Muscle fatigue in frog semitendinosus: alterations in contractile function L. V. THOMPSON,
E. M. BALOG,
D. A. RILEY,
AND R. H. FITTS
Biology Department/Biological and Biomedical Research Institute, Milwaukee 53233; and Cellular Biology and Anatomy Department, Milwaukee, Wisconsin 53226 Thompson, L. V., E. M. Balog, D. A. Riley, and R. H. Fitts. Muscle fatigue in frog semitendinosus: alterations in contractile function. Am. J. Physiol. 262 (CeZZPhysioZ. 31): Cl500C1506,1992.-The purpose of this study was to characterize the contractile properties of the frog semitendinosus (ST) muscle before and during recovery from fatigue, to relate the observed functional changes to alterations in specific steps in the crossbridge model of muscle contraction, and to determine how fatigue affects the force-frequency relationship. The frog ST (22OC) was fatigued by direct electrical stimulation with lOO-ms 150-Hz trains at l/s for 5 min. The fatigue protocol reduced peak twitch (P,) and tetanic (P,) force to 32 and 8.5% of initial force, respectively. The decline in P, was less than P,, in part due to a prolongation in the isometric contraction time (CT), which increased to 300% of the initial value. The isometric twitch duration was greatly prolonged as reflected by the lengthened CT and the 800% increase in the one-half relaxation time (XRT). Both P, and P, showed a biphasic recovery, a rapid initial phase (2 min) followed by a slower (40 min) return to the prefatigue force. CT and 1/2RT also recovered in two phases, returning to 160 and 265% of control in the first 5 min. CT returned to the prefatigue value between 35 and 40 min, whereas even at 60 min l/2RT was 133% of control. The maximal velocity of shortening, determined by the slack test, was significantly reduced [from 6.7 t 0.5 to 2.5 of: 0.4 optimal muscle length/s] at fatigue. The force-frequency relationship was shifted to the left, so that optimal frequency for generating P, was reduced. In conclusion, the biphasic recovery of P, and P, suggests that fatigue was caused by multiple factors with varying recovery times. The fast phase probably mirrors a rapid change in one or more of the steps of excitation-contraction coupling, whereas the slow phase may be mediated by cellular recovery from contraction-induced increases in H+ and inorganic phosphate. contractile properties; peak twitch force; peak tetanic force; maximal velocity of shortening; contraction time; one-half relaxation time
NUMEROUS INVESTIGATIONS have been conducted over the last century in an attempt to elucidate the etiology of muscle fatigue. Despite these efforts the precise cause of muscle fatigue remains unknown. Alterations in the functional capacity of the central nervous system (central fatigue), the peripheral a-motor nerves, the neuromuscular junctions, or the skeletal muscles could all contribute to fatigue. However, the preponderance of experimental evidence suggests that the etiology of muscle fatigue is primarily confined to the peripheral skeletal muscle cells (4, 10, 18). The problem is complex inasmuch as the degree and cellular mechanisms of muscle fatigue vary with the type of contractile activity and the fiber type composition of the muscle. Fatigue, defined as a reduction in muscle force and power (8, 12, 18), probably results from multiple factors, including alterations in excitation-contraction coupling (ECC) (18), cross-bridge events (27, 29), and cell metabolism (28) . Cl500
0363-6143/92
$2.00
Copyright
Marquette University, Medical College of Wisconsin,
Several experimental models have been utilized to examine the mechanisms of muscle fatigue (18, 22). In addition to a reduced peak tetanic and twitch force, fatigue is generally associated with a prolonged isometric twitch contraction and relaxation time (17,24) and a reduced maximal velocity of shortening ( Vmax) and peak power (8). The prolonged twitch duration reduces the optimal frequency for peak tetanic force and should shift the entire force-frequency relationship to the left toward lower frequencies (24). Edwards et al. (13) observed force elicited by low-frequency stimulation (20 Hz) to be depressed for hours after fatigue in the adductor pollicis muscle of humans (low-frequency fatigue). These authors’ conclusion that the force-frequency relationship shifts right with fatigue was based on the response to a single stimulus frequency (20 Hz). Perhaps more importantly, these data do not describe the force-frequency relationship in the contracting fatigued muscle uninfluenced by periods of recovery. Edwards (12) suggested that the force-frequency relationship may shift left if muscle relaxation is slowed, and we have observed a prolonged relaxation time after fatigue produced by both high- and low-frequency stimulation (24). To assess the functional effects of muscular fatigue (degree of force and power loss), it is important to know whether the force-frequency relation shifts left or right with fatigue and to determine the frequency at which peak force is elicited. Because of the prolonged twitch duration associated with fatigue, one would expect the forcefrequency relation to shift left; however, to our knowledge, this has not been experimentally established. Furthermore, no information exists on the effect of fatigue on the optimal stimulation frequency. Because the firing rate of a-motoneurons has been reported to decrease with fatigue (4), it is important to establish the extent to which the optimal frequency is altered. Consequently, one objective of this work was to compare the force-frequency relationship and to determine the optimal stimulation frequency in control and fatigued skeletal muscle. Our working hypothesis is that fatigue results from a combination of factors ranging from a disruption in ECC to events acting directly on the cross bridge (18). Consequently, a solution to the etiology of fatigue will require the employment of a preparation in which each of these causative factors can be systematically studied. In our view, the dorsal head of the frog semitendinosus (ST) muscle is a good choice for the following reasons: 1) ease of isolation of both the intact muscle and the single fiber; 2) good viability in vitro; 3) a parallel fiber alignment allowing the isotonic and isometric contractile properties to be easily and reproducibly measured; and
0 1992 the American
Physiological
Society
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MUSCLE
FATIGUE:
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4) a high
(90%) fast-twitch fiber population, which ensures a high glycolytic capacity and easily fatigable preparation. Our long-term goal is to elucidate the cellular sites and mechanisms of muscle fatigue and to understand the relative importance of each in affecting the functional capacity of skeletal muscle. The specific purpose of this work was to study the isometric and isotonic contractile properties of the frog ST muscle before and during recovery from fatigue and to relate the observed functional changes to alterations in specific steps in the cross-bridge model of muscle contraction (27). A second objective was to establish the effect of fatigue on the force-frequency relationship of limb skeletal muscle. MATERIALS
AND
METHODS
Animals. Northern frogs (Rana pipiens pipiens) were purchased (Kons Scientific, Germantown, WI) and housed in a large aquarium with continuously filtered and aerated water (22OC). To maintain high metabolic stores (glycogen, ATP), they were fed live crickets every 2 days (16). Muscle preparation. The ST muscle was removed from double-pithed frogs and placed in a dissection chamber containing a frog Ringer solution (in mM: 83 NaCl, 2.5 KCl, 1.8 CaCl,, 1.0 MgC12, and 37 NaHCO,). The dorsal head was isolated along its entire length so that it remained connected to its tendinous attachments. Silk threads (4-O) were tied to the proximal and distal tendons at the fiber-tendon junction and small loops formed. Additional threads were tied to the proximal and distal tendons, and knots were formed distal to the loops to prevent slippage. The muscle was then transferred to an experimental chamber and suspended between a stationary post and a dualmode length and force transducer (Cambridge model 352 ergometer). The experimental chamber contained frog Ringer solution (gassed with 95% O&% C02, pH 7.5), maintained at 22°C by a Cambion bipolar temperature controller (model 8093011-01) and thermoelectric module (model 806-1036-04). The bath temperature was monitored by a telethermometer (Yellow Springs model 46 TUC). Fiber type classification and distribution. The dorsal heads of ST muscles were removed from three double-pithed frogs. The excised muscles were laid flat and straight on pieces of index card and quick frozen in a Freon 12 slurry cooled by liquid nitrogen. Cryostat cross sections (10 pm) were cut serially through the proximal, central, and distal portions of the muscle. The deep (lateral or bone side) and superficial (medial or skin side) orientation of the muscle was carefully preserved to permit assignment of fiber type regional distribution. Sections were picked up on 1 mM CrK(SO& (chrome alum) 0.5% gelatincoated slides for histochemical staining of myofibrillar (MF) adenosinetriphosphatase (ATPase) activities (19). A cross section of a rat plantaris muscle was included on each slide. This provided a known standard for histochemical staining because the methods utilized were optimized for mammalian tissues (19). The fibers were classified as slow or fast twitch based upon their stability in acid or base reaction media, respectively, using the methods of Guth and Samaha (19) with slight modification as described here. Cold fixation followed by alkaline preincubation reduced staining to unacceptably low levels. Fixing, but omitting the alkaline preincubation step before incubating at pH 9.2, produced adequate staining for fiber typing. Acid preincubation at pH 4.3 was shortened to 2 min at room temperature to achieve the desired reversal effect; longer preincubations eliminated staining. The MF ATPase-stained sections from the midbelly of the muscles were utilized to quantitate the occurrence and regional
IN CONTRACTILE
FUNCTION
