Cordotomy-denervation interactions on contractile and myofibrillar properties of fast and slow muscles in the rat

EXPERIMENTAL

NEUROLOGY

loo,2

16-236 ( 1988)

Cordotomy-Denervation Interactions on Contractile and Myofibrillar Properties of Fast and Slow Muscles in the Rat M. MIDRIO, D. DANIELI BETTO, R. BETTO,* D. NOVENTA, AND F. ANTICO’ Institute of Human Physiology, University of Padova, I-35131 Padova. and *National Research Council Unit for Muscle Biology and Physiopathology, Institute of General Pathology, I-35131 Padova, Italy Received January 8, 1987; revision received September 3, 1987 Cordotomy-denervation interactions were studied on contractile and myofibrillar properties of slow (soleus) and fast (extensor digitorum longus) muscles of the rat. The spinal cord was transected midthoracically in neonatal (2-day-old) animals. Two months after birth, a unilateral transection of the sciatic nerve was carried out in both cordotomized and control animals. Five weeks after denervation, contractile properties were tested isometrically in vitro; myofibrillar properties were assessedby histochemical staining of the muscle fibers and by electrophoretic analysis ofthe myosin heavy chain composition. The following results were obtained: (i) In cordotomized animals the contraction time of the soleus was significantly shorter (-23.3% on average) than that in the control animals and this shortening was accompanied by a proportional slow-to-fast shift in myofibrillar properties. (ii) The extensor digitorum longus properties were not significantly different in the control and cordotomized animals. (iii) Denervation in control animals was followed by a marked increase of contraction and half-relaxation times in the extensor digitorum longus, whereas in the soleus only the half-relaxation time was significantly increased; myofibrillar prop erties in the soleus showed an appreciable slow-to-fast shift, whereas in the fast muscle the main change was an increase in type 2A fibers to the detriment of type 2B. (iv) In

Abbreviations: EDL-extensor digitorum longus muscle; SOL-soleus muscle; C, D-muscles from innervated and denervated legs of control animals, respectively; S, SD-muscles from innervated and denervated legs of spinal cord-transected animals, respectively; MHC-myosin heavy chains. ’ The authors thank Professors Andrea Corsi and Giovanni Salviati for their critical review of the manuscript, and Dr. Mario Bolzan for his help with statistics. This work was supported by grants from the Minister0 della Pubblica Istruzione, Fondi 40%, and, in part, by institutional funds from the Consiglio Nazionale delle Bicerche. 216 0014-4886188 $3.00 Copyright 0 1988 by Academic Press, Inc. All rights of reproduction in any form reserved.

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cordotomized animals, denervation caused the soleus contraction time to increase to control values, whereas myotibrillar properties shifted to an even faster pattern; in the extensor digitorum longus denervation caused the same changes seen in the control animals. The results showed that cordotomy at birth caused the soleus to develop as a faster muscle than in the control animals. The concurrent effects of cordotomy and denervation on the myofibrillar properties of the soleus suggest that the slow-to-fast change in these properties is a common consequence of the reduction in the level of motor activity. The opposite effects of the two experimental conditions in the soleus contraction time support the view that the contractile alterations that follow denervatiOn mainly reflect alterations in the mUScle activation process. 0 1988 Academic Pwss, Inc.

INTRODUCTION According to many authors the reduction of motor activity by tenotomy (9,65), limb immobilization (3,25,43), or spinal cord transection (6, 17,26, 32) has a “speeding” effect on slow muscle, with a shortening of the twitch contraction time, and no effects on fast muscle. On the other hand, the abolition of motor activity by denervation is classically reported to have a “slowing” effect on muscles [see (40) for references]. Those observations seem to substantiate the statement by Fischbach and Robbins (25) that disuse and denervation have opposite effects on contractile properties and support the view that neuromotor activity is not the only factor regulating those properties [see (30)]. However, it was also reported that disuse had a slowing effect on fast muscle resembling the effect of denervation (18), and long-term denervation had a speeding effect on slow (3 1,62) or mixed ( 12) muscles. In a previous work (15) we confirmed that cordotomy and denervation have different and partially contrasting effects on muscle contractile properties, by carrying out the two experimental interventions in series in the same animal. In the rat, cordotomy at birth caused the slow muscle (soleus, SOL) to become faster than that in the control animals, with a significant shortening of the twitch contraction time, while leaving unchanged the properties of the fast muscle (extensor digitorum longus, EDL); on the other hand, denervation 3 to 6 months after cordotomy was followed by a marked lengthening of the contraction time in both muscles. The analysis of mechanical properties alone cannot, however, give sufficient information on the actual changes which occurred in the muscle, especially in the contractile proteins. Under physiologic conditions the expression of the “fast” or the “slow” isoform of myofibrillar proteins correlates very well with the contractile properties of the muscle, but after experimental manipulations this normal relation may be altered [see (37)]. After cordotomy in weanling or adult animals the speeding of the slow muscle is not accompanied by a proportional slow-to-fast change in myosin properties ( 14, 22, 37,41,42, 52); on the other hand, during the first weeks after denerva-

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ET AL.

tion, when the lengthening of the contraction time is maximal, myosin properties change very little (23,3 1,35,38). Such discrepancies probably depend on the fact that the time course of muscle contraction is controlled not only by the properties of myofibrillar proteins, but by the properties of the excitation-contraction coupling system as well [see (36, 54)], and that changes in each of these two systems may occur independently of one another (48,5 1). In order to more accurately compare the effects of cordotomy and denervation, in the present work the study of mechanical properties of muscles was complemented by the study of myosin histochemical ATPase activity and of myosin heavy chain isoforms. As in the preceding work, cordotomy was carried out in newborn animals. In this experimental model the muscles of paraplegic limbs develop in a condition of reduced motor activity and do not have to adapt to a new functional situation, as is the case when cordotomy is in adult or weanling animals. This initial condition can help in the interpretation of the results, as it is not complicated by a possible incomplete adaptation of already differentiated muscle to the change of neuromotor activity [see, for instance, (14)]. Denervation was done 2 months after birth, both in cordotomized and control animals. The results showed that cordotomy and denervation had concurrent effects on myosin properties of slow muscle, in spite of causing opposite effects on the contraction time. MATERIALS

AND

METHODS

Wistar rats were used in all experiments. Spinal cord was transected in 2day-old animals; the transection was at the midthoracic level under aseptic conditions following the technique described by Stelzner et al. (6 1). In some experiments we used anesthesia as described elsewhere (15), but in most cases we preferred general hypothermia for anesthesia (6 1) because this reduced cannibalism by mothers. Two or three animals from each litter were not operated and were used as controls of cordotomized littermates. By using iridectomy scissors a small fragment of the cord was isolated and gently removed. Bleeding was controlled with hemostatic cellulose (Surgicel) and the wound was sutured. No general antibiotic treatment was administered. Two months after birth, the sciatic nerve was cut unilaterally near the trochanter under ether anesthesia in both cordotomized and control animals, To avoid reinnervation, about 1.5 cm of the distal stump of the nerve was removed. Muscle properties were investigated 5 weeks after denervation. The muscles were excised from living animals under ether anesthesia and immersed in the bathing solution for mechanical measurements or in liquid nitrogen for histochemical processing; the animals were afterward killed by increasing ether administration. Four groups of muscles were analyzed: muscles from the innervated leg of control animals (C), muscles from the dener-

