peak twitch tension was significantly elevated in atrophied muscles immediately after suspension (Table 1). Twitch contraction time and hdf-relaxation time were decreased, as previously reported by Elder and McComas (1987) md Winiarski et al. (1987) (Table I), and regained baseline values within 2 weeks (Table 2). Weight-specific peak tetanic tension (though it tended to increase after suspension) remained statistically unchanged through all experimental procedures; when expressed in absolute terns, peak tetanic tension was depressed immediately
FIG.2. Percent distribution of fibers (I, na, and IHc) in soleus muscles. Abbreviations: PREVENTION, control group ( C ), 5-week hidlimb-suspended rats untreated (S) or treated with torbafylline (S T) . TREATMENT, rats recovering 2 or 4 weeks, after a 5-week period of hindlimb suspension, with (SIX2 +T, SR4 +T, respectively) or without torbafylline (SIC?, SR4). Values rare means k SE (n = 5). "Values are significantly different between control and suspended rats. bValues are significantly different between control and torbafylline-treated suspended rats.
+
after suspension (Table 1). These results confirm earlier observations by Elder and McComas (1987) and Winiarski et id.(1987). Similar to twitch contraction time, there was a significant decrease in half rise time of maximal tetanus as well as in tetanic half-relaxation time immediately after hindlimb suspension (Table %). Treatment with torbafgrIline induced an increase in peak twitch tension (expressed in absolute units, g ) with respect to baseline controls, just for SW2 group (Tables 1 and 2). Some signs of "overcompensation9' in twitch half-relaxation time were only observed in the untreated SR4 group, while an overcompensation of tehnic half-relaxation time was shown in untreated as well as in treated SR4 groups (Table 2). The fatigability index (tension decline during tetmic train stimulation over a period of 4 min) remained unchanged in treated and untreated rats immediately after hindlimb suspension at all intervals measured (Fig. I). Other investigators have previ-
CAN. J. PWYSIOL. BHARMACOL. VOL. 70, 1992
TABLE3. Mean cr~ss-sectionalarea, capillaries per fiber, and capillary density of soleaas muscle
Treatment Prevention
2 weeks recovery
Suspended
+
Contr~l
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-
-
Suspended
torbafylline
2 weeks recovery
+
torbafylline
-
p p
Area, pn2 Capillaries per fiber Capillary density9 m-'
1653fHM
4 weeks recovery
4 weeks recovery
+
torbafylline
-
504f76'
430f47'
2.32k0.08
H.28f 0.10"
1.03f O.Mb
H429f 101
2663f 227"
2492f 24Sb
1438)98
1139+73d
1 . 8 ~ _ O . H 4 ~1.61f 0.09*
B248f 56
1428f73
1109P27F
1385k48
l . 9 7 f 0.06'
2 . 2 3 f 0.09
1773 f3gf
1610$51
NOTE:Values are meam +SE ( n = 5 ) .
'Significantly different between control and suspended rats. ?3ignificantIydifferent between control and suspended rats treated with torbafylline. * ~ i ~ n i f i c a different nd~ between ccsntrol rats and rats recovering after kindlimb suspension with torbafylline for 2 weeks. 'Significantly different between control rats sand rats recovering after hindlimb suspension for 4 weeks. h~ignificandy different between control rats and rats recovering aker hindlimb suspension for 2 weeks.
ously mentioned no change in the fatigability of the SOL muscle whatever the duration of the suspension, 1, 2, or 4 weeks (Fell et al. 1985; Winiarski et d. 1987). In the SR4 group, torbafylline induced a significantly decrease in tension decline during the 4-min fatigability test period (Fig. 1). In ED%,muscles, a smdl muscle atrophy occurred after 5 weeks 3.1 to 96.5 f 1.9 mg) . of hindlimb suspension (1 15.6 Much smaller but qualitatively similar changes in muscle twitch and tetanic characteristics as described for soleus muscles were observed, Only significant results are therefore reported below. Peak twitch tension in absolute units (11.8 f 0.8 g) did not change with hindlirnb suspension or recovery except for the S +T group, which had values higher than baseline controls (15.3 8.9 $). Weight-specific peak twitch tension was only increased after suspension (from 103.0 8.8 to 146.5 16.6 g/g in the S group, and to 173.3 12.4 glg in the S +T group). No change occurred in peak tetanic tension. It is noteworthy that there seemed to be a slowing of muscle contraction in the SR4+T group, consistent with the observations of the SOL muscle. Twitch contraction time (51.0 f 4.8 ms) and hdf-relaxation time (7'9.0 2.0 ms) were increased (65.0 2.0 and 92.5 _+ 3.2 ms, respectively). Similarly, M f rise time s f maximal tetanus (93.0 2.8 ms) and tetanic half-relaxation time (78.0 f 3.4 ms) were elevated (103.8 1.3 and 87.5 3.2 ms, respectively ). There were no significant changes in the fatigability index of any of the groups analyzed in EDE muscles (from 1043 f 61 to 114 18 g/g before and after 4 min of eIectrostimulatiora).
+
+
+
+
+
+ +
+
+
+
+
Fiber type dstrkbeetion Fiber type distribution was significantly changed aker hindlimb suspension in SOL muscle both in treated and untreated animals (Fig. 2). The decrease in type 1 fibers (90.0 3.6 to 60 3.9%) was due to a similar increase both in type IIc (3.4 f 1.5 to 21.0 5.6%) and IIa (6.6 _+ 2.1 to 19.8 2 3.1 %) fibers. Two weeks of recovery were sufficient for a return to control conditions. It is likely that, owing to the small sample size, the differences between treated and untreated a n h d s were only of marginal statistical significance. The reversibility of the transition sf the fibers after hindlimb suspension is in accordance with our previous results (Desplarashes et d. 198'9b) and those of Thomason et d. (1987). After 4 weeks of recovery there was a tendency for an overcompensation, in particul& with torbafyllineltreated
+
+
+
animals (180% sf type I fibers). We observed no significant changes in fiber type distribution in any group sf animals analyzed in the ED%muscle (8, 20, 5, 67% for type I, IIa, IIab, and %I%, respectively). Fiber size and capikl~rity Fiber size and capillarity were only analyzed in SOL muscles (Table 3). Fiber size was reduced by 70% immediately after hindlirnb suspension. Vdues were similar for treated and untreated animals. Fiber size essentially returned to normal vdues after two weeks of recovery. Significantly smaller values noted for this variable in some instances (SW +T and SR4 groups) can currently not be explained and may be due to measurement difficulties sf this parameter and are not considered to have a functional significance (Zurnstein et d. 1983). Capillary to fiber ratios were significantly reduced by close to 50% in treated and untreated animals after suspension. Recovery was incomplete after 2 weeks and even after 4 weeks except for the group treated with torbafylline. As the fiber cross-sectional area was decreased to a larger extent than the capillary to fiber ratio, there was a similar close to 80% increase in capillary density in both groups immediately following suspension, in accord with previous results (Musacchia et d. 1988; Desplanches et al. 1987~; Desplmches et d. 1990). The recovery of capillary density was completed after 2 weeks in all groups. Myo$bri&s and mitochondp.a'a There was a close to 18% decrease in myofibrillar volume density in both groups immediately after suspension (Table 4). Muscles s d y hHly recovered to baseline values after 4 weeks. There were no differences between the treatment groups. There was a close to 60% reduction in absolute myofibrillar volume immediately after suspension in both groups of animals. Even after 4 weeks myofibrililar volume had not fully reversed in either of the groups. Mitochondria1 volume density was increased by 50 % immediately after suspension in treated and control animals. With torbafyuine, recovery was complete after 2 weeks; without the drug, animals took 4 weeks to recover. Absolute vdues for mitochcsndrid volumes were reduced by 40% both in treated and untreated animals immediately after suspension. Because s f the rapid recovery of muscle mass, mitochondristl volumes fully recovered after only 2 weeks in all groups.
