Coexistence of potentiation and low-frequency fatigue during ...

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Coexistence of potentiation and low-frequency fatigue during voluntary exercise in human skeletal muscle J.R. Fowles and H.J. Green

Abstract: The role of muscle potentiation in overcoming low-frequency fatigue (LFF) as it developed during submaximal voluntary exercise was investigated in eight males (age 26.4 ± 0.7 years, mean ± SE) performing isometric leg extension at ~30% of maximal voluntary contraction for 60 min using a 0.5-duty cycle (1 s contraction, 1 s rest). At 5, 20, 40, and 60 min, exercise was interrupted for 3 min, and the maximum positive rate of force development (+dF/dtmax) and maximal twitch force (Pt) were measured in maximal twitch contractions at 0, 1, 2, and 3 min of rest (R0, R1, R2, R3); they were also measured at 15 min of recovery following the entire 60-min exercise period. These measures were compared with pre-exercise (PRE) as an indicator of potentiation. Force at low frequency (10 Hz) was also measured at R0, R1, R2, and R3, and at 15 min of recovery, while force at high frequency (100 Hz) was measured only at R0 and R3 and in recovery. Voluntary exercise increased twitch +dF/dtmax at R0 following 5, 20, 40, and 60 min of exercise, from 2553 ± 150 N/s at PRE to 39%, 41%, 42%, and 36% above PRE, respectively (P < 0.005). Twitch +dF/dtmax decayed at brief rest (R3) following 20, 40, and 60 min of exercise (P < 0.05). Pt at R0 following 5 and 20 min of exercise was above that at PRE (P < 0.05), indicating that during the early phase of moderateintensity repetitive exercise, potentiation occurs in the relative absence of LFF. At 40 and 60 min of exercise, Pt at R0 was unchanged from PRE. The LFF (10 Hz) induced by the protocol was evident at 40 and 60 min (R0–R3; P < 0.05) and at 15 min following exercise (P < 0.05). High-frequency force was not significantly compromised by the protocol. Since twitch force was maintained, these results suggest that as exercise progresses, LFF develops, which can be compensated for by potentiation. Key words: excitability, myosin light chain, phosphorylation, isometric exercise. Résumé : Le rôle de la potentialisation musculaire dans le renversement de la fatigue à basse fréquence (FBF), qui se développe durant un exercice volontaire sous-maximal, a été examiné chez 8 hommes (âge 26,4 ± 0,7 ans, moyenne ± ÉT) effectuant une extension isométrique de la jambe à ~30 % d'une contraction maximale volontaire, pendant 60 min, selon un cycle de travail de 0,5 (contraction 1 s, repos 1 s). À 5, 20, 40 et 60 min, l'exercice a été interrompu pendant 3 min, et la force maximale développée (+dF/dtmax) ainsi que la force maximale de la secousse (Pt ) ont été mesurées en contractions maximales à 0, 1, 2 et 3 min de repos (R0, R1, R2, R3), ainsi qu'à 15 min de la récupération suivant la période d'exercice de 60 min. Ces mesures ont été comparées aux valeurs de pré-exercice (PRE) à titre d'indicateur de potentialisation. La force à basse fréquence (10 Hz) a aussi été mesurée à R0, R1, R2 et R3, et à 15 min de la récupération, alors que la force à haute fréquence (100 Hz) a été mesurée uniquement à R0 et R3, et durant la récupération. L'exercice volontaire a augmenté la +dF/dtmax à R0, après 5, 20, 40 et 60 min d'exercice, de 2553 ± 150 N/s valeur PRE à 39 %, 41 %, 42 % et 36 % au-dessus de la valeur PRE, respectivement (P < 0,005). La +dF/dtmax a diminué lors d'un bref repos (R3) après 20, 40 et 60 min d'exercice (P < 0,05). La Pt à R0, après 5 et 20 min d'exercice, a été supérieure à la valeur PRE (P < 0,05), ce qui indique que durant la phase initiale d'un exercice répété d'intensité moyenne, il se produit une potentialisation en l'absence relative de FBF. Après 40 et 60 min d'exercice, la Pt à R0 n'a pas différé de la valeur PRE. La FBF (10 Hz) induite par le protocole a été manifeste à 40 et 60 min (R0–R3; P < 0,05) et à 15 min post-exercice (P < 0,05). La force à haute fréquence n'a pas été affectée de manière significative par le protocole. La force de la secousse ayant été maintenue, ces résultats donnent à penser que la FBF se développe au cours de l'exercice, ce qui pourrait être compensé par la potentialisation. Mots clés : excitabilité, chaîne légère de myosine, phosphorylation, exercice isométrique. [Traduit par la Rédaction]

Fowles and Green

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Introduction Received 16 January 2003. Published on the NRC Research Press Web site at http://cjpp.nrc.ca on 17 November 2003. J.R. Fowles and H.J. Green.1 Department of Kinesiology, University of Waterloo, Waterloo, ON N2L 3G1, Canada. 1

Corresponding author (e-mail: [email protected]).