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vated leg of control animals (D), muscles from the innervated leg of the spinal cord-transected animals (S), and muscles from the denervated leg of the spinal cord-transected animals (SD). Contractile Properties. For stimulation and recording, the muscles were mounted in a vertical position between two platinum plates, in a chamber filled with 100 ml of a solution containing (in mM) 120 NaCI, 4.7 KCl, 2.5 CaC12, 3.15 MgC&, 1.3 NaH2P04, 25 NaHC03, 11.1 glucose, and 3.75 PM d-tubocurarine. The solution was continuously bubbled with a mixture of 02 (95%) and CO2 (5%) and maintained at 37°C; the pH was 7.2 to 7.4. The distal tendon of the EDL, or a piece of tendo calcaneus in the case of the SOL, was firmly tied to a glass hook at the bottom of the chamber, and the proximal tendon was connected to an isometric transducer (Harvard, 507947). The muscles were stimulated through the bathing solution by means of the two platinum plates. Supramaximal square stimuli (0.2-ms duration) were derived from a booster amplifier driven by an electronic stimulator (Grass S 88). The output from the transducer was amplified and recorded on a storage oscilloscope (Tektronix 5 103 N/DII). The muscles were gently stretched to the optimal length for twitch tension. The following parameters were measured: contraction time (CT) and halfrelaxation time ($ RT) of the twitch; twitch tension (PJ and tetanic tension (PO)normalized to 1 g wet tissue. The twitch:tetanus ratio (PJP,,) was calculated from these values. The maximum rate of tension development was also measured. This was expressed as the percentage of the maximum tetanic tension developed per millisecond (%PJms), according to the criterion suggested by Buller and Lewis (8) for C and S muscles. In the case of D and SD muscles, and especially in the case of SOL, we found it difficult to apply this criterion because of the low tension the muscles developed during tetanic stimulation (see Results and Discussion). To be able to compare the data obtained under the different experimental conditions (C, D, S, and SD) we chose to express the rate of tension development as the tension developed in one millisecond (g/ms) during the steepest phase of the tetanus rise. The values were normalized to 1 g wet muscle. Histochemical Analysis. Every muscle was weighed and quickly frozen in a stretched position in liquid nitrogen. Serial cross sections were cut from the widest portion of the muscle belly at -23°C (?2”C) in a cryostat microtome (Slee Pearson) and were stained for myofibrillar ATPase after acid (pH 4.35 and 4.6) or alkaline (pH 10.4) preincubation, using the Padikula and Hermann method (47) modified by Guth and Samaha (28). Fiber types were identified as type 1 (dark after acid and light after alkaline preincubation), type 2A (dark after alkaline and light after acid preincubation), type 2B (dark or intermediate after both alkaline and pH 4.6 preincubation, light after pH 4.35 preincubation), and type 2C (dark or intermediate after all preincuba-

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TABLE I Atrophy of Soleus Muscle after Denervation, Cordotomy, or Cordotomy-Denervation’ C

D

Animal weight(g)

329 + 28 (175-465,11)

329 f 28 (175-465,11)

Muscle weight (mg)

158 + 8.3 (107-196,ll)

56 + 3.6 (39-71,ll) c

96 + 6.0 (63-120,5) d

40 k 7.0 (21-65,5) e

Muscle/animal wt X lo-’

0.51 * 0.03 (0.4-0.61,ll)

0.18 f 0.02 (O.l-0.3,ll) c

0.49 + 0.04 (0.4-61,5)

0.23 k 0.04 (0.16-0.30,5) e

2746 f 181 (2162-3248,5)

2347 -+ 172 (1734-2802,5)

2591+ 175 (2130-3080,5)

2475 +- 106 (2134-2800,5)

451 f67 (308-671,5)

1365 + 138 (1002-1862,5) f

800+45 (687-919,5) g

1848 + 207 (1370-2565,5) h

Total fiber number Fibers/mm’

S 196+22 (149-2885) b

SD 196+22 (149-288,5)

a C, D-muscles from innervated and denervated legs of control animals, respectively; S, SD-muscles from innervated and denervated legs of spinal cord-transected animals, respectively. Values are means f SE. Numbers in parenthesis are range and N, respectively. * P < 0.005 with respect to C, Wilcoxon test for unpaired groups. cP < 0.0005 with respect to C, Student’s t test for paired groups. d P < 0.0005 with respect to C, Wilcoxon test for unpaired groups. e P < 0.0005 with respect to S, Wilcoxon test for paired groups. rP < 0.0005 with respect to C, Wilcoxon test for paired groups. 8P < 0.0025 with respect to C, Wilcoxon test for unpaired groups. h P < 0.00 1 with respect to S, Wilcoxon test for paired groups.

tions) according to the terminology of Brooke and Kaiser (4). The total number of fibers was counted from a photomontage of the entire transverse section of the muscle. The percentage fiber composition was obtained by identifying the different types of fibers contained in randomly selected sample areas of enlarged photographs. At least 500 fibers (15 to 20% of the total population) were counted. Ekctrophoretic Analysis. The histochemical identification of muscle fibers as slow (type 1) or fast (types 2A, 2B, and 2C) fibers was complemented with the electrophoretic analysis of the myosin heavy chain (MHC) composition. Myosin was extracted from some of the muscle cryostat sections used for histochemistry and electrophoresis was carried out on a 6% polyacrylamide slab gel using the Laemmli method (39), as described by Carraro et al. ( 13).

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FIG. 1. Cross sections of soleus (SOL) and extensor digitorum longus (EDL) muscles from innervated (C) and denervated (D) legs of control rats, and innervated (S) and denervated (SD) legs of spinal cord-transected rats, stained for myofibrillar ATPase (X 150). Only the sections stained after alkaline preincubation are shown. The identification of fiber subtypes was obtained by comparison with the sections stained after acid preincubation (see Materials and Methods).

Densitometry of protein bands was as described by Volpe et al. (64). This method allows a quantitative evaluation of the slow (type 1) and fast (type 2) isoforms of myosin in the muscle specimen, without distinguishing the fast myosin in subtypes corresponding to those of the histochemical classification. StatisticalAnalysis. Means f SE were calculated, and the results were analyzed with the Student t test or the Wilcoxon test for paired or unpaired data as appropriate. The Student t test was used when the distribution of data (analyzed with the Kolmogorov-Smirnov test) could be proved to be normal and the variances for the groups under comparison (analyzed with the Snedecar-Fisher F test) were not significantly different. This was generally the case with groups having N > 10. In all the other cases, the nonparametric method of analysis was used. The 0.05 level of probability was established for statistical significance.

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EDL %

’ 12R2B2C

12A2B2C

12A2BZC

12R262C

FIG. 2. Histograms showing the different fiber type compositions of EDL and SOL from innervated (C) and denervated (D) legs of control rats, and innervated (S) and denervated (SD) legs of spinal cord-transected rats.