ABOUDMR BT AE.
TABLE4. Volume density (%) and absolute volume -
-
-
(X
cm" of myofibrils and total mitochondria
-
Treatment Revention
2 weeks recovery
Suspended
+
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Control Myofilsrils 88.9f 0.7 Volume density (9%) A b s o l u t e ~ 0 l ~ m e ( x 1 0 - ~ c m90.2k3.8 ~) Mitochondria 7.2f 0.5 Volume density ( %) Absolute volume ( x ern" 7.2f 0.4
2 weeks recovery
Suspended
torbafylline
79.0f 0.8" 31.3k3.7"
78.4f 0.9"$3,7f 0.4h 31.9f3.%b 76.5f2.7k
10.7k0.5" 4.3k8.7"
10.7f0.3' 4.1 f8.5h
9.0fQ.2k 5.2f 0.3
4 weeks recoveq
+
4 weeks recovery
ttsrbafygrlline
86.2f Q.7d 87.9f 0.5 66.2f3.2"71.9f2.3e
84.9f0.7 77.1k3.7f
f
torbafylline
7.7f 0.4' 5.9f 0.4
7.$k0.3
6.4f84.5
8.4f0.2 7.6f 0.4
NOTE:Values are means fSE (it = 5). USignificantlydifferent between control and suspended rds. %gnraificantly different between control and suspended rats treated with torbafylline. d~ignificantlydifferent between control rats and rats recovering after hindlimb suspension with torbaijlline for 2 weeks. "Significantly different between control rats and rats recovering afer hindlimb suspensican for 4 weeks. f~i~plificantl~ different between cuntml rats and rats recovering after hindlimb suspension with torbafylline for 4 weeks. ' ~ i ~ n i f i c a n tdifferent l~ between control rats and rats recovering after kindlimb suspension for 2 weeks. 'Significantly different between rats recovering after hindlimb suspension with or without torbafjlline for 2 weeks.
Discussion Muscle mass, body mass No measurable influence of the drug on the hindlimb suspension induced SOL muscle atrophy, nor on muscle mass during the recovery from atrophy, was evidenced. Surprisingly, it was found that the recovery of muscle mass was very rapid and dready complete afier only 2 weeks of recovery. Thomason et d.(1987) had previously reported a nearly csmplete recovery of soleus muscle weight only at 28 days following hindlimb suspension, while Kaspers et al. (1990) observed that muscle wet mass m d overdl fiber cross-sectional area were nearly 14%less than control values, after a comparable period of recovery. Judged by the animals' weight, we could growth in not detect any influence of torbafylline on this study. H1'stochemist~ In SOL, even if it did not reach the level of statistical significance, a tendency for torbafylline to increase the number of slow-twitch fibers, during reconversion, was demonstrated by an almost complete reversal to 100% type I fibers at 2 and 4 weeks of recovery. In EDL neither hindlimb suspension nor torbafyg.llline showed any influence on fiber type distribution under any of the experimental conditions analyzed. Fiber size and capiikrity Recovery of fiber size was essentially completed after 2 weeks. This was not the case for the recovery of the capillary to fiber ratio. Full recovery of the capillary to fiber ratio seemed to be helped by tohafylline as evidenced at 4 weeks of recovery. In a previous study, it had been shown that, without dmg, capillaries returned to the normal range after 8 weeks of sponbneous recovery (Desplsanches et al. 1987b). Owing to the faster rate sf the recovery of the muscle fiber size, capillary density was normd dready after 2 weeks. Structural csntposition ol$ muscle fibers (soleus only)) The recovery of myofibrillar volume was not complete even after 4 weeks. There was no apparent effect sf torbawlline on myofibriliar volume or volume density under any of the experimental conditions. Under torbafylline, there was a faster return of mitochondria1 volume density to baseline
values than without the drug. Recovery was complete in 2 weeks with and in 4 weeks without the drug, supporting the tendencies observed in the fiber type data. With respect to absolute mitochondria volumes, recovery was complete after 2 w e e k with no apparent influence of the administered drug. The observed changes in mitschondrid volume densities were mainly due to changes of the interfibrillar population of mitochondria while subsarcolernrnd mitochondria remined relatively unaffected (data not reported).
Functional measurenwnts and faligabiia'~ Despite the Iarge drop in muscle mass and the close to 60% reduction of myofibrillar volume and cross-sectional area, peak twitch tension remained unaffected during suspension and was even increased with torbafylline at 2 weeks of recovery. After 2 weeks of recsvery, there was a normalization of the twitch contraction time while the hdf-relaxation time was even increased beyond normah values at 4 weeks recovery, similar to the findings of the 100% type 1 fibers composition. In contrast to peak twitch tension, peak tetmic tension was influenced by h e loss in myofibrillar volume. There was full recovery sf peak tetanic tension closely matched by the absolute myofibrillar volumes, after 2 weeks, without any detectable effect of torbafylline on these parameters. A speeding up s f contraction and relaxation rates immediately after suspension, a recovery in 2 weeks, and an overcompensation at 4 weeks after cessation of hindlimb suspension were reported, again compatible with the structural findings. Interestingly torbafylline induced a significant increase in fatigue resistance compared with untreated controls at 4 weeks of recovery. Moreover, fatigue resistance of these muscles was significantly elevated above baseline control values. Because both the mass and the fiber type distribution s f EDL muscles are, if at all, only marginally affected, it was not surprising to find only minimal changes in physiological twitch and tetanus characteristics of this muscle. In general? trends point in the s m e direction as those observed in the SOL muscle, but the differences did not reach the level of significance. There was n~ apparent or consistent effect of torbafylline on any of the twitch or fatigue characteristics analyzed in EDL muscles.
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820
CAN. J. PHYSIOL. PHARMACOL. VOL. 40, I992
In conclusion, we found that torbafylline has no effect in preventing hindlimb suspension induced atrophy of rat soleus muscle. In contrast, it was found that recovery sf a number of structural and functional parmeters was accelerated in the group sf treated a n i d s at 2 and 4 weeks after cessation sf hindlimb suspension. Notably, we found a faster return to baseline values of mitmhondrid volume density estimates with torbafylline . Furthermore, tsrbafy$rllime significantly increased fatigue resistance after 4 weeks sf recovery. It seems justified to further evaluate the potentid sf torbafylllline particularly in recovery from muscle atrophy.
