Force-velocity shifts with repetitive isometric and isotonic contractions of canine gastrocnemius in situ B. T. AMEREDES, W. F. BRECHUE, G. M. ANDREW, AND W. N. STAINSBY Departments of Physiology and Pharmacology, College of Medicine, University of Florida, Gainesville, Florida 32610 AMEREDES,B. T., W. F. BRECHUE,G. M. ANDREW,AND isometric contractions (3), indicating an effect of muscle W. N. STAINSBY.Force-velocity shifts with repetitive isometric shortening on the fatigue process. Furthermore, these and isotonic
contructions
of
canine
gastrocnemius
in situ. J.
Appl. Physiol. 73(5): 2105-2111, 1992.-The force-velocity (FV) relationships of canine gastrocnemius-plantarismusclesat optimal musclelength in situ were studied before and after 10 min of repetitive isometric or isotonic tetanic contractions induced by electrical stimulation of the sciatic nerve (200-ms trains, 50 impulses/s, 1 contraction/s). F-V relationships and maximal velocity of shortening ( Vmax) were determined by curve fitting with the Hill equation. Mean Vmax before fatigue was 3.8 t 0.2 (SE) average fiber lengths/s; mean maximal isometric tension (P,) was 508 t 15 g/g. With a significant decrease of force development during isometric contractions (-27 t 4%, P < 0.01, n = 5), Vmaxwas unchanged. However, with repetitive isotonic contractions at a low load (P/P, = 0.25, n = 5), a significant decreasein Vmag was observed(-21 & 2%, P < O.Ol), whereasP, wasunchanged.Isotonic contractions at an intermediate load (P/P, = 0.5, n = 4) resulted in significant decreasesin both Vmax(-26 t 6%, P c 0.05) and P, (-12 k 2%, P < 0.01). These results show that repeated contractions of canine skeletal muscleproduce specific changesin the F-V relationship that are dependenton the type of contractions being performed and indicate that decreasesin other contractile properties, such asvelocity development and shortening, can occur independently of changesin isometric tension. fatigue; Hill equation power; shortening; maximal velocity of shortening
MUSCLE FATKXJX can be described
pected muscle served series
as a loss of the experformance of a muscle fiber, motor unit, or group (9). This loss of performance can be obduring a single sustained contraction or during a of repeated shorter contractions. The latter has
been shown to be affected by both metabolic factors, such
as oxygen availability (4,15, 16), and mechanical factors, such as duty cycle (5). Two measures of muscle performance that are of interest are force production and velocity development. Also, implicit in the measurement of velocity development is the amount of muscle shortening or displacement. Although classically defined as a drop in force production, we propose that fatigue may also be defin ed as a decrease in the ability to sho irten and develop velocity or power (12), as in the case of isotonic contractions where the force production is constant. Previous studies of the canine diaphragm muscle in situ demonstrated a greater loss of force development during isovelocity muscle contractions compared with
results suggested an effect of muscle shortening on the force-velocity (F-V) characteristics of fatigued muscle (3). Thus the purpose of the following study was to 1) measure changes in the F-V relationship that occur with repetitive shortening (isotonic) and nonshortening (isometric) contractions and 2) determine whether the mechanical conditions of fatiguing contractions have specific effects on the ability of the muscle to develop the full range of force and velocity. The studies were performed with the canine gastrocnemius-plantaris (GP) muscle, prepared with a minimum of surgery (20), in which mechanical performance was recorded using a lever system specifically designed for this purpose (10). METHODS
General methods. Fourteen mongrel dogs (16 t 3 kg) were anesthetized with pentobarbital sodium (30 mg/kg iv), followed by maintenance doses of 60 mg. The animals were ventilated through an endotracheal tube with a Harvard respirator (tidal volume = 25 ml/kg). End-tidal CO, was maintained at 4.5-5% by adjustment of pump frequency. The right femoral artery was catheterized to provide arterial blood pressure measurements and blood sampling capability, and the right femoral vein was catheterized to allow administration of fluids and anesthetic. Body temperature was monitored with an intraesophageal probe and was maintained at 37-40°C with a heating pad on the body. Surgical methods. A longitudinal incision was made in the posterior thigh to gain access to the sciatic notch between the muscles. The sciatic nerve was located, cleared, and cut, and the distal end was placed into an electrode holder. The incision was closed with wound clips so that the electrode holder remained in place, and the wires were exited through the remaining slit in the skin. Another small incision was made on the lateral side of the popliteal area through which the peroneal nerve was ligated so as to avoid dorsiflexion of the foot during sciatic nerve stimulation. A third incision was made in the skin over the area at which the medial gastrocnemius muscle connects to the calcaneus tendon to gain access to the tendon and the lower portion of the GP muscle group. The tendon was cleared and cut close to the calcaneus and was placed into a tendon clamp that was later connected to a pneumatic lever (10). The origin of the GP
0161-7567/92 $2.00 Copyright 0 1992 the American Physiological Society
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was anchored by bone nails placed in the femur and tibia, and a strut was placed between the lever and bone nails to minimize flexing. A thermoprobe was inserted into the connective tissue between the muscle and the tibia to provide a measure of muscle temperature, which was maintained at 37-39*C with a heat lamp overhead. Saline-soaked gauze and plastic wrap were placed over the incision to maintain muscle moisture. Optimal muscle length. Optimal muscle length was defined as the length at which maximal isometric force was developed using twitch contractions (0.2 ms, 4 V) and was found to correspond to within 2 t 2% of the length (L,) at which maximal tetanic isometric tension (P,) was developed. L, was measured as the length from the tibia1 origin of the medial head of the muscle to the point at which the visible muscle fibers terminated on the calcaneus tendon [ 12.3 t 0.5 (SE) cm]. This value was used to calculate the average fiber length (.& = 3.5 +- 0.1 cm) within the muscle at optimal muscle length (2). As will be described, the normalization of shortening velocity between each muscle studied relied on calculation of E, within each muscle. The GP muscle is pennate, having a significant number of fibers at an angle to the long axis of the muscle. Previous studies in this laboratory, using light and electron microscopy on fixed canine GP muscle sections at L,, have shown that the angle of pennation averages 20° (2) and that average muscle fiber length at L, can be calculated by dividing whole muscle length by a factor of 3.5 (2). Thus, although some inaccuracies may exist for a given population of fibers within a muscle, this method of calculation results in accurate fiber length values for the average of all fibers within the canine GP muscle. Force and velocity measurements. This study utilized a muscle preparation that was created in situ with a minimum of surgery to investigate the fatigue characteristics of a skeletal muscle in a situation as close to in vivo conditions as possible, with the exception of electrical stimulation of a cut nerve. One possible complication of this approach is that of contributions of nearby muscles to the mechanics of contractions assumed to be solely a product of the GP muscle. Therefore a series of control isometric (72 = 3) and isotonic (n = 3) experiments was performed to assess these effects. With each major muscle removal (semitendinosus, gracilis, sartorius, and semimembranosus), force or shortening was reassessed to determine the magnitude of change in the measured mechanical characteristics of the GP muscle. Furthermore, nerves exiting the muscle on the tibia1 side were also ligated. These experiments typically took IO-30 min, during which neither isometric force nor isotonic shortening changed ~2%. These studies indicated that the measured mechanical performance of the minimally prepared GP muscle was an accurate depiction of events occurring in that muscle group alone. Measurements of muscle force and shortening were accomplished with a pneumatic muscle lever in which compressed air was used to produce force loads on the muscle (9). This lever system had an air-expandable metal bellows attached to a pivot arm and could be filled to any desired pressure up to 50 psi. Typically, 2 psi was enough to expand the bellows and move the pivot arm up
REPETITIVE
CONTRACTIONS
