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Effects of immobilization on the isometric contractile properties of embryonic avian skeletal muscle
EXPERIMENTAL
NEUROLOGY
99,59-72 (1988)
Effects of Immobilization on the Isometric Contractile Properties of Embryonic Avian Skeletal Muscle PETER J. REISER, BRADFORD
T. STOKES, AND PATRICIA
J. WALTERS’
Department of Physiology, The Ohio State University, Columbus, Ohio 43210 ReceivedMarch 12, 1987; revision receivedJune 11, 1987 Chicken embryos were chronically immobilized by applying a neuromuscular blocking agent, curare, to the chorioallantoic membrane from day 8 through day 16 of incubation to study the effects of a deficit in motor activity on the development of contractile properties of skeletal muscle. Compared with control embryos, spontaneous embryonic motor activity was depressed by 60 to 90% in the curare-treated animals during the treatment period. Growth of the posterior latissimus dorsi muscle, a fast-twitch muscle in the adult, was greatly affected by immobilization. The average blotted mass of the muscles from curare-treated 1S- to 19-day embryos was approximately 20% of that from control embryos. The isometric contractile properties of posterior latissimus dorsi muscles isolated from control and curare-treated embryos were compared at I8 to 19 days of incubation. The times to peak tension and to onehalf relaxation of the twitch and tetanic responses were significantly greater for the muscles from the immobilized embryos. The peak twitch and tetanic tensions, normalized for muscle cross-sectional area, were significantly less than control values for the muscles from curare-treated embryos. The maximal rate of tetanic tension production was, however, unaffected by immobilization. The results of this study demonstrate that the development of isometric contractile properties of embryonic skeletal muscle is significantly altered by an experimentally induced reduction of spontaneous motor activity. A disruption in the functional development of the sarcoplasmic reticulum following a similar decrease in motor activity, as reported by others, is discussed as a potential mechanism for the altered contractile properties of muscles from the curare-treated embryos. 0 1988 Academic PUSS. I~C.
Abbreviations: ALD, PLD-anterior, posterior latissimus dorsi; SR-sarcoplasmic reticulum. ’ The authors thank Dr. James A. Grossie for valuable discussions and Susan Krey for typing the manuscript. This study was supported by the Muscular Dystrophy Association and by grant BNS-7.905756 from the National Science Foundation. The present address of P. J. R. is Depart59 0014-4886/88 $3.00 Copyright 0 1988 by Academic Press, Inc. All rights of reproduction in any form reserved.
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INTRODUCTION The isometric contractile properties of the chicken posterior latissimus dorsi (PLD) muscle undergo rapid differentiation during the final week of embryonic development toward those characteristic of the adult fast-twitch muscle (8, 15, 17). This late embryonic period follows a time during which the frequency of normal, spontaneous embryonic body movements reaches a maximum (13), suggesting that motor activity might, in some way, be a requisite for the functional differentiation of fast-twitch skeletal muscle. It has been demonstrated that the activity patterns of muscle have a marked influence on the contractile characteristics of adult skeletal muscle [for review, see (lo)]. It was shown elsewhere that chronic immobilization of chicken embryos, achieved through applications of the neuromuscular blocking agent, curare, resulted in several modifications of the developing avian motor system including a decrease in the extent of motoneuron cell death (14), a normal event in the maturation of motor systems, and a delay in the maturation of the motor end plates (19). More recently, Roufa and Martonosi (18) demonstrated that similar treatments with curare during embryonic development resulted in alterations in both the myofibrillar and sarcoplasmic reticulum protein composition of embryonic muscle. Tate et al. (23) reported that surgical denervation of the adult pectoralis muscle, also fast-twitch, led to functional impairment of the isolated sarcoplasmic reticulum. Reiser and Stokes (16) reported that alterations in the contractile properties of skeletal muscles from chicken embryos bred for avian muscular dystrophy are temporally related to abnormal electrical discharge patterns in the motoneuronal pool innervating the muscles (21). Shear (20) demonstrated that not only is motor activity essential for normal myofiber growth in neonatal PLD muscles but that the timing of experimentally induced immobilization has profound effects on the ability of the myofibers to recover when activity is restored. Our purpose was to determine if chronic immobilization during chicken embryonic development leads to altered differentiation of specific contractile properties of developing skeletal muscle. The results are interpreted on the basis of other studies which have demonstrated that decreases in normal activity affect several biochemical parameters of developing and adult skeletal muscle, especially those related to the sarcoplasmic reticulum. A preliminary report of this study was presented earlier (22).
ment of Physiology, School of Medicine, University of Wisconsin, Madison, WI 53706, where reprint requests should be addressed.
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MATERIALS AND METHODS Fertile White Leghorn chicken eggs were obtained from the Department of Poultry Science at Ohio State University and maintained in a forced-draft incubator at 38°C and 60 to 70% relative humidity. One group of embryos (N = 11) received 3.0 mg d-tubocurarine chloride (Sigma) in 100 to 250 ~1 saline applied to the chorioallantoic membrane through a small window in the egg shell as described by Pittman and Oppenheim ( 14). Treatments were administered on days 8, 10, 12, 14, and 16 of incubation. The choices of treatment dosage and frequency were based on the results of Pittman and Oppenheim ( 14) who showed that even at 4 days after the last of a daily dose of 2.5 mg curare, the motor activity of chicken embryos was still suppressed by 70% compared with the control level. The mortality rate of the curaretreated embryos prior to day 18 was approximately 30%. Another group of embryos in the present study received saline in a manner identical to the curare treatments. Several muscles from nontreated (neither saline nor curare) embryos were also studied. The contractile properties of the muscles from the embryos that received saline and the nontreated control embryos did not differ, and therefore, the data from these muscles (N = 9) were pooled. Embryonic motility measurements were made using a candling method in a humidified, temperature-controlled Plexiglas box. The eggs were held close to a light source and the silhouette of the embryo was observed. The number of movements in a 3-min period was counted. All observable limb and trunk movements were counted on the assumption that all skeletal muscles were similarly affected by the curare treatments. These measurements were made immediately prior to each drug treatment when the effect of the previous drug treatment was expected to be lowest. Thus, the measured reductions in motility were conservative estimates of the effect of the curare treatments. PLD muscles were isolated from embryos on incubation day 18 or 19. The beak length and extent of yolk absorption of all embryos confirmed that they were at stages consistent with embryonic day 18 or 19 (9). The methods of dissection, mounting the muscle in the experimental chamber, and recording the isometric contractile properties were as described in detail elsewhere ( 15). Briefly, a muscle was mounted between two platinum stimulating electrodes in the experimental chamber and tied with surgical silk thread on one end to a rigid post and on the other end to an isometric tension transducer (model AE 875, Aksjeselskapet Mikro-Electronikk, Horton, Norway). A small amount of tendon was included between the knots to ensure that the ends of the muscle fibers were not damaged. The muscle was tied close to the myotendonous region on either end with short pieces of thread to minimize the external compliance. The muscle was then set to the optimal length, LO, which was determined as the length at which the greatest twitch tension was
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generated. Previously, it was reported that the greatest tetanic tension was generated at the same muscle length (17). After measuring the contractile properties, LQ was measured using a dissecting microscope by subtracting any tendonous material included between the silk thread knots when the knots were held apart the same distance as they were in the experimental chamber. Electrical stimulation was provided through a Grass S48 stimulator connected to a DC power supply ( 15). A stimulation frequency of 40 Hz was used to elicit the tetanic response as the results of an earlier study (15) showed that this frequency elicited the maximal rate and magnitude of tension development in normal embryonic muscles. The stimulus protocol for each muscle consisted of four cycles of one tetanus (5 s duration) followed by two twitches, each spaced several minutes apart to ensure complete relaxation between responses. A typical protocol required 90 min to complete. Measurements on the isometric responses included the time to peak twitch tension, the time to one-half peak tetanic tension, magnitude of peak twitch and tetanic tensions, maximal rates of twitch and tetanic tension development, and the time to one-half relaxation for both the twitches and tetani. Except for the rates of tension development, all parameters were read directly from the screen of a storage oscilloscope (model 2090, Nicolet Instrument Corp., Madison, Wisconsin). The rates of tension development were determined with a DECLAB-1 l/MING computer (Digital Equipment Corp., Marlboro, Massachusetts) after transfer of the records from the oscilloscope to the computer. The bathing solution in the experimental chamber consisted of the following (in mM): NaCl, 118.4; KCl, 4.7; NaHC03, 25.0; KH2PO4, 1.11; CaC&, 2.5; MgS04, 1.2; glucose, 5.53. The solution was gassed with a 95% 02-5% COz mixture (pH = 7.40). d-Tubocurarine chloride was added to the bathing solution at a final concentration of 20 mg/liter to ensure that stimulation of the muscle was direct rather than through the nerve terminals. The experiments were conducted at 22.6 f 0.2”C (mean f SE). Following an experiment, the blotted muscle mass was measured after removing fluid which was clinging to the external surface of the muscle, using absorbent paper. The dry muscle mass was obtained after the muscle had been stored in a desiccator for several days. Tests for statistical significance were based on two-tailed Student’s t-tests for unpaired observations. RESULTS
Decrease in Embryonic Motility. To verify the effectiveness of the dosage of curare in reducing spontaneous motor activity, the frequency of embryonic body movements was measured every 48 h just prior to the next treat-
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FIG. 1. Embryonic motility between days 10 and 18 in ovu for control (9) and curare-treated (11) embryos. Plotted points represent mean + SE.