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distribution of the slow- and fast-twitch fiber types. At this level, the muscle contained close to 1,000 fibers; fewer fibers were present in the distal third of the muscle. An average of 330 fibers were sampled per muscle for profiling the fiber type distribution. Optimal length and measurement of isometric contractile properties. Each preparation was adjusted to its optimal length (L,) at which maximal twitch force was elicited (mean resting tension = 11.8 mN). L,, was measured from the fiber-tendon junction at the origin to the fiber-tendon junction at the insertion by microcalipers. Each muscle was stimulated along its entire length with platinum wire electrodes. An isometric twitch contraction was elicited via a supramaximal0.5-ms square-wave pulse (Grass Instruments S48 stimulator) and peak tetanic contraction with 0.5-ms supramaximal pulses at 150 Hz. The transducer output was amplified, displayed on a Tektronix 5111 storage oscilloscope, and sent to a Commodore 64 microcomputer via a universal input-output board consisting of an eight-bit lo-kHz analog-to-digital converter (Microworld Computers, Lakewood, CO). Twitch contraction time (CT), defined as the time from onset of force to peak force, one-half relaxation time (XRT), defined as the time from peak force to 50% decline in peak force, peak twitch force (P,), peak rate of force development and decline for a twitch (P,, *dP/dt), peak tetanic force (P,), and peak rate of force development and decline for a tetanus (P,, &dP/dt) were computer stored and analyzed by custom-designed software. Measurement of muscle Vmax. V,,, was determined by the slack-test technique as described previously (15). P, was elicited by direct electrical stimulation, and the preparation rapidly released to a shorter length so that force fell to baseline. The activated muscle shortened, taking up the slack after which force redeveloped. The duration of unloaded shortening, defined as the time between onset of slack and redevelopment of force, was determined by computer analysis of the force record. Eight different length steps were used for each muscle, and the slack distance was plotted as a function of the duration of the unloaded shortening [mean r = 0.995 t 0.001 (SE)]. V,,, (I&) was determined by dividing the slope of the fitted line by L,,. Fatigue studies: isometric twitch and tetanic contractile properties during recovery. The muscles were fatigued by direct electrical stimulation with lOO-ms 150-Hz trains at l/s for 5 min. The muscles were allowed to recover under aerobic conditions (95% oz-5% CO,), and the isometric contractile properties were determined at 0, 10, and 60 s and 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, and 60 min of recovery. Fatigue studies: force-frequency relationship and maximum isotonic shortening velocity. The force-frequency relationship was determined before and immediately after 5 min of electrical stimulation by an isometric contraction elicited by supramaxima1 pulses at one of the following frequencies: 5, 10, 20, 40, 60, 80, 100, 150, 200, and 250 Hz. We studied only one frequency per preparation to prevent fatigue of the muscle during the prefatigue test and recovery from influencing the poststimulation measurement. This technique produced a prefatigue forcefrequency relationship that was not significantly different from that obtained with the conventional procedure of measuring the force response of multiple frequencies per preparation (Fig. 5A). V max values of the fatigued preparations were determined by the slack-test technique as described above. To prevent recovery, five rather than eight length steps spaced 15 s apart were employed. Data analysis. Statistical significance was determined by a one-way analysis of variance with Students’s unpaired twotailed t test performed on the means. A level of P < 0.05 was chosen as significant. RESULTS
Based on the dorsal head of the ST muscle
Fiber type classification and distribution.
enzyme histochemistry
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Cl502
MUSCLE
FATIGUE:
ALTERATIONS
of the frog (R. pipiens pipiens) contained 89% fast- and 11% slow-twitch fibers. The fast-twitch fibers all showed high base MF ATPase activity but demonstrated some variation from low to moderately high stain intensity after acid MF ATPase. The slow-twitch fibers showed either a low or moderate base MF ATPase activity. The fibers with the lowest base MF ATPase activity are probably slow-nontwitch fibers (14). The slow-twitch fibers were confined to a peripheral zone along the deep (lateral) side and were absent entirely from the distal third of the muscle. Isometric twitch contractile properties. Representative isometric twitches immediately before and after the stimulation period are shown in Fig. 1. The mean Pt was 128 +- 9 mN prefatigue and declined with fatigue to 41 t 2 mN, which was 32% of initial force. The isometric CT increased from 21.3 t 0.4 to 63.1 t 1.8 ms and l/2RT from 13.3 t 0.4 to 110.0 t 8.1 ms, which were -300 and 800% of the initial values, respectively. +dP/dt declined from 7 t 1 to 1 t 1 mN/ms (14% of initial value). Corrected for the change in force [dP/dt PC’], the pre- and postfatigue values were 56.6 t 1.3 and 31.3 t 0.9 mN.ms-l*rnN-l (55% of initial value), respectively. -dP/dt decreased from 5 t 1 to 0.3 t 0.1 mN/ms (6% of initial value). Corrected for the change in force (-dP/dt PC’), the values were 40.9 t 1.2 and 7.4 t 0.4 mN*ms-l rnN-l (18% of initial value), respectively. The isometric contractile properties during recovery from fatigue are displayed in Table 1, and Fig. 2 (inset) shows representative twitch records at specific time points of recovery. Twitch force recovered in two phases, a rapid initial phase, from 32 to 62% of control in the first 2 min followed by a slower phase requiring 15-20 min for complete recovery (Fig. 2). The best-fit linear regression lines for the rapid and slow phases were y = 5.28 + 1.15 (log X) and y = -8.38 + 6.70 (log x), respectively, where y l
l
l
150.
1 3
I 40
1 80
Time
I 120
I 160
I 200
(msec)
Fig. 1. Original records of an isometric twitch contraction immediately before (prefatigue) and after (fatigue) stimulation period. Determination of isometric twitch contractile properties was performed by computer analysis. Prefatigue peak twitch force (P,) was 123 mN, contraction time (CT) was 19.5 ms, and one-half relaxation time (%RT) was 13.6 ms. Fatigue P, was 44 mN, and CT and %RT were 56.6 and 87.8 ms, respectively.
IN CONTRACTILE
FUNCTION
is force and x is minutes of recovery. CT and VRT showed a rapid recovery in the first 5 min, returning to 160 and 265% of control, respectively. Thereafter, recovery slowed with CT returning to the prefatigue value between 35 and 40 min, whereas even at 60 min XRT was 133% of normal (Table 1). Isometric tetanic contractde properties. The prefatigue P, was 575 t 22 mN and at fatigue was 55 t 2 mN, which was 8.5% of the initial force. Figure 3 shows representative prefatigue and fatigue tetanic contractions. Recovery of P, occurred in two phases. A rapid increase representing ~25% of the total occurred within 2 min. The best-fit regression line was y = 8.16 + 8.55 (log x), where y is force and x is minutes of recovery. During the next 38 min the recovery of P, was exponential, reaching -90% of the prefatigue value by 40 min (Fig. 4). The best-fit linear regression line was y = -102 + 55.8 (log x). The means t SE for P, during recovery are shown in Table 1. Figure 4 (inset) shows representative tetanic contractions at 10 and 60 s and 5 and 20 min of recovery. Isotonic Vmax. Vmax of the prefatigue ST was 6.7 t 0.5 L,/s compared with a Vmax of 2.5 t 0.4 L,/s after 5 min of stimulation (mean t SE for 8 muscles). Force-frequency relationship. The force-frequency relations for the prefatigue and fatigue preparations are shown in Fig. 5B. In the control preparation, the ~-HZ stimulation produced little or no fusion and the optimal frequency for eliciting P, was 150 Hz. With fatigue, the force-frequency curve shifted to the left, so that the ~-HZ stimulation yielded a partially fused contraction. Additionally, the optimal frequency for generating P, was reduced to 60 Hz. DISCUSSION
Elucidating the cellular mechanisms of muscle fatigue has proved difficult due in part to the multiplicity of changes associated with fatigue (12, 18). In this work, we selected a stimulation procedure intense enough to produce extensive fatigue (P, < 10% of control) but not so intense as to elicit irreversible muscle damage. The fact that the preparation recovered after fatigue demonstrates that the decline in force with stimulation represented true fatigue and not cell death. The observation that the dorsal head of the frog ST muscle contained primarily fast-twitch fibers (89%) agreed with the published finding of Engel and Irwin (14). The variation in acid MF ATPase activity within the fast-twitch fiber group suggests that more than one isozyme of fast myosin exists. The limited distribution of the slow-nontwitch fibers to the deep proximal and midbelly portions and their relative low percentage of occurrence would account for their rare sampling during single fiber analysis and minor influence on whole muscle contractile physiology. In an attempt to relate the alterations in contractile function observed with fatigue to specific cellular or molecular sites, we have redrawn a schematic model of the kinetics of the actomyosin ATP hydrolysis reaction (cross-bridge cycling) in skeletal muscle (Fig. 6). The scheme was recently published by Metzger and Moss (27) and represented their adaptation of the current models of
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MUSCLE
Table 1. Isometric Group
ALTERATIONS
IN CONTRACTILE
Cl503
FUNCTION
contractile properties n
Prefatigue Fatigue Recovery 10 s 1 min 2 min 5 min 10 min 15 min 20 min 25 min 30 min 35 min 40 min 45 min 50 min 55 min 60 min
FATIGUE:
Pt,
CT
mN
ms
30 11
128&9 4lt2*
21.3~10.4 63.ltl.8*
13 19 11 18 13 12 9 8 8 9 9 9 9 9 9
63t3* 66t4* 79t5* 87t6* 103&B* 116t8 115t8 126t5 137t5 136rt5 138t6 138k6 138t7 138&B 139tlO
59.3&1.8* 45.6tl.6” 41.7tl.6* 34.2tl.l* 3O.lH.3” 28.2tl.2* 25.4tl.2” 23.7t0.7* 22.8k0.8 22.721.0 21.7t0.5 20.6t0.5 20.6t0.4 19.9kO.4 21.4t0.5
%RT, ms
13.3t0.4 1 lO.Ot8.1” 93.3t4.0* 57.9t3.1* 49.lt3.1* 36.lt2.2* 33.6t3.0* 35.0t3.6* 28.9t2.9* 29.lt3.7* 26.0t2.6* 22.7tl.4” 20.9zk1.1* 19.5t0.9* 18.6t0.8* 18.3t0.8* 18.lt0.6*
+dP/dt, mN/ms
-dP/dt, mN/ms
7tl l*l*
5tl 0.3tO.l*
2&l* 3tl* 3tl* 4tl* 5tl* 6tl 6tl 721 8tl 8tl 8tl 8tl 8tl 8tl 8kl
PO mN
ltl* ltl* ltl* 2tl* 2tl* 2tl* 2tl* 3tl* 3&l* 4tl* 4tl* 4tl* 4tl 4-+1 5tl
n
575t22 55t2*
44 6
97t5* 120t4* 147t6* 201&7* 261&B* 308&10* 352tl2* 391Ik12* 420&15* 480tl7* 489t20* 500t20 5llt21 516t23 516t22
22 26 20 22 21 21 21 26 25 17 18 19 20 20 21
Values are means t SE. II, No. of muscles/group except for P,, where n is given separately; Pt, peak twitch force; CT, contraction -dP/dt, peak rate of twitch force decline; P,,, peak one-half relaxation time; +dP/dt, peak rate of twitch force development; * Significantly different from control, P < 0.05.
