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Ca2+ capacity and uptake rate in skinned fibers of myodystrophic muscle
EXPERIMENTAL
87,
NEUROLOGY
137-146 (1985)
Ca2’ Capacity and Uptake Rate in Skinned Fibers of Myodystrophic Muscle BERTA. Department
of Physiology of Oklahoma,
Received
April
MOBLEY’
and Biophysics, College of Medicine, Oklahoma City, Oklahoma 73190
25. 1984: revision
received
September
University
10, 1984
Parameters related to the capacity and the rate of uptake of calcium ions by the sarcoplasmic reticulum were measured in skinned extensor digitorum longus fibers of control and myodystrophic mice. Single fibers were isolated and skinned in a relaxing solution and mounted on a force transducer and apparatus for changing the bathing solution (T = 25°C). To test the capacity of the sarcoplasmic reticulum, fibers were placed in a solution for maximal loading and then moved to a test solution in which the major anion in the relaxing solution, gluconate, was replaced by chloride. In the resulting contractures, the means of the forces produced by 10 control and myodystrophic fibers were not significantly different. The conclusion is that the capacities of sarcoplasmic reticulum for calcium in control and myodystrophic fibers are equivalent. To test the rate of loading of sarcoplasmic reticulum, 1 I control and myodystrophic fibers were depleted of calcium with caffeine and EGTA. Then they were placed in a solution with pCa = 5.5, and the delay before a contracture began was recorded. The delay was the time required for the sarcoplasmic reticulum to load calcium and attain a threshold for calcium-induced calcium release. The mean delay for the control fibers was significantly less than the mean delay in myodystrophic fibers. The disparity of loading times probably reflected a difference in the activities of the calcium pumps or a difference in the number of pump sites; 5 PM valinomycin did not significantly alter the loading times of either type or fiber. 0 1985 Academw
Press, Inc.
INTRODUCTION The myodystrophic mouse [Lane et al. (9)] is a relatively new preparation for studying muscular dystrophy in animals, and it may provide insights ’ I thank Dr. Arthur Peskoff for helpful discussions on the problem of diffusion in skinned fibers. I also thank Mr. John Welch for constructing the apparatus for changing the bathing solution of the skinned fibers. This work was supported by U.S. Public Health Service grant NS16989.
137 0014-4886185 $3.00 Copyright 0 1985 by Academic Press. Inc. All rights of reproductmn in any form reserved
138
BERT
A. MOBLEY
into the dystrophic diseases that afflict humans. Mobley et al. (11) investigated the contractile apparatus of myodystrophic mice and litter-mate controls by comparing the myofibrillar ATPase in muscles of the legs and the force produced in skinned fibers as a function of the free calcium concentration. They found no significant difference between the force produced in myodystrophic fibers and controls at given calcium concentrations, but the myodystrophic ATPase activity was found to be significantly smaller than the ATPase of control muscles. However, the investigators were unable to ascribe the difference in ATPase activity to a general defect in myodystrophic proteins because a histomorphometric study showed that the cross-sectional area of myodystrophic muscle contained a larger fraction of slow fibers than control muscles in that whereas the relative numbers of fast and slow fibers per muscle was unchanged with dystrophy, the fast fibers seemed to be atrophied more than the slow fibers. I studied the sarcoplasmic reticulum in skinned fibers from extensor digitorum longus muscles in myodystrophic and control mice. The investigation included a comparison of the capacity of the sarcoplasmic reticulum for calcium ions as judged indirectly by the force produced by chloride contractures in maximally loaded fibers. Also compared were the rates at which sarcoplasmic reticulum accumulated calcium ions; this too was deduced indirectly from the timed delay before calcium-induced calcium release produced a contracture in the fibers. Neymark et al. (12) investigated preparations of sarcoplasmic reticulum membranes in myodystrophic and control mice. They found that the basal ATPase of the myodystrophic sarcoplasmic reticulum was elevated compared with controls, but that the calcium-activated ATPase of the two types of muscles were equivalent. The results of this study indirectly indicate that the capacities of sarcoplasmic reticulum are equivalent but that myodystrophic skinned fibers accumulate calcium ions into the sarcoplasmic reticulum at a slower rate than control fibers. The potassium ionophore, valinomycin, was shown to have no significant effect on the uptake rate of calcium ions in either myodystrophic or control fibers. METHODS Single fibers were isolated from the extensor digitorum longus muscles of myodystrophic mice and their littermate controls (Jackson Laboratories, Bar Harbor, Me.). The myodystrophic mice were homozygous mutants, and the litter-mate controls were heterozygotes or homozygous normal. The phenotype was described by Lane et al. (9). The dissection was carried out in a relaxing solution (see Table 1). The solution was warmed to 30°C and bubbled with oxygen. The fibers were physically skinned in the relaxing
CALCIUM
IN SKINNED
MYODYSTROPHIC
TABLE
139
FIBERS
1
Solutions” Ca*+ Capacity experiments Car+ uptake rate experiments
K gluconate K Cl Mg ‘32 K2 EGTA K2 Ca EGTA Caffeine
Relaxing solution
Loading solution
Chloride contracture solution
94.8 7.1 0.5 -
93.2 7.1 OS9 0.41 -
94.8 7.1 0.5 -
Relaxing solution
Depletion solution
Ca*+ loading and releasing solution’
65.2 a.2 10.0 -
65.2 8.2 9.78 0.22 25.0
94.8 7.0 0.06 0.44 -
‘All concentrations are mM; all solutions were pH 7.0 and also contained Na2ATP, 3.3 mM; imidazole; 20 mM; creatine phosphate, 12 mM, creatine phosphokinase, 15 units/ml. b Valinomycin at 5 j1J4 was included in some of these solutions.
