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The development of compensatory hypertrophy in the plantaris muscle of the rat
=========ANNALS
or ANATOMY = = = = = = = = =
The development of compensatory hypertrophy in the plantaris muscle of the rat H. Degens*, N. E. L. Meessen*, P. Wirtz** and R. A. Binkhorst* *Department of Physiology and **Department of Cytology and Histology, University of Nijmegen, P. O. Box 9101, NL-6500 HB Nijmegen, The Netherlands
Summary. The aim of this investigation was to study the time course of compensatory hypertrophy (CH) over a seven week period after its surgical induction in the lower limb of the rat. CH of the left plantaris muscle of the rat was induced by denervation of the ipsilateral gastrocnemius and soleus muscles. Muscle fibres were classifed as type I, I c, II a and II b. Hypertrophy of the muscle was first observed about ten days after induction of CH. All fibre types appeared to contribute to this hypertrophy. During the period between four and twenty eight days there was a marked increase in the percentage of type I fibres, mainly at the expense of type II a, as compared with control muscles. During this CH period so called 'intermediate' I c fibres were found, indicating fibre type transition taking place. The isometric twitch time to peak tension (TPT) of the plantaris muscle was studied in situ. The TPT of CH muscles remained the same during the experimental period of seven weeks. This might be explained by the effect of the increase in type I (slow) fibres being masked by the far larger number of fast fibres, which still accounted for approximately 790/0 of the total number of fibres after CH.
tion of compensatory hypertrophy, and no information was obtained during the earlier period of the development of hypertrophy. Since Gollnick et al. (1981) found, as we did, that the number of fibres did not change as a result of compensatory hypertrophy, the increase in the percentage of type I fibres obviously occurred in expense of type II fibres. Baldwin et al. (1982) already suggested that a fibre type conversion occurred during compensatory hypertrophy. Since little is known about the time course of fibre type conversion during the development of compensatory hypertrophy, we decided to study the fibre type composition and fibre crosssectional areas during the development of compensatory hypertrophy from two days up to seven weeks postoperatively. In addition we measured twitch time to peak tension to relate this parameter to changes in fibre type composition.
Materials and methods Animals and experiments
Key words: Compensatory hypertrophy - Fibre types Twitch time to peak tension - Plantaris muscle - Rat
Introduction Previous studies have shown an increase in the percentage of type I fibres (Baldwin et al. 1982; Degens et al. 1992) and a concomitant increase in the twitch time to peak tension with compensatory hypertrophy (Degens et al. 1993; Roy et al. 1982). Those data were collected several weeks after inducCorrespondence: H. Degens, Department of Human Anatomy and Cell Biology, University of Liverpool, P. O. Box 147, L69 3BX Liverpool, United Kingdom
)~I£ SEMPER
Ann Anat (1995) 177: 285 - 289 Gustav Fischer Verlag Jena
Female Wistar rats (seven weeks old; 100 - 125 g) were classed at random in a control group (CO: 56 animals) and an experimental group (EO: 60 animals). They were kept in a room maintained at 22°C with 12 h light and 12 h darkness per 24 h period. Compensatory hypertrophy was obtained by denervating the gastrocnemius and the soleus muscles of the left leg under general anaesthesia (45 mg/kg Nembutal i. p.). All the branches of the n. tibialis to these two muscles were cut and sutured to the semimembranosus or the semitendinosus muscles. This denervation procedure was followed by an operation in which either the tendon of the gastrocnemius muscle was cut (for the twitch time to peak tension study), or in which the tendon of the plantaris muscle was attached to the tendon of the denervated gastrocnemius muscle (for the histochemical studies). These operation methods have been found not to lead to different results during contractile measurements (Binkhorst and van 't Hof 1973).
Histochemistry
Results
In a second group, analyses were made of groups of five muscles of the CO group and five of the EO group at 2, 4, 7, 10, 14, 28, 42 and 49 days post-operatively. The muscles were removed from the animals under ether anaesthesia, weighed and rapidly frozen in isopentane cooled in liquid nitrogen. Serial cross-sections, perpendicular to the long axis of the muscle, of 7 !Jm were cut using a Walter Dittes cryostat at - 25°C, from the mid belly of the muscle. Sections were stained for haematoxylin-eosin, succinic dehydrogenase (SDH) according to Nachlas et al. (1957) and myofibrillar adenosine triphosphatase (ATPase) according to Dubowitz and Brook (1973) after pre-incubation at pH 4.35. The latter enzyme reaction enables the distinction between type I (dark) and type II (light) fibres. 'Intermediate' fibres (I c) showed an intermediate staining. With the SDH reaction a sub-classification can be made of the type II fibres. Type II fibres staining dark for SDH were classified as type II a, and type II fibres staining weakly for SDH were classified as type II b.