time; tetanic
%RT, force.
600
lOOi
1 150
>re-fatigue
7
Time (msec) I
10
20
,
,
I
30
40
Recovery
50
I
60
Time
(msec)
(mid
Fig. 2. Recovery of twitch force after stimulation. Values are mean ME; n = B-30. Inset: representative twitch records at prefatigue (solid line) and at 10 s (dashed-dotted line), 60 s (dotted line), and 5 min (dashed line) of recovery. P, recovered to prefatigue value in 15 min.
Fig. 3. Original records of isometric tetanic contractions immediately before (prefatigue) and after (fatigue) stimulation. Determination of isometric tetanic contractile properties was performed by. computer analysis. Prefatigue peak tetanic force (P,) was 461 mN; fatigue value was 59 mN.
ATP hydrolysis. Force production depends on the binding of the myosin head (M) to actin (A). Inorganic phosphate (Pi) release (Fig. 6, step 5) is thought to be coupled to the transition(s) in actomyosin binding from a weakly bound, low-force state (AM Pi) to the strongly bound, high-force state (AM’ ADP). This latter state is probably the dominant cross bridge formed during a peak isometric contraction (27). The transitions from the lowto high-force state are thought to limit +dP/dt, and Metzger and Moss (27) recently observed the transition rate constant to be sevenfold higher in fast- compared with slow-twitch fibers. In contrast to +dP/dt where the rate of cross-bridge attachment appears to be limiting, skeletal muscle Vmax is thought to be limited by the rate of cross-bridge dissociation. The rate-limiting step in
cross-bridge detachment is unknown, but the possibilities include steps 6, 7,1, and 2 of the scheme shown in Fig. 6. In this work both P, and P, showed a biphasic recovery, a rapid initial phase (2 min) followed by a slower (40 min) return to the prefatigue force. However, the recovery curves did not mirror each other, primarily because PO declined substantially more with stimulation (8.5 vs. 32% of initial force). P, is the best indicator of fatigue, because at a constant temperature it is affected only by the number of cross bridges in the high-force state (6). In contrast, Pt is influenced by additional factors such as +dP/dt and the duration of the Ca2+ transient. In this study, the increased CT allowed more time for force development and in this way probably tempered the decline in P, (Fig. 2). The CT, along with the relaxation
l
ADP
l
l
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Cl504
MUSCLE
FATIGUE:
ALTERATIONS
IN CONTRACTILE
FUNCTION
Pre-fatigue v Method l Method 400
1 2
600
(msec)
0
10
20
Minutes
30
40
50
60
of Recovery Frequency
(pps)
Fig. 4. Recovery of force production (% of initial force) after stimulation. Values are means & SE; n = 6-44. Inset: representative tetanus records at prefatigue (a) and at 10 (b) and 60 s (c) and 5 (d) and 20 (e) min of recovery. P,, returned to prefatigue value by 45 min.
time, reflects the time course of the Ca2+ transient. In 1978, Blinks et al. (5) and more recently Allen et al. (1) and Westerblad et al. (31) have demonstrated that the amplitude of the Ca2+ transient decreased while the duration of the transient increased with maintained contractile activity. Therefore it is likely that the large increase twitch contraction and relaxation time associated with muscle fatigue in this preparation can be attributed to a prolongation of the Ca2+ transient. As stated above, it is generally thought that +dP/dt is limited by the cross-bridge transition rate from the weakly bound, low force state to the strongly bound, high force state or step 5 in Fig. 6 (6,27). In fatigue +dP/dt was depressed (Fig. 1). If +dP/dt in an intact muscle is limited by the cross-bridge transition rate (as it appears to be in fully activated single fibers), then the rate constant for cross-bridge binding may be reduced in the fatigued cell. Alternatively, the rate-limiting step in the rate of force development in intact muscles may be limited by other factors (such as rate of Ca2+ release) or the limiting step may switch with fatigue from cross-bridge binding to Ca2+ release. This could occur if the ECC process was altered such that fewer SR Ca2+ release channels were opened (18). The reduced +dP/dt was in part caused by fewer active cross bridges acting in parallel. Consequently, when the data were corrected for the fall in force (+dp/dt P;l), the observed decrease in +dP/dt was attenuated. The rapid phase of recovery in P, may be related to alterations in ECC. Possible candidates include 1) altered sarcolemma action potential (3, 23, 24); 2) blockage or altered action potential propagation into the T tubule (20, 21); 3) altered T-tubular charge movement (18); and 4) a reduced Ca2+ release from the SR (1, 5, 31). In contrast, the slow phase of force recovery may be best explained bY alterations in metabolic processes, particularly an elevated H+ and Pi concentration within the cell (25, 29, 30). The fatigue-induced prolongation of the twitch duration caused an increased fusion of force at low frequencies and a leftward shift in the force-frequency curve. Consel
Y
g 60 z
0
Fatigue
Lo a” 20 11
1
Oh 0
100
150
Frequency
200
250
300
(pps)
Fig. 5. Force-frequency relationship. A: prefatigue force-frequency relationship determined by 2 different methods. Method 1: preparation (n = 6) was stimulated at each specific frequency (5, 10, 20, 40, 60, 80, 100, 150,200, and 250 Hz) and force was expressed as % of P, (mean t SE). Method 2: preparation (n = 3-12/frequency) was stimulated at 1 frequency and value was expressed as % of P, (mean t SE). There were no significant differences in force-frequency relationship between methods 1 and 2. B: force-frequency relationship determined by method 2 (A) for prefatigue and fatigue. Values are means t SE for 3-12 preparations/frequency. Optimal prefatigue frequency for P, was 150 Hz. Force-frequency curve shifted to left at fatigue and optimal frequency for P, decreased to 60 Hz (although no significant differences in force were observed between 60 and 150 Hz in fatigued preparation). I
i
ATP
AM 4
1 p
AMeATP ZAM-ADP.5 1
3’
5
2 I
:
SAkADP 4
Tl
;
MeADP.P,
/ I
Tl
M.ATP C weak
3
binding
ADP
-Z+AM-ADP 6
;“;AM
/
1
7
I I strong
binding
Fig. 6. Schematic model of kinetics of actomyosin ATP hydrolysis reaction during contraction in skeletal muscle, where A is actin and M is heavy meromyosin or myosin Sl. Scheme is redrawn from Metzger and Moss (27).
