Rat locomotory during trotting
muscle fiber activity and galloping
T. E. SULLIVAN AND R. B. ARMSTRONG Department of Biolugy, Boston University, Boston,
SULLIVAN, T. El., AND IL B. ARMSTRONG. Rat locomotory muscle fiber activity during trotting and galloping. J. Appl. Physiol.: Respirat. Environ. Exercise Physiol. 44(S): 358-363, 1978. -This study was designed to investigate the hypothesis that the trot-gallop transition in running quadrupeds occurs when active cross-sectional areas of muscles, or fiber populations within muscles, reach some critical point as animals increase speed within trotting. Rats (Rattu norvegicus) were used as experimental animals, and glycogen depletion was used to estimate patterns of fiber activity. Our results indicate that 1) the contribution to power output by the front limb muscles was less than that of the hind limb muscles during trotting and galloping; 2) the active cross-sectional area of plantaris muscles peaked immediately prior to the transition in gait; 3) the ankle plantar flexor group of muscles as a whole did not attain a maximum active cross-sectional area during fast trotting; and 4) no major discontinuities in whole muscle or fiber type glycogen depletion rates occurred across the gait change. Although these findings do not prove the hypothesis, they support the concept that the trot-gallop transition follows the attainment of peak active muscle cross-sectional areas as animals increase trotting speed, muscle fiber t,ypes; glycogen; locomotion
exercise; motor unit recruitment;
LOCOMOTORY PATTERNSEXHIBITED
byterrestrialanimals
have been categorized into gaits from the movements of limbs and the sequence of supporting stages in the stride (5). Cavagna et al. (4) described three basic quadrupedal gaits based on measurements of the forces applied to the ground: the walk, trot, and gallop. The precise physiological significance of the gaits is unknown. However, empirical observations show that gait changes occur consistently at about the same speed in an individual animal, and that the speed, stride length, and stride frequency at the trot-gallop transition vary in a regular manner with the animal’s size (7). It was h ypothesized from this evidence and from theoretical considerations suggested by the mathematical models of McMahon (8) that the trot-gallop transition occurs when the active cross-sectional areas of muscles (1, 7), or fiber populations within muscles (l), reach a “critical point” for optimal muscle contraction. We previously tested this hypothesis by studying patterns of glycogen loss in several lion muscles through the trot-gallop transition (1). No evidence was found to support the concept that gait changes occur when whole muscles or specific fiber populations within muscles 358
Massachusetts
02215
reach maximal active cross-sectional areas. It was observed that the proportional contributions by motor unit types to whole muscle power output changed when the animals changed gaits. Results from the study using lions are useful, but it is necessary to extend the experiments to other species. This is particularly true considering the obvious shortcomings of using lions as experimental subjects. As previously discussed (l-3, 6), the use of glycogen loss as an indicator of fiber recruitment requires an experimental situation in which all fibers will utilize glycogen as a substrate during activity. Employing exercise intensities above maximal oxygen consumption (Vo, maX) levels is one way of achieving this. Comparison of the data of Shepherd and Gollnick (14) and Heglund et al. (7) indicates that th e t ro t -gallop transition in laboratory rats occurs well above VoZmax. It has also been demonstrated that rats running at these speeds metabolize significant amounts of glycogen in all their muscle fiber types (2). Rats therefore appeared to be suitable test animals for the study of muscle activity across the trotgallop transition. This study was designed to further test the hypothesis that the trot-gallop transition in running animals occurs when the active muscles attain a critical point in active cross-sectional area. We were also interested in extending the information we previously obtained on discontinuities in fiber type activity across the gait change. METHODS
Animals and training. Male Sprague-Dawley rats with an initial average body weight of 233 t 4 (SE) g (145-304 g) were housed in individual cages in a room maintained at 23 -+ 1°C. Days were artificially divided into 12 h each of light and darkness. Food (commercial pellets) and water were provided ad lib&urn. All animals were run lo-15 min/day 5 days/wk for 2-3 wk on a motor-driven treadmill. During these training sessions the running speedswere increased until speeds near the trot-gallop transition (48-60 mmmin-I) were obtained (7-10 days). At this time animals were assigned to one of four exercise groups based on their ability to slow trot (36.2 m min-I), fast trot (47.7 m min-I), slow gallop (60.1 m minP), or fast gallop (73.3 mmmin-l). These respective speeds corresponded to metabolic power outputs of 76, 101, 126, and 153% voZmax as calculated from
0021~8987/78/0000-0000$01.25
l
l
l
Copyright
0 1978 the American
Physiological
Society
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ACTIVITY
OF
RAT
MUSCLE
FIBERS
DURING
359
RUNNING
the data of Shepherd and Gollnick (14) for rats running in work wheels. Experimental protocol. Twenty-four hours after the last training session animals in the respective groups were run 200 m without electrical shock at 36.2, 47.7, 60.1, or 73.3 m min. Exercise times at these respective speeds were 5.7, 4.2, 3.3, an.d 2.7 min. After running the rats were killed by decapitation following I min of ether anesthesia. Randomly assigned rats that had rested for 24 h were killed without exercise to serve as controls. In half of the animals from each group the pectoralis major, latissimus dorsi, biceps brachii (long head), and triceps brachii (long head) muscles were studied. From the other half of the rats the soleus (S), plantaris (P), deep axial portion of the lateral head of gastrocnemius, or red gastrocnemius (R), and peripheral portion of the medial head of gastrocnemius, or white gastrocnemius (W) muscles were used. In four animals all of these muscles were taken to verify there were no systematic differences between groups. All muscles were immediately divided into two parts. One was quickly frozen in liquid nitrogen or weighed and placed in hot 30% KOH for biochemistry and the other was mounted on a specimen holder with OCT mounting medium and frozen in 2-methylbutane cooled with liquid nitrogen for histochemistry. Animal weights at the time of death averaged 360 t 5 g (243-433 g). All samples were extirpated and processed within 8 min of decapitation, Mz~cZe analysis. Samples removed for biochemistry were assayed for glycogen with the anthrone technique (13). Serial sections were cut from the tissue mounted for histochemistry in a cryostat at -2OOC. Sections were mounted on coverslips and stained for myofibrillar adenosine triphosphatase (ATPase) (lo), nicotinamide adenine dinucleotide-diaphorase (NADH-D) (9), and glycogen with the periodic acid-Schiff (PAS) stain (11). Muscle fibers were classified as slow-twitch (SO), fasttwitch-oxidative-glycolytic (FOG), or fast-twitch-glycolytic (FG) from the ATPase and NADH-D stains as described by Peter et al. (12). Fiber populations were determined by examining loo-150 fibers in fascicles randomly distributed throughout the muscle cross section. Fiber diameters were measured on the cross sections stained for NADH-D using a micrometer eyepiece. Histochemical glycogen contents were estimated from the PAS sections in at least 50 fibers of each type and were subjectively rated as dark, moderate, light, or negative. Values of 4, 3, 2, and 1, respectively, were assigned for mathematical analysis. All determinations were done using a light microscope. Biochemical rates of glycogen depletion. Whole muscle biochemical glycogen depletion rates in mmol of glucose units kg+ min-* (AG min-I) were determined by subtracting individual postexercise glycogen concentrations (G,,) from the mean for all resting glycogen concentrations (c,) and dividing by the time of the exercise l
l
l
AG*
Histochemical
l
min-3
=
GT
determinations.