ment in the control and curare-treated embryos (Fig. 1). At all times during the treatment period, embryonic motility was significantly depressed in those embryos receiving curare compared with the control group. The average decrease in motility ranged from 60% on day 10 to 90% on day 18. Changes in Physical Properties. Associated with the decrease in embryonic motility, there were marked differences in the physical properties of the PLD muscles between the control and curare-treated embryos (Table 1). The mass of the skeletal musculature of the entire body, as assessedby visual inspection during the dissections, and quantitated for the PLD muscle in particular, was less in the curare-treated embryos compared with the control embryos. The mean dry and blotted masses of the PLD muscles from embryos treated with
TABLE
1
Mass, Length, and Cross-sectional Area of Posterior Latisimus Dorsi Muscles from Control and Curare-Treated Embryos” Parameter
Control
Curare-treated
Dry ma= (W Blotted mass (mg) Dry mass:blotted mass Lo (mm) Estimated cross-sectional area (mm2)b
1.0 f0.1 (8) 7.0 f 0.4 (9) 0.15+0.01(8) 13.7 + 0.3 (9) 0.50 + 0.03 (9)
0.4 +0.1** (11) 1.5 -+0.3+* (11) 0.27 + O.OS* (11) 9.1 *0.33**(11) 0.16t0.02**(11)
’ Values are expressed as mean + SE (N). b See text for details of calculation. * Indicates significant difference from control value at P < 0.05, **P < 0.01
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1 FIG. 2. Examples of the twitch response of muscles from control and curare-treated embryos. The vertical scales of the responses were adjusted to superimpose the records and thereby illustrate the differences in time to peak tension and the time to one-half relaxation. Horizontal bar represents 200 ms. Bottom trace: vertical bar indicates time at which the stimulus pulse was applied.
curare were reduced by approximately 60% and 80%, respectively, compared with control muscles (P < 0.0 1). The mean dry mass:blotted mass ratio for the PLD muscles was greater (P -C0.0 1) for the curare-treated embryos compared with control embryos. Longitudinal growth of the muscles was affected by the curare treatments as the mean length (&) of PLD muscles in the experimental group was reduced by 34% compared with the control group (P < 0.0 I). The average cross-sectional area of the muscles, calculated as (A&,/ L.&D (1) where Mb is blotted muscle mass, Lo is muscle length, and D is tissue density of 1.06 g/cm3, was reduced by about 70% compared with the control value (P < 0.0 1).
Changesin Contractile Properties Several parameters of the isometric twitch and tetanic responses of the PLD muscle were studied to assess the effects of immobilization on the differentiation of contractile properties. Because the contractile properties of the muscles changed during the stimulation protocol, especially for the curare-treated group, a comparison of the responses of the muscles at the beginning of the protocol (i.e., following a period of rest) is first presented followed by a description of changes during the protocol. Contractile Properties at Beginning of Stimulation Protocol. Examples of the twitch responses from the control and curare-treated groups are shown in Fig. 2. Previously, we reported that the twitch response of embryonic PLD muscles consists of an initial rapid, or phasic, component followed by a much slower tonic component ( 15). We also reported that the contribution of the tonic component to the overall twitch response decreases during the final week in ova. Because the contribution of the tonic component was negh-
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TABLE 2 Characteristics of First Isometric Twitch and Tetanic Responses during the Stimulus Protocol for Muscles from Control and Curare-Treated Embryos” Parameter Twitch P (kN/m’) (max dP/dt)/P(% t(P) (ms) t(fR) (ms) Tetanus PO W/m*) (max dPo/dt)/Po W0) (4 t(fR) (ms)
Control
P/ms)
(%Po/ms)
41.8 k 2.7 t 77 2 93 +
3.019) 0.3 (9) 3 (9) 7 (9)
82.2 + 6.2 (9) 1.5 + 0.3 (7) 48 + 4 (8) 168 228 (6)
Curare-treated 22.6 + 2.3-c 110 f 182 +
5.2** 0.1: 7** 19**
(IO) (II) (II) (9)
31.4f 6.6**(10) 1.7 + 0.1 (8) 43 AZ 3 (8) 244 + 32* (10)
a Values are expressed as mean + SE (A’). Abbreviations: P-peak twitch tension; (max dP/ dt)/P-maximal rate of twitch tension development normalized with peak twitch tension; t(P)-time to peak twitch tension; t( fR)-time to one-half relaxation; PO-peak tetanic tension; (max dPO/dt)/P,,-maximal rate of tetanic tension development normalized with peak tetanic tension; t( i PO)-time to one-half peak tetanic tension. * Indicates significantly different from control value at P -c 0.05, **P < 0.0 I.
gible at day 18 in the present study for both the control and the curare-treated muscles, the results described here pertain to the rapid phasic component of the twitch. The mean peak isometric tension, normalized with muscle crosssectional area, was reduced by 46% for the muscles isolated from the curaretreated embryos compared with the control muscles (Table 2). The maximal rate of twitch tension production, normalized with peak twitch tension, was slightly lower for the experimental muscles compared with the control value. The average time to peak twitch tension was 43% longer for the muscles from the curare-treated embryos compared with those from the control embryos. The time to one-half relaxation from the peak of the twitch response for the experimental muscles was approximately twofold longer than the control value. Examples of the isometric tetanic responses of muscles from control and curare-treated embryos are shown in Fig. 3. Several qualitative differences were present in the responses between the control and experimental muscles. The profile of the tetanus was more consistent among the control muscles than among the experimental group. After peak tension was attained in the control muscles, there was a slight decline in tension while the stimulus train was maintained. After the last stimulus pulse, tension rapidly returned to the baseline in this group of muscles. For the muscles from the curare-treated
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control
\
FIG. 3. Examples of tetanic responses of muscles from one control and two curare-treated embryos. Horizontal bar represents 2 s for each record; vertical bar represents 32 kN/m’, 7 kN/ m2, and 4 kN/m’ for the top, middle, and bottom records, respectively. Bottom trace indicates time and duration of stimulus train.