quently, the optimal frequency for eliciting P, decreased from 150 to 60 Hz. This observation supports the finding of Metzger and Fitts (24), who observed an increased force when the stimulation frequency was reduced from 75 to 5 Hz. Additionally, Bigland-Ritchie et al. (4)
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MUSCLE
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observed a reduced motor nerve firing rate as muscle fatigue developed. Edwards et al. (13) found fatigue to shift the force-frequency curve to the right so that force produced at low frequencies was reduced. However, to our knowledge a complete characterization of the force-frequency relationship in fatigued muscle has not been carried out. It is clear from our results that fatigue produced by high-frequency stimulation shifts the force-frequency relationship to the left (Fig. 5). Consequently, when the stimulation frequency of a fatigued .muscle was reduced, the force produced increased (24). Regardless of the stimulation frequency (high or low) employed to elicit fatigue, a prolonged twitch duration (which probably reflects a prolonged Ca2+ transient) accompanies muscle fatigue (24), and thus one would expect the force-frequency relationship to undergo a leftward shift. It is not clear why Edwards et al. (13) found a right shift; however, their measurements were restricted to a few stimulation frequencies elicited after 21 h of recovery. Consequently the prolonged relaxation time associated with fatigue and responsible for the left shift in the force-frequency relationship may have recovered (Table 1). The decreased Vmax confirms the published report of Edman and Mattiazzi (11). They suggested that the decline in Vmax was mediated by an increased myoplasmic H+ concentration, since unstimulated fibers incubated in high CO2 tension (and thus low pH) showed a similar reduction in force and Vmax. Donaldson and Hermansen (9) showed an elevated H+ to depress P, in the skinned fiber, and Metzger and Moss (26) recently demonstrated H+ to depress P, and Vmax, with the inhibitory effect greater in fast- than slow-twitch fibers. Fiber Vmax is proportional to the myofibrillar ATPase activity (2). An increased H+ has recently been shown to depress both fiber Vmax and ATPase (7), presumably by a direct inhibitory effect of H+ on the ATPase thus slowing the crossbridge cycle rate (Fig. 6). Previously published work by Fitts and Holloszy, utilizing the frog sartorius (17) and rat soleus (16) muscles, showed Vmax to be resistant to fatigue induced change. However, P, decreased only 52 and 30%, respectively, whereas in this study P, decreased 92%. In the Edman and Mattiazzi study (1 I), Vmax did not decline until P, fell by 2 10%. Apparently, the activity of myofibrillar ATPase is unaltered until force falls by ~10%; however, the exact relationship between force, V max9 and ATPase during fatigue seems dependent on the fiber type and activation frequency. In conclusion, the considerable greater fall in P, relative to P, reflects the fact that the decline in P, was tempered by a prolonged contraction time, which probably resulted from a prolongation in the intracellular Ca2+ transient. The biphasic recovery of P, and P, suggests that fatigue was caused by multiple factors with varying recovery times. The results of this study clearly demonstrate that muscle fatigue is associated with a reduced Vmax and a shift in the force-frequency relation to the left. The lower force and velocity will lead to a reduced power output in the fatigued muscle. We thank manuscript.
Barbara
DeNoyer
for
help
in the
preparation
of the
IN
CONTRACTILE
Cl505
FUNCTION
This research was supported in part by National Institute of Arthritis and Musculoskeletal and Skin Diseases Grant AR-39894. L. V. Thompson was supported by Predoctoral Fellowship 89-FA-15 from the American Heart Association-Wisconsin Affiliate and in part by a scholarship from the Foundation for Physical Therapy. Address for reprint requests: R. H. Fitts, Biology Dept., Marquette University, Milwaukee, WI 53233. Received
4 November
1991; accepted
in final
form
13 January
1992.
REFERENCES D. G., J. A. Lee, and H. Westerblad. Intracellular 1. Allen, calcium and tension during fatigue in isolated single muscle fibres from Xenopus laevis. J. Physiol. Lond. 415: 433-458, 1989. 2. Barany, M. ATPase activity of myosin correlated with speed of muscle shortening. J. Gen. Physiol. 50: 197-218, 1967. 3. Bezanilla, F., C. Caputo, H. Gonzalez-Serratos, and R. A. Venosa. Sodium dependence of the inward spread of activation in isolated twitch muscle fibres of the frog. J. Physiol. Lond. 223: 507-523, 1972. 4. Bigland-Ritchie, B., N. J. Dawson, R. S. Johansson, and 0. C. J. Lippold. Reflex origin for the slowing of motoneurone firing rates in fatigue of human voluntary contractions. J. PhysioL. Lond. 379: 451-459, 1986. 5. Blinks, J. R., R. Rudel, and S. R. Taylor. Calcium transients in isolated amphibian skeletal muscle fibres: detection with aequorin. J. Physiol. Lond. 277: 291-323, 1978A 6. Brenner, B. Effect of Ca2+ on cross-bridge turnover kinetics in skinned single rabbit psoas fibers: implications for regulation of muscle contraction. Proc. Natl. Acad. Sci. USA 85: 3265-3269, 1988. 7. Chase, P. B., and M. J. Kushmerick. Effects of pH on contraction of rabbit fast and slow skeletal muscle fibers. Biophys. J. 53: 935-946, 1988. 8. De Haan, A., D. A. Jones, and A. J. Sargeant. Changes in velocity of shortening, power output and relaxation rate during fatigue of rat medial gastrocnemius muscle. Pfluegers Arch. 413: 422-428, 1989. 9. Donaldson, S. K. B., and L. Hermansen. Differential, direct effects of H+ on Ca2+ -activated force of skinned fibers from the soleus, cardiac and adductor magnus muscles of rabbits. Pfluegers Arch. 376: 55-65, 1978. 10. Eberstein, A., and A. Sandow. Fatigue mechanisms in muscle fibers. In: The Effect of Use and Disuse on Neuromuscular Function, edited by E. Gutmann and P. Hnik. Prague: Czechoslovak Acad. of Sci., 1963, p. 515-526. 11. 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 fibres. J. Muscle Res. CeZZ MotiZ. 2: 321-334, 1981. 12. Edwards, R. H. T. New techniques for studying human muscle function, metabolism, and fatigue. Muscle Nerve 7: 599-609, 1984. R. H. T., D. K. Hill, D. A. Jones, and P. A. 13. Edwards, Merton. Fatigue of long duration in human skeletal muscle after exercise. J. Physiol. Lond. 277: 769-778, 1977. 14. Engel, W. K., and R. L. Irwin. A histochemical-physiological correlation of frog skeletal muscle fibers. Am. J. Physiol. 213: 511-518, 1967. 15. Fitts, R. H., D. L. Costill, and P. R. Gardetto. Effect of swim exercise training on human muscle fiber function. J. AppZ. PhysioZ. 66: 465-475, 1989. 16. Fitts, R. H., and J. 0. Holloszy. Contractile properties of rat soleus muscle: effects of training and fatigue. Am. J. Physiol. 233 (CeZZ Physiol. 2): C86-C91, 1977. 17. Fitts, R. H., and J. 0. Holloszy. Effects of fatigue and recovery on contractile properties of frog muscle. J. Appl. PhysioZ. 45: 899902, 1978. 18. Fitts, R. H., and J. M. Metzger. Mechanisms of muscular fatigue. In: Principles of Exercise Biochemistry, edited by J. R. Poortmans. Basel: Karger, 1988, vol 27, p. 212-229. 19. Guth, L., and F. J. Samaha. Procedure for the histochemical demonstration of actomyosin ATPase. Exp. NeuroZ. 28: 365-367, 1970. 20. Howell, J. N., and H. Oetliker. Effects of repetitive activity, ruthenium red, and elevated extracellular calcium on frog skeletal muscle: implications for T-tubule conduction. Can. J. Physiol.
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FATIGUE:
ALTERATIONS
Pharmacol. 65: 691-696, 1986. 21. Howell, J. N., and K. W. Snowdowne. Inhibition of tetanus tension by elevated extracellular calcium concentration. Am. J. Physiol. 240 (Cell Physiol. 9): Cl93-C200, 1981. 22. Jones, D. A. Muscle fatigue due to changes beyond the neuromuscular junction. In: Human Muscle Fatigue: Physiological Mechanisms. Ciba Foundation Symposium 82, edited by R. Porter and J. Whelan. London: Pitman, 1981, p. 178-192. 23. Metzger, J. M., and R. H. Fitts. Fatigue from high- and lowfrequency muscle stimulation: role of sarcolemma action potentials. Exp. Neurol. 93: 320-333, 1986. 24. Metzger, J. M., and R. H. Fitts. Fatigue from high- and lowfrequency muscle stimulation: contractile and biochemical alterations. J. Appl. Physiol. 62: 2075-2082, 1987. 25. Metzger, J. M., and R. H. Fitts. Role of intracellular pH in muscle fatigue. J. Appl. Physiol. 62: 1392-1397, 1987. 26. Metzger, J. M., and R. L. Moss. Greater hydrogen ion-induced depression of tension and velocity in skinned single fibres of rat
IN CONTRACTILE
FUNCTION
fast than slow muscles. J. Physiol. Lond. 393: 727-742, 1987. 27. Metzger, J. M., and R. L. Moss. pH modulation of the kinetics of a Ca2+ sensitive cross-bridge state transition in mammalian single skeletal muscle fibres. J. Physiol. Lond. 428: 7X-764, 1990. 28. Nassar-Gentina, V., J. V. Passoneau, J. L. Vergara, and S. I. Rapoport. Metabolic correlates of fatigue and of recovery from fatigue in single frog muscle fibers. J. Gen. PhysioZ. 72: 593-606, 1978. 29. 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. 30. Thompson, L. V., E. M. Balog, and R. H. Fitts. Muscle fatigue in frog semitendinosus: role of intracellular pH. Am. J. Physiol. 262 (Cell Physiol. 31): Cl507-Cl512, 1992. 31. Westerblad, H., J. A. Lee, A. G. Lamb, S. R. Bolsover, and D. G. Allen. Spatial gradients of intracellular calcium in skeletal muscle during fatigue. Pfluegers Arch. 415: 734-740, 1990.