RESULTS

Soleus Muscle: Cordotomy. Body development of cordotomized animals was consistently less than that of the controls. The weight of the S muscle was reduced proportionally to the body weight, so that the muscle-to-body ratio was normal (Table 1). The total number of muscle fibers in the crosssectional area was within the control range. The fibers appeared to be smaller than those in the controls (Fig. 1); this was also revealed by the fact that the average number of fibers counted per unit area in S muscles was about twice the number in C muscles (Table 1). The fiber type composition of the experimental muscles was very different from that of the C muscles. The percentage of type 1 fibers was significantly reduced and that of type 2A fibers was significantly increased (Figs. 1 and 2, and Table 2). The electrophoretic analysis of myosin from a single cryostat section showed a parallel variation in the relative proportion of type 1 and type 2 MHCs (Fig. 3 and Table 2). Mechanical properties also changed significantly. Contraction time was reduced and the maximum rate of tension development was increased. In absolute values, twitch and tetanic tensions were lower than those in C muscles; however, when normalized to 1 g wet weight, the twitch tension was greater and the tetanic tension was as great as that of C muscles; the PJP,, ratio was correspondingly increased (Table 3). Denervation. Denervation was followed by a great reduction of muscle weight. All fiber types showed severe atrophy (Fig. l), without significant changes in their total number: correspondingly, the number of fibers per unit area increased, becoming on average three times as great as in the controls (Table 1).

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EFFECTS ON MUSCLES

TABLE 2 Fiber Type and Myosin Heavy Chain (MHC) Composition (W) of the Soleus Muscle after Denervation, Cordotomy, or Cordotomy-Denervation“ C Type 1 fibers

85.4 k 3.5 (75.9-93.25)

D

s

SD

64.4 + 5.6 (47.0-73.5,5) b

53.3 + 5.3* (44.0-73.9,5) c

40.5 + 9.1 (7.8-62.1,5)

18.4 + 3.4 (lO.l-28.0,5) d

40.9 + 5.1 (23.1-53.6,5) e

50.2 + 12.4 (29.0-89.1,5)

Type 2A fibers

9.3t3.1 (2.6-16.5,5)

Type 2C fibers

5.3 + 1.4 (0.4-8.0,s)

17.2 t4.1 (3.2-25.8,5) f

5.7 -t 2.9 (O-15.4,5)

9.2 +- 3.2 (3.1-21.5,5)

Type 1 MHC

93.6

89.7

54.7

40.6

Type 2 MHC

6.4

10.3

45.3

59.4

a C, D-muscles from innervated and denervated legs of control animals, respectively; S, SD-muscles from innervated and denervated legs of spinal cord-transected animals, respectively. Values are means + SE. Numbers in parenthesis are range and N, respectively. Data of MHC composition are from one experiment. b P < 0.0 1 with respect to C, Wilcoxon test for paired groups. ’ P < 0.00 1 with respect to C, Wilcoxon test for unpaired groups. d P < 0.05 with respect to C, Wilcoxon test for paired groups. e P < 0.0005 with respect to C, Wilcoxon test for unpaired groups. ‘P < 0.025 with respect to C, Wilcoxon test for paired groups. * Values not normally distributed (median = 50.9).

The histochemical profile was significantly altered, with reduction of type 1 fibers and increase of type 2A and type 2C fibers (Figs. 1 and 2). These changes were paralleled by the reduction of type 1 MHC and the increase of type 2 MHC (Fig. 3 and Table 2). As far as mechanical changes are concerned, contraction time was only slightly increased, while the half-relaxation time was markedly lengthened. The twitch tension was not significantly changed, whereas the tetanic tension was reduced to less than half the C value, with a corresponding increase of the PJPO ratio. Contraction speed was not significantly changed (Table 3). Cordotomy-Denervation. Denervation in cordotomized animals induced muscular atrophy comparable to that observed in control animals, as indicated by the similar muscle-to-body ratio in the two experimental conditions. The total number of SD muscle fibers was in the range of S and C muscles, but the number of fibers per unit area was more than two and four times greater, respectively (Fig. 1 and Table 1).

224

MIDRIO EDL C

D

S

SD

SOL C

ET AL.

D

S

SD +-Ml-c2 tMHC-I

FIG. 3. A 6% SDS-polyacrylamide gel electrophoresis of myofibrillar proteins from cryostat sections of EDL and SOL muscles from innervated (C) and denervated (D) legs of control rats, and innervated (S) and denervated (SD) legs of spinal cord-transected rats. Only the myosin heavy chain (MHC) region is shown.

Histochemically, type 1 fibers decreased even more than in S muscles, and 2A fibers became the predominant type (Figs. 1 and 2). Parallel changes were observed in the MHC composition, with type 2 myosin becoming prevalent (Fig. 3 and Table 2). Both contraction time and half-relaxation time were lengthened in comparison with S muscles. Twitch tension was higher than in C muscles, whereas tetanic tension was greatly reduced, with a PJP, ratio (0.49) quite comparable to that of D muscles (0.47). Contraction speed was not significantly different from that calculated for S muscles (Table 3). Extensor Digitorum Longus muscle: Cordotomy. Development of the muscle was reduced in proportion to body weight, so that the muscle-tobody ratio remained in the range of the controls. Atrophy was evident in type 2B fibers, whereas type 1 and type 2A fibers appeared to be unaffected (Fig. 1). The total number of fibers was unchanged. The number of fibers per unit area was about 1.5 times the value of C muscles (Table 4). Fiber type composition (Fig. 2 and Table 5), MHC composition (Fig. 3 and Table 5), and contractile properties (Table 6) were not significantly different from those of the C muscles. Denervation. Denervation caused a severe atrophy of the muscles, affecting to the same degree all kinds of fibers (Fig. l), with a doubling of the average number of fibers counted per unit area (Table 4). Both type 1 and type 2A fibers significantly increased, and type 2B fibers correspondingly decreased. The occurrence of a few 2C fibers, which were virtually absent in C muscles, was also observed (Fig. 2 and Table 5). The relative proportion of type 1 and type 2 MHCs was not significantly different from that in the controls (Fig. 3 and Table 5). The contractile properties showed an increase of both contraction time and half-relaxation time. Twitch tension also increased, with an increase in PJPO ratio, whereas the maximum rate of tension development was unchanged (Table 6). Cordotomy-denervation. The effects of denervation in cordotomized animals were the same as those in the controls, with nonsignificant differences between the two groups of muscles (Figs. l-3 and Tables 4-6).