embryos, the tetanic response was more variable as illustrated in Fig. 3. In some of those muscles, tension was maintained fairly steady near the peak level during the stimulus train while in others tension declined to a submaximal level where it remained until the stimulus was terminated. The relaxation phase was also more variable among the experimental muscles compared to the control group. In some instances, relaxation occurred in a clearly biphasic fashion with an initial rapid phase followed by a much slower phase. The bottom record of Fig. 3 shows an extreme case but illustrates a close similarity of some of the curare-treated muscles with previously reported observations on the contractile properties of muscles from younger embryos ( 15). In most experimental muscles, relaxation occurred in an approximately exponential manner, similar to the control muscles, but was
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significantly slower. The mean time to one-half relaxation during the exponential phase was 244 -t 32 ms for the curare-treated group and 168 +- 28 ms for the control group (P < 0.05) for the first tetanus of a protocol. The mean maximal rate of tetanic tension development, normalized with peak tension (PO), and the mean time to one-half PO at the beginning of the tetanus were not significantly different between the experimental and control muscles. PO, normalized with muscle cross-sectional area, was reduced by 62% in the muscles from the curare-treated embryos compared to the control muscles.
Changes in Contractile Properties during the Stimulation Protocol. Changes occurred in contractile properties during the stimulation protocol such that, in general, the muscles generated less tension and the response became slower compared with the responses at the beginning of the protocol. These changes were markedly greater for the muscles from the curare-treated embryos compared with the control muscles. For example, the time to peak twitch tension increased by an average 15% and 50% for the control and experimental muscles, respectively, and the time to one-half relaxation of the twitch response increased 4.4-fold for the muscles from the curare-treated muscles and only 0.3-fold for the control muscles during the stimulus protocol. All these changes in the kinetics of the twitch response during the stimulation protocol were significantly different (P < 0.01) between the experimental and control groups of muscles. The maximal rate of twitch tension production decreased slightly during the protocol for the control (8 + 3%) and curare-treated ( 12 + 4%) groups. On the average, the peak twitch tension decreased by 12% during the protocol for the muscles from the curare-treated embryos whereas peak tension increased by an average 6% for the control muscles. This difference between the control and experimental groups was also statistically significant (P < 0.0 1). On the average, POfor the muscles from curare-treated embryos decreased by 11% between the first and fourth tetanus of the protocol, whereas POfor the muscles from control embryos did not change significantly. However, the maximal rates of tetanic tension production and the time to half peak tetanic tension were virtually unchanged during the stimulus protocol for either the control or curare-treated groups. DISCUSSION The effects of chronic immobilization on the differentiation of isometric contractile properties during embryonic development of a vertebrate fasttwitch muscle have been examined. Spontaneous motor activity was significantly decreased in the presence of the neuromuscular blocking agent, curare, between days 8 and 18 in ovo. The results demonstrate that this decrease in muscle activity was accompanied by significant alterations in the contractile and physical properties of the embryonic avian PLD muscle.
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The growth of the PLD muscle was significantly affected by the decreased muscle activity in the presence of curare. The dependence of skeletal muscle growth on motor activity during development was previously noted by Roufa and Martonosi (18) who also applied curare to induce a decrease in muscle activity of avian embryos. Drachman (5), using botulinum toxin injections to block neuromuscular activity, demonstrated an atrophy of embryonic chicken skeletal muscle rather than a simple slowing of muscle growth when muscle activity is reduced. Histological evidence showed an infiltration of fat cells into skeletal muscle during the immobilization period (5) which is characteristic of adult muscle atrophy. It is likely, therefore, that muscle activity during embryonic development is necessary not only for normal muscle growth but also for the maintenance of the myofibrillar and cytoskeletal elements of skeletal muscle [see (4) for additional evidence]. Alterations in several isometric contractile properties of developing muscle were found to be associated with the decrease in embryonic motor activity. In general, the lower tension development per unit of cross-sectional area and slower kinetics of the isometric twitch response suggest that the muscles from the curare-treated group were delayed in maturation since their properties were similar to those of normal muscles from younger embryos ( 15). The fact that the kinetics of the twitch were more profoundly affected than those of the tetanus may indicate that a sustained level of activation, as occurs during a tetanus, could at least partially compensate for whatever is the primary cause of the alterations in the twitch response of the immobilized muscles. One obvious possibility is that the transient in intracellular calcium concentration during the twitch response is altered in the immobilized muscle, perhaps due to a change in the calcium sequestration and/or release from the sarcoplasmic reticulum (SR). A deficiency in the functions of the SR could account for the longer times to peak twitch tension and to one-half relaxation of the twitch tetanus as observed for the immobilized muscles in the present study on the assumption that the rate of Ca2’ sequestration is rate-limiting for isometric relaxation. During tetanic stimulation, it is possible that the repetitive nature of the stimulus results in a normal rate of release of calcium from the SR in immobilized muscles such that the time to onehalf peak tension and the maximal rate of tension development are not affected, provided the underlying rate-limiting mechanism, presumably the rate of cross-bridge attachment as discussed below [see also ( 1 l)], is unaltered by immobilization. However, a slower rate of calcium sequestration by the SR could account for the longer than normal times to one-half relaxation of the twitch and tetanus and the longer time to peak twitch tension as observed for the immobilized muscles. Evidence does exist to support the hypothesis that an alteration in the function of the SR of immobilized muscle is responsible for the differences in
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the kinetics of the isometric contractile properties that we observed between immobilized and control embryonic muscles. Roufa and Martonosi ( 18) reported lower than normal concentrations of myosin, Ca*+-ATPase of the sarcoplasmic reticulum and ATP:creatine phosphotransferase at 15 to 2 1 days in ova in pectoralis and leg muscles of chicken embryos which had been treated with curare between days 7 and 14. Those authors did not observe any differences between the total Ca*+ content or ATP-mediated, oxalatedependent Ca” transport of muscle homogenates from curare-treated embryos compared with muscle homogenates from control embryos. However, the dosage of curare was lower and the treatment period was shorter in that study (18) than ours and, as the authors pointed out, a difference in the rate of Ca*+ uptake between control and immobilized muscles may have been undetected by a relatively large scatter in their data. Less direct evidence to support the hypothesis that the altered contractile properties of immobilized muscle are due to an alteration of the SR comes from a study by Tate et al. (23) in which several characteristics of the SR isolated from adult chicken pectoralis muscle (also fast-twitch) after 20 days of surgical denervation were studied. Those authors reported distinct functional alterations in the Ca*’ sequestering ability of the SR from the denervated pectoralis muscles, including a lower affinity of the Ca*+-ATPase for Ca*+, lower rate of Ca*+ uptake, and lower ATP-dependent Ca*+ accumulation. If similar changes occurred in the SR of immobilized embryonic muscles in our study, then the observed changes in contractile properties could be explained by an alteration in the function of the SR. The basis for the much slower second phase of relaxation of the tetanic response exhibited by some of the muscles from the curare-treated group (bottom trace of Fig. 3) is not obvious. However, the slower relaxation is a characteristic of PLD muscles from normal embryos at earlier ages as demonstrated elsewhere ( 15). One possibility is that the efflux of IQ during the action potential into the lumen of the transverse tubular system might lead to a large increase in [K+], if the lumen volume is relatively small in early embryonic muscles and in muscles from the curare-treated group, This could lead to a state of prolonged membrane depolarization due to the restricted diffusion of K+ out of the narrow tubules which, in turn, could impede the sequestration of Ca*+ by the SR. Ziskind and Dennis (24) reported that curare, at concentrations from 1 to 10 PM, depolarizes embryonic rat skeletal muscle in vitro, an effect possibly mediated through acetylcholine receptors as suggested by the authors. It has not been demonstrated that curare has a similar effect on embryonic chicken skeletal muscle. Both the control and the curare-treated muscles were bathed in Ringer’s solution containing curare in the present study. Therefore, the presence of curare could not by itself account for the prolonged relaxation of the tetanic response exhibited by some