solution and then segments of about 1 mm in length were mounted on the force transducer. The experiments were conducted at 25°C. A capacitance force transducer (Photocon Systems, Arcadia, Calif.) and an apparatus for changing the solutions bathing the skinned fibers was mounted on the stage of a compound microscope (Leitz Dialux, West Germany) equipped with Hoffman Modulation Contrast Optics (McBain Instruments, Chatsworth, Calif.). The head of the microscope could be swung away from the stage to allow the segment to be mounted on the transducer, and when the head of the microscope was down, the sarcomere lengths in the segments were adjusted and measured and the diameters of the fibers were measured with a micrometer eyepiece (total magnifications 800 and 80X, respectively). The sarcomere length was determined by measuring the total length of 10 sarcomeres and dividing by 10. Release of calcium from the sarcoplasmic reticulum was manifested as force produced by the segments. In the set of experiments in which the peak force of contractures was measured, the waveform of the contractures was recorded on a Nicolet 1074 Instrument Computer (Madison, Wis.) and a Gould Brush 220 recorder (Cleveland, Ohio), and the peak force was read directly from the computer. In the other set of experiments in which the delay before the contracture was measured, the recorder was used and the times were measured from the chart paper. Free calcium concentrations in the different solutions were calculated with the computer program and binding constants from Fabiato and Fabiato (5). Because each skinned fiber probably differed in the relative fraction of
140
BERT
A. MOBLEY
myofibrils that were removed with the surface membrane, nonparametric tests were used to assess the significance of the differences between means. When means of parameters from different fibers were compared, the Mann-Whitney U test was employed. When means of parameters from the same fibers were compared, the Wilcoxon matched-pairs signed rank test was used. RESULTS Table 2 shows results from 10 control and 10 myodystrophic fibers which were tested to determine a maximal amount of calcium that could be accumulated by the sarcoplasmic reticulum in the two types of fibers. Each fiber was mounted on the transducer, the sarcomere length was set, and the diameter was measured while the segment was in relaxing solution. Then the segment was moved to the loading solution (&a = 6.5, see Table 1) where it remained for 20 min. Next the segment was moved to a solution which was identical to the relaxing solution except that the anion, gluconate, was replaced by chloride (Table 1). Such a change in ionic composition has been shown to release calcium ions from the sarcoplasmic reticulum in skinned fibers and to produce a contracture [(3); see Fig. 11, and the calcium released was shown to be a constant fraction of the total calcium in the saroplasmic reticulum prior to stimulation (15). The relative quantity of TABLE Capacity
of the Sarcoplasmic
2
Reticulum Determined from the Maximum Chloride Contractures of Skinned Fibers
Control Diameter (Pm) I.
51
2. 3. 4. 5. 6. 7. 8. 9. IO.
62 77 56 70 54 51 77 57 63
Mean
62
SD
10 3
SE
Force
Produced
Myodystrophic
Sarcomere length (rm)
Diameter (we
Sarcomere length (4
2.5 2.6 2.8 2.7 2.4 2.5 2.7 2.7 2.1 2.1
66 19 37 22 60 80 118 74 81 58
65 54 75 64 47 38 37 44 54 47
2.5 2.5 2.8 2.4 2.1 2.7 2.1 2.6 2.7 2.7
70 42 33 66 19 56 22 112 118 90
2.6 0.1 0.0
62 30 9
53 12 4
2.6 0. I 0.0
63 35 II
in
CALCIUM
IN SKINNED
MYODYSTROPHIC
FIBERS
141
0.2 mN
FIG. 1. A chloride contracture recorded from control fiber 8 in Table 2. The maximum force was 0.34 mN.
calcium ions loaded into the sarcoplasmic reticulum was judged from the magnitude of the maximum force produced by the segment during the contracture. However, to compensate for the different sizes of segments and their corresponding abilities to generate different amounts of force, absolute force was normalized by dividing it by the presumed circular cross-sectional area of the respective segment. Earlier experiments were carried out on four skinned fibers in which contractures were elicited by caffeine or chloride from the same segment after different periods of the loading solution. These experiments revealed that 10 min of loading was sufficient to produce a maximum contracture. However, to allow a significant safety margin, 20 min of loading was used in these experiments. Table 2 shows that the skinned myodystrophic fibers had a smaller mean diameter than the control fibers. A range of sarcomere lengths was used for the segments, but corresponding numbers of segments of each type were set at each different sarcomere length. Table 2 also shows that after the normalization procedure, the means of the contractures from the two types of segments were virtually identical, and it was concluded that the capacity of the sarcoplasmic reticulum in control and myodystrophic fibers is the same. Table 3 gives results from 11 control and 11 myodystropic fibers which were used to determine parameters related to the rate of calcium accumulation by the sarcoplasmic reticulum in skinned fibers. These skinned segments were mounted at a sarcomere length of 2.7 pm, and their diameters were measured while the segments were bathed in the relaxing solution. The segments were placed for 5 min in a relaxing solution to which 25 mM caffeine had been added (pCa = 8.0, see Table 1). This solution was designed to remove calcium from the sarcoplasmic reticulum in the skinned fibers (4) so that all fibers would begin accumulating calcium from a comparable state. Then the fibers were moved to relaxing solution for 1 min and then to a solution (&a = 5.5, see Table 1) which would allow the sarcoplasmic reticulum to load and later release calcium when a “threshold” level of loading was reached (4). The events were recorded on the chart recorder, and the time until the production of force was initiated
142
BERT A. MOBLEY TABLE 3 Calcium Uptake Rate of Sarcoplasmic Reticulum Determined by Calcium-Induced Calcium Release in Skinned Fibers
Control”
Myodystrophic’
Zero Time (s) Diameter Zero Time (s) Diamete? (pm*) Diameter Zero Time (s) Diamete? (pm2) (Valinomycin) (w@ bon’) Diamete? (rm*) I. 2. 3. 4. 5. 6. 7. 8. 9. 10. II. Mean
SD SE
Zero Time (s) Diamete? (pm’) (Valinomycin)
52 63 70 62 68 52 47 55 49 59 41
0.005 0.018 0.035 0.037 0.045 0.013 0.033 0.012 0.010 0.036 0.066
0.049 0.006 0.047 0.04 I 0.051 0.038 0.007 0.005 0.060 0.040 0.026
33 32 49 41 53 71 48 38 49 45 65
0.022 0.135 0.074 0.100 0.058 0.046 0.024 0.106 0.072 0.080 0.058
0.033 0.143 0.060 0.034 0.021 0.049 0.057 0.096 0.053 0.069 0.057
58 8 2
0.028 0.018 0.005
0.034 0.020 0.006
48 12 4
0.070 0.034 0.010
0.061 0.034
0.010
a Sarcomerelength of all fiberswas 2.7 pm.
