Acta Physiol Scand 1993, 147, 347-355

Age-related changes in force and efficiency in rat skeletal muscle

A. DE H A A N ' , C. J. DE R U I T E R l , A. LIND' and A. J. SARGEANT'

Vrije Universiteit Amsterdam, T h e Netherlands ; Department of Neurophysiology, Faculty of Medicine, University of Amsterdam, T h e Netherlands

Department of Muscle and Exercise Physiology, Faculty of Human Movement Sciences,

DE HAAN, A., DE RUITER, C. J., LIND, A. & SARGEANT, A. J. 1993. Age-related changes in force and efficiency in rat skeletal muscle. Acta Physiol Scand 147, 347-355. Received 16 July 1992, accepted 13 November 1992. ISSN 0001-6772. Department of Muscle and Exercise Physiology, Vrije Universiteit and Department of Neurophysiology, University of Amsterdam, The Netherlands.

We investigated the effect of age on (the reduction of) work output, efficiency and muscle fibre type composition. Rat medial gastrocnemius muscles of three age-groups performed a series of 15 repeated contractions within 6 s (blood flow was arrested). Stimulation and shortening velocities were chosen as optimal for each group, while all muscles shortened over the same relative fibre lengths. The fibre type composition showed a higher proportion of the oxidative type IIBd fibres in the middle-aged group [Smonthsold; 39.8k6.8 vs. 23.6+4.2YOofthefibreareain theyoungrats(1.3 months old)] in contrast to the type IIBm fibres (52.9 vs. 67.9%, respectively), while the old group (22 months old) was not different from the middle-aged group. Work output in the last contraction (relative to the first contraction) was not different between the age- groups (53.1 * 18.1; 48.0k6.5 and 61.1 +6.2%, respectively). High-energy phosphate utilization was not different between the groups (150.61- 11.2; 154.61 15.6 and 157.2 7.0 pmol g-' dry wt, respectively). However, the efficiency was - 30% lower in the muscles of the youngest group, which corresponds with a lower specific power and specific tension. Since the change in fibre type composition is unlikely to be the cause of the low efficiency in the young animals, the causes remain unclear, but may be related to the rapid growth of the young rats in our study.

Key words : age, skeletal muscle, specific force, work output, energy metabolism, efficiency.

Many changes in muscle morphological and physiological parameters occur during growth (see e.g. Brooks & Faulkner 1988, Fitts et al. 1984, Goldspink 1978). In rat muscles differen- tiation into muscle fibre types has finished after N 30 d of age (Close 1964). Thereafter, only minor changes in contractile properties are observed, while muscle dimensions may increase enormously. Muscle economy of repeated iso- metric contractions was observed to decrease

Correspondence: Arnold de Haan, Department of Muscle and Exercise Physiology, Faculty of Human Movement Sciences, Vrije Universiteit, Meibergdreef 15, 1105 A2 Amsterdam, The Netherlands.

with increasing age (de Haan et al. 1988a). However, when muscle dimensions were taken into account no difference in economy was found.

Fatigue as a result of repeated isometric contractions was found to be more pronounced in muscles from young ( - 40-day-old) rats com- pared to mature ( N 125-day-old) rats (de Haan et al. 1989). The difference was only present in the first second of the contractions, which indicate that differences may exist in the speed of contraction. Using contractions containing a dynamic (shortening) phase, it was shown that reduction of power output was greater in the younger animals (de Haan et al. 1988b).

347

348 A. De Haan et al.

Differences in fatiguability of skeletal muscles with age, might indicate that there are differences in efficiency during growth. Therefore, effects of age were investigated on muscle efficiency of successive dynamic contractions. Conditions were chosen such that muscles in each group could produce their highest power output.

The used medial gastrocnemius muscle has a mixed fibre type composition (Ariano et al. 1973). Changes in fibre type composition during growth (Kovanen & Suominen 1987, Sillau & Banchero 1977) may affect fatiguability (Burke et al. 1973) and perhaps efficiency (Goldspink 1978). Therefore also the fibre type composition was measured in the used age-groups. A classification into 4 fibre types was used accord- ing to Lind & Kernel1 (1991). They suggested that the three type I1 subgroups (IIA, IIBd and IIBm) are similar to the types IIA, IIX and IIB, respectively as reported by Schiaffino et al. (1989).

