J. Anat. (1977), 124, 2, pp. 371-381 With 4 figures Printed in Great Britain
371
Myosin ATPase activity after strengthening exercise M. MAZHER JAWEED, GERALD J. HERBISON AND JOHN F. DITUNNO
The Department of Rehabilitation Medicine, Thomas Jefferson University Hospital, Philadelphia, Pa. 19107
(Accepted 20 August 1976) INTRODUCTION
Recognition of a relationship between the kind of physical activity and the type of long term response of the skeletal muscles has led to the terms 'high intensityshort duration' exercises and 'low intensity-prolonged duration' exercises (Gordon, Kowalski & Fritts, 1967; Holloszy, 1967; Herbison & Gordon, 1973; Jaweed, Gordon, Herbison & Kowalski, 1974a). The former appear to enhance the strength, while the latter increase the endurance of the muscles. Although knowledge of the metabolic adaptations to such long-term endurance activities as free running and swimming has advanced a great deal in recent years (Gordon et al. 1967; Barnard, Edgerton & Peter, 1970; Baldwin et al. 1972; Gallispie, Simpson & Edgerton, 1974; Kowalski, Gordon, Martinez & Adamek, 1969; Jaweed et al. 1974a; Saubert, Armstrong, Shepherd & Gollnick, 1973; Armstrong et al. 1974), little is known about the mechanisms operative in strengthening exercises such as weight lifting (Gordon et al. 1967; Kowalski et al. 1969; Goldspink, 1970; Jaweed et al. 1974a) and isometric exercises (Exner, Staudte & Pette, 1973) in small laboratory animals. In part this may be due to the difficulties involved in developing a suitable experimental model for strengthening exercises. Forceful weight lifting increases the concentration of myofibrillar proteins (MP) in the rectus femoris (Gordon et al. 1967; Kowalski et al. 1969), vastus medialis, and soleus muscles of the rat, representing white, red and intermediate muscles respectively (Jaweed et al. 1974a). The rise in MP was similar in all three types of muscle, regardless of their innate fibre composition. This corroborates the concept that weight lifting exercises primarily affect the contractile mechanism of mnuscles (Goldspink, 1970; Goldspink & Howells, 1974). Histochemically, Kowalski et al. (1969) observed elevation of phosphorylase and succinic dehydrogenase activities after extensive weight lifting, which led them to suggest that metabolic demand in weight lifting has an incremental influence upon the enzyme activities of rat muscles. Since the activities of the oxidative and glycolytic enzymes primarily reflect the dynamic metabolic state, and may not necessarily represent the contractile state, of the muscle, it is essential to examine either the specific activity of myosin ATPase, or the speed of contraction, to determine whether or not there has been a change in the intrinsic properties of a muscle after exercise. Several investigators have attempted to demonstrate changes in the histochemical activity of myosin ATPase (Barnard et al. 1970; Saubert et al. 1973) following running: but there has been no report yet which documents changes in the fibre types, as evidenced by ATPase, as a consequence of running or weight lifting exercises in either man or experimental
372 M. MAZHER JAWEED AND OTHERS animals. The present histochemical study, therefore, was designed to look for alterations in myosin ATPase activity at pH 9 4, in response to two workloads of weight lifting exercise of differing intensity and duration. The soleus was used because of its known physiological, biochemical and histological characteristics, and because of its involvement in maintaining posture and its weight bearing function. MATERIALS AND METHODS
Animal groups Three groups (n = 8) of adult female Wistar rats, weighing between 200 and 225 g, were acclimatized for three days under standard laboratory conditions and were maintained on ad libitum purina chow and water. One group was kept sedentary, and therefore served as the inactive control (C); whereas the other two groups (A and B) were trained for different intensities and durations of weight lifting exercises (Gordon et al. 1967; Jaweed et al. 1974a), either once (A) or twice (B) each day. Exercise In group 'A' the animals daily climbed 25 times on a 45 cm long ladder set at a 50° incline, carrying weight up to 150 g on their backs, as described previously (Gordon et al. 1967; Jaweed et al. 1974a). Each session of 25 climbs was divided into 5 bouts of 5 climbs, allowing a rest period of 3 minutes between two successive bouts. The period of exercise extended over 5 days per week for 6 weeks. The rats were trained to lift weights gradually. During the first week the rats climbed the ladder without any weight. During the second week the animals carried 40 g on their backs, followed by 60 g in the third, and 100 g in the fourth week. During the final two weeks the rats climbed the ladder with 150 g on their backs. This exercise, therefore, was considered a high intensity activity for short durations. The exercise for group 'B' was based on twice daily climbing on a 90 cm long ladder at a 50° incline (Jaweed, Herbison, Ditunno & Gordon, 1974 b). The animals were trained to climb 50 times each day, one session of 25 climbs in the morning and the other 6 hours later in the afternoon, 5 days a week for a period of 6 weeks. Each session was divided into two bouts of 12 and 13 climbs, allowing a rest period of 5 minutes between bouts. During the first week the rats climbed the ladder without weights. During the second week the animals carried 40 g, followed by 100 gm in the third, and 150 g in the fourth week. During the final two weeks the animals carried 200 g on their backs. In this exercise the animals carried more weights on a longer ladder (twice the length in group A) and more frequently, twice each day. The activity thus represented a high intensity and prolonged duration exercise.