Histochemical and morphometrical analysis During the first ten days the relative muscle weight (muscle weight/rat weight) of the EG was somewhat lower in comparison with the CG (Fig. 1). After 10 days, hypertrophy developed gradually to a maximum of about 1500,10 after 6 weeks. In figure 2 the development of hypertrophy of the fibres of each type in both regions is shown. During the first ten days in the EG there appeared to be a tendency for the fibres to be slightly smaller, compared with the CG. After this period the fibres of the EG were larger, compared with the CG. Simultaneously, the percentage of type I fibres increased in the EG (p < 0.001) (Fig. 3). The total number of fibres, however, did not differ significantly between the CG (3608 ± 377; mean ± sd.) and the EG (3287 ± 388; mean ± sd.). Figure 3 also shows a transient occurrence of type I c fibres (intermediately stained), with a maximum during the period from 14 to 28 days post-operatively. Data presented in figure 4 suggest that in the deep area (A) of the muscle mainly type II a fibres passed into type I fibres.
Morphometry Because fibre type distribution in the muscle was not homogeneous (Degens et al. 1992), the deep (area A) and superficial (area B) regions were evaluated separately. In each region about 100 fibres were classified and their cross-sectional area measured. To measure the cross-sectional areas of the fibres, sections of the muscles were projected on a screen. The magnification factor was 200. A transparant gauge with 10 holes of different known diameters was laid over individual fibres on the screen. The fibres of control and hypertrophied muscles did not have an elongated appearance in the muscle sections. The hole with the diameter that most closely resembled a particular fibre was used to calculate from the magnification factor the cross-sectional area of that particular fibre. The diameters of the holes corresponded to absolute diameters of the fibres of 10 !Jm increasing with lO!Jm to 100 !Jm. An equal magnification factor was used in all muscles analysed. One may argue, that the sections are not perpendicular to the long axis of the fibres, since the angle of pinnation of the fibres may be 23 ° for control and 25 ° for hypertrophied rat plantaris muscle (Binkhorst and Van 't Hof 1973) or 15° and 17° respectively (Roy et al. 1982). The real fibre cross-sectional areas can be estimated by multiplying the measured cross-sectional areas by the cosine of the angle of pinnation. In the present study a comparison is made between the control and hypertrophied plantaris muscles and the difference in the cosine of the angle of pinnation between both cases is less than 1070. Therefore, it is reasonable to use the measured fibre cross-sectional areas for comparison between control and hypertrophied muscles.
~
0.18
~
0.16
i
0.14
...
EG
.~
III
:;:;>
0.12
CG
0.10
~
~
0.08
0
10
20
30
40
50
60 days
Fig. 1. Development of muscle hypertrophy expressed as relative weight (muscle weight per body weight) of experimental (EO) and control group (CO) during 49 days after induction of compensatory hypertrophy (each point n = 5).
Twitch time to peak tension (TPT)
Twitch time to peak tension (TPT) In 16 rats of the CO group and 20 rats of the EO group this parameter was evaluated during in situ measurements, under general anaesthesia (45 mg/g i. p. Nembutal) in a liquid paraffin pool at 35°C. TPT was determined at La, the length of the muscle at which the twitch force is maximal and TPT minimal. The apparatus and the method used are described earlier (Binkhorst and Van 't Hof 1973; Degens et al. 1993).
The relation between TPT (ms) and post-operative age in days could be expressed in a regression equation: TPT = O.013*days + 12.9 (r = 0.22) for the CG and TPT = 0.020*days + 12.9 (r = 0.38) for the EG, but both were not significant. No significant differences were found during the first post-operative days: the mean values for the TPT of muscles analyzed over the first ten days was 13.0 ± 1.1 ms (n = 6) for the CG and 12.9 ± 1.0ms (n = 10) for the EG.