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EFFECTS ON MUSCLES

TABLE 3 Contractile Properties of the Soleus Muscle after Denervation, Cordotomy, or Cordotomy-Denervation” C

D

S

SD

C-r (msf

32.4 + 0.7 (27.9-37.0,13)

35.2 * 1.8 (26.7 iz 44.4,13)

25.1 2 2.6 (19.0-3 1.8,5) b

31.Ok3.1 (21.0-41.5,5) c

l/2 RT (ms)

39.0 f 2.7 (28%66.513)

62.2 + 6.7* (36.5-115.0,13) d

38.9 + 3.9 (31.0-52.5,5)

51.6k8.8 (30.0-73.0,5) e

pt (9)

154&7 (110.3-181.5,13)

163 f 24 (90.6-299.3,13)

235 + 40 (96.0-322.6,s)

POk)

8Olk 42 (541-1096,13)

348 f. 45 (125-587,13) ET

205+31 (154.6-3 16.9,5) f 857 + 81 (721-1120,5)

PtlPo

0.19 kO.01 (0.13-0.29,13)

0.47 k 0.02 (0.32-0.58,13) g

0.24 + 0.01 (0.22-0.27,5)

0.49 f 0.03 (0.38-0.56,5) h

1.59 + 0.28 (0.90-2.3 1,5) i 13.4 + 2.5 (6.9-20.5,5)

-

Rate of rise of tetanic tension WP,/ms

g/ms

0.76 + 0.06 (0.43-1.23,13)

-

5.97 5 0.5 (3.03-9.52,13)

8.77 + 1.5 (2.57-18.72,13)

463 zk 72 (240-645,5) h

12.2 & 1.9 (7.8-17.7,5)

“C, D-muscles from innervated and denervated legs of control animals, respectively; S, SD-muscles from innervated and denervated legs of spinal cord-transected animals, respectively. Values are means -+ SE. Numbers in parenthesis are range and N, respectively. b P < 0.0025 with respect to C, Wilcoxon test for unpaired groups. ’ P < 0.0 1 with respect to S, Wilcoxon test for paired groups. d P < 0.0005 with respect to C, Wilcoxon test for paired groups. ’ P < 0.05 with respect to S, Wilcoxon test for paired groups. IP i 0.025 with respect to C, Wilcoxon test for unpaired groups. 8 P < 0.0005 with respect to C, Student’s t test for paired groups. h P < 0.0005 with respect to S, Wilcoxon test for paired groups. i P < 0.00 1 with respect to C, Wilcoxon test for unpaired groups. ’ P < 0.0005 with respect to C, Wilcoxon test for unpaired groups. * Values not normally distributed (median = 60.0).

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TABLE 4 Atrophy of the Extensor Digitorum Longus after Denervation, Cordotomy, or Cordotomy-Denervation” C

D

331 f 30 (175-472,ll)

Animal weight(g)

331 + 30 (175-472,ll)

S 183 f 19 (ill-279,8)

SD 183k 19 (11 l-279,8)

b

Muscle weight(g)

Muscle/animal wt

Total finer number Fibers/mm’

155 f 13 (84-208, I 1) X

lo-’

68*6 (46-94,ll) c

94+ 10 (52-153,8)

0.42 f 0.02 (0.35-0.62,ll)

0.23 + 0.02 (0.16-0.36,11) c

0.5 1 + 0.02 (0.46-0.60,8)

0.23 f 0.02 (0.15-0.28,8) f

3396 + 366 (2906-4482,6)

3443 + 176 (2942-3914,6)

3230 f 174 (2657-3724,6)

3386 f 504 (3053-3719,5)

458 + 44 (363-625,6)

833+ 111 (539-1272,6) g

652 + 45 (504-820,6)

1084 + 178 (657-1523,4)

d

h

51+6 (24-75,8) e

i

’ C, D, muscles from innervated and denervated legs ofcontrol animals, respectively; S, SDmuscles from innervated and denervated legs of spinal cord-transected animals, respectively. Values are means + SE. Numbers in parenthesis are range and N, respectively. b P c 0.001 with respect to C, Wilcoxon test for unpaired groups. ’ P < 0.0005 with respect to C, Student’s t test for paired groups. dP < 0.0025 with respect to C, Wilcoxon test for unpaired groups. eP < 0.0025 with respect to S, Wilcoxon test for paired groups. f P < 0.0005 with respect to S, Wilcoxon test for paired groups. g P < 0.0 1 with respect to C, Wilcoxon test for paired groups. ’ P < 0.0 1 with respect to C, Wilcoxon test for unpaired groups. i P < 0.05 with respect to S, Wilcoxon test for unpaired groups.

DISCUSSION E_tTects of Cordotomy. The results of this work confirm that spinal cord transection at birth causes the slow muscle of the rat to develop as a faster muscle than in the control animals, while leaving unchanged the properties of the fast muscle. The contraction time of the SOL was significantly shorter in S than in C muscles, with a concomitant increase of the maximal rate of tension development and a shift of both the histochemical profile and MHC composition toward a fast pattern. The shortening of the contraction time we observed in the SOL is comparable to that reported in our previous paper (I 5) (-23.3 and - 19.2%, respec-

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TABLE 5 Fiber type and Myosin Heavy Chain (MHC) Composition (W) of the Extensor Digitorium Longus after Denervation, Cordotomy, or Cordotomy-DenervatiotP C Type 1 fibers

Type 2A fibers

D

S

SD

4.1 + 0.4 (2.9-5.55)

8.1 -to.7 (5.1-9.8,5) b

3.3 f 0.8 (1.3-6.7,5)

5.9 + 1.9 (3.1-11.1,5)

37.8 + 3.1 (27.6-41.6,5)

59.4 + 4.3 (55.8-69.1,5)

31.9 + 3.8 (19.0-40.3,5)

56.3 + 1.3 (53.3-58.4,5) d

C

Type 2B fibers

57.9 k 2.7 (5 1.2-66.8,5)

31.3 f 4.4 (25.5-34.8,5) e

63.9 + 4.0 (52.9-78.6,5)

36.9 + 2.7 (29.6-42.9,5) d

Type 2C fibers

0.2 k 0.18 (O-0.7,5)

1.2 kO.6 (O-2.1,5)

0.7 f 0.3 (O-1.5,5)

0.9 f 0.1 (0.7-1.2,5)

1.6 98.4

3.1 96.3

3.2 96.8

8.7 91.3

Type 1 MHC Type 2 MHC

’ C, D-muscles from innervated and denervated legs of control animals, respectively; S, SD-muscles from innervated and denervated legs of spinal cord-transected animals, respectively. Values are means f SE. Numbers in parenthesis are range and N, respectively. Data of MHC composition are from one experiment. b P i 0.0025 with respect to C, Wilcoxon test for paired groups. ‘P < 0.001 with respect to C, Wilcoxon test for paired groups. d P i 0.0 1 with respect to S, Wilcoxon test for paired groups. ‘P < 0.0005 with respect to C, Wilcoxon test for paired groups. * Values not normally distributed (median = 0).

tively). In spite of such a change, the contraction time of the SOL muscle remained markedly longer than that of the EDL, with a persisting clear-cut difference between the mechanical properties of the two muscles (cf. Tables 3 and 6). This is partially contrasting with the results of Buller et al. (6) and Gallego et al. (26) in the cat, and of Davey et al. (17) and Hoh and Dunlop (32) in the rat. According to those authors, cordotomy shortened the SOL contraction time to a typical fast muscle value. These differences may be accounted for by the differences in the animal age at the time of cordotomy. The spinal cord transection has been considered by several authors to be an experimental model of muscle disuse ( 18,26,29,45,46), and the speeding of the slow muscle has been interpreted as a consequence of the reduced motor activity (1, 25, 45) or of the change of the pattern of motor activity, from a tonic to a phasic one (1, 20, 32). According to Stelzner et al. (6 I),