METHODS Preparation of the animals. Male Wistar rats of three

different ages (1.3, 5 and 22 months old; called young, middle-aged and old, respectively) were anaesthetized with pentobarbitone (i.p. 60 mg kg-' body mass). The rat was placed on a heated pad (35 "C) to maintain body temperature. The gastrocnemius muscle of the right leg was exposed and the medial and lateral heads were separated carefully. Proximally the origin on the femur was left intact. The femur was fixed in a vertical position, the muscle was held horizontally, surrounded by a diffusing jacket through which passed heated vapour from a nebulizer to keep the temperature around the muscle at 36°C. Measurements with a needle probe showed that muscle temperature did not deviate more than 1 "C from the surrounding tem- perature. The distal tendon of the medial gastro- cnemius muscle (GM) was connected firmly to a steel link, that was attached to a force transducer. The force transducer is part of an isovelocity measuring system of which a more detailed description has been given elsewhere (de Haan et B E . 1988a). The force transducer is mounted on the lever-arm of a computer-controlled stepping motor (Compumotor Co., Petaluma, CA, USA) and is held horizontally by an air bearing. Displacements of the lever-arm were derived from the output of a potentiometer mounted on the motor axis. Force and displacement signals were digitized (fre- quency; 1OOOHz) and stored on an Apple IIe microcomputer for subsequent analysis. Contractions were induced by supramaximal stimulation of the severed sciatic nerve (Neurolog Systems; pulse height, 1 mA; pulse width, 0.5 ms) with only the branch

leading to the G M muscle left intact. Deep anaesthesia was maintained by administration of pentobarbitone as necessary.

Tetanic optimum length. Muscle twitch-optimum length (Ltw = the length with highest active twitch force) was obtained using 10 twitches at different lengths of the muscle-tendon complex (one per 30 s). The relation between tetanic optimum length (Lo), L,, and passive force exerted at L,+2.5 mm was established in a pilot study. Both twitch optimum- length and the passive force exerted by the muscle- tendon complex were used to get a first approximation of Lo. Three subsequent isometric contractions (8 min rest in between) at lengths around the approximated Lo were used to obtain Lo (within 0.5 mm). The durations and frequencies of the stimulations during these isometric contractions were 100 ms, 120 Hz (young) and 150 ms, 100 Hz (middle-aged and old). Muscle length ( L J , length of the total muscletendon complex and most distal fibre-bundle length (LJ at Lo were measured with a pair of compasses. This procedure was followed by a 10-min resting period.

Isovelocity releases. After the blood flow to the GM had been arrested, the muscle-tendon complexes (n = 6 for each age-group) performed 15 repetitive tetanic contractions within 6 s. Blood flow was arrested to prevent removal of metabolites by the blood. The stimulation frequencies used were 120 Hz (young) and 100 Hz for the middle-aged and old rats. These frequencies produced 95 yo of the maximal isometric force at Lo in the pilot study and were chosen to minimize high-frequency fatigue.

During the 15 contractions the muscles were allowed to shorten at the velocities which produced the highest power output in the pilot study. These optimal velocities of shortening (V,,,) were (mm s-I);

60 (young), 65 (middle-aged) and 75 (old rats). The sequence of stimulation, and length and force changes are shown in Figure 1. First the muscle was stretched to (Lo + 22% Lf), then stimulation started. After 20 ms of isometric force generation the muscle was allowed to shorten at a constant speed (60, 65 or 75 mm s-I). In order to prevent large influences of series-elasticity on muscle performance the isometric phase lasted only 20 ms. During this isometric phase the tension could build up to slightly over one third of the maximal isometric tension. The stimulation stopped at (L,-34% LJ . After stimulation had stopped the muscle-tendon complex was allowed to continue shortening for 30 ms. During these 30 ms the muscle relaxed and the force dropped to zero. The relaxed muscle was then stretched back to (L,+22% L,). This cycle (lasting 400 ms) was repeated 15 times.