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of the curare-treated muscles. As the in uvo curare treatments were applied to the chorioallantoic membrane in the present study, the concentration of curare in the interstitial space of skeletal muscle following its absorption is not known. The elimination of a depolarizing effect of curare as being at least partially responsible for the alteration in contractile properties of the curaretreated muscles must be reserved until this potential effect is studied. For instance, if the in ova curare treatments result in an increase in the number of acetylcholine receptors in the immobilized muscles, then the curare in the Ringer’s solution could have a greater depolarizing effect on these muscles compared with the control group if, in fact, curare has a depolarizing effect on this tissue that is mediated through acetylcholine receptors. The decrease in normalized peak twitch and tetanic tension and the unusual force maintenance during the tetanus that we observed for the immobilized muscles could be due to a decrease in the amount of calcium available for activating the myofilaments, through either an alteration in SR function or failure of the immobilized muscles to respond synchronously with action potentials to 40 Hz tetanic stimulation. Alternatively, the decrease in normalized tension could be explained by the large increase in fat tissue with the resultant decrease in myofibrillar protein density that occurs in immobilized embryonic muscles as demonstrated by Drachman (5). Light micrographs published by Crow and Stockdale (4) also show a less than normal density of contractile material in muscles of embryos treated with curare suggesting the infiltration of some other tissue. Roufa and Martonosi (18) reported a decrease in the concentration of myosin of skeletal muscle of embryos chronically immobilized with curare. All these alterations could independently result in less than normal tension development through a reduction in the number of force generating cross-bridges per unit of muscle cross-sectional area. Numerous studies have demonstrated a dependence of developmental transitions in myosin isozymes on contractile activity. For instance, Cemy and Bandman (2) reported that when spontaneous contractions of embryonic chicken muscle cultures are blocked, neonatal myosin heavy chain is lost. In another study, Cemy and Bandman (3) demonstrated that the type of myosin heavy chain in regenerating myotubes in chicken pectoral muscles following cold injury is dependent on the presence of intact innervation. Recently, several workers examined the effects of chronic curarization on the distribution of fast and slow cell types and myosin isozymes of embryonic chicken skeletal muscle in ova. McLennan ( 12) using myosin ATPase histochemical staining to identify different fiber types, reported that immobilization of embryonic muscle with curare beginning at day 4 in ovo resulted in the elimination of embryonic type I (i.e., slow) fibers but did not seem to affect the presence of embryonic type II fibers as late as day 16 in ova. This
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result provided indirect evidence that slow type, but not fast type, myosin was lost as a result of inactivity. Crow and Stockdale (4) also used curare treatments to chronically immobilize chicken embryos beginning at day 4 and examined cross sections of hind limb muscles with monoclonal antibodies against fast and slow myosin heavy and light chains. Those authors determined that the fast and slow types of myosin isozymes and the spatial distribution of cell types in thigh muscles are not affected at day 12 in ova. By day 16, however, slow myosin was lost from those muscles that normally contain the slow isozyme with a relative sparing of the fast isozyme. They concluded that the maintenance of different cell types in embryonic muscle is dependent on normal neuromuscular activity. Using antibodies against fast and slow myosin isozymes in another study in which chicken embryos were immobilized with curare between days 7 and 12 in ovo, Gauthier et al. (7) demonstrated that, although the slow anterior latissimus dorsi (ALD) muscle at day 18 was strikingly different from control muscle (i.e., more fibers reacted positively with antibodies against fast myosin from adult pectoralis muscle and fewer reacted with antibodies against slow myosin from adult ALD), the PLD muscle was virtually unaffected. However, as stated by those authors, the antibodies used in their study might not have revealed changes that occurred in the myosin of the fasttwitch PLD. All three of these latter studies demonstrate that, with regard to myosin composition, fast embryonic chicken muscle is relatively spared when motor activity is experimentally reduced, suggesting that the altered contractile properties that we observed are due to something other than a change in the myosin isozymes. Furthermore, the maximal rate of tetanic tension development, which is presumably determined by the rate of cross-bridge attachment, was unaltered by curarization in the present study. Drachman and Johnston (6) demonstrated that changes in max dP/dt are well correlated with changes in the enzymatic activity of myosin of developing fast-twitch mammalian muscle. It can be inferred, therefore, that the ATPase activity of myosin in the curarized muscles is similar to that of control muscles and, therefore, is not responsible for the altered twitch kinetics that follow immobilization. Thus, support is given to the hypothesis that the altered isometric contractile properties of immobilized muscles are due not to changes in the contractile proteins but rather to some other factor(s), perhaps an alteration in the function of the sarcoplasmic reticulum. REFERENCES 1. CARLSON. F. D., AND D. R. WILKIE. 1974. Muscle Physi0log.v. Prentice-Hall, Englewood Cliffs, New Jersey. 2. CERNY, L. C., AND E. BANDMAN. 1986. Contractile activity is required for neonatal myosin heavy chain in embryonic chick pectoral muscle cultures. J. Cell Biol. 103: 2 153-2 16 1.
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3. CERNY’, L. C., AND E. BANDMAN. 1987. Expression of myosin heavy chain isofonns in regenerating myotubes of innervated and denervated chicken pectoral muscle. Dev. Biol. 119: 350-362.
4. CROW, M. T., AND F. E. STOCKDALE. 1986. Myosin expression and specialization among the earliest muscle fibers of the developing avian limb. Der. Biol. 113: 238-254. 5. DRACHMAN, D. B. 1964. Atrophy of skeletal muscle in chick embryos treated with botulinum toxin. Science 145: 7 19-72 1. 6. DRACHMAN, D. B., AND D. M. JOHNSTON. 1973. Development of a mammalian fast muscle: dynamic and biochemical properties correlated. J. Physiol. (London) 234: 29-42. 7. GAUTHIER, G. F., R. D. ONO, AND A. W. HOBBS. 1984. Curare-induced transformation of myosin pattern in developing skeletal muscle. Dev. Biol. 105: 144-154. 8. GORDON, T., R. D. PURVES, AND G. VRBOVA. 1977. Differentiation of electrical and contractile properties of slow and fast muscle fibres. J. Physiol. (London) 269: 535-547. 9. HAMBURGER, V.,, AND H. L. HAMILTON. 195 1. A series of normal stages in the develop ment of the chick embryo. J. Morphol. 88: 49-92. 10. JOLESZ, F., AND F. A. SRETER. 198 1. Development, innervation, and activity-pattern induced changes in skeletal muscle. Annu. Rev. Physiol. 43: 53 l-552. 11. JULIAN, F. J., AND R. L. Moss. 1976. The concept of active state in striated muscle. Circ. Rex 38: 53-59. 12. MCLENNAN, I. S. 1983. Differentiation of muscle fiber types in the chicken hindlimb. Dev. Biol. 97: 222-228. 13. OPPENHEIM, R. W. 1972. The embryology of behavior in birds: a critical review of the role of sensory stimulation in embryonic movement. Pages 258-277 in K. H. VOOUS, Ed., Proceedings of the XVth International Ornithological Congress. Brill, Leiden. 14. PIT~MAN, R., AND R. W. OPPENHEIM. 1979. Cell death in motoneurons in the chick embryo spinal cord. IV. Evidence that a functional neuromuscular interaction is involved in the regulation of naturally occurring cell death and the stabilization of synapses. J. Comp. Neurol. 187: 425-446. 15. REISER, P. J., AND B. T. STOKES. 1982a. Development of contractile properties in avian embryonic skeletal muscle. Am. J. Physiol. 242: C52-C58. 16. RAISER, P. J., AND B. T. STOKES. 1982b. Comparison of the development of isometric contractile properties of embryonic avian normal and dystrophic skeletal muscle. Exp. Neural. 77: 505-5 18. 17. REISER, P. J., B. T. STOKES, AND J. A. RALL. 1982. Isometric contractile properties and velocity of shortening during avian myogenesis. Am. J. Physiol. 243: C 177-C 183. 18. ROUFA, D., AND A. N. MARTONOSI. 198 1. Effect of curare on the development of chicken embryo skeletal muscle in OYO.B&hem. Pharmacol. 30: 150 1- 1505. 19. SRIHARI, T., AND G. VRBOVA. 1978. The role of muscle activity in the differentiation of neuromuscular junction in slow and fast chick muscles. J. Neurocytol. 7: 529-540. 20. SHEAR, C. R. 198 1. Effects of disuse on growing and adult chick skeletal muscle. J. CellSci. 48: 35-54. 2 1.