G ‘X min -
(1)
The percentage of a
given fiber type per cross-sectional area of each muscle (Pf, where f -= SO, FOG, or FG) was calculated by dividing the product of the percentage fiber population (%f) of the fb1 er t ype in the muscle and the average cross-sectional area of fibers of that type (A,) by the sum of these values for all fiber types in that muscle Af*%f-* Pf - C(A,*%,)
100
Average histochemical stain intensity of each fiber type (I& in each sample was calculated by dividing the intensities of all fibers of a given fiber type by the total number of fibers of that type in that sample ( IZ$. Fiber glycogen depletion rates (AIaf min-I) were then calculated by subtracting individual animal exercise values (I., e,) from the mean resting value and dividing by the time of the exercise l
I af
=
11 f
(3)
nf
I AI,,
l
min-1
=
I min
-af’I
(4
The sum of the average staining intensity of all fiber types in a sample (Iaf) multiplied by each type’s percentage of whole muscle cross-sectional area (P,> divided by 100 gives the histochemical stain intensity of the whole muscle (I,). Whole muscle histochemical glycogen depletion rates (AI, min-I) were determined by subtracting individual animal exercise values for each muscle (I, ,,) from the mean resting value and dividing this by the time of the exercise l
Im AI,,min-1
=
=
CCIaf’
I mr-
pf)
100
(5)
I mex min
(6)
The maximal and minimal percentages of fibers of each type showing glycogen depletion (%G,) were determined for each muscle at every speed. The sum of the products of the %G/s for all three fiber types and the proportion of the total cross-sectional area of each fiber type was divided by 100 to give the maximum and minimum percentages of muscle cross-sectional area showing glycogen depletion (%Gm) %G, =
CWG,-P3
100
(7)
Statistical analysis. Statistical significance of differences in biochemical glycogen concentrati .ons was determined by use of a one-way analysis of variance and Scheffe’s multiple comparison procedure (IS), RESULTS
No significant differences were found among resting and postexercise glycogen concentrations in pectoralis, latissimus dorsi, biceps brachii, or triceps brachii muscles (Table l), so further computation of active cross-
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360
T. E. SULLIVAN
TABLE 1. Glycogen concentrations muscles at rest and after exercise
uf rat
Condition Muscle Soleus n Plantaris n Red gastrocnemius
White
n gastrocnemius
fectoralis
n major n
Latissimus
dorsi x
Biceps brachii head) n Triceps brachii head) n
(long
(long
Rest
Slow trot
Fast trot
Slow gallop
Fast gallop
31.75 22.11 8 35.53 k3.14 9 40.13 k 1.71 9 44.23 + 1.77 9 34.60 22.77 13 36.30 22.36 13 23.00 22.34 13 32.22 k2.82 II
17.09* 51.77 6 29.46 52.71 6 24.67* 53.27 6 39.29 22.94 5 29.29 41.91 a 39.44 k1.84 7 22.66 k1.46 7 26.69 20.97 8
19.84* 22.01 7 26.83 13.01 7 21.21* 21.77 6 36.87 k2.02 7 29.65 k2.06 11 36.90 k-3.04 12 22.54 k 1.81 12 27.68 ~1.76 11
21.36 k1.97 5 19.99* k4.08 6 22.41* 24.21 6 27,58* k5.79 6 29.76 -+ I.54 7 40.42 23.93 8 22.45 k3.14 8 25.27 23.12 8
28.04 k3.47 7 24.92 k3.28 7 21.44* 52.67 6 26.79* k2.63 6 28.81 42.14 12 41.03 k2.81 11 19.82 22.10 11 26.53 21.77 13
Values are means + SE, given in mmol of glucose unitsno. of animals in group. * Postexercise value significantly < 0.05).
AND
R. B. ARMSTRONG
chemically estimated whole muscle glycogen depletion rates displayed the same general patterns as the rates calculated from biochemistry (Figs. l-4). The only exception was in W during slow galloping (Fig. 4). The validity of estimating muscle glycogen levels from histochemistry was further demonstrated by regressing whole muscle histochemical staining intensity (&-,, Eq. 5) against actual glycogen concentration for each muscle. This regression yielded a significant correlation (P < 0.01) ofr = 0.80 (72 = 106). Histochemical glycogen depletion rates within fiber populations. All three fiber types increased their estimated rates of glycogen depletion across the transition SOLEUS
Glycogen Depletion Rate 0 l-l
M.
% of Muscle Showing Depletion
kg-l wet muscle weight; n = lower than resting value (P
sectional areas and glycogen depletion rates were not made for the front limb muscles. The minimal loss of glycogen in these muscles was restricted to the SO and FOG fibers. Proportiuns of muscle cross-sectional ureas showing glycogen loss. From 75 to 100% of the cross-sectional areas of S and P (Figs. 1 and 2) displayed glycogen loss after fast trotting. In P this proportion increased from about 50% during slow trotting, then leveled off through slow and fast galloping. The percentage of S showing depletion was high throughout trotting, and therefore did not reach a maximum just prior to the transition in gait. Although the proportion of R (Fig. 3) showing glycogen loss increased to about 100% during fast trotting, the white portion of the same muscle (W) only had about 25% of its area depleted (Fig. 4). Thus, a significant percentage of whole gastrocnemius muscle displayed no glycogen loss during fast trotting. No discontinuities in proportions of muscles losing glycogen occurred at the trot-gallop transition. Proportions of fiber populations showing glycogen loss. The data summarized in T&Jr fi +V-&e t.hat at most of the running speeds studied nearly all of the high oxidative (SO and FQG) fibers showed glycogen loss. Thus, increases in the proportions of the total muscle cross sections losing glycogen resulted primarily from changes in the number of FG fibers that showed depletion. Whole muscle glycogen depletion rates. In the hind limb muscles considerable variation in the patterns of glycogen depletion rates were observed when the rates were plotted as a function of speed (Figs. l-4). However, no discontinuities were observed across the trot-gallop transition in any of these muscles, and rates of depletion increased through the transition in all muscles. Histo-
’
t
+6
A -A m--m
SO Fibers FOG Fibers
T
C
36
48
60
Trot
73 Gallop
Speed
(m x rnifl’)
FIG. 1. Biochemical and histochemical glycogen data for soleus muscle. Upper graph: biochemically (B) and histochemically (H) determined glycogen depletion rates (Zefi ordinate), and percentages of muscle cross-sectional areas showing glycogen depletion (right ordinate) plotted as a function of running speed. Depletion rates were calculated from Eq. 1 and Eqs. 2-6 for B and H, respectively. Ordinate units are mmol glucose units. kg-’ (wet muscle wt) . min-l (B) and histochemical units mmin- l (H) (see METHoDs for explanation of histochemical units). Percentages of depleted muscle &oss-sectional area were calculated from Eq. 7. Depicted range represents maximum and minimum percentages of muscle showing depletion. Lower graph: histochemically determined glycogen depletion rates within fiber types of muscles as a function of speed, These values were calculated from Eg. 4. Qrdinate units are histochemical units. min-I.
