Eur J Appl Physiol (2014) 114:113–121 DOI 10.1007/s00421-013-2757-x
Original Article
Assessment of calf muscle fatigue during submaximal exercise using transcranial magnetic stimulation versus transcutaneous motor nerve stimulation Simon Green · Emily Robinson · Emily Wallis
Received: 24 July 2013 / Accepted: 15 October 2013 / Published online: 23 October 2013 © Springer-Verlag Berlin Heidelberg 2013
Abstract Purpose Few studies have assessed the time-dependent response of fatigue (i.e., loss of force) during submaximal exercise without the use of maximum contractions. There is unexplored potential in the use of the superimposed muscle twitch (SIT), evoked by transcranial magnetic stimulation (TMS) or motor nerve stimulation (MNS), to assess fatigue during voluntary submaximal contractions. For the human triceps surae muscles, there are also no data on TMSevoked twitches. Methods To optimise the TMS stimulus for assessment of fatigue, we first tested the effects of TMS power (40, 55, 70, 85, 100 % max) on SIT force during contractions (0– 100 % MVC in 10 % increments) in six subjects. Then, we compared SIT responses (TMS and MNS) during submaximal contractions and MVCs (all at 60 s intervals) during a continuous protocol of intermittent contractions (30 % MVC) consisting of consecutive 5 min periods of baseline, fatigue (ischaemia) and recovery. Results For TMS, SIT force increased as a diminishing function of TMS power (P 50 % MVC). The SIT stimulus was delivered mid-way through the contraction when the force was stable and at the target force, whereas the POT stimulus was delivered soon after each contraction. The amplitude of the twitches was calculated as the difference between the peak twitch force and the baseline force at the moment the electrical stimulus was applied. Each subject performed a total of 33 contractions, performed in a randomised order, and averaged responses from triplicate measurements were used to establish the relationships between voluntary contractile force and the MNS-evoked (SIT and POT) forces. On a separate day, the effect of voluntary contraction force and TMS power on the SIT amplitude was tested. Voluntary contractions (5 s duration) at 11 forces (0–100 % MVC in 10 % increments) were performed while a single stimulus at one of five TMS power levels (40, 55, 70, 85 and 100 %) was applied mid-way during the contraction. This yielded 55 combinations of contractile force and TMS power, and they were presented in a random order with 15 s rest (0–50 % MVC) or 30 s rest (>50 % MVC) between contractions. Three of the subjects did not tolerate the highest stimulus intensity (i.e., 100 % power) and completed 44 rather than 55 contractions.
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Experiment 2 Since fatigue is a time-dependent and reversible process, we compared MVC and MNS- and TMS-evoked twitch responses during a continuous bout of submaximal exercise when there was no fatigue, significant fatigue induced by ischaemia, and reversal of this fatigue (Fig. 2). This submaximal exercise was performed continuously for 15 min and consisted of two ‘control’ periods of exercise, ‘baseline’ (t = 0–5 min) and ‘recovery’ (t = 10– 15 min), separated by a period of ‘ischaemia’ (Fig. 2). Throughout this exercise, moderate intermittent contractions (30 % MVC) of the right calf muscle were performed. Subjects performed two familiarisation sessions focused on learning how to perform repeated and consistent “square-wave” contractions at 30 % MVC, where each contraction required a rapid rise in force to the target force, stable force at the target value, and rapid decline to the relaxed state, as described elsewhere (Donnelly and Green 2013; Reeder and Green 2012). This intensity was chosen so that all subjects could maintain the target force during the entire protocol and consistently perform stable contractions upon which twitch responses could be easily discerned, and the outcome of Experiment 1 revealed that TMS-evoked twitches were maximised at this force. Contractions were sustained for 2 s and separated by 4 s of rest. During ischaemia a cuff, wrapped around the right thigh, was inflated to 30 mmHg above the systolic blood pressure to completely eliminate arterial blood flow to the contracting calf muscle. Blood pressure was measured non-invasively at heart level (Finometer Midi, Finapres Medical Systems, Amsterdam, The Netherlands) and its continuous display enabled the cuff pressure to be adjusted upwards and maintenance of ischaemia as blood pressure increased during the submaximal protocol. At the end of the tenth minute the thigh cuff was deflated and the subject continued contracting until the end of the protocol. This submaximal protocol was repeated on 3 days, separated by 7–14 days, during which maximum voluntary
Fig. 2 Schematic diagram of the submaximal protocol used in Experiment 2. Throughout this protocol either MVCs, TMS-evoked twitches or MNSevoked twitches were performed at 60 s intervals and on separate days
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contractions, TMS-evoked twitches or MNS-evoked twitches were performed at 60 s intervals (Fig. 2). No MVCs were performed during submaximal exercise involving twitches. TMS twitches (SIT) were evoked using a TMS power level that varied slightly between subjects (75–85 % max) but which maximised the SIT amplitude during mild contractions (10 % MVC) performed before the submaximal protocol. To establish the effect of repeated maximum efforts or twitches on the fatigue inherent in these responses, subjects also completed two series of 15 MVCs and 15 pairs of MNS-evoked twitches (4 s interval between the pair) at 1 min intervals, performed on separate days. Statistical analyses All data were checked for normality and homoscedasticity before parametric tests were applied. Contrasts involving more than two levels were made using either a repeated measures ANOVA (main factor is time) or twoway repeated measures ANOVA (main factors are time and technique), and significant differences were then located using the Holm–Sidak test. The level of significance was set at P ≤ 0.05. All data are expressed as mean and SD (text and tables) or SE (figures).