to a length stop above the pivot arm, placing the muscle at L,, as previously determined by isometric twitch contractions. This resulted in a passive tension of ~45 g/g within the muscle when set to L,. An air pressure load of -3 psi was usually sufficient to produce a minimally afterloaded isotonic contraction on stimulation. Isometric contractions could be produced by the placement of a stop beneath the pivot arm that prohibited shortening of the stimulated muscle. In later experiments, an electronic solenoid was used to switch between predetermined air pressures, producing either afterloaded isotonic or maximally loaded isometric contractions. The maximal isometric tetanic tension was in the range of 20-30 kg of unnormalized force, and the minimally afterloaded isotonic contraction was 0.6-0.9 kg, varying with the size of the muscle. Calibration of the force transducer was done by hanging known weights on the muscle lever. Contraction force (F) measurements were normalized by muscle wet weight, yielding force values expressed per gram of muscle tissue. Calibration of the length transducer was done by moving the pivot arm in Z-mm increments along a ruler. The instantaneous velocity of shortening (V) was measured by placing a tangent on the maximal slope of the initial portion of the shortening record of isotonic contractions, as recorded on a Gould 2600s rectilinear strip chart recorder. This point of tangent placement on the shortening record corresponded with the time that the prescribed load was reached. Significant deviation of the shortening signal away from the tangent (i.e., a slowing of the velocity) began at ~66% of the total signal height. For the lightest afterloads, the load plateau was reached in -8 ms, which was within the lower limits of the recorder at these signal amplitudes (100 Hz, ~4 ms). The most rapid upswings of the shortening records during these contractions typically required 40-80 ms to reach 66% of total signal amplitude. Again, these times were within the frequency response (30 Hz) and rise time (~8 ms) specifications of the recorder for signals of this amplitude. The velocity values obtained under these conditions were expressed in terms of average muscle fiber lengths per second (&ls). ProtocoZ. After determination of L,, the pretrial F- V relationship was obtained by filling the expandable bellows with step increments of air pressure to produce increasingly afterloaded isotonic contractions. These increments produced loads that ranged from 0.2 to 30 kg, depending on muscle size. In initial isotonic experiments with afterloads of 25% (low load) and 50% (intermediate load) of P,, a large plastic tank (Nalge) served as an in-line reservoir to damp pressure transients within the metal bellows and to keep the isotonic load plateau constant. However, this tank could only withstand 15-18 psi, which resulted in an inability to test isotonic loads above ~0.40P, in the initial fatigue studies of the 25% afterload, because the muscle sizes of this group were slightly larger than the others (Table 1). In the case of the OJOP, fatigue studies, slightly smaller animals were used (Table l), enabling achievement of loads up to 0.50-0.6OP,. For the isometric contraction series, the smaller animals (Table 1) in combination with a large metal reservoir tank enabled achievement of isotonic afterloads up to P,. We
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FORCE-VELOCITY TABLE 1. Characteristics preparations
Isotonic Isotonic Isometric
CHANGES
WITH
of the experimental
n
Load
Muscle Wet Weight, g
Lf, cm
&w/R
5 4 5
0.25 0.50 1
TO-+4 53t4 53t2
3.8&O. 1 3.7tO.2 3.ltO.l
0.24t0.01 0.22t0.02 0.22rt0.02
Values are means t SE; n, no. of preparations. L,, calculated mean fiber length; P,, isometric tetanic tension at optimal fiber length (L,); P,, isometric twitch tension at L,.
attempted to counter potential problems resultant of these methodological differences by collecting many points in the low-load region during the isotonic experiments so that regression errors would be minimized when curve fitting to obtain maximal velocity of shortening ( V,,,), as described below. During the repetitive contraction trials, muscles were stimulated to produce either brief tetanic isometric or isotonic contractions (200-ms train duration, 50 impulses/s, 2-4 V), l/s, over a period of 10 min. Immediately after the repetitive contraction trial, the F-V relationship was determined again, as quickly as possible (60-90 s). In the case of isotonic contraction trials, the last contraction was switched to isometric, providing the final isometric tension value. For isometric contraction trials, a minimal load (0.03 t O.OlP,) was utilized to induce isotonic contractions of high velocity. These contractions were performed just before the trial and then again as the last contraction of the trial. These protocols provided reproducible measures of the pretrial and final dynamics of the extreme ends of the F-V relationship. The whole muscle was then exposed surgically while still connected to the lever system, and L, was measured. The muscle was then excised, trimmed, and weighed. Data analysis and statistics. To determine the full F-V curve and to calculate Vmax, the force and velocity data were subjected to iterative least-squares curve fitting by computer using the hyperbolic relationship described by a rearranged version of the Hill equation V p = b[(K + a/P + a) - 11 where VP is velocity at any given load (P), a and b are asymptotes of the hyperbolic relationship in force and velocity units, and K is a constant corresponding to the measured value of pretrial or final isometric tension, expressed as a fraction of P,. This equation constrained the curve to pass through P,, for which K was entered as a value of 1 for the pretrial curves, and the fraction of 1 corresponded to the measured final isometric tension value for the posttrial curves. Vmaxwas calculated in each case by setting P = 0, after a and b had been determined by curve fitting. This hyperbolic form of the Hill equation and the iterative method of regression allow velocity and load to vary directly, rather than having a transform of velocity (P, - P/V) vary with load as in the case of the straight-line form of the Hill equation (14). A comparison of both regression methods resulted in ~1.5% overall difference in calculated V,,, values. One-tailed paired t tests were used to compare pretrial and final values of both P, and Vmax in the isotonic con-
REPETITIVE
CONTRACTIONS
2107
traction experiments, whereas both P, and Vat the minimal load were compared in the isometric contraction experiments. This statistical treatment determined whether respective final P, or Vvalues were significantly less than their pretrial counterparts, where each individual muscle was its own control. RESULTS
Characteristics of the experimental muscle preparations, such as muscle weight, calculated fiber length, and twitch-to-tetanic ratios are given in Table 1, grouped by experiment. Mean muscle wet weight for all experiments was 59 t 3 (SE) g; mean calculated fiber length was 3.5 t 0.1 cm. Mean isometric pretrial tension development was 508 t 15 g/g; mean twitch-to-tetanic ratio was 0.23 t 0.01. The mean calculated Vmax was 3.8 t 0.2 &/s. The average initial amount of whole muscle shortening was 1.04 t 0.04 and 0.55 t 0.04 cm in the low- and interme-
diate-load isotonic contraction experiments, respectively. Figure 1 illustrates recordings of typical isometric and intermediate-load isotonic contraction experiments. The low-load isotonic contraction experiments looked similar to those of the intermediate load, with less of a decrement in muscle shortening over time. The time course of changes in performance (either as force development or shortening) in all experiments was similar, in that the initial decline observed within the first 3 min was rather rapid, followed by a slowly declining phase for the rest of the trial. In each case, significant fatigue was observed at 10 min of contraction, measured as decreased force in the isometric case C-27 t 4 (SE)%, P -c 0.011 or decreased shortening (or a drop of power production) in respective low-load and intermediate-load isotonic cases (-19 t 2%, P < 0.01, and -31 t 7%, P < 0.05). These relative decreases were not statistically different from each other, as determined by independent sample t test comparisons performed between the fatigue indexes of all three groups. Figure 2 shows pre- and posttrial F-V data for an isometric contraction experiment (P/P, = 1) and for separate isotonic contraction experiments with an intermediate load (P/P, = 0.50) and a low load (P/P, = 0.25). The best-fit Hill equation curves are drawn through each set of data. Table 2 contains observed means of pretrial and final isometric tension, velocity of shortening at the minimal load, and calculated VImax. Repetitive isometric contractions resulted in a significant decrement of the highforce, low-velocity end of the curve; however, at this same point, velocity development measured at the minimal load (0.03PJ was unchanged (+l t 1%; NS), and
calculated
Vmax was not decreased. Repetitive isotonic resulted in effects opposite to those mentioned above, namely that significant decreases in both velocity development at the minimal load (-22 t 3%, P < 0.01) and calculated Vmax (-21 t 2%, P -c 0.05) were demonstrated, with no significant change in the ability to develop isometric force (-8 t 4%; NS). Repetitive isotonic contractions at the intermediate load (0.5PJ resulted in decreases at both ends of the F-V relationship; velocity development at the minimal load contractions at the low load (0.25P,)
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2108
FORCE-VELOCITY
0I,~
o.irr B
1
% 0 0
CHANGES
WITH
REPETITIVE
CONTRACTIONS
----- ---j----3.11r-q Time
10
0.2
irninl
1A
fcml 0 J r-j
-I-------
0
m----4
10
Time IminI
FIG. 1. A: recording of repetitive isometric contraction trial, starting at time 0 and ending at 10 min, with individual contractions shown at fast paper speed at minutes 5 and 10. Isotonic contractions at a minimal load are shown by arrows, both before and as final contraction of isometric contraction trial, with corresponding displacement (D) signals shown below. Time between pretrial isotonic contraction and first isometric contraction was 1-5 min. B: recording of intermediate-load repetitive isotonic contraction trial [tension-to-maximal isometric tension ratio (P/P,) = 0.51, with individual contractions shown at fast paper speed at minutes 5 and IQ. Pretrial and final isometric contractions are shown as first and last contractions of recording.