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ACTIVITY
OF
RAT
MUSCLE
FIBERS
PLANTARIS
Glycogen Depletion Rote B H
DURING M.
361
RUNNING % of Muscle Showing Depletion
.
A--rB
T -100
-50
.6
A-A m--m M
SO Fibers FOG Fibers FG Fibers T
mation about the spatial recruitment of motor units within a given muscle, and the relative rates of activity of a particular muscle or fiber population within the muscle. The front limb muscles generally had higher proportions of low-oxidative (FG) fibers than the hind limb muscles (Table 3), and during running all limbs cycle at the same frequency. The absence of significant glycogen loss in the front limb muscles therefore indicates their contributions to force or power output were relatively low. From comparative studies on the modes of terrestrial locomotion in mammals, Gvambaryan (5) has suggested that rat hind limbs are particularly well placed for propulsion and provide most of the power for acceleration. On the other hand, the disparities in glycogen use between the front and hind limb muscles may represent differences in the types of contraction the muscles undergo during the step cycle. The total cross-sectional areas of several of the hindlimb muscles (R and S) were active during fast trotting, suggesting that at some point during the stride all motor units with .in the muscles may have been active. Care must be observed in this interpretation of the data because of the possibility that recruitment of units RED
Gtycogen Depletion Rate B t-i
GASTROCNEMIUS
M.
% of Muscle Showing Depletion
f
L-J,,, 36
A--r8
648
60
Trot 2. Biochemical muscle (see Fig.
FIG.
taris
73
e
Gallop Speed
.6
- e-4-l m % 1-
ISE
T
’
1
(m x mine’)
and histochemical 1 for legend).
glycogen
data
for
plan-
4-
.4 I
from trotting to galloping without any discontinuities except in W (Figs. l-4). In W, the FOG fibers showed a large increment in glycogen depletion rate during slow galloping, while the rate in the FG fibers remained low. The greatest changes in fiber type depletion rates in the muscles between slow trotting and-fast galloping occurred in the FG fibers.
2-
.2l c
oL
047
.6 DISCUSSION
In this experiment we tested the hypothesis that the -trot-gallop transition occurs when the active cross-sectional areas of locomotory muscles reach a critical point necessitating the recruitment of additional muscles or muscle groups (1, 7). Precise measurements of tensions and/or power outputs in the muscles of the running animals are not technically possible. However, glycogen depletion may be used to estimate roughly the proportion of a given muscle that is active under the proper experimental conditions. Thus, we have utilized glycogen depletion 1) to estimate the proportions of active muscle cross-sectional areas through the trot-gallop transition, and 2) to estimate rates of glycogen depletion in whole muscles and fiber populations across the erait change. These resDective estimates provide infor-
A -A m--m M
SO Fibers FOG Fibers FG Fibers
36
48
I
k _---
do
-a
T
73 Gallop
Speed 3. Biochemical gastrocnemius muscle
__--
60
Trot FIG.
’
(m x min-II
and hi stochemical (see Fig. 1 for legend).
glycogen
data
for
red
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362
T. E. SULLIVAN WHITE
Glycogen Depletion Rote B l-l
.
a--m
FOG
GASTROCNEMIUS
M.
% of Muscle Showing Depletion
Fibers
36
48
60
73
Trot Speed
3. Fiber compositions
data
for
Characteristic
Soieus ! ; Plantaris i ’ I f
Exercise Type
Slow trot
I-Soleus
SO FOG
so
Plantaris
Red gastrocnemius
gastrocnemius
99
70 96
97
97
8 100
89
94
73 25
100 100
25
1
93 5
72 5
Slow gallop
Fast gallop
84
77 70 7
99
so
FQG FG
Max
1 86
FOG FG White
Min
100
FOG FG
Fast trot
Max
97 55
70 68 15
Red gastrocnemius
White
gastrocnemius ’
77 86 50
Pectoralis
major
88 84 70
Latissimus
dorsi ’ ’ I I i ’ :
51 15
it
Because fiber glycogen contents are assigned one of four categories, is not possible to compute a single mean value for proportions of fiber populations showing glycogen loss. Thus the maximum (max> and minimum (min) possible percentages are presented.
Biceps
Triceps
within muscle fiber populations may cycle either during different phases of a stride or during successive step cycles. In either of these cases, glycogen loss in all fibers would not indicate that simultaneous activity in all units had been achieved at any given time. In fact,
brachii
brachii ;
Values diameters. text by&.
are
Area,, pm2 Populations, % Est proportion of cross sections* Area, pm2 Populations, % Est proportion of cross sections Area, pm” Populations, % Est proportion of cross sections Area, pm2 Populations, % Est proportion of cross sections Area, pm’ Populations, % Est proportion of cross sections Area, pm’ Populations, % Est proportion of cross sections Area, pm’ Populations, 8 Est proportion of cross sections Area, pm” Populations, % Est proportion of cross sections
means 2 SE. Area * Calculation of estimated
Type
so
white
2. Percentage of fiber pop&ions showing glycogen Loss
Fiber
rat muscles Fiber
and histochemical glycogen (see Fig. 1 for legend).
TABLE
Muscle
of the
(mx min-1 1 Muscle
4. Biochemical gastrocnemius muscle FIG.
R. B. ARMSTRONG
when whole muscle histochemical rates of depletion are compared with percentages of depleted areas, the depletion rates are relatively lower during trotting than during galloping (Figs. l-4). This consistent difference could have resulted from changes in the number of units contracting simultaneously, from variation in the time the units were active during each stride, or from alterations in reliance on glycogen and anaerobic metabolism. Indeed, most of the depletion occurs in the high-oxidative fibers during trotting, whereas during galloping a higher proportion of FG fibers are active. Thus one would expect the “depletion rate per active cross-sectional area” to be lower during trotting. Nevertheless, when all fibers in a muscle have lost glycogen after a brief, high-intensity bout of running, it may indicate that at some point during the step cycle all motor units were simultaneously active. For the purpose of testing the present hypothesis, this assumption will be made. Nearly all fibers in S showed glycogen loss during fast trotting. This would not seem to represent a critical point necessitating a gait change, however, for the muscle was similarly depleted during slow trotting. In R the proportion of the muscle losing glycogen increased during trotting to near lOO%, then remained constant through the galloping speeds. This pattern indicates R may have attained some optimum just prior to the gait change. The difficulty in extending this interpretation to support the hypothesis is that R is only one portion of gastrocnemius muscle. Only 25% of the cross-sectional TABLE
Gallop
AND
muscle
3,132 74.24 5 0.25 78.5
muscle
2,098 8.75 5 0.75 6.8
muscle
i
25.75'5 0.25 ) 21.5 i I 2,304 I 3,553 50.08 4 0.91 ,41.25 10.91 41,2 j 52.1 I
3,053 3,133 3,729 30.50 2 0.50 54.00 2 17.0 i 16.00 f 1.50 31.3 52.8 : 18.6
muscle
1,375 9.04 f 1.90 4.3
1,778 I 3,818 24.50 A 3,07 175.50 k 3.07 12.8 ; 87.2 r 1,675 : 4,186 41,70 L 2.92 I 49.38 k 1.34 24.2 I 71.5
muscle
1,375 6.85 k 1.34 2.2
2,444 5,607 33.45 ir 1.55 159.58 k 2.42 19.3 ! 78.3
muscle
1,485 8.50& 0.60 0.6
2,516 4,376 51.53 + 1.41 39.93+ 1.04 40.9 I 54.8
muscle
1,321 2.48 4 0.11 0.6
2,098 5,180 26.0 2 1.48 ] 71.60 + 1.50 12.6 i 86.8
muscle
I of single proportion
fibers was calculated of muscle cross sections
from fiber is described in
2.