Results Experiment 1 Examples of superimposed and potentiated twitch responses to MNS during and following a submaximal contraction, as well as a superimposed twitch response to TMS during a submaximal contraction, are shown in Fig. 3. The relationship between voluntary contractile force and MNS-induced SIT force (Fig. 4) was well described by a logistic function [y = a/(1 + e−(x − b)/c); R2 = 0.99] with a sigmoidal shape. Compared with resting twitch
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Fig. 5 Superimposed twitch (SIT) forces evoked by transcranial magnetic stimulation at five power levels (40–100 % max) of the MagStim200. For each TMS power, averaged data were fitted to a quadratic function (see text) and these fits are illustrated as five regression lines Fig. 3 Raw traces of superimposed and potentiated twitch responses to motor nerve stimulation (top trace) and superimposed twitch response to transcranial magnetic stimulation (bottom trace) during and following a submaximal contraction (30 % MVC)
Fig. 4 Experiment 1. Superimposed twitch (SIT) and potentiated (POT) forces evoked by motor nerve stimulation as a function of voluntary force (% MVC). The line of best fit was generated by fitting the average responses to a sigmoidal function (see text). The inset illustrates the M-wave responses for TA and GM as a function of voluntary force
force (9.3 ± 3.8 % MVC), there was a significant reduction in SIT force at all voluntary forces beyond 30 % MVC, whereas the potentiated twitch did not change significantly. The amplitudes of the M-wave during the SIT for GM and TA were not significantly affected by voluntary force (see inset in Fig. 4). For TMS, a quadratic function (y = a + bx − cx2; 2 R = 0.88–0.97) described the relationship between voluntary and SIT force at all power levels (Fig. 5). MEP amplitudes for GM exhibited a similar relationship; whereas for TA there was no effect of voluntary force on the MEP amplitude (Fig. 5). Visual inspection of these data suggests there is an optimum voluntary force (~20–40 % MVC) at which SIT force is maximised, that this force is similar between the power levels, and that there is a positive and diminishing effect of TMS power on the maximum SIT force. SIT force averaged across all voluntary forces was significantly higher (P 0.05).
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Fig. 6 Experiment 2. a Force responses during maximum voluntary contractions (MVC) and MNS-induced twitch responses (SIT and POT) during a 15 min period without intervening submaximal contractions. b Force responses during MVCs and evoked by MNS and TMS during a 15 min submaximal protocol illustrated in Fig. 2. The asterisk indicates that these values are significantly different (P 0.05). Force responses evoked by maximum voluntary effort, MNS (SIT and POT) and TMS during the 15 min submaximal protocol are shown in Fig. 6b. Responses were expressed relative to the average response during the ‘baseline’ period, and there was no significant change in responses during this period. The coefficient of variation of these force responses during the baseline period was lowest for the MVCs (3.7 ± 1.9 %), intermediate for MNS (SIT: 8.0 ± 4.5 %; POT = 6.9 ± 4.6 %) and highest for TMS (11.7 ± 5.1 %), and the MVC and TMS values were significantly different (paired t test, P 0.05). M-wave and MEP amplitudes for GM, SOL and TA during MNS- and TMS-evoked twitches did not change significantly as a function of time throughout the submaximal protocol (Fig. 7).