was decreased significantly (-28 t 5%, P < O.Ol), as was calculated Vmax (-26 t 6%, P < 0.05), and the final isometric tension value was also decreased significantly (-12 t 2%, P < 0.01). These overall changes in the F-V relationship are illustrated in Fig. 3, which shows the mean Hill equation curves for each set of experiments on a normalized scale,
nificantly changed; and 3) with significant decrements of the ability to shorten and attain velocity due to repetitive isotonic contractions at a load that is one-half of maximal isometric tension, there was also a significant loss of the ability to develop maximal isometric tension. Thus the loss of performance capacity, or fatigue, was specific to either end of the F-V range when the muscles repetitively performed contractions that were mechanically limited toward the extremes of the F-V range. Critique of methods. Changes in the ability of the muscle to attain high initial shortening velocities during the isometric contraction experiments were monitored using a reproducible minimal isotonic afterload that was typically 3% of maximum isometric tension. However, a problem exists when using a hyperbolic relationship to extrapolate toward the ordinate and obtain Vmax, because very small changes in the light-load region of the F- Vrelationship result in very large changes in the veloc-
DISCUSSION
The main findings of this study were 1) with a significant decrement of isometric tension development during repetitive isometric contractions over a lo-min period, the canine GP muscle in situ retained the ability to attain high shortening velocities at very light loads; 2) with significant decrements in the ability to shorten and attain velocity due to repetitive isotonic contractions at a light load, the ability to develop isometric tension was not sig-
P/P,=
0 %
0
OS2
d4
0.6 %
0
P/$*=0.25
0.50
CL6
1.0
0 %
0
2. Pre- (0) and posttrial (Cl) force-velocity (F-V) relationship data for individual experiments of fatigue with isometric contractions (P/P, = l), intermediate-load isotonic contractions (P/P, = 0.5), and low-load isotonic contractions (P/P, = 0.25), with best-fit Hill equation regression curves drawn through data points. E,, average fiber length. FIG.
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FORCE-VELOCITY
CHANGES
WITH
REPETITIVE
2109
CONTRACTIONS
2. Pretrial and final values of isometric tension, velocities, and Hill equation slope
TABLE
Velocity at Minimal Load, Isometric
Isotonic Isotonic Isometric
n
Load
5 4
0.25 0.50
5
1.00
Tension,
Pre
g/g
Final
495t35 503t21 524t19
Calculated
Pre
454k30 445t29* 384+28*
Final
3.5t0.3 3.2t0.2 4.0t0.4
Vmax, a/P,
EflS
&f/S
Pre
2.7_+0.2* 2.3tO.l* 4.OkO.3
Final
3.9kO.2 3.4-t0.2 4.1t0.4
Pre
3.0t0.2* 2.5+0.2"r 4.5+0.3?
l.Ot0.2 1.6~0.3 1.3t0.3
Final
1.2t0.3 1.6t0.4 0.8+0.4$
Values are means + SE; n no. of preparations, Vmax,maximal velocity of shortening; a/P,, Hill equation slope ratio. * P < 0.01(l-tailed paired t test); t P < 0.05 (l-tailed paired t test); $ P < 0.05 (Mailed paired t test).
ity values. As mentioned, we addressed this problem by collecting many values at loads from 3 to 40% of P, in an attempt to minimize possible deviations of the curve fit extrapolations in the problem region. Furthermore, it has been observed previously that the F- V relationship of the canine GP muscle in situ has less curvature (2) than those reported for muscles and fibers in vitro (23). This flatter curve further minimizes the possibility of extrapolation errors at the high-velocity end of the F- V relationship, at least for these cases in the whole muscle. Previous studies of muscle contractile properties have utilized an approach in which an absolute amount of fatigue was matched within experiments, measured as either a percentage loss of shortening (50%) in the case of isotonic contractions or a percentage loss of force (70%) in the case of isometric contractions (17). However, this approach typically requires a different total number of contractions between experiments and therefore a different number of times that the muscle is activated. This difference in activation history may bias the comparison between experiments in a way that makes interpretation of mechanical effects difficult. In contrast, we fixed the number of stimuli and contractions across all experiments to limit the changes observed to those due to loadinduced mechanics. Thus the difference in the specificity of fatigue shown by our results cannot be ascribed to a difference in the number of times the muscle was activated, because the duration of the trials and the contraction stimulation scheme were the same. F-V shifts: isometric contractions. The results of the present study display preservation of the ability of whole muscle to attain high velocities of shortening when significant fatigue of maximal isometric force production was
present, These data indicate that fatigue produced by repetitive isometric contractions mainly affects the high force-producing ability of the muscle. This force-producing ability is thought to be an index of cross-bridge interaction, i.e., the number of cross bridges formed with activation via the excitation-contraction coupling mechanism (23). If this index is valid, the present study supports the idea that the mechanism of fatigue, in the case of repetitive isometric contractions, produced a decrement in the total number of cross bridges formed with each contraction, which may not be closely related to mechanisms responsible for cross-bridge cycling changes during shortening contractions. F-Vshifts: isotonic contractions. The results of the present study indicate little change in the ability to generate maximal isometric force when a significant decrement of the ability to shorten and develop velocity was present during repetitive low-load isotonic contractions. If it is true that velocity development is an index of cross-bridge cycling rate (23), then these data suggest that the observed shift in the F-V relationship was due to a decreased rate of cross-bridge cycling within the muscle, as a result of repetitive isotonic contractions. Furthermore, these repetitive low-load isotonic contractions had little effect on the ability to form the maximal number of cross bridges in response to activation during an isometric contraction at L,. However, in the intermediate-load isotonic fatigue experiments, both Vmagand P, were decreased, suggesting that both cross-bridge cycling and numbers were affected by the mechanics of these contractions. Further implications of F- Vshifts. The dissimilar shifts in the F-V relationship after repetitive contractions in P/P,=
0.50
1-0
P/P,=0.25 n
as
0.8
0.6
Q8
Kl
0.6
‘v~x~~.~~~~,~::,“i:.a 0
0.2
OA
0.6
a8
1.0
0
0.2
3. Mean
normalized
pretrial
and final
0.6
0.6
1.0
0
W
F-V curves
after
repetitive
isometric
44
Q6
Q8
1.0
% 0
% 0
% 0
FIG.
0.4
contractions
(P/P, =
I>, interme-
diate-load isotonic contractions (P/P, = 0.5), and low-load isotonic contractions (P/P, = 0.25). Fractional values for maximal velocity of shortening ( VmaK)and P, points correspond to ratio of final to pretrial values shown in Table 2 for each repetitive contraction case. Downloaded from www.physiology.org/journal/jappl by ${individualUser.givenNames} ${individualUser.surname} (018.218.056.169) on September 29, 2018. Copyright © 1992 American Physiological Society. All rights reserved.