STOKES, B. T. 1977. Multiunit activity patterns in the brachial spinal cord of dystrophic chick embryos. Exp. Neural. 56: 179-I 88. 22. STOKES, B. T., P. J. WALTERS, AND P. J. REISER. 1981. Twitch and tetanic responses of skeletal muscle from paralyzed chick embryos. Sot. Neurosci. Abstr. 7: 769. 23. TATE, C. A., R. J. BICK, T. D. MYERS, B. J. R. Prm, W. B. VAN WINKLE, AND M. L. ENTMAN. 1983. Alteration of sarcoplasmic reticulum after denervation of chicken pectoralis muscle. B&hem. J. 210: 339-344. 24. ZISKIND, L., AND M. J. DENNIS. 198 1. Depolarizing effects of curare on embryonic rat muscles. Nature 276: 622-623.
NEUROLOGY
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Effects of Immobilization on the Isometric Contractile Properties of Embryonic Avian Skeletal Muscle PETER J. REISER, BRADFORD
T. STOKES, AND PATRICIA
J. WALTERS’
Department of Physiology, The Ohio State University, Columbus, Ohio 43210 ReceivedMarch 12, 1987; revision receivedJune 11, 1987 Chicken embryos were chronically immobilized by applying a neuromuscular blocking agent, curare, to the chorioallantoic membrane from day 8 through day 16 of incubation to study the effects of a deficit in motor activity on the development of contractile properties of skeletal muscle. Compared with control embryos, spontaneous embryonic motor activity was depressed by 60 to 90% in the curare-treated animals during the treatment period. Growth of the posterior latissimus dorsi muscle, a fast-twitch muscle in the adult, was greatly affected by immobilization. The average blotted mass of the muscles from curare-treated 1S- to 19-day embryos was approximately 20% of that from control embryos. The isometric contractile properties of posterior latissimus dorsi muscles isolated from control and curare-treated embryos were compared at I8 to 19 days of incubation. The times to peak tension and to onehalf relaxation of the twitch and tetanic responses were significantly greater for the muscles from the immobilized embryos. The peak twitch and tetanic tensions, normalized for muscle cross-sectional area, were significantly less than control values for the muscles from curare-treated embryos. The maximal rate of tetanic tension production was, however, unaffected by immobilization. The results of this study demonstrate that the development of isometric contractile properties of embryonic skeletal muscle is significantly altered by an experimentally induced reduction of spontaneous motor activity. A disruption in the functional development of the sarcoplasmic reticulum following a similar decrease in motor activity, as reported by others, is discussed as a potential mechanism for the altered contractile properties of muscles from the curare-treated embryos. 0 1988 Academic PUSS. I~C.
Abbreviations: ALD, PLD-anterior, posterior latissimus dorsi; SR-sarcoplasmic reticulum. ’ The authors thank Dr. James A. Grossie for valuable discussions and Susan Krey for typing the manuscript. This study was supported by the Muscular Dystrophy Association and by grant BNS-7.905756 from the National Science Foundation. The present address of P. J. R. is Depart59 0014-4886/88 $3.00 Copyright 0 1988 by Academic Press, Inc. All rights of reproduction in any form reserved.
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INTRODUCTION The isometric contractile properties of the chicken posterior latissimus dorsi (PLD) muscle undergo rapid differentiation during the final week of embryonic development toward those characteristic of the adult fast-twitch muscle (8, 15, 17). This late embryonic period follows a time during which the frequency of normal, spontaneous embryonic body movements reaches a maximum (13), suggesting that motor activity might, in some way, be a requisite for the functional differentiation of fast-twitch skeletal muscle. It has been demonstrated that the activity patterns of muscle have a marked influence on the contractile characteristics of adult skeletal muscle [for review, see (lo)]. It was shown elsewhere that chronic immobilization of chicken embryos, achieved through applications of the neuromuscular blocking agent, curare, resulted in several modifications of the developing avian motor system including a decrease in the extent of motoneuron cell death (14), a normal event in the maturation of motor systems, and a delay in the maturation of the motor end plates (19). More recently, Roufa and Martonosi (18) demonstrated that similar treatments with curare during embryonic development resulted in alterations in both the myofibrillar and sarcoplasmic reticulum protein composition of embryonic muscle. Tate et al. (23) reported that surgical denervation of the adult pectoralis muscle, also fast-twitch, led to functional impairment of the isolated sarcoplasmic reticulum. Reiser and Stokes (16) reported that alterations in the contractile properties of skeletal muscles from chicken embryos bred for avian muscular dystrophy are temporally related to abnormal electrical discharge patterns in the motoneuronal pool innervating the muscles (21). Shear (20) demonstrated that not only is motor activity essential for normal myofiber growth in neonatal PLD muscles but that the timing of experimentally induced immobilization has profound effects on the ability of the myofibers to recover when activity is restored. Our purpose was to determine if chronic immobilization during chicken embryonic development leads to altered differentiation of specific contractile properties of developing skeletal muscle. The results are interpreted on the basis of other studies which have demonstrated that decreases in normal activity affect several biochemical parameters of developing and adult skeletal muscle, especially those related to the sarcoplasmic reticulum. A preliminary report of this study was presented earlier (22).
ment of Physiology, School of Medicine, University of Wisconsin, Madison, WI 53706, where reprint requests should be addressed.