(zero time) was measured. The zero time (see Fig. 2) was assumed to be inversely related to the level of activity of the calcium pump and/or the number of pump sites in the sarcoplasmic reticulum. The absolute zero times were quite variable, ranging from 0.24 to 4.1 min with a mean of 2.13 + 1.23 min. In order to normalize the process of diffusion of calcium
Force (mN)
o,ol
kzero
t I
“rl/
I 0
I 1
lime
(min.)
FIG. 2. An illustration of the term “zero time,” the elapsed time between the entry of the skinned fiber into the loading and releasing solution and the beginning of the development of force. The trace was taken from the experiment on myodystrophic fiber 7 in Table 3. The zero time for that fiber was 56 s. Experiments were oRen terminated, as in this case, before the maximum force developed: this was done to avoid possibly damaging the fiber prior to the experiment with valinomycin.
CALCIUM
IN SKINNED
MYODYSTROPHIC
FIBERS
143
into fibers of different diameters, the zero times were each divided by the squares of the measured diameters of the respective fibers; the resulting ratios obtained in control and myodystrophic fibers were then compared. The null hypothesis that control and myodystrophic fibers were independent samples drawn from populations having the same mean was rejected (P < 0.002). The zero times of the control fibers were therefore significantly less than the zero times of the myodystrophic fibers. This probably means that the myodystrophic fibers accumulated calcium ions at pCa = 5.5 into the sarcoplasmic reticulum at a slower rate than the control fibers if it is assumed that the two types of fibers had the same relative threshold of release (4). The effect of the potassium ionophore, valinomycin, on the zero times of control and myodystrophic fibers was determined by repeating the procedure used to obtain the first contracture in each fiber in Table 3. The solution used to elicit the second contracture was the same as that used to elicit the first except for the addition of 5 PM valinomycin (see Table 1). The results are shown in Table 3. Valinomycin did not cause a significant change in the zero times of control or myodystrophic fibers. DISCUSSION The experiments showed that chloride contractures elicited in maximally loaded skinned fibers of myodystrophic and control mice produced equivalent magnitudes of force. The indirect conclusion from these experiments is that the sarcoplasmic reticulum in both myodystrophic and control fibers has equivalent capacities for calcium ions. A critical assumption for this conclusion is that a given concentration of calcium will produce an equivalent force in both types of fibers. This assumption was found to be valid by Mobley et al. (1 l), who tested both types of skinned fibers in solutions of different buffered calcium concentrations. The conclusion above is also based on the work of Endo (4), who showed that chloride contractures of the type elicited in these experiments release a constant fraction of the amount of calcium that was stored in the sarcoplasmic reticulum prior to release. An assumption that must be made, therefore, is that the fraction of total calcium that was released in the chloride contractures was the same for both control and myodystrophic fibers. It is unlikely that a difference in maximum loading of the two types of fibers would be exactly compensated by a difference in the fraction of total calcium released in one type of fiber to produce the results of Table 2. The second set of experiments provided indirect evidence that the turnover or activity of the calcium pump in the sarcoplasmic reticulum of
144
BERT
A. MOBLEY
myodystrophic fibers was less than that in the control fibers or that the number of pump sites was less. The fibers were depleted of calcium with caffeine and EGTA, and then they were placed in a lightly buffered loading and releasing solution with pCa = 5.5. That concentration of calcium was sufficient to release calcium by the mechanism known as calcium-induced calcium release; however, first the sarcoplasmic reticulum in the fibers had to load a threshold concentration of calcium (4). The delay between the time the depleted fiber entered the loading and releasing solution and the beginning of the regenerative release of calcium, as evidenced by the rapid rise in force (see Fig. 2), must bear a relation to the rapidity of calcium loading of the sarcoplasmic reticulum and hence to the activity of the pump and/or the density of pump sites. Ford and Podolsky (6, 7) investigated and explained the delay in the production of force as being due to diffusion and uptake of calcium by the sarcoplasmic reticulum and the eventual rise in force as being due to calcium-induced calcium release. An implicit assumption in the conclusion relating to the zero time to the calcium pump is that the threshold concentrations for release of calcium are the same for both myodystrophic and control fibers. If this latter assumption is not valid, then the conclusion from these experiments would become more complicated and involve not only conclusions about calcium uptake by the pump, but about calcium release by the calcium-induced release mechanism. It should also be noted that in these experiments the loading and release occurred at pCa = 5.5. Investigations at other concentrations of calcium might conceivably produce different results. Mobley et al. (11) found that approximately 70% of the fibers in these control extensor digitorum longus muscles were fast fibers, and that that fraction was unchanged in dystrophic muscles. Therefore, while it is surely true that some of the fibers investigated in this study were slow fibers which had a reduced calcium uptake rate compared with fast fibers (l), there is no reason to assume that more slow fibers were isolated for skinning from myodystrophic muscles than were isolated from the control muscles. Duggan (2) found that the concentration of potassium in the bathing solution was a factor in the uptake rate of the calcium pump of sarcoplasmic reticulum. Although Duggan’s observation does not necessarily mean that potassium is transported during the uptake of calcium, such transport was considered. Somlyo et al. (13) showed that potassium was taken up by the sarcoplasmic reticulum during a tetanus, and Kitazawa et al. (8) showed that the potassium ionophore, valinomycin, increased the uptake of potassium during a tetanus, although it had no apparent effect on the calcium that was released or on the calcium, potassium, or other ionic concentrations in the terminal cisternae of resting muscles.