Acknowledgments We would like to thank H. Claassen and K. Bab1 for exeellent technical assistance. This work was supported by a grmt of Hoechst AG, Werk Kdle Albert, Wiesbaden, Germany. The care and use of animals conformed to the World Health Organization. Comte, J., Gautheron, D. C., Godinot, C., and Okyayuz-Baklouti, I. 1990. Effects of chronic torbafylline treatment on energy metabolism of ischemic skeletal muscle. Drug Dev. Res. 20: 291 299. Codey, K. E., Ksyar, S . R., Rosler, K., et ak. 1987. Adaptative variation in the mammalian respiratory system in relation to energetic demand: IV. Capillaries and h e i r relationship to oxidative capaeity. Respir. Physiol. 69: 47-64. Desplanches, D., Mayet, M, H., Sempore, B., and Flandrois, R. B987a. Stmctural and functional responses to prolonged kindlimb suspension in rat muscle. J. Appl. Pkysiol. 63: 558 -563. Desplanches, D., Mayet, M. H., Sempore, B., et a / . 1987b. Effect s f spontaneous recovery OH retraining after hindlimb suspension on aerobic capacity. 9. Appl. Physioi. 63: 1739 - 1743. Desplanches, D., Kayar, S. R., Sempore, B., st a!. 1990. Rat soleus muscle ultrastmcture following hindlimb suspension. S. Appl. Physiol. 69: 504 -5898. Elder, G. C. B., and McComas, A. J. 1987. Development of rat muscle during short- and long-term hindlimb suspension. J. Appl. Physiol. 62: 1917-1923. Fdl, R. D., Gladden, L. B., Steffen, J. M . , and Musacchia, X. J. 1985. Fatigue and contraction of slow m d fast muscles in hypoldnetic/Rypodynamic rats. J. Appl. Bhysiol. 58: 65 -69. Fitts, W. H.,Metzger, J. M., Riley, D. A., and UnswoHth, B. R. 1986. Models of disuse: a comparison of hindlimb suspension and immobilization. J. Appl. Bhysiol. 68: 1946- 1953. Fitts, R. H., Brimmer, C. J., Heywod-Cooksey, A., and T i m e r man, R. J. 1989. Single muscle fiber enzyme shifts with hindlimb suspension and immobilization. Am. J. Physiol. 256: @ 1082CB09l. Grandmontagne, M., Vaage, O., Vollestadt, N. K., and Hermansen, N . 1982. Confrontation de mCthodes histschimiqaaes et d'immnofluorescence p u r le typage des fibres QU muscle squelettique de rat. J. Physiol. (London), 78: 14. (Abstr.)
Herbert, M. E., Roy, R. R., and Edgertow, &I. R. 1988. Influence of one-week hindlimb suspnsion and intermittent high load exercise om rat muscles. Exp. Neurol. 1102: 190- 198. Wemansen, L., VolBestadt, N. K., Staff, P. H.,et a!. 1983. The effect of immobilization and training on strength and composition of human skeletal muscle. In Space physiology. CNRS, Lyon, France. pp. 255 -266. HoppeBer, H.,Mathieu, O . , Krauer, R., et ark. 1981. Design sf the mammalian respiratory system. VI. Distribution of mitochondria and capillaries in various muscles. Respir . Physiol. 44: 87 - 111. Hudlicka, O . , and Brice, S. 1990. Effects of torbafqrlline, pewtoxifylline and buflomedil on vascularisation and fibre type of rat skeletal muscles subjected to limited blood flow. Br. 3. Phamacol. 99: 786 -790. Hudlicka, O., and Torres, S. H.1990. Collateral circulation in skeletal muscles: effect of pentoxifgrlline and torbafylline. S. Med. (Westbury, NY), 21: 165- 180. Kaspers, C. E., White, T. P., and Maxwell, L. @. 1990. Running during recovery from hindlimb suspension induces transient muscle injury. J. Appl. Physioll. 68: 533 -539. Morey, E. W. 1979. Spaceflight and bone turnover: correlation with a new rat model of weightlessness. Bioscience. 29: % 68 - 172. Musacchia, X. J . , Steffen, 5 . M., Fell, R. D., and Dombrowski, M. J. 1988. Comparative moqhometry s f fibers and capillaries in soleus following weightlessness (SL-3) and suspension. Physiologist, 31: S28-S29. Okayayuz-Bakloaati, I. 1989. The effects of torbafgrlline on blood flow, ps, and function of rat ischaernic skeletal muscle. Ear. J. PharmacoH. 166: 75 - 86. Pierobon-Bonarioli, S . , Sartore, S., Daila Libera, L., et al. 1981. "Fast" isomyosins and fiber types in mammalian skeletal muscle. J. Histockern. Cytochem. 29: 1179- 1188. Reiser, P. J., Masper, C. E., and Moss, I%. k. 1987. Myosin subunits and contractile properties of single fibers from hypokinetic rat muscles. J. Appl. Physiol. 63: 2293 -2300. Templeton, G . H., Sweeney, H. L., Timson, B. F., et wk. 1988. Changes in fiber composition of soleus muscle during rat hindlimb suspension. J. Appl. Physiol. 65: 1191-1195. Thomason, D. B., and Booth, H. W. 1998. Atrophy sf the soleus muscle by hindlimb unweighting. J. Appl. Physiol. 68: 1- 12. Thomason, D. B., Herrick, R. E., Surdyka, D., and Baldwin, K. M. 1987. Time course of soleus muscle myosin expression during hindlimb suspension and recovery. J. Appl. Physiol. 63: 130 137. Weibel, E. R. 1979. In Stereological methods. Practical methods for biological mophometry. Vol. 1. Chap. 4 and 4 . Academic Press, London, New York, Toronto. Winiarski, A. M., Roy, R. R.,Alford, E. K., et ak. 1987. Mechanica1 properties of rat skeletal muscle after hindlimb suspension. Exp. Neaarol. 96: 650-660. Zumstein, A., Mathieu, O.,Howald, H., and Hoppeler, H. 1983. Morphometric analysis of the capillary supply in skeletal muscles of trained and untrained subjects. Its limitations in muscle biopsies. Pfluegers Arch. 397: 277 -283.
FwA~zrGRABER-VON BERGEN Can. J. Physiol. Pharmacol. Downloaded from www.nrcresearchpress.com by Simon Fraser University on 11/10/14 For personal use only.
Depastment of Anatomy, Unt~~ersikg! of Bern, Bern, Switzerland
ROLANDFAVIER U M 1341 CNRS, kboraeoire de ghj~siologde,f i e ~ l t iBe mkdecinc, &yon Grange-Blarlckae, 693 73 Lyun , c e h 88, Francs
AND
HANSHOPPELER Department- sf Anatomy, Uuiversn'ty of Bern, Bern, Switzerland Received September 24, 1991 ABOUDRAW, S., DESPLANCHES, B.,GRABER-$78~ BERGEN,E, FAVIER, R . , OKYAYUZ-BAK~UTI, I., and HOPPELBW, W. 1992. Effects of torbafylline on muscle atrophy: prevention and recovery. Can. 5. Physiol. Pharmacol. 70: 814-820. The effects of torbafylline on the prevention of and the recovery from 5 weeks of hindlimb suspension induced atrophy were analyzed in rat soleus and extensor digitomm longus muscles. Muscle alterations were investigated by determining a suite of electrophysiologisal, histochemical, and muscle ultrastmctural characteristics. Administration of torbafylline during the suspension period was ineffective in preventing any sf the observed muscle atrophic changes. Application of torbafylline during the recovery period resulted in a faster recovery of some soleus muscle structural and functional properties. Mitochondrial volume densities and capillary to fiber ratios returned towards baseline values earlier in the recovery process with tsrbafylline. Furthemore, the drug significantly improved soleus muscle fatigue resistance 4 weeks after cessation of hindlimb suspension. Key w~urds:xanthine, rat muscle contraction, histocytochernistry, mitochondria, capillaries. ABOUBRAR, S., BESPLANCHES, B., GRABER-VON BERGEN, E, FAVIER, R., OKYAYWZ-BAK~UTI, I., et HOPPELEW, H. 1992. Effects of torbafylline on muscle atrophy: prevention md recovery. Can. 5. Physiol. Phamacol, 70 : 814-820. Les effets d'une substance phamcologique (la torbafyIline) sur la prevention du dkveloppement d'une atrophie musculaire ou sur sa r6cupCration, onat tt6 6tudiCes, chez Be rat. Cette arnyotrophie a kt6 induite, au niveau des muscles solCaire et extenseur commun des doigts, par 5 sernaines de suspension Qu train postkrieur de 19animal.Les alterations musculaires ont Ct6 quamtifit5es par la mesure de certain$ paramktres Clectrophgrsiologiques, histoehimiques et ultra,atmctrmraux. L'administration de torbafylline, lors de la pkriode de suspension, s'est rkvklCe inefficace pour prkvenir Bes modifications likes B l'amyotrsphie. En revanche, lors de la ptriode Be rCcupCration, un retour plus rapide 2 la normale de certaines propri6tCs fonctionnelles (densitt volumttrique mitschondriaTe, nombre de capillaires par fibre) a kt6 obsemke, dam le muscle soldaire, aprks administration de torbafylline. De plus, cete substance augmente de facon significative la r6sismnce B la fatigue du muscle soltaire, 4 semaines aprks 19arrCtde la suspension. Mots el& : xanthine, propriktks csntractiles du muscle de rat, histocytochlmie, rnitochondries, capillaires.