2110
FORCE-VELOCITY
CHANGES
WITH
these studies could possibly be explained by selective fatigue or derecruitment of different fiber populations within the whole muscle, which may be dependent on the me& .anics of contraction. For instance, &nificant fatigue of th .e high-force-generating fiber population (6) could result in depression of the isometric-f&&e capacity of the whole muscle, as seen in the isometric contraction experiments. However, this explanation presents a dilemma, because the fibers typically classified as highforce generators are the fast-twitch fibers (6). If the contribution of these fibers was reduced or eliminated, it should theoretically produce a decrease in Vmax (l3), which was not observed in the F- V relationships obtained in our isometric contraction experiments. Conversely, significant fatigue of the fast-twitch fiber population (6) during repetitive shortening contractions could result in a depression of V,,, as observed in the isotonic contraction experiments. However, one might also expect the loss of these high-force-generating fibers to produce a significant decrease in the ability to develop maximal isometric tension, which was not observed in the low-load isotonic contraction experiments. An alternative explanation of the observed results may be related to calcium release and uptake by the sarcoplasmic reticulum during repetitive contractions. For example, during fatigue of repetitive maximal isometric contractions, calcium release may be declining with each contraction (1). However, the amounts may be great enough to allow sufficient numbers of cross bridges to form to produce a lightly loaded isotonic contraction with a high rate of cross-bridge cycling, even though this amount of calcium is not sufficient to produce maximum cross-bridge numbers and development of prefatigue isometric tension levels. This situation could theoretically produce the shift in the F-V relationship observed in the repetitive isometric contraction experiments. Alternatively, if calcium uptake by the sarcoplasmic reticulum is slowed with repetitive isotonic contractions, perhaps due to local deformations caused by shortening (20), the possible result would be prolonged disinhibition of actin binding sites, which could allow the maximum number of cross bridges to be formed with a subsequent isometric contraction at L,. This reasoning suggests a calcium “priming” effect of low-load isotonic contractions, with regard to actin binding site availability and maximal cross-bridge interaction, and could theoretically produce the F-V shift observed in the repetitive low-load isotonic contraction experiments. Another alternative explanation of the observed results may be that repetitive isometric contractions produced a reduction in blood flow compared with the isotunic contraction cases. This situation could result in a decrease in phosphocreatine replacement and accumulation of Pi, which has been shown to inhibit force production in skeletal muscle (18). Furthermore, it has been shown that down to a pH of 7.0, increases in Pi produce no change in Vmaxin the glycerinated rabbit psoas muscle (7). Under repetitive contraction conditions, GP muscle pH has been shown to fall to only 7.2 (13), and venous effluent pH has been measured as 7.1(21). Therefore this phosphate-dependent mechanism may be a factor producing the F- V shifts we observed as a result of repetitive isometric contractions. Comparisons with previous studies. Our finding of the
REPETITIVE
CONTRACTIONS
preservation of Vmaxwith a decrease of P, during isometric contractions agrees with previous investigations of frog sartorius muscle in vitro (11) and cat soleus muscle in situ (14). However, this finding only partially agrees with the results of Edman and Mattiazzi (8) in the frog anterior tibialis muscle fiber in vitro in that they also found a pre lservation of Vmaxbut only when isometric tension was reduced bY 40%. They further reported that Vmaxwas reduced if isometric tension was depressed by > 10% (8), whereas we observed preservation of Vmax when isometric tension was depressed by >25%. These differences may be ascribed to differences in experimental conditions, such as stimulus frequency, temperature, and species studied; however, a clear possibility may be that they are due to differences inherent between muscle preparations in situ and in vitro, such as the presence or absence of the circulation. Our findings in the isotonic contraction experiments agree with prior isovelocity fatigue studies of canine diaphragm in situ (3), as well as isotonic contraction studies of mouse diaphragm strips in vitro (19). Thus our results and others indicate that the effects of repetitive shortening contractions on the F- V relationship are complex and partially dependent on developed load and muscle shortening. In summary, these studies investigated the mechanical effects of repetitive contractions on the F-V relationship of in situ mammalian skeletal muscle, using a preparation in which surgery and manipulation were minimal and normal circulation was present. The results indicate that the effects of repetitive contractions on the F- Vprofile of canine skeletal muscle are specific to the mechanics of contraction occurring with repetitive electrical stimulation. Whether these differences are due to changes in blood flow or membrane activation that are related to the mechanics of contraction or whether they are due to an alteration of a mechanism that is present with repeated contractions, such as a decrement in calcium release over time, is unknown. However, the fact that these differences can be demonstrated indicates that the typical use of isometric contractions as an experimental marker of fatigue can be misleading, especially in terms of overall muscle mechanical performance decrements, which include the ability to shorten, develop velocity, and produce power. We thank Jeffrey Daniel, Viet Pham, Laura Koweek, and Kimberly Hess for their technical assistance. We also thank Pia Jacobs for secretarial assistance with the preparation of this manuscript. B. T. Ameredes is a Postdoctoral Research Fellow of the American Heart Association, Florida Affiliate (Award 90F/lO). This study was also supported by National Institute of Arthritis and Metabolic Diseases Grant AR-39378. Present address and address for reprint requests: B. T. Ameredes, Div. of Pulmonary, Allergy, and Critical Care Medicine, University of Pittsburgh, 440 Scaife Hall, 3550 Terrace St., Pittsburgh, PA 15261. Received 15 July 1991; accepted in final form 7 May 1992. REFERENCES 1. ALLEN, D. G., J. A. LEE, AND H. WESTERBLAD. Intracellular calcium and tension during fatigue in isolated single muscle fibers from Xenopus Zueuis.J. Physiol. Land. 415: 433-458, 1989. 2. ALLEN, P. D. The MechanicaL Properties of In Situ Dog Skeletal Muscle (PhD thesis). Gainesville: Univ. of Florida, 1973. 3. AMEREDES,~ T., AND T. LCLANTON. Increased fatigue ofisove-
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locity vs. isometric contractions of canine diaphragm. J. Appl. Physiol. 69: 740-746, 4.
5.
6.
7.
8.
1990.
BARK, H., G. SUPINSKI, R. BIJNDY, AND S. KELSEN. Effects of hypoxia on diaphragm blood flow, oxygen uptake, and contractility. Am. Rev. Respir. Dis. 138: 1535-1541, 1988. BELLEMARE, F., D. WIGHT, C. M. LAVIGNE, AND A. GRASSING. Effect of tension and timing of contraction on blood flow of the diaphragm. J. Appl. Physiol. 54: 1597-1606,1983. BURKE, R. E., D. N. LEVINE, P. TSAIRIS, AND F. E. ZAJAC III. Physiological types and histochemical profiles in motor units of the cat gastrocnemius. J. Physiol. Lond. 234: 723-748, 1973. COOKE, R., K. FRANKS, G. B. LUCIANI, AND E. PATE. The inhibition of rabbit skeletal muscle contraction by hydrogen ions and phosphate. J. Physiol. Lond. 395: 77-97, 1988. EDMAN, K. A. P., AND A. R. MATTIAZZI. Effects of fatigue and altered pH on isometric force and velocity of shortening at zero load in frog muscle fibers. J. Muscle Res. CeZl Motil. 2: 321-334, 1981.
EDWARDS, R. H. T. Human muscle function and fatigue. In: Human Muscle Fatigue: Physiological Mechanisms. London: Pitman, 1981, p. l-18. (Ciba Found. Symp. 82) 10. FALES, J. T., S. A. LILLENTHAL, S. A. TALBOT, AND K. L. ZIERLER. A pneumatic isotonic lever system for dog skeletal muscle. J. Appl. Physiol. 13: 307-308, 1958. 11. FITTS, R. H., AND J. 0. HOLLOSZY. Effects of fatigue and recovery on contractile properties of frog muscle. J. Appl. Physiol. 45: 8999.
902,1978.