Discussion In the present study, twitches were evoked for the first time from the human calf muscle using TMS. These twitch responses varied as a parabolic function of voluntary force at all TMS powers tested, which differed from the inverse, sigmoidal response observed for MNS. TMSevoked twitches were highest at mild–moderate voluntary forces, and the potentiating effect of TMS power on twitch amplitude diminished progressively so that smaller differences in twitch amplitude were observed between the highest power levels. On this basis, we used relatively high, but submaximal, TMS power (70–85 %) and supramaximal MNS stimuli to evoke twitches during intermittent, submaximal contractions when there was no fatigue, substantial fatigue, and recovery from fatigue. This behaviour of fatigue was reflected in responses of the MVC, and the twitch responses behaved similarly to the MVC throughout the submaximal protocol. This suggests that either twitch technique could be used to assess dynamic response
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Fig. 7 Effect of motor nerve stimulation (MNS) and transcranial magnetic stimulation (TMS) on M-wave and MEP amplitudes in soleus (SOL), gastrocnemius medialis (GM) and tibialis anterior (TA) muscles during the submaximal protocol in Experiment 2
characteristics of fatigue during intermittent, submaximal contractions of the human calf muscle. Despite the greater distance between the TMS coil and cortical neurons projecting to the lower limbs compared with upper limbs, TMS has been used to evoke twitch responses in lower limb muscles such as the quadriceps (Sidhu et al. 2009), dorsiflexors (Knorr et al. 2011; Mileva
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et al. 2012) and, in the present study, plantar flexors. The amplitude of a TMS-evoked, superimposed twitch varies as a parabolic function of voluntary contractile force in muscles of the upper (Todd et al. 2003) and lower (Sidhu et al. 2009) limbs. In the elbow flexors, increase in TMS power raises the superimposed twitch force in a diminishing manner until a plateau was reached (Molenaar et al. 2012). The effects of both voluntary force and TMS power were tested in the present study and the results can be viewed as a family of parabolic curves (Fig. 5). They show that for any TMS power the twitch force is greatest at lower voluntary forces (~20–40 % MVC), and that this response increases as a function of TMS power but in a diminishing manner, so that there is little effect of raising TMS power beyond ~85 % maximum. There are theoretical and practical implications of these findings. The parabolic relationships observed for TMS differ markedly from the inverse, sigmoidal relationship between voluntary force and force of a superimposed twitch evoked by MNS (Belanger and McComas 1981; Herbert and Gandevia 1999), demonstrated again in the present study. Theoretically, the rise and fall of the parabola implies two or more simultaneous, underlying mechanisms affecting TMS-evoked twitches. In common between TMS and MNS is the diminishing effect of voluntary effort on the superimposed twitch, manifest only at higher forces (>50 % MVC) for TMS, due to progressively less reserve of force as more of the total α-motoneuron pool is recruited voluntarily. The rising phase of the TMS parabola implies a separate, simultaneous process of increasing potentiation of the twitch by voluntary effort and cortical activation which exceeds this effect common to both techniques. Activation of antagonists occurs with TMS, as well as MNS, and this will diminish twitch force. Consequently, for TMS there are at least three simultaneous processes acting across the full range of voluntary force which contribute to its parabolic influence on the superimposed twitch. This has practical implications for the assessment of fatigue using twitch responses. Ideally, an evoked twitch should be maximal and establishing this is straightforward for MNS. For TMS, there is a more challenging problem of optimising the stimulus intensity and voluntary force to maximise the twitch. The process of optimisation needs to also account for the diminishing effect of TMS power on SIT force, variation in subjective tolerance of TMS, antagonist coactivation, maximisation of the EMG (i.e., MEP) or twitch response, as well as the ultimate application of the twitch response. Investigators have focused on maximising the MEP, rather than twitch, in agonists while restricting antagonist coactivation below ~15 % Mmax (Molenaar et al. 2012; Sidhu et al. 2009; Todd et al. 2003). In the present study, MEP responses in an agonist (GM) tracked the twitch
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responses (Fig. 6), whereas antagonist coactivation was high and only remained low (~20 % Mmax) at the lowest TMS power. Although such high coactivation should suppress the twitch, the extent to which it does so is not clear given the coexisting influences of rising potentiation and falling force ‘reserve’ mentioned above. Across the range of voluntary force, antagonist activation was constant while agonist MEPs tracked the twitch responses, suggesting that twitch behaviour accurately reflected underlying activation of the calf muscle. Moreover, twitches evoked at high TMS power revealed fatigue behaviour similar to that seen in the MVCs (Fig. 7), and this suggests that a process of optimisation focused on maximising the twitch as opposed to MEP response is effective when using TMS to assess fatigue during submaximal contractions. A primary objective of this study was to compare TMSand MNS-evoked twitch responses with MVCs during fatiguing, submaximal exercise. For this comparison to be valid, we limited the frequency of MVCs to a level (one per minute) that, when