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TABLE 6 Contractile Properties of the Extensor Digitorum Longus after Denervation, Cordotomy, or Cordotomy-Denervation” C

D

S

CT (ms)

11.2kO.7 (9.0-14.0,6)

17.5 f 0.5 (16.0-19.5,6) b

l/2 RT (ms)

9.3 f 0.8 (7.5-12.5,6)

19.5 + 1.7 (14.0-25.5,6) d

9.3 + 0.5 (7.6-l 1.0,5)

15.3 + 1.1 (12.0-17.5,5) f?

pt (9)

279 f 15 (218-331,6)

403 f 42 (270-534,6) f

267 +- 29 (203-354.5)

466 + 48 (314-595,5) g

PO(g)

973 f 141 (552-1356,6)

802 L!C95 (535-l 120,6)

976 + 127 (691-1367,5)

903 + 85 (706-l 190,5)

0.29 + 0.02 (0.24-0.33,6)

0.50 k 0.03 (0.42-0.60,6) b

0.27 AI0.0 1 (0.25-0.30,5)

0.50 + 0.03 (0.44-0.64,5)

-

2.90 f 0.21 (2.04-3.15,5) 33.9 f 9.5 (15.6-57.9,4)

Rate of rise of tetanic tension %P,/ms g/ms

3.03 f0.17 (2.49-3.61,6) 29.7 f 8.0 (20.2-45.4,3)

31.7 + 5.6 (lO.O-42.7,6)

11.5kO.3 (1 l.O-12.5,5)

SD 17.2 k 0.5 (15.8-18.6,5) c

C

36.7 k 4.8 (18.6-44.6,5)

“C, D-muscles from innervated and denervated legs of control animals, respectively; S, SD-muscles from innervated and denervated legs of spinal cord-transected animals, respectively. Values are means k SE. Numbers in parenthesis are range and N, respectively. * P < 0.00 1 with respect to C, Wilcoxon test for paired groups. ’ P < 0.0005 with respect to S, Wilcoxon test for paired groups. d P < 0.0025 with respect to C, Wilcoxon test for paired groups. ’ P < 0.001 with respect to S, Wilcoxon test for paired groups. /P i 0.025 with respect to C, Wilcoxon test for paired groups. 8P < 0.005 with respect to S, Wilcoxon test for paired groups.

the survival of motor function is very different in rats spinal cord-transected immediately after birth (0 to 5 days of age) or in older rats (2 1 to 26 days of age): in the first case, the hind limbs support the hindquarters and step during locomotion, whereas in the second case the hind limbs are passively extended and do not participate in locomotion. It appears, therefore, that the level of spontaneous motor activity is higher in rats spinal cord-transected at birth

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than in rats transected 3 to 4 weeks later. Consequently, muscle disuse should be less pronounced in rats spinal cord-transected at birth. In our experiments, cordotomy was carried out in 2-day-old rats, and their motor behavior during development was similar to that described by Stelzner et al. (6 1) and confirmed also by Margreth et al. (44). Hoh and Dunlop (32) and Davey et al. (17) transected the spinal cords in 3- to 4-week-old rats. Thus, it is possible that the greater speeding of the SOL observed in their experiments depended on a greater degree of muscle inactivity. Different effects of cordotomy on slow muscle according to the animal’s age were also reported for the cat, but the data are not comparable to those observed in the rat: the shortening of SOL contraction time is maximal when cordotomy is at birth (6, 26), minimal when cordotomy is in 2- to 4-week-old animals (7, 37), and well evident when cordotomy is in 12-week-old (37) or in adult (14,45) animals. According to the data reported by Smith et al. (59), the level of spontaneous motor activity seems to be higher in the 2-week than in the 12-week spinal cord-transected animals. However, sufficient observations on the motor behavior of the cat in all the different experimental conditions are not available, and a correlation with the differences in the contraction time is not possible. Our results show a good correlation between the mechanical and the histochemical changes of S muscles with respect to C muscles, differing with what has been reported for adult cordotomized animals ( 11,22,37,4 1,42). In our cordotomized animals, SOL contraction time was shortened by an average 8.6 ms, which means a reduction of about 40% of the difference (2 1.5 ms, cf. Tables 3 and 6) between contraction times of the SOL and EDL in control animals. The maximal rate of tension development (expressed as %P,lms) increased by 0.83, corresponding to 38% of the difference (2.12) with the EDL; on the other hand, the percentage of type 1 fibers decreased from 85.4 to 5 3, which corresponds to a 40% reduction in the difference (8 1.3) between control SOL and control EDL (cf. Tables 2 and 4), and a similar difference (-42%) can be calculated for the slow MHC content. It may be that the discrepancies between myofibrillar ATPase and contractile properties observed when cordotomy is carried out in adult animals depend on an incomplete adaptation of the already differentiated muscles to the new functional situation, with nonparallel changes of the multiple factors which control the time course of the muscle contraction-relaxation cycle [see (36, 54)], Also Cope et al. (14) recently pointed to the possibility that the coordination of the changes following spinal transection in adult animals requires a long period to be completed and that some motor unit properties are not sensitive to the regulating factors. When the spinal cord is transected at birth, the muscles develop without undergoing any change in the level or pattern of activity, and it may be that the consistency between myofibrillar and contrac-

230

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ET AL.

tile properties we observed reflects a very well-coordinated differentiation of muscles according to the neuromotor activity which persists after cordotomy. E&zts of Denervation. The main result of the present work appears to be the concurrence of the effects of denervation and of cordotomy on the myosin properties of the SOL, contrasting with the differences observed in the mechanical properties. This contrast was particularly evident when denervation was in cordotomized animals and the conclusive discussion of the results is in the section devoted to the cordotomy-denervation interactions. The discussion is limited here to some particular aspects of muscle response to denervation. The contraction time of the denervated SOL did not increase significantly, although the half-relaxation time was markedly prolonged. The results concerning the contraction time differ from those of our previous paper (15) in which a significant increase after denervation was reported. This may be ascribed to the longer interval between denervation and killing the animal in this work (5 weeks) compared with that in the previous one (3 weeks). In the SOL of the rat, Finol et al. (24), at 42 days, and Dulhunty (19), at 48 days after denervation, observed a reversal of the changes that occurred at an earlier stage, with shortening of the contraction time toward the control values. According to Dulhunty ( 19), this late speeding may be due to the synthesis of fast contractile proteins. Such a hypothesis is consistent with several data in the literature showing that the histochemical profile (27, 35, 38,63), myosin biochemical properties (62), and myosin composition ( 12, 13, 27) are modified after long intervals from denervation into a fast pattern and it is also supported by the histochemical and electrophoretic results of this work. From the histochemical point of view, a significant increase of type 2C fibers was observed only in the denervated SOL. Type 2C fibers are considered immature (4,5) or intermediate (2,4,55,56) fibers, able to differentiate into either the slow or the fast type ( 16). It seems reasonable to assume that in the rearrangement of SOL fiber type composition, which occurs after denervation, type 2C fibers represent transitional stages of the differentiation of type 1 fibers into type 2A fibers ( 16). Intrinsic contractile properties of the EDL, as revealed by the rate of tension development, appeared to be substantially unchanged after denervation, in spite of the significant alterations in the histochemical profile. The greatest percentage increase concerned type 1 fibers, which almost doubled: their final value remained, however, very low (8%) and for this reason possibly no appreciable changes in the contraction speed were observed. A remarkable shift was also observed from type 2B to type 2A fibers. However, since type 2A and type 2B fibers, though differing in resistance to fatigue