12. GIBSON, H., AND R. H. T. EDWARDS. Muscular exercise and fatigue. Sports Med. 2: 120-132, 1985. 13. GIGER, U., Z. ARGOR, M. SCHNALL, W. J. BANK, AND B. CHANCE. Metabolic myopathy in canine muscle-type phosphofructokinase deficiency. Muscle Nerve 11: 1260-1265, 1988.
REPETITIVE
CONTRACTIONS
2111
14. HATCHER, D. D., AND A. R. LUFF. Contractile properties of cat skeletal muscle after repetitive stimulation. J. Appl. Physiot. 64: 502-510,
1988.
HOGAN, M. C., J. ROCA, P. D. WAGNER, AND J. B. WEST. Limitation of maximal O2 uptake and performance by acute hypoxia in dog muscle in situ. J. Appl. Physiol. 65: 815-821, 1988. 16. KING, C. E., S. L. DODD, AND S. M. CAIN. Muscle O2 deficit during hypoxia and two levels of 0, demand. J. Appl. Physiol. 62: 1384-
15.
1389,1987.
17. MARDINI, I. A., AND R. J. M. MCCARTER. Contractile properties of the shortening rat diaphragm in vitro. J. Appl. Physiol. 62: llll1116, 1987. 18. NOSEK, T. M., K. Y. FENDER, AND R. E. GODT. It is diprotonated inorganic phosphate that depresses force in skinned skeletal muscle fibers, Science Wash. DC 236: 191-193, 1987. 19 SEOW, C. Y., AND N. L. STEPHENS. Fatigue of mouse diaphragm muscle in isometric and isotonic contractions. J. Appl. Physiol. 64: l
2388-2393,
1988.
2. STAINSBY, W. N., AND G. M. ANDREW. Maximal blood flow and power output of dog muscle in situ. Med. Sci. Sports Exercise 20, Suppl.: Slog-S112, 1988. 21* STAINSBY, W. N., AND P, D. EITZMAN. Roles of COa, 0,, and acid in arteriovenous [H+] difference during muscle contractions. J. Appl. PhysioL. 65: 1803-1810, 1988. 22* TAYLOR, S. R., AND R. RUDEL. Striated muscle fibers: inactivation of contraction induced by shortening. Science Wash. DC 167: 882l
884,197O. 23.
WOLEDGE, R., N. A. CURTIN, AND E. HOMSHER. Mechanics of contraction. In: Energetic Aspects of Muscle Contraction, edited by R. Woledge, N. A. Curtin, and E. Homsher. London: Academic, 1985, chapt. 2, p. 27-117.
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contructions
of
canine
gastrocnemius
in situ. J.
Appl. Physiol. 73(5): 2105-2111, 1992.-The force-velocity (FV) relationships of canine gastrocnemius-plantarismusclesat optimal musclelength in situ were studied before and after 10 min of repetitive isometric or isotonic tetanic contractions induced by electrical stimulation of the sciatic nerve (200-ms trains, 50 impulses/s, 1 contraction/s). F-V relationships and maximal velocity of shortening ( Vmax) were determined by curve fitting with the Hill equation. Mean Vmax before fatigue was 3.8 t 0.2 (SE) average fiber lengths/s; mean maximal isometric tension (P,) was 508 t 15 g/g. With a significant decrease of force development during isometric contractions (-27 t 4%, P < 0.01, n = 5), Vmaxwas unchanged. However, with repetitive isotonic contractions at a low load (P/P, = 0.25, n = 5), a significant decreasein Vmag was observed(-21 & 2%, P < O.Ol), whereasP, wasunchanged.Isotonic contractions at an intermediate load (P/P, = 0.5, n = 4) resulted in significant decreasesin both Vmax(-26 t 6%, P c 0.05) and P, (-12 k 2%, P < 0.01). These results show that repeated contractions of canine skeletal muscleproduce specific changesin the F-V relationship that are dependenton the type of contractions being performed and indicate that decreasesin other contractile properties, such asvelocity development and shortening, can occur independently of changesin isometric tension. fatigue; Hill equation power; shortening; maximal velocity of shortening
MUSCLE FATKXJX can be described
pected muscle served series
as a loss of the experformance of a muscle fiber, motor unit, or group (9). This loss of performance can be obduring a single sustained contraction or during a of repeated shorter contractions. The latter has
been shown to be affected by both metabolic factors, such
as oxygen availability (4,15, 16), and mechanical factors, such as duty cycle (5). Two measures of muscle performance that are of interest are force production and velocity development. Also, implicit in the measurement of velocity development is the amount of muscle shortening or displacement. Although classically defined as a drop in force production, we propose that fatigue may also be defin ed as a decrease in the ability to sho irten and develop velocity or power (12), as in the case of isotonic contractions where the force production is constant. Previous studies of the canine diaphragm muscle in situ demonstrated a greater loss of force development during isovelocity muscle contractions compared with
results suggested an effect of muscle shortening on the force-velocity (F-V) characteristics of fatigued muscle (3). Thus the purpose of the following study was to 1) measure changes in the F-V relationship that occur with repetitive shortening (isotonic) and nonshortening (isometric) contractions and 2) determine whether the mechanical conditions of fatiguing contractions have specific effects on the ability of the muscle to develop the full range of force and velocity. The studies were performed with the canine gastrocnemius-plantaris (GP) muscle, prepared with a minimum of surgery (20), in which mechanical performance was recorded using a lever system specifically designed for this purpose (10). METHODS
General methods. Fourteen mongrel dogs (16 t 3 kg) were anesthetized with pentobarbital sodium (30 mg/kg iv), followed by maintenance doses of 60 mg. The animals were ventilated through an endotracheal tube with a Harvard respirator (tidal volume = 25 ml/kg). End-tidal CO, was maintained at 4.5-5% by adjustment of pump frequency. The right femoral artery was catheterized to provide arterial blood pressure measurements and blood sampling capability, and the right femoral vein was catheterized to allow administration of fluids and anesthetic. Body temperature was monitored with an intraesophageal probe and was maintained at 37-40°C with a heating pad on the body. Surgical methods. A longitudinal incision was made in the posterior thigh to gain access to the sciatic notch between the muscles. The sciatic nerve was located, cleared, and cut, and the distal end was placed into an electrode holder. The incision was closed with wound clips so that the electrode holder remained in place, and the wires were exited through the remaining slit in the skin. Another small incision was made on the lateral side of the popliteal area through which the peroneal nerve was ligated so as to avoid dorsiflexion of the foot during sciatic nerve stimulation. A third incision was made in the skin over the area at which the medial gastrocnemius muscle connects to the calcaneus tendon to gain access to the tendon and the lower portion of the GP muscle group. The tendon was cleared and cut close to the calcaneus and was placed into a tendon clamp that was later connected to a pneumatic lever (10). The origin of the GP
0161-7567/92 $2.00 Copyright 0 1992 the American Physiological Society
2105
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2106
FORCE-VELOCITY
CHANGES
WITH
was anchored by bone nails placed in the femur and tibia, and a strut was placed between the lever and bone nails to minimize flexing. A thermoprobe was inserted into the connective tissue between the muscle and the tibia to provide a measure of muscle temperature, which was maintained at 37-39*C with a heat lamp overhead. Saline-soaked gauze and plastic wrap were placed over the incision to maintain muscle moisture. Optimal muscle length. Optimal muscle length was defined as the length at which maximal isometric force was developed using twitch contractions (0.2 ms, 4 V) and was found to correspond to within 2 t 2% of the length (L,) at which maximal tetanic isometric tension (P,) was developed. L, was measured as the length from the tibia1 origin of the medial head of the muscle to the point at which the visible muscle fibers terminated on the calcaneus tendon [ 12.3 t 0.5 (SE) cm]. This value was used to calculate the average fiber length (.& = 3.5 +- 0.1 cm) within the muscle at optimal muscle length (2). As will be described, the normalization of shortening velocity between each muscle studied relied on calculation of E, within each muscle. The GP muscle is pennate, having a significant number of fibers at an angle to the long axis of the muscle. Previous studies in this laboratory, using light and electron microscopy on fixed canine GP muscle sections at L,, have shown that the angle of pennation averages 20° (2) and that average muscle fiber length at L, can be calculated by dividing whole muscle length by a factor of 3.5 (2). Thus, although some inaccuracies may exist for a given population of fibers within a muscle, this method of calculation results in accurate fiber length values for the average of all fibers within the canine GP muscle. Force and velocity measurements. This study utilized a muscle preparation that was created in situ with a minimum of surgery to investigate the fatigue characteristics of a skeletal muscle in a situation as close to in vivo conditions as possible, with the exception of electrical stimulation of a cut nerve. One possible complication of this approach is that of contributions of nearby muscles to the mechanics of contractions assumed to be solely a product of the GP muscle. Therefore a series of control isometric (72 = 3) and isotonic (n = 3) experiments was performed to assess these effects. With each major muscle removal (semitendinosus, gracilis, sartorius, and semimembranosus), force or shortening was reassessed to determine the magnitude of change in the measured mechanical characteristics of the GP muscle. Furthermore, nerves exiting the muscle on the tibia1 side were also ligated. These experiments typically took IO-30 min, during which neither isometric force nor isotonic shortening changed ~2%. These studies indicated that the measured mechanical performance of the minimally prepared GP muscle was an accurate depiction of events occurring in that muscle group alone. Measurements of muscle force and shortening were accomplished with a pneumatic muscle lever in which compressed air was used to produce force loads on the muscle (9). This lever system had an air-expandable metal bellows attached to a pivot arm and could be filled to any desired pressure up to 50 psi. Typically, 2 psi was enough to expand the bellows and move the pivot arm up