Introduction A new xanhine derivative, torbaQllline, 7-ethoxymethy1- 1(5-hydroxy-%-me~yhexy1)-3-me~yhmthine, has k e n recently developed. This dmg has shown promise for the treatment of peripheral vvasculiar disease. After acute occlusion sf the femoral artery, torbafylline ameliorated the decreased skeletal muscle blood flow, red cell flux, and surface Po2. It reduced fatigability and increased the release of lactate from the ischemic muscle during and after nerve stimulation (OkyayuzBzddouti 1989). Moreover, Hudicka and Price (1990) showed that ttsrbafylline significantly decreased the proportion of glycolytic fibers in ligated fast-twitch skeletal muscles such as extensor digitomm longus and tibidis anterior (glycolytic cortex, white part). Five weeks after unilateral ligation of the common iliac artery in rat, administration sf torbafylline slightly diminished the relative loss of weight in soleus of the ligated leg compared with the contralateral control leg 'Author for correspondence. Printed in Canada ! lmprimt au Canada
(Hudlich and Torres 1990). These findings were confirmed by results of Comte et al. (1990). Loss of muscle mass and fiber type shifts are prominent events occurring during imm&~bilizaticsn-induced muscle atrophy. It therefore appeared promising to further explore the therapeutic potential of torbafylline in muscle atrophy. This was done using the headdown suspension model, developed by Morey (1979). In this model, rats are allowed to move freely, while their hindlimb muscles experience a decreased mechanical loading (hypodynamia). In general, it was found that after hindlimb suspension, slow-twitch muscles such as the soleus (SOL), containing predominantly slow-twitch, oxidative fibers (type 1) develop a greater atrophy (up to 50%) than fast-twitch muscles such as the extensor digitorurn lsngus (EDE) containing mainly fast-twitch, oxidative-glycolytic (IIa) and fasttwitch, oxidative-glycolytk (Ib)fibers (Thomason and Booth 1998). In addition, an increased expression sf fast-type myosin in slow-type fibers (Fitts et d. 1986; Reiser et d. 1987; Templeton ei HI. 1988) and changes in the major componems
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ABOUDWAW BT AL.
of the respiratory system (decrease in capillary per fiber ratio and increase in mitochondrial volume density and oxidative enzyme activities in single muscle fibers) were d s o reported (Desplanches et d.1990; Fitts et d. 1989). The purpose of the study was to explore possible effects of torbafjlline on the prevention of and the recovery from hindimb suspension induced muscle atrophy. Specificdly , we tested the hypothesis that torbafylline is capable of preventing some or all of the structural and hnctiond changes induced by 5 weeks sf hindlimb suspension when administered during the suspension period. The second hypothesis tested was that torbafylline given during the recovery period would accelerate the recovery of muscle tissue from the atrophic state. This was done by monitoring muscle fatigability, contractile properties, and the major stmcturd components of the muscle's respiratory system (capillarity and mitochondria~volume) in the SOL and the EDL muscles s f rats.
Materials and methods Thirty-five female pathogen-free Wistar rats, weighing 150 g, were housed in a temperature-controlled room (22 -24°C) with a 12-h dark-light cycle. They were provided b r i n a laboratory chow and water ad libitum. Hive rats were used as baseline controls and no manipulations were perfomed on these animals (baseline controls; C). Afier 1 week of acclimatization to the new environment, 30 rats were suspended (S) for a period of 5 weeks in individual cages using Morey's tail-suspension model (Marey 1979). These animals were assigned randomly to one of three experimental conditions. In each condition five rats were given torbafylline (25 mglkg, 1 % aqueous solution twice per day, 6 days per week) by gavage (%f T) while the remaining five hats were given comparable volumes of water (%).The first group of animals received torbafylline during the 5-week suspension period to evaluate whether the drug would prevent muscle atrophy (prevention group, S +T) . These animals were sacrificed immediately after the 5-week suspension period. The second and third groups of animals were suspended for 5 weeks without drug intervention. Thereafter, they were allowed to recover either with ar without the drug (SR),Ten animals were sacrificed after 2 weeks (treatment 2 weeks; S U ) and the remaining 10 animals after 4 weeks of recovery (treatment 4 weeks; SR4). During the 5 weeks s f suspension, the growth rate was similar in all rats. The baseline control animals were sacrificed after 5 weeks ( i s . , at the time when the animals of the prevention group were sacrificed).
Contmcile properties All the rats were anesthetized by inhalation of halothane. Halothane (2.5 % , Fluothane, Pitman-Moore, NJ) in oxygen was introduced through an inlet in the rats' Plexiglas container at a rate of 2.5 Llmin. After 3 -4 rnin of exposure to 2.5 % halothane, anesthesia was maintained with a 1.5 % hdothane -oxygen mixture introduced by face m s k (funnel in glass), throughout the recording of the electrophysiological parameters and all the muscular sampling procedure. The SOL of the right leg was exposed amad dissected free of surrounding tissue, leaving its nerve and blood supply intact. The rat was then placed ventrally in a chamber maintained at 97'6. For muscle stimulation, the leg was fixed in a cradle with steel pins through the ankle and h e h e e joints. After section of the plantaris and gastrocnemius tendons, a small steel hook was tied to the distal tendon of the SOL with a silk suture for connection to a Grass FT03 force transducer. The sciatic nerve was severed in the gluteal region, and the distal end of the nerve was drawn in a suction electrode. To prevent the muscles from drying out, the preparation was humidified with a Krebs solution a d covered with a small piece of parafilm. Supramaximd square-wave pulses of 0.5 ms duration were administered with a Grass 548 stimulator. The resting tension of the muscles was adjusted so that twitch tension was maximal. After a 5 4 1 1 rest period to dlow the muscle to recover from the adjustment to optimal length, the
8 15
muscle was stimulated via the nerve. An isometric twitch contraction was produced with supramaximal 0.5-ms square-wave pulse and peak tetanic contraction with a 338-ms supramaximal pulse at 100 Hz. Isometric fatigue properties were determined by 330-rns trains of 40 Hz (SOL) or 50 Hz (EDL), delivered 11s for 4 min. Twitch contraction time (TPT), half-relaxation time (1I2 RT), peak twitch tension during a twitch, peak tetanic tension, half rise time of maxim1 tetanus (112 TPT), and the tetanus 112 RT were recorded on a high speed galvansmetric recorder (Eastman Ksdak, Rochester, NY). The electrostimulation protocol led to some edema, as previously described by Herbert et al. (1988). It induced an acute gain in muscle mass of the order of 15 (for baseline control rats) to 40% (for suspended rats) with regard to the contralateral (unstimulated) muscles. For the calculations of weight-specific values of the hnctiond parameters, we used the muscle mass of the contralateral muscles for normalization. The weight gain was probably due to an increased fluid volun~e,with no change in the contractile protein mass, which might bias some of the electrophysiological results. At the end of the physiological measurements, the stimulated SOL and EDL, muscles were rapidly removed from the hindlimbs, and were weighed and processed for histochemical analysis as described below. The same muscles from the contralateral leg were processed for morphometric analysis as described below.