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E. M. BALOG,
D. A. RILEY,
AND R. H. FITTS
Biology Department/Biological and Biomedical Research Institute, Milwaukee 53233; and Cellular Biology and Anatomy Department, Milwaukee, Wisconsin 53226 Thompson, L. V., E. M. Balog, D. A. Riley, and R. H. Fitts. Muscle fatigue in frog semitendinosus: alterations in contractile function. Am. J. Physiol. 262 (CeZZPhysioZ. 31): Cl500C1506,1992.-The purpose of this study was to characterize the contractile properties of the frog semitendinosus (ST) muscle before and during recovery from fatigue, to relate the observed functional changes to alterations in specific steps in the crossbridge model of muscle contraction, and to determine how fatigue affects the force-frequency relationship. The frog ST (22OC) was fatigued by direct electrical stimulation with lOO-ms 150-Hz trains at l/s for 5 min. The fatigue protocol reduced peak twitch (P,) and tetanic (P,) force to 32 and 8.5% of initial force, respectively. The decline in P, was less than P,, in part due to a prolongation in the isometric contraction time (CT), which increased to 300% of the initial value. The isometric twitch duration was greatly prolonged as reflected by the lengthened CT and the 800% increase in the one-half relaxation time (XRT). Both P, and P, showed a biphasic recovery, a rapid initial phase (2 min) followed by a slower (40 min) return to the prefatigue force. CT and 1/2RT also recovered in two phases, returning to 160 and 265% of control in the first 5 min. CT returned to the prefatigue value between 35 and 40 min, whereas even at 60 min l/2RT was 133% of control. The maximal velocity of shortening, determined by the slack test, was significantly reduced [from 6.7 t 0.5 to 2.5 of: 0.4 optimal muscle length/s] at fatigue. The force-frequency relationship was shifted to the left, so that optimal frequency for generating P, was reduced. In conclusion, the biphasic recovery of P, and P, suggests that fatigue was caused by multiple factors with varying recovery times. The fast phase probably mirrors a rapid change in one or more of the steps of excitation-contraction coupling, whereas the slow phase may be mediated by cellular recovery from contraction-induced increases in H+ and inorganic phosphate. contractile properties; peak twitch force; peak tetanic force; maximal velocity of shortening; contraction time; one-half relaxation time
NUMEROUS INVESTIGATIONS have been conducted over the last century in an attempt to elucidate the etiology of muscle fatigue. Despite these efforts the precise cause of muscle fatigue remains unknown. Alterations in the functional capacity of the central nervous system (central fatigue), the peripheral a-motor nerves, the neuromuscular junctions, or the skeletal muscles could all contribute to fatigue. However, the preponderance of experimental evidence suggests that the etiology of muscle fatigue is primarily confined to the peripheral skeletal muscle cells (4, 10, 18). The problem is complex inasmuch as the degree and cellular mechanisms of muscle fatigue vary with the type of contractile activity and the fiber type composition of the muscle. Fatigue, defined as a reduction in muscle force and power (8, 12, 18), probably results from multiple factors, including alterations in excitation-contraction coupling (ECC) (18), cross-bridge events (27, 29), and cell metabolism (28) . Cl500
0363-6143/92
$2.00
Copyright
Marquette University, Medical College of Wisconsin,
Several experimental models have been utilized to examine the mechanisms of muscle fatigue (18, 22). In addition to a reduced peak tetanic and twitch force, fatigue is generally associated with a prolonged isometric twitch contraction and relaxation time (17,24) and a reduced maximal velocity of shortening ( Vmax) and peak power (8). The prolonged twitch duration reduces the optimal frequency for peak tetanic force and should shift the entire force-frequency relationship to the left toward lower frequencies (24). Edwards et al. (13) observed force elicited by low-frequency stimulation (20 Hz) to be depressed for hours after fatigue in the adductor pollicis muscle of humans (low-frequency fatigue). These authors’ conclusion that the force-frequency relationship shifts right with fatigue was based on the response to a single stimulus frequency (20 Hz). Perhaps more importantly, these data do not describe the force-frequency relationship in the contracting fatigued muscle uninfluenced by periods of recovery. Edwards (12) suggested that the force-frequency relationship may shift left if muscle relaxation is slowed, and we have observed a prolonged relaxation time after fatigue produced by both high- and low-frequency stimulation (24). To assess the functional effects of muscular fatigue (degree of force and power loss), it is important to know whether the force-frequency relation shifts left or right with fatigue and to determine the frequency at which peak force is elicited. Because of the prolonged twitch duration associated with fatigue, one would expect the forcefrequency relation to shift left; however, to our knowledge, this has not been experimentally established. Furthermore, no information exists on the effect of fatigue on the optimal stimulation frequency. Because the firing rate of a-motoneurons has been reported to decrease with fatigue (4), it is important to establish the extent to which the optimal frequency is altered. Consequently, one objective of this work was to compare the force-frequency relationship and to determine the optimal stimulation frequency in control and fatigued skeletal muscle. Our working hypothesis is that fatigue results from a combination of factors ranging from a disruption in ECC to events acting directly on the cross bridge (18). Consequently, a solution to the etiology of fatigue will require the employment of a preparation in which each of these causative factors can be systematically studied. In our view, the dorsal head of the frog semitendinosus (ST) muscle is a good choice for the following reasons: 1) ease of isolation of both the intact muscle and the single fiber; 2) good viability in vitro; 3) a parallel fiber alignment allowing the isotonic and isometric contractile properties to be easily and reproducibly measured; and
0 1992 the American
Physiological
Society
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MUSCLE
FATIGUE:
ALTERATIONS
4) a high
(90%) fast-twitch fiber population, which ensures a high glycolytic capacity and easily fatigable preparation. Our long-term goal is to elucidate the cellular sites and mechanisms of muscle fatigue and to understand the relative importance of each in affecting the functional capacity of skeletal muscle. The specific purpose of this work was to study the isometric and isotonic contractile properties of the frog ST muscle before and during recovery from fatigue and to relate the observed functional changes to alterations in specific steps in the cross-bridge model of muscle contraction (27). A second objective was to establish the effect of fatigue on the force-frequency relationship of limb skeletal muscle. MATERIALS
AND
METHODS
Animals. Northern frogs (Rana pipiens pipiens) were purchased (Kons Scientific, Germantown, WI) and housed in a large aquarium with continuously filtered and aerated water (22OC). To maintain high metabolic stores (glycogen, ATP), they were fed live crickets every 2 days (16). Muscle preparation. The ST muscle was removed from double-pithed frogs and placed in a dissection chamber containing a frog Ringer solution (in mM: 83 NaCl, 2.5 KCl, 1.8 CaCl,, 1.0 MgC12, and 37 NaHCO,). The dorsal head was isolated along its entire length so that it remained connected to its tendinous attachments. Silk threads (4-O) were tied to the proximal and distal tendons at the fiber-tendon junction and small loops formed. Additional threads were tied to the proximal and distal tendons, and knots were formed distal to the loops to prevent slippage. The muscle was then transferred to an experimental chamber and suspended between a stationary post and a dualmode length and force transducer (Cambridge model 352 ergometer). The experimental chamber contained frog Ringer solution (gassed with 95% O&% C02, pH 7.5), maintained at 22°C by a Cambion bipolar temperature controller (model 8093011-01) and thermoelectric module (model 806-1036-04). The bath temperature was monitored by a telethermometer (Yellow Springs model 46 TUC). Fiber type classification and distribution. The dorsal heads of ST muscles were removed from three double-pithed frogs. The excised muscles were laid flat and straight on pieces of index card and quick frozen in a Freon 12 slurry cooled by liquid nitrogen. Cryostat cross sections (10 pm) were cut serially through the proximal, central, and distal portions of the muscle. The deep (lateral or bone side) and superficial (medial or skin side) orientation of the muscle was carefully preserved to permit assignment of fiber type regional distribution. Sections were picked up on 1 mM CrK(SO& (chrome alum) 0.5% gelatincoated slides for histochemical staining of myofibrillar (MF) adenosinetriphosphatase (ATPase) activities (19). A cross section of a rat plantaris muscle was included on each slide. This provided a known standard for histochemical staining because the methods utilized were optimized for mammalian tissues (19). The fibers were classified as slow or fast twitch based upon their stability in acid or base reaction media, respectively, using the methods of Guth and Samaha (19) with slight modification as described here. Cold fixation followed by alkaline preincubation reduced staining to unacceptably low levels. Fixing, but omitting the alkaline preincubation step before incubating at pH 9.2, produced adequate staining for fiber typing. Acid preincubation at pH 4.3 was shortened to 2 min at room temperature to achieve the desired reversal effect; longer preincubations eliminated staining. The MF ATPase-stained sections from the midbelly of the muscles were utilized to quantitate the occurrence and regional
IN CONTRACTILE
FUNCTION
Cl501