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MATERIALS AND METHODS Fertile White Leghorn chicken eggs were obtained from the Department of Poultry Science at Ohio State University and maintained in a forced-draft incubator at 38°C and 60 to 70% relative humidity. One group of embryos (N = 11) received 3.0 mg d-tubocurarine chloride (Sigma) in 100 to 250 ~1 saline applied to the chorioallantoic membrane through a small window in the egg shell as described by Pittman and Oppenheim ( 14). Treatments were administered on days 8, 10, 12, 14, and 16 of incubation. The choices of treatment dosage and frequency were based on the results of Pittman and Oppenheim ( 14) who showed that even at 4 days after the last of a daily dose of 2.5 mg curare, the motor activity of chicken embryos was still suppressed by 70% compared with the control level. The mortality rate of the curaretreated embryos prior to day 18 was approximately 30%. Another group of embryos in the present study received saline in a manner identical to the curare treatments. Several muscles from nontreated (neither saline nor curare) embryos were also studied. The contractile properties of the muscles from the embryos that received saline and the nontreated control embryos did not differ, and therefore, the data from these muscles (N = 9) were pooled. Embryonic motility measurements were made using a candling method in a humidified, temperature-controlled Plexiglas box. The eggs were held close to a light source and the silhouette of the embryo was observed. The number of movements in a 3-min period was counted. All observable limb and trunk movements were counted on the assumption that all skeletal muscles were similarly affected by the curare treatments. These measurements were made immediately prior to each drug treatment when the effect of the previous drug treatment was expected to be lowest. Thus, the measured reductions in motility were conservative estimates of the effect of the curare treatments. PLD muscles were isolated from embryos on incubation day 18 or 19. The beak length and extent of yolk absorption of all embryos confirmed that they were at stages consistent with embryonic day 18 or 19 (9). The methods of dissection, mounting the muscle in the experimental chamber, and recording the isometric contractile properties were as described in detail elsewhere ( 15). Briefly, a muscle was mounted between two platinum stimulating electrodes in the experimental chamber and tied with surgical silk thread on one end to a rigid post and on the other end to an isometric tension transducer (model AE 875, Aksjeselskapet Mikro-Electronikk, Horton, Norway). A small amount of tendon was included between the knots to ensure that the ends of the muscle fibers were not damaged. The muscle was tied close to the myotendonous region on either end with short pieces of thread to minimize the external compliance. The muscle was then set to the optimal length, LO, which was determined as the length at which the greatest twitch tension was
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generated. Previously, it was reported that the greatest tetanic tension was generated at the same muscle length (17). After measuring the contractile properties, LQ was measured using a dissecting microscope by subtracting any tendonous material included between the silk thread knots when the knots were held apart the same distance as they were in the experimental chamber. Electrical stimulation was provided through a Grass S48 stimulator connected to a DC power supply ( 15). A stimulation frequency of 40 Hz was used to elicit the tetanic response as the results of an earlier study (15) showed that this frequency elicited the maximal rate and magnitude of tension development in normal embryonic muscles. The stimulus protocol for each muscle consisted of four cycles of one tetanus (5 s duration) followed by two twitches, each spaced several minutes apart to ensure complete relaxation between responses. A typical protocol required 90 min to complete. Measurements on the isometric responses included the time to peak twitch tension, the time to one-half peak tetanic tension, magnitude of peak twitch and tetanic tensions, maximal rates of twitch and tetanic tension development, and the time to one-half relaxation for both the twitches and tetani. Except for the rates of tension development, all parameters were read directly from the screen of a storage oscilloscope (model 2090, Nicolet Instrument Corp., Madison, Wisconsin). The rates of tension development were determined with a DECLAB-1 l/MING computer (Digital Equipment Corp., Marlboro, Massachusetts) after transfer of the records from the oscilloscope to the computer. The bathing solution in the experimental chamber consisted of the following (in mM): NaCl, 118.4; KCl, 4.7; NaHC03, 25.0; KH2PO4, 1.11; CaC&, 2.5; MgS04, 1.2; glucose, 5.53. The solution was gassed with a 95% 02-5% COz mixture (pH = 7.40). d-Tubocurarine chloride was added to the bathing solution at a final concentration of 20 mg/liter to ensure that stimulation of the muscle was direct rather than through the nerve terminals. The experiments were conducted at 22.6 f 0.2”C (mean f SE). Following an experiment, the blotted muscle mass was measured after removing fluid which was clinging to the external surface of the muscle, using absorbent paper. The dry muscle mass was obtained after the muscle had been stored in a desiccator for several days. Tests for statistical significance were based on two-tailed Student’s t-tests for unpaired observations. RESULTS
Decrease in Embryonic Motility. To verify the effectiveness of the dosage of curare in reducing spontaneous motor activity, the frequency of embryonic body movements was measured every 48 h just prior to the next treat-
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FIG. 1. Embryonic motility between days 10 and 18 in ovu for control (9) and curare-treated (11) embryos. Plotted points represent mean + SE.
ment in the control and curare-treated embryos (Fig. 1). At all times during the treatment period, embryonic motility was significantly depressed in those embryos receiving curare compared with the control group. The average decrease in motility ranged from 60% on day 10 to 90% on day 18. Changes in Physical Properties. Associated with the decrease in embryonic motility, there were marked differences in the physical properties of the PLD muscles between the control and curare-treated embryos (Table 1). The mass of the skeletal musculature of the entire body, as assessedby visual inspection during the dissections, and quantitated for the PLD muscle in particular, was less in the curare-treated embryos compared with the control embryos. The mean dry and blotted masses of the PLD muscles from embryos treated with
TABLE
1
Mass, Length, and Cross-sectional Area of Posterior Latisimus Dorsi Muscles from Control and Curare-Treated Embryos” Parameter
Control
Curare-treated
Dry ma= (W Blotted mass (mg) Dry mass:blotted mass Lo (mm) Estimated cross-sectional area (mm2)b
1.0 f0.1 (8) 7.0 f 0.4 (9) 0.15+0.01(8) 13.7 + 0.3 (9) 0.50 + 0.03 (9)
0.4 +0.1** (11) 1.5 -+0.3+* (11) 0.27 + O.OS* (11) 9.1 *0.33**(11) 0.16t0.02**(11)
’ Values are expressed as mean + SE (N). b See text for details of calculation. * Indicates significant difference from control value at P < 0.05, **P < 0.01
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1 FIG. 2. Examples of the twitch response of muscles from control and curare-treated embryos. The vertical scales of the responses were adjusted to superimpose the records and thereby illustrate the differences in time to peak tension and the time to one-half relaxation. Horizontal bar represents 200 ms. Bottom trace: vertical bar indicates time at which the stimulus pulse was applied.
curare were reduced by approximately 60% and 80%, respectively, compared with control muscles (P < 0.0 1). The mean dry mass:blotted mass ratio for the PLD muscles was greater (P -C0.0 1) for the curare-treated embryos compared with control embryos. Longitudinal growth of the muscles was affected by the curare treatments as the mean length (&) of PLD muscles in the experimental group was reduced by 34% compared with the control group (P < 0.0 I). The average cross-sectional area of the muscles, calculated as (A&,/ L.&D (1) where Mb is blotted muscle mass, Lo is muscle length, and D is tissue density of 1.06 g/cm3, was reduced by about 70% compared with the control value (P < 0.0 1).
Changesin Contractile Properties Several parameters of the isometric twitch and tetanic responses of the PLD muscle were studied to assess the effects of immobilization on the differentiation of contractile properties. Because the contractile properties of the muscles changed during the stimulation protocol, especially for the curare-treated group, a comparison of the responses of the muscles at the beginning of the protocol (i.e., following a period of rest) is first presented followed by a description of changes during the protocol. Contractile Properties at Beginning of Stimulation Protocol. Examples of the twitch responses from the control and curare-treated groups are shown in Fig. 2. Previously, we reported that the twitch response of embryonic PLD muscles consists of an initial rapid, or phasic, component followed by a much slower tonic component ( 15). We also reported that the contribution of the tonic component to the overall twitch response decreases during the final week in ova. Because the contribution of the tonic component was negh-
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TABLE 2 Characteristics of First Isometric Twitch and Tetanic Responses during the Stimulus Protocol for Muscles from Control and Curare-Treated Embryos” Parameter Twitch P (kN/m’) (max dP/dt)/P(% t(P) (ms) t(fR) (ms) Tetanus PO W/m*) (max dPo/dt)/Po W0) (4 t(fR) (ms)
Control
P/ms)
(%Po/ms)
41.8 k 2.7 t 77 2 93 +
3.019) 0.3 (9) 3 (9) 7 (9)
82.2 + 6.2 (9) 1.5 + 0.3 (7) 48 + 4 (8) 168 228 (6)
Curare-treated 22.6 + 2.3-c 110 f 182 +
5.2** 0.1: 7** 19**
(IO) (II) (II) (9)
31.4f 6.6**(10) 1.7 + 0.1 (8) 43 AZ 3 (8) 244 + 32* (10)
a Values are expressed as mean + SE (A’). Abbreviations: P-peak twitch tension; (max dP/ dt)/P-maximal rate of twitch tension development normalized with peak twitch tension; t(P)-time to peak twitch tension; t( fR)-time to one-half relaxation; PO-peak tetanic tension; (max dPO/dt)/P,,-maximal rate of tetanic tension development normalized with peak tetanic tension; t( i PO)-time to one-half peak tetanic tension. * Indicates significantly different from control value at P -c 0.05, **P < 0.0 I.