CALCIUM
IN
SKINNED
MYODYSTROPHIC
FIBERS
145
Calcium-induced contractures were elicited in the presence of valinomycin to test whether increased potassium permeability of the sarcoplasmic reticulum membrane would appear to affect the activity of the calcium pump. As the addition of valinomycin caused no significant change in the zero times of either type of fiber, it was concluded that potassium permeability and the transport of potassium is not a limiting factor in the rate of calcium uptake in skinned fibers. An explanation for the apparent difference in these results on skinned fibers and those of Neymark et al. ( 12) on the ATPase activity of sarcoplasmic reticulum membranes is not obvious. Neymark et al. obtained qualitatively consistent results at both 4 and 30°C. The concentration of calcium in their experiments was 0.1 mM, whereas the sarcoplasmic reticulum in the skinned fibers of this study were loaded at a calcium concentration of 3 PM. Some other investigations of sarcoplasmic reticulum in other types of dystrophic mice ( 10, 14) showed that the rate of calcium uptake was less in dystrophic sarcoplasmic reticulum than in controls. This work furnishes independent physiologic support to those direct biochemical investigations. REFERENCES 1. BRIGGS,F. N., J. L. POLAND, AND R. J. SOLARO. 1977. Relative capabilities ofsarcoplasmic reticulum in fast and slow mammalian skeletal muscles. J. Phyyiol. (London) 266: 587594.
DUGGAN, P. F. 1977. Ca uptake and associated ATPase activity in fragmented sarcoplasmic reticulum-requirement for potassium ions. J. Biol. Chem. 252: 1620-1627. 3. ENDO, M., AND J. R. BLINKS. 1973. Inconstant association of aequotin luminescence with tension during calcium release in skinned muscle fibres. Nature (London) 246: 2 IS-22 1. 4. ENDO, M. 1977. Calcium release from the sarcoplasmic reticulum. Physiol. Rev. 57: 7l2.
108.
5. FABIATO, A., AND F. FABIATO. 1979. Calculator programs for computing the composition of the solutions containing multiple metals and ligands used for experiments in skinned muscle cells. J. Physiol. (Paris) 75: 463-505. 6. FORD, L. E., AND R. J. PODOLSKY. 1972. Calcium uptake and force development by skinned muscle fibres in EGTA buffered solutions. J. Physiol. (London) 223: l-19. 7. FORD, L. E., AND R. J. PODOLSKY. 1972. Intracellular calcium movements in skinned muscle fibres. J. Physiol. (London) 223: 2 l-33. 8. KITAZAWA, T., A. P. SOMLYO, AND A. V. SOMLYO. 1984. The effects of valinomycin on ion movements across the sarcoplasmic reticulum in frog muscle. J. Physiol (London) 350: 253-268.
LANE, P. W., T. C. BEAMER, AND D. D. MYERS. 1976. Myodystrophy, a new myopathy on chromosome 8 of the mouse. J. Hered. 67: 135-138. 10. MARTONOSI, A. 1968. Sarcoplasmic reticulum. VI. Microsomal Ca*’ transport in genetic muscular dystrophy of mice. Proc. Sot. Exp. Biol. Med. 127: 824-828. 9.
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Il. MOBLEY, B. A., Y. S. REDDY, D. P. FEEBACK, J. B. BODENSTEINER,M. BOKHARI, R. D. ROBINSON,AND R. CLARK. 1985. Control of myofibrillar ATPase activity and force in myodystrophic muscle. Muscle Nerve (In press). 12. NEYMARK, M. A., S. J. KOPACZ, AND CH-P. LEE. 1980. Characterization of ATPase in sarcoplasmic reticulum from two strains of dystrophic mice. Muscle Nerve 3: 3 16-325. 13. SOMLYO, A. V., H. GONZALEZ-SERRATOS, H. SHUMAN, G. MCCLELLAN, AND A. P. SOMLYO. 198 1. Calcium release and ionic changes in the sarcoplasmic reticulum of tetanized muscle: an electron probe study. J. Cell. Biol. 90: 517-594. 14. SRETER, F. A., N. IKEMOTO, AND J. GERGELY. 1967. Studies on the fragmented sarcoplasmic reticulum of normal and dystrophic mouse muscle. Pages 289-298, A. T. MILHORAT, Ed., in Exploratory Concepts in Muscular Dystrophy and Related Disorders. Edited by Excerpta Medica, Amsterdam, New York. 15. THORENS, S., AND M. ENDO. 1975. Calcium-induced calcium release and “depolarization”induced calcium release: their physiological significance. Proc. Japan Acad. 51: 473478.