Can. J. Physiol. Pharmacol. 81: 1092–1100 (2003)

I:\cjpp\Cjpp-8112\Y03-114.vp November 21, 2003 9:39:14 AM

Repetitive submaximal activity results in two welldocumented effects on the mechanical behaviour of skeletal muscle. When assessed under standardized conditions using evoked contractions, repetitive activity can result in an elevation of force, a phenomenon that has been termed potentiation (Krarup 1981). Conversely, repetitive activity can result

doi: 10.1139/Y03-114

© 2003 NRC Canada


Fowles and Green

in a reduction in force, which is defined as fatigue. A number of studies have indicated both potentiation and fatigue in animal skeletal muscle (Barclay 1992; Bruton et al. 1996; Krarup 1981; Rankin et al. 1988; Vandenboom et al. 1997). Despite suggestions that potentiation and fatigue may occur concurrently with voluntary exercise in humans (Garner et al. 1989; Grange and Houston 1991; Green and Jones 1989; Houston and Grange 1990; Rassier and MacIntosh 2000; Skurvydas and Zachovajevas 1998), no study has clearly documented these phenomenon to coexist during activity at an intensity typical to that achieved during submaximal voluntary exercise. It has been observed (Vandervoort and McComas 1983) that potentiation of the ankle plantar flexors and dorsiflexors does not occur following brief contractions below 70% of maximum force, and therefore, most researchers use maximal contractions to induce potentiation (Alway et al. 1987; Grange and Houston 1991; Green and Jones 1989; Houston and Grange 1990; Skurvydas and Zachovajevas 1998). Of particular interest is whether these two phenomena can coexist in voluntary contracting muscle at submaximal intensity and, if so, whether potentiation can obviate the effects of fatigue. In unfatigued muscle, potentiation induced by brief voluntary activity or brief repetitive stimulation results in an elevation of twitch force and force at low frequencies of tetanic stimulation following the conditioning contraction (Grange et al. 1993; Vandervoort and McComas 1983). The potentiated response has been attributed to the phosphorylation of a specific class of myosin light chains (MLC-2) by myosin light chain kinase, which alters cross-bridge interactions between thick and thin filaments (Metzger et al. 1989), increasing the proportion of myosin heads in the forcegenerating state at submaximal levels of Ca2+ activation (Sweeney and Stull 1990). In effect, potentiation shifts the force–pCa relationship to the left, allowing more force to be generated at a given submaximal level of cytosolic free Ca2+ ([Ca2+]i) (Sweeney and Kushmerick 1985). Potentiation, as evidenced by the incorporation of phosphate into myosin light chains (Houston and Grange 1990; Vandenboom et al. 1997), has been strongly correlated (r = 0.97; P < 0.001) to the rate of positive force development (+dF/dtmax) in rat extensor digitorum longus (EDL) muscle (Vandenboom et al. 1995). The relationship between potentiation and +dF/dtmax has also been observed in human skeletal muscle (O’Leary et al. 1997). Interestingly, a particular type of fatigue referred to as low-frequency fatigue (LFF) is observed by a reduction in force observed at low frequencies of stimulation (Edwards et al. 1977). This type of fatigue occurs within the range where potentiation is believed to enhance force output (Sweeney and Kushmerick 1985). Conceivably, during submaximal fatiguing contractions, intrinsic mechanisms of potentiation could help to minimize the decline in force without alterations in extrinsic control mechanisms, such as modifying recruitment and rate coding of motor units. Alternatively, intrinsic compensation may not occur during the repetitive activity itself but during the recovery period where LFF may exist for an extended period (Edwards et al. 1977). This is what we have observed in a previous study using repetitive voluntary isometric contractions of the quadriceps muscles (Green and Jones 1989). A brief tetanic contraction shown

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to elevate force at low frequencies of stimulation in the prefatigued state had no effect when performed immediately following the exercise. However, the potentiating contraction was able to fully alleviate LFF when examined during a 4-h recovery period. When only LFF is present, Ca2+ release from the sarcoplasmic reticulum is reduced (Westerblad et al. 1993) by mechanisms associated with excitation–contraction coupling (Lännergen et al. 1996). This compromises submaximal force but not maximal force, because a small change in [Ca2+]i has a major effect on the steep portion of the force– pCa curve, whereas maximal force is unaffected, owing to the saturating levels of Ca2+ at maximal activation (Allen et al. 1995). Conceivably, potentiation mediated by myosin light chain phosphorylation may compensate for the reduced release of Ca2+ and reduced submaximal forces characteristic to LFF, which could help to maintain submaximal force in vivo. In effect, the increased sensitivity to Ca2+ with potentiation (Metzger et al. 1989; Sweeney and Stull 1990) may balance the reduced availability of [Ca2+]i to provide the same submaximal force during repetitive submaximal contractions. The objective of the study was to determine if during the course of voluntary submaximal exercise potentiation occurs in muscle to a degree sufficient to mask the development of LFF. Our hypothesis was that potentiation would help to minimize the loss of force generated by LFF during a submaximal isometric leg-extension protocol. Our results suggest that repeated submaximal contraction does result in potentiation in human vastus lateralis and that the potentiation helps offset LFF as it develops during exercise (Fowles and Green 1999).