gible at day 18 in the present study for both the control and the curare-treated muscles, the results described here pertain to the rapid phasic component of the twitch. The mean peak isometric tension, normalized with muscle crosssectional area, was reduced by 46% for the muscles isolated from the curaretreated embryos compared with the control muscles (Table 2). The maximal rate of twitch tension production, normalized with peak twitch tension, was slightly lower for the experimental muscles compared with the control value. The average time to peak twitch tension was 43% longer for the muscles from the curare-treated embryos compared with those from the control embryos. The time to one-half relaxation from the peak of the twitch response for the experimental muscles was approximately twofold longer than the control value. Examples of the isometric tetanic responses of muscles from control and curare-treated embryos are shown in Fig. 3. Several qualitative differences were present in the responses between the control and experimental muscles. The profile of the tetanus was more consistent among the control muscles than among the experimental group. After peak tension was attained in the control muscles, there was a slight decline in tension while the stimulus train was maintained. After the last stimulus pulse, tension rapidly returned to the baseline in this group of muscles. For the muscles from the curare-treated
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control
\
FIG. 3. Examples of tetanic responses of muscles from one control and two curare-treated embryos. Horizontal bar represents 2 s for each record; vertical bar represents 32 kN/m’, 7 kN/ m2, and 4 kN/m’ for the top, middle, and bottom records, respectively. Bottom trace indicates time and duration of stimulus train.
embryos, the tetanic response was more variable as illustrated in Fig. 3. In some of those muscles, tension was maintained fairly steady near the peak level during the stimulus train while in others tension declined to a submaximal level where it remained until the stimulus was terminated. The relaxation phase was also more variable among the experimental muscles compared to the control group. In some instances, relaxation occurred in a clearly biphasic fashion with an initial rapid phase followed by a much slower phase. The bottom record of Fig. 3 shows an extreme case but illustrates a close similarity of some of the curare-treated muscles with previously reported observations on the contractile properties of muscles from younger embryos ( 15). In most experimental muscles, relaxation occurred in an approximately exponential manner, similar to the control muscles, but was
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significantly slower. The mean time to one-half relaxation during the exponential phase was 244 -t 32 ms for the curare-treated group and 168 +- 28 ms for the control group (P < 0.05) for the first tetanus of a protocol. The mean maximal rate of tetanic tension development, normalized with peak tension (PO), and the mean time to one-half PO at the beginning of the tetanus were not significantly different between the experimental and control muscles. PO, normalized with muscle cross-sectional area, was reduced by 62% in the muscles from the curare-treated embryos compared to the control muscles.
Changes in Contractile Properties during the Stimulation Protocol. Changes occurred in contractile properties during the stimulation protocol such that, in general, the muscles generated less tension and the response became slower compared with the responses at the beginning of the protocol. These changes were markedly greater for the muscles from the curare-treated embryos compared with the control muscles. For example, the time to peak twitch tension increased by an average 15% and 50% for the control and experimental muscles, respectively, and the time to one-half relaxation of the twitch response increased 4.4-fold for the muscles from the curare-treated muscles and only 0.3-fold for the control muscles during the stimulus protocol. All these changes in the kinetics of the twitch response during the stimulation protocol were significantly different (P < 0.01) between the experimental and control groups of muscles. The maximal rate of twitch tension production decreased slightly during the protocol for the control (8 + 3%) and curare-treated ( 12 + 4%) groups. On the average, the peak twitch tension decreased by 12% during the protocol for the muscles from the curare-treated embryos whereas peak tension increased by an average 6% for the control muscles. This difference between the control and experimental groups was also statistically significant (P < 0.0 1). On the average, POfor the muscles from curare-treated embryos decreased by 11% between the first and fourth tetanus of the protocol, whereas POfor the muscles from control embryos did not change significantly. However, the maximal rates of tetanic tension production and the time to half peak tetanic tension were virtually unchanged during the stimulus protocol for either the control or curare-treated groups. DISCUSSION The effects of chronic immobilization on the differentiation of isometric contractile properties during embryonic development of a vertebrate fasttwitch muscle have been examined. Spontaneous motor activity was significantly decreased in the presence of the neuromuscular blocking agent, curare, between days 8 and 18 in ovo. The results demonstrate that this decrease in muscle activity was accompanied by significant alterations in the contractile and physical properties of the embryonic avian PLD muscle.
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The growth of the PLD muscle was significantly affected by the decreased muscle activity in the presence of curare. The dependence of skeletal muscle growth on motor activity during development was previously noted by Roufa and Martonosi (18) who also applied curare to induce a decrease in muscle activity of avian embryos. Drachman (5), using botulinum toxin injections to block neuromuscular activity, demonstrated an atrophy of embryonic chicken skeletal muscle rather than a simple slowing of muscle growth when muscle activity is reduced. Histological evidence showed an infiltration of fat cells into skeletal muscle during the immobilization period (5) which is characteristic of adult muscle atrophy. It is likely, therefore, that muscle activity during embryonic development is necessary not only for normal muscle growth but also for the maintenance of the myofibrillar and cytoskeletal elements of skeletal muscle [see (4) for additional evidence]. Alterations in several isometric contractile properties of developing muscle were found to be associated with the decrease in embryonic motor activity. In general, the lower tension development per unit of cross-sectional area and slower kinetics of the isometric twitch response suggest that the muscles from the curare-treated group were delayed in maturation since their properties were similar to those of normal muscles from younger embryos ( 15). The fact that the kinetics of the twitch were more profoundly affected than those of the tetanus may indicate that a sustained level of activation, as occurs during a tetanus, could at least partially compensate for whatever is the primary cause of the alterations in the twitch response of the immobilized muscles. One obvious possibility is that the transient in intracellular calcium concentration during the twitch response is altered in the immobilized muscle, perhaps due to a change in the calcium sequestration and/or release from the sarcoplasmic reticulum (SR). A deficiency in the functions of the SR could account for the longer times to peak twitch tension and to one-half relaxation of the twitch tetanus as observed for the immobilized muscles in the present study on the assumption that the rate of Ca2’ sequestration is rate-limiting for isometric relaxation. During tetanic stimulation, it is possible that the repetitive nature of the stimulus results in a normal rate of release of calcium from the SR in immobilized muscles such that the time to onehalf peak tension and the maximal rate of tension development are not affected, provided the underlying rate-limiting mechanism, presumably the rate of cross-bridge attachment as discussed below [see also ( 1 l)], is unaltered by immobilization. However, a slower rate of calcium sequestration by the SR could account for the longer than normal times to one-half relaxation of the twitch and tetanus and the longer time to peak twitch tension as observed for the immobilized muscles. Evidence does exist to support the hypothesis that an alteration in the function of the SR of immobilized muscle is responsible for the differences in