The muscles in the three groups shortened actively over the same relative fibre distance as muscle performance and energy utilization are functions of relative muscle length (de Haan et al. 1986). Given the active shortening distances, shortening velocities and

Age-related changes in muscle ejiciency 349

I I

7 - 1 I I

I I

L I

I O V I I I I 1 7 1 I L A

0 50 100 150 200 250

Time (ms)

Fig. 1. The sequence of length changes and stimulation used during the dynamic contractions. Muscles were stretched before stimulation to Lo + 22 % L,:L,+2.6 mm (young), Lo + 3.2 mm (middle-aged) and Lo+3.5 mm (old). At t = 0 stimulation started and after 20 ms of tension generation, muscles were allowed to shorten with constant velocities. Stimu- lation stopped after 113 ms (young), 123 ms (middle- aged) and 119 ms (old) of active shortening (as. period). At the end of stimulation (at L,-34% LJ, muscle lengths were Lo-4.2 mm, Lo-4.8 mm and Lo- 5.5 mm for the young, middle-aged and old muscles, respectively. The black horizontal bar represents the stimulation period. Force (in N) is shown in the lower trace. Length (in mm) is shown in the upper trace and expressed relative to muscle optimum length. The traces were redrawn from a contraction with an old rat.

the used stimulation frequencies, the young, middle- aged and old muscles, respectively received 16, 15 and 14 stimulation pulses during one contraction. The stimulation durations ( = active shortening period + 20 ms, see Fig. 1) were 133, 143 and 139 ms for the young, middle-aged and old muscles re- spectively. Work per contraction was calculated by integrating force over shortening distance. Physio- logical cross-sectional area was calculated as the quotient of muscle volume and fibre length (Woittiez et al, 1983). Muscle volume was calculated as muscle mass divided by the density, 1.072 g cm-3 according to Gollnick et af. (1981).

Biochemical analysis. Immediately after the 15th contraction, the diffusing jacket was removed and the muscle was freeze-clamped with a pair of tongs precooled in liquid nitrogen. The contralateral GM was also freeze-clamped. After weighing, the muscle was ground in a mortar while continuously adding liquid nitrogen. The powdered muscle tissue was freeze-dried and stored at -80 "C until analysis.

Duplicate extractions were made by homogenizing drymuscletissue(7.5rfr0.5 mg)in375 pl5%(vol/vol) perchloric acid. The homogenate was centrifuged for

15 min at 0 "C (MSE l8000g). Of the supernatant 265 pl was neutralized after adding 35 pl potassium carbonate (K,CO,; 5 mol I-'). The neutralized extract was centrifuged for 3 min to remove potassium- perchlorate (Eppendorf 10.000 rpm) and stored at - 18 "C till analysis.

Enzymatic determination of phosphocreatine (PC) and ATP were performed as described by Bergmeyer (1970) on a double beam spectrofotometer (UV-190, Shimadzu, Tokyo, Japan). Lactate (LAC) was de- termined using a lactate-analyser (YSI, model 23L). From the difference (A) in concentrations of ATP, PC and LAC between the experimental and contralateral (resting) GM, high energy phosphate consumption (HEPC) was calculated (HEPC = - APC - 2 x AATP + 1.5 x ALAC). (During exhaustive exercise total ADP and AMP concentrations remain fairly unchanged. The decrease of ATP corresponds with an increase of inosine-5'-monophosphate (IMP). There- fore two high-energy phosphates hydrolysed were taken per ATP decreased). A dry wt/wet wt ratio of 0.23 was used (de Haan et al. 1986). Efficiency (mJ pmol-') is defined as the ratio work produced by the muscle (mJ) and high-energy phosphate consumed (pmol) for the whole muscle (HEPC).

Fibretype composition. Three groups (n = 4) of male Wistar rats were used to reveal possible changes in fibre type composition of the medial gastrocnemius muscle (GM) with increasing age. The first group contained young animals ( - 45 d old; mean body mass (& SD) 153 & 13 g), the rats in the second group were 'middle-aged' (- 5 months old; mean body mass 423 + 10 g) and the third group consisted of old animals (- 22 months old; mean body mass 653 140 9). In each age group the G M of the right hindlimb was excised and frozen rapidly in isopentane cooled in liquid nitrogen according to standard procedures, and stored at -80 "C for subsequent analysis. From these muscles 10 pm-thick serial cross- sections from the thickest point of the muscle belly were cut in a cryotome at - 20 "C. The cross-sections were pre-incubated in acid (pH 4.7; Brooke & Kaiser 1970) or alkaline, after pretreatment with paraform- aldehyde (pH 10.4; Guth & Samaha 1970), before staining for myosine-ATPase activity (pH 9.4). Com- bination of these two stainings show four different fibre types: I, IIA, IIBd and IIBm (Lind & Kernell 1991).