Methods Subjects Eight healthy active males, age 26.4 ± 0.7 years and weight 79.6 ± 1.1 kg (mean ± SE), participated in the study. The study carried the approval of the Office of Human Research at the University of Waterloo, and all participants were fully informed of all experimental procedures and associated risks prior to obtaining written consent. Experimental design To investigate the coexistence of potentiation and LFF, a protocol involving repeated voluntary submaximal isometric knee extension exercise was employed (Fig. 1). The exercise, which consisted of alternating 1-s contraction with 1-s relaxation, was performed at 30% of the maximal voluntary contraction (30% MVC) for 60 min. A trained research assistant ensured that each participant followed the contractile schedule. The contraction schedule was established based on pilot testing and was selected to produce a predominate LFF during the early phase of repetitive exercise. Target force was displayed on an oscilloscope screen during exercise, and contractions were timed by a metronome. Force was assessed throughout the 60-min protocol. To assess the existence of potentiation and fatigue induced by the repetitive exercise schedule, exercise was interrupted at 5, 20, 40, and 60 min for a period of 3 min, and the force characteristics of the quadriceps were assessed using electrical stimulation at © 2003 NRC Canada



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Can. J. Physiol. Pharmacol. Vol. 81, 2003

Fig. 1. Voluntary exercise protocol with measurement sequences indicated at specific times. Exercise was conducted at 30% of maximal voluntary contraction (30% MVC) using an intermittent schedule of 1 s contraction and 1 s relaxation (1 s on, 1 s off). Exercise was interrupted at 5, 20, 40, and 60 min for 3 min. During the rest periods, measurement sequences were performed at 0 (R0), 1 (R1), 2 (R2), and 3 (R3) min. The ranges 0–5, 8–20, 23–20, and 43–60 represent blocks of intermittent exercise. Note that the MVC at PRE was performed 3–4 days prior to the repetitive exercise protocol. PRE, pre-exercise; POST, post-exercise; C, contraction; R, relaxation; Pt, twitch force; P10, P20, P50, and P100, force elicited at 10, 20, 50, and 100 Hz, respectively.

different frequencies and compared with pre-exercise (PRE). These measurements were recorded immediately after the last repetitive contraction (R0) and at 1 (R1), 2 (R2), and 3 (R3) min of rest. Following the measurements, the subjects resumed the schedule of repetitive voluntary contractions. After 60 min, recovery was followed over a 15-min rest period (POST15). The rational for selecting a 3-min period was that potentiation should be nearly completely reversed during this time period, allowing for the detection of LFF (Green and Jones 1989). The subjects reported to the laboratory 3–4 days prior to the experimental day. This visit was used as an initial accommodation and testing session for measurement of MVC force and the forces at different electrical stimulation frequencies. Following the preliminary testing, the subjects practiced the repetitive exercise task. Prior to exercise, on the experimental day, the force–frequency measures were repeated (to account for differences in stimulating electrode placement between days). At this time, MVCs were not performed to avoid confounding effects of postactivation potentiation on the exercise task. Therefore, PRE measures for MVC contractions are from the accommodation and testing before the experimental day, whereas evoked PRE measures are from the values obtained directly prior to exercise. Subjects were allowed 10 min of rest after PRE testing before beginning the 60 min of exercise. Throughout the experiment, including POST15, the testing was completed without alteration of the electrode or subject positioning. Force measurements Subjects were first assessed for maximal twitch properties, obtained by supramaximal stimulation followed by 3 MVCs with twitch interpolation (IT) (Fig. 1). The MVC measures were used as a reference to adjust stimulation voltage to a force output of approximately 55% of MVC at a stimulation frequency of 100 Hz. Using this voltage, 1-s