distribution of the slow- and fast-twitch fiber types. At this level, the muscle contained close to 1,000 fibers; fewer fibers were present in the distal third of the muscle. An average of 330 fibers were sampled per muscle for profiling the fiber type distribution. Optimal length and measurement of isometric contractile properties. Each preparation was adjusted to its optimal length (L,) at which maximal twitch force was elicited (mean resting tension = 11.8 mN). L,, was measured from the fiber-tendon junction at the origin to the fiber-tendon junction at the insertion by microcalipers. Each muscle was stimulated along its entire length with platinum wire electrodes. An isometric twitch contraction was elicited via a supramaximal0.5-ms square-wave pulse (Grass Instruments S48 stimulator) and peak tetanic contraction with 0.5-ms supramaximal pulses at 150 Hz. The transducer output was amplified, displayed on a Tektronix 5111 storage oscilloscope, and sent to a Commodore 64 microcomputer via a universal input-output board consisting of an eight-bit lo-kHz analog-to-digital converter (Microworld Computers, Lakewood, CO). Twitch contraction time (CT), defined as the time from onset of force to peak force, one-half relaxation time (XRT), defined as the time from peak force to 50% decline in peak force, peak twitch force (P,), peak rate of force development and decline for a twitch (P,, *dP/dt), peak tetanic force (P,), and peak rate of force development and decline for a tetanus (P,, &dP/dt) were computer stored and analyzed by custom-designed software. Measurement of muscle Vmax. V,,, was determined by the slack-test technique as described previously (15). P, was elicited by direct electrical stimulation, and the preparation rapidly released to a shorter length so that force fell to baseline. The activated muscle shortened, taking up the slack after which force redeveloped. The duration of unloaded shortening, defined as the time between onset of slack and redevelopment of force, was determined by computer analysis of the force record. Eight different length steps were used for each muscle, and the slack distance was plotted as a function of the duration of the unloaded shortening [mean r = 0.995 t 0.001 (SE)]. V,,, (I&) was determined by dividing the slope of the fitted line by L,,. Fatigue studies: isometric twitch and tetanic contractile properties during recovery. The muscles were fatigued by direct electrical stimulation with lOO-ms 150-Hz trains at l/s for 5 min. The muscles were allowed to recover under aerobic conditions (95% oz-5% CO,), and the isometric contractile properties were determined at 0, 10, and 60 s and 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, and 60 min of recovery. Fatigue studies: force-frequency relationship and maximum isotonic shortening velocity. The force-frequency relationship was determined before and immediately after 5 min of electrical stimulation by an isometric contraction elicited by supramaxima1 pulses at one of the following frequencies: 5, 10, 20, 40, 60, 80, 100, 150, 200, and 250 Hz. We studied only one frequency per preparation to prevent fatigue of the muscle during the prefatigue test and recovery from influencing the poststimulation measurement. This technique produced a prefatigue forcefrequency relationship that was not significantly different from that obtained with the conventional procedure of measuring the force response of multiple frequencies per preparation (Fig. 5A). V max values of the fatigued preparations were determined by the slack-test technique as described above. To prevent recovery, five rather than eight length steps spaced 15 s apart were employed. Data analysis. Statistical significance was determined by a one-way analysis of variance with Students’s unpaired twotailed t test performed on the means. A level of P < 0.05 was chosen as significant. RESULTS
Based on the dorsal head of the ST muscle
Fiber type classification and distribution.
enzyme histochemistry
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Cl502
MUSCLE
FATIGUE:
ALTERATIONS
of the frog (R. pipiens pipiens) contained 89% fast- and 11% slow-twitch fibers. The fast-twitch fibers all showed high base MF ATPase activity but demonstrated some variation from low to moderately high stain intensity after acid MF ATPase. The slow-twitch fibers showed either a low or moderate base MF ATPase activity. The fibers with the lowest base MF ATPase activity are probably slow-nontwitch fibers (14). The slow-twitch fibers were confined to a peripheral zone along the deep (lateral) side and were absent entirely from the distal third of the muscle. Isometric twitch contractile properties. Representative isometric twitches immediately before and after the stimulation period are shown in Fig. 1. The mean Pt was 128 +- 9 mN prefatigue and declined with fatigue to 41 t 2 mN, which was 32% of initial force. The isometric CT increased from 21.3 t 0.4 to 63.1 t 1.8 ms and l/2RT from 13.3 t 0.4 to 110.0 t 8.1 ms, which were -300 and 800% of the initial values, respectively. +dP/dt declined from 7 t 1 to 1 t 1 mN/ms (14% of initial value). Corrected for the change in force [dP/dt PC’], the pre- and postfatigue values were 56.6 t 1.3 and 31.3 t 0.9 mN.ms-l*rnN-l (55% of initial value), respectively. -dP/dt decreased from 5 t 1 to 0.3 t 0.1 mN/ms (6% of initial value). Corrected for the change in force (-dP/dt PC’), the values were 40.9 t 1.2 and 7.4 t 0.4 mN*ms-l rnN-l (18% of initial value), respectively. The isometric contractile properties during recovery from fatigue are displayed in Table 1, and Fig. 2 (inset) shows representative twitch records at specific time points of recovery. Twitch force recovered in two phases, a rapid initial phase, from 32 to 62% of control in the first 2 min followed by a slower phase requiring 15-20 min for complete recovery (Fig. 2). The best-fit linear regression lines for the rapid and slow phases were y = 5.28 + 1.15 (log X) and y = -8.38 + 6.70 (log x), respectively, where y l
l
l
150.
1 3
I 40
1 80
Time
I 120
I 160
I 200
(msec)
Fig. 1. Original records of an isometric twitch contraction immediately before (prefatigue) and after (fatigue) stimulation period. Determination of isometric twitch contractile properties was performed by computer analysis. Prefatigue peak twitch force (P,) was 123 mN, contraction time (CT) was 19.5 ms, and one-half relaxation time (%RT) was 13.6 ms. Fatigue P, was 44 mN, and CT and %RT were 56.6 and 87.8 ms, respectively.
IN CONTRACTILE
FUNCTION
is force and x is minutes of recovery. CT and VRT showed a rapid recovery in the first 5 min, returning to 160 and 265% of control, respectively. Thereafter, recovery slowed with CT returning to the prefatigue value between 35 and 40 min, whereas even at 60 min XRT was 133% of normal (Table 1). Isometric tetanic contractde properties. The prefatigue P, was 575 t 22 mN and at fatigue was 55 t 2 mN, which was 8.5% of the initial force. Figure 3 shows representative prefatigue and fatigue tetanic contractions. Recovery of P, occurred in two phases. A rapid increase representing ~25% of the total occurred within 2 min. The best-fit regression line was y = 8.16 + 8.55 (log x), where y is force and x is minutes of recovery. During the next 38 min the recovery of P, was exponential, reaching -90% of the prefatigue value by 40 min (Fig. 4). The best-fit linear regression line was y = -102 + 55.8 (log x). The means t SE for P, during recovery are shown in Table 1. Figure 4 (inset) shows representative tetanic contractions at 10 and 60 s and 5 and 20 min of recovery. Isotonic Vmax. Vmax of the prefatigue ST was 6.7 t 0.5 L,/s compared with a Vmax of 2.5 t 0.4 L,/s after 5 min of stimulation (mean t SE for 8 muscles). Force-frequency relationship. The force-frequency relations for the prefatigue and fatigue preparations are shown in Fig. 5B. In the control preparation, the ~-HZ stimulation produced little or no fusion and the optimal frequency for eliciting P, was 150 Hz. With fatigue, the force-frequency curve shifted to the left, so that the ~-HZ stimulation yielded a partially fused contraction. Additionally, the optimal frequency for generating P, was reduced to 60 Hz. DISCUSSION
Elucidating the cellular mechanisms of muscle fatigue has proved difficult due in part to the multiplicity of changes associated with fatigue (12, 18). In this work, we selected a stimulation procedure intense enough to produce extensive fatigue (P, < 10% of control) but not so intense as to elicit irreversible muscle damage. The fact that the preparation recovered after fatigue demonstrates that the decline in force with stimulation represented true fatigue and not cell death. The observation that the dorsal head of the frog ST muscle contained primarily fast-twitch fibers (89%) agreed with the published finding of Engel and Irwin (14). The variation in acid MF ATPase activity within the fast-twitch fiber group suggests that more than one isozyme of fast myosin exists. The limited distribution of the slow-nontwitch fibers to the deep proximal and midbelly portions and their relative low percentage of occurrence would account for their rare sampling during single fiber analysis and minor influence on whole muscle contractile physiology. In an attempt to relate the alterations in contractile function observed with fatigue to specific cellular or molecular sites, we have redrawn a schematic model of the kinetics of the actomyosin ATP hydrolysis reaction (cross-bridge cycling) in skeletal muscle (Fig. 6). The scheme was recently published by Metzger and Moss (27) and represented their adaptation of the current models of
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MUSCLE
Table 1. Isometric Group
ALTERATIONS
IN CONTRACTILE
Cl503
FUNCTION
contractile properties n
Prefatigue Fatigue Recovery 10 s 1 min 2 min 5 min 10 min 15 min 20 min 25 min 30 min 35 min 40 min 45 min 50 min 55 min 60 min
FATIGUE:
Pt,
CT
mN
ms
30 11
128&9 4lt2*
21.3~10.4 63.ltl.8*
13 19 11 18 13 12 9 8 8 9 9 9 9 9 9
63t3* 66t4* 79t5* 87t6* 103&B* 116t8 115t8 126t5 137t5 136rt5 138t6 138k6 138t7 138&B 139tlO
59.3&1.8* 45.6tl.6” 41.7tl.6* 34.2tl.l* 3O.lH.3” 28.2tl.2* 25.4tl.2” 23.7t0.7* 22.8k0.8 22.721.0 21.7t0.5 20.6t0.5 20.6t0.4 19.9kO.4 21.4t0.5
%RT, ms
13.3t0.4 1 lO.Ot8.1” 93.3t4.0* 57.9t3.1* 49.lt3.1* 36.lt2.2* 33.6t3.0* 35.0t3.6* 28.9t2.9* 29.lt3.7* 26.0t2.6* 22.7tl.4” 20.9zk1.1* 19.5t0.9* 18.6t0.8* 18.3t0.8* 18.lt0.6*
+dP/dt, mN/ms
-dP/dt, mN/ms
7tl l*l*
5tl 0.3tO.l*
2&l* 3tl* 3tl* 4tl* 5tl* 6tl 6tl 721 8tl 8tl 8tl 8tl 8tl 8tl 8kl
PO mN
ltl* ltl* ltl* 2tl* 2tl* 2tl* 2tl* 3tl* 3&l* 4tl* 4tl* 4tl* 4tl 4-+1 5tl
n
575t22 55t2*
44 6
97t5* 120t4* 147t6* 201&7* 261&B* 308&10* 352tl2* 391Ik12* 420&15* 480tl7* 489t20* 500t20 5llt21 516t23 516t22
22 26 20 22 21 21 21 26 25 17 18 19 20 20 21
Values are means t SE. II, No. of muscles/group except for P,, where n is given separately; Pt, peak twitch force; CT, contraction -dP/dt, peak rate of twitch force decline; P,,, peak one-half relaxation time; +dP/dt, peak rate of twitch force development; * Significantly different from control, P < 0.05.