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(lo), have similar contractile properties, it is not surprising that the rate of tension development was not affected. A separate comment must be reserved for the changes in the PJP, ratio after denervation. Both in the SOL and EDL, the ratio increased markedly in comparison with that in the C muscles. Increase in the ratio in the SOL was essentially due to reduction of tetanic tension without significant change in twitch tension, whereas in the EDL it was essentially due to the increase in twitch tension (see Tables 3 and 6). As suggested by Finol et al. (24), a reduction of tetanic tension without parallel reduction of twitch tension may be explained by failure of some denervated fibers to respond to high-frequency stimulation, as a consequence of decreased excitability of cells. In our experiments, the slow tetanic output of denervated muscles was not associated with a slower rate of tension increase during stimulation: this fact made it problematic to comparatively evaluate the contraction speed of C and D muscles as %P,,/ms and forced us to use another criterion for the measurement (see Methods). Cordotomy-Denervation Interactions. The pattern of motor activity has been considered by many authors to be the main factor controlling the development of myofibrillar and metabolic characteristics of muscles, the slowfrequency activation favoring the differentiation toward the slow muscle type and the high-frequency toward the fast type [see (36, 50, 53)] for review). Recent evidence has been adduced that the total amount of activity, rather than the activity pattern, is important in affecting the properties of muscles, high levels of activity favoring transformation of the muscle into the slow type (33,34,49,50,60). If this is the case, then a reduction of motor activity should favor the transformation of slow muscles into the fast type (53). Our results appear to be in agreement with such a hypothesis: indeed, both the abolition of neuromotor activity by denervation and the reduction of the same activity by cordotomy ( 1,42) were associated with the development of fast myosin properties in the SOL, and this effect was further enhanced when the two experimental procedures were carried out in series in the same animal. The view that muscle inactivity alone is sufficient to cause the slow-to-fast transformation of muscles has been questioned especially on the basis of observations that the histochemical changes in denervated slow muscles are very late (38) or even absent 1 to 8 months after denervation (23) whereas the changes are well evident after comparable intervals from cordotomy or cord isolation: some involvement of innervation in the process of muscle speeding has therefore been postulated (23). This possibility cannot be ruled out, but it may also be suggested that the effects of neuromotor deprivation are delayed or masked, at least to some extent, by the effects of the fibrillation activity of denervated muscle. Fibrillation activity, resembling tonic activity

232

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ET AL.

of postural muscles, has been postulated also to play a role in changing the fiber type composition of denervated EDL ( 19). According to the view that the increase in the level of activity is associated with a fast-to-slow transformation, one would expect that fibrillation would cause, in the muscle, a shift of the histochemical profile toward the slow pattern; in fact, we found that after denervation the percentage of type 1 fibers doubled, with a consistent increase also in type 2A fibers, to the detriment of type 2B fibers. It is of interest that these changes are rather similar to those observed by Salviati et al. (57) in the EDL of myotonic rats. The discrepancies between mechanical changes and myosin properties observed after denervation, especially in the SOL of cordotomized animals, do not necessarily refute a prominent role for motor activity in determining muscle contractile properties. As was pointed out in the Introduction, the time course of muscle contraction depends not only on the nature of contractile proteins, but also on the kinetics of the excitation-contraction coupling process, and it is generally assumed (19, 20, 24, 40) that after denervation the changes in contractile properties are caused primarily by alterations of this process. Dulhunty and Gage found in rat SOL and EDL (20, 21) that both the asymmetrical charge movements, which are assumed (58) to be generated during exitation-contraction coupling, and the number of indentations in the terminal cisternae, which is correlated with the amount of charge movement, changed after denervation and after cordotomy: after denervation the EDL was affected the most, and its properties approached those of the SOL; the reverse effect, i.e., an approaching of the SOL properties to the EDL properties, was found in chronically cordotomized rats, together with a marked shortening of the SOL contraction time. Dulhunty and Gage suggested (20,2 1) that these changes depend on changes in the activity pattern of the muscles. The EDL is normally subjected to a high-frequency, though sporadic, activation, and this would maintain the “fast” charge movement which is lost upon denervation. In the SOL, the normal tonic activity pattern does not appear to control the characteristics of the charge movement, but after cordotomy the pattern becomes similar to that of the EDL; thus the charge movement of the SOL adopts the characteristics of the fast muscle. There is an evident parallelism between the results obtained by Dulhunty and Gage and our own results on contraction time in the same muscles. Denervation had almost no effect on contraction time of the slow muscle, but it did markedly lengthen the contraction time of the fast muscle; on the contrary, cordotomy significantly shortened the contraction time of the slow muscle, without affecting the fast muscle. Dulhunty and Gage did not study the cordotomy-denervation interactions, but it seems likely that, were denervation carried out when the SOL is differentiated as a fast muscle, the effects on charge movement and cisternae indentations should be similar to

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those observed in the EDL, with the acquisition or the reacquisition by the SOL of the slow muscle features. In our experiments denervation in cordotomized animals was effective in lengthening the contraction time of the SOL as well as that of the EDL: this effect on the SOL could be explained by assuming that it reflected the loss after denervation of the “fast” activation characteristics acquired by the muscle under the condition determined by cordotomy at birth. REFERENCES 1. ALAIMO, M. A., J. L. SMITH, R. R. ROY, AND V. R. EDGERTON. 1984. EMG activity of slow and fast ankle extensors following spinal cord transection. .I. Appl. Physiol. 56: 16081613. 2. BILLETER, R., H. WEBER, A. LUTZ, H. HOWALD, H. M. EPPENBERGER,AND E. JENNY. 1980. Myosin types in human skeletal muscle fibers. Histochemistry65: 249-259. 3. BOOTH, F. W., AND J. R. KELSO. 1973. Effect of hind-limb immobilization on contractile and histochemical properties of skeletal muscle. Pfriierrs Arch. 342: 23 l-238. 4. BROOKE, M. H., AND K. K. KAISER. 1970. Three “myosin adenosinetriphosphatase” systems: the nature of their pH lability and sulphydryl dependence. J. Histochem. Cytothem. 18:670-672. 5. BROOKE, M. H., E. WILLIAMSON, AND K. K. KAISER. 197 1. The behavior ofthe four principal muscle fiber types in the developing rat and reinnervated muscle. Arch. Neural. 25: 360-366.