Histochemistry A 5-mm thick block from the midportion of each muscle was mounted in an embedding medium (TEK ACT compound) and stored at -80°C. Serial transverse sections (18 pm) were cut on a microtome at --3Q°C and were stained by the myosin - adenosine triphosphatase (ATPase) method (Grandmontagne et al. 1982). After preincubation at pH 4.4 in acid buffer (50 n M acetic acid) with 25 mM CaCl, for 4 min at 25'6, the ATPase reaction was carried out in buffer (pH 9.4) with 18 mM CaC1, and 2.7 mM ATP at 37°C for 28 rnin. Muscle fibers were classified into three major types (I, IIa, and I&) and intermediate type fibers (Int 1 or IIc, Int I%or IIab) (Hemansen et d. 1983; Pierobon-Bomioli et d o 1981). The histochemical method used in this report was based on the observed difference in pH lability of the myosin A T P a e activity of the isomyosins in the different fibers. A comparative imunohistochemical and enzyme histochemical analysis had been previously performed (Grandmantagme et al. 1982; Pierobon-%rmioli et aI. 1981) to validate the histochemical method used. Fiber type distribution was expressed as number of fibers of each type relative to the total number of fibers. Measurements were made on some 200 fibers from each muscle. Moiphsrnetry Samples for EM morphometry were processed as previously described in detail (Woppeler et al. 1981). Carefully the total muscle was removed, weighed, and cut longitudinally into thin strips. The strips were aligned in linear sequence and a total of six relatively large blocks were taken at random intervals. They were immersion fixed in a 6.25 % solution of glutaraldehyde in 0.1 M sodium cacodylate buffer adjusted to 438 mosmol with NaCl, total osmolarity 1100 mosmol, pH 7.4. After 1 h of fixation, samples were cut into small blocks suitable for electron microscopy and fixed for mother 5 h. After rinsing in sodium cacodylate buffer the tissue was postfmed in a solution of 1% osmic acid in 0.06 M veronal acetate buffer for 2 h. After block staining in 8.5% uranyl acetate in 0.05 M maleate buffer and dehydration in increasing concentrations of ethanol, the samples were finally embedded in Epon. For the stereological amlysis two tissue blocks per muscle and animal were transversely sectioned. Capillary to fiber ratio, capillary density, and mean fiber cross-sectional area were estimated at a final magnification of x 85W.Six pictures per block and, hence, 12 pictures per animal were taken in consecgltive frames of slotted grids (type R 100A; Veco Company, Netherlands) yielding in each animal some 150 fiber profiles for analysis. Point counting was done on a grid A 100 (1W test points). A final magnification of x 24 OOCB was used for the estimation of the volume of mitochondria, myofibrils, and residual sarcoplasmic
CAN. J. PHYSIOL. PHARMACOL. VOL. 70, 1992
TABLEI. Prevention: muscle weight and isometric contractile characteristics of soleus muscle in control rats, 5-week hindlimb-suspended rats treated with or without torbsQlline
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Control group
Suspended group
Suspended group torbafylline
+
Muscle weight, rng Twitch TPT, rns 112 RT, m Peak tension, g Specific tension, g/g Tetanus 1/2TPT, ms 112 RT, ms Peak tension, g Specific tension, g/g NOTE:Values are means f SE (n = 5). Abbreviations: TPT, twitch contraction time; 112 RT, half-relaxation time; 112 TPT,half rise time of maximal tetanus. aSignificantly different between control and suspended rats. '~i~nificantlydifferent between control and suspended rats treated with torbafylline. "Significantly different between suspended rats untreated or treated with torbafjlline.
TABLE2. Treatment: muscle weight and isometric contractile characteristics of soleus muscle in rats recavering 2 or 4 weeks, with or without torbafylline, after a 5-week period of hindlimb suspension 2 weeks recovery
2 weeks recovery torbafylline
+
4 weeks recovery torbafylline
+
4 weeks recovery
Muscle weight, rang Twitch TPT, rns 112 RT, ms Peak tension, g Specific tension, g/g Tetanus 112 TPT, ms 112 RT, rns Peak tension, g Specific tension, g/g NOTE:Values are means f SB (n = 5). Abbreviations: TPT,twitch csntraction time; 112 RT,half-relaxationtime; 112 TPT, hdf rise time s f maximal tetanus. d~ignificantlydifferent between control rats and rats recovering afger hindlimb suspension with torbafylline for 2 weeks, "Significantly different between control rats and rats recovering after kindlimb suspension for 4 weeks. f~i~nificantly different between control rats and rats recovering after hindlimb suspension with torba%ylline for 4 weeks. gSignificantly different between rats recovering after hindlimb suspension with or without torbafjlline for 4 weeks.
components per unit volume of muscle fiber. Systematic sampling with a random stating point was done in consecutive frames of 200-square mesh grids. Twenty micrographs per block and hence 40 micrographs per were taken and analys& by p i n t counting with a grid C 16 (144 test points). All stereological variables were calculated by applying standard procedures (Weibel 1979) previously established (Hoppeler et al. 1981). Morphological parameters are expressed in relative and absolute terns. For example, the absolute volume of mitochondria (Vmt in mL) was calculated from the mass of the soleus (Ms in g) and its fractional volume of mitochondria (Vv(mt,f), unitless) as V(mt) = Vv(mt,fl x Ms/l .M, whereby the interstitial space is neglected and the muscle is assumed to consist entirely of muscle fibers and 1.06 g/mL is the specific density of muscle tissue (see Csdey et al. 1987). statisfics
For dl data, intergroup differences were analysed with a two-way analysis of variance and an appropriate (Sheffk) post-hoe test for muZtiple comparison. Data are expressed as means SE; statistical significance was accepted at p < 0.05.