performed in isolation, did not induce fatigue (Fig. 6a). Further evidence of the lack of fatigue inherent in performing MVCs was observed during the baseline period of submaximal contractions at 30 % MVC (Fig. 6b). During the following periods of ischaemia and recovery, the MVC and twitch responses declined to a substantial and similar extent before recovering with similar time-courses to baseline levels. These data demonstrate that the fatigue inherent in maximum voluntary efforts is reflected well in the TMS and MNS twitch responses evoked during submaximal contractions, as well as ‘potentiated’ twitches evoked by MNS after contractions. These data provide a basis for further assessment of dynamic response characteristics of fatigue using more frequent twitch responses and empirical modelling. In the present study, the frequency of twitch responses was limited to 60 s for comparison with MVCs, yet there is potential for increasing this frequency. In a preliminary experiment (data not shown), we used TMS and MNS on separate occasions to evoke twitch responses during submaximal intermittent and sustained contractions at a higher frequency (15 s intervals) in two subjects. This higher frequency of twitch responses was tolerated well by subjects and revealed more subtle, time-dependent features of fatigue than observed in the present study. Such features can be quantified using empirical modelling and might shed light on the timing and extent of contribution of underlying mechanisms to the overall fatigue response. The accuracy with which a fatigue response can be described empirically depends on the amplitude and variability (i.e., signal–noise ratio) of the fatigue variable. TMS-evoked twitches were similar in amplitude to MNS-evoked twitches (Experiment 1), consistent with other observations for knee extensor and elbow flexor muscles (Sidhu et al. 2009; Todd et al. 2003), and their
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variabilities during submaximal exercise (baseline) were similar (c.v. = 7–11 %). However, the variability of TMSevoked twitches was significantly greater than the variability of MVCs, although less variation (c.v. = 6 %) and clearer individual profiles of fatigue (data not shown) were observed in subjects with higher absolute twitch responses (~20 % MVC). The sources of this higher variability with TMS require further investigation, but these observations also suggest that the variability and accuracy of fatigue assessment can be enhanced by prudent selection of experimental subjects on the basis of a high twitch amplitude. For TMS, this selection can be made when trying to locate a stimulus ‘hotspot’ because responsive subjects will very quickly yield large and clear twitches to moderate TMS power (~70 % max) during mild contractions, whereas this will be a more difficult and protracted problem in less responsive subjects. The use of TMS and MNS has the potential to shed light on different aspects of fatigue (see “Introduction”). In the present study, ischaemia was used to induce fatigue and, although this effect is well established in the contracting calf muscle (Egana and Green 2005), the underlying mechanisms during voluntary exercise are less clear. There is strong evidence for involvement of metabolic and ionic mechanisms within contracting myocytes in this effect (Barclay 1986; Hogan et al. 1994), and their effect is reflected in the decline in MNS-evoked twitches and socalled ‘peripheral fatigue’. There is also some evidence of involvement of ischaemia-sensitive type III and IV muscle nerve afferents and their inhibition of α-motoneurons in fatigue during intense exercise (Amann et al. 2011), and their contribution might be reflected in a greater decline in TMS-evoked twitches than MNS-evoked twitches. However, the similarity of decline in TMS and MNS twitches during ischaemia suggests minimal involvement of any mechanism proximal to the motor nerve. In conclusion, this study demonstrates that muscle twitches evoked by TMS or MNS provide similar descriptions of fatigue during intermittent submaximal contractions compared with maximum voluntary efforts. This evidence supports the use of a twitch-based approach to the assessment of dynamic response characteristics of muscle fatigue during submaximal exercise. Acknowledgments The authors thank Professors Simon Gandevia and Janet Taylor from Neuroscience Research Australia for their help and advice in the early phase of this project.
References Amann M, Blain GM, Proctor LT, Sebranek JJ, Pegelow DF, Dempsey JA (2011) Implications of group III and IV muscle afferents for high-intensity endurance exercise performance in humans. J Physiol 589:5299–5309
Eur J Appl Physiol (2014) 114:113–121 Barclay JK (1986) A delivery-independent blood flow effect on skeletal muscle fatigue. J Appl Physiol 61:1084–1090 Belanger AY, McComas AJ (1981) Extent of motor unit activation during effort. J Appl Physiol 51:1131–1135 Bylund-Fellenius AC, Walker PM, Elander A, Holm S, Schersten T (1981) Energy metabolism in relation to oxygen partial pressure in human skeletal muscle during exercise. Biochem J 200:247–255 Donnelly J, Green S (2013) Effect of hypoxia on the dynamic response characteristics of hyperaemia in the contracting human calf muscle. Exp Physiol 98(1):81–93 Egana M, Green S (2005) Effect of body tilt on calf muscle performance and blood flow in humans. J Appl Physiol 98:2249–2258 Egana M, Green S (2007) Intensity-dependent effect of body tilt angle on calf muscle fatigue in humans. Eur J Appl Physiol 99:1–9 Egana M, Ryan K, Warmington S, Green S (2010) Effect of body tilt angle on fatigue and EMG activities in lower limbs during cycling. Eur J Appl Physiol 108:649–656 Enoka RM, Duchateau J (2008) Muscle fatigue: what, why and how it