Experimental design All three groups were killed under the influence of anaesthesia (SodiumNembutal 50 mg/kg BW), 6 weeks after the start of the experiment. To stabilize the metabolism a rest period of 48 hours was allowed after the end of training before they were killed. The soleus from one side was used for wet and dry weights, while the other was utilized for histochemical procedures. The muscles were frozen in liquid nitrogen-cooled isopentane and 6,m sections were cut in a cryostat at -25 °C and stained to show myosin ATPase activity at pH 9.4, according to the method of Padykula & Herman (1955). The diameters of approximately 200-250
373 Myosin A TPase and exercise fibres of type I (= slow twitch-oxidative, SO) and type II (= fast twitch-oxidativeglycolytic, FOG) were measured by means of a filar micrometer fixed on a Zeiss microscope according to the method of Brooke (1973). The selection of the field was based on the procedure detailed elsewhere (Jaweed, Herbison & Gordon, 1974c). To quantitate the degree of reduction or enlargement of fibre size, the 'atrophic and hypertrophic' (A-H) factors were calculated for the individual rats (Brooke, 1973). The histograms for the control and exercising groups were drawn from 200 or more fibres for both fibre types from the diameter ranges arranged 8 ,um apart. The data on muscle weight, fibre diameter, and percent composition, plus the atrophic and hypertrophic factors, were analysed by Neuman-Keul's analyses of variance. RESULTS
The data on muscle weight (MW), muscle weight/body weight (MW/BW), dry weight (DW), fibre diameter and fibre type composition (type I, SO; and type II, FOG) fibres are given in Table 1 and Figure 1. Figures 2-4 illustrate the histochemical changes in the muscle fibres of the three experimental groups. Body weights Pre-experiment BW of the three groups under study were similar, and ranged between 207 and 214 g. There was likewise no significant difference in the postexperiment BW (range between 259-261). Members of the control (C) group gained 24 g in average over their original weight, whereas those of groups A and B gained 21 g and 29 g, respectively. Muscle weights Compared to control group (C), both the exercising groups showed an increase in muscle mass: group A gained 22 9 % (P < 0-05) in wet weight and 16-5 in dry weight; whereas group B increased 26-4% (P < 0-05) in wet weight and 27 5% (P < 0-05) in dry weight. The MW/BW ratios indicated parallel changes (see Table 1). The two exercising groups were non-significant in their relative increments. Percent hydration in the control and exercising groups was similar, indicating that the gain in muscle weight was not due to oedema.
Fibre diameter and fibre types; control soleus The soleus of control animals consisted of 83 % type I (SO) fibres with a fibre diameter of 42-9 ± 1.8 ,um; and 17 % type II (FOG) fibres with a fibre diameter of 37-9 ± 3-4 ,m (Table 1). Experimental soleus The fibre diameter of type I (SO) fibre in group A enlarged 13-7% (P < 0 05) over controls, along with an increase in the number of type II (FOG) fibres from 6.5 %, in the control, to 21 % in the experimental soleus. The percentage composition of the two fibres, and the fibre diameter of type II (FOG) remained unchanged as compared to controls. Group B, on the other hand, did not show any significant enlargement in the diameters of either fibre type. An increment of 8 2 % in the number of type I (SO) fibres and a similar decrease in the number of type II (FOG) fibres was significant (P < 0-01). The type II (FOG) fibres fell from 16-5 % in the control soleus to a mere 8-1 % (P < 0-01) in this experimental group, indicating a conversion of type II (FOG) fibres to type I (SO) fibres (Table 1).