+
Results Baseline control rats and rats after 5 weeks of tail suspension with and without torbafyl'ine had weights f After weeks of 6 , Ig7 f and 217 recovery the body weights for unveated and treated rats were 224 5 and 216 3 g, respectively; after 4 weeks of recovery- they- were 233 _+ 8 and 248 f 5 g. There were no diferences in the growth rates of animals with or without the drug. 99
+
g9
+
Muscle mass and finetional memurements Muscle mass and bnctional measurements of soleus muscles are reported in Table I . There was a similar, close to 60% decrease in SOL muscle mass for both treated and untreated hindlimb suspended animals. Afer 2 weeks of recovery, muscle mass had dready returned to control values but not for the treated animals (Table 2). Ht has to be comsidered that for this study, we only had age-matched baseline controls (i.e., animals that were not hindlimb suspended) for the
ABQUDRWR ET AL.
Fatigability
% Fibers
index(g/g)
PREVENTION
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PREVENTION
I
"
a
.
I
.
1
.
I
TREATMENT ( 2 Weeks ) TREATMENT (2 Weeks)
IOOr
[LIZ.
TREATMENT (4 Weeks)
I
TREATMENT (4 Weeks)
FIG. 1. Fatigability i d e x (g/g) In SOL muscle. Abbreviations: PREVENTION, control group (G), 5-week hindlimbsuspended rats untreated ( S ) or treated with torbafylline ( S f T). TREATMENT, rats recovering 2 or 4 weeks, after a 5-week period of hindlimb suspension, with (SW +T, SR4 +T, respectively) or without torbafylline (SW, SR4). Values are means f SE (n = 5). Values are significantly different between rats recovering 4 weeks with or without torbafylline.
11-week-old animals (immediately after suspension). However, data from the literature show that this recovery was at least close to complete because SOL muscle mass in female Wistar control rats of age 19 weeks has been reported to be 1 12 f 5 rng (Besplmches et al. 1987b). As a consequence of muscle atrophy, weight-specific peak twitch tension was significantly elevated in atrophied muscles immediately after suspension (Table 1). Twitch contraction time and hdf-relaxation time were decreased, as previously reported by Elder and McComas (1987) md Winiarski et al. (1987) (Table I), and regained baseline values within 2 weeks (Table 2). Weight-specific peak tetanic tension (though it tended to increase after suspension) remained statistically unchanged through all experimental procedures; when expressed in absolute terns, peak tetanic tension was depressed immediately
FIG.2. Percent distribution of fibers (I, na, and IHc) in soleus muscles. Abbreviations: PREVENTION, control group ( C ), 5-week hidlimb-suspended rats untreated (S) or treated with torbafylline (S T) . TREATMENT, rats recovering 2 or 4 weeks, after a 5-week period of hindlimb suspension, with (SIX2 +T, SR4 +T, respectively) or without torbafylline (SIC?, SR4). Values rare means k SE (n = 5). "Values are significantly different between control and suspended rats. bValues are significantly different between control and torbafylline-treated suspended rats.
+
after suspension (Table 1). These results confirm earlier observations by Elder and McComas (1987) and Winiarski et id.(1987). Similar to twitch contraction time, there was a significant decrease in half rise time of maximal tetanus as well as in tetanic half-relaxation time immediately after hindlimb suspension (Table %). Treatment with torbafgrIline induced an increase in peak twitch tension (expressed in absolute units, g ) with respect to baseline controls, just for SW2 group (Tables 1 and 2). Some signs of "overcompensation9' in twitch half-relaxation time were only observed in the untreated SR4 group, while an overcompensation of tehnic half-relaxation time was shown in untreated as well as in treated SR4 groups (Table 2). The fatigability index (tension decline during tetmic train stimulation over a period of 4 min) remained unchanged in treated and untreated rats immediately after hindlimb suspension at all intervals measured (Fig. I). Other investigators have previ-
CAN. J. PWYSIOL. BHARMACOL. VOL. 70, 1992
TABLE3. Mean cr~ss-sectionalarea, capillaries per fiber, and capillary density of soleaas muscle
Treatment Prevention
2 weeks recovery
Suspended
+
Contr~l
Can. J. Physiol. Pharmacol. Downloaded from www.nrcresearchpress.com by Simon Fraser University on 11/10/14 For personal use only.
-
-
Suspended
torbafylline
2 weeks recovery
+
torbafylline
-
p p
Area, pn2 Capillaries per fiber Capillary density9 m-'
1653fHM
4 weeks recovery
4 weeks recovery
+
torbafylline
-
504f76'
430f47'
2.32k0.08
H.28f 0.10"
1.03f O.Mb
H429f 101
2663f 227"
2492f 24Sb
1438)98
1139+73d
1 . 8 ~ _ O . H 4 ~1.61f 0.09*
B248f 56
1428f73
1109P27F
1385k48
l . 9 7 f 0.06'
2 . 2 3 f 0.09
1773 f3gf
1610$51
NOTE:Values are meam +SE ( n = 5 ) .
'Significantly different between control and suspended rats. ?3ignificantIydifferent between control and suspended rats treated with torbafylline. * ~ i ~ n i f i c a different nd~ between ccsntrol rats and rats recovering after kindlimb suspension with torbafylline for 2 weeks. 'Significantly different between control rats sand rats recovering after hindlimb suspension for 4 weeks. h~ignificandy different between control rats and rats recovering aker hindlimb suspension for 2 weeks.
ously mentioned no change in the fatigability of the SOL muscle whatever the duration of the suspension, 1, 2, or 4 weeks (Fell et al. 1985; Winiarski et d. 1987). In the SR4 group, torbafylline induced a significantly decrease in tension decline during the 4-min fatigability test period (Fig. 1). In ED%,muscles, a smdl muscle atrophy occurred after 5 weeks 3.1 to 96.5 f 1.9 mg) . of hindlimb suspension (1 15.6 Much smaller but qualitatively similar changes in muscle twitch and tetanic characteristics as described for soleus muscles were observed, Only significant results are therefore reported below. Peak twitch tension in absolute units (11.8 f 0.8 g) did not change with hindlirnb suspension or recovery except for the S +T group, which had values higher than baseline controls (15.3 8.9 $). Weight-specific peak twitch tension was only increased after suspension (from 103.0 8.8 to 146.5 16.6 g/g in the S group, and to 173.3 12.4 glg in the S +T group). No change occurred in peak tetanic tension. It is noteworthy that there seemed to be a slowing of muscle contraction in the SR4+T group, consistent with the observations of the SOL muscle. Twitch contraction time (51.0 f 4.8 ms) and hdf-relaxation time (7'9.0 2.0 ms) were increased (65.0 2.0 and 92.5 _+ 3.2 ms, respectively). Similarly, M f rise time s f maximal tetanus (93.0 2.8 ms) and tetanic half-relaxation time (78.0 f 3.4 ms) were elevated (103.8 1.3 and 87.5 3.2 ms, respectively ). There were no significant changes in the fatigability index of any of the groups analyzed in EDE muscles (from 1043 f 61 to 114 18 g/g before and after 4 min of eIectrostimulatiora).