Discussion After surgical intervention to induce compensatory hypertrophy the plantaris muscle is forced to take over part of the
286
area a
areab
fibre diameter )..J
25
type I
EG 20
ACG 1
'--1T----...1. . . . type II - a
25
type II - a
20
r--r
r
G
type II - b
EG 25
EG
I
20
15
q:
,
10
,
20
MG
I
30
,
I
10
50
40
20
fibre - type I %oftotal 25
20 15 10
5
intermediate 10
20
30
40
50 days
Fig. 3. Percentage of type I fibres in cross-sections of the total muscle of the control (CO) and experimental (EO) group and type Ic (intermediate) fibres in the EO during 49 days after induction of compensatory hypertrophy, starting from 7 days after surgery (each point n = 5). functions of the fast gastrocnemius as well as of the slow soleus muscles. It seems reasonable to assume that an increased effort is demanded of both the fast twitch and the slow twitch fibres in the plantaris muscle. The present results show how the muscle achieves to meet this extra loading. In agreement with another study (Gollnick et al. 1981), we
30
40
50 days
Fig. 2. Diameters of type I, IIa and II b fibres in the deep (A) and superficial (B) area of the control group (CO) and the experimental group (EO) during 49 days after induction of compensatory hypertrophy. Number of type I fibres in area B are too small for reliable counting. (Values are mean ± sd.); 1): not significant; all other cases p < 0.01).
found no significant change in the total number of fibres in the muscles after the induction of compensatory hypertrophy, implicating that: 1. hypertrophy of the muscle was paralleled by an equivalent increase in the size of individual fibres, amounting to a maximum of about 50070 at six weeks after surgery, and 2. that transformation accounted for the changes in fibre type proportion. In the present study the increase in type I fibres, due to compensatory hypertrophy, an effect which was also observed previously at a later stage (Baldwin et al. 1982; Degens et al. 1992), was found to be evident already at two weeks post-operatively. This is contrary to the study of Oakley and Gollnick (1985), in which an increase was not found until the fourth week after induction of compensatory hypertrophy. The difference in results may be attributed to the fact that Oakley and Gollnick ablated the gastrocnemius muscle leaving the soleus muscle intact. In this study both muscles were denervated, forcing the plantaris muscle not only to take over the load of the gastrocnemius, but also the postural function of the soleus muscle. This is reflected by an almost three-fold rise in the number of type I fibres in our study against a two-fold rise in the other study. The decrease of type II a fibres in the deep region accompanying the increase in type I fibres suggests, that at least in this area type II a fibres transform into type I fibres. At the level of the whole muscle this observation is supported by an increase in the content of slow (type I) myosin heavy-chains, while the amount of fast type II b myosin heavy-chain
287
area a areab fili~-~----------------------------------------------------------,
.,.
CG
CG
80
n·b
0.-.0.. _-0 ....... ____ .. _ ....... _0 __ .. - - - - ... _ ... _ .. -0 .................
60
n-a_ _x:---_.x_
40 20
00
··. . .L----.. _._ . -..-._.-·-·---·-....
filire-~
80 60
EG
EG
~
40
n-b
'><
----x
_._._.-,.
/p---o---::;::;:;--·~..().----
20
.........
0/
I ..,-'
. . ---..--..
o.. ___ ......
/
10
20
30
40
50
lI-a
~x:----:x-x
.. ""._'_.-._.-..-.--'--._..--10
20
decreased with compensatory hypertrophy as found by Kandarian et al. (1992). The presence of intermediate (type I c) fibres, especially between 14 and 28 days post-operatively, as was also observed by Oakley and Gollnick (1985), corresponds with the period of an increase of type I fibres. It is reasonable to assume that these fibres are 'transitional' fibres, i. e. being a transition from type II a to type I. On grounds of our morphometric and histochemical results revealing an increase in type I fibres one might expect to find an increase in twitch time to peak tension. No such change was found in the muscles undergoing compensatory hypertrophy up to seven weeks post-operatively, this being in contrast with the increase in twitch time to peak tension observed as a result of compensatory hypertrophy in other studies (Degens et al. 1993; Roy et al. 1982). Possibly the increase in number and size of the type I fibres is too small to measurably influence twitch time to peak tension of the muscle (Biscoe and Taylor 1967). Brody (1976) provided evidence, that the twitch time to peak tension is related more closely to the sarcotubular Ca2 + uptake than to the activity of myosin ATPase, a parameter that was not included in our study. In conclusion the present study shows that in the experimental model used, compensatory hypertrophy is preceded by a short post-surgical period in which no hypertrophy is found. During the development of compensatory hypertrophy an intermediate fibre type (I c) is observed, intermediate in myosin ATPase activity between type II and type I. After six weeks fibre type transformation is completed. No change was found, not even temporarily, in the twitch time to peak tension. It is discussed that different ex-
30
40
-50 days
Fig. 4. General trend in fibre type distribution in the deep (A) and the superficial (B) area for the control (CG) and the experimental (EG) group during 49 days after induction of compensatory hypertrophy, starting from 7 days after surgery (each point n = 5).