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ACTIVITY
OF
RAT
MUSCLE
FIBERS
DURING
363
RUNNING
area of W showed glycogen loss after fast trotting, so gastrocnemius muscle as a whole was far from 100% active cross-sectional area at this speed. The depletion pattern in P was similar to that of R. The proportion of the muscle losing glycogen increased during trotting, and extrapolation of the line (Fig. 2) indicates at the actual gait change (about 55 m*min+) the muscle reached a peak active cross-sectional area. In other words, during galloping the proportion of the muscle losing glycogen did not increase above that observed during fast trotting. Although the proportion of active cross-sectional area did not attain 100% in P, this distinctive pattern suggests the muscle may have reached a critical point immediately before the animals changed gait w There are differences in individual actions of S, P, and gastrocnemius muscle, but one major group function of the muscles during locomotion is plantar flexion at the ankle. The glycogen depletion data for these muscles demonstrate a hierarchy of muscle fiber recruitment, not only within the individual muscles, but also among muscles within the group. Thus, as the animals increased their running speed, there was a progressively greater reliance on fibers with lower oxidative capacities that were situated in more peripheral muscles and more peripheral areas of muscle cross sections, respectively. Considering the ankle plantar flexor group as a whole, no critical point in active crosssectional area appears to have been reached immediately prior to the transition to galloping, since a large proportion of gastrocnemius muscle (which has 3-4 times the cross-sectional area of P and S combined) was inactive during fast trotting. On the other hand, P functions not only in ankle plantar flexion, but serves as an important digital flexor. If this muscle (as well as other possible “rate-limiting” muscles in the limbs that
were not studied) did reach some optimum in fast trotting, increased running speeds in the trotting mode would become dificult if not impossible. It therefore seems reasonable that the gait change might represent a distribution of power output to a larger muscle mass through recruitment of different muscles or muscular woups (1, 7). Nd major discontinuities in whole muscle or individual fiber type glycogen depletion rates occurred across the trot-gallop transition, except in the FOG population of W. This gait change in rats takes place at running if major speeds above Vo, max- (14), so it is doubtful alterations in substrate utilization occur in the muscles between fast trotting and slow galloping. We therefore feel reasonably confident that no major discontinuities in power output occurred with the gait transition either at the whole muscle or fiber type level in the muscles studied. These findings contrast with those from lion
(1)
l
In summary, the design of this experiment and the technical limitations inherent in the use of glycogen depletion to estimate fiber use prevent us from either proving or disproving the concept that gait changes result from muscles reaching peak contractile conditions. However, the data support the idea that some muscles do attain some sort of maximum immediately prior to the trot-gallop transition. An absolute demonstration of the validity of the hypothesis awaits the development of more sophisticated apparatus and techniques than are now available, and a systema tic study of all major locomotory muscles in an animal running through the gait transition. This work AMl-81123-02. Received
was supported
for publication
by National 2 May
Institutes
of Health
Grant
1977,
REFERENCES 1. ARMSTRONG, R, B., P. MARUM, C. W. SAUBERT IV, H. J. SEEHERMAN, AND C. R. TAYLOR. Muscle fiber activity as a function of speed and gait. J, Appl. Physiol.: Respirut. Environ. Exercise Physiol. 43: 672-677, 1977. 2. ARMSTRONG, R. B., C. W. SAUBERT IV, W. L. SEMBROWICH, R. E. SHEPHERD, AND P. D. GOLLNICK. Glycogen depletion in rat skeletal muscle fibers at different intensities and durations of exercise. Pfluegers Arch. 352: 243-256, 1974. 3. BURKE, R. E., AND V. R. EDGERTON. Motor unit properties and selective involvement in movement. Exercise Sport Sci. Rev. 3: 31-81, 1975, 4. CAVAGNA, G. A., N. C. HEGLUND, AND C. R. TAYLOR, Mechanical work in terrestrial locomotion: two basic mechanisms for minimizing energy expenditure. Am. J. Physiol. 233: R243-R261, 1977 or Am. J. Physiol.: ReguZatory Integrative Comp. Physiol. 2: R243-R261, 1977. 5, GAMBARYAN, I? D. How Mammals Run. New York: Halstead, 1974, 6. GOLLNICK, P, D., R. B. ARMSTRONG, C. W, SAUBERT IV, W. L, SEMBROWICH, AND R. E. SHEPHERD. Glycogen depletion patterns in human skeletal muscle fibers during prolonged work. Pfluegers Arch. 344: l-12, 1973. 7. HEGLUND, N. C., C. R. TAYLOR, AND T. A. MCMAM~N. Scaling stride frequency and gait to animal size: mice to horses. Science
186: 1112-1113, 1974. 8. MCMAHON, T. A. Using body size to understand the structural design of animals: quadrupedal locomotion. J. AppL Physiol. 39: 619-627, 1975. 9. NOVIKOFF, A. V., W. SHIN, AND J. DRUCKER. Mitochondrial localization of oxidative enzymes: staining results with two tetrazolium salts. J. Biophys. Biochem. Cytol. 9: 47-61, 1961, 10. PADYKULA, H. A., AND E. HERMAN, The specificity of the histochemical method of adenosine triphosphatase. J. Histothem. Cytochem. 3: 170-195, 1955. 11. PEARSE, A. G. E. Histochemistry-Theoretical and Applied. Boston, Mass.: Little, Brown, 1961, p. 832. 12. PETER, J, B,, R. J. BARNARD, V. R, EDGERTON, C. A. GILLESPIE, AND K. E. STEMPEL. Metabolic profiles of three types of skeletal muscle in guinea pigs and rabbits. Biochemistry 11: 2627-2633, 1972. 13. SELFTER, S., S. DAYTON, B. NOVIC, AND E, MUNTWYLER. The estimation of glycogen with the anthrone reagent. Arch. Biothem. 25: 191-200, 1950. 14. SHEPHERD, R, E., AND P. D. GOLLNICK. Oxygen uptake of rats at different work intensities. Pfluegers Arch. 362: 219-222, 1976. 15. SNEDECOR, G, W., AND W. G. C~CHRAN. StatisticaL Methods. Ames, Iowa: Iowa State Univ. Press, 1967, p. 271.