REPETITIVE
CONTRACTIONS
to a length stop above the pivot arm, placing the muscle at L,, as previously determined by isometric twitch contractions. This resulted in a passive tension of ~45 g/g within the muscle when set to L,. An air pressure load of -3 psi was usually sufficient to produce a minimally afterloaded isotonic contraction on stimulation. Isometric contractions could be produced by the placement of a stop beneath the pivot arm that prohibited shortening of the stimulated muscle. In later experiments, an electronic solenoid was used to switch between predetermined air pressures, producing either afterloaded isotonic or maximally loaded isometric contractions. The maximal isometric tetanic tension was in the range of 20-30 kg of unnormalized force, and the minimally afterloaded isotonic contraction was 0.6-0.9 kg, varying with the size of the muscle. Calibration of the force transducer was done by hanging known weights on the muscle lever. Contraction force (F) measurements were normalized by muscle wet weight, yielding force values expressed per gram of muscle tissue. Calibration of the length transducer was done by moving the pivot arm in Z-mm increments along a ruler. The instantaneous velocity of shortening (V) was measured by placing a tangent on the maximal slope of the initial portion of the shortening record of isotonic contractions, as recorded on a Gould 2600s rectilinear strip chart recorder. This point of tangent placement on the shortening record corresponded with the time that the prescribed load was reached. Significant deviation of the shortening signal away from the tangent (i.e., a slowing of the velocity) began at ~66% of the total signal height. For the lightest afterloads, the load plateau was reached in -8 ms, which was within the lower limits of the recorder at these signal amplitudes (100 Hz, ~4 ms). The most rapid upswings of the shortening records during these contractions typically required 40-80 ms to reach 66% of total signal amplitude. Again, these times were within the frequency response (30 Hz) and rise time (~8 ms) specifications of the recorder for signals of this amplitude. The velocity values obtained under these conditions were expressed in terms of average muscle fiber lengths per second (&ls). ProtocoZ. After determination of L,, the pretrial F- V relationship was obtained by filling the expandable bellows with step increments of air pressure to produce increasingly afterloaded isotonic contractions. These increments produced loads that ranged from 0.2 to 30 kg, depending on muscle size. In initial isotonic experiments with afterloads of 25% (low load) and 50% (intermediate load) of P,, a large plastic tank (Nalge) served as an in-line reservoir to damp pressure transients within the metal bellows and to keep the isotonic load plateau constant. However, this tank could only withstand 15-18 psi, which resulted in an inability to test isotonic loads above ~0.40P, in the initial fatigue studies of the 25% afterload, because the muscle sizes of this group were slightly larger than the others (Table 1). In the case of the OJOP, fatigue studies, slightly smaller animals were used (Table l), enabling achievement of loads up to 0.50-0.6OP,. For the isometric contraction series, the smaller animals (Table 1) in combination with a large metal reservoir tank enabled achievement of isotonic afterloads up to P,. We
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FORCE-VELOCITY TABLE 1. Characteristics preparations
Isotonic Isotonic Isometric
CHANGES
WITH
of the experimental
n
Load
Muscle Wet Weight, g
Lf, cm
&w/R
5 4 5
0.25 0.50 1
TO-+4 53t4 53t2
3.8&O. 1 3.7tO.2 3.ltO.l
0.24t0.01 0.22t0.02 0.22rt0.02
Values are means t SE; n, no. of preparations. L,, calculated mean fiber length; P,, isometric tetanic tension at optimal fiber length (L,); P,, isometric twitch tension at L,.
attempted to counter potential problems resultant of these methodological differences by collecting many points in the low-load region during the isotonic experiments so that regression errors would be minimized when curve fitting to obtain maximal velocity of shortening ( V,,,), as described below. During the repetitive contraction trials, muscles were stimulated to produce either brief tetanic isometric or isotonic contractions (200-ms train duration, 50 impulses/s, 2-4 V), l/s, over a period of 10 min. Immediately after the repetitive contraction trial, the F-V relationship was determined again, as quickly as possible (60-90 s). In the case of isotonic contraction trials, the last contraction was switched to isometric, providing the final isometric tension value. For isometric contraction trials, a minimal load (0.03 t O.OlP,) was utilized to induce isotonic contractions of high velocity. These contractions were performed just before the trial and then again as the last contraction of the trial. These protocols provided reproducible measures of the pretrial and final dynamics of the extreme ends of the F-V relationship. The whole muscle was then exposed surgically while still connected to the lever system, and L, was measured. The muscle was then excised, trimmed, and weighed. Data analysis and statistics. To determine the full F-V curve and to calculate Vmax, the force and velocity data were subjected to iterative least-squares curve fitting by computer using the hyperbolic relationship described by a rearranged version of the Hill equation V p = b[(K + a/P + a) - 11 where VP is velocity at any given load (P), a and b are asymptotes of the hyperbolic relationship in force and velocity units, and K is a constant corresponding to the measured value of pretrial or final isometric tension, expressed as a fraction of P,. This equation constrained the curve to pass through P,, for which K was entered as a value of 1 for the pretrial curves, and the fraction of 1 corresponded to the measured final isometric tension value for the posttrial curves. Vmaxwas calculated in each case by setting P = 0, after a and b had been determined by curve fitting. This hyperbolic form of the Hill equation and the iterative method of regression allow velocity and load to vary directly, rather than having a transform of velocity (P, - P/V) vary with load as in the case of the straight-line form of the Hill equation (14). A comparison of both regression methods resulted in ~1.5% overall difference in calculated V,,, values. One-tailed paired t tests were used to compare pretrial and final values of both P, and Vmax in the isotonic con-
REPETITIVE
CONTRACTIONS
2107
traction experiments, whereas both P, and Vat the minimal load were compared in the isometric contraction experiments. This statistical treatment determined whether respective final P, or Vvalues were significantly less than their pretrial counterparts, where each individual muscle was its own control. RESULTS
Characteristics of the experimental muscle preparations, such as muscle weight, calculated fiber length, and twitch-to-tetanic ratios are given in Table 1, grouped by experiment. Mean muscle wet weight for all experiments was 59 t 3 (SE) g; mean calculated fiber length was 3.5 t 0.1 cm. Mean isometric pretrial tension development was 508 t 15 g/g; mean twitch-to-tetanic ratio was 0.23 t 0.01. The mean calculated Vmax was 3.8 t 0.2 &/s. The average initial amount of whole muscle shortening was 1.04 t 0.04 and 0.55 t 0.04 cm in the low- and interme-