perimental models to induce compensatory hypertrophy have different effects on its development and final state. Acknowledgements. The authors wish to thank Mrs. HMTh Loermans for her accurate technical assistance.
References Baldwin KM, Valdez V, Herrick RE, MacIntosh AM, Roy RR (1982) Biochemical properties of overloaded fast-twitch skeletal muscle. J Appl Physiol 52: 467 -472 Binkhorst RA, Van't HofMA (1973) Force-velocity relationship and contraction time of the rat fast plantaris muscle due to compensatory hypertrophy. Pflugers Arch 342: 145 -158 Biscoe TJ, Taylor A (1967) The effect of admixture of fast and slow muscle in determining the form of the muscle twitch. Med Bioi Eng 5: 473 -479 Brody IA (1976) Regulation of isometric contraction in skeletal muscle. Exp Neurol 50: 673 - 683 Degens H, Turek Z, Binkhorst RA (1993) Compensatory hypertrophy and training effects on the functioning of ageing rat m. plantaris. Mech Ageing Dev 66: 299 - 311 Degens H, Turek Z, Hoofd LlC, Van't Hof MA, Binkhorst RA (1992) The relationship between capillarisation and fibre types during compensatory hypertrophy of the plantaris muscle in the rat. J Anat 180: 455 - 463 Dubowitz V, Brook MH (1973) Muscle biopsy: a modern approach. Saunders, London Gollnick PD, Timson BF, Moore RL, Riedy M (1981) Muscular enlargement and number of fibres in skeletal muscles of rats. J Appl Physiol 50: 936 - 943
288
Kandarian SC, Schulte LM, Esser KA (1992) Age effects on myosin subunit and biochemical alterations with skeletal muscle hypertrophy. J Appl Physiol 72: 1934-1939 Nachlas MM, Tsou KC, Souza E, de Cheng CS, Seligman AM (1957) Cytochemical demonstration of succinic dehydrogenase by the use of a new P-nitrophenyl substituted ditetrazole. J Histochem Cytochem 5: 420-436
289
Oakley CR, Gollnick PD (1985) Conversion of rat muscle fibre types. A time course study. Histochemistry 83: 555 - 560 Roy RR, Meadows ID, Baldwin KM, Edgerton VR (1982) Functional significance of compensatory overloaded rat fast muscle. J Appl Physiol 52: 473 -478 Accepted August 1, 1994
or ANATOMY = = = = = = = = =
The development of compensatory hypertrophy in the plantaris muscle of the rat H. Degens*, N. E. L. Meessen*, P. Wirtz** and R. A. Binkhorst* *Department of Physiology and **Department of Cytology and Histology, University of Nijmegen, P. O. Box 9101, NL-6500 HB Nijmegen, The Netherlands
Summary. The aim of this investigation was to study the time course of compensatory hypertrophy (CH) over a seven week period after its surgical induction in the lower limb of the rat. CH of the left plantaris muscle of the rat was induced by denervation of the ipsilateral gastrocnemius and soleus muscles. Muscle fibres were classifed as type I, I c, II a and II b. Hypertrophy of the muscle was first observed about ten days after induction of CH. All fibre types appeared to contribute to this hypertrophy. During the period between four and twenty eight days there was a marked increase in the percentage of type I fibres, mainly at the expense of type II a, as compared with control muscles. During this CH period so called 'intermediate' I c fibres were found, indicating fibre type transition taking place. The isometric twitch time to peak tension (TPT) of the plantaris muscle was studied in situ. The TPT of CH muscles remained the same during the experimental period of seven weeks. This might be explained by the effect of the increase in type I (slow) fibres being masked by the far larger number of fast fibres, which still accounted for approximately 790/0 of the total number of fibres after CH.