For determination of the relative (to the total cross- section) fibre area of each fibretype, fibres of 15 sections of the same size were randomly chosen from the muscle cross-section. The fibres in each of the fifteen sections (each containing - 50 fibres) were drawn manually from a microscope extended with a drawing-tube, by tracing the fibre contours. The fibre areas were calculated semi-automatically by manual tracing the microscope-drawings on a X- Y tablet. The total area of each fibretype was expressed as a

13-2

350 A . De Haan et al.

percentage of the total area. Fibres that did not match any of the standard staining intensities (probably 'type IIC' fibres) were excluded from the calculations. The error made by omitting these fibres was very small as the highest relative area occupied by ' I IC' fibres was 2.7% in the one muscle cross-section containing the most of these 'IIC' fibres.

Statistics. The one way-factor analysis of variance (ANOVA) was used to test for differences between the three age groups. The Scheffk multiple-comparison procedure was used to test for significant differences between individual group means ( P < 0.05).

RESULTS

Fibre type distribution

The different fibre types are not randomly spread over the entire muscle cross-section.

(u - E 6ooo 5000 F T 3

4000 m 2 3000 Q

2000

v

L LL P 1000

0 I I I A IIBd llBm

Fibre type

Fig. 2. Mean fibre area's of type I, IIA, IIBd, IIBm fibres for the young (a), middle-aged (a) and old group (w) (n = 4). * Denotes significant difference ( P < 0.05) between the young and the other two groups.

....... 3 llBd

- I+IIA

0 5 10 1 5 2 0 25 5, , 2 *."---. _"I_______^__--------------

0 ~ ~ ~ 1 ' ' ' '

Age (months) Fig. 3. Fibre area's (relative to total) of type I , HA, IIBd and IIBm fibres in relation to age. The type IIBd area was significantly different ( P < 0.05) between the young and the middle-aged group.

Table 1. Some morphological and force data of medial gastrocnemius muscles of young, middle- aged and old male Wistar rats (n = 6)

Young Middle-aged Old ~

Age (months)

Body mass (9)

Muscle mass (9)

Fibre length (mm)

(mm) Muscle length

Complex length

CSA

Force (N)

Specific force

(mm)

( x m')

(kN m-')

1.3 (0.2)

(17) 133

0.383 (0.045) 11.4 (0.4) 26.4 (0.5) 33.5 (2.2) 0.031

(0.004) 5.1

162.2 (12.5)

(0.6)

5.0 (0.4)

418" (4) 1.245

(0.054) 14.3"

36.4"

46.4"

(0.3)

(0.8)

(1 .O) 0.081

(0.003) 18.0 (1.1)

222.2 (13.8)

22.0 (1.0)

718 (33)

1.242 (0.092) 15.9 (1.2) 41.6 (1.1) 50.4 (1.7) 0.073

(0.00s)

(1.1) 17.0

234.4 (24.5)

Mean (and standard deviations) are given for all parameters.

All data from the young rats were significantly different ( P < 0.05) from the middle-aged and old rats.

* Denotes significant difference between the middle-aged and old rats ( P < 0.05).

Physiological cross-sectional area (CSA) was calculated as described in Methods.

Type I and IIA fibres were only located in the deep portion of the GM and were not found in the superficial region, whereas type IIBd fibres were seen in the whole muscle cross-section, with a decrease in number towards the outer regions. Type IIBm fibres were scarcely present in the deep portion of the GM but were the dominant type in the more superficial regions.

The mean fibre areas of type I, IIA and IIBd fibres were similar. Within each age-group the mean fibre area was greatest in the type IIBm fibres (Fig. 2). There was no difference with respect to the relative increase in fibre area's between the fibre types during growth.

Expressing the muscle fibre type composition as a percentage of the total area, type I and IIA fibres accounted each for only - 5 yo of the total fibre area and this did not change with age (Fig. 3). The relative total area of type IIBd fibres increased from young to middle-aged rats (P < 0.05; from 23.6k4.2 to 39.8+6.8y0),

Age-related changes in muscle eficiency 35 1

while the results for the old rats (35.5 & 6.1 yo) were not different from either the young or the middle-aged rats. Similar, but reverse, changes as seen for the IIBd fibres seemed to be present for the IIBm fibres, however no significant changes were found for these fibres (Fig. 3).

Morphology and force

The differences in muscle dimensions are summarized in Table 1. Muscle mass and cross- sectional area were higher (P < 0.05) in the middle-aged compared to the young rats, but were not different from the old rats. In contrast, muscle and fibre lengths in the old group were higher (P < 0.05) compared to the middle-aged group.