tetanic trains of 10 Hz (P10), 20 Hz (P20), 50 Hz (P50), and 100 Hz (P100) were administered, and the forces were recorded. The voltage was kept constant throughout the testing for each individual subject. Pretesting, prior to the experimental day, also included an MVC held for 5 s, which was used as a conditioning stimulus to assess post-tetanic potentiation (PTP). The force elicited by a maximal twitch (Pt) 3 s following the conditioning stimulus was used as the measure of PTP. The experimental setup and instrumentation for measuring muscle contractile characteristics in isometric knee extension has been described previously (Green and Jones 1989). Twitches and tetani were delivered to the quadriceps from a Grass Model S48 stimulator (Grass Instruments Co., Quincy, Mass.). A linear variable differential transducer (Schaevitz, Camden, N.J.) was amplified by a Daytronic carrier preamplifier (Daytronic Corporation, Dayton, Ohio) at 1 kHz, converted to a digital signal, and fed into a 12-bit A/D converter; data were then imported into a PC computer for mechanical measurements. Calibration was performed prior to each test session with weights of known amounts. On the experimental day, the resting measures used to obtain the force–frequency response were administered in the same order, with identical rest periods between specific assessments for all subjects. The complete set of measurements, which included Pt and force at P10, P20, P50, P100, and MVC with IT and PTP, were only performed at PRE, at immediately following the 60-min exercise bout (POST), and at POST15. During the 3-min rest breaks, the properties of only twitch contraction and contraction at P10 were recorded at R0, R1, R2, and R3. Maximal twitch properties were used to assess both PTP and LFF. In addition, the properties of contraction at P100 were assessed at R0 and R3 of each rest break. These stimulation frequencies allowed detection of both low- and high-frequency fatigue. Because of the time constraints and to avoid confounding © 2003 NRC Canada



Fowles and Green

effects of potentiation induced by repeated testing, only single trials of each measure were performed at each time point. This protocol, distributed over 3 min, does not result in either PTP or LFF (J.R. Fowles and H.J. Green, personal communication). Operational definitions Potentiation is indicated by the increase in twitch +dF/dtmax (and Pt ) that occurred compared with PRE. The relationship of +dF/dtmax to the potentiation of twitch forces has been observed in animal (Vandenboom et al. 1997) and human (O’Leary et al. 1997) skeletal muscle. Increases in +dF/dtmax would be expected to occur with phosphorylation, since myosin light chain phosphorylation results in increased rates of weak-to-strong cross bridge formation and force development (Metzger et al. 1989). Moreover, +dF/dtmax is used as the best indicator of potentiation, because it is relatively insensitive to the effects of fatigue (Vandenboom et al. 1997). This is of particular importance considering that elevated Pt, or other indicators of potentiation such as reduced contraction and relaxation time, may be affected by LFF. The effects of potentiation on +dF/dtmax decay over time as myosin light chains lose phosphate content (Houston and Grange 1990; Vandenboom et al. 1995). In rat EDL muscle after a maximal stimulus, this period can last >6 min, whereas in human quadriceps potentiated force normally declines back to baseline within 3 min (Green and Jones 1989; Rutherford et al. 1986). In contrast, LFF is indicated by a reduction in twitch and low-frequency stimulated force. This type of fatigue is long lasting (Jones 1996). Therefore, assessment of twitch and 10-Hz force directly following contractions (R0) and a brief rest was interpreted to indicate the presence and decay of potentiation, whereas prolonged reduction (assessed at R3 of each rest interval during exercise) in twitch and lowfrequency force indicated the presence of LFF. The force behavior of Pt at R0 at each time point indicated the balance of these two processes during exercise. Maximal twitches were evoked using a single supramaximal (approximately 150 V) impulse of 50 µs duration while tetani at low (10 and 20 Hz) and high (50 and 100 Hz) frequencies were induced using a voltage that elicited approximately 55% of MVC at 100 Hz with a pulse duration of 50 µs and train duration of 1 s. The maximal twitch properties (obtained by progressive increases in voltage until a plateau in force was obtained) measured included Pt, contraction time (CT), half relaxation time (RT1/2), and +dF/dtmax. Tetanic force, regardless of frequency of stimulation, was taken as the peak force recorded during the 1 s train duration. CT was calculated as the time elapsed for force to increase from 0% to 100% of peak force. RT1/2 was calculated at the time elapsed for force to fall by 50% from peak force. Twitch +dF/dtmax contractions were calculated by taking the maximum slope in the first differential of the force profile. The interpolated twitch (IT), which consists of supramaximal twitches superimposed during an MVC (Behm et al. 1996), was used to assess motor unit activation (MUA) (voluntary activation). For each MVC, MUA was calculated according to Rutherford et al. (1986). The formula used to calculate MUA is (potentiated IT/Pt) × 100.

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Data analysis Statistical analysis was performed on Statistica for Windows R.4.5 software (Statsoft 7 Inc., Tulsa, Okla.). Descriptive statistics included means and SE. One-way analysis of variance (ANOVA) with repeated measures was used to analyze performance measures at PRE and R0 over time. Twofactor ANOVAs were used to compare differences in force measures over the rest breaks at each time point. Correlation coefficients were determined by linear regression. Post-hoc analysis of mean values was performed using the Scheffe’s test. The probability level for statistical significance was accepted at P < 0.05.