influences muscle function. J Physiol 586:11–23 Fitzpatrick R, Taylor JL, McCloskey DI (1996) Effects of arterial perfusion pressure on force production in working human hand muscles. J Physiol 495(3):885–891 Fulco CS, Lewis SF, Frykman PN, Boushel R, Smith S, Harman EA, Cymerman A, Pandolf KB (1996) Muscle fatigue and exhaustion during dynamic leg exercise in normoxia and hypobaric hypoxia. J Appl Physiol 81:1891–1900 Gandevia SC (2001) Spinal and supraspinal factors in human muscle fatigue. Physiol Rev 81:1725–1789 Green S, Mehlsen J (1999) Peripheral arterial disease. In: Saltin B, Boushel R, Secher N, Mitchell J (eds) Exercise and circulation in health and disease. Human Kinetics, Champaign, pp 283–290 Herbert RD, Gandevia SC (1999) Twitch interpolation in human muscles: mechanisms and implications for measurement of voluntary activation. J Neurophysiol 82:2271–2283 Hogan MC, Richardson RS, Kurdak SS (1994) Initial fall in skeletal muscle force development during ischemia is related to oxygen availability. J Appl Physiol 77:2380–2384
121 Keller ML, Pruse J, Yoon T, Schlinder-Delap B, Harkins A, Hunter SK (2011) Supraspinal fatigue is similar in men and women for a low-force fatiguing contraction. Med Sci Sports Exerc 43:1873–1883 Knorr S, Ivanova TD, Doherty TJ, Campbell JA, Garland SJ (2011) The origins of neuromuscular fatigue post-stroke. Exp Brain Res 214:303–315 Lamarra N (1990) Variables, constants, and parameters: clarifying the system structure. Med Sci Sports Exerc 22:88–95 Leyk D, Baum K, Wamser P, Wackerhage H, Essfeld D (1999) Cardiac output, leg blood flow and oxygen uptake during foot plantar flexions. Int J Sports Med 20:510–515 McCreary CR, Chilibeck PD, Marsh GD, Paterson DH, Cunningham DA, Thompson RT (1996) Kinetics of pulmonary oxygen uptake and muscle phosphates during moderate-intensity exercise. J Appl Physiol 81:1331–1338 Mileva KN, Sumners DP, Bowtell JL (2012) Decline in voluntary activation contributes to reduced maximal performance of fatigued lower limb muscles. Eur J Appl Physiol 112:3959–3970 Molenaar JP, McNeil CJ, Bredius MS, Gandevia SC (2012) Effects of aging and sex on voluntary activation and peak relaxation rate of human elbow flexors studied with motor cortical stimulation. Age. doi:10.1007/s11357-012-9435-5 Reeder E, Green S (2012) Dynamic response characteristics of muscle hyperaemia: effect of exercise intensity and relation to electromyographic activity. Eur J Appl Physiol 112:3997–4013 Sidhu SK, Bentley DJ, Carroll TJ (2009) Cortical voluntary activation of the human knee extensors can be reliably estimated using transcranial magnetic stimulation. Muscle Nerve 39:186–196 Todd G, Taylor JL, Gandevia SC (2003) Measurement of voluntary activation of fresh and fatigued human muscles using transcranial magnetic stimulation. J Physiol 551:661–671 Tucker KJ, Tuncer M, Turker KS (2005) A review of the H-reflex and M-wave in the human triceps surae. Hum Mov Sci 24:667–688 van Duinen H, Gandevia SC, Taylor JL (2010) Voluntary activation of the different compartments of the flexor digitorum profundus. J Neurophysiol 104:3213–3221
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Original Article
Assessment of calf muscle fatigue during submaximal exercise using transcranial magnetic stimulation versus transcutaneous motor nerve stimulation Simon Green · Emily Robinson · Emily Wallis
Received: 24 July 2013 / Accepted: 15 October 2013 / Published online: 23 October 2013 © Springer-Verlag Berlin Heidelberg 2013
Abstract Purpose Few studies have assessed the time-dependent response of fatigue (i.e., loss of force) during submaximal exercise without the use of maximum contractions. There is unexplored potential in the use of the superimposed muscle twitch (SIT), evoked by transcranial magnetic stimulation (TMS) or motor nerve stimulation (MNS), to assess fatigue during voluntary submaximal contractions. For the human triceps surae muscles, there are also no data on TMSevoked twitches. Methods To optimise the TMS stimulus for assessment of fatigue, we first tested the effects of TMS power (40, 55, 70, 85, 100 % max) on SIT force during contractions (0– 100 % MVC in 10 % increments) in six subjects. Then, we compared SIT responses (TMS and MNS) during submaximal contractions and MVCs (all at 60 s intervals) during a continuous protocol of intermittent contractions (30 % MVC) consisting of consecutive 5 min periods of baseline, fatigue (ischaemia) and recovery. Results For TMS, SIT force increased as a diminishing function of TMS power (P 50 % MVC). The SIT stimulus was delivered mid-way through the contraction when the force was stable and at the target force, whereas the POT stimulus was delivered soon after each contraction. The amplitude of the twitches was calculated as the difference between the peak twitch force and the baseline force at the moment the electrical stimulus was applied. Each subject performed a total of 33 contractions, performed in a randomised order, and averaged responses from triplicate measurements were used to establish the relationships between voluntary contractile force and the MNS-evoked (SIT and POT) forces. On a separate day, the effect of voluntary contraction force and TMS power on the SIT amplitude was tested. Voluntary contractions (5 s duration) at 11 forces (0–100 % MVC in 10 % increments) were performed while a single stimulus at one of five TMS power levels (40, 55, 70, 85 and 100 %) was applied mid-way during the contraction. This yielded 55 combinations of contractile force and TMS power, and they were presented in a random order with 15 s rest (0–50 % MVC) or 30 s rest (>50 % MVC) between contractions. Three of the subjects did not tolerate the highest stimulus intensity (i.e., 100 % power) and completed 44 rather than 55 contractions.