The relative increase in isometric force from young to middle-aged was greater than in cross- sectional area. As a result also specific force (force per cross-sectional area) was greater (P < 0.05) in the middle-aged compared to the young group. No significant difference in specific force was observed between middle-aged and old animals.

Work output

Work output in first contraction of the young, middle-aged and old group was 11.3f 1.6, 51.6k5.2 and 53.0f5.5 mJ, respectively.

Changes in work output during the series of 15 contractions were similar for all three groups

120 r

4 0 ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' 0 2 4 6 8 1 0 1 2 1 4 1 6

Contraction no Fig. 4. Changes in work output per contraction during the 15 successive contractions. Work is expressed as a percentage of the work output in the first contraction for each muscle. Mean data (n = 6) are presented for young (A), middle-aged (0) and old rats 1.).

(Fig. 4). After an initial enhancement of 7-12Y0, work per contraction decreased to 53.1 f 18.1, 48.0k6.5 and 61.1_+6.2y0 of the first con- traction for the young, middle-aged and old rats, respectively. There was no significant difference in reduction of work output between the three groups.

Total work output of the 15 contractions was not different between the middle-aged and old group, but the total output of the young group was significantly lower ( P < 0.05; Table 2). This difference was not only due to different muscle dimensions since the specific power was also lower in the youngest group ( P < 0.05; Table 2) .

Table 2. Total work output, specific power, total high-energy phosphate consumption (HEPC), mean rate of high-energy phosphate consumption (dHEPC) and efficiency of the GM from young, middle-aged and old rats

Young Middle-aged Old (n = 6) (n = 6) (n = 6)

Total work output (mJ) 155.5" (28.6)

Specific power (kW m-3) 256.2" (27.0)

HEPC @no1 muscle-') 13.3" (2.2) 75.5 (5.6)

(1.3)

dHEPC (pmol g-' dry wt s-l)

Efficiency (mJ pmol-') 11.7*

706.4 (77.1) 329.4 (29.8) 44.7 (5.1) 72.7 (6.7) 15.9 (0.8)

740.0 (82.0) 358.5 (28.5) 45.0

75.4

16.6

(4.9)

(3.4)

(1.8) ~

Means and (standard deviations) are given for all three groups. * Denotes significantly different ( P < 0.05) from the middle-aged and old rats.

352 A. De Haan et al.

Table 3. Metabolite concentrations of rat medial gastrocnemius muscles from the young, middle-aged and the old rats in resting muscles (Control) and after 15 repetitive isovelocity contractions (Exp)

Old (n = 6) Young (n = 6)

Control Exp A Control Exp A Control Exp A

Middle-aged (n = 6)

ATP 28.0 14.0 14.0 29.1 13.3 15.8 28.1 14.0 14.1 (1.3) (1.8) (1.8) (1.3) (2.1) (2.0) (0.7) (2.7) (2.7)

PC 87.5 33.1 54.4 85.9 33.7 52.2 92.3 40.1 52.2 (2.0) (4.2) (4.0) (7.4) (4.6) (11.9) (3.3) (9.8) (8.1)

Lactate 13.0+ 58.4 45.4 6.8 58.2 51.4 7.1 58.3 51.2 (2.0) (5.5) (7.1) (2.0) (5.1) (6.3) (1.0) (2.3) (2.8)

Mean data (and standard deviations) are given in pmol g-' dry muscle. * Denotes significant difference between young and the other two groups. A denotes the concentration difference between the control (resting) and experimental (exercised) muscle

metabolites.

Energy utilization and eficiency

Muscle concentrations of ATP, phosphocreatine (PC) and lactate of control (resting) muscles and of experimental muscles at the end of the exercise are shown in Table 3. Only the resting concentration of lactate was significantly higher (P < 0.05) in the young compared to the other groups. In all three groups the ATP con- centration decreased from - 28 to - 14 ymol g-l dry wt and PC from - 90 to 35 pmol g-l dry wt. The lactate concentration increased by - 50 pmol g-l dry wt.

Total high-energy phosphate consumption (HEPC) was significantly lower in the young rats (Table 2). However, during active periods the mean rate of energy utilization was not different between the groups (Table 2).

Efficiency (total work output/high-energy phosphate consumption) was 26-30 yo lower (P < 0.05) in the young group compared to the middle-aged and old group (Table 2).