Results Characterization of low-frequency fatigue Pt and force at P10 and P20 were depressed at R3 below PRE values by 15%, 36%, and 18% respectively, following 60 min of exercise (Fig. 2). There was no recovery evident during the 15 min of rest. Force at both P50 and P100 were not different from those at PRE, 60 min of exercise, and POST15. The fact that Pt and force at P10 and P20 did not recover in the 15-min period following the exercise protocol provides evidence that LFF was induced. Moreover, since the 60-min exercise protocol did not result in significant reductions (P > 0.05) in force at the high stimulation frequencies (50 and 100 Hz), it would appear that a predominant LFF resulted from this protocol. Potentiation during exercise Twitch +dF/dtmax was significantly elevated above PRE values at R0 after 5, 20, 40, and 60 min of exercise by 39%, 41%, 42%, and 36%, respectively (P < 0.005) (Fig. 3A). Twitch +dF/dtmax decayed at R3 at 20, 40, and 60 min (P < 0.05). Resultant twitch force was potentiated at R0 following 5 and 20 min of voluntary exercise by 17% and 13%, respectively (Fig. 3B). At 40 min (140 ± 6.9 N) and at 60 min (137 ± 5.3 N), Pt at R0 was not different from that at PRE (132 ± 4.9 N). During the 3-min rest periods, Pt decayed following 20, 40, and 60 min of exercise, reducing force below R0 by 14%, 17%, and 20%, respectively. At all of these exercise durations, the decline in Pt was only significant at R3, although it is evident that the decline was progressive. No decline in Pt was observed during rest following 5 min of exercise. Pt and twitch +dF/dtmax were significantly correlated (r = 0.87; P < 0.05) for all time points. Twitch contraction and relaxation times corroborated the presence and decay of potentiation, as indicated by the +dF/dtmax measure (results not presented). Unlike Pt, exercise-induced potentiation did not result in increased force at P10 at R0 of any time interval during exercise (Fig. 4). Rather, a depression of force was observed at R0 following 20 min of exercise. A progressive reduction was also observed at 40 and 60 min of exercise and at POST15. Following 5 min of exercise, force at P10 at R0 was unchanged from that at PRE. At 5 min, force at P10 at R1 and R2 was higher than that at R0. At 20 min and beyond, force at P10 during the recovery period was not different from that at R0. During this period, force at P10 was persistently lower than that at 5 min, regardless of the duration of recovery. The force behaviour measured in a 10-Hz © 2003 NRC Canada



1096 Fig. 2. Change in force elicited at different frequencies of stimulation prior to exercise (PRE), following 60 min of exercise (60), and after 15 min of rest (POST15). Data represent means ± SE; n = 8. *, significantly different from PRE (P < 0.05).

Fig. 3. Twitch maximum rate of force development (+dF/dtmax) (A) and twitch force (B) measured prior to exercise (PRE) and during three 1-min rest intervals (R0, 0 min rest; R1, 1 min rest; R2, 2 min rest; R3, 3 min rest), at 5, 20, 40, and 60 min of the exercise protocol, and following 15 min of recovery (POST15). Values are means ± SE; n = 8. *, significantly different from PRE (P < 0.05); +, significantly different from R0 (P < 0.05).

Can. J. Physiol. Pharmacol. Vol. 81, 2003 Fig. 4. Tetanic 10-Hz stimulation and force measured prior to exercise (PRE) and during three 1-min rest intervals (R0, 1 min rest; R1, 1 min rest; R2, 2 min rest; R3, 3 min rest), at 5, 20, 40, and 60 min of the exercise protocol, and following 15 min of recovery (POST15). Values are means ± SE; n = 8. *, significantly different from PRE (P < 0.05); +, significantly different from R0 (P < 0.05).

Fig. 5. Force oscillations recorded during an unfused tetanus at 10 Hz prior to exercise (A) and at 60 min of exercise (B). Tracing is from a representative subject. Prior to exercise, ∆F contributes 21% of force above the lowest mean oscillation, whereas at 60 min ∆F contributes to 67% of force above the lowest mean oscillation. ∆F, force oscillation; Fm, mean force at lowest point in oscillation.

unfused tetanus and the different oscillations that occur from PRE to 60 min are presented in Figs. 5A and 5B. As is evident from the tracings prior to (Fig. 5A) and following exercise (Fig. 5B), force oscillations (∆F) were considerably increased with exercise. © 2003 NRC Canada



Fowles and Green Fig. 6. Tetanic force measured at 100 Hz (P100) prior to exercise (PRE) and during the 3 1-min rest intervals (R0, 0 min rest; R3, 3 min rest) at 5, 20, 40, and 60 min of exercise and following 15 min of recovery (POST15). *, significantly different from PRE (P < 0.05).

1097 Table 1. Effects of exercise and potentiation on maximal voluntary contractile (MVC) force and motor unit activation (MUA). Measurement

PRE

60 min

POST15

MVC (N) MUA (%) PTP (N) Rel PTP (%) PTP +dF/dtmax (N/s)

699±40 90±7 198±8 53±9 5221±267

598±47* 82±9* 177±9* 60±5 4835±358

568±47* 84±8 179±10* 71±8* 4960±394

Note: Data are means ± SE; n = 8 subjects. PRE, prior to exercise; 60 min, following 60 min of exercise; POST15, after 15 min rest; PTP, post-tetanic potentiation; Rel PTP, PTP relative to resting twitch; PTP +dF/dtmax, maximum rate of force development in a PTP twitch. *Significantly different from PRE (P < 0.05).