87,
NEUROLOGY
137-146 (1985)
Ca2’ Capacity and Uptake Rate in Skinned Fibers of Myodystrophic Muscle BERTA. Department
of Physiology of Oklahoma,
Received
April
MOBLEY’
and Biophysics, College of Medicine, Oklahoma City, Oklahoma 73190
25. 1984: revision
received
September
University
10, 1984
Parameters related to the capacity and the rate of uptake of calcium ions by the sarcoplasmic reticulum were measured in skinned extensor digitorum longus fibers of control and myodystrophic mice. Single fibers were isolated and skinned in a relaxing solution and mounted on a force transducer and apparatus for changing the bathing solution (T = 25°C). To test the capacity of the sarcoplasmic reticulum, fibers were placed in a solution for maximal loading and then moved to a test solution in which the major anion in the relaxing solution, gluconate, was replaced by chloride. In the resulting contractures, the means of the forces produced by 10 control and myodystrophic fibers were not significantly different. The conclusion is that the capacities of sarcoplasmic reticulum for calcium in control and myodystrophic fibers are equivalent. To test the rate of loading of sarcoplasmic reticulum, 1 I control and myodystrophic fibers were depleted of calcium with caffeine and EGTA. Then they were placed in a solution with pCa = 5.5, and the delay before a contracture began was recorded. The delay was the time required for the sarcoplasmic reticulum to load calcium and attain a threshold for calcium-induced calcium release. The mean delay for the control fibers was significantly less than the mean delay in myodystrophic fibers. The disparity of loading times probably reflected a difference in the activities of the calcium pumps or a difference in the number of pump sites; 5 PM valinomycin did not significantly alter the loading times of either type or fiber. 0 1985 Academw
Press, Inc.
INTRODUCTION The myodystrophic mouse [Lane et al. (9)] is a relatively new preparation for studying muscular dystrophy in animals, and it may provide insights ’ I thank Dr. Arthur Peskoff for helpful discussions on the problem of diffusion in skinned fibers. I also thank Mr. John Welch for constructing the apparatus for changing the bathing solution of the skinned fibers. This work was supported by U.S. Public Health Service grant NS16989.
137 0014-4886185 $3.00 Copyright 0 1985 by Academic Press. Inc. All rights of reproductmn in any form reserved
138
BERT
A. MOBLEY
into the dystrophic diseases that afflict humans. Mobley et al. (11) investigated the contractile apparatus of myodystrophic mice and litter-mate controls by comparing the myofibrillar ATPase in muscles of the legs and the force produced in skinned fibers as a function of the free calcium concentration. They found no significant difference between the force produced in myodystrophic fibers and controls at given calcium concentrations, but the myodystrophic ATPase activity was found to be significantly smaller than the ATPase of control muscles. However, the investigators were unable to ascribe the difference in ATPase activity to a general defect in myodystrophic proteins because a histomorphometric study showed that the cross-sectional area of myodystrophic muscle contained a larger fraction of slow fibers than control muscles in that whereas the relative numbers of fast and slow fibers per muscle was unchanged with dystrophy, the fast fibers seemed to be atrophied more than the slow fibers. I studied the sarcoplasmic reticulum in skinned fibers from extensor digitorum longus muscles in myodystrophic and control mice. The investigation included a comparison of the capacity of the sarcoplasmic reticulum for calcium ions as judged indirectly by the force produced by chloride contractures in maximally loaded fibers. Also compared were the rates at which sarcoplasmic reticulum accumulated calcium ions; this too was deduced indirectly from the timed delay before calcium-induced calcium release produced a contracture in the fibers. Neymark et al. (12) investigated preparations of sarcoplasmic reticulum membranes in myodystrophic and control mice. They found that the basal ATPase of the myodystrophic sarcoplasmic reticulum was elevated compared with controls, but that the calcium-activated ATPase of the two types of muscles were equivalent. The results of this study indirectly indicate that the capacities of sarcoplasmic reticulum are equivalent but that myodystrophic skinned fibers accumulate calcium ions into the sarcoplasmic reticulum at a slower rate than control fibers. The potassium ionophore, valinomycin, was shown to have no significant effect on the uptake rate of calcium ions in either myodystrophic or control fibers. METHODS Single fibers were isolated from the extensor digitorum longus muscles of myodystrophic mice and their littermate controls (Jackson Laboratories, Bar Harbor, Me.). The myodystrophic mice were homozygous mutants, and the litter-mate controls were heterozygotes or homozygous normal. The phenotype was described by Lane et al. (9). The dissection was carried out in a relaxing solution (see Table 1). The solution was warmed to 30°C and bubbled with oxygen. The fibers were physically skinned in the relaxing
CALCIUM
IN SKINNED
MYODYSTROPHIC
TABLE
139
FIBERS
1
Solutions” Ca*+ Capacity experiments Car+ uptake rate experiments
K gluconate K Cl Mg ‘32 K2 EGTA K2 Ca EGTA Caffeine
Relaxing solution
Loading solution
Chloride contracture solution
94.8 7.1 0.5 -
93.2 7.1 OS9 0.41 -
94.8 7.1 0.5 -
Relaxing solution
Depletion solution
Ca*+ loading and releasing solution’
65.2 a.2 10.0 -
65.2 8.2 9.78 0.22 25.0
94.8 7.0 0.06 0.44 -
‘All concentrations are mM; all solutions were pH 7.0 and also contained Na2ATP, 3.3 mM; imidazole; 20 mM; creatine phosphate, 12 mM, creatine phosphokinase, 15 units/ml. b Valinomycin at 5 j1J4 was included in some of these solutions.