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+
+
+
+
+ +
+
+
+
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Fiber type dstrkbeetion Fiber type distribution was significantly changed aker hindlimb suspension in SOL muscle both in treated and untreated animals (Fig. 2). The decrease in type 1 fibers (90.0 3.6 to 60 3.9%) was due to a similar increase both in type IIc (3.4 f 1.5 to 21.0 5.6%) and IIa (6.6 _+ 2.1 to 19.8 2 3.1 %) fibers. Two weeks of recovery were sufficient for a return to control conditions. It is likely that, owing to the small sample size, the differences between treated and untreated a n h d s were only of marginal statistical significance. The reversibility of the transition sf the fibers after hindlimb suspension is in accordance with our previous results (Desplarashes et d. 198'9b) and those of Thomason et d. (1987). After 4 weeks of recovery there was a tendency for an overcompensation, in particul& with torbafyllineltreated
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animals (180% sf type I fibers). We observed no significant changes in fiber type distribution in any group sf animals analyzed in the ED%muscle (8, 20, 5, 67% for type I, IIa, IIab, and %I%, respectively). Fiber size and capikl~rity Fiber size and capillarity were only analyzed in SOL muscles (Table 3). Fiber size was reduced by 70% immediately after hindlirnb suspension. Vdues were similar for treated and untreated animals. Fiber size essentially returned to normal vdues after two weeks of recovery. Significantly smaller values noted for this variable in some instances (SW +T and SR4 groups) can currently not be explained and may be due to measurement difficulties sf this parameter and are not considered to have a functional significance (Zurnstein et d. 1983). Capillary to fiber ratios were significantly reduced by close to 50% in treated and untreated animals after suspension. Recovery was incomplete after 2 weeks and even after 4 weeks except for the group treated with torbafylline. As the fiber cross-sectional area was decreased to a larger extent than the capillary to fiber ratio, there was a similar close to 80% increase in capillary density in both groups immediately following suspension, in accord with previous results (Musacchia et d. 1988; Desplanches et al. 1987~; Desplmches et d. 1990). The recovery of capillary density was completed after 2 weeks in all groups. Myo$bri&s and mitochondp.a'a There was a close to 18% decrease in myofibrillar volume density in both groups immediately after suspension (Table 4). Muscles s d y hHly recovered to baseline values after 4 weeks. There were no differences between the treatment groups. There was a close to 60% reduction in absolute myofibrillar volume immediately after suspension in both groups of animals. Even after 4 weeks myofibrililar volume had not fully reversed in either of the groups. Mitochondria1 volume density was increased by 50 % immediately after suspension in treated and control animals. With torbafyuine, recovery was complete after 2 weeks; without the drug, animals took 4 weeks to recover. Absolute vdues for mitochcsndrid volumes were reduced by 40% both in treated and untreated animals immediately after suspension. Because s f the rapid recovery of muscle mass, mitochondristl volumes fully recovered after only 2 weeks in all groups.
ABOUDMR BT AE.
TABLE4. Volume density (%) and absolute volume -
-
-
(X
cm" of myofibrils and total mitochondria
-
Treatment Revention
2 weeks recovery
Suspended
+
Can. J. Physiol. Pharmacol. Downloaded from www.nrcresearchpress.com by Simon Fraser University on 11/10/14 For personal use only.
Control Myofilsrils 88.9f 0.7 Volume density (9%) A b s o l u t e ~ 0 l ~ m e ( x 1 0 - ~ c m90.2k3.8 ~) Mitochondria 7.2f 0.5 Volume density ( %) Absolute volume ( x ern" 7.2f 0.4
2 weeks recovery
Suspended
torbafylline
79.0f 0.8" 31.3k3.7"
78.4f 0.9"$3,7f 0.4h 31.9f3.%b 76.5f2.7k
10.7k0.5" 4.3k8.7"
10.7f0.3' 4.1 f8.5h
9.0fQ.2k 5.2f 0.3
4 weeks recoveq
+
4 weeks recovery
ttsrbafygrlline
86.2f Q.7d 87.9f 0.5 66.2f3.2"71.9f2.3e
84.9f0.7 77.1k3.7f
f
torbafylline
7.7f 0.4' 5.9f 0.4
7.$k0.3
6.4f84.5
8.4f0.2 7.6f 0.4
NOTE:Values are means fSE (it = 5). USignificantlydifferent between control and suspended rds. %gnraificantly different between control and suspended rats treated with torbafylline. d~ignificantlydifferent between control rats and rats recovering after hindlimb suspension with torbaijlline for 2 weeks. "Significantly different between control rats and rats recovering afer hindlimb suspensican for 4 weeks. f~i~plificantl~ different between cuntml rats and rats recovering after hindlimb suspension with torbafylline for 4 weeks. ' ~ i ~ n i f i c a n tdifferent l~ between control rats and rats recovering after kindlimb suspension for 2 weeks. 'Significantly different between rats recovering after hindlimb suspension with or without torbafjlline for 2 weeks.
Discussion Muscle mass, body mass No measurable influence of the drug on the hindlimb suspension induced SOL muscle atrophy, nor on muscle mass during the recovery from atrophy, was evidenced. Surprisingly, it was found that the recovery of muscle mass was very rapid and dready complete afier only 2 weeks of recovery. Thomason et d.(1987) had previously reported a nearly csmplete recovery of soleus muscle weight only at 28 days following hindlimb suspension, while Kaspers et al. (1990) observed that muscle wet mass m d overdl fiber cross-sectional area were nearly 14%less than control values, after a comparable period of recovery. Judged by the animals' weight, we could growth in not detect any influence of torbafylline on this study. H1'stochemist~ In SOL, even if it did not reach the level of statistical significance, a tendency for torbafylline to increase the number of slow-twitch fibers, during reconversion, was demonstrated by an almost complete reversal to 100% type I fibers at 2 and 4 weeks of recovery. In EDL neither hindlimb suspension nor torbafyg.llline showed any influence on fiber type distribution under any of the experimental conditions analyzed. Fiber size and capiikrity Recovery of fiber size was essentially completed after 2 weeks. This was not the case for the recovery of the capillary to fiber ratio. Full recovery of the capillary to fiber ratio seemed to be helped by tohafylline as evidenced at 4 weeks of recovery. In a previous study, it had been shown that, without dmg, capillaries returned to the normal range after 8 weeks of sponbneous recovery (Desplsanches et al. 1987b). Owing to the faster rate sf the recovery of the muscle fiber size, capillary density was normd dready after 2 weeks. Structural csntposition ol$ muscle fibers (soleus only)) The recovery of myofibrillar volume was not complete even after 4 weeks. There was no apparent effect sf torbawlline on myofibriliar volume or volume density under any of the experimental conditions. Under torbafylline, there was a faster return of mitochondria1 volume density to baseline
values than without the drug. Recovery was complete in 2 weeks with and in 4 weeks without the drug, supporting the tendencies observed in the fiber type data. With respect to absolute mitochondria volumes, recovery was complete after 2 w e e k with no apparent influence of the administered drug. The observed changes in mitschondrid volume densities were mainly due to changes of the interfibrillar population of mitochondria while subsarcolernrnd mitochondria remined relatively unaffected (data not reported).
Functional measurenwnts and faligabiia'~ Despite the Iarge drop in muscle mass and the close to 60% reduction of myofibrillar volume and cross-sectional area, peak twitch tension remained unaffected during suspension and was even increased with torbafylline at 2 weeks of recovery. After 2 weeks of recsvery, there was a normalization of the twitch contraction time while the hdf-relaxation time was even increased beyond normah values at 4 weeks recovery, similar to the findings of the 100% type 1 fibers composition. In contrast to peak twitch tension, peak tetmic tension was influenced by h e loss in myofibrillar volume. There was full recovery sf peak tetanic tension closely matched by the absolute myofibrillar volumes, after 2 weeks, without any detectable effect of torbafylline on these parameters. A speeding up s f contraction and relaxation rates immediately after suspension, a recovery in 2 weeks, and an overcompensation at 4 weeks after cessation of hindlimb suspension were reported, again compatible with the structural findings. Interestingly torbafylline induced a significant increase in fatigue resistance compared with untreated controls at 4 weeks of recovery. Moreover, fatigue resistance of these muscles was significantly elevated above baseline control values. Because both the mass and the fiber type distribution s f EDL muscles are, if at all, only marginally affected, it was not surprising to find only minimal changes in physiological twitch and tetanus characteristics of this muscle. In general? trends point in the s m e direction as those observed in the SOL muscle, but the differences did not reach the level of significance. There was n~ apparent or consistent effect of torbafylline on any of the twitch or fatigue characteristics analyzed in EDL muscles.