At P100, the initial reduction in force at R0 was not observed until 40 min of exercise (Fig. 6). No further reduction in force at P100 at R0 was observed at 60 min. At 40 and 60 min, force at P100 had recovered by R3, such that no differences were found with force at P100 measured at PRE. At POST15, force at P100 was also unchanged from PRE. The exercise protocol also resulted in a decrease in MVC of approximately 14% (Table 1). The depression in MVC persisted throughout the 15 min recovery period (POST15). The depression in MVC at 60 min appears to be partly explained by increased central inhibition, since MUA, measured using the interpolated twitch, was also reduced. At POST15, MUA was not different from that at PRE. By 60 min, the absolute amount of twitch force potentiation following an MVC decreased by 15%, concurrent with the reduced MVC used to potentiate the twitch. PTP +dF/dtmax was unchanged, however, confirming that +dF/dtmax is a good measure of potentiation and is unchanged by this low-frequency fatiguing exercise protocol (unlike the absolute force output, which varied). When presented relative to the depressed twitch force at postexercise time points, the relative enhancement of force in a PTP actually increased between PRE and POST15 from 53% to 71%, respectively (P < 0.05) (Table 1).

Discussion In this study, our objective was to investigate the hypothesis that potentiation, as measured primarily by increased +dF/dtmax and Pt induced during the voluntary exercise protocol, would be sufficient to increase low-frequency force and offset the development of LFF. We provided a brief rest period of 3 min at selected intervals during our exercise protocol to dissociate potentiation from LFF. Our findings strongly suggest that potentiation can, in fact, offset LFF. However, such a conclusion needs qualification. During prolonged submaximal exercise, two stages of interaction between potentiation and fatigue were observed. The first stage was when potentiation dominated in the general absence of fatigue; the second was when potentiation occurred in the presence of fatigue.

Potentiation during submaximal exercise With the exercise protocol employed in this study, potentiation is clearly indicated by the 39% increase in twitch +dF/dtmax observed immediately following 5 min of intermittent, submaximal contractions. Given that Vandenboom and co-workers (Vandenboom et al. 1993; Vandenboom et al. 1995) found high correlations between phosphate incorporation into myosin light chains and increased +dF/dtmax in maximal twitch, our data suggest that potentiation is occurring with submaximal exercise. This increase is considerably below the 82%–97% increase in twitch +dF/dtmax observed in this study when a sustained MVC was used to induce potentiation. The resultant effect of increased +dF/dtmax, which elevates Pt at 5 min by 17%, is also less than that observed in a previous study following a sustained MVC performed by nonexercised muscle (Green and Jones 1989). The smaller potentiation observed with 5 min exercise could be due either to fatigue or to the fact that 30% MVC is inadequate as a conditioning stimulus, as previously indicated (Vandervoort and McComas 1983). Since there is no evidence of either high- or low-frequency fatigue at this time point, the latter explanation would appear more probable. Since potentiation is a phenomenon that occurs primarily in fast-twitch fibres in animal muscle (Moore and Stull 1984), it is understandable that the extent of force potentiation is also related to a greater percentage of fast-twitch fibres in the human leg extensors (Hamada et al. 2000). The fact that potentiation can occur during submaximal exercise with this muscle group in humans is supported by Gollnick et al. (1974), who observed that fast-twitch fibres are used at lower force levels than generally believed, and by the previous work by Rutherford et al. (1986), who also observed potentiation early in repetitive exercise performed at 30% and 45% MVC. Interestingly, the potentiation of Pt observed following the first 5 min of submaximal exercise in this study persisted throughout the 3-min rest period. This was unexpected, since we have previously shown that the effects of potentiation are essentially lost during the first 3 min of recovery (Green and Jones 1989). The prolonged potentiation could be due to a sustained phosphorylation of MLC-2 caused by the repeated submaximal contractions. This mechanism is supported by a lack of decay in twitch +dF/dtmax from R1 to R3. The putative sustained phosphorylation during this period cannot be due to the stimulation sequence, which involved a maximal © 2003 NRC Canada