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Experiment 2 Since fatigue is a time-dependent and reversible process, we compared MVC and MNS- and TMS-evoked twitch responses during a continuous bout of submaximal exercise when there was no fatigue, significant fatigue induced by ischaemia, and reversal of this fatigue (Fig. 2). This submaximal exercise was performed continuously for 15 min and consisted of two ‘control’ periods of exercise, ‘baseline’ (t = 0–5 min) and ‘recovery’ (t = 10– 15 min), separated by a period of ‘ischaemia’ (Fig. 2). Throughout this exercise, moderate intermittent contractions (30 % MVC) of the right calf muscle were performed. Subjects performed two familiarisation sessions focused on learning how to perform repeated and consistent “square-wave” contractions at 30 % MVC, where each contraction required a rapid rise in force to the target force, stable force at the target value, and rapid decline to the relaxed state, as described elsewhere (Donnelly and Green 2013; Reeder and Green 2012). This intensity was chosen so that all subjects could maintain the target force during the entire protocol and consistently perform stable contractions upon which twitch responses could be easily discerned, and the outcome of Experiment 1 revealed that TMS-evoked twitches were maximised at this force. Contractions were sustained for 2 s and separated by 4 s of rest. During ischaemia a cuff, wrapped around the right thigh, was inflated to 30 mmHg above the systolic blood pressure to completely eliminate arterial blood flow to the contracting calf muscle. Blood pressure was measured non-invasively at heart level (Finometer Midi, Finapres Medical Systems, Amsterdam, The Netherlands) and its continuous display enabled the cuff pressure to be adjusted upwards and maintenance of ischaemia as blood pressure increased during the submaximal protocol. At the end of the tenth minute the thigh cuff was deflated and the subject continued contracting until the end of the protocol. This submaximal protocol was repeated on 3 days, separated by 7–14 days, during which maximum voluntary
Fig. 2 Schematic diagram of the submaximal protocol used in Experiment 2. Throughout this protocol either MVCs, TMS-evoked twitches or MNSevoked twitches were performed at 60 s intervals and on separate days
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contractions, TMS-evoked twitches or MNS-evoked twitches were performed at 60 s intervals (Fig. 2). No MVCs were performed during submaximal exercise involving twitches. TMS twitches (SIT) were evoked using a TMS power level that varied slightly between subjects (75–85 % max) but which maximised the SIT amplitude during mild contractions (10 % MVC) performed before the submaximal protocol. To establish the effect of repeated maximum efforts or twitches on the fatigue inherent in these responses, subjects also completed two series of 15 MVCs and 15 pairs of MNS-evoked twitches (4 s interval between the pair) at 1 min intervals, performed on separate days. Statistical analyses All data were checked for normality and homoscedasticity before parametric tests were applied. Contrasts involving more than two levels were made using either a repeated measures ANOVA (main factor is time) or twoway repeated measures ANOVA (main factors are time and technique), and significant differences were then located using the Holm–Sidak test. The level of significance was set at P ≤ 0.05. All data are expressed as mean and SD (text and tables) or SE (figures).
Results Experiment 1 Examples of superimposed and potentiated twitch responses to MNS during and following a submaximal contraction, as well as a superimposed twitch response to TMS during a submaximal contraction, are shown in Fig. 3. The relationship between voluntary contractile force and MNS-induced SIT force (Fig. 4) was well described by a logistic function [y = a/(1 + e−(x − b)/c); R2 = 0.99] with a sigmoidal shape. Compared with resting twitch
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Fig. 5 Superimposed twitch (SIT) forces evoked by transcranial magnetic stimulation at five power levels (40–100 % max) of the MagStim200. For each TMS power, averaged data were fitted to a quadratic function (see text) and these fits are illustrated as five regression lines Fig. 3 Raw traces of superimposed and potentiated twitch responses to motor nerve stimulation (top trace) and superimposed twitch response to transcranial magnetic stimulation (bottom trace) during and following a submaximal contraction (30 % MVC)
Fig. 4 Experiment 1. Superimposed twitch (SIT) and potentiated (POT) forces evoked by motor nerve stimulation as a function of voluntary force (% MVC). The line of best fit was generated by fitting the average responses to a sigmoidal function (see text). The inset illustrates the M-wave responses for TA and GM as a function of voluntary force
force (9.3 ± 3.8 % MVC), there was a significant reduction in SIT force at all voluntary forces beyond 30 % MVC, whereas the potentiated twitch did not change significantly. The amplitudes of the M-wave during the SIT for GM and TA were not significantly affected by voluntary force (see inset in Fig. 4). For TMS, a quadratic function (y = a + bx − cx2; 2 R = 0.88–0.97) described the relationship between voluntary and SIT force at all power levels (Fig. 5). MEP amplitudes for GM exhibited a similar relationship; whereas for TA there was no effect of voluntary force on the MEP amplitude (Fig. 5). Visual inspection of these data suggests there is an optimum voluntary force (~20–40 % MVC) at which SIT force is maximised, that this force is similar between the power levels, and that there is a positive and diminishing effect of TMS power on the maximum SIT force. SIT force averaged across all voluntary forces was significantly higher (P 0.05).