374 374
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371
Myosin ATPase activity after strengthening exercise M. MAZHER JAWEED, GERALD J. HERBISON AND JOHN F. DITUNNO
The Department of Rehabilitation Medicine, Thomas Jefferson University Hospital, Philadelphia, Pa. 19107
(Accepted 20 August 1976) INTRODUCTION
Recognition of a relationship between the kind of physical activity and the type of long term response of the skeletal muscles has led to the terms 'high intensityshort duration' exercises and 'low intensity-prolonged duration' exercises (Gordon, Kowalski & Fritts, 1967; Holloszy, 1967; Herbison & Gordon, 1973; Jaweed, Gordon, Herbison & Kowalski, 1974a). The former appear to enhance the strength, while the latter increase the endurance of the muscles. Although knowledge of the metabolic adaptations to such long-term endurance activities as free running and swimming has advanced a great deal in recent years (Gordon et al. 1967; Barnard, Edgerton & Peter, 1970; Baldwin et al. 1972; Gallispie, Simpson & Edgerton, 1974; Kowalski, Gordon, Martinez & Adamek, 1969; Jaweed et al. 1974a; Saubert, Armstrong, Shepherd & Gollnick, 1973; Armstrong et al. 1974), little is known about the mechanisms operative in strengthening exercises such as weight lifting (Gordon et al. 1967; Kowalski et al. 1969; Goldspink, 1970; Jaweed et al. 1974a) and isometric exercises (Exner, Staudte & Pette, 1973) in small laboratory animals. In part this may be due to the difficulties involved in developing a suitable experimental model for strengthening exercises. Forceful weight lifting increases the concentration of myofibrillar proteins (MP) in the rectus femoris (Gordon et al. 1967; Kowalski et al. 1969), vastus medialis, and soleus muscles of the rat, representing white, red and intermediate muscles respectively (Jaweed et al. 1974a). The rise in MP was similar in all three types of muscle, regardless of their innate fibre composition. This corroborates the concept that weight lifting exercises primarily affect the contractile mechanism of mnuscles (Goldspink, 1970; Goldspink & Howells, 1974). Histochemically, Kowalski et al. (1969) observed elevation of phosphorylase and succinic dehydrogenase activities after extensive weight lifting, which led them to suggest that metabolic demand in weight lifting has an incremental influence upon the enzyme activities of rat muscles. Since the activities of the oxidative and glycolytic enzymes primarily reflect the dynamic metabolic state, and may not necessarily represent the contractile state, of the muscle, it is essential to examine either the specific activity of myosin ATPase, or the speed of contraction, to determine whether or not there has been a change in the intrinsic properties of a muscle after exercise. Several investigators have attempted to demonstrate changes in the histochemical activity of myosin ATPase (Barnard et al. 1970; Saubert et al. 1973) following running: but there has been no report yet which documents changes in the fibre types, as evidenced by ATPase, as a consequence of running or weight lifting exercises in either man or experimental
372 M. MAZHER JAWEED AND OTHERS animals. The present histochemical study, therefore, was designed to look for alterations in myosin ATPase activity at pH 9 4, in response to two workloads of weight lifting exercise of differing intensity and duration. The soleus was used because of its known physiological, biochemical and histological characteristics, and because of its involvement in maintaining posture and its weight bearing function. MATERIALS AND METHODS
Animal groups Three groups (n = 8) of adult female Wistar rats, weighing between 200 and 225 g, were acclimatized for three days under standard laboratory conditions and were maintained on ad libitum purina chow and water. One group was kept sedentary, and therefore served as the inactive control (C); whereas the other two groups (A and B) were trained for different intensities and durations of weight lifting exercises (Gordon et al. 1967; Jaweed et al. 1974a), either once (A) or twice (B) each day. Exercise In group 'A' the animals daily climbed 25 times on a 45 cm long ladder set at a 50° incline, carrying weight up to 150 g on their backs, as described previously (Gordon et al. 1967; Jaweed et al. 1974a). Each session of 25 climbs was divided into 5 bouts of 5 climbs, allowing a rest period of 3 minutes between two successive bouts. The period of exercise extended over 5 days per week for 6 weeks. The rats were trained to lift weights gradually. During the first week the rats climbed the ladder without any weight. During the second week the animals carried 40 g on their backs, followed by 60 g in the third, and 100 g in the fourth week. During the final two weeks the rats climbed the ladder with 150 g on their backs. This exercise, therefore, was considered a high intensity activity for short durations. The exercise for group 'B' was based on twice daily climbing on a 90 cm long ladder at a 50° incline (Jaweed, Herbison, Ditunno & Gordon, 1974 b). The animals were trained to climb 50 times each day, one session of 25 climbs in the morning and the other 6 hours later in the afternoon, 5 days a week for a period of 6 weeks. Each session was divided into two bouts of 12 and 13 climbs, allowing a rest period of 5 minutes between bouts. During the first week the rats climbed the ladder without weights. During the second week the animals carried 40 g, followed by 100 gm in the third, and 150 g in the fourth week. During the final two weeks the animals carried 200 g on their backs. In this exercise the animals carried more weights on a longer ladder (twice the length in group A) and more frequently, twice each day. The activity thus represented a high intensity and prolonged duration exercise.