diate-load isotonic contraction experiments, respectively. Figure 1 illustrates recordings of typical isometric and intermediate-load isotonic contraction experiments. The low-load isotonic contraction experiments looked similar to those of the intermediate load, with less of a decrement in muscle shortening over time. The time course of changes in performance (either as force development or shortening) in all experiments was similar, in that the initial decline observed within the first 3 min was rather rapid, followed by a slowly declining phase for the rest of the trial. In each case, significant fatigue was observed at 10 min of contraction, measured as decreased force in the isometric case C-27 t 4 (SE)%, P -c 0.011 or decreased shortening (or a drop of power production) in respective low-load and intermediate-load isotonic cases (-19 t 2%, P < 0.01, and -31 t 7%, P < 0.05). These relative decreases were not statistically different from each other, as determined by independent sample t test comparisons performed between the fatigue indexes of all three groups. Figure 2 shows pre- and posttrial F-V data for an isometric contraction experiment (P/P, = 1) and for separate isotonic contraction experiments with an intermediate load (P/P, = 0.50) and a low load (P/P, = 0.25). The best-fit Hill equation curves are drawn through each set of data. Table 2 contains observed means of pretrial and final isometric tension, velocity of shortening at the minimal load, and calculated VImax. Repetitive isometric contractions resulted in a significant decrement of the highforce, low-velocity end of the curve; however, at this same point, velocity development measured at the minimal load (0.03PJ was unchanged (+l t 1%; NS), and
calculated
Vmax was not decreased. Repetitive isotonic resulted in effects opposite to those mentioned above, namely that significant decreases in both velocity development at the minimal load (-22 t 3%, P < 0.01) and calculated Vmax (-21 t 2%, P -c 0.05) were demonstrated, with no significant change in the ability to develop isometric force (-8 t 4%; NS). Repetitive isotonic contractions at the intermediate load (0.5PJ resulted in decreases at both ends of the F-V relationship; velocity development at the minimal load contractions at the low load (0.25P,)
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2108
FORCE-VELOCITY
0I,~
o.irr B
1
% 0 0
CHANGES
WITH
REPETITIVE
CONTRACTIONS
----- ---j----3.11r-q Time
10
0.2
irninl
1A
fcml 0 J r-j
-I-------
0
m----4
10
Time IminI
FIG. 1. A: recording of repetitive isometric contraction trial, starting at time 0 and ending at 10 min, with individual contractions shown at fast paper speed at minutes 5 and 10. Isotonic contractions at a minimal load are shown by arrows, both before and as final contraction of isometric contraction trial, with corresponding displacement (D) signals shown below. Time between pretrial isotonic contraction and first isometric contraction was 1-5 min. B: recording of intermediate-load repetitive isotonic contraction trial [tension-to-maximal isometric tension ratio (P/P,) = 0.51, with individual contractions shown at fast paper speed at minutes 5 and IQ. Pretrial and final isometric contractions are shown as first and last contractions of recording.
was decreased significantly (-28 t 5%, P < O.Ol), as was calculated Vmax (-26 t 6%, P < 0.05), and the final isometric tension value was also decreased significantly (-12 t 2%, P < 0.01). These overall changes in the F-V relationship are illustrated in Fig. 3, which shows the mean Hill equation curves for each set of experiments on a normalized scale,
nificantly changed; and 3) with significant decrements of the ability to shorten and attain velocity due to repetitive isotonic contractions at a load that is one-half of maximal isometric tension, there was also a significant loss of the ability to develop maximal isometric tension. Thus the loss of performance capacity, or fatigue, was specific to either end of the F-V range when the muscles repetitively performed contractions that were mechanically limited toward the extremes of the F-V range. Critique of methods. Changes in the ability of the muscle to attain high initial shortening velocities during the isometric contraction experiments were monitored using a reproducible minimal isotonic afterload that was typically 3% of maximum isometric tension. However, a problem exists when using a hyperbolic relationship to extrapolate toward the ordinate and obtain Vmax, because very small changes in the light-load region of the F- Vrelationship result in very large changes in the veloc-
DISCUSSION
The main findings of this study were 1) with a significant decrement of isometric tension development during repetitive isometric contractions over a lo-min period, the canine GP muscle in situ retained the ability to attain high shortening velocities at very light loads; 2) with significant decrements in the ability to shorten and attain velocity due to repetitive isotonic contractions at a light load, the ability to develop isometric tension was not sig-
P/P,=
0 %
0
OS2
d4
0.6 %
0
P/$*=0.25
0.50
CL6
1.0
0 %
0
2. Pre- (0) and posttrial (Cl) force-velocity (F-V) relationship data for individual experiments of fatigue with isometric contractions (P/P, = l), intermediate-load isotonic contractions (P/P, = 0.5), and low-load isotonic contractions (P/P, = 0.25), with best-fit Hill equation regression curves drawn through data points. E,, average fiber length. FIG.
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FORCE-VELOCITY
CHANGES
WITH
REPETITIVE
2109
CONTRACTIONS
2. Pretrial and final values of isometric tension, velocities, and Hill equation slope
TABLE
Velocity at Minimal Load, Isometric
Isotonic Isotonic Isometric
n
Load
5 4
0.25 0.50
5
1.00
Tension,
Pre
g/g
Final
495t35 503t21 524t19
Calculated
Pre
454k30 445t29* 384+28*
Final
3.5t0.3 3.2t0.2 4.0t0.4
Vmax, a/P,
EflS
&f/S
Pre
2.7_+0.2* 2.3tO.l* 4.OkO.3
Final
3.9kO.2 3.4-t0.2 4.1t0.4
Pre
3.0t0.2* 2.5+0.2"r 4.5+0.3?
l.Ot0.2 1.6~0.3 1.3t0.3
Final
1.2t0.3 1.6t0.4 0.8+0.4$
Values are means + SE; n no. of preparations, Vmax,maximal velocity of shortening; a/P,, Hill equation slope ratio. * P < 0.01(l-tailed paired t test); t P < 0.05 (l-tailed paired t test); $ P < 0.05 (Mailed paired t test).
ity values. As mentioned, we addressed this problem by collecting many values at loads from 3 to 40% of P, in an attempt to minimize possible deviations of the curve fit extrapolations in the problem region. Furthermore, it has been observed previously that the F- V relationship of the canine GP muscle in situ has less curvature (2) than those reported for muscles and fibers in vitro (23). This flatter curve further minimizes the possibility of extrapolation errors at the high-velocity end of the F- V relationship, at least for these cases in the whole muscle. Previous studies of muscle contractile properties have utilized an approach in which an absolute amount of fatigue was matched within experiments, measured as either a percentage loss of shortening (50%) in the case of isotonic contractions or a percentage loss of force (70%) in the case of isometric contractions (17). However, this approach typically requires a different total number of contractions between experiments and therefore a different number of times that the muscle is activated. This difference in activation history may bias the comparison between experiments in a way that makes interpretation of mechanical effects difficult. In contrast, we fixed the number of stimuli and contractions across all experiments to limit the changes observed to those due to loadinduced mechanics. Thus the difference in the specificity of fatigue shown by our results cannot be ascribed to a difference in the number of times the muscle was activated, because the duration of the trials and the contraction stimulation scheme were the same. F-V shifts: isometric contractions. The results of the present study display preservation of the ability of whole muscle to attain high velocities of shortening when significant fatigue of maximal isometric force production was
present, These data indicate that fatigue produced by repetitive isometric contractions mainly affects the high force-producing ability of the muscle. This force-producing ability is thought to be an index of cross-bridge interaction, i.e., the number of cross bridges formed with activation via the excitation-contraction coupling mechanism (23). If this index is valid, the present study supports the idea that the mechanism of fatigue, in the case of repetitive isometric contractions, produced a decrement in the total number of cross bridges formed with each contraction, which may not be closely related to mechanisms responsible for cross-bridge cycling changes during shortening contractions. F-Vshifts: isotonic contractions. The results of the present study indicate little change in the ability to generate maximal isometric force when a significant decrement of the ability to shorten and develop velocity was present during repetitive low-load isotonic contractions. If it is true that velocity development is an index of cross-bridge cycling rate (23), then these data suggest that the observed shift in the F-V relationship was due to a decreased rate of cross-bridge cycling within the muscle, as a result of repetitive isotonic contractions. Furthermore, these repetitive low-load isotonic contractions had little effect on the ability to form the maximal number of cross bridges in response to activation during an isometric contraction at L,. However, in the intermediate-load isotonic fatigue experiments, both Vmagand P, were decreased, suggesting that both cross-bridge cycling and numbers were affected by the mechanics of these contractions. Further implications of F- Vshifts. The dissimilar shifts in the F-V relationship after repetitive contractions in P/P,=
0.50
1-0
P/P,=0.25 n
as
0.8
0.6
Q8
Kl
0.6
‘v~x~~.~~~~,~::,“i:.a 0
0.2
OA
0.6
a8
1.0
0
0.2
3. Mean
normalized
pretrial
and final
0.6
0.6
1.0
0
W
F-V curves
after
repetitive
isometric
44
Q6
Q8
1.0
% 0
% 0
% 0
FIG.