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muscle fiber activity and galloping
T. E. SULLIVAN AND R. B. ARMSTRONG Department of Biolugy, Boston University, Boston,
SULLIVAN, T. El., AND IL B. ARMSTRONG. Rat locomotory muscle fiber activity during trotting and galloping. J. Appl. Physiol.: Respirat. Environ. Exercise Physiol. 44(S): 358-363, 1978. -This study was designed to investigate the hypothesis that the trot-gallop transition in running quadrupeds occurs when active cross-sectional areas of muscles, or fiber populations within muscles, reach some critical point as animals increase speed within trotting. Rats (Rattu norvegicus) were used as experimental animals, and glycogen depletion was used to estimate patterns of fiber activity. Our results indicate that 1) the contribution to power output by the front limb muscles was less than that of the hind limb muscles during trotting and galloping; 2) the active cross-sectional area of plantaris muscles peaked immediately prior to the transition in gait; 3) the ankle plantar flexor group of muscles as a whole did not attain a maximum active cross-sectional area during fast trotting; and 4) no major discontinuities in whole muscle or fiber type glycogen depletion rates occurred across the gait change. Although these findings do not prove the hypothesis, they support the concept that the trot-gallop transition follows the attainment of peak active muscle cross-sectional areas as animals increase trotting speed, muscle fiber t,ypes; glycogen; locomotion
exercise; motor unit recruitment;
LOCOMOTORY PATTERNSEXHIBITED
byterrestrialanimals
have been categorized into gaits from the movements of limbs and the sequence of supporting stages in the stride (5). Cavagna et al. (4) described three basic quadrupedal gaits based on measurements of the forces applied to the ground: the walk, trot, and gallop. The precise physiological significance of the gaits is unknown. However, empirical observations show that gait changes occur consistently at about the same speed in an individual animal, and that the speed, stride length, and stride frequency at the trot-gallop transition vary in a regular manner with the animal’s size (7). It was h ypothesized from this evidence and from theoretical considerations suggested by the mathematical models of McMahon (8) that the trot-gallop transition occurs when the active cross-sectional areas of muscles (1, 7), or fiber populations within muscles (l), reach a “critical point” for optimal muscle contraction. We previously tested this hypothesis by studying patterns of glycogen loss in several lion muscles through the trot-gallop transition (1). No evidence was found to support the concept that gait changes occur when whole muscles or specific fiber populations within muscles 358
Massachusetts
02215
reach maximal active cross-sectional areas. It was observed that the proportional contributions by motor unit types to whole muscle power output changed when the animals changed gaits. Results from the study using lions are useful, but it is necessary to extend the experiments to other species. This is particularly true considering the obvious shortcomings of using lions as experimental subjects. As previously discussed (l-3, 6), the use of glycogen loss as an indicator of fiber recruitment requires an experimental situation in which all fibers will utilize glycogen as a substrate during activity. Employing exercise intensities above maximal oxygen consumption (Vo, maX) levels is one way of achieving this. Comparison of the data of Shepherd and Gollnick (14) and Heglund et al. (7) indicates that th e t ro t -gallop transition in laboratory rats occurs well above VoZmax. It has also been demonstrated that rats running at these speeds metabolize significant amounts of glycogen in all their muscle fiber types (2). Rats therefore appeared to be suitable test animals for the study of muscle activity across the trotgallop transition. This study was designed to further test the hypothesis that the trot-gallop transition in running animals occurs when the active muscles attain a critical point in active cross-sectional area. We were also interested in extending the information we previously obtained on discontinuities in fiber type activity across the gait change. METHODS
Animals and training. Male Sprague-Dawley rats with an initial average body weight of 233 t 4 (SE) g (145-304 g) were housed in individual cages in a room maintained at 23 -+ 1°C. Days were artificially divided into 12 h each of light and darkness. Food (commercial pellets) and water were provided ad lib&urn. All animals were run lo-15 min/day 5 days/wk for 2-3 wk on a motor-driven treadmill. During these training sessions the running speedswere increased until speeds near the trot-gallop transition (48-60 mmmin-I) were obtained (7-10 days). At this time animals were assigned to one of four exercise groups based on their ability to slow trot (36.2 m min-I), fast trot (47.7 m min-I), slow gallop (60.1 m minP), or fast gallop (73.3 mmmin-l). These respective speeds corresponded to metabolic power outputs of 76, 101, 126, and 153% voZmax as calculated from
0021~8987/78/0000-0000$01.25
l
l
l
Copyright
0 1978 the American
Physiological
Society
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ACTIVITY
OF
RAT
MUSCLE
FIBERS
DURING
359
RUNNING
the data of Shepherd and Gollnick (14) for rats running in work wheels. Experimental protocol. Twenty-four hours after the last training session animals in the respective groups were run 200 m without electrical shock at 36.2, 47.7, 60.1, or 73.3 m min. Exercise times at these respective speeds were 5.7, 4.2, 3.3, an.d 2.7 min. After running the rats were killed by decapitation following I min of ether anesthesia. Randomly assigned rats that had rested for 24 h were killed without exercise to serve as controls. In half of the animals from each group the pectoralis major, latissimus dorsi, biceps brachii (long head), and triceps brachii (long head) muscles were studied. From the other half of the rats the soleus (S), plantaris (P), deep axial portion of the lateral head of gastrocnemius, or red gastrocnemius (R), and peripheral portion of the medial head of gastrocnemius, or white gastrocnemius (W) muscles were used. In four animals all of these muscles were taken to verify there were no systematic differences between groups. All muscles were immediately divided into two parts. One was quickly frozen in liquid nitrogen or weighed and placed in hot 30% KOH for biochemistry and the other was mounted on a specimen holder with OCT mounting medium and frozen in 2-methylbutane cooled with liquid nitrogen for histochemistry. Animal weights at the time of death averaged 360 t 5 g (243-433 g). All samples were extirpated and processed within 8 min of decapitation, Mz~cZe analysis. Samples removed for biochemistry were assayed for glycogen with the anthrone technique (13). Serial sections were cut from the tissue mounted for histochemistry in a cryostat at -2OOC. Sections were mounted on coverslips and stained for myofibrillar adenosine triphosphatase (ATPase) (lo), nicotinamide adenine dinucleotide-diaphorase (NADH-D) (9), and glycogen with the periodic acid-Schiff (PAS) stain (11). Muscle fibers were classified as slow-twitch (SO), fasttwitch-oxidative-glycolytic (FOG), or fast-twitch-glycolytic (FG) from the ATPase and NADH-D stains as described by Peter et al. (12). Fiber populations were determined by examining loo-150 fibers in fascicles randomly distributed throughout the muscle cross section. Fiber diameters were measured on the cross sections stained for NADH-D using a micrometer eyepiece. Histochemical glycogen contents were estimated from the PAS sections in at least 50 fibers of each type and were subjectively rated as dark, moderate, light, or negative. Values of 4, 3, 2, and 1, respectively, were assigned for mathematical analysis. All determinations were done using a light microscope. Biochemical rates of glycogen depletion. Whole muscle biochemical glycogen depletion rates in mmol of glucose units kg+ min-* (AG min-I) were determined by subtracting individual postexercise glycogen concentrations (G,,) from the mean for all resting glycogen concentrations (c,) and dividing by the time of the exercise l
l
l
AG*
Histochemical
l
min-3
=
GT
determinations.