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Fig. 6 Experiment 2. a Force responses during maximum voluntary contractions (MVC) and MNS-induced twitch responses (SIT and POT) during a 15 min period without intervening submaximal contractions. b Force responses during MVCs and evoked by MNS and TMS during a 15 min submaximal protocol illustrated in Fig. 2. The asterisk indicates that these values are significantly different (P 0.05). Force responses evoked by maximum voluntary effort, MNS (SIT and POT) and TMS during the 15 min submaximal protocol are shown in Fig. 6b. Responses were expressed relative to the average response during the ‘baseline’ period, and there was no significant change in responses during this period. The coefficient of variation of these force responses during the baseline period was lowest for the MVCs (3.7 ± 1.9 %), intermediate for MNS (SIT: 8.0 ± 4.5 %; POT = 6.9 ± 4.6 %) and highest for TMS (11.7 ± 5.1 %), and the MVC and TMS values were significantly different (paired t test, P 0.05). M-wave and MEP amplitudes for GM, SOL and TA during MNS- and TMS-evoked twitches did not change significantly as a function of time throughout the submaximal protocol (Fig. 7).
Discussion In the present study, twitches were evoked for the first time from the human calf muscle using TMS. These twitch responses varied as a parabolic function of voluntary force at all TMS powers tested, which differed from the inverse, sigmoidal response observed for MNS. TMSevoked twitches were highest at mild–moderate voluntary forces, and the potentiating effect of TMS power on twitch amplitude diminished progressively so that smaller differences in twitch amplitude were observed between the highest power levels. On this basis, we used relatively high, but submaximal, TMS power (70–85 %) and supramaximal MNS stimuli to evoke twitches during intermittent, submaximal contractions when there was no fatigue, substantial fatigue, and recovery from fatigue. This behaviour of fatigue was reflected in responses of the MVC, and the twitch responses behaved similarly to the MVC throughout the submaximal protocol. This suggests that either twitch technique could be used to assess dynamic response
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Fig. 7 Effect of motor nerve stimulation (MNS) and transcranial magnetic stimulation (TMS) on M-wave and MEP amplitudes in soleus (SOL), gastrocnemius medialis (GM) and tibialis anterior (TA) muscles during the submaximal protocol in Experiment 2
characteristics of fatigue during intermittent, submaximal contractions of the human calf muscle. Despite the greater distance between the TMS coil and cortical neurons projecting to the lower limbs compared with upper limbs, TMS has been used to evoke twitch responses in lower limb muscles such as the quadriceps (Sidhu et al. 2009), dorsiflexors (Knorr et al. 2011; Mileva
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et al. 2012) and, in the present study, plantar flexors. The amplitude of a TMS-evoked, superimposed twitch varies as a parabolic function of voluntary contractile force in muscles of the upper (Todd et al. 2003) and lower (Sidhu et al. 2009) limbs. In the elbow flexors, increase in TMS power raises the superimposed twitch force in a diminishing manner until a plateau was reached (Molenaar et al. 2012). The effects of both voluntary force and TMS power were tested in the present study and the results can be viewed as a family of parabolic curves (Fig. 5). They show that for any TMS power the twitch force is greatest at lower voluntary forces (~20–40 % MVC), and that this response increases as a function of TMS power but in a diminishing manner, so that there is little effect of raising TMS power beyond ~85 % maximum. There are theoretical and practical implications of these findings. The parabolic relationships observed for TMS differ markedly from the inverse, sigmoidal relationship between voluntary force and force of a superimposed twitch evoked by MNS (Belanger and McComas 1981; Herbert and Gandevia 1999), demonstrated again in the present study. Theoretically, the rise and fall of the parabola implies two or more simultaneous, underlying mechanisms affecting TMS-evoked twitches. In common between TMS and MNS is the diminishing effect of voluntary effort on the superimposed twitch, manifest only at higher forces (>50 % MVC) for TMS, due to progressively less reserve of force as more of the total α-motoneuron pool is recruited voluntarily. The rising phase of the TMS parabola implies a separate, simultaneous process of increasing potentiation of the twitch by voluntary effort and cortical activation which exceeds this effect common to both techniques. Activation of antagonists occurs with TMS, as well as MNS, and this will diminish twitch force. Consequently, for TMS there are at least three simultaneous processes acting across the full range of voluntary force which contribute to its parabolic influence on the superimposed twitch. This has practical implications for the assessment of fatigue using twitch responses. Ideally, an evoked twitch should be maximal and establishing this is straightforward for MNS. For TMS, there is a more challenging problem of optimising the stimulus intensity and voluntary force to maximise the twitch. The process of optimisation needs to also account for the diminishing effect of TMS power on SIT force, variation in subjective tolerance of TMS, antagonist coactivation, maximisation of the EMG (i.e., MEP) or twitch response, as well as the ultimate application of the twitch response. Investigators have focused on maximising the MEP, rather than twitch, in agonists while restricting antagonist coactivation below ~15 % Mmax (Molenaar et al. 2012; Sidhu et al. 2009; Todd et al. 2003). In the present study, MEP responses in an agonist (GM) tracked the twitch