BULLER, A. J., J. C. ECCLES, AND R. M. ECCLES. 1960. Differentiation of fast and slow muscles in the cat hind limb. J. Physiol. (London) 150: 399-4 16. 7. BULLER, A. J., J. C. ECCLES,AND R. M. ECCLES. 1960. Interaction between motoneurones and muscles in respect of the characteristic speeds of their responses. J. Physiol. (London)

6.

150: 417-439.

8. BULLER, A. J., AND D. M. LEWIS. 1965. The rate of tension development in isometric tetanic contractions of mammalian fast and slow skeletal muscle. J. Physiol. (London) 176:337-354.

9. BULLER, A. J., AND D. M. LEWIS. 1965. Some observations on the effects of tenotomy in the rabbit. J. Physiol. (London) 178: 326-342. 10. BURKE, R. E., AND P. TSAIRIS. 1974. The correlation ofphysiological properties with histochemical characteristics in single muscle units. Pages 145-159 in D. DRACHMAN, Ed., Trophicfunction of the nerve. New York Academy of Science. 11. CACCIA, M. R., G. MEOLA, G. BRIGNOLI, L. ANDREUSSI, AND G. SCARLATO. 1978. Physiological and histochemical changes of the extensor digitorum longus and soleus muscles after lateral cordotomy in the albino rat. Exp. Neural. 62: 647-657. 12. CARRARO, U., L. DALLA LIBERA, C. CATANI, AND D. DANIELI BEI-~o. 1982. Chronic denervation of rat diaphragm: selective maintenance of adult fast myosin heavy chains. Muscle Nerve 5: 5 15-524. 13. CARRARO, U., D. MORALE, I. MUSSINI, S. LUCKE, M. CANTINI, R. BEI-~o, C. CATANI, L. DALLA LIBERA, D. DANIELI BETTO, AND D. NOVENTA. 1985. Chronic denervation of rat hemidiaphragm: maintenance of fiber heterogeneity with associated increasing uniformity of myosin isoforms. J. Cell Biol. 100: 16 l-174. 14. COPE, T. C., S. C. BODINE, M. FOURNIER, AND V. R. EDGERTON. 1986. Soleus motor units in chronic spinal transected cats: physiological and morphological alterations. J. Neurophysiol. 55: 1202- 1220.

234

MIDRIO

ET AL.

15. DANIELI BETTO, D., AND M. MIDRIO. 1978. Effects of the spinal cord section and of subsequent denervation on the mechanical properties of fast and slow muscles. Experientia 34: 55-56.

16. DANIELI BE~o, D., E. ZERBATO, AND R. BETTO. 1986. Type 1,2A, and 2B myosin heavy chain electrophoretic analysis of rat muscle fibers. Biochern. Biophys. Res. Commun. 138: 981-987.

17. DAVEY, D. F., C. DUNLOP, J. F. Y. HOH, AND S. Y. P. WONG. 198 1. Contractile properties and ultrastructure of extensor digitorum longus and soleus muscles in spinal cord transected rats. Aust. J. Biol. Med. Sci. 59: 393-404. 18. DAVIS, C. J. F., AND A. MONTGOMERY. 1977. The effect of prolonged inactivity upon the contraction characteristics of fast and slow mammalian twitch muscle. J. Physiol. (London) 270: 58 l-594. 19. DULHUNTY, A. F. 1985. Excitation-contraction coupling properties in denervated EDL and soleus muscles. J. Muscle Res. Cell Motil. 6: 207-225. 20. DULHUNTY, A. F., AND P. W. GAGE. 1983. Asymmetrical charge movement in slow- and fast-twitch mammalian muscle fibres in normal and paraplegic rats. J. Physiot. (London) 341:213-231. 21.

22.

23.

24. 25. 26.

DULHUNTY, A. F., AND P. W. GAGE. 1985. Excitation-contraction coupling and charge movement in denervated rat extensor digitorum longus and soleus muscles. J. Physiol. (London) 358: 75-89. EDGERTON, V. R., L. A. SMITH, E. ELDRED, T. C. COPE, AND L. M. MENDELL. 198 1. Muscle and motor unit properties of exercised and non-exercised chronic spinal cats. Pages 355-37 1 in D. PETTE, Ed., Plasticity ofMuscle. deGruyter, New York. ELDRIDGE, L., AND W. MOMMAERTS. 1981. Ability of electrically silent nerves to specify fast and slow muscle characteristics. Pages 325-337 in D. PETTE, Ed., Plasticity ofMusde. deGruyter, New York. FINOL, H. J., D. M. LEWIS, AND R. OWENS. 198 1. The effects of denervation on contractile properties of rat skeletal muscle. J Physiol. (London) 319: 8 l-92. FISCHBACH,G. D., AND N. ROBBINS. 1969. Changes in contractile properties of disused soleus muscle. J. Physiol. (London) 201: 305-320. GALLEGO, R., P. HUIZAR, N. KUDO, AND M. KUNO. 1978. Disparity of motoneurone and muscle differentiation following spinal transection in the kitten. J. Physiol. (London) 281: 253-265.

27.

28. 29. 30.

3 1. 32.

GAUTHIER, G. F., AND A. W. HOBBS. 1982. Effects of denervation on the distribution of myosin isoenzymes in skeletal muscle fibers. Exp. Neural. 76: 33 l-346. GUTH, L., AND F. S. SAMAHA. 1969. Qualitative differences between actomyosin ATPase of slow and fast mammalian muscles. Exp. Neural. 25: 138-l 52. GUTH, L., V. F. KEMERER, T. A. SAMARAS, J. E. WARNICK, AND E. X. ALBUQUERQUE. 198 1. The roles of disuse and loss of neurotrophic function in denervation atrophy of skeletal muscle. Exp. Neural. 73: 20-36. GUTMANN, E. 1976. Problems in differentiating trophic relationships between nerve and muscle ceils. Pages 323-343 in S. Thesleff, Ed., Motor Innervation ofMuscle. Academic Press, London/New York/San Francisco. GUTMANN, E., J. MELICHNA, AND I. SYROW. 1972. Contraction properties and ATPase activity in fast and slow muscle of the rat during denervation. Exp. Neural. 36: 488-497. HOH, J. F. Y., AND C. DUNLOP. 1974. Transformation of slow-twitch muscle to fast-twitch muscle in paraplegic rat. Pages 50-5 1 in ZZZInternational Congress on Muscle Diseases. Congress Series No. 334. Excerpta Medica, Amsterdam.