time; tetanic
%RT, force.
600
lOOi
1 150
>re-fatigue
7
Time (msec) I
10
20
,
,
I
30
40
Recovery
50
I
60
Time
(msec)
(mid
Fig. 2. Recovery of twitch force after stimulation. Values are mean ME; n = B-30. Inset: representative twitch records at prefatigue (solid line) and at 10 s (dashed-dotted line), 60 s (dotted line), and 5 min (dashed line) of recovery. P, recovered to prefatigue value in 15 min.
Fig. 3. Original records of isometric tetanic contractions immediately before (prefatigue) and after (fatigue) stimulation. Determination of isometric tetanic contractile properties was performed by. computer analysis. Prefatigue peak tetanic force (P,) was 461 mN; fatigue value was 59 mN.
ATP hydrolysis. Force production depends on the binding of the myosin head (M) to actin (A). Inorganic phosphate (Pi) release (Fig. 6, step 5) is thought to be coupled to the transition(s) in actomyosin binding from a weakly bound, low-force state (AM Pi) to the strongly bound, high-force state (AM’ ADP). This latter state is probably the dominant cross bridge formed during a peak isometric contraction (27). The transitions from the lowto high-force state are thought to limit +dP/dt, and Metzger and Moss (27) recently observed the transition rate constant to be sevenfold higher in fast- compared with slow-twitch fibers. In contrast to +dP/dt where the rate of cross-bridge attachment appears to be limiting, skeletal muscle Vmax is thought to be limited by the rate of cross-bridge dissociation. The rate-limiting step in
cross-bridge detachment is unknown, but the possibilities include steps 6, 7,1, and 2 of the scheme shown in Fig. 6. In this work both P, and P, showed a biphasic recovery, a rapid initial phase (2 min) followed by a slower (40 min) return to the prefatigue force. However, the recovery curves did not mirror each other, primarily because PO declined substantially more with stimulation (8.5 vs. 32% of initial force). P, is the best indicator of fatigue, because at a constant temperature it is affected only by the number of cross bridges in the high-force state (6). In contrast, Pt is influenced by additional factors such as +dP/dt and the duration of the Ca2+ transient. In this study, the increased CT allowed more time for force development and in this way probably tempered the decline in P, (Fig. 2). The CT, along with the relaxation
l
ADP
l
l
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Cl504
MUSCLE
FATIGUE:
ALTERATIONS
IN CONTRACTILE
FUNCTION
Pre-fatigue v Method l Method 400
1 2
600
(msec)
0
10
20
Minutes
30
40
50
60
of Recovery Frequency
(pps)
Fig. 4. Recovery of force production (% of initial force) after stimulation. Values are means & SE; n = 6-44. Inset: representative tetanus records at prefatigue (a) and at 10 (b) and 60 s (c) and 5 (d) and 20 (e) min of recovery. P,, returned to prefatigue value by 45 min.
time, reflects the time course of the Ca2+ transient. In 1978, Blinks et al. (5) and more recently Allen et al. (1) and Westerblad et al. (31) have demonstrated that the amplitude of the Ca2+ transient decreased while the duration of the transient increased with maintained contractile activity. Therefore it is likely that the large increase twitch contraction and relaxation time associated with muscle fatigue in this preparation can be attributed to a prolongation of the Ca2+ transient. As stated above, it is generally thought that +dP/dt is limited by the cross-bridge transition rate from the weakly bound, low force state to the strongly bound, high force state or step 5 in Fig. 6 (6,27). In fatigue +dP/dt was depressed (Fig. 1). If +dP/dt in an intact muscle is limited by the cross-bridge transition rate (as it appears to be in fully activated single fibers), then the rate constant for cross-bridge binding may be reduced in the fatigued cell. Alternatively, the rate-limiting step in the rate of force development in intact muscles may be limited by other factors (such as rate of Ca2+ release) or the limiting step may switch with fatigue from cross-bridge binding to Ca2+ release. This could occur if the ECC process was altered such that fewer SR Ca2+ release channels were opened (18). The reduced +dP/dt was in part caused by fewer active cross bridges acting in parallel. Consequently, when the data were corrected for the fall in force (+dp/dt P;l), the observed decrease in +dP/dt was attenuated. The rapid phase of recovery in P, may be related to alterations in ECC. Possible candidates include 1) altered sarcolemma action potential (3, 23, 24); 2) blockage or altered action potential propagation into the T tubule (20, 21); 3) altered T-tubular charge movement (18); and 4) a reduced Ca2+ release from the SR (1, 5, 31). In contrast, the slow phase of force recovery may be best explained bY alterations in metabolic processes, particularly an elevated H+ and Pi concentration within the cell (25, 29, 30). The fatigue-induced prolongation of the twitch duration caused an increased fusion of force at low frequencies and a leftward shift in the force-frequency curve. Consel
Y
g 60 z
0
Fatigue
Lo a” 20 11
1
Oh 0
100
150
Frequency
200
250
300
(pps)
Fig. 5. Force-frequency relationship. A: prefatigue force-frequency relationship determined by 2 different methods. Method 1: preparation (n = 6) was stimulated at each specific frequency (5, 10, 20, 40, 60, 80, 100, 150,200, and 250 Hz) and force was expressed as % of P, (mean t SE). Method 2: preparation (n = 3-12/frequency) was stimulated at 1 frequency and value was expressed as % of P, (mean t SE). There were no significant differences in force-frequency relationship between methods 1 and 2. B: force-frequency relationship determined by method 2 (A) for prefatigue and fatigue. Values are means t SE for 3-12 preparations/frequency. Optimal prefatigue frequency for P, was 150 Hz. Force-frequency curve shifted to left at fatigue and optimal frequency for P, decreased to 60 Hz (although no significant differences in force were observed between 60 and 150 Hz in fatigued preparation). I
i
ATP
AM 4
1 p
AMeATP ZAM-ADP.5 1
3’
5
2 I
:
SAkADP 4
Tl
;
MeADP.P,
/ I
Tl
M.ATP C weak
3
binding
ADP
-Z+AM-ADP 6
;“;AM
/
1
7
I I strong
binding
Fig. 6. Schematic model of kinetics of actomyosin ATP hydrolysis reaction during contraction in skeletal muscle, where A is actin and M is heavy meromyosin or myosin Sl. Scheme is redrawn from Metzger and Moss (27).