D I S C U S S I O N

The rats in the young group are approximately 40 d old. By this age the muscles of the rat are fully differentiated, mature contractile properties of rat hindlimb muscles are reached within 30 days (Close 1964). Nevertheless these rats are very young compared to the full lifespan of Wistar rats. Male Wistar rats have a mean life time of 24 months and may live for approximately 36 months (see Larsson & Edstrom 1986). The

old (22 months, body mass 718 g) rats in this study were past the age at which their body mass reached maximum values. These old rats were characterized by a lack of spontaneous movement and obesitas.

Fibre-type distribution

The muscle cross-sections for fibre-staining were taken from the thickest point of the muscle belly. In the pennate G M it is very unlikely that all the fibres cross this point of the muscle (Gollnick et al. 1981). Furthermore there seemed to be a decrease in the relative (to the whole muscle cross-section) cross-sectional area of the deep portion of the G M (containing type I and IIA fibres) from proximal to distal parts of the GM. The deep portion of the muscle can be assumed to be cone-shaped. Although great care has been taken to cut the muscle cross-sections from the same part of the G M in each age-group, for the above mentioned reasons the data about area of fibres, have to be considered with caution. There is an increase in the relative total area of type IIBd fibres during the first months of life of the Wistar rat (Fig. 3). The total proportion of type I IB (= IIBd + IIBm) fibres does not change, indicating that the increase in Yo IIBd fibres is probably the result of a fibre-type transformation from type IIBm (young) to type IIBd (middle- aged) fibres. An age-related redistribution (from 2 to 24 months) of subtypes of type I1 fibres has also been reported by Marini et al. (1989) and Larsson et al. (1991). Unfortunately, at present

Age-related changes an muscle eficiency 353

it is unclear whether the observed shift in histochemically determined fibre type compo- sition (from young to middle-aged rats) in our experiments was accompanied by changes in oxidative capacity, fatiguability and/or myosine ATPase activity.

In all three age-groups the individual areas of the type IIBm fibres were larger than those of the IIBd fibres, type I and IIA had the smallest areas. Atrophic processes are reflected by the relatively large standard deviations of the in- dividual fibre areas in old age (Fig. 2). Con- sidering the data on relative area, there is no indication that a re-innervation process of fast twitch fibres by slow twitch neurons had occurred in the muscles of the old rats in the present study, as suggested by others (Kanda & Hashizume 1989; Kovanen & Suominen 1987). Perhaps the old rats in this study were not aged enough for such processes to take place. On the other hand there was some fibre-type grouping in the old GM, probably due to some kind of re- innervation process, but this fibre-type grouping was not limited to one particular fibre type.

SpeciJic force

An interesting result is the lower specific force in the G M of the young as compared to the middle- aged and old rats. In a recent study by de Haan et al. (1988a) a slight, but significant positive correlation ( r = 0.64) was found between body mass (range 95-490 g) and specific force (range 171-319 kN m-') for the G M of male Wistar rats. It is difficult to compare the different studies in the literature that dealt with the effect of aging on specific muscle force, because of differences in relative age, species, strains, muscles studied and methods used. Brooks & Faulkner (1988) reported a lower specific force in aged mice. In contrast in many studies no difference was found in specific muscle force between rats varying from 6 months to old age (Fitts et al. 1984, Larsson & Edstrom 1986, Larsson et al. 1991, Walters et al. 1990). We are not aware of any studies that include animals of the same relative age as the young rats in the present study.

There are a number of possible errors which might have induced the observed difference in specific force. (1) Errors in the calculation of the cross-sectional area (CSA) could have been made

owing to the fact the distal fibre bundle length was assumed to be a good representation of the mean fibre length of the G M in all three age groups. Such errors might explain part of the difference in specific force, particularly if fibre length in the young group is underestimated leading to an overestimation of CSA and therefore reducing values for specific force. However, the data on efficiency, which parameter is independent of the length measurement show similar changes to the specific force. (2) In theory it is possible that a more parallel arrangement of the muscle fibres of the G M to the line of force measurement could account for a somewYlat higher specific force of the G M in the middle-aged and old rats. Such a decrease in the angle of pennation has not been reported in the literature and even a maximal decrease from about 20" (Woittiez er al. 1986) to 0" would only account for 20% of the difference in specific force observed. Moreover, in an earlier study a 40% increase in specific force was found for the EDL-muscle (an almost parallel fibred muscle) in middle-aged and old rats compared to young animals (Lodder et al. 1991). (3) I n calculating the CSA the same value for density of the muscle tissue (I .072 g ~ m - ~ ) was used for all three age groups, which might not be entirely correct. However small changes in muscle density can not account for our results.