Experimental design All three groups were killed under the influence of anaesthesia (SodiumNembutal 50 mg/kg BW), 6 weeks after the start of the experiment. To stabilize the metabolism a rest period of 48 hours was allowed after the end of training before they were killed. The soleus from one side was used for wet and dry weights, while the other was utilized for histochemical procedures. The muscles were frozen in liquid nitrogen-cooled isopentane and 6,m sections were cut in a cryostat at -25 °C and stained to show myosin ATPase activity at pH 9.4, according to the method of Padykula & Herman (1955). The diameters of approximately 200-250
373 Myosin A TPase and exercise fibres of type I (= slow twitch-oxidative, SO) and type II (= fast twitch-oxidativeglycolytic, FOG) were measured by means of a filar micrometer fixed on a Zeiss microscope according to the method of Brooke (1973). The selection of the field was based on the procedure detailed elsewhere (Jaweed, Herbison & Gordon, 1974c). To quantitate the degree of reduction or enlargement of fibre size, the 'atrophic and hypertrophic' (A-H) factors were calculated for the individual rats (Brooke, 1973). The histograms for the control and exercising groups were drawn from 200 or more fibres for both fibre types from the diameter ranges arranged 8 ,um apart. The data on muscle weight, fibre diameter, and percent composition, plus the atrophic and hypertrophic factors, were analysed by Neuman-Keul's analyses of variance. RESULTS
The data on muscle weight (MW), muscle weight/body weight (MW/BW), dry weight (DW), fibre diameter and fibre type composition (type I, SO; and type II, FOG) fibres are given in Table 1 and Figure 1. Figures 2-4 illustrate the histochemical changes in the muscle fibres of the three experimental groups. Body weights Pre-experiment BW of the three groups under study were similar, and ranged between 207 and 214 g. There was likewise no significant difference in the postexperiment BW (range between 259-261). Members of the control (C) group gained 24 g in average over their original weight, whereas those of groups A and B gained 21 g and 29 g, respectively. Muscle weights Compared to control group (C), both the exercising groups showed an increase in muscle mass: group A gained 22 9 % (P < 0-05) in wet weight and 16-5 in dry weight; whereas group B increased 26-4% (P < 0-05) in wet weight and 27 5% (P < 0-05) in dry weight. The MW/BW ratios indicated parallel changes (see Table 1). The two exercising groups were non-significant in their relative increments. Percent hydration in the control and exercising groups was similar, indicating that the gain in muscle weight was not due to oedema.
Fibre diameter and fibre types; control soleus The soleus of control animals consisted of 83 % type I (SO) fibres with a fibre diameter of 42-9 ± 1.8 ,um; and 17 % type II (FOG) fibres with a fibre diameter of 37-9 ± 3-4 ,m (Table 1). Experimental soleus The fibre diameter of type I (SO) fibre in group A enlarged 13-7% (P < 0 05) over controls, along with an increase in the number of type II (FOG) fibres from 6.5 %, in the control, to 21 % in the experimental soleus. The percentage composition of the two fibres, and the fibre diameter of type II (FOG) remained unchanged as compared to controls. Group B, on the other hand, did not show any significant enlargement in the diameters of either fibre type. An increment of 8 2 % in the number of type I (SO) fibres and a similar decrease in the number of type II (FOG) fibres was significant (P < 0-01). The type II (FOG) fibres fell from 16-5 % in the control soleus to a mere 8-1 % (P < 0-01) in this experimental group, indicating a conversion of type II (FOG) fibres to type I (SO) fibres (Table 1).
374 374
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