- Email: [email protected]
Membrane cable properties of normal and dystrophic chicken muscle fibers
EXPERIMENTAL
NEUROLOGY
Membrane
47,
Cable
544-557
(lws)
Properties of Normal Chicken Muscle Fibers
and
Dystrophic
F. J. LEBEDA AND E. X. ALBUQUERQUE* Defiartment of Pharmacology avid Experimental Therapeutics, University Maryland School of Medicine, Baltimore, Maryland 21201 Received
February
of
5, 1975
Values of fiber radius obtained by square pulse analysis and histological measurements indicated that the innervated dystrophic fibers which had been examined with microelectrodes were hypertrophic. The specific membrane resistance of dystrophic fibers was also greater than normal. In addition, experimentally induced compensatory hypertrophy of innervated nondystrophic (normal) fibers of the posterior latissimus dorsi led to alterations in several membrane characteristics which resulted in values resembling those of innervated dystrophic fibers. Twenty-one days after denervation, the values for the cable properties of normal and dystrophic fibers were increased, yet similar values were attained for the space constant, specific membrane resistance, and membrane capacitance. In both normal and dystrophic muscles which were denervated for 21 days the fiber radius decreased 40%. To study the mechanism underlying the increase of the specific membrane resistance after denervation, the resting membrane conductance was selectively altered. In solutions of low pH (5.0) where chloride conductance was presumably reduced, the space constant, time constant and specific membrane resistance of innervated normal and dystrophic fibers were increased and approached values obtained from al-day denervated muscles. In contrast, solutions of low pH had no marked effects on 21-day denervated normal and dystrophic muscles. It is suggested that the increased values for these cable properties from denervated normal and dpstrophic posterior latissimus dorsi muscles may be partially due to reduced potassium and chloride conductances. Furthermore, the presence of hypertorphic fibers may be a significant morphological adaptation in dystrophic muscles. 1 Send reprint requests to Dr. Edson X. Albuquerque, Department of Pharmacology and Experimental Therapeutics, University of Maryland, School of Medicine, Baltimore, MD 21201. A preliminary report of these results was presented at the Fifth Annual Meeting for the Society for Neuroscience, St. Louis, MO, 1974 (18). This work was supported by U.S. Public Health Service Grant NS-12063. The authors are grateful to Miss Mabel A. Zelle for excellent technical and secretarial assistance during the course of this study. 544 Copyright 0 All
rights
of
1975
by Academic
Press,
reproduction in any form
Inc. reserved.
DYSTROPHIC
MUSCLE
545
INTRODUCTION Skeletal muscle provides a convenient model for studying the ionic conductances of electrogenic membranes. In frog sartorius muscles, about twothirds of the total membrane conductance is due to chloride ions traversing the membrane; the other one-third is due mostly to potassium ions (10). At rest, most chloride ions cross only at the sarcolemma while potassium ions go through both the sarcolemmal and the transverse-tubule membranes (5). The changes occurring postsynaptically after denervation of mammalian “fast” skeletal muscles include depolarization, extrajunctional acetycholine sensitivity, tetrodotoxin-resistant action potentials, and increased specific membrane resistance (1,2,4). The increase of specific membrane resistance following denervation cannot be attributed to muscle inactivity since innervated mammalian muscles subjected to chronic immobilization do not exhibit changes in this membrane property (7). Upon denervation of frog muscle preparations, the specific membrane resistance increased less than twofold (12) ; in denervated rat muscles resistance increased threefold (1)) but preliminary experiments with denervated chicken muscles revealed a fivefold increase (18). Assuming that a decrease in potassium conductance occurred in denervated chicken muscle as in denervated rat muscle, another ion, possibly chloride, would have to contribute significantly to the total decrease in membrane conductance. The objective of the present investigation is to estimate the contributions of potassium and chloride ions to the total membrane conductance in normal and dystrophic, innervated and chronically denervated posterior latissimus dorsi of the chicken. In addition, the changes in the cable properties occurring in normal fibers induced to become hypertrophic were observed in order to obtain further information regarding the large fibers encountered in dystrophic chicken muscles. METHODS Animals and Prepurations. Both normal (line 200) and genetically dystrophic (line 304) New Hampshire chickens were purchased from the University of California at Davis. Animals between the ages of 10 to 20 weeks ex OZJO were used in all of the experiments. The “fast” posterior latissimus dorsi muscles were used for this study; the denervation procedure has been described by Lebeda, Warnick and Albuquerque (19). Contralateral muscles served either as intact or sham operated controls. To induce fibers of the posterior latissimus dorsi muscle to hypertrophy. several muscles of the wing were denervated. An incision was made along the dorsal surface, 5 cm rostra1 to the anterior latissimus dorsi. The two largest and most dorsal branches of the brachial nerve complex were exposed ; the medial nerve was doubly ligated, and a 1 cm portion
546
LEBEDA
AND
ALBUQUERQUE
was removed. During this procedure the surrounding vasculature remained intact. Upon recovery, pronounced wing droop was noted and was still evident at the time of autopsy up to two weeks later. The animals were killed 1 to 21 days after denervation by cervical dislocation, decapitation, and exsaguination. Muscles were carefully removed under a continuous flow of physiological solution. Individual muscles were mounted at approximately their in situ length by stainless steel insect pins to a paraffin lined Plexiglass plate in the center of which was a planoconvex lens. Recording and Stimulating Techniques. Glass microelectrodes filled with 3 M KC1 and having tip resistances between 15-20 Mohm were used for recording and intracellular stimulation. Conventional methods for square pulse analysis were used ( 1, 6). The stimulating electrode was usually placed 50-100, 300, 600 and 1000 pm away from the recording electrode. At each distance, several anodal current pulses with durations of 300 msec were passed through the stimulating electrode to hyperpolarize the membrane by 5-10, 15-20 or 25-30 mv. All voltage and current displacements were monitored on a Mingograf 81 polygraph, a Tektr,onix 565 oscilloscope and recorded on film with a Grass model 4 kymograph camera. The logarithm of the voltage to current ratio was plotted against the interelectrode distance. On semilogarithmic coordinates, at least three of the four points had to fall on a straight line, otherwise the data from a particular fiber were discarded. The internal resistivity of the myoplasm (Ri) was assumed to be 160 Ohm cm. Solutions and Drugs. The composition of the physiological solution for chickens was obtained from Ginsborg (8). The following concentrations are expressed in millimolar units : NaCI, 150 ; KCl, 5 ; MgClz, 2 ; CaClz, 5 ; NaHC03, 20 ; glucose, 11. Solutions were continuously bubbled with a 95% O,-5 % COa gas mixture, and were maintained at 23 C. Solutions of low pH (5.0 f 0.1) were prepared by adding 5 N HCI (about 2 mM) to normal chicken physiological solution. It was necessary to periodically check pH and prepare fresh low pH solution during the course of an experiment. Histological Procedures. Muscles were fixed either in 10% buffered neutral formalin or in a glutaraldehyde-cacodylate buffer solution. The muscles were then ,dehydrated, embedded in paraffin, and sectioned. Fiber diameters of innervated and denervated muscles were measured with the aid of a camera lucida (450X) and were expressed as the percentage of control values. Atrophic fibers in dystrophic muscles were not measured since these were assumed not to be readily accessible with microelectrodes. Statistical Analysis. All tabulated values were expressed as the mean * the standard error of the mean ( SEM) . Student’s t-test was performed and P values less than 0.01 were considered to be statistically significant.
DYSTROPHIC
MUSCLE
547
RESULTS Membrane Cable Properties of Innervated and Denervated Normal and Dystrophic Muscle Fibers. As noted in a previous study ( 19), resting membrane potentials of innervated normal and dystrophic posterior latissimus dorsi fibers were similar (ca. -72 mv) . Twenty-one days after denervation the resting membrane potentials of both groups of muscles declined to about -60 mv. From the data shown in Tables 1 and 2, innervated normal muscle fibers had a larger mean input resistance (Xin) than innervated dystrophic fibers at 12-20 weeks ex oz’o. There were other differences between the cable properties of innervated normal and clystrophic muscles: the space constant (A), the specific membrane resistance (R,,), the time constant ( rm), and the membrane capacitance ( C,,,) were all significantly larger than normal in dystrophic fibers. After denervation, mean values of Ri,, R,, and ~~ from 21-day denervated normal muscles increased about fivefold; A increased by a factor of two and the fiber radius p declined about 40% in denervated normal m’uscles (Table 1). C,, however, remained constant. The dystrophic fibers did show marked alterations 21 days after denervation (Table 2). Input resistance underwent a fivefold increase as in normal fibers, but Rm and Trn only increased by a factor of three after denervation. As in denervated normal fibers, C, remained unchanged and the fiber radius also decreased 40%. Unlike denervated normal fibers, the space constant of denervated dystrophic fibers did not increase significantly. The calculated values for innervated and denervated normal and dystrophic fiber radius were consistent with the histological measurements of fiber size (Table 3). The data from Tables 1 and 2 also show that high values of R, (ca. 2100 ohm*cm2) were measured by day 6 in both normal and dystrophic fibers. This increase in membrane resistance, however, was not accompanied by a large change in the radius of normal or dystrophic muscle fibers. An important finding was that some of the values for cable properties recorded from 21-day denervated normal and dystrophic fibers were quite similar. Although denervated dystrophic fibers had a lower value for Ri, than denervated normal fibers, this result could be accounted for by noting that the chronically denervated dystrophic fibers were larger than chronically denervated normal fibers. Nevertheless, denervated normal and dystrophic fibers atrophied to similar extents (about 40%). It is possible that with a longer denervation period, dystrophic fibers would atrophy further and would have radii similar to denervated normal fibers. Effects of Low pH Sollttions on the Mmzbrane Cable Properties. To investigate the underlying mechanism for the increase in R, after denervation (Tables 1 and 2)) the selective alteration of the total membrane conductance of innervated and denervated posterior latissimus dorsi muscles
TABLE
1
15 13 16 9 14
Control 3 6 14 21
0.35 0.55 1.06 1.55 1.60
f f f f f
f
Ri, (Mohm)
0 Values are expressed as the mean b P < 0.01 with respect to control.
Number of fibers
Days after denervation
SEM.
0.02 0.09 0.09b 0.19b O.llb
0.66 0.85 1.28 1.06 1.10
f f f f f
x (mm) 0.03 0.09 0.08” 0.14b 0.12b
3.4 4.3 10.8 17.8 15.4
f f f f f
7m (msec) 0.4 0.3 l.lb 3.0b 1.3b
22.2 20.3 18.1 13.7 13.4
f f f f f
64
P
1.0 1.3 l.lb 1.6b l.Ob
634 1208 2037 2636 2958
(ohm f f f f f
43 210b 273b 447b 4.59b
.cm*)
J&l
MEMBRANE CABLE PROPERTIES OF INNERVATED AND CHRONICALLY DENERVATED NORMAL (LINE 200) POSTERIOR LATISSIMUS DORSI MUSCLES OF THE CHICKENS
5.4 4.5 3.8 9.0 7.2
f 0.5 f 0.6 ZIZ 0.3 f 2.0 f 1.3
(rF/cm2)
Cm
% m
s 4 !z
Ii
5
E
r iz
23 9
9 6
7
Control 3
6 14
21
f f
1.12 f
0.47 f 0.84 f
0.26 0.40
0.2oc
0.16 0.13c
0.02* 0.06
0.04* 0.04
1.25 f
0.24
1.40 f 0.16c 1.25 32 0.11
1.02 f 0.93 f
x (mm)
PROPERTIES OF INNERVATED POSTERIOK LATISSIMUS
Rin (Mohm)
CABLE
a Values are expressed as the mean f SEM. b P < 0.01 with respect to line 200 control. c P < 0.01 with respect to line 304 control.
Number of fibers
Days after denervation
MEMBRANE
2
Tm (msec)
18.2 f
19.3 f 13.7 f 2.oc
1.4c 1.3
0.4* 1.6’
18.4 f
28.7 f 20.6 f
33.1 f 25.8 f
P (ml
2.7c
2.7 2.2c
1.6* 1.8
CHRONICALLY DENERVATED MUSCLES OF THE CHICKEN'
6.8 f 14.1 f
AND DORSI
TABLE
2891
2308 2452
f
f f
10.58 f 1130 f
RIO (ohm.cm2)
DYSTROPHIC
726c
448c 232c
78b 128
(LINE
304)
8.3 f
10.3 f 5.8 f
7.1 f 12.9 f
2.4
1.6 0.7
0.6b 1.4c
s m
5
zl z
550
LEBEDA
AND
TABLE
ALBUQUERQUE
3
COMPARISON OF INNERVATED AND CHRONICALLY DENERVATED NORMAL (LINE 200) AND DYSTROPHIC (LINE 304) FIBER RADII MEASURED HISTOLOGICALLY AND CALCULATED BY SQUARE PULSE ANALYSIS Days after denervation
(meas.)O
(talc.)”
(meas.)
(talc.)
Control
lOO%c
100%
100%
100%
72 66
92 a2 62 60
-
78 82 63 56
3 6 14 21 0 Radii measured b Calculated radii c Values expressed
Normal
Dystrophic
at 450X from at least 30 fibers. expressed as percentages from Tables as percentages of respective control.
71 53
1 and 2.
was attempted. Specifically, chloride conductance was reduced by bathing muscles in low pH (5.0) physiological solution. The effects of this procedure have been studied by Hutter and Warner ( 13-15) and Warner As shown in Table 4, innervated normal posterior latissimus dorsi muscles in solutions of low pH had elevated values for Rin, X, R, and r,,,. The mean values for x and R, in low pH solution approached the mean values obtained from 21-day denervated normal muscles ‘bathed in normal physiological solution. The input resistance, however, increased about fivefold 21 days after denervation of normal muscle, yet increased only two to threefold in innervated normal muscles bathed in low pH solutions. The values of Ri, and R, (Table 5) underwent similar changes when innervated dystrophic muscles were bathed in low pH solutions. As with normal fibers, values of R, approached but did not attain values calculated from 21-day denervated dystrophic fibers in normal pH solutions. Inherent errors arising from the following assumptions are expected, but the general conclusions derived from the calculations appear reasonable and should provide a basis for more accurate ion conductance measurements in chicken muscles. First it was assumed that sodium conductance at rest was small compared to the total membrane conductance and was therefore ignored (10). Second, the total membrane conductance G (M) was defined (see Ref. (11)) as the sum of the chloride conductance G (CZ) and the potassium conductance G(K) at pH 7.0 (Table 6). For the innervated muscles, G(M) was taken as the reciprocal of the mean specific membrane resistance : G(M) “l/R,. Values of G(M), for innervated normal and dystrophic fibers were 1.58 mmho/cm2 and 1.06 mmho/cm2, respectively. The data for G(M), G( Cl) and G(K) were
denervated = 5.0
21-day pH
4
14
5
1.5
Number of fibers
1.88 f
1.60 f
0.81 f
0.35 f
Rin (Mohm)
0.4.5c
0.11
0.07b
0.02
f
0.97 f
1.10 f
1.12 f
0.66
(mm)
x
O.lf+
0.12
TABLE
4
16.2 f
15.4 f
7.5 f
3.4 f
Tnl (msec)
2.4c
1.3
0.7b
0.4
2.1c
1.0
1.4c
1.0
7.0 solution
12.1 f
13.4 f
19.1 f
22.2 f
P (rm)
R,
are from
2600
2958
2119
666<
459
1.
(LINE
161b
43
.cm”)
Table
f
f
f
634 f
(ohm
AND 21-DAY CHRONICALLY DENERVATED NORMAL MUSCLES IN NORMAL AND Low pH SOLUTIONS
0.8Ob
0.03
CABLE PROPERTIES OF INNERVATED POSTERIOR LATISSIMUS DORSI
a Values are expressed as the mean + SEM. Data obtained from muscles bathed in pH * P < 0.01 with respect to corresponding muscle in pH 7.0 solution. c Value is not significantly different from corresponding muscle in pH 7.0 solution.
denervated = 7.0
21-day pH
innervated pH = 5.0
Innervated pH = 7.0
PH
Muscle/solution
MEMBRANE
7.2 f
3.6 f
5.4 f
12.1 f
200)
2.3c
1.3
0.4<
0.5
5 % rl
g 22 =I n
4
Zl-day pH
TABLE
5
f
1.34 f
1.12 f
0.52
0.26 f
0.3oc
0.30
0.10*
0.02
0.98
f
1.25 f
1.45 f
1.02 f
x (mm)
Ri,
(Mohm)
0.09
0.24
0.15”
0.04
20.2
f
18.2 f
19.5 f
6.8 f
(msec)
3.45
3.0
3.3*
0.3
i-m
l.lC
2.7
2.ac
1.6
f
are from
2255 f
2891
2443 f
Table
412c
726
414*
78
.cmz)
R,
(LINE
1058 f
(ohm
DYSTROPHIC pH SOLUTIONS
7.0 solution
14.1 f
18.4 f
29.3 f
33.1 f
6.4
P
CABLE PROPERTIES OF INNERVATED AND Zl-DAY DENERVATED POSTERIOR LATISSIMUS DORSI MUSCLES IN NORMAL AND Low
a Values are expressed as the mean f SEM. Data obtained from muscles bathed in pH *P < 0.01 with respect to corresponding muscle in pH 7.0 solution. c Value is not significantly different from corresponding muscle in pH 7.0 solution.
denervated = 5.0
7
10
Innervated pH = 5.0
21-day denervated pH = 7.0
23
Number of fibers
Innervated pH = 7.0
Muscle/solution PH
MEMBRANE
2.
304)
9.5 f
8.3 f
8.5 f
7.1 f
1.9
2.4
2.1c
0.6
2 m
iz
:: !2
DYSTROPHIC
TABLE ESTIMATED (LINE
IONIC
304)
CONDUCTANCES
POSTERIOR
Fibers
6 NORMAL DORSI
(LINE MUSCLES
G(M) = (l/h at pH = 7.0)
G(K) ‘v (l/R,,, at pH = 5.0)
mmho/cm2
mmho/cm?
Innervated normal 21-day denervated normal Innervated dystrophic LI Conductance values membrane resistance.
FOR
LATISSIMUS
553
MUSCLE
1.58
0.34 1.06 estimated
from
0.47 0.38 0.41 the reciprocals
200)
AND
DYSTROPHIC
OF THE
CHICKEN=
G(U) ‘v G(M) G(K)
G(n) G(M)
mmho/cm2 1.10 -0.04 0.65 of the mean
values
0.70 =O 0.61 for specific
derived from different fibers and the reciprocal of the means were used in these calculations, therefore, only estimates could be made of the individual ionic conductances. In addition, when muscleswere bathed in low pH solutions, potassium and sodium conductances were assumed to be unaltered, while the chloride conductance was assumedto approach zero. Thus at a pH of 5.0, G(M) N G(K). As seen in Table 6, innervated normal fibers had a value for G(K) of 0.47 mmho/cm” while in innervated dystrophic fibers G(K) was 0.41 mmho/cm?. The chloride conductance of these fibers was then calculated : G (Cl) N G(M) -G(K). Th e values for G (CLj for innervated normal and dystrophic fibers were 1.10 and 0.65 mmho/cm’, respectively, indicating that the chloride conductance of the dystrophic fibers was about 40% less than normal. Another important finding was that the ratio of chloride conductance to the total membrane conductance was 70% in normal posterior latissimus dorsi fibers: the chloride contribution to the total membrane conductance was estimated to be 61% in dystrophic preparations. These values are in close agreement with data obtained from frog muscles (10). Finally, it is important to stress that no significant change in resistance occurred in Z-day denervated normal muscles (Table 4) and 21-day denervated dystrophic muscles (Table 5) w hen bathed in low pH solutions. It is apparent that chloride conductance was substantially reduced following denervation. Further, it was estimated that G(M) decreased by 80% while G(K) declined by about 2070 in the 21-day denervated normal muscles. dembrane Cable Properties of Norvnal Fibers Undergoing Conzpematory Hypertrophy. The presence of larger than normal fibers in dystrophic muscles raised the possibility that a compensatory mechanism may be involved. After cutting two nerve branches of the brachial plexus,
554
LEBEDA
AND
ALBUQUERQUE
pronounced wing droop occurred which placed an excess load on the posterior latissimus ‘dorsi muscle and induced the fibers to hypertrophy. The mean values of h, fiber radius and R, are presented in Fig. 1 for innervated normal (N) and dystrophic (D) muscles and normal posterior latissimus dorsi muscles that had undergone compenstatory hypertrophy for two weeks (H). The hypertrophic fibers had larger than normal radii which approached those of innervated dystrophic fibers and were not significantly different. Similarly, the higher than normal values for A and R, of dystrophic fibers were not significantly different from values displayed by the hypertrophic posterior latissimus dorsi fibers. The mean values for Ri, and C, of the hypertrophic posterior latissimus dorsi fibers were not significantly different from values of either innervated normal and dystrophic muscles (Tables 1 and 2). The 7m of these hypertrophic fibers was similar to dystrophic fibers but significantly different from the mean values of innervated normal fibers. DISCUSSION Several important findings were obtained in the low pH studies. First, chloride conductance was estimated to contribute about 70% to the total resting membrane conductance in normal posterior latissimus dorsi muscles (Table 6). These findings are consistent with those observed in the frog sartorius muscles ( 10). The potassium conductance was approximately the same in normal and dystrophic posterior latissimus dorsi muscles, 0.47 and 0.41 mmho/cnP, respectively. In contrast, the chloride conductance of innervated dystrophic muscles was 40% less than normal. It is possible that such a reduction is correlated with the 49% larger than normal radius of dystrophic fibers. Second, the 20% reduction in potassium conductance after denervation of chicken posterior latissimus dorsi muscles is similar to the results obtained from denervated rat diaphragm (17) and denervated frog sartorius muscles (9). In denervated chicken muscles, however, the fivefold increase in the specific membrane resistance cannot be accounted for by a fall of the potassium conductance alone. A reduction in the conductance of another ionic species, mainly, chloride, may also be responsible for the large post-denervation increase in R,. For instance, the values of R, from innervated normal posterior latissimus dorsi fibers in low pH solutions approached those of 21-day denervated muscles in normal medium; that is, when chloride conductance was reduced by low pH solutions, a marked increase in R, resulted. Further, the cable properties of 21-day denervated fibers were not markedly affected by low pH solutions, implying that chloride conductance was greatly reduced in these muscles and that low pH solutions had no additional effects. Based on this evidence, the reduction
DYSTROPHIC
555
MUSCLE
5.0
1.00
0.75
35
7
m
hsec)
1.0
0.50
/J (w)
1D1A!Jl-ll ho
A (~4
N
H
N
D
H
2.0
I250
k
30 25 LhL 20 N
D
R,bhm
cm2)
loo0
750
500 H
N
D
H
FIG. 1. Selected cable properties of innervated normal (N) and dystropbic fibers (D) compared with the properties of normal fibers undergoing compensatory hypertrophy (H) (n = 5). Mean values * SEM for the space constant (A), time constant (TV), the fiber radius (p) and the specific membrane resistance (IL) for normal and dystrophic fibers were obtained from Tables 1 and 2, respectively.
of chloride conductance may be of significant importance in describing one of the post-denervation alterations in chicken skeletal muscles. It is also apparent from Tables 4 and 5 that values of R, from innervated normal and dystrophic muscles bathed in low pH solutions approached but did not attain those values obtained from 21-day denervated muscles. One explanation involves the relationship between Ri, and fiber radius; Ri" increasesin a nonlinear manner as fiber radius decreases( 16). Since no decrease in fiber radius occurred in low pH solutions (Tables 4 and 5 ; Ref. (5) ) , the Rin under these conditions would not have been expected to equal the large Ri" values obtained from the smaller 21-day denervated muscles. Furthermore, potassium conductance declined 20% after denervation (Table 6) and was assumed not to become altered in low pH medium (13, 14). An altered chloride conductance may also be associated with innervated dystrophic fibers. From the membrane resistance calculations of innervated dystrophic fibers, the 67% larger than normal R, only approximates the 49% larger fiber radius. One possibility is that the greater fiber size alone
556
LEBEDA
AND
ALBUQUERQUE
could account for the augmented values of R,. Yet, up to 13 weeks ex ovo when normal and dystrophic fibers were equal in size (18 pm), dystrophic fibers were found to have a twofold larger than normal R, values (3). In addition, results from the compensatory hypertrophy experiments demonstrated that fibers underwent a 26% increase in fiber radius, but at the same time exhibited a 100% increase in R, (Fig. 1). These results indicate that a larger fiber radius along with reduced membrane conductance may contribute to the increased value of R, of the hypertrophic fibers examined in dystrophic and functionally overloaded normal muscles. Note that hypertrophic fibers are also found in muscles from patients with Duchenne dystrophy (20). The presence of large fibers in dystrophic muscles may be the result of a compensatory mechanism initiated by the disease process. REFERENCES 1. ALBUQUERQUE, E. X., and R. J. MCISAAC. 1970. Fast and slow mammalian muscles after denervation. Exp. Neural. 26 : 183-202. 2. ALBUQUERQUE, E. X., F. T. SCHUH, and F. C. KAUFFMAN. 1971. Early membrane depolarization of the fast mammalian muscle after denervation. PfEiigers Arch. 328: 36-50. 3. ALBUQUERQUE, E. X., and J. E. WARNICK. 1971. Electrophysiological observations in normal and dystrophic chicken muscles. Science 172: 1260-1263. 4. ALBUQUERQUE, E. X., and J. E. WARNICK. 1972. The pharmacology of batrachotoxin. IV. Interaction with tetrodotoxin on innervated and chronically denervated rat skeletal muscle. J. Pharmacol. Exp. They. 180: 683697. 5. EISENBERG, R. S., and P. W. GAGE. 1969. Ionic conductances of the surface and transverse tubular membranes of frog sartorius fibers. J. Gen. Pkysiol. 53: 279297. 6. FATT, P., and B. KATZ, 1951. An analysis of the end-plate potential recorded with an intracellular electrode. J. Pkysiol. (London) 115 : 320-370. 7. FISCRBACH, G. D., and N. ROBBINS. 1971. Effect of chronic disuse of rat soleus neuromuscular junctions on postsynaptic membrane. J. Neuropkysiol 34 : 562-569. 8. GINSBORG, B. L. 1960. Some properties of avian skeletal muscle fibres with multiple neuromuscular junctions. J. Physiol. (London) 154 : 581-598. 9. HARRIS, E. J., and J. G. NICHOLIS. 1956. The effect of denervation on the rate of entry of potassium into frag muscle. I. Physiol. (London) 131: 473-476. 10. HODGKIN, A. L., and P. HOROWICZ. 1959. The influence of potassium and chloride ions on the membrane potential of single muscle tibres. J. Pkysiol. (London) 148 : 127-160. 11. Hooo, J., E. J. WILLIAMS, and R. J. JOHNSTON. 1969. The membrane electrical parameters of Nitella translucens. J. Tkeor. Biol. 24: 317-334. 12. HUBBARD, S. J. 1963. The electrical constants and the component conductances of frog skeletal muscle after denervation. I. Pkysiol. (London) 165: 443-456. 13. HUTTER, 0. F., and A. E. WARNER. 1967. The pH sensitivity of the chloride conductance of frog skeletal muscle. J. Pkysiol. (London) 189: M-425. 14. HUTTER, 0. F., and A. E. WARNER. 1967. The effect of pH on the Yl efflux from frog skeletal muscle. J. Pkysiol. (London) 189: 427443.
DYSTROPRIC
MUSCLE
557
15. HUTTER, 0. F., and A. E. WARNER. 197.2. The voltage dependence of the chloride conductance of frog muscle. J. Physiol. (London) 227: 275-290. 16. KATZ, B., and S. THESLEFF. 1957. On factors which determine the amplitude of the ‘miniature end-plate potential’. J. Phgsiol. (London) 137: 267-278. 17. KLAUS, W., H. L~LLYANN, and E. MUSCHOLL. 1960. Der kalium-flux des normafen und denervierten rattenzwerchfells. Pfiiigers Arch. 271: 761-77.5. 18. LEBEDA, F. J., and E. X. ALBUQUERQUE. 1973. Membrane electrical constants of innervated and denervated, normal and dystrophic chicken muscles. In. Abstracts of the 4th Annual Meeting of the Society for Neuroscience, October 2&23, St. Louis, MO. 19. LEBEDA, F. J., J. E. WARNICI, and E. X. ALBUQUERQUE. 1974. Electrical and chemosensitive properties of normal and dystrophic chicken muscle. Exp. Nmrol. 43 : 21-37. 20. PEARSON, C. M. 1962. Histopathological features of muscle in the preclinical stages of muscular dystrophy. Brain 85 : 109-120. 21. WARNER, A. E. 1972. Kinetic properties of the chloride conductance of frog muscle. J. Physiol. (Lordon) 227 : 291-312.
NEUROLOGY
Membrane
47,
Cable
544-557
(lws)
Properties of Normal Chicken Muscle Fibers
and
Dystrophic
F. J. LEBEDA AND E. X. ALBUQUERQUE* Defiartment of Pharmacology avid Experimental Therapeutics, University Maryland School of Medicine, Baltimore, Maryland 21201 Received
February
of
5, 1975
Values of fiber radius obtained by square pulse analysis and histological measurements indicated that the innervated dystrophic fibers which had been examined with microelectrodes were hypertrophic. The specific membrane resistance of dystrophic fibers was also greater than normal. In addition, experimentally induced compensatory hypertrophy of innervated nondystrophic (normal) fibers of the posterior latissimus dorsi led to alterations in several membrane characteristics which resulted in values resembling those of innervated dystrophic fibers. Twenty-one days after denervation, the values for the cable properties of normal and dystrophic fibers were increased, yet similar values were attained for the space constant, specific membrane resistance, and membrane capacitance. In both normal and dystrophic muscles which were denervated for 21 days the fiber radius decreased 40%. To study the mechanism underlying the increase of the specific membrane resistance after denervation, the resting membrane conductance was selectively altered. In solutions of low pH (5.0) where chloride conductance was presumably reduced, the space constant, time constant and specific membrane resistance of innervated normal and dystrophic fibers were increased and approached values obtained from al-day denervated muscles. In contrast, solutions of low pH had no marked effects on 21-day denervated normal and dystrophic muscles. It is suggested that the increased values for these cable properties from denervated normal and dpstrophic posterior latissimus dorsi muscles may be partially due to reduced potassium and chloride conductances. Furthermore, the presence of hypertorphic fibers may be a significant morphological adaptation in dystrophic muscles. 1 Send reprint requests to Dr. Edson X. Albuquerque, Department of Pharmacology and Experimental Therapeutics, University of Maryland, School of Medicine, Baltimore, MD 21201. A preliminary report of these results was presented at the Fifth Annual Meeting for the Society for Neuroscience, St. Louis, MO, 1974 (18). This work was supported by U.S. Public Health Service Grant NS-12063. The authors are grateful to Miss Mabel A. Zelle for excellent technical and secretarial assistance during the course of this study. 544 Copyright 0 All
rights
of
1975
by Academic
Press,
reproduction in any form
Inc. reserved.
DYSTROPHIC
MUSCLE
545
INTRODUCTION Skeletal muscle provides a convenient model for studying the ionic conductances of electrogenic membranes. In frog sartorius muscles, about twothirds of the total membrane conductance is due to chloride ions traversing the membrane; the other one-third is due mostly to potassium ions (10). At rest, most chloride ions cross only at the sarcolemma while potassium ions go through both the sarcolemmal and the transverse-tubule membranes (5). The changes occurring postsynaptically after denervation of mammalian “fast” skeletal muscles include depolarization, extrajunctional acetycholine sensitivity, tetrodotoxin-resistant action potentials, and increased specific membrane resistance (1,2,4). The increase of specific membrane resistance following denervation cannot be attributed to muscle inactivity since innervated mammalian muscles subjected to chronic immobilization do not exhibit changes in this membrane property (7). Upon denervation of frog muscle preparations, the specific membrane resistance increased less than twofold (12) ; in denervated rat muscles resistance increased threefold (1)) but preliminary experiments with denervated chicken muscles revealed a fivefold increase (18). Assuming that a decrease in potassium conductance occurred in denervated chicken muscle as in denervated rat muscle, another ion, possibly chloride, would have to contribute significantly to the total decrease in membrane conductance. The objective of the present investigation is to estimate the contributions of potassium and chloride ions to the total membrane conductance in normal and dystrophic, innervated and chronically denervated posterior latissimus dorsi of the chicken. In addition, the changes in the cable properties occurring in normal fibers induced to become hypertrophic were observed in order to obtain further information regarding the large fibers encountered in dystrophic chicken muscles. METHODS Animals and Prepurations. Both normal (line 200) and genetically dystrophic (line 304) New Hampshire chickens were purchased from the University of California at Davis. Animals between the ages of 10 to 20 weeks ex OZJO were used in all of the experiments. The “fast” posterior latissimus dorsi muscles were used for this study; the denervation procedure has been described by Lebeda, Warnick and Albuquerque (19). Contralateral muscles served either as intact or sham operated controls. To induce fibers of the posterior latissimus dorsi muscle to hypertrophy. several muscles of the wing were denervated. An incision was made along the dorsal surface, 5 cm rostra1 to the anterior latissimus dorsi. The two largest and most dorsal branches of the brachial nerve complex were exposed ; the medial nerve was doubly ligated, and a 1 cm portion
546
LEBEDA
AND
ALBUQUERQUE
was removed. During this procedure the surrounding vasculature remained intact. Upon recovery, pronounced wing droop was noted and was still evident at the time of autopsy up to two weeks later. The animals were killed 1 to 21 days after denervation by cervical dislocation, decapitation, and exsaguination. Muscles were carefully removed under a continuous flow of physiological solution. Individual muscles were mounted at approximately their in situ length by stainless steel insect pins to a paraffin lined Plexiglass plate in the center of which was a planoconvex lens. Recording and Stimulating Techniques. Glass microelectrodes filled with 3 M KC1 and having tip resistances between 15-20 Mohm were used for recording and intracellular stimulation. Conventional methods for square pulse analysis were used ( 1, 6). The stimulating electrode was usually placed 50-100, 300, 600 and 1000 pm away from the recording electrode. At each distance, several anodal current pulses with durations of 300 msec were passed through the stimulating electrode to hyperpolarize the membrane by 5-10, 15-20 or 25-30 mv. All voltage and current displacements were monitored on a Mingograf 81 polygraph, a Tektr,onix 565 oscilloscope and recorded on film with a Grass model 4 kymograph camera. The logarithm of the voltage to current ratio was plotted against the interelectrode distance. On semilogarithmic coordinates, at least three of the four points had to fall on a straight line, otherwise the data from a particular fiber were discarded. The internal resistivity of the myoplasm (Ri) was assumed to be 160 Ohm cm. Solutions and Drugs. The composition of the physiological solution for chickens was obtained from Ginsborg (8). The following concentrations are expressed in millimolar units : NaCI, 150 ; KCl, 5 ; MgClz, 2 ; CaClz, 5 ; NaHC03, 20 ; glucose, 11. Solutions were continuously bubbled with a 95% O,-5 % COa gas mixture, and were maintained at 23 C. Solutions of low pH (5.0 f 0.1) were prepared by adding 5 N HCI (about 2 mM) to normal chicken physiological solution. It was necessary to periodically check pH and prepare fresh low pH solution during the course of an experiment. Histological Procedures. Muscles were fixed either in 10% buffered neutral formalin or in a glutaraldehyde-cacodylate buffer solution. The muscles were then ,dehydrated, embedded in paraffin, and sectioned. Fiber diameters of innervated and denervated muscles were measured with the aid of a camera lucida (450X) and were expressed as the percentage of control values. Atrophic fibers in dystrophic muscles were not measured since these were assumed not to be readily accessible with microelectrodes. Statistical Analysis. All tabulated values were expressed as the mean * the standard error of the mean ( SEM) . Student’s t-test was performed and P values less than 0.01 were considered to be statistically significant.
DYSTROPHIC
MUSCLE
547
RESULTS Membrane Cable Properties of Innervated and Denervated Normal and Dystrophic Muscle Fibers. As noted in a previous study ( 19), resting membrane potentials of innervated normal and dystrophic posterior latissimus dorsi fibers were similar (ca. -72 mv) . Twenty-one days after denervation the resting membrane potentials of both groups of muscles declined to about -60 mv. From the data shown in Tables 1 and 2, innervated normal muscle fibers had a larger mean input resistance (Xin) than innervated dystrophic fibers at 12-20 weeks ex oz’o. There were other differences between the cable properties of innervated normal and clystrophic muscles: the space constant (A), the specific membrane resistance (R,,), the time constant ( rm), and the membrane capacitance ( C,,,) were all significantly larger than normal in dystrophic fibers. After denervation, mean values of Ri,, R,, and ~~ from 21-day denervated normal muscles increased about fivefold; A increased by a factor of two and the fiber radius p declined about 40% in denervated normal m’uscles (Table 1). C,, however, remained constant. The dystrophic fibers did show marked alterations 21 days after denervation (Table 2). Input resistance underwent a fivefold increase as in normal fibers, but Rm and Trn only increased by a factor of three after denervation. As in denervated normal fibers, C, remained unchanged and the fiber radius also decreased 40%. Unlike denervated normal fibers, the space constant of denervated dystrophic fibers did not increase significantly. The calculated values for innervated and denervated normal and dystrophic fiber radius were consistent with the histological measurements of fiber size (Table 3). The data from Tables 1 and 2 also show that high values of R, (ca. 2100 ohm*cm2) were measured by day 6 in both normal and dystrophic fibers. This increase in membrane resistance, however, was not accompanied by a large change in the radius of normal or dystrophic muscle fibers. An important finding was that some of the values for cable properties recorded from 21-day denervated normal and dystrophic fibers were quite similar. Although denervated dystrophic fibers had a lower value for Ri, than denervated normal fibers, this result could be accounted for by noting that the chronically denervated dystrophic fibers were larger than chronically denervated normal fibers. Nevertheless, denervated normal and dystrophic fibers atrophied to similar extents (about 40%). It is possible that with a longer denervation period, dystrophic fibers would atrophy further and would have radii similar to denervated normal fibers. Effects of Low pH Sollttions on the Mmzbrane Cable Properties. To investigate the underlying mechanism for the increase in R, after denervation (Tables 1 and 2)) the selective alteration of the total membrane conductance of innervated and denervated posterior latissimus dorsi muscles
TABLE
1
15 13 16 9 14
Control 3 6 14 21
0.35 0.55 1.06 1.55 1.60
f f f f f
f
Ri, (Mohm)
0 Values are expressed as the mean b P < 0.01 with respect to control.
Number of fibers
Days after denervation
SEM.
0.02 0.09 0.09b 0.19b O.llb
0.66 0.85 1.28 1.06 1.10
f f f f f
x (mm) 0.03 0.09 0.08” 0.14b 0.12b
3.4 4.3 10.8 17.8 15.4
f f f f f
7m (msec) 0.4 0.3 l.lb 3.0b 1.3b
22.2 20.3 18.1 13.7 13.4
f f f f f
64
P
1.0 1.3 l.lb 1.6b l.Ob
634 1208 2037 2636 2958
(ohm f f f f f
43 210b 273b 447b 4.59b
.cm*)
J&l
MEMBRANE CABLE PROPERTIES OF INNERVATED AND CHRONICALLY DENERVATED NORMAL (LINE 200) POSTERIOR LATISSIMUS DORSI MUSCLES OF THE CHICKENS
5.4 4.5 3.8 9.0 7.2
f 0.5 f 0.6 ZIZ 0.3 f 2.0 f 1.3
(rF/cm2)
Cm
% m
s 4 !z
Ii
5
E
r iz
23 9
9 6
7
Control 3
6 14
21
f f
1.12 f
0.47 f 0.84 f
0.26 0.40
0.2oc
0.16 0.13c
0.02* 0.06
0.04* 0.04
1.25 f
0.24
1.40 f 0.16c 1.25 32 0.11
1.02 f 0.93 f
x (mm)
PROPERTIES OF INNERVATED POSTERIOK LATISSIMUS
Rin (Mohm)
CABLE
a Values are expressed as the mean f SEM. b P < 0.01 with respect to line 200 control. c P < 0.01 with respect to line 304 control.
Number of fibers
Days after denervation
MEMBRANE
2
Tm (msec)
18.2 f
19.3 f 13.7 f 2.oc
1.4c 1.3
0.4* 1.6’
18.4 f
28.7 f 20.6 f
33.1 f 25.8 f
P (ml
2.7c
2.7 2.2c
1.6* 1.8
CHRONICALLY DENERVATED MUSCLES OF THE CHICKEN'
6.8 f 14.1 f
AND DORSI
TABLE
2891
2308 2452
f
f f
10.58 f 1130 f
RIO (ohm.cm2)
DYSTROPHIC
726c
448c 232c
78b 128
(LINE
304)
8.3 f
10.3 f 5.8 f
7.1 f 12.9 f
2.4
1.6 0.7
0.6b 1.4c
s m
5
zl z
550
LEBEDA
AND
TABLE
ALBUQUERQUE
3
COMPARISON OF INNERVATED AND CHRONICALLY DENERVATED NORMAL (LINE 200) AND DYSTROPHIC (LINE 304) FIBER RADII MEASURED HISTOLOGICALLY AND CALCULATED BY SQUARE PULSE ANALYSIS Days after denervation
(meas.)O
(talc.)”
(meas.)
(talc.)
Control
lOO%c
100%
100%
100%
72 66
92 a2 62 60
-
78 82 63 56
3 6 14 21 0 Radii measured b Calculated radii c Values expressed
Normal
Dystrophic
at 450X from at least 30 fibers. expressed as percentages from Tables as percentages of respective control.
71 53
1 and 2.
was attempted. Specifically, chloride conductance was reduced by bathing muscles in low pH (5.0) physiological solution. The effects of this procedure have been studied by Hutter and Warner ( 13-15) and Warner As shown in Table 4, innervated normal posterior latissimus dorsi muscles in solutions of low pH had elevated values for Rin, X, R, and r,,,. The mean values for x and R, in low pH solution approached the mean values obtained from 21-day denervated normal muscles ‘bathed in normal physiological solution. The input resistance, however, increased about fivefold 21 days after denervation of normal muscle, yet increased only two to threefold in innervated normal muscles bathed in low pH solutions. The values of Ri, and R, (Table 5) underwent similar changes when innervated dystrophic muscles were bathed in low pH solutions. As with normal fibers, values of R, approached but did not attain values calculated from 21-day denervated dystrophic fibers in normal pH solutions. Inherent errors arising from the following assumptions are expected, but the general conclusions derived from the calculations appear reasonable and should provide a basis for more accurate ion conductance measurements in chicken muscles. First it was assumed that sodium conductance at rest was small compared to the total membrane conductance and was therefore ignored (10). Second, the total membrane conductance G (M) was defined (see Ref. (11)) as the sum of the chloride conductance G (CZ) and the potassium conductance G(K) at pH 7.0 (Table 6). For the innervated muscles, G(M) was taken as the reciprocal of the mean specific membrane resistance : G(M) “l/R,. Values of G(M), for innervated normal and dystrophic fibers were 1.58 mmho/cm2 and 1.06 mmho/cm2, respectively. The data for G(M), G( Cl) and G(K) were
denervated = 5.0
21-day pH
4
14
5
1.5
Number of fibers
1.88 f
1.60 f
0.81 f
0.35 f
Rin (Mohm)
0.4.5c
0.11
0.07b
0.02
f
0.97 f
1.10 f
1.12 f
0.66
(mm)
x
O.lf+
0.12
TABLE
4
16.2 f
15.4 f
7.5 f
3.4 f
Tnl (msec)
2.4c
1.3
0.7b
0.4
2.1c
1.0
1.4c
1.0
7.0 solution
12.1 f
13.4 f
19.1 f
22.2 f
P (rm)
R,
are from
2600
2958
2119
666<
459
1.
(LINE
161b
43
.cm”)
Table
f
f
f
634 f
(ohm
AND 21-DAY CHRONICALLY DENERVATED NORMAL MUSCLES IN NORMAL AND Low pH SOLUTIONS
0.8Ob
0.03
CABLE PROPERTIES OF INNERVATED POSTERIOR LATISSIMUS DORSI
a Values are expressed as the mean + SEM. Data obtained from muscles bathed in pH * P < 0.01 with respect to corresponding muscle in pH 7.0 solution. c Value is not significantly different from corresponding muscle in pH 7.0 solution.
denervated = 7.0
21-day pH
innervated pH = 5.0
Innervated pH = 7.0
PH
Muscle/solution
MEMBRANE
7.2 f
3.6 f
5.4 f
12.1 f
200)
2.3c
1.3
0.4<
0.5
5 % rl
g 22 =I n
4
Zl-day pH
TABLE
5
f
1.34 f
1.12 f
0.52
0.26 f
0.3oc
0.30
0.10*
0.02
0.98
f
1.25 f
1.45 f
1.02 f
x (mm)
Ri,
(Mohm)
0.09
0.24
0.15”
0.04
20.2
f
18.2 f
19.5 f
6.8 f
(msec)
3.45
3.0
3.3*
0.3
i-m
l.lC
2.7
2.ac
1.6
f
are from
2255 f
2891
2443 f
Table
412c
726
414*
78
.cmz)
R,
(LINE
1058 f
(ohm
DYSTROPHIC pH SOLUTIONS
7.0 solution
14.1 f
18.4 f
29.3 f
33.1 f
6.4
P
CABLE PROPERTIES OF INNERVATED AND Zl-DAY DENERVATED POSTERIOR LATISSIMUS DORSI MUSCLES IN NORMAL AND Low
a Values are expressed as the mean f SEM. Data obtained from muscles bathed in pH *P < 0.01 with respect to corresponding muscle in pH 7.0 solution. c Value is not significantly different from corresponding muscle in pH 7.0 solution.
denervated = 5.0
7
10
Innervated pH = 5.0
21-day denervated pH = 7.0
23
Number of fibers
Innervated pH = 7.0
Muscle/solution PH
MEMBRANE
2.
304)
9.5 f
8.3 f
8.5 f
7.1 f
1.9
2.4
2.1c
0.6
2 m
iz
:: !2
DYSTROPHIC
TABLE ESTIMATED (LINE
IONIC
304)
CONDUCTANCES
POSTERIOR
Fibers
6 NORMAL DORSI
(LINE MUSCLES
G(M) = (l/h at pH = 7.0)
G(K) ‘v (l/R,,, at pH = 5.0)
mmho/cm2
mmho/cm?
Innervated normal 21-day denervated normal Innervated dystrophic LI Conductance values membrane resistance.
FOR
LATISSIMUS
553
MUSCLE
1.58
0.34 1.06 estimated
from
0.47 0.38 0.41 the reciprocals
200)
AND
DYSTROPHIC
OF THE
CHICKEN=
G(U) ‘v G(M) G(K)
G(n) G(M)
mmho/cm2 1.10 -0.04 0.65 of the mean
values
0.70 =O 0.61 for specific
derived from different fibers and the reciprocal of the means were used in these calculations, therefore, only estimates could be made of the individual ionic conductances. In addition, when muscleswere bathed in low pH solutions, potassium and sodium conductances were assumed to be unaltered, while the chloride conductance was assumedto approach zero. Thus at a pH of 5.0, G(M) N G(K). As seen in Table 6, innervated normal fibers had a value for G(K) of 0.47 mmho/cm” while in innervated dystrophic fibers G(K) was 0.41 mmho/cm?. The chloride conductance of these fibers was then calculated : G (Cl) N G(M) -G(K). Th e values for G (CLj for innervated normal and dystrophic fibers were 1.10 and 0.65 mmho/cm’, respectively, indicating that the chloride conductance of the dystrophic fibers was about 40% less than normal. Another important finding was that the ratio of chloride conductance to the total membrane conductance was 70% in normal posterior latissimus dorsi fibers: the chloride contribution to the total membrane conductance was estimated to be 61% in dystrophic preparations. These values are in close agreement with data obtained from frog muscles (10). Finally, it is important to stress that no significant change in resistance occurred in Z-day denervated normal muscles (Table 4) and 21-day denervated dystrophic muscles (Table 5) w hen bathed in low pH solutions. It is apparent that chloride conductance was substantially reduced following denervation. Further, it was estimated that G(M) decreased by 80% while G(K) declined by about 2070 in the 21-day denervated normal muscles. dembrane Cable Properties of Norvnal Fibers Undergoing Conzpematory Hypertrophy. The presence of larger than normal fibers in dystrophic muscles raised the possibility that a compensatory mechanism may be involved. After cutting two nerve branches of the brachial plexus,
554
LEBEDA
AND
ALBUQUERQUE
pronounced wing droop occurred which placed an excess load on the posterior latissimus ‘dorsi muscle and induced the fibers to hypertrophy. The mean values of h, fiber radius and R, are presented in Fig. 1 for innervated normal (N) and dystrophic (D) muscles and normal posterior latissimus dorsi muscles that had undergone compenstatory hypertrophy for two weeks (H). The hypertrophic fibers had larger than normal radii which approached those of innervated dystrophic fibers and were not significantly different. Similarly, the higher than normal values for A and R, of dystrophic fibers were not significantly different from values displayed by the hypertrophic posterior latissimus dorsi fibers. The mean values for Ri, and C, of the hypertrophic posterior latissimus dorsi fibers were not significantly different from values of either innervated normal and dystrophic muscles (Tables 1 and 2). The 7m of these hypertrophic fibers was similar to dystrophic fibers but significantly different from the mean values of innervated normal fibers. DISCUSSION Several important findings were obtained in the low pH studies. First, chloride conductance was estimated to contribute about 70% to the total resting membrane conductance in normal posterior latissimus dorsi muscles (Table 6). These findings are consistent with those observed in the frog sartorius muscles ( 10). The potassium conductance was approximately the same in normal and dystrophic posterior latissimus dorsi muscles, 0.47 and 0.41 mmho/cnP, respectively. In contrast, the chloride conductance of innervated dystrophic muscles was 40% less than normal. It is possible that such a reduction is correlated with the 49% larger than normal radius of dystrophic fibers. Second, the 20% reduction in potassium conductance after denervation of chicken posterior latissimus dorsi muscles is similar to the results obtained from denervated rat diaphragm (17) and denervated frog sartorius muscles (9). In denervated chicken muscles, however, the fivefold increase in the specific membrane resistance cannot be accounted for by a fall of the potassium conductance alone. A reduction in the conductance of another ionic species, mainly, chloride, may also be responsible for the large post-denervation increase in R,. For instance, the values of R, from innervated normal posterior latissimus dorsi fibers in low pH solutions approached those of 21-day denervated muscles in normal medium; that is, when chloride conductance was reduced by low pH solutions, a marked increase in R, resulted. Further, the cable properties of 21-day denervated fibers were not markedly affected by low pH solutions, implying that chloride conductance was greatly reduced in these muscles and that low pH solutions had no additional effects. Based on this evidence, the reduction
DYSTROPHIC
555
MUSCLE
5.0
1.00
0.75
35
7
m
hsec)
1.0
0.50
/J (w)
1D1A!Jl-ll ho
A (~4
N
H
N
D
H
2.0
I250
k
30 25 LhL 20 N
D
R,bhm
cm2)
loo0
750
500 H
N
D
H
FIG. 1. Selected cable properties of innervated normal (N) and dystropbic fibers (D) compared with the properties of normal fibers undergoing compensatory hypertrophy (H) (n = 5). Mean values * SEM for the space constant (A), time constant (TV), the fiber radius (p) and the specific membrane resistance (IL) for normal and dystrophic fibers were obtained from Tables 1 and 2, respectively.
of chloride conductance may be of significant importance in describing one of the post-denervation alterations in chicken skeletal muscles. It is also apparent from Tables 4 and 5 that values of R, from innervated normal and dystrophic muscles bathed in low pH solutions approached but did not attain those values obtained from 21-day denervated muscles. One explanation involves the relationship between Ri, and fiber radius; Ri" increasesin a nonlinear manner as fiber radius decreases( 16). Since no decrease in fiber radius occurred in low pH solutions (Tables 4 and 5 ; Ref. (5) ) , the Rin under these conditions would not have been expected to equal the large Ri" values obtained from the smaller 21-day denervated muscles. Furthermore, potassium conductance declined 20% after denervation (Table 6) and was assumed not to become altered in low pH medium (13, 14). An altered chloride conductance may also be associated with innervated dystrophic fibers. From the membrane resistance calculations of innervated dystrophic fibers, the 67% larger than normal R, only approximates the 49% larger fiber radius. One possibility is that the greater fiber size alone
556
LEBEDA
AND
ALBUQUERQUE
could account for the augmented values of R,. Yet, up to 13 weeks ex ovo when normal and dystrophic fibers were equal in size (18 pm), dystrophic fibers were found to have a twofold larger than normal R, values (3). In addition, results from the compensatory hypertrophy experiments demonstrated that fibers underwent a 26% increase in fiber radius, but at the same time exhibited a 100% increase in R, (Fig. 1). These results indicate that a larger fiber radius along with reduced membrane conductance may contribute to the increased value of R, of the hypertrophic fibers examined in dystrophic and functionally overloaded normal muscles. Note that hypertrophic fibers are also found in muscles from patients with Duchenne dystrophy (20). The presence of large fibers in dystrophic muscles may be the result of a compensatory mechanism initiated by the disease process. REFERENCES 1. ALBUQUERQUE, E. X., and R. J. MCISAAC. 1970. Fast and slow mammalian muscles after denervation. Exp. Neural. 26 : 183-202. 2. ALBUQUERQUE, E. X., F. T. SCHUH, and F. C. KAUFFMAN. 1971. Early membrane depolarization of the fast mammalian muscle after denervation. PfEiigers Arch. 328: 36-50. 3. ALBUQUERQUE, E. X., and J. E. WARNICK. 1971. Electrophysiological observations in normal and dystrophic chicken muscles. Science 172: 1260-1263. 4. ALBUQUERQUE, E. X., and J. E. WARNICK. 1972. The pharmacology of batrachotoxin. IV. Interaction with tetrodotoxin on innervated and chronically denervated rat skeletal muscle. J. Pharmacol. Exp. They. 180: 683697. 5. EISENBERG, R. S., and P. W. GAGE. 1969. Ionic conductances of the surface and transverse tubular membranes of frog sartorius fibers. J. Gen. Pkysiol. 53: 279297. 6. FATT, P., and B. KATZ, 1951. An analysis of the end-plate potential recorded with an intracellular electrode. J. Pkysiol. (London) 115 : 320-370. 7. FISCRBACH, G. D., and N. ROBBINS. 1971. Effect of chronic disuse of rat soleus neuromuscular junctions on postsynaptic membrane. J. Neuropkysiol 34 : 562-569. 8. GINSBORG, B. L. 1960. Some properties of avian skeletal muscle fibres with multiple neuromuscular junctions. J. Physiol. (London) 154 : 581-598. 9. HARRIS, E. J., and J. G. NICHOLIS. 1956. The effect of denervation on the rate of entry of potassium into frag muscle. I. Physiol. (London) 131: 473-476. 10. HODGKIN, A. L., and P. HOROWICZ. 1959. The influence of potassium and chloride ions on the membrane potential of single muscle tibres. J. Pkysiol. (London) 148 : 127-160. 11. Hooo, J., E. J. WILLIAMS, and R. J. JOHNSTON. 1969. The membrane electrical parameters of Nitella translucens. J. Tkeor. Biol. 24: 317-334. 12. HUBBARD, S. J. 1963. The electrical constants and the component conductances of frog skeletal muscle after denervation. I. Pkysiol. (London) 165: 443-456. 13. HUTTER, 0. F., and A. E. WARNER. 1967. The pH sensitivity of the chloride conductance of frog skeletal muscle. J. Pkysiol. (London) 189: M-425. 14. HUTTER, 0. F., and A. E. WARNER. 1967. The effect of pH on the Yl efflux from frog skeletal muscle. J. Pkysiol. (London) 189: 427443.
DYSTROPRIC
MUSCLE
557
15. HUTTER, 0. F., and A. E. WARNER. 197.2. The voltage dependence of the chloride conductance of frog muscle. J. Physiol. (London) 227: 275-290. 16. KATZ, B., and S. THESLEFF. 1957. On factors which determine the amplitude of the ‘miniature end-plate potential’. J. Phgsiol. (London) 137: 267-278. 17. KLAUS, W., H. L~LLYANN, and E. MUSCHOLL. 1960. Der kalium-flux des normafen und denervierten rattenzwerchfells. Pfiiigers Arch. 271: 761-77.5. 18. LEBEDA, F. J., and E. X. ALBUQUERQUE. 1973. Membrane electrical constants of innervated and denervated, normal and dystrophic chicken muscles. In. Abstracts of the 4th Annual Meeting of the Society for Neuroscience, October 2&23, St. Louis, MO. 19. LEBEDA, F. J., J. E. WARNICI, and E. X. ALBUQUERQUE. 1974. Electrical and chemosensitive properties of normal and dystrophic chicken muscle. Exp. Nmrol. 43 : 21-37. 20. PEARSON, C. M. 1962. Histopathological features of muscle in the preclinical stages of muscular dystrophy. Brain 85 : 109-120. 21. WARNER, A. E. 1972. Kinetic properties of the chloride conductance of frog muscle. J. Physiol. (Lordon) 227 : 291-312.