Fatiguing protocol

In order to make a fair as possible comparison between the three age-groups with respect to the fatiguability of the GM, the muscles were stimulated at the optimum stimulation frequency, contracting at the optimal shortening velocity (b&) and shortening over the same distance relative to fibre optimum length (LJ . In this study the mean forces during the active shortening time of the first contraction as a percentage of isometric force were: 32.8k1.8, 35.4k2.5 and 35.2_+2.3% for the young, middle-aged and old G M muscles respectively. These values indicate that the relative stress upon the GM with respect to force-velocity characteristics has indeed been comparable in all three groups. Given the differences in KPt and distance of shortening between the age groups, small differences in stimulation time occurred. This fact, together with the different optimal

354 A. De Haan et al.

stimulation frequencies, introduced a small difference in the total amount of electrical stimuli the muscles received over the 15 isovelocity contractions, i.e. 240, 225 and 210 for the young, middle-aged and old animals, respectively. These differences are inevitable as we wanted the relative shortening length to be the same for all muscles, but this might have lead to a somewhat higher stress upon the G M of the young (and middle-aged) rats relatively to the old GM.

Work a n d fat iguabi l i ty

The G M of the middle-aged and old rats produced 4.5 times and 4.8 times more work than the G M of the young animals (Table 2 ) . The difference in specific power indicates that the differences in work are related to the differences in specific force (Table 2).

If fatiguability is defined by the decrease in work performed by the GM in the 15th contraction relatively to the 1st contraction, there were no differences in fatiguability between the age-groups (Fig. 3). The reason for the large standard deviation in the relative work of the 15th contraction in the young group is unclear. In earlier work fatiguability was reported to be age-related (de Haan e t a l . 1988b). Whether the different results are due to the difference in the type of contractions (mainly isometric vs. pure dynamic) has to be investigated.

Chemical data and e&ciency

The concentrations of ATP and PC in the resting G M are in accordance with values for rats (body mass range 95-490 g) as described elsewhere (de Haan e t al. 1988 a). The mean rate of high-energy phosphate consumption (HEPC) is similar for all three groups (Table 2). This is a further indication that the relative stress upon the G M in each age-group has indeed been comparable; per gram of muscle tissue the same amounts of high-energy phosphates have been used. The differences in efficiency seem to be caused by a relative low work output in the young G M as a consequence of a lower specific force.

In conclusion it can be stated that there were relatively small differences in fibre-type dis- tribution of the medial gastrocnemius muscle, between the young and the older rats. These

differences were not related to any differences in fatiguability during the high intensity short duration exercise in the present study. The efficiency of this type of exercise was N 30% lower in the young animals. This lower efficiency was associated with a lower specific tension. The causes for the less efficient muscles in young rats remain unclear but may be related to the rapid growth of the young rats in our study.

R E F E R E N C E S

ARIANO, M.A., ARMSTRONG, R.B. & EDGERTON, V.R. 1973. Hindlimb muscle fiber population of five mammals. 3 Histochem Cytochem 21, 51-55.

RERGMEYER, H.V. 1970. Methoden der Enxymatischen Analyse. Verlag Chemie, Weinheim.

BROOKE, M.H. & KAISER, K.K. 1970. Muscle fibre types: How many and what kind? Arch Neurol23,

BROOKS, S.V. & FAULKNER, J.A. 1988. Contractile properties of skeletal muscles from young, adult and aged mice. 3 Physiol404, 71-82.

BURKE, R. E., ZEVINE, D. N., TSAIRIS, P. & ZAJAC, F.E. 1973. Physiological types and histochemical profiles in motor units of the cat gastrocnemius. 3’ Physiol234, 723-748.

CLOSE, R. 1964. Dynamic properties of fast and slow skeletal muscle of the rat during development. 3 Physiol 173, 74-95.

FITTS, R.H., TROUP, J.P., WITZMANN, F.A. & HOLLOSZY, J.O. 1984. The effect of aging and exercise on skeletal muscle function. Mechan Aging Deve l27 , 161-172.

GOLDSPINK, G. 1978. Energy turnover during con- traction of different types of muscle. In: E. Asmussen & C. Jorgensen (eds), Biomechanics V I A , International Series on Biomechanics, pp. 27-39. University Park Press, Baltimore, USA.