G ‘X min -
(1)
The percentage of a
given fiber type per cross-sectional area of each muscle (Pf, where f -= SO, FOG, or FG) was calculated by dividing the product of the percentage fiber population (%f) of the fb1 er t ype in the muscle and the average cross-sectional area of fibers of that type (A,) by the sum of these values for all fiber types in that muscle Af*%f-* Pf - C(A,*%,)
100
Average histochemical stain intensity of each fiber type (I& in each sample was calculated by dividing the intensities of all fibers of a given fiber type by the total number of fibers of that type in that sample ( IZ$. Fiber glycogen depletion rates (AIaf min-I) were then calculated by subtracting individual animal exercise values (I., e,) from the mean resting value and dividing by the time of the exercise l
I af
=
11 f
(3)
nf
I AI,,
l
min-1
=
I min
-af’I
(4
The sum of the average staining intensity of all fiber types in a sample (Iaf) multiplied by each type’s percentage of whole muscle cross-sectional area (P,> divided by 100 gives the histochemical stain intensity of the whole muscle (I,). Whole muscle histochemical glycogen depletion rates (AI, min-I) were determined by subtracting individual animal exercise values for each muscle (I, ,,) from the mean resting value and dividing this by the time of the exercise l
Im AI,,min-1
=
=
CCIaf’
I mr-
pf)
100
(5)
I mex min
(6)
The maximal and minimal percentages of fibers of each type showing glycogen depletion (%G,) were determined for each muscle at every speed. The sum of the products of the %G/s for all three fiber types and the proportion of the total cross-sectional area of each fiber type was divided by 100 to give the maximum and minimum percentages of muscle cross-sectional area showing glycogen depletion (%Gm) %G, =
CWG,-P3
100
(7)
Statistical analysis. Statistical significance of differences in biochemical glycogen concentrati .ons was determined by use of a one-way analysis of variance and Scheffe’s multiple comparison procedure (IS), RESULTS
No significant differences were found among resting and postexercise glycogen concentrations in pectoralis, latissimus dorsi, biceps brachii, or triceps brachii muscles (Table l), so further computation of active cross-
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360
T. E. SULLIVAN
TABLE 1. Glycogen concentrations muscles at rest and after exercise
uf rat
Condition Muscle Soleus n Plantaris n Red gastrocnemius
White
n gastrocnemius
fectoralis
n major n
Latissimus
dorsi x
Biceps brachii head) n Triceps brachii head) n
(long
(long
Rest
Slow trot
Fast trot
Slow gallop
Fast gallop
31.75 22.11 8 35.53 k3.14 9 40.13 k 1.71 9 44.23 + 1.77 9 34.60 22.77 13 36.30 22.36 13 23.00 22.34 13 32.22 k2.82 II
17.09* 51.77 6 29.46 52.71 6 24.67* 53.27 6 39.29 22.94 5 29.29 41.91 a 39.44 k1.84 7 22.66 k1.46 7 26.69 20.97 8
19.84* 22.01 7 26.83 13.01 7 21.21* 21.77 6 36.87 k2.02 7 29.65 k2.06 11 36.90 k-3.04 12 22.54 k 1.81 12 27.68 ~1.76 11
21.36 k1.97 5 19.99* k4.08 6 22.41* 24.21 6 27,58* k5.79 6 29.76 -+ I.54 7 40.42 23.93 8 22.45 k3.14 8 25.27 23.12 8
28.04 k3.47 7 24.92 k3.28 7 21.44* 52.67 6 26.79* k2.63 6 28.81 42.14 12 41.03 k2.81 11 19.82 22.10 11 26.53 21.77 13
Values are means + SE, given in mmol of glucose unitsno. of animals in group. * Postexercise value significantly < 0.05).
AND
R. B. ARMSTRONG
chemically estimated whole muscle glycogen depletion rates displayed the same general patterns as the rates calculated from biochemistry (Figs. l-4). The only exception was in W during slow galloping (Fig. 4). The validity of estimating muscle glycogen levels from histochemistry was further demonstrated by regressing whole muscle histochemical staining intensity (&-,, Eq. 5) against actual glycogen concentration for each muscle. This regression yielded a significant correlation (P < 0.01) ofr = 0.80 (72 = 106). Histochemical glycogen depletion rates within fiber populations. All three fiber types increased their estimated rates of glycogen depletion across the transition SOLEUS
Glycogen Depletion Rate 0 l-l
M.
% of Muscle Showing Depletion
kg-l wet muscle weight; n = lower than resting value (P
sectional areas and glycogen depletion rates were not made for the front limb muscles. The minimal loss of glycogen in these muscles was restricted to the SO and FOG fibers. Proportiuns of muscle cross-sectional ureas showing glycogen loss. From 75 to 100% of the cross-sectional areas of S and P (Figs. 1 and 2) displayed glycogen loss after fast trotting. In P this proportion increased from about 50% during slow trotting, then leveled off through slow and fast galloping. The percentage of S showing depletion was high throughout trotting, and therefore did not reach a maximum just prior to the transition in gait. Although the proportion of R (Fig. 3) showing glycogen loss increased to about 100% during fast trotting, the white portion of the same muscle (W) only had about 25% of its area depleted (Fig. 4). Thus, a significant percentage of whole gastrocnemius muscle displayed no glycogen loss during fast trotting. No discontinuities in proportions of muscles losing glycogen occurred at the trot-gallop transition. Proportions of fiber populations showing glycogen loss. The data summarized in T&Jr fi +V-&e t.hat at most of the running speeds studied nearly all of the high oxidative (SO and FQG) fibers showed glycogen loss. Thus, increases in the proportions of the total muscle cross sections losing glycogen resulted primarily from changes in the number of FG fibers that showed depletion. Whole muscle glycogen depletion rates. In the hind limb muscles considerable variation in the patterns of glycogen depletion rates were observed when the rates were plotted as a function of speed (Figs. l-4). However, no discontinuities were observed across the trot-gallop transition in any of these muscles, and rates of depletion increased through the transition in all muscles. Histo-
’
t
+6
A -A m--m
SO Fibers FOG Fibers
T
C
36
48
60
Trot
73 Gallop
Speed
(m x rnifl’)
FIG. 1. Biochemical and histochemical glycogen data for soleus muscle. Upper graph: biochemically (B) and histochemically (H) determined glycogen depletion rates (Zefi ordinate), and percentages of muscle cross-sectional areas showing glycogen depletion (right ordinate) plotted as a function of running speed. Depletion rates were calculated from Eq. 1 and Eqs. 2-6 for B and H, respectively. Ordinate units are mmol glucose units. kg-’ (wet muscle wt) . min-l (B) and histochemical units mmin- l (H) (see METHoDs for explanation of histochemical units). Percentages of depleted muscle &oss-sectional area were calculated from Eq. 7. Depicted range represents maximum and minimum percentages of muscle showing depletion. Lower graph: histochemically determined glycogen depletion rates within fiber types of muscles as a function of speed, These values were calculated from Eg. 4. Qrdinate units are histochemical units. min-I.