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the kinetics of the isometric contractile properties that we observed between immobilized and control embryonic muscles. Roufa and Martonosi ( 18) reported lower than normal concentrations of myosin, Ca*+-ATPase of the sarcoplasmic reticulum and ATP:creatine phosphotransferase at 15 to 2 1 days in ova in pectoralis and leg muscles of chicken embryos which had been treated with curare between days 7 and 14. Those authors did not observe any differences between the total Ca*+ content or ATP-mediated, oxalatedependent Ca” transport of muscle homogenates from curare-treated embryos compared with muscle homogenates from control embryos. However, the dosage of curare was lower and the treatment period was shorter in that study (18) than ours and, as the authors pointed out, a difference in the rate of Ca*+ uptake between control and immobilized muscles may have been undetected by a relatively large scatter in their data. Less direct evidence to support the hypothesis that the altered contractile properties of immobilized muscle are due to an alteration of the SR comes from a study by Tate et al. (23) in which several characteristics of the SR isolated from adult chicken pectoralis muscle (also fast-twitch) after 20 days of surgical denervation were studied. Those authors reported distinct functional alterations in the Ca*’ sequestering ability of the SR from the denervated pectoralis muscles, including a lower affinity of the Ca*+-ATPase for Ca*+, lower rate of Ca*+ uptake, and lower ATP-dependent Ca*+ accumulation. If similar changes occurred in the SR of immobilized embryonic muscles in our study, then the observed changes in contractile properties could be explained by an alteration in the function of the SR. The basis for the much slower second phase of relaxation of the tetanic response exhibited by some of the muscles from the curare-treated group (bottom trace of Fig. 3) is not obvious. However, the slower relaxation is a characteristic of PLD muscles from normal embryos at earlier ages as demonstrated elsewhere ( 15). One possibility is that the efflux of IQ during the action potential into the lumen of the transverse tubular system might lead to a large increase in [K+], if the lumen volume is relatively small in early embryonic muscles and in muscles from the curare-treated group, This could lead to a state of prolonged membrane depolarization due to the restricted diffusion of K+ out of the narrow tubules which, in turn, could impede the sequestration of Ca*+ by the SR. Ziskind and Dennis (24) reported that curare, at concentrations from 1 to 10 PM, depolarizes embryonic rat skeletal muscle in vitro, an effect possibly mediated through acetylcholine receptors as suggested by the authors. It has not been demonstrated that curare has a similar effect on embryonic chicken skeletal muscle. Both the control and the curare-treated muscles were bathed in Ringer’s solution containing curare in the present study. Therefore, the presence of curare could not by itself account for the prolonged relaxation of the tetanic response exhibited by some
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of the curare-treated muscles. As the in uvo curare treatments were applied to the chorioallantoic membrane in the present study, the concentration of curare in the interstitial space of skeletal muscle following its absorption is not known. The elimination of a depolarizing effect of curare as being at least partially responsible for the alteration in contractile properties of the curaretreated muscles must be reserved until this potential effect is studied. For instance, if the in ova curare treatments result in an increase in the number of acetylcholine receptors in the immobilized muscles, then the curare in the Ringer’s solution could have a greater depolarizing effect on these muscles compared with the control group if, in fact, curare has a depolarizing effect on this tissue that is mediated through acetylcholine receptors. The decrease in normalized peak twitch and tetanic tension and the unusual force maintenance during the tetanus that we observed for the immobilized muscles could be due to a decrease in the amount of calcium available for activating the myofilaments, through either an alteration in SR function or failure of the immobilized muscles to respond synchronously with action potentials to 40 Hz tetanic stimulation. Alternatively, the decrease in normalized tension could be explained by the large increase in fat tissue with the resultant decrease in myofibrillar protein density that occurs in immobilized embryonic muscles as demonstrated by Drachman (5). Light micrographs published by Crow and Stockdale (4) also show a less than normal density of contractile material in muscles of embryos treated with curare suggesting the infiltration of some other tissue. Roufa and Martonosi (18) reported a decrease in the concentration of myosin of skeletal muscle of embryos chronically immobilized with curare. All these alterations could independently result in less than normal tension development through a reduction in the number of force generating cross-bridges per unit of muscle cross-sectional area. Numerous studies have demonstrated a dependence of developmental transitions in myosin isozymes on contractile activity. For instance, Cemy and Bandman (2) reported that when spontaneous contractions of embryonic chicken muscle cultures are blocked, neonatal myosin heavy chain is lost. In another study, Cemy and Bandman (3) demonstrated that the type of myosin heavy chain in regenerating myotubes in chicken pectoral muscles following cold injury is dependent on the presence of intact innervation. Recently, several workers examined the effects of chronic curarization on the distribution of fast and slow cell types and myosin isozymes of embryonic chicken skeletal muscle in ova. McLennan ( 12) using myosin ATPase histochemical staining to identify different fiber types, reported that immobilization of embryonic muscle with curare beginning at day 4 in ovo resulted in the elimination of embryonic type I (i.e., slow) fibers but did not seem to affect the presence of embryonic type II fibers as late as day 16 in ova. This
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result provided indirect evidence that slow type, but not fast type, myosin was lost as a result of inactivity. Crow and Stockdale (4) also used curare treatments to chronically immobilize chicken embryos beginning at day 4 and examined cross sections of hind limb muscles with monoclonal antibodies against fast and slow myosin heavy and light chains. Those authors determined that the fast and slow types of myosin isozymes and the spatial distribution of cell types in thigh muscles are not affected at day 12 in ova. By day 16, however, slow myosin was lost from those muscles that normally contain the slow isozyme with a relative sparing of the fast isozyme. They concluded that the maintenance of different cell types in embryonic muscle is dependent on normal neuromuscular activity. Using antibodies against fast and slow myosin isozymes in another study in which chicken embryos were immobilized with curare between days 7 and 12 in ovo, Gauthier et al. (7) demonstrated that, although the slow anterior latissimus dorsi (ALD) muscle at day 18 was strikingly different from control muscle (i.e., more fibers reacted positively with antibodies against fast myosin from adult pectoralis muscle and fewer reacted with antibodies against slow myosin from adult ALD), the PLD muscle was virtually unaffected. However, as stated by those authors, the antibodies used in their study might not have revealed changes that occurred in the myosin of the fasttwitch PLD. All three of these latter studies demonstrate that, with regard to myosin composition, fast embryonic chicken muscle is relatively spared when motor activity is experimentally reduced, suggesting that the altered contractile properties that we observed are due to something other than a change in the myosin isozymes. Furthermore, the maximal rate of tetanic tension development, which is presumably determined by the rate of cross-bridge attachment, was unaltered by curarization in the present study. Drachman and Johnston (6) demonstrated that changes in max dP/dt are well correlated with changes in the enzymatic activity of myosin of developing fast-twitch mammalian muscle. It can be inferred, therefore, that the ATPase activity of myosin in the curarized muscles is similar to that of control muscles and, therefore, is not responsible for the altered twitch kinetics that follow immobilization. Thus, support is given to the hypothesis that the altered isometric contractile properties of immobilized muscles are due not to changes in the contractile proteins but rather to some other factor(s), perhaps an alteration in the function of the sarcoplasmic reticulum. REFERENCES 1. CARLSON. F. D., AND D. R. WILKIE. 1974. Muscle Physi0log.v. Prentice-Hall, Englewood Cliffs, New Jersey. 2. CERNY, L. C., AND E. BANDMAN. 1986. Contractile activity is required for neonatal myosin heavy chain in embryonic chick pectoral muscle cultures. J. Cell Biol. 103: 2 153-2 16 1.
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