tion of compensatory hypertrophy, and no information was obtained during the earlier period of the development of hypertrophy. Since Gollnick et al. (1981) found, as we did, that the number of fibres did not change as a result of compensatory hypertrophy, the increase in the percentage of type I fibres obviously occurred in expense of type II fibres. Baldwin et al. (1982) already suggested that a fibre type conversion occurred during compensatory hypertrophy. Since little is known about the time course of fibre type conversion during the development of compensatory hypertrophy, we decided to study the fibre type composition and fibre crosssectional areas during the development of compensatory hypertrophy from two days up to seven weeks postoperatively. In addition we measured twitch time to peak tension to relate this parameter to changes in fibre type composition.
Materials and methods Animals and experiments
Key words: Compensatory hypertrophy - Fibre types Twitch time to peak tension - Plantaris muscle - Rat
Introduction Previous studies have shown an increase in the percentage of type I fibres (Baldwin et al. 1982; Degens et al. 1992) and a concomitant increase in the twitch time to peak tension with compensatory hypertrophy (Degens et al. 1993; Roy et al. 1982). Those data were collected several weeks after inducCorrespondence: H. Degens, Department of Human Anatomy and Cell Biology, University of Liverpool, P. O. Box 147, L69 3BX Liverpool, United Kingdom
)~I£ SEMPER
Ann Anat (1995) 177: 285 - 289 Gustav Fischer Verlag Jena
Female Wistar rats (seven weeks old; 100 - 125 g) were classed at random in a control group (CO: 56 animals) and an experimental group (EO: 60 animals). They were kept in a room maintained at 22°C with 12 h light and 12 h darkness per 24 h period. Compensatory hypertrophy was obtained by denervating the gastrocnemius and the soleus muscles of the left leg under general anaesthesia (45 mg/kg Nembutal i. p.). All the branches of the n. tibialis to these two muscles were cut and sutured to the semimembranosus or the semitendinosus muscles. This denervation procedure was followed by an operation in which either the tendon of the gastrocnemius muscle was cut (for the twitch time to peak tension study), or in which the tendon of the plantaris muscle was attached to the tendon of the denervated gastrocnemius muscle (for the histochemical studies). These operation methods have been found not to lead to different results during contractile measurements (Binkhorst and van 't Hof 1973).
Histochemistry
Results
In a second group, analyses were made of groups of five muscles of the CO group and five of the EO group at 2, 4, 7, 10, 14, 28, 42 and 49 days post-operatively. The muscles were removed from the animals under ether anaesthesia, weighed and rapidly frozen in isopentane cooled in liquid nitrogen. Serial cross-sections, perpendicular to the long axis of the muscle, of 7 !Jm were cut using a Walter Dittes cryostat at - 25°C, from the mid belly of the muscle. Sections were stained for haematoxylin-eosin, succinic dehydrogenase (SDH) according to Nachlas et al. (1957) and myofibrillar adenosine triphosphatase (ATPase) according to Dubowitz and Brook (1973) after pre-incubation at pH 4.35. The latter enzyme reaction enables the distinction between type I (dark) and type II (light) fibres. 'Intermediate' fibres (I c) showed an intermediate staining. With the SDH reaction a sub-classification can be made of the type II fibres. Type II fibres staining dark for SDH were classified as type II a, and type II fibres staining weakly for SDH were classified as type II b.
Histochemical and morphometrical analysis During the first ten days the relative muscle weight (muscle weight/rat weight) of the EG was somewhat lower in comparison with the CG (Fig. 1). After 10 days, hypertrophy developed gradually to a maximum of about 1500,10 after 6 weeks. In figure 2 the development of hypertrophy of the fibres of each type in both regions is shown. During the first ten days in the EG there appeared to be a tendency for the fibres to be slightly smaller, compared with the CG. After this period the fibres of the EG were larger, compared with the CG. Simultaneously, the percentage of type I fibres increased in the EG (p < 0.001) (Fig. 3). The total number of fibres, however, did not differ significantly between the CG (3608 ± 377; mean ± sd.) and the EG (3287 ± 388; mean ± sd.). Figure 3 also shows a transient occurrence of type I c fibres (intermediately stained), with a maximum during the period from 14 to 28 days post-operatively. Data presented in figure 4 suggest that in the deep area (A) of the muscle mainly type II a fibres passed into type I fibres.
