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. 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