GOLLNICK, P. D., TIMSON, B. F., MOORE, L. F. & RIEDY, M. 1981. Muscular enlargement and number of fibres in skeletal muscles of rats. 3 Appl Physiol 50, 936-943.

GUTH, L. & SAMAHA, F.J. 1970. Procedure for the histochemical demonstration of actomysin ATPase. Exp Neurol28, 365-367.

HAAN, A. DE, JONG, J. DE, DOORN, J.E. VAN, HUIJING, P.A., WOITTIEZ, R.D. & WESTRA, H.G. 1986. Muscle economy of isometric contractions as a function of stimulation time and relative muscle length. Pfiigers Arch 407, 445-450.

HAAN, A. DE, REXWINKEL, R., DOORN, J.E. VAN, WESTRA, H.G., HOLLANDER, A.P., HUIJING, P.A., WOITTIEZ, R.D. & SARGEANT, A.J. 1988a. Influence of muscle dimensions on economy of isometric exercise in rat medial gastrocnemius muscles in situ. Eur 1 Appl Physiol 57, 6669.

369-379.

Age-related changes in muscle eficiency 355

1991. The effect of growth on specific tetanic force in skeletal muscle of the anaesthetized rat. 3 Physiol 438, 151P.

MARINI, J.F., PONS, F., ANOAL, M., LEGER, J. & LEGER, J.J. 1989. Anti-myosine Heavy Chain monoclonal antibodies reveal two IIB(fast) fibre subtypes. 3 Histochem Cytochem 37, 1721-1729.

SCHIAFFINO, S., GORZA, L., SARTORE, S., SAGGIN, J., AUSINO, S., VIANELLO, M., GUNDERSEN, K. & LOMO, T. 1989. Three myosine heavy chain isoforms in type 2 skeletal muscle fibres. 3 Muscle Res Cell Motil 10, 197-205.

SILLAU, A.H. & BANCHERO, N. 1977. Effect of maturation on capillary density, fibre size and composition in rat skeletal muscle. Proc Soc Exp Biol Med 154, 461-466.

WALTERS, T.J., SWEENEY, H.L. & FARRAR, R.P. 1990. Aging does not affect contractile properties of type IIb FDL muscle in Fischer 344 rats. A m 3 Physiol

WOITTIEZ, R.D., HUIJING, P.A. & ROZENDAL, R.H. 1983. Influence of muscle architecture on the length-force diagram in mammalian muscle. Pjliigers Arch 399, 275-279.

WOITTIEZ, R.D., HEERKENS, Y.F., HUIJING, P.A., RIJNSBURGER, W.H. & ROZENDAL, R.H. 1986. Functional morphology of the m. gastrocnemius medialis of the rat during growth. 3 Morph 187,

258, C103 1-C1035.

247-258.

HAAN, A. DE, DOORN, J.E. VAN & SARGEANT, A.J. 1988 b. Age-related changes in power output during repetitive contractions of rat medial gastrocnemius muscle. Pfiigers Arch 412, 665-667.

HAAN, A. DE, LODDER, M.A.N. & SARGEANT, A.J. 1989. Age-related effects of fatigue and recovery from fatigue in rat medial gastrocnemius muscle. Quart 3 Exp Physiol74, 715-726.

KANDA, K. & HASHIZUME, K. 1989. Changes in properties of the medial gastrocnemius motor units in aging rats. 3 Neurophysiol61, 737-746.

KOVANEN, V. & SUOMINEN, H. 1987. Effects of age and life time physical training on fibre composition of slow and fast skeletal muscle in rats. P’ugers Arch 408, 543-551.

LARSSON, L. & EDSTROM, E. 1986. Effects of age on enzyme-histochemical fibre spectra and contractile properties of fast- and slow-twitch skeletal muscles in the rat. 3 Neurol Sci 76, 69-89.

LARSSON, L., ANSVED, T., EDSTROM, L., GORZA, L. & SCHIAFFINO, S. 1991. Effects ofage on physiological, immunohistochemical and biochemical properties of fast-twitch single motor units in the rat. 3 Physiol

LIND, A. & KERNELL, D. 1991. Myofibrillar ATPase histochemistry of rat’s skeletal muscles: a ‘two- dimensional’ quantitative approach. 3 Histochem Cytochem 39, 589-597.

LODDER, M.A.N., HAAN, A. DE & SARGEANT, A.J.

443, 257-276.