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responses (Fig. 6), whereas antagonist coactivation was high and only remained low (~20 % Mmax) at the lowest TMS power. Although such high coactivation should suppress the twitch, the extent to which it does so is not clear given the coexisting influences of rising potentiation and falling force ‘reserve’ mentioned above. Across the range of voluntary force, antagonist activation was constant while agonist MEPs tracked the twitch responses, suggesting that twitch behaviour accurately reflected underlying activation of the calf muscle. Moreover, twitches evoked at high TMS power revealed fatigue behaviour similar to that seen in the MVCs (Fig. 7), and this suggests that a process of optimisation focused on maximising the twitch as opposed to MEP response is effective when using TMS to assess fatigue during submaximal contractions. A primary objective of this study was to compare TMSand MNS-evoked twitch responses with MVCs during fatiguing, submaximal exercise. For this comparison to be valid, we limited the frequency of MVCs to a level (one per minute) that, when performed in isolation, did not induce fatigue (Fig. 6a). Further evidence of the lack of fatigue inherent in performing MVCs was observed during the baseline period of submaximal contractions at 30 % MVC (Fig. 6b). During the following periods of ischaemia and recovery, the MVC and twitch responses declined to a substantial and similar extent before recovering with similar time-courses to baseline levels. These data demonstrate that the fatigue inherent in maximum voluntary efforts is reflected well in the TMS and MNS twitch responses evoked during submaximal contractions, as well as ‘potentiated’ twitches evoked by MNS after contractions. These data provide a basis for further assessment of dynamic response characteristics of fatigue using more frequent twitch responses and empirical modelling. In the present study, the frequency of twitch responses was limited to 60 s for comparison with MVCs, yet there is potential for increasing this frequency. In a preliminary experiment (data not shown), we used TMS and MNS on separate occasions to evoke twitch responses during submaximal intermittent and sustained contractions at a higher frequency (15 s intervals) in two subjects. This higher frequency of twitch responses was tolerated well by subjects and revealed more subtle, time-dependent features of fatigue than observed in the present study. Such features can be quantified using empirical modelling and might shed light on the timing and extent of contribution of underlying mechanisms to the overall fatigue response. The accuracy with which a fatigue response can be described empirically depends on the amplitude and variability (i.e., signal–noise ratio) of the fatigue variable. TMS-evoked twitches were similar in amplitude to MNS-evoked twitches (Experiment 1), consistent with other observations for knee extensor and elbow flexor muscles (Sidhu et al. 2009; Todd et al. 2003), and their
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variabilities during submaximal exercise (baseline) were similar (c.v. = 7–11 %). However, the variability of TMSevoked twitches was significantly greater than the variability of MVCs, although less variation (c.v. = 6 %) and clearer individual profiles of fatigue (data not shown) were observed in subjects with higher absolute twitch responses (~20 % MVC). The sources of this higher variability with TMS require further investigation, but these observations also suggest that the variability and accuracy of fatigue assessment can be enhanced by prudent selection of experimental subjects on the basis of a high twitch amplitude. For TMS, this selection can be made when trying to locate a stimulus ‘hotspot’ because responsive subjects will very quickly yield large and clear twitches to moderate TMS power (~70 % max) during mild contractions, whereas this will be a more difficult and protracted problem in less responsive subjects. The use of TMS and MNS has the potential to shed light on different aspects of fatigue (see “Introduction”). In the present study, ischaemia was used to induce fatigue and, although this effect is well established in the contracting calf muscle (Egana and Green 2005), the underlying mechanisms during voluntary exercise are less clear. There is strong evidence for involvement of metabolic and ionic mechanisms within contracting myocytes in this effect (Barclay 1986; Hogan et al. 1994), and their effect is reflected in the decline in MNS-evoked twitches and socalled ‘peripheral fatigue’. There is also some evidence of involvement of ischaemia-sensitive type III and IV muscle nerve afferents and their inhibition of α-motoneurons in fatigue during intense exercise (Amann et al. 2011), and their contribution might be reflected in a greater decline in TMS-evoked twitches than MNS-evoked twitches. However, the similarity of decline in TMS and MNS twitches during ischaemia suggests minimal involvement of any mechanism proximal to the motor nerve. In conclusