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Surgically induced hypertrophy in skeletal muscles of the laboratory mouse.

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Surgically Induced Hypertrophy in Skeletal
Muscles of the Laboratory Mouse
~-
R. W. D. ROWE A N D G. GOLDSPINK
Department of Zoology, University of H u l l , England
ABSTRACT
Two surgical techniques were used to increase the work load per fiber in
mouse skeletal muscles. These were, part removal of the muscle and incapacitation of a
synergetic muscle. Both techniques were used for the soleus muscle. They resulted in the
fiber size distribution of this muscle, which was normally unimodal, becoming distinctly
bimodal. The same effect was obtained for the part removal of the anterior tibialis
muscle. For the above mentioned muscles the extent of hypertrophy was very considerable. The recorded increase in the mean fiber cross-sectional area was in the region of
56% to 92%. Part removal of the biceps brachii however produced only a comparatively
slight increase i n fiber size (9% ). In this case the normal muscle was bimodal and the
effect of part removal was to slightly increase the size of the second peak. The reason
for the two peaks occurring in the distributions of fiber size in some muscles was due
to the presence of fibers at a basic level of development and also the presence of hypertrophied fibers. The increase in mean fiber size resulting from a n increased work load
was in all cases, due to a proportion of the basic fibers undergoing hypertrophy and not
due to a gradual increase i n the size of all the fibers.
Several workers have used various experimental procedures to alter the normal
growth of muscles in laboratory animals.
Denervation has been extensively studied
(Tower, '39; Banker and Denny Brown, '59;
Alder, Crawford and Edwards, '60). It results in a reduction of the gross muscle
bulk due to the atrophy and degeneration
of the constituent muscle fibers. Tenotomy, as reported by McMinn and Vrbova
('62), also results in a decrease in fiber
size and in some cases degeneration of
the fibers. Cross union of the nerve supply
of two different muscles has been carried
out by Buller, Eccles and Eccles ('60a,b)
and the transfer of the tendon attachment
to another location from its normal location has been employed by Crawford ('6 l ) .
The effects of surgical incapacitation of
synergetic muscles has been studied to
some extent by Denny Brown ('60), Crawford ('61), Van Linge ('62). Non-surgical
methods that have been used include
forced exercise and immobilization. Considerable hypertrophy of the muscle fibers
resulting from forced exercise of a muscle
has been reported by Goldspink ('64) and
Walker ('66). The general conclusion
that has emerged from this previous work
is that postnatal growth of muscles is very
dependent on its work load and functional
activity.
ANAT. REC.,161: 69-76.
The purpose of the present investigation
was to investigate the relationship between the work load imposed on a muscle
and the growth and development of its
constituent fibers. It was decided that two
techniques that could be conveniently used
to alter the work load per fiber of a muscle
were the part removal of the inuscle and
the incapacitation of a synergetic muscle.
The part removal method was particularly
convenient as the work load per fiber could
be adjusted by the quantity of muscle tissue removed. It was felt that a quantitative study of the changes at the cellular
level resulting from the application of
these techniques was particularly required.
It is well established that the constituent
fibers of different muscles are not uniform
in size (Fernand, '49; Walls, '60; Rowe,
'67), chemical composition (Stein and
Padykula, '62; George and Susheela, '61;
Dubowitz, '65; Drews and Engel, '66) or
in their physiological characteristics
(Denny Brown, '29; Buller et al., '60a,b;
Close, '64). In view of this knowledge it
was decided that the muscles chosen for
study should be of different fiber composition, different physiological characteristics and from different anatomical locations.
69
70
R . W. D. ROWE A N D G. GOLDSPINK
MATERIALS AND METHODS
The mice used were of the 129/Re
strain. They were fed on breeding diet
cube (Messrs Heygate and Sons Ltd.,
Northampton, England) and supplied with
food and water ad libitum.
Part removal. Three muscles were used
in this investigation; the soleus, the biceps
brachii, and the anterior tibialis. They
were chosen for the reasons mentioned
above. An additional consideration was
that they possess a relatively simple structure which permitted transverse sections
to be prepared i n which all the fibers
were present exhibiting their true crosssectional area (Rowe, '67). Thus, it was
possible to count all the fibers of the
muscle and to obtain reproducible measurements of fiber size.
The surgical procedure was as follows.
Female mice were anaethetised by intraperitoneal injection of Nembutal using
the dosages recommended by Pilgrim and
DeOme ('55). A n incision was made in
the skin to expose the appropriate muscle
and a proportion of the muscle fibers was
separated along their length from the rest
of the muscle, and then excised as near
as possible to the insertion and origin tendons. Care was taken during the operation not to damage the blood and nerve
supply of the remaining fibers. The incision was then sutured with Chinese silk
and the wound sprayed with Nobecutane
plastic skin (Evans Medical Ltd., Liverpool). This procedure was carried out on
the muscles of both side of the mouse.
Only one of the three different muscles
was operated on i n each mouse. Normal
mice from the stock colony of the same
sex, age and weight provided control muscles. After a number of weeks had elapsed
(see table 1) the mice were sacrificed and
weighed. The muscles were then excised,
weighed on a torsion balance and cytologically analysed.
Incapacitation of synergetic muscle.
The gastrocnemius muscle was incapacitated by tenotomy and the effect of this
procedure on the underlying soleus muscle was investigated in mice of both sexes.
Mice four to five weeks of age were anaesthetised as described above and a n incision made in the skin to reveal the tendon
achillis. A short length of the gastrocne-
mius tendon, approximately 3 mm, was
removed just proximal to its junction with
the soleus tendon. Care was taken during
the operation not to damage the nerve or
blood supply of the soleus. The incision
in the skin was sutured and then sprayed
with Nobecutane. The procedure was carried out on both hind limbs of each experimental mouse. Control muscles were
taken from littermates which had been
kept under identical environmental condiditions. Approximately fifteen weeks after
the operation the mice were sacrificed and
both soleus muscles were then dissected
out, superficially dried and weighed on a
torsion balance. The muscles were then
fixed after ensuring that the fibers were
lying as straight as possible. To ascertain
the effects of fixing the muscles off the
bone, two muscles that had been subjected
to the same experimental procedure were
fixed on the bone with the foot in a position intermediate between full extension
and full flexion.
Histological methods. The fixative used
in this investigation was Fleming's solution without acetic acid (Gatenby, ' 3 7 ) .
Goldspink ('61 ) has previously found this
fixative to be particularly useful for small
skeletal muscles as it causes very little
distortion of muscle fibers. All muscles
were fixed for 30 hours and then washed
in running water for a minimum of 12
hours foIlowing which they were dehydrated in ethanol and embedded i n Ester
wax (Steedman, '60). Transverse sections
were cut at 6
and mounted serially.
The sections were stained in Mallorys
triple stain and finally mounted using
Canada Balsam.
The total number of fibers in each
muscle was determined by counting all
the fibers in a transverse section taken
from a predetermined level (Rowe, '67).
The fiber counts were made by projecting
the sections at a magnification of approximately 100 x and then counting the fibers
using a n electric pen counter.
The distribution of fiber sizes was obtained €or each muscle by measuring the
diameters o f a sample of 100 fibers. It
has previously been shown that this size
of sample is statistically valid for the measurement of fibers (Meara, '47; Joubert,
'56). The transverse sections were projected a t a magnification of 450 x and
71
INDUCED MUSCLE FIBER HYPERTROPHY
the diameter of the fibers measured using
a pair of micrometer calipers previously
calibrated for this magnification. Fernand
('49) and other workers have shown that
different muscles of the same animal have
fibers of different cross-sectional area arranged in different spatial configurations.
The method of sampling the fibers was
therefore important. The sample was
taken in the same way for all the muscles
so that it included fibers from all regions
of the transverse section. This was done
by measuring all the fibers that were transected by several parallel lines drawn
across the projected image. The measurements of the individual fibers were assigned to appropriate size groups. The
size groups were as follows: 2.6-7.5
(midpoint 5 p ) ; 7.6-12.5
(midpoint 10
p ) ; 12.6-17.5
(midpoint 15 p ) etc.
The fiber diameter distributions for
these muscles are given in figure 1. The
normal or control anterior tibialis and soleus muscles showed a unimodal distribution with a peak at 25 p whereas the
experimental anterior tibialis and soleus
muscles exhibited a distinctly bimodal distribution of fiber sizes. The effect of part
removal in the case of these two muscles
was therefore to induce some of the fibers
which normally occur at 25 p to undergo
hypertrophy to a diameter of 35-40 p.
The effect of this apparent jump in size
of some of the fibers was to cause the
emergence of the second peak in the distribution.
In the case of the biceps brachii the
distribution of fiber diameters of the control muscles was bimodal. At stated above
the effect on the fibers of part removal of
the muscle was comparatively slight but
RESULTS
nevertheless noticeable. It resulted in the
Part removal. The results of the part second distribution peak (35-40
beremoval experiment are presented in ta- coming more prominent. This must have
ble 1 and figure 1. It will be seen from been due to some of the 25
diameter
table 1 that removal of part of the muscle fibers undergoing hypertrophy to the 35resulted in a considerably reduced fiber 40 size; hence it caused the second peak
number. The mean percentage reduction to increase at the expense of the first.
varied from muscle to muscle, however it
Incapacitation of synergetic muscle.
was highly significant for all three mus- The results of this experiment are precles (P < 0.001). It will also be seen from sented in table 2 and figure 2. As a result
table 1 that the mean fiber diameters of of the incapacitation of the gastrocnethe experimental anterior tibialis and so- mius, the weight of the soleus muscles in
leus showed a very marked increase over both sexes of experimental animals, were
the normal values (P < 0.001). Part re- considerably higher than their controls
moval of the biceps brachii resulted in (P < 0.001). A statistical comparison of
only a comparatively slight increase in the total fiber numbers in the experimenmean fiber diameter ( P < 0.2). When ex- tal and control soleus muscles revealed
pressed as a percentage of the normal that there was a slight increase in the
mean values the increases in mean fiber number of fibers in the experimental musdiameter were as follows, anterior tibialis cles from female mice (P < 0.02). In the
38.33%; soleus 25.30% ; biceps brachii case of the males, the difference in the
fiber number was not significant (P < 0.7).
3.62%.
TABLE 1
D a t a o n the n u m b e r a n d size of fibers of control a n d experimental m u s c l e s
after part removal
Muscle
No. of
muscles
Postoperative
Final body
period in days weights in gm
E x p . anterior tibialis
9
61
Cont. anterior tibialis
E x p . biceps brachii
Cont. biceps brachii
E x p . soleus
Cont. soleus
5
-
9
5
11
5
61
1
Standard errors given in parentheses
61
-
25.12(*0.67)l
26.01(21.14)
24.02(*0.55)
26.01(21.14)
24.92(20.60)
26.01(k1.14)
Total no.
of fibers
2038(23)
2835( k4)
1668(%3)
2140(23)
577(*2)
737( 23 )
Mean fiber
diameter p
33.90(*0.85)
24.53(20.50)
34.05(*0.34)
32.86(*1.06)
30.99(*0.60)
24.73( 21.02)
45-
I
30
No.
351
FIBER DIAM. I N MICRONS
BICEPS BRACHll
EXPERIMENTAL
- - - - - - - _CONTROL
._____
No.
0
5
10
15
2c
2!
3(
3:
4(
4.
51
Fig. 1 Fiber diameter distributions of the anterior tibialis, biceps brachii and soleus muscles for the experiment in which part of the muscle was removed. The mean distributions of the experimental muscles are given
by the soiid lines and those of the normal or control animals by the stippled lines. Twice the standard errors is
given for each point.
FIBER D I A M . IN MICRONS
ANTERIOR TlBlALlS
?
73
INDUCED MUSCLE FIBER HYPERTROPHY
The effect of the incapacitation of the gastrocnemius on the diameters of the fibers
in the soleus was very pronounced. Both
sexes of experimental animals showed a
significant increase in the mean fiber diameter over the control muscles (males
P < 0.001; females P < 0.01). The results
of plotting the fiber diameter distributions
show that the control animals of both
sexes exhibited a normal unimodal distribution (Rowe, '67) whereas the muscles
from the experimental animals exhibited
a bimodal distribution. In other words the
effect on the distribution of fiber sizes
after the incapacitation of the gastrocnemius was the same as for the part removal
of this muscle. It had previously been the
practice of the authors to fix the soleus
muscle whilst it was still in situ, i.e. on
the bone. However in this case the insertion tendon had to be cut in order to
carry out physiological measurements (to
be published elsewhere) and therefore
this was not possible. To test the effects
of fixation off the bone, several experimental muscles were fixed in situ. The
fiber size distribution of two of these muscles are given in the top right hand corner
of figure 2. It will be seen that these muscles still showed a bimodal distribution
but because of the more stretched position
of the fibers the diameters at which the
peaks occurred were somewhat lower 25
and 35 as compared with 35 and 50 p,
on the bone and off the bone respectively.
DISCUSSION
The data presented here for the effect
of increasing the work load of a muscle
is in general agreement with the findings
of previous workers. That is to say, certain types of exercise induce muscle fibers
to enlarge. In addition to confirming this
generally held view, certain new findings
emerged concerning the nature of muscle
fiber hypertrophy.
From the plots of the frequency of fibers at different sizes it appears that the
fibers of all three muscles studied may
exist at one of two levels. There is a basic
level of development at 20-25
diameter,
and a hypertrophied level of development
at 35-40
diameter. The effect of the increased work load on the muscle was to
cause a proportion of the fibers at the
basic level to undergo further hypertrophy.
The hypertrophy of the muscle as a whole
therefore, was due to a jump in the size
of some of the fibers and not a gradual
increase in the size of all the fibers.
The degree of hypertrophy resulting
from the use of surgical methods for increasing the work load of a muscle was
very considerable for the soleus and anterior tibialis muscles. For instance, in the
case of part removal of the soleus and
incapacitation of the gostrocnernius, the
soleus fibers increased in diameter by approximately 25% and 32% respectively.
Expressed as an increase in the mean
cross-sectional area of the fibers the increase is very considerable indeed; 56%
and 75% respectively. After part removal
of the anterior tibialis the recorded increase in the mean cross-sectional area of
the fibers was even greater (92% increase). O n the other hand, the biceps
brachii muscle did not respond to anything like the same extent ( 9 % increase).
One of the reasons for this smaller response by the biceps brachii was the presence in the experimental muscles of a
higher proportion of small diametered, regenerating fibers. This resulted in more
small fibers being included in the 100
fiber samples, which in turn reduced the
mean fiber diameters. One other reason
for the lower percentage increase in mean
fiber diameter may be that the biceps
brachii has less potential to develop because it is normally a bimodal muscle. In
other words approximately half the fibers
TABLE 2
Data o n the number and size o f fibers o f the soleus after the incapacitation o f the gastrocnemius
Muscle
No. of Postoperative
Final body
muscles period in days weights in gm.
Male exp. soleus
6
Male cont. soleus
10
Female exp. soleus
5
Female cont. soleus 5
106
-
107
-
31.82(*1.30)1
29.03( 20.71)
25.04(20.50)
25.40( 20.50)
Muscle
weights mg.
Total no.
of fibers
Mean fiber
diameter IL
12.08(*1.22)
7.53(*0.34)
11.24(*0.57)
6.15(*0.35)
784(*3)
767(23)
842(23)
730(--f3)
46.46(21.16)
37.32(*0.69)
44.37(20.68)
36.38(21.50)
_
~
1
Standard errors given in parentheses
_
~
~
~
74
R. W. D. ROWE AND G. GOLDSPINK
of this muscle had already undergone further hypertrophy and thus the maximum
potential to develop had been reduced by
50% before it was subjected to the in-
creased work load. The fibers of the normal soleus and anterior tibialis, unlike
the biceps brachii were all still at the basic
level of development before the increased
-
40
A
J
35 -
30 -
25 -
T
p 2015 1
FIBER DIAM. I N MICRONS
Fig. 2 Fiber diameter distributions of the soleus muscles for the experiment in which
the gastrocnemius muscle was incapacitated. The mean distribution of the experimental
muscles is shown by the solid lines and that of the normal or control muscles by the stippled
line. Twice the standard error is given for each point.
INDUCED MUSCLE FIBER HYPERTROPHY
work load was applied. The much greater
response of these muscles may have been
due to the fact that all the fibers have the
potential to undergo further hypertrophy.
In the experiment involving the incapacitation of the gastrocnemius a slight
but significant increase was recorded in
the number of fibers in female soleus muscles. Van Linge ('62) also recorded
hyperplasia in the plantaris following the
denervation of the triceps surae and the
implantation of the plantaris tendon into
the tuberosity of the calcaneum. In this
present investigation, as well as the recorded increase in the total number of fibers in the muscle, the formation of new
fibers was seen to be taking place by the
process of longitudinal splitting and budding. It would seem therefore that under
conditions of extreme work load the number of fibers may increase. This may arise
due to damage caused by imposing the
extreme work load and this may in some
way, stimulate hyperplasia as well as regeneration.
The exact nature of the stimulus that
induces muscle fibers to undergo hypertrophy is not known. However, the data
presented here demonstrates that the work
load per fiber a muscle is subjected to
constitutes a part, or even the whole, of
the stimulus.
ACKNOWLEDGEMENTS
This work was supported by a Research
grant from the Muscular Dystrophy Group
of Great Britain.
LITERATURE CITED
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517-530.
Buller, A. J., J. C. Eccles and R. M. Eccles 1960a
Differentiation of fast and slow muscles i n the
cat hind limb. J. Physiol., 150: 399-416.
1960b Interactions between motoneurones and muscles in respect of the characteristic
speeds of their responses. J. Physiol., 150: 417439.
Close, R. 1964 Dynamic properties of fast and
slow skeletal muscles of the rat during development. J. Physiol., 173: 74-95.
75
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the ATP-ase reaction in muscle fibres by E.D.T.A.
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Steedman, H. F. 1960 Section Cutting in Microscopy. Blackwell Scientific publication, Oxford.
Stein, J. M., and H. A. Padykula 1982 Histochemical classification of individual skeletal
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