Morphometry Because fibre type distribution in the muscle was not homogeneous (Degens et al. 1992), the deep (area A) and superficial (area B) regions were evaluated separately. In each region about 100 fibres were classified and their cross-sectional area measured. To measure the cross-sectional areas of the fibres, sections of the muscles were projected on a screen. The magnification factor was 200. A transparant gauge with 10 holes of different known diameters was laid over individual fibres on the screen. The fibres of control and hypertrophied muscles did not have an elongated appearance in the muscle sections. The hole with the diameter that most closely resembled a particular fibre was used to calculate from the magnification factor the cross-sectional area of that particular fibre. The diameters of the holes corresponded to absolute diameters of the fibres of 10 !Jm increasing with lO!Jm to 100 !Jm. An equal magnification factor was used in all muscles analysed. One may argue, that the sections are not perpendicular to the long axis of the fibres, since the angle of pinnation of the fibres may be 23 ° for control and 25 ° for hypertrophied rat plantaris muscle (Binkhorst and Van 't Hof 1973) or 15° and 17° respectively (Roy et al. 1982). The real fibre cross-sectional areas can be estimated by multiplying the measured cross-sectional areas by the cosine of the angle of pinnation. In the present study a comparison is made between the control and hypertrophied plantaris muscles and the difference in the cosine of the angle of pinnation between both cases is less than 1070. Therefore, it is reasonable to use the measured fibre cross-sectional areas for comparison between control and hypertrophied muscles.
~
0.18
~
0.16
i
0.14
...
EG
.~
III
:;:;>
0.12
CG
0.10
~
~
0.08
0
10
20
30
40
50
60 days
Fig. 1. Development of muscle hypertrophy expressed as relative weight (muscle weight per body weight) of experimental (EO) and control group (CO) during 49 days after induction of compensatory hypertrophy (each point n = 5).
Twitch time to peak tension (TPT)
Twitch time to peak tension (TPT) In 16 rats of the CO group and 20 rats of the EO group this parameter was evaluated during in situ measurements, under general anaesthesia (45 mg/g i. p. Nembutal) in a liquid paraffin pool at 35°C. TPT was determined at La, the length of the muscle at which the twitch force is maximal and TPT minimal. The apparatus and the method used are described earlier (Binkhorst and Van 't Hof 1973; Degens et al. 1993).
The relation between TPT (ms) and post-operative age in days could be expressed in a regression equation: TPT = O.013*days + 12.9 (r = 0.22) for the CG and TPT = 0.020*days + 12.9 (r = 0.38) for the EG, but both were not significant. No significant differences were found during the first post-operative days: the mean values for the TPT of muscles analyzed over the first ten days was 13.0 ± 1.1 ms (n = 6) for the CG and 12.9 ± 1.0ms (n = 10) for the EG.
Discussion After surgical intervention to induce compensatory hypertrophy the plantaris muscle is forced to take over part of the
286
area a
areab
fibre diameter )..J
25
type I
EG 20
ACG 1
'--1T----...1. . . . type II - a
25
type II - a
20
r--r
r
G
type II - b
EG 25
EG
I
20
15
q:
,
10
,
20
MG
I
30
,
I
10
50
40
20
fibre - type I %oftotal 25
20 15 10
5
intermediate 10
20
30
40
50 days
Fig. 3. Percentage of type I fibres in cross-sections of the total muscle of the control (CO) and experimental (EO) group and type Ic (intermediate) fibres in the EO during 49 days after induction of compensatory hypertrophy, starting from 7 days after surgery (each point n = 5). functions of the fast gastrocnemius as well as of the slow soleus muscles. It seems reasonable to assume that an increased effort is demanded of both the fast twitch and the slow twitch fibres in the plantaris muscle. The present results show how the muscle achieves to meet this extra loading. In agreement with another study (Gollnick et al. 1981), we
30
40
50 days
Fig. 2. Diameters of type I, IIa and II b fibres in the deep (A) and superficial (B) area of the control group (CO) and the experimental group (EO) during 49 days after induction of compensatory hypertrophy. Number of type I fibres in area B are too small for reliable counting. (Values are mean ± sd.); 1): not significant; all other cases p < 0.01).