solution and then segments of about 1 mm in length were mounted on the force transducer. The experiments were conducted at 25°C. A capacitance force transducer (Photocon Systems, Arcadia, Calif.) and an apparatus for changing the solutions bathing the skinned fibers was mounted on the stage of a compound microscope (Leitz Dialux, West Germany) equipped with Hoffman Modulation Contrast Optics (McBain Instruments, Chatsworth, Calif.). The head of the microscope could be swung away from the stage to allow the segment to be mounted on the transducer, and when the head of the microscope was down, the sarcomere lengths in the segments were adjusted and measured and the diameters of the fibers were measured with a micrometer eyepiece (total magnifications 800 and 80X, respectively). The sarcomere length was determined by measuring the total length of 10 sarcomeres and dividing by 10. Release of calcium from the sarcoplasmic reticulum was manifested as force produced by the segments. In the set of experiments in which the peak force of contractures was measured, the waveform of the contractures was recorded on a Nicolet 1074 Instrument Computer (Madison, Wis.) and a Gould Brush 220 recorder (Cleveland, Ohio), and the peak force was read directly from the computer. In the other set of experiments in which the delay before the contracture was measured, the recorder was used and the times were measured from the chart paper. Free calcium concentrations in the different solutions were calculated with the computer program and binding constants from Fabiato and Fabiato (5). Because each skinned fiber probably differed in the relative fraction of
140
BERT
A. MOBLEY
myofibrils that were removed with the surface membrane, nonparametric tests were used to assess the significance of the differences between means. When means of parameters from different fibers were compared, the Mann-Whitney U test was employed. When means of parameters from the same fibers were compared, the Wilcoxon matched-pairs signed rank test was used. RESULTS Table 2 shows results from 10 control and 10 myodystrophic fibers which were tested to determine a maximal amount of calcium that could be accumulated by the sarcoplasmic reticulum in the two types of fibers. Each fiber was mounted on the transducer, the sarcomere length was set, and the diameter was measured while the segment was in relaxing solution. Then the segment was moved to the loading solution (&a = 6.5, see Table 1) where it remained for 20 min. Next the segment was moved to a solution which was identical to the relaxing solution except that the anion, gluconate, was replaced by chloride (Table 1). Such a change in ionic composition has been shown to release calcium ions from the sarcoplasmic reticulum in skinned fibers and to produce a contracture [(3); see Fig. 11, and the calcium released was shown to be a constant fraction of the total calcium in the saroplasmic reticulum prior to stimulation (15). The relative quantity of TABLE Capacity
of the Sarcoplasmic
2
Reticulum Determined from the Maximum Chloride Contractures of Skinned Fibers
Control Diameter (Pm) I.
51
2. 3. 4. 5. 6. 7. 8. 9. IO.
62 77 56 70 54 51 77 57 63
Mean
62
SD
10 3
SE
Force
Produced
Myodystrophic
Sarcomere length (rm)
Diameter (we
Sarcomere length (4
2.5 2.6 2.8 2.7 2.4 2.5 2.7 2.7 2.1 2.1
66 19 37 22 60 80 118 74 81 58
65 54 75 64 47 38 37 44 54 47
2.5 2.5 2.8 2.4 2.1 2.7 2.1 2.6 2.7 2.7
70 42 33 66 19 56 22 112 118 90
2.6 0.1 0.0
62 30 9
53 12 4
2.6 0. I 0.0
63 35 II
in
CALCIUM
IN SKINNED
MYODYSTROPHIC
FIBERS
141
0.2 mN
FIG. 1. A chloride contracture recorded from control fiber 8 in Table 2. The maximum force was 0.34 mN.
calcium ions loaded into the sarcoplasmic reticulum was judged from the magnitude of the maximum force produced by the segment during the contracture. However, to compensate for the different sizes of segments and their corresponding abilities to generate different amounts of force, absolute force was normalized by dividing it by the presumed circular cross-sectional area of the respective segment. Earlier experiments were carried out on four skinned fibers in which contractures were elicited by caffeine or chloride from the same segment after different periods of the loading solution. These experiments revealed that 10 min of loading was sufficient to produce a maximum contracture. However, to allow a significant safety margin, 20 min of loading was used in these experiments. Table 2 shows that the skinned myodystrophic fibers had a smaller mean diameter than the control fibers. A range of sarcomere lengths was used for the segments, but corresponding numbers of segments of each type were set at each different sarcomere length. Table 2 also shows that after the normalization procedure, the means of the contractures from the two types of segments were virtually identical, and it was concluded that the capacity of the sarcoplasmic reticulum in control and myodystrophic fibers is the same. Table 3 gives results from 11 control and 11 myodystropic fibers which were used to determine parameters related to the rate of calcium accumulation by the sarcoplasmic reticulum in skinned fibers. These skinned segments were mounted at a sarcomere length of 2.7 pm, and their diameters were measured while the segments were bathed in the relaxing solution. The segments were placed for 5 min in a relaxing solution to which 25 mM caffeine had been added (pCa = 8.0, see Table 1). This solution was designed to remove calcium from the sarcoplasmic reticulum in the skinned fibers (4) so that all fibers would begin accumulating calcium from a comparable state. Then the fibers were moved to relaxing solution for 1 min and then to a solution (&a = 5.5, see Table 1) which would allow the sarcoplasmic reticulum to load and later release calcium when a “threshold” level of loading was reached (4). The events were recorded on the chart recorder, and the time until the production of force was initiated
142
BERT A. MOBLEY TABLE 3 Calcium Uptake Rate of Sarcoplasmic Reticulum Determined by Calcium-Induced Calcium Release in Skinned Fibers
Control”
Myodystrophic’
Zero Time (s) Diameter Zero Time (s) Diamete? (pm*) Diameter Zero Time (s) Diamete? (pm2) (Valinomycin) (w@ bon’) Diamete? (rm*) I. 2. 3. 4. 5. 6. 7. 8. 9. 10. II. Mean
SD SE
Zero Time (s) Diamete? (pm’) (Valinomycin)
52 63 70 62 68 52 47 55 49 59 41
0.005 0.018 0.035 0.037 0.045 0.013 0.033 0.012 0.010 0.036 0.066
0.049 0.006 0.047 0.04 I 0.051 0.038 0.007 0.005 0.060 0.040 0.026
33 32 49 41 53 71 48 38 49 45 65
0.022 0.135 0.074 0.100 0.058 0.046 0.024 0.106 0.072 0.080 0.058
0.033 0.143 0.060 0.034 0.021 0.049 0.057 0.096 0.053 0.069 0.057
58 8 2
0.028 0.018 0.005
0.034 0.020 0.006
48 12 4
0.070 0.034 0.010
0.061 0.034
0.010
a Sarcomerelength of all fiberswas 2.7 pm.