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ACTIVITY
OF
RAT
MUSCLE
FIBERS
PLANTARIS
Glycogen Depletion Rote B H
DURING M.
361
RUNNING % of Muscle Showing Depletion
.
A--rB
T -100
-50
.6
A-A m--m M
SO Fibers FOG Fibers FG Fibers T
mation about the spatial recruitment of motor units within a given muscle, and the relative rates of activity of a particular muscle or fiber population within the muscle. The front limb muscles generally had higher proportions of low-oxidative (FG) fibers than the hind limb muscles (Table 3), and during running all limbs cycle at the same frequency. The absence of significant glycogen loss in the front limb muscles therefore indicates their contributions to force or power output were relatively low. From comparative studies on the modes of terrestrial locomotion in mammals, Gvambaryan (5) has suggested that rat hind limbs are particularly well placed for propulsion and provide most of the power for acceleration. On the other hand, the disparities in glycogen use between the front and hind limb muscles may represent differences in the types of contraction the muscles undergo during the step cycle. The total cross-sectional areas of several of the hindlimb muscles (R and S) were active during fast trotting, suggesting that at some point during the stride all motor units with .in the muscles may have been active. Care must be observed in this interpretation of the data because of the possibility that recruitment of units RED
Gtycogen Depletion Rate B t-i
GASTROCNEMIUS
M.
% of Muscle Showing Depletion
f
L-J,,, 36
A--r8
648
60
Trot 2. Biochemical muscle (see Fig.
FIG.
taris
73
e
Gallop Speed
.6
- e-4-l m % 1-
ISE
T
’
1
(m x mine’)
and histochemical 1 for legend).
glycogen
data
for
plan-
4-
.4 I
from trotting to galloping without any discontinuities except in W (Figs. l-4). In W, the FOG fibers showed a large increment in glycogen depletion rate during slow galloping, while the rate in the FG fibers remained low. The greatest changes in fiber type depletion rates in the muscles between slow trotting and-fast galloping occurred in the FG fibers.
2-
.2l c
oL
047
.6 DISCUSSION
In this experiment we tested the hypothesis that the -trot-gallop transition occurs when the active cross-sectional areas of locomotory muscles reach a critical point necessitating the recruitment of additional muscles or muscle groups (1, 7). Precise measurements of tensions and/or power outputs in the muscles of the running animals are not technically possible. However, glycogen depletion may be used to estimate roughly the proportion of a given muscle that is active under the proper experimental conditions. Thus, we have utilized glycogen depletion 1) to estimate the proportions of active muscle cross-sectional areas through the trot-gallop transition, and 2) to estimate rates of glycogen depletion in whole muscles and fiber populations across the erait change. These resDective estimates provide infor-
A -A m--m M
SO Fibers FOG Fibers FG Fibers
36
48
I
k _---
do
-a
T
73 Gallop
Speed 3. Biochemical gastrocnemius muscle
__--
60
Trot FIG.
’
(m x min-II
and hi stochemical (see Fig. 1 for legend).
glycogen
data
for
red
Downloaded from www.physiology.org/journal/jappl by ${individualUser.givenNames} ${individualUser.surname} (129.186.138.035) on January 13, 2019.
362
T. E. SULLIVAN WHITE
Glycogen Depletion Rote B l-l
.
a--m
FOG
GASTROCNEMIUS
M.
% of Muscle Showing Depletion
Fibers
36
48
60
73
Trot Speed
3. Fiber compositions
data
for
Characteristic
Soieus ! ; Plantaris i ’ I f
Exercise Type
Slow trot
I-Soleus
SO FOG
so
Plantaris
Red gastrocnemius
gastrocnemius
99
70 96
97
97
8 100
89
94
73 25
100 100
25
1
93 5
72 5
Slow gallop
Fast gallop
84
77 70 7
99
so
FQG FG
Max
1 86
FOG FG White
Min
100
FOG FG
Fast trot
Max
97 55
70 68 15
Red gastrocnemius
White
gastrocnemius ’
77 86 50
Pectoralis
major
88 84 70
Latissimus
dorsi ’ ’ I I i ’ :
51 15
it
Because fiber glycogen contents are assigned one of four categories, is not possible to compute a single mean value for proportions of fiber populations showing glycogen loss. Thus the maximum (max> and minimum (min) possible percentages are presented.
Biceps
Triceps
within muscle fiber populations may cycle either during different phases of a stride or during successive step cycles. In either of these cases, glycogen loss in all fibers would not indicate that simultaneous activity in all units had been achieved at any given time. In fact,
brachii
brachii ;
Values diameters. text by&.
are
Area,, pm2 Populations, % Est proportion of cross sections* Area, pm2 Populations, % Est proportion of cross sections Area, pm” Populations, % Est proportion of cross sections Area, pm2 Populations, % Est proportion of cross sections Area, pm’ Populations, % Est proportion of cross sections Area, pm’ Populations, % Est proportion of cross sections Area, pm’ Populations, 8 Est proportion of cross sections Area, pm” Populations, % Est proportion of cross sections
means 2 SE. Area * Calculation of estimated
Type
so
white
2. Percentage of fiber pop&ions showing glycogen Loss
Fiber
rat muscles Fiber
and histochemical glycogen (see Fig. 1 for legend).
TABLE
Muscle
of the
(mx min-1 1 Muscle
4. Biochemical gastrocnemius muscle FIG.
R. B. ARMSTRONG
when whole muscle histochemical rates of depletion are compared with percentages of depleted areas, the depletion rates are relatively lower during trotting than during galloping (Figs. l-4). This consistent difference could have resulted from changes in the number of units contracting simultaneously, from variation in the time the units were active during each stride, or from alterations in reliance on glycogen and anaerobic metabolism. Indeed, most of the depletion occurs in the high-oxidative fibers during trotting, whereas during galloping a higher proportion of FG fibers are active. Thus one would expect the “depletion rate per active cross-sectional area” to be lower during trotting. Nevertheless, when all fibers in a muscle have lost glycogen after a brief, high-intensity bout of running, it may indicate that at some point during the step cycle all motor units were simultaneously active. For the purpose of testing the present hypothesis, this assumption will be made. Nearly all fibers in S showed glycogen loss during fast trotting. This would not seem to represent a critical point necessitating a gait change, however, for the muscle was similarly depleted during slow trotting. In R the proportion of the muscle losing glycogen increased during trotting to near lOO%, then remained constant through the galloping speeds. This pattern indicates R may have attained some optimum just prior to the gait change. The difficulty in extending this interpretation to support the hypothesis is that R is only one portion of gastrocnemius muscle. Only 25% of the cross-sectional TABLE
Gallop
AND
muscle
3,132 74.24 5 0.25 78.5
muscle
2,098 8.75 5 0.75 6.8
muscle
i
25.75'5 0.25 ) 21.5 i I 2,304 I 3,553 50.08 4 0.91 ,41.25 10.91 41,2 j 52.1 I
3,053 3,133 3,729 30.50 2 0.50 54.00 2 17.0 i 16.00 f 1.50 31.3 52.8 : 18.6
muscle
1,375 9.04 f 1.90 4.3
1,778 I 3,818 24.50 A 3,07 175.50 k 3.07 12.8 ; 87.2 r 1,675 : 4,186 41,70 L 2.92 I 49.38 k 1.34 24.2 I 71.5
muscle
1,375 6.85 k 1.34 2.2
2,444 5,607 33.45 ir 1.55 159.58 k 2.42 19.3 ! 78.3
muscle
1,485 8.50& 0.60 0.6
2,516 4,376 51.53 + 1.41 39.93+ 1.04 40.9 I 54.8
muscle
1,321 2.48 4 0.11 0.6
2,098 5,180 26.0 2 1.48 ] 71.60 + 1.50 12.6 i 86.8
muscle
I of single proportion
fibers was calculated of muscle cross sections
from fiber is described in
2.