found no significant change in the total number of fibres in the muscles after the induction of compensatory hypertrophy, implicating that: 1. hypertrophy of the muscle was paralleled by an equivalent increase in the size of individual fibres, amounting to a maximum of about 50070 at six weeks after surgery, and 2. that transformation accounted for the changes in fibre type proportion. In the present study the increase in type I fibres, due to compensatory hypertrophy, an effect which was also observed previously at a later stage (Baldwin et al. 1982; Degens et al. 1992), was found to be evident already at two weeks post-operatively. This is contrary to the study of Oakley and Gollnick (1985), in which an increase was not found until the fourth week after induction of compensatory hypertrophy. The difference in results may be attributed to the fact that Oakley and Gollnick ablated the gastrocnemius muscle leaving the soleus muscle intact. In this study both muscles were denervated, forcing the plantaris muscle not only to take over the load of the gastrocnemius, but also the postural function of the soleus muscle. This is reflected by an almost three-fold rise in the number of type I fibres in our study against a two-fold rise in the other study. The decrease of type II a fibres in the deep region accompanying the increase in type I fibres suggests, that at least in this area type II a fibres transform into type I fibres. At the level of the whole muscle this observation is supported by an increase in the content of slow (type I) myosin heavy-chains, while the amount of fast type II b myosin heavy-chain
287
area a areab fili~-~----------------------------------------------------------,
.,.
CG
CG
80
n·b
0.-.0.. _-0 ....... ____ .. _ ....... _0 __ .. - - - - ... _ ... _ .. -0 .................
60
n-a_ _x:---_.x_
40 20
00
··. . .L----.. _._ . -..-._.-·-·---·-....
filire-~
80 60
EG
EG
~
40
n-b
'><
----x
_._._.-,.
/p---o---::;::;:;--·~..().----
20
.........
0/
I ..,-'
. . ---..--..
o.. ___ ......
/
10
20
30
40
50
lI-a
~x:----:x-x
.. ""._'_.-._.-..-.--'--._..--10
20
decreased with compensatory hypertrophy as found by Kandarian et al. (1992). The presence of intermediate (type I c) fibres, especially between 14 and 28 days post-operatively, as was also observed by Oakley and Gollnick (1985), corresponds with the period of an increase of type I fibres. It is reasonable to assume that these fibres are 'transitional' fibres, i. e. being a transition from type II a to type I. On grounds of our morphometric and histochemical results revealing an increase in type I fibres one might expect to find an increase in twitch time to peak tension. No such change was found in the muscles undergoing compensatory hypertrophy up to seven weeks post-operatively, this being in contrast with the increase in twitch time to peak tension observed as a result of compensatory hypertrophy in other studies (Degens et al. 1993; Roy et al. 1982). Possibly the increase in number and size of the type I fibres is too small to measurably influence twitch time to peak tension of the muscle (Biscoe and Taylor 1967). Brody (1976) provided evidence, that the twitch time to peak tension is related more closely to the sarcotubular Ca2 + uptake than to the activity of myosin ATPase, a parameter that was not included in our study. In conclusion the present study shows that in the experimental model used, compensatory hypertrophy is preceded by a short post-surgical period in which no hypertrophy is found. During the development of compensatory hypertrophy an intermediate fibre type (I c) is observed, intermediate in myosin ATPase activity between type II and type I. After six weeks fibre type transformation is completed. No change was found, not even temporarily, in the twitch time to peak tension. It is discussed that different ex-
30
40
-50 days
Fig. 4. General trend in fibre type distribution in the deep (A) and the superficial (B) area for the control (CG) and the experimental (EG) group during 49 days after induction of compensatory hypertrophy, starting from 7 days after surgery (each point n = 5).
perimental models to induce compensatory hypertrophy have different effects on its development and final state. Acknowledgements. The authors wish to thank Mrs. HMTh Loermans for her accurate technical assistance.
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