CORDOTOMY-DENERVATION

EFFECTS ON MUSCLES

235

33. HUDLICKA, O., T. AITMAN, A. HEILIG, E. LEBERER, K. R. TYLER, AND D. PETTE. 1984. Effects of different patterns of long-term stimulation on blood flow, fuel uptake and enzyme activities in rabbit fast skeletal muscles. pfligers Arch. 402: 306-3 12. 34. HUDLICKA, O., K. R. TYLER, T. SRIHARI, A. HEILIG, AND D. PETTE. 1982. The effect of different patterns of long-term stimulation on contractile properties and myosin light chains in rabbit fast muscles. Pfliigers Arch. 393: 164- 170. 35. JAWEED, M. M., G. J. HERBISON, AND J. F. DITUNNO. 1975. Denervation and reinnervation of fast and slow muscles. A histochemical study in rats. J. Histochem. Cytochem. 23: 808-827. 36. JOLESZ, F., AND F. A. SRETER. 198 1. Development, innervation, and activity-pattern induced changes in skeletal muscle. Annu. Rev. Physiol. 43: 53 1-552. 37. JOHNSON, D. J., L. A. SMITH, E. ELDRED, AND V. R. EDGERTON. 1982. Exercise-induced changes of biochemical, histochemical, and contractile properties of muscle in cordoto mized kittens. Exp. Neurol. 76: 414-427. 38. KARPATI, G., AND W. K. ENGEL. 1968. Correlative histochemical study of skeletal muscle after suprasegmental denervation, peripheral nerve section, and skeletal fixation. Neuralogy 18: 68 l-692. 39. LAEMMLI, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680-685. 40. LEWIS, D. M. 1972. The effect of denervation on the mechanical and electrical responses of fast and slow mammalian twitch muscle. J. Physiol. (London) 222: 5 l-75. 4 1. LIEBER, R. L., J. 0. FRIDEN, A. R. HARGENS, AND E. R. FERINGA. 1986. Long-term effects of spinal cord transection on fast and slow rat skeletal muscle. II. Morphometric properties. Exp. Neurol. 91: 435-448. 42. LIEBER, R. L., C. B. JOHANSSON, H. L. VAHLSING, A. R. HARGSEN, AND E. R. FERINGA. 1986. Long-term effects of spinal cord transection on fast and slow rat skeletal muscle. 1. Contractile properties. Exp. Neural. 91: 423-434. 43. MAIER, A., J. L. CROCKETT, D. R. SIMPSON, C. W. SAUBERT, IV, AND V. R. EDGERTON. 1976. Properties of immobilized guinea pig hindlimb muscles. Am. J. Physiol. 231: 15201526. 44. MARGRETH, A., L. DALLA LIBERA, G. SALVIATI, AND N. ISCHIA. 1980. Spinal transection and the postnatal differentiation of slow myosin isoenzymes. Muscle Nerve 3: 483-486. 45. MAYER, R. F., R. E. BURKE, J. TOOP, B. WALMSLEY, AND J. A. HODGSON. 1984. The effect of spinal cord transection on motor units in cat medial gastrocnemius muscles. Muscle Nerve 7: 23-3 I. 46. MUNSON, J. B., R. C. FOEHRING, S. A. LOF~ON, J. E. ZENGEL, AND G. W. SYPERT. 1986. Plasticity of medial gastrocnemius motor units following cordotomy in the cat. J. Neurophysiol. 55: 6 19-634. 47. PADIKULA, H. A., AND HERMANN, E. 1955. Factors affecting the activity of adenosinephosphatase and other phosphataseses as measured by histochemical techniques. J. Histochem. Cytochem. 3: 161-165. 48. PETE, D., W. MULLER, E. LEISNER, AND G. VRBOV.& 1976. Time dependent effects on contractile properties, fibre population, myosin light chains and enzymes of energy metabolism in intermittently and continuously stimulated fast twitch muscles of the rabbit. PfltigersArch. 364: 103-l 12. 49. PE’ITE, D., AND K. R. TYLER. 1983. Response of succinate dehydrogenase. activity in fibres of rabbit tibialis anterior muscle to chronic nerve stimulation. J. Physiol. (London) 338: l-9. 50. PET~E, D., AND G. VRBOV~. 1985. Neural control ofphenotypic expression in mammalian muscle fibers. Muscle Nerve 8: 676-689.

236

53. 54. 55. 56. 57. 58. 59. 60. 61. 62. 63. 64. 65.

ET AL.

AND R. C. WHALEN. 1979. Independent development ofcontractile properties and myosin light chains in embryonic chick fast and slow muscle. pfliigrs Arch. 378: 251-257. ROY, R. R., R. D. SACKS, K. M. BALDWIN, M. SHORT, AND V. R. EDGERTON. 1984. Interrelationships of contraction time, Va and myosin ATPase after spinal transection. J. Appl. Physiol. 56: l594- 160 1. SALMONS, S., AND J. HENRIKSSON. 198 1. The adaptive response of skeletal muscle to increased use. Muscle Nerve 4: 94-105. SALTIN, B., AND P. D. GOLLNICK. 1983. Skeletal muscle adaptability: significance for metabolism and perfomnnce. Pages 555-63 1 in Handbook of Physiology. Amer. Physiol. Sot., Bethesda, MD. SALVIATI, G., R. BE-~TO, AND D. DANIELI BETTO. 1982. Polymorphism of myofibrillar proteins of rabbit skeletal-muscle fibres. An electrophoretic study of single fibres. Biothem. J. 207: 261-272. SALVIATI, G., R. BE~o, D. DANIELI BE~TO, AND M. ZEVIANI. 1984. Myofibrillar-protein isoforms and sarcoplasmic-reticulum Ca-transport activity of single human muscle fibres. Biochem. J. 224: 2 15-225. SALVIATI, G., E. BIASIA, R. BETTO, AND D. DANIELI BE~o. 1986. Fast to slow transition induced by experimental myotonia in rat EDL muscle. Pfiigers Arch. 406: 266-272. SCHNEIDER,M. F., AND W. K. CHANDLER. 1973. Voltage dependent charge movement in skeletal muscle: a possible step in exitation-contraction coupling. Nature (London) 242: 244-246. SMITH, J. L., L. A. SMITH, R. F. ZERNICKE, AND M. HOY. 1982, Locomotion in exercised and nonexercised cats cordotomized at 2 or 12 weeks of age. Exp. Neural. 76: 393-4 13. SRETER, F. A., K. PINTER, F. JOLESZ,AND K. MABUCHI. 1982. Fast to slow transformation of fast muscles in response to long-term phasic stimulation. Exp. Neural. 75: 92- 102. STELZNER, D. J., W. B. ERSHLER, AND E. D. WEBER. 1975. Effects of spinal transection in neonatal and weanling rats: survival of function. Exp. Neural. 46: 156- 177. SYROW, I., E. GUTMANN, AND J. MELICHNA. 1971. Differential response of myosin ATPase activity and contraction properties of fast and slow rabbit muscles following denervation. Experientia 27: 1426-1427. TOMANEK, R. J., AND D. D. LUND. 1973. Degeneration ofdifferent types of skeletal muscle fibers. I. Denervation. J. Anat. 116: 395-407. VOLPE, P., D. BIRAL, E. DAMIANI, AND A. MARGRETH. 198 1. Characterization of human muscle myosins with respect to the light chains. Biochem. J. 195: 25 l-258. VRBOV& G. 1963. The effect of motoneurone activity on the speed of contraction of striated muscle. J. Physiol. (London) 169: 5 13-526.

5 1. PETTE, D., G. VRBOVA,

52.

MIDRIO