(zero time) was measured. The zero time (see Fig. 2) was assumed to be inversely related to the level of activity of the calcium pump and/or the number of pump sites in the sarcoplasmic reticulum. The absolute zero times were quite variable, ranging from 0.24 to 4.1 min with a mean of 2.13 + 1.23 min. In order to normalize the process of diffusion of calcium
Force (mN)
o,ol
kzero
t I
“rl/
I 0
I 1
lime
(min.)
FIG. 2. An illustration of the term “zero time,” the elapsed time between the entry of the skinned fiber into the loading and releasing solution and the beginning of the development of force. The trace was taken from the experiment on myodystrophic fiber 7 in Table 3. The zero time for that fiber was 56 s. Experiments were oRen terminated, as in this case, before the maximum force developed: this was done to avoid possibly damaging the fiber prior to the experiment with valinomycin.
CALCIUM
IN SKINNED
MYODYSTROPHIC
FIBERS
143
into fibers of different diameters, the zero times were each divided by the squares of the measured diameters of the respective fibers; the resulting ratios obtained in control and myodystrophic fibers were then compared. The null hypothesis that control and myodystrophic fibers were independent samples drawn from populations having the same mean was rejected (P < 0.002). The zero times of the control fibers were therefore significantly less than the zero times of the myodystrophic fibers. This probably means that the myodystrophic fibers accumulated calcium ions at pCa = 5.5 into the sarcoplasmic reticulum at a slower rate than the control fibers if it is assumed that the two types of fibers had the same relative threshold of release (4). The effect of the potassium ionophore, valinomycin, on the zero times of control and myodystrophic fibers was determined by repeating the procedure used to obtain the first contracture in each fiber in Table 3. The solution used to elicit the second contracture was the same as that used to elicit the first except for the addition of 5 PM valinomycin (see Table 1). The results are shown in Table 3. Valinomycin did not cause a significant change in the zero times of control or myodystrophic fibers. DISCUSSION The experiments showed that chloride contractures elicited in maximally loaded skinned fibers of myodystrophic and control mice produced equivalent magnitudes of force. The indirect conclusion from these experiments is that the sarcoplasmic reticulum in both myodystrophic and control fibers has equivalent capacities for calcium ions. A critical assumption for this conclusion is that a given concentration of calcium will produce an equivalent force in both types of fibers. This assumption was found to be valid by Mobley et al. (1 l), who tested both types of skinned fibers in solutions of different buffered calcium concentrations. The conclusion above is also based on the work of Endo (4), who showed that chloride contractures of the type elicited in these experiments release a constant fraction of the amount of calcium that was stored in the sarcoplasmic reticulum prior to release. An assumption that must be made, therefore, is that the fraction of total calcium that was released in the chloride contractures was the same for both control and myodystrophic fibers. It is unlikely that a difference in maximum loading of the two types of fibers would be exactly compensated by a difference in the fraction of total calcium released in one type of fiber to produce the results of Table 2. The second set of experiments provided indirect evidence that the turnover or activity of the calcium pump in the sarcoplasmic reticulum of
144
BERT
A. MOBLEY
myodystrophic fibers was less than that in the control fibers or that the number of pump sites was less. The fibers were depleted of calcium with caffeine and EGTA, and then they were placed in a lightly buffered loading and releasing solution with pCa = 5.5. That concentration of calcium was sufficient to release calcium by the mechanism known as calcium-induced calcium release; however, first the sarcoplasmic reticulum in the fibers had to load a threshold concentration of calcium (4). The delay between the time the depleted fiber entered the loading and releasing solution and the beginning of the regenerative release of calcium, as evidenced by the rapid rise in force (see Fig. 2), must bear a relation to the rapidity of calcium loading of the sarcoplasmic reticulum and hence to the activity of the pump and/or the density of pump sites. Ford and Podolsky (6, 7) investigated and explained the delay in the production of force as being due to diffusion and uptake of calcium by the sarcoplasmic reticulum and the eventual rise in force as being due to calcium-induced calcium release. An implicit assumption in the conclusion relating to the zero time to the calcium pump is that the threshold concentrations for release of calcium are the same for both myodystrophic and control fibers. If this latter assumption is not valid, then the conclusion from these experiments would become more complicated and involve not only conclusions about calcium uptake by the pump, but about calcium release by the calcium-induced release mechanism. It should also be noted that in these experiments the loading and release occurred at pCa = 5.5. Investigations at other concentrations of calcium might conceivably produce different results. Mobley et al. (11) found that approximately 70% of the fibers in these control extensor digitorum longus muscles were fast fibers, and that that fraction was unchanged in dystrophic muscles. Therefore, while it is surely true that some of the fibers investigated in this study were slow fibers which had a reduced calcium uptake rate compared with fast fibers (l), there is no reason to assume that more slow fibers were isolated for skinning from myodystrophic muscles than were isolated from the control muscles. Duggan (2) found that the concentration of potassium in the bathing solution was a factor in the uptake rate of the calcium pump of sarcoplasmic reticulum. Although Duggan’s observation does not necessarily mean that potassium is transported during the uptake of calcium, such transport was considered. Somlyo et al. (13) showed that potassium was taken up by the sarcoplasmic reticulum during a tetanus, and Kitazawa et al. (8) showed that the potassium ionophore, valinomycin, increased the uptake of potassium during a tetanus, although it had no apparent effect on the calcium that was released or on the calcium, potassium, or other ionic concentrations in the terminal cisternae of resting muscles.