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twitch and a 10-Hz tetanus at 0, 1, 2, and 3 min with a 100Hz tetanus at 0 and 3 min, since we have observed that this schedule has no effect on potentiation and occurred only with the interaction of 5 min of exercise (J.R. Fowles and H.J. Green, personal communication). Also contrary to expectations was that the twitch potentiation that we observed immediately following 5 min of repetitive activity was not accompanied by an increase in force at P10. At this point, force at P10 was similar to that at PRE. However, a significant increase of force at P10 was observed at 1 and 2 min of recovery. These results indicate that unlike the twitch, potentiation measured with low-frequency stimulation is not observed immediately following the repetitive activity but only during recovery. Conceivably, there is an early inhibitory effect preventing the potentiating effects of MLC-2 phosphorylation from being realized. Bruton et al. (1996) have also observed augmented tetanic force in the minute immediately following 10 contractions of isolated frog muscle fibres, an event that they attributed to improved cross-bridge kinetics (Barclay 1992). The improved crossbridge kinetics were attributed to a reduction of inorganic phosphate (Bruton et al. 1996; Cooke and Pate 1990), which would occur early in recovery as creatine phosphate is restored (Sahlin et al. 1979). It is possible that during this early period of voluntary contractile activity, potentiation may be enough to increase +dF/dtmax and force in a single twitch but not in an unfused tetanus. With an unfused tetanus, a faster relaxation rate could also mask the effects of increases in +dF/dtmax (Vollestad et al. 1997). Alternatively, the failure to find a potentation in force at P10 similar to what was found in Pt could be due to differences in the stimulation protocol. Twitch characteristics were assessed using supramaximal voltage, whereas force at P10 was measured using submaximal voltage, with activation only a fraction of the motor unit potential. Although the selective activation of motor units at 10 Hz could be a factor in the failure to observe potentiation at R0, this seems unlikely given the overshoot that occurs in recovery. Coexistence of potentiation and fatigue As exercise progressed, potentiation of Pt measured immediately following repetitive exercise was obscured. The loss of Pt could occur as a consequence of a disturbance in excitation–contraction coupling, culminating in a reduction in peak [Ca2+]i transient or as a consequence of a rightward shift in the force–pCa relationship. Since the +dF/dtmax remained elevated during this time period, suggesting that MLC-2 phosphorylation remained viable, the loss of potentiation would appear as a result of depressions in peak [Ca2+]i. Additional evidence of the existence of fatigue mediated by excitation–contraction coupling disturbances was provided during the recovery period. In contrast to the early phase of exercise, where Pt remained potentiated during the recovery period, Pt was observed to be depressed late in recovery. This observation suggests that as potentiation is lost, LFF fatigue becomes conspicuous. The reversal of the increase in +dF/dtmax during recovery late in the exercise protocol is further evidence of the loss of MLC-2-mediated potentiation during this time period. The onset of LFF is even more clearly indicated by a sustained impairment of force at P10 at 40 and 60 min of the ex-

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ercise protocol. Since force at P100 is not significantly reduced at R3 throughout the exercise, our protocol was effective in producing primarily LFF. At both of these time points, force at P10 was depressed immediately following the exercise and remained depressed during the 3 min of recovery and even 15 min after exercise. This sustained force loss is characteristic of LFF (Edwards et al. 1977). Our results also indicate that the existence of LFF prevents a potentiation-induced net elevation of force at P10 during recovery, as observed early in exercise. Although phosphorylation of MLC-2 probably occurs during this period, as evidenced by a significantly increased R0 twitch +dF/dtmax at 40 and 60 min, it cannot offset the lower [Ca2+]i–time integral, characteristic of LFF, resulting in a net decrease in force. The fact that Pt is maintained while force at P10 is depressed at 40 and 60 min highlights the importance of the lowered [Ca2+]i–time integral in LFF, which would affect the 1-s unfused tetanus more than a single 150-ms twitch. It has been established that LFF is due primarily to a reduction in sarcoplasmic reticulum Ca2+ release (Chin et al. 1997; Westerblad et al. 1993), lowering the Ca2+ transient for a given electrical input (Chin et al. 1997). Force is compromised more at low activation levels, which is on the steep portion of the force–pCa curve, than at high levels of activation, where the curve plateaus (Westerblad et al. 1993). The total [Ca2+]i required to phosphorylate myosin light chains and increase +dF/dtmax may be different than that required to achieve sustained force output. In this regard, it has been shown that moderate levels of fatigue as measured during tetany can result in increases in both Pt and +dF/dtmax without impairing MLC-2 phosphorylation (Vandenboom and Houston 1996). With our submaximal exercise protocol, the persistence of LFF in the general absence of reductions in P100 suggests only moderate reductions in [Ca2+]i. It is clear that at P10 following 40 and 60 min of exercise, LFF dominates during recovery. In support of our interpretation that potentiation is viable and present during fatigue, however, is the additional observation that force oscillation during an unfused 10-Hz tetanus was greater at points of fatigue than early in exercise (Fig. 5). A greater force at the peak of oscillation (produced by potentiation) above a reduced mean force (produced by LFF) may indicate that of the fibres activated, potentiation is supplying a greater amount of submaximal force later in exercise. Ratkevicius et al. (1995) termed a greater peak force over the mean force in a 10-Hz stimulation as “torque ripple” and observed it during 3 min of 10-Hz stimulation following 30% MVC contractions for 20 min. Vollestad et al. (1997) termed the oscillation “∆F” and observed a greater ∆F following 31 min of exercise at 30% MVC, which was similar to the intensity used in our study. In our study, we have measured force oscillation (∆F) as the mean oscillation in force assessed during 1 s stimulation at 10 Hz, using the mean force (Fm) calculated from the lowest forces observed during the oscillations as the baseline. Our method, which produces results similar to those previously observed using the average force at 10 Hz to calculate ∆F (Vollestad et al. 1997), illustrates the effects more dramatically. We observed that as LFF developed, greater ∆F and faster contractile speed elicited by increased +dF/dtmax (and increased –dF/dt, results not presented) observed at 40 and 60 min of exercise provided a © 2003 NRC Canada