Downloaded from www.physiology.org/journal/jappl by ${individualUser.givenNames} ${individualUser.surname} (129.186.138.035) on January 13, 2019.
ACTIVITY
OF
RAT
MUSCLE
FIBERS
DURING
363
RUNNING
area of W showed glycogen loss after fast trotting, so gastrocnemius muscle as a whole was far from 100% active cross-sectional area at this speed. The depletion pattern in P was similar to that of R. The proportion of the muscle losing glycogen increased during trotting, and extrapolation of the line (Fig. 2) indicates at the actual gait change (about 55 m*min+) the muscle reached a peak active cross-sectional area. In other words, during galloping the proportion of the muscle losing glycogen did not increase above that observed during fast trotting. Although the proportion of active cross-sectional area did not attain 100% in P, this distinctive pattern suggests the muscle may have reached a critical point immediately before the animals changed gait w There are differences in individual actions of S, P, and gastrocnemius muscle, but one major group function of the muscles during locomotion is plantar flexion at the ankle. The glycogen depletion data for these muscles demonstrate a hierarchy of muscle fiber recruitment, not only within the individual muscles, but also among muscles within the group. Thus, as the animals increased their running speed, there was a progressively greater reliance on fibers with lower oxidative capacities that were situated in more peripheral muscles and more peripheral areas of muscle cross sections, respectively. Considering the ankle plantar flexor group as a whole, no critical point in active crosssectional area appears to have been reached immediately prior to the transition to galloping, since a large proportion of gastrocnemius muscle (which has 3-4 times the cross-sectional area of P and S combined) was inactive during fast trotting. On the other hand, P functions not only in ankle plantar flexion, but serves as an important digital flexor. If this muscle (as well as other possible “rate-limiting” muscles in the limbs that
were not studied) did reach some optimum in fast trotting, increased running speeds in the trotting mode would become dificult if not impossible. It therefore seems reasonable that the gait change might represent a distribution of power output to a larger muscle mass through recruitment of different muscles or muscular woups (1, 7). Nd major discontinuities in whole muscle or individual fiber type glycogen depletion rates occurred across the trot-gallop transition, except in the FOG population of W. This gait change in rats takes place at running if major speeds above Vo, max- (14), so it is doubtful alterations in substrate utilization occur in the muscles between fast trotting and slow galloping. We therefore feel reasonably confident that no major discontinuities in power output occurred with the gait transition either at the whole muscle or fiber type level in the muscles studied. These findings contrast with those from lion
(1)
l
In summary, the design of this experiment and the technical limitations inherent in the use of glycogen depletion to estimate fiber use prevent us from either proving or disproving the concept that gait changes result from muscles reaching peak contractile conditions. However, the data support the idea that some muscles do attain some sort of maximum immediately prior to the trot-gallop transition. An absolute demonstration of the validity of the hypothesis awaits the development of more sophisticated apparatus and techniques than are now available, and a systema tic study of all major locomotory muscles in an animal running through the gait transition. This work AMl-81123-02. Received
was supported
for publication
by National 2 May
Institutes
of Health
Grant
1977,
REFERENCES 1. ARMSTRONG, R, B., P. MARUM, C. W. SAUBERT IV, H. J. SEEHERMAN, AND C. R. TAYLOR. Muscle fiber activity as a function of speed and gait. J, Appl. Physiol.: Respirut. Environ. Exercise Physiol. 43: 672-677, 1977. 2. ARMSTRONG, R. B., C. W. SAUBERT IV, W. L. SEMBROWICH, R. E. SHEPHERD, AND P. D. GOLLNICK. Glycogen depletion in rat skeletal muscle fibers at different intensities and durations of exercise. Pfluegers Arch. 352: 243-256, 1974. 3. BURKE, R. E., AND V. R. EDGERTON. Motor unit properties and selective involvement in movement. Exercise Sport Sci. Rev. 3: 31-81, 1975, 4. CAVAGNA, G. A., N. C. HEGLUND, AND C. R. TAYLOR, Mechanical work in terrestrial locomotion: two basic mechanisms for minimizing energy expenditure. Am. J. Physiol. 233: R243-R261, 1977 or Am. J. Physiol.: ReguZatory Integrative Comp. Physiol. 2: R243-R261, 1977. 5, GAMBARYAN, I? D. How Mammals Run. New York: Halstead, 1974, 6. GOLLNICK, P, D., R. B. ARMSTRONG, C. W, SAUBERT IV, W. L, SEMBROWICH, AND R. E. SHEPHERD. Glycogen depletion patterns in human skeletal muscle fibers during prolonged work. Pfluegers Arch. 344: l-12, 1973. 7. HEGLUND, N. C., C. R. TAYLOR, AND T. A. MCMAM~N. Scaling stride frequency and gait to animal size: mice to horses. Science
186: 1112-1113, 1974. 8. MCMAHON, T. A. Using body size to understand the structural design of animals: quadrupedal locomotion. J. AppL Physiol. 39: 619-627, 1975. 9. NOVIKOFF, A. V., W. SHIN, AND J. DRUCKER. Mitochondrial localization of oxidative enzymes: staining results with two tetrazolium salts. J. Biophys. Biochem. Cytol. 9: 47-61, 1961, 10. PADYKULA, H. A., AND E. HERMAN, The specificity of the histochemical method of adenosine triphosphatase. J. Histothem. Cytochem. 3: 170-195, 1955. 11. PEARSE, A. G. E. Histochemistry-Theoretical and Applied. Boston, Mass.: Little, Brown, 1961, p. 832. 12. PETER, J, B,, R. J. BARNARD, V. R, EDGERTON, C. A. GILLESPIE, AND K. E. STEMPEL. Metabolic profiles of three types of skeletal muscle in guinea pigs and rabbits. Biochemistry 11: 2627-2633, 1972. 13. SELFTER, S., S. DAYTON, B. NOVIC, AND E, MUNTWYLER. The estimation of glycogen with the anthrone reagent. Arch. Biothem. 25: 191-200, 1950. 14. SHEPHERD, R, E., AND P. D. GOLLNICK. Oxygen uptake of rats at different work intensities. Pfluegers Arch. 362: 219-222, 1976. 15. SNEDECOR, G, W., AND W. G. C~CHRAN. StatisticaL Methods. Ames, Iowa: Iowa State Univ. Press, 1967, p. 271.
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