0.4
contractions
(P/P, =
I>, interme-
diate-load isotonic contractions (P/P, = 0.5), and low-load isotonic contractions (P/P, = 0.25). Fractional values for maximal velocity of shortening ( VmaK)and P, points correspond to ratio of final to pretrial values shown in Table 2 for each repetitive contraction case. Downloaded from www.physiology.org/journal/jappl by ${individualUser.givenNames} ${individualUser.surname} (018.218.056.169) on September 29, 2018. Copyright © 1992 American Physiological Society. All rights reserved.
2110
FORCE-VELOCITY
CHANGES
WITH
these studies could possibly be explained by selective fatigue or derecruitment of different fiber populations within the whole muscle, which may be dependent on the me& .anics of contraction. For instance, &nificant fatigue of th .e high-force-generating fiber population (6) could result in depression of the isometric-f&&e capacity of the whole muscle, as seen in the isometric contraction experiments. However, this explanation presents a dilemma, because the fibers typically classified as highforce generators are the fast-twitch fibers (6). If the contribution of these fibers was reduced or eliminated, it should theoretically produce a decrease in Vmax (l3), which was not observed in the F- V relationships obtained in our isometric contraction experiments. Conversely, significant fatigue of the fast-twitch fiber population (6) during repetitive shortening contractions could result in a depression of V,,, as observed in the isotonic contraction experiments. However, one might also expect the loss of these high-force-generating fibers to produce a significant decrease in the ability to develop maximal isometric tension, which was not observed in the low-load isotonic contraction experiments. An alternative explanation of the observed results may be related to calcium release and uptake by the sarcoplasmic reticulum during repetitive contractions. For example, during fatigue of repetitive maximal isometric contractions, calcium release may be declining with each contraction (1). However, the amounts may be great enough to allow sufficient numbers of cross bridges to form to produce a lightly loaded isotonic contraction with a high rate of cross-bridge cycling, even though this amount of calcium is not sufficient to produce maximum cross-bridge numbers and development of prefatigue isometric tension levels. This situation could theoretically produce the shift in the F-V relationship observed in the repetitive isometric contraction experiments. Alternatively, if calcium uptake by the sarcoplasmic reticulum is slowed with repetitive isotonic contractions, perhaps due to local deformations caused by shortening (20), the possible result would be prolonged disinhibition of actin binding sites, which could allow the maximum number of cross bridges to be formed with a subsequent isometric contraction at L,. This reasoning suggests a calcium “priming” effect of low-load isotonic contractions, with regard to actin binding site availability and maximal cross-bridge interaction, and could theoretically produce the F-V shift observed in the repetitive low-load isotonic contraction experiments. Another alternative explanation of the observed results may be that repetitive isometric contractions produced a reduction in blood flow compared with the isotunic contraction cases. This situation could result in a decrease in phosphocreatine replacement and accumulation of Pi, which has been shown to inhibit force production in skeletal muscle (18). Furthermore, it has been shown that down to a pH of 7.0, increases in Pi produce no change in Vmaxin the glycerinated rabbit psoas muscle (7). Under repetitive contraction conditions, GP muscle pH has been shown to fall to only 7.2 (13), and venous effluent pH has been measured as 7.1(21). Therefore this phosphate-dependent mechanism may be a factor producing the F- V shifts we observed as a result of repetitive isometric contractions. Comparisons with previous studies. Our finding of the
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preservation of Vmaxwith a decrease of P, during isometric contractions agrees with previous investigations of frog sartorius muscle in vitro (11) and cat soleus muscle in situ (14). However, this finding only partially agrees with the results of Edman and Mattiazzi (8) in the frog anterior tibialis muscle fiber in vitro in that they also found a pre lservation of Vmaxbut only when isometric tension was reduced bY 40%. They further reported that Vmaxwas reduced if isometric tension was depressed by > 10% (8), whereas we observed preservation of Vmax when isometric tension was depressed by >25%. These differences may be ascribed to differences in experimental conditions, such as stimulus frequency, temperature, and species studied; however, a clear possibility may be that they are due to differences inherent between muscle preparations in situ and in vitro, such as the presence or absence of the circulation. Our findings in the isotonic contraction experiments agree with prior isovelocity fatigue studies of canine diaphragm in situ (3), as well as isotonic contraction studies of mouse diaphragm strips in vitro (19). Thus our results and others indicate that the effects of repetitive shortening contractions on the F- V relationship are complex and partially dependent on developed load and muscle shortening. In summary, these studies investigated the mechanical effects of repetitive contractions on the F-V relationship of in situ mammalian skeletal muscle, using a preparation in which surgery and manipulation were minimal and normal circulation was present. The results indicate that the effects of repetitive contractions on the F- Vprofile of canine skeletal muscle are specific to the mechanics of contraction occurring with repetitive electrical stimulation. Whether these differences are due to changes in blood flow or membrane activation that are related to the mechanics of contraction or whether they are due to an alteration of a mechanism that is present with repeated contractions, such as a decrement in calcium release over time, is unknown. However, the fact that these differences can be demonstrated indicates that the typical use of isometric contractions as an experimental marker of fatigue can be misleading, especially in terms of overall muscle mechanical performance decrements, which include the ability to shorten, develop velocity, and produce power. We thank Jeffrey Daniel, Viet Pham, Laura Koweek, and Kimberly Hess for their technical assistance. We also thank Pia Jacobs for secretarial assistance with the preparation of this manuscript. B. T. Ameredes is a Postdoctoral Research Fellow of the American Heart Association, Florida Affiliate (Award 90F/lO). This study was also supported by National Institute of Arthritis and Metabolic Diseases Grant AR-39378. Present address and address for reprint requests: B. T. Ameredes, Div. of Pulmonary, Allergy, and Critical Care Medicine, University of Pittsburgh, 440 Scaife Hall, 3550 Terrace St., Pittsburgh, PA 15261. Received 15 July 1991; accepted in final form 7 May 1992. REFERENCES 1. ALLEN, D. G., J. A. LEE, AND H. WESTERBLAD. Intracellular calcium and tension during fatigue in isolated single muscle fibers from Xenopus Zueuis.J. Physiol. Land. 415: 433-458, 1989. 2. ALLEN, P. D. The MechanicaL Properties of In Situ Dog Skeletal Muscle (PhD thesis). Gainesville: Univ. of Florida, 1973. 3. AMEREDES,~ T., AND T. LCLANTON. Increased fatigue ofisove-
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