quently, the optimal frequency for eliciting P, decreased from 150 to 60 Hz. This observation supports the finding of Metzger and Fitts (24), who observed an increased force when the stimulation frequency was reduced from 75 to 5 Hz. Additionally, Bigland-Ritchie et al. (4)
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MUSCLE
FATIGUE:
ALTERATIONS
observed a reduced motor nerve firing rate as muscle fatigue developed. Edwards et al. (13) found fatigue to shift the force-frequency curve to the right so that force produced at low frequencies was reduced. However, to our knowledge a complete characterization of the force-frequency relationship in fatigued muscle has not been carried out. It is clear from our results that fatigue produced by high-frequency stimulation shifts the force-frequency relationship to the left (Fig. 5). Consequently, when the stimulation frequency of a fatigued .muscle was reduced, the force produced increased (24). Regardless of the stimulation frequency (high or low) employed to elicit fatigue, a prolonged twitch duration (which probably reflects a prolonged Ca2+ transient) accompanies muscle fatigue (24), and thus one would expect the force-frequency relationship to undergo a leftward shift. It is not clear why Edwards et al. (13) found a right shift; however, their measurements were restricted to a few stimulation frequencies elicited after 21 h of recovery. Consequently the prolonged relaxation time associated with fatigue and responsible for the left shift in the force-frequency relationship may have recovered (Table 1). The decreased Vmax confirms the published report of Edman and Mattiazzi (11). They suggested that the decline in Vmax was mediated by an increased myoplasmic H+ concentration, since unstimulated fibers incubated in high CO2 tension (and thus low pH) showed a similar reduction in force and Vmax. Donaldson and Hermansen (9) showed an elevated H+ to depress P, in the skinned fiber, and Metzger and Moss (26) recently demonstrated H+ to depress P, and Vmax, with the inhibitory effect greater in fast- than slow-twitch fibers. Fiber Vmax is proportional to the myofibrillar ATPase activity (2). An increased H+ has recently been shown to depress both fiber Vmax and ATPase (7), presumably by a direct inhibitory effect of H+ on the ATPase thus slowing the crossbridge cycle rate (Fig. 6). Previously published work by Fitts and Holloszy, utilizing the frog sartorius (17) and rat soleus (16) muscles, showed Vmax to be resistant to fatigue induced change. However, P, decreased only 52 and 30%, respectively, whereas in this study P, decreased 92%. In the Edman and Mattiazzi study (1 I), Vmax did not decline until P, fell by 2 10%. Apparently, the activity of myofibrillar ATPase is unaltered until force falls by ~10%; however, the exact relationship between force, V max9 and ATPase during fatigue seems dependent on the fiber type and activation frequency. In conclusion, the considerable greater fall in P, relative to P, reflects the fact that the decline in P, was tempered by a prolonged contraction time, which probably resulted from a prolongation in the intracellular Ca2+ transient. The biphasic recovery of P, and P, suggests that fatigue was caused by multiple factors with varying recovery times. The results of this study clearly demonstrate that muscle fatigue is associated with a reduced Vmax and a shift in the force-frequency relation to the left. The lower force and velocity will lead to a reduced power output in the fatigued muscle. We thank manuscript.
Barbara
DeNoyer
for
help
in the
preparation
of the
IN
CONTRACTILE
Cl505
FUNCTION
This research was supported in part by National Institute of Arthritis and Musculoskeletal and Skin Diseases Grant AR-39894. L. V. Thompson was supported by Predoctoral Fellowship 89-FA-15 from the American Heart Association-Wisconsin Affiliate and in part by a scholarship from the Foundation for Physical Therapy. Address for reprint requests: R. H. Fitts, Biology Dept., Marquette University, Milwaukee, WI 53233. Received
4 November
1991; accepted
in final
form
13 January
1992.
REFERENCES D. G., J. A. Lee, and H. Westerblad. Intracellular 1. Allen, calcium and tension during fatigue in isolated single muscle fibres from Xenopus laevis. J. Physiol. Lond. 415: 433-458, 1989. 2. Barany, M. ATPase activity of myosin correlated with speed of muscle shortening. J. Gen. Physiol. 50: 197-218, 1967. 3. Bezanilla, F., C. Caputo, H. Gonzalez-Serratos, and R. A. Venosa. Sodium dependence of the inward spread of activation in isolated twitch muscle fibres of the frog. J. Physiol. Lond. 223: 507-523, 1972. 4. Bigland-Ritchie, B., N. J. Dawson, R. S. Johansson, and 0. C. J. Lippold. Reflex origin for the slowing of motoneurone firing rates in fatigue of human voluntary contractions. J. PhysioL. Lond. 379: 451-459, 1986. 5. Blinks, J. R., R. Rudel, and S. R. Taylor. Calcium transients in isolated amphibian skeletal muscle fibres: detection with aequorin. J. Physiol. Lond. 277: 291-323, 1978A 6. Brenner, B. Effect of Ca2+ on cross-bridge turnover kinetics in skinned single rabbit psoas fibers: implications for regulation of muscle contraction. Proc. Natl. Acad. Sci. USA 85: 3265-3269, 1988. 7. Chase, P. B., and M. J. Kushmerick. Effects of pH on contraction of rabbit fast and slow skeletal muscle fibers. Biophys. J. 53: 935-946, 1988. 8. De Haan, A., D. A. Jones, and A. J. Sargeant. Changes in velocity of shortening, power output and relaxation rate during fatigue of rat medial gastrocnemius muscle. Pfluegers Arch. 413: 422-428, 1989. 9. Donaldson, S. K. B., and L. Hermansen. Differential, direct effects of H+ on Ca2+ -activated force of skinned fibers from the soleus, cardiac and adductor magnus muscles of rabbits. Pfluegers Arch. 376: 55-65, 1978. 10. Eberstein, A., and A. Sandow. Fatigue mechanisms in muscle fibers. In: The Effect of Use and Disuse on Neuromuscular Function, edited by E. Gutmann and P. Hnik. Prague: Czechoslovak Acad. of Sci., 1963, p. 515-526. 11. 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 fibres. J. Muscle Res. CeZZ MotiZ. 2: 321-334, 1981. 12. Edwards, R. H. T. New techniques for studying human muscle function, metabolism, and fatigue. Muscle Nerve 7: 599-609, 1984. R. H. T., D. K. Hill, D. A. Jones, and P. A. 13. Edwards, Merton. Fatigue of long duration in human skeletal muscle after exercise. J. Physiol. Lond. 277: 769-778, 1977. 14. Engel, W. K., and R. L. Irwin. A histochemical-physiological correlation of frog skeletal muscle fibers. Am. J. Physiol. 213: 511-518, 1967. 15. Fitts, R. H., D. L. Costill, and P. R. Gardetto. Effect of swim exercise training on human muscle fiber function. J. AppZ. PhysioZ. 66: 465-475, 1989. 16. Fitts, R. H., and J. 0. Holloszy. Contractile properties of rat soleus muscle: effects of training and fatigue. Am. J. Physiol. 233 (CeZZ Physiol. 2): C86-C91, 1977. 17. Fitts, R. H., and J. 0. Holloszy. Effects of fatigue and recovery on contractile properties of frog muscle. J. Appl. PhysioZ. 45: 899902, 1978. 18. Fitts, R. H., and J. M. Metzger. Mechanisms of muscular fatigue. In: Principles of Exercise Biochemistry, edited by J. R. Poortmans. Basel: Karger, 1988, vol 27, p. 212-229. 19. Guth, L., and F. J. Samaha. Procedure for the histochemical demonstration of actomyosin ATPase. Exp. NeuroZ. 28: 365-367, 1970. 20. Howell, J. N., and H. Oetliker. Effects of repetitive activity, ruthenium red, and elevated extracellular calcium on frog skeletal muscle: implications for T-tubule conduction. Can. J. Physiol.
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Cl506
MUSCLE
FATIGUE:
ALTERATIONS
Pharmacol. 65: 691-696, 1986. 21. Howell, J. N., and K. W. Snowdowne. Inhibition of tetanus tension by elevated extracellular calcium concentration. Am. J. Physiol. 240 (Cell Physiol. 9): Cl93-C200, 1981. 22. Jones, D. A. Muscle fatigue due to changes beyond the neuromuscular junction. In: Human Muscle Fatigue: Physiological Mechanisms. Ciba Foundation Symposium 82, edited by R. Porter and J. Whelan. London: Pitman, 1981, p. 178-192. 23. Metzger, J. M., and R. H. Fitts. Fatigue from high- and lowfrequency muscle stimulation: role of sarcolemma action potentials. Exp. Neurol. 93: 320-333, 1986. 24. Metzger, J. M., and R. H. Fitts. Fatigue from high- and lowfrequency muscle stimulation: contractile and biochemical alterations. J. Appl. Physiol. 62: 2075-2082, 1987. 25. Metzger, J. M., and R. H. Fitts. Role of intracellular pH in muscle fatigue. J. Appl. Physiol. 62: 1392-1397, 1987. 26. Metzger, J. M., and R. L. Moss. Greater hydrogen ion-induced depression of tension and velocity in skinned single fibres of rat
IN CONTRACTILE
FUNCTION
fast than slow muscles. J. Physiol. Lond. 393: 727-742, 1987. 27. Metzger, J. M., and R. L. Moss. pH modulation of the kinetics of a Ca2+ sensitive cross-bridge state transition in mammalian single skeletal muscle fibres. J. Physiol. Lond. 428: 7X-764, 1990. 28. Nassar-Gentina, V., J. V. Passoneau, J. L. Vergara, and S. I. Rapoport. Metabolic correlates of fatigue and of recovery from fatigue in single frog muscle fibers. J. Gen. PhysioZ. 72: 593-606, 1978. 29. 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. 30. Thompson, L. V., E. M. Balog, and R. H. Fitts. Muscle fatigue in frog semitendinosus: role of intracellular pH. Am. J. Physiol. 262 (Cell Physiol. 31): Cl507-Cl512, 1992. 31. Westerblad, H., J. A. Lee, A. G. Lamb, S. R. Bolsover, and D. G. Allen. Spatial gradients of intracellular calcium in skeletal muscle during fatigue. Pfluegers Arch. 415: 734-740, 1990.
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