Fowles and Green

means to increase force above what would be allowed in the presence of LFF alone. The ∆F observed in this experiment accounted for only 19% of relative force output (∆F/Fm) at PRE, but contributed 39% and 50% of unfused force directly following contractions at 40 and 60 min, respectively. This interpretation, based on less fusion in low-frequency stimulation, which increases force, warrants further investigation. It appears that our exercise protocol elicited a predominant potentiation early in exercise, which occurred in the relative absence of LFF. Moreover, it is evident that LFF was conspicuous late in exercise. We have also provided evidence, albeit indirect, based on twitch characteristics measured at rest and during brief recovery, of the existence of potentiation during LFF. However, since we used voluntary submaximal exercise to induce potentiation and LFF, it is possible that a variety of extrinsic mechanisms may be involved. As evidenced by the potentiation in twitch characteristics observed early, the generation of 30% MVC involved recruitment of a pool of fast-twitch fibres. As the exercise progressed, the ability to sustain 30% MVC in the face of developing LFF could have been mediated primarily by additional compensatory potentiation of active fast-twitch motor units. If potentiation is the primary mechanism for maintaining 30% MVC throughout exercise, then the changes in the contractile characteristics observed could be credited to the muscle fibres specifically recruited during the activity. Alternatively, given the submaximal nature of the task, as LFF fatigue progresses extrinsic control mechanisms may result in greater additional recruitment of fast-twitch motor units. Fast-twitch fibres possess the potential to contribute to greater potentiation (Grange et al. 1993; Howlett et al. 1999; Sweeney et al. 1993) and greater LFF (Rankin et al. 1988). If this is the case, the changes in the mechanical properties assessed over the course of the exercise protocol could reflect the changes occurring in muscle fibres, presumably fast twitch, that are being progressively recruited. Changes in rate coding could represent another extrinsic control strategy, which could be invoked either during the potentiation itself or during the course of LFF. It is possible that reductions in rate coding could offset potentiation or reduce fatigue (Garland and Gossen 2002). An additional factor that could potentially influence the interpretation of our results is the increase in muscle temperature that probably occurs during the exercise protocol. Increases in temperature mediated by passive warming of the muscle have been shown to increase Pt and force at P10 and P100 at submaximal stimulation voltage but not at supramaximal voltages (Davies and Young 1983). Since we have used maximal twitch properties in this study, PTP is clearly indicated by the increased in Pt. In the case of the +dF/dtmax, the increases were evident very early in the exercise and persisted throughout the protocol, which suggests that myosin light chain phosphorylation is the dominant influence. It is possible that the reduction in +dF/dtmax during the 3-min recovery period late in exercise could have been influenced by decreases in muscle temperature during this period. However, increases in muscle temperature would be expected to have the greatest effect on our tetanic measures. If such is the case, the decrease in force at P10 that we have observed with exercise and that we have used to document LFF would be expected to be even greater.

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Functional implications for the coexistence of potentiation and fatigue In summary, the results of this experiment indicate that repeated low-intensity contractions can produce potentiation in human muscle. The presence of potentiation may have two important influences during submaximal voluntary exercise, in either the absence or the presence of LFF. In the absence of fatigue, potentiation elevates submaximal force so that a given force may be achieved at a lower Ca2+ activation, which may lead to an alteration of extrinsic recruitment strategies early in exercise or help delay the onset of fatigue. In the presence of LFF, potentiation may restore force to near-prefatigue levels, which may allow forces to be produced with lower Ca2+ cycling later in exercise. What these data indicate, in combination with other studies identifying the coexistence of potentiation and fatigue during exercise (Behm and St-Pierre 1997; Ratkevicius et al. 1995; Skurvydas and Zachovajevas 1998; Vollestad et al. 1997), is that the interaction of potentiation and fatigue during voluntary activity is complex. The prevailing influence of one effect seems to be specific to the nature of the task, the tension–time integral producing potentiation, and fatigue, and possibly fibre-type distribution or the training level of the subject.

Acknowledgement Research grant support from the Natural Sciences and Engineering Research Council of Canada is gratefully acknowledged.

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