CALCIUM
IN
SKINNED
MYODYSTROPHIC
FIBERS
145
Calcium-induced contractures were elicited in the presence of valinomycin to test whether increased potassium permeability of the sarcoplasmic reticulum membrane would appear to affect the activity of the calcium pump. As the addition of valinomycin caused no significant change in the zero times of either type of fiber, it was concluded that potassium permeability and the transport of potassium is not a limiting factor in the rate of calcium uptake in skinned fibers. An explanation for the apparent difference in these results on skinned fibers and those of Neymark et al. ( 12) on the ATPase activity of sarcoplasmic reticulum membranes is not obvious. Neymark et al. obtained qualitatively consistent results at both 4 and 30°C. The concentration of calcium in their experiments was 0.1 mM, whereas the sarcoplasmic reticulum in the skinned fibers of this study were loaded at a calcium concentration of 3 PM. Some other investigations of sarcoplasmic reticulum in other types of dystrophic mice ( 10, 14) showed that the rate of calcium uptake was less in dystrophic sarcoplasmic reticulum than in controls. This work furnishes independent physiologic support to those direct biochemical investigations. REFERENCES 1. BRIGGS,F. N., J. L. POLAND, AND R. J. SOLARO. 1977. Relative capabilities ofsarcoplasmic reticulum in fast and slow mammalian skeletal muscles. J. Phyyiol. (London) 266: 587594.
DUGGAN, P. F. 1977. Ca uptake and associated ATPase activity in fragmented sarcoplasmic reticulum-requirement for potassium ions. J. Biol. Chem. 252: 1620-1627. 3. ENDO, M., AND J. R. BLINKS. 1973. Inconstant association of aequotin luminescence with tension during calcium release in skinned muscle fibres. Nature (London) 246: 2 IS-22 1. 4. ENDO, M. 1977. Calcium release from the sarcoplasmic reticulum. Physiol. Rev. 57: 7l2.
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5. FABIATO, A., AND F. FABIATO. 1979. Calculator programs for computing the composition of the solutions containing multiple metals and ligands used for experiments in skinned muscle cells. J. Physiol. (Paris) 75: 463-505. 6. FORD, L. E., AND R. J. PODOLSKY. 1972. Calcium uptake and force development by skinned muscle fibres in EGTA buffered solutions. J. Physiol. (London) 223: l-19. 7. FORD, L. E., AND R. J. PODOLSKY. 1972. Intracellular calcium movements in skinned muscle fibres. J. Physiol. (London) 223: 2 l-33. 8. KITAZAWA, T., A. P. SOMLYO, AND A. V. SOMLYO. 1984. The effects of valinomycin on ion movements across the sarcoplasmic reticulum in frog muscle. J. Physiol (London) 350: 253-268.
LANE, P. W., T. C. BEAMER, AND D. D. MYERS. 1976. Myodystrophy, a new myopathy on chromosome 8 of the mouse. J. Hered. 67: 135-138. 10. MARTONOSI, A. 1968. Sarcoplasmic reticulum. VI. Microsomal Ca*’ transport in genetic muscular dystrophy of mice. Proc. Sot. Exp. Biol. Med. 127: 824-828. 9.
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Il. MOBLEY, B. A., Y. S. REDDY, D. P. FEEBACK, J. B. BODENSTEINER,M. BOKHARI, R. D. ROBINSON,AND R. CLARK. 1985. Control of myofibrillar ATPase activity and force in myodystrophic muscle. Muscle Nerve (In press). 12. NEYMARK, M. A., S. J. KOPACZ, AND CH-P. LEE. 1980. Characterization of ATPase in sarcoplasmic reticulum from two strains of dystrophic mice. Muscle Nerve 3: 3 16-325. 13. SOMLYO, A. V., H. GONZALEZ-SERRATOS, H. SHUMAN, G. MCCLELLAN, AND A. P. SOMLYO. 198 1. Calcium release and ionic changes in the sarcoplasmic reticulum of tetanized muscle: an electron probe study. J. Cell. Biol. 90: 517-594. 14. SRETER, F. A., N. IKEMOTO, AND J. GERGELY. 1967. Studies on the fragmented sarcoplasmic reticulum of normal and dystrophic mouse muscle. Pages 289-298, A. T. MILHORAT, Ed., in Exploratory Concepts in Muscular Dystrophy and Related Disorders. Edited by Excerpta Medica, Amsterdam, New York. 15. THORENS, S., AND M. ENDO. 1975. Calcium-induced calcium release and “depolarization”induced calcium release: their physiological significance. Proc. Japan Acad. 51: 473478.