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CAN. J. PHYSIOL. PHARMACOL. VOL. 40, I992
In conclusion, we found that torbafylline has no effect in preventing hindlimb suspension induced atrophy of rat soleus muscle. In contrast, it was found that recovery sf a number of structural and functional parmeters was accelerated in the group sf treated a n i d s at 2 and 4 weeks after cessation sf hindlimb suspension. Notably, we found a faster return to baseline values of mitmhondrid volume density estimates with torbafylline . Furthermore, tsrbafy$rllime significantly increased fatigue resistance after 4 weeks sf recovery. It seems justified to further evaluate the potentid sf torbafylllline particularly in recovery from muscle atrophy.
Acknowledgments We would like to thank H. Claassen and K. Bab1 for exeellent technical assistance. This work was supported by a grmt of Hoechst AG, Werk Kdle Albert, Wiesbaden, Germany. The care and use of animals conformed to the World Health Organization. Comte, J., Gautheron, D. C., Godinot, C., and Okyayuz-Baklouti, I. 1990. Effects of chronic torbafylline treatment on energy metabolism of ischemic skeletal muscle. Drug Dev. Res. 20: 291 299. Codey, K. E., Ksyar, S . R., Rosler, K., et ak. 1987. Adaptative variation in the mammalian respiratory system in relation to energetic demand: IV. Capillaries and h e i r relationship to oxidative capaeity. Respir. Physiol. 69: 47-64. Desplanches, D., Mayet, M, H., Sempore, B., and Flandrois, R. B987a. Stmctural and functional responses to prolonged kindlimb suspension in rat muscle. J. Appl. Pkysiol. 63: 558 -563. Desplanches, D., Mayet, M. H., Sempore, B., et a / . 1987b. Effect s f spontaneous recovery OH retraining after hindlimb suspension on aerobic capacity. 9. Appl. Physioi. 63: 1739 - 1743. Desplanches, D., Kayar, S. R., Sempore, B., st a!. 1990. Rat soleus muscle ultrastmcture following hindlimb suspension. S. Appl. Physiol. 69: 504 -5898. Elder, G. C. B., and McComas, A. J. 1987. Development of rat muscle during short- and long-term hindlimb suspension. J. Appl. Physiol. 62: 1917-1923. Fdl, R. D., Gladden, L. B., Steffen, J. M . , and Musacchia, X. J. 1985. Fatigue and contraction of slow m d fast muscles in hypoldnetic/Rypodynamic rats. J. Appl. Bhysiol. 58: 65 -69. Fitts, W. H.,Metzger, J. M., Riley, D. A., and UnswoHth, B. R. 1986. Models of disuse: a comparison of hindlimb suspension and immobilization. J. Appl. Bhysiol. 68: 1946- 1953. Fitts, R. H., Brimmer, C. J., Heywod-Cooksey, A., and T i m e r man, R. J. 1989. Single muscle fiber enzyme shifts with hindlimb suspension and immobilization. Am. J. Physiol. 256: @ 1082CB09l. Grandmontagne, M., Vaage, O., Vollestadt, N. K., and Hermansen, N . 1982. Confrontation de mCthodes histschimiqaaes et d'immnofluorescence p u r le typage des fibres QU muscle squelettique de rat. J. Physiol. (London), 78: 14. (Abstr.)
Herbert, M. E., Roy, R. R., and Edgertow, &I. R. 1988. Influence of one-week hindlimb suspnsion and intermittent high load exercise om rat muscles. Exp. Neurol. 1102: 190- 198. Wemansen, L., VolBestadt, N. K., Staff, P. H.,et a!. 1983. The effect of immobilization and training on strength and composition of human skeletal muscle. In Space physiology. CNRS, Lyon, France. pp. 255 -266. HoppeBer, H.,Mathieu, O . , Krauer, R., et ark. 1981. Design sf the mammalian respiratory system. VI. Distribution of mitochondria and capillaries in various muscles. Respir . Physiol. 44: 87 - 111. Hudlicka, O . , and Brice, S. 1990. Effects of torbafqrlline, pewtoxifylline and buflomedil on vascularisation and fibre type of rat skeletal muscles subjected to limited blood flow. Br. 3. Phamacol. 99: 786 -790. Hudlicka, O., and Torres, S. H.1990. Collateral circulation in skeletal muscles: effect of pentoxifgrlline and torbafylline. S. Med. (Westbury, NY), 21: 165- 180. Kaspers, C. E., White, T. P., and Maxwell, L. @. 1990. Running during recovery from hindlimb suspension induces transient muscle injury. J. Appl. Physioll. 68: 533 -539. Morey, E. W. 1979. Spaceflight and bone turnover: correlation with a new rat model of weightlessness. Bioscience. 29: % 68 - 172. Musacchia, X. J . , Steffen, 5 . M., Fell, R. D., and Dombrowski, M. J. 1988. Comparative moqhometry s f fibers and capillaries in soleus following weightlessness (SL-3) and suspension. Physiologist, 31: S28-S29. Okayayuz-Bakloaati, I. 1989. The effects of torbafgrlline on blood flow, ps, and function of rat ischaernic skeletal muscle. Ear. J. PharmacoH. 166: 75 - 86. Pierobon-Bonarioli, S . , Sartore, S., Daila Libera, L., et al. 1981. "Fast" isomyosins and fiber types in mammalian skeletal muscle. J. Histockern. Cytochem. 29: 1179- 1188. Reiser, P. J., Masper, C. E., and Moss, I%. k. 1987. Myosin subunits and contractile properties of single fibers from hypokinetic rat muscles. J. Appl. Physiol. 63: 2293 -2300. Templeton, G . H., Sweeney, H. L., Timson, B. F., et wk. 1988. Changes in fiber composition of soleus muscle during rat hindlimb suspension. J. Appl. Physiol. 65: 1191-1195. Thomason, D. B., and Booth, H. W. 1998. Atrophy sf the soleus muscle by hindlimb unweighting. J. Appl. Physiol. 68: 1- 12. Thomason, D. B., Herrick, R. E., Surdyka, D., and Baldwin, K. M. 1987. Time course of soleus muscle myosin expression during hindlimb suspension and recovery. J. Appl. Physiol. 63: 130 137. Weibel, E. R. 1979. In Stereological methods. Practical methods for biological mophometry. Vol. 1. Chap. 4 and 4 . Academic Press, London, New York, Toronto. Winiarski, A. M., Roy, R. R.,Alford, E. K., et ak. 1987. Mechanica1 properties of rat skeletal muscle after hindlimb suspension. Exp. Neaarol. 96: 650-660. Zumstein, A., Mathieu, O.,Howald, H., and Hoppeler, H. 1983. Morphometric analysis of the capillary supply in skeletal muscles of trained and untrained subjects. Its limitations in muscle biopsies. Pfluegers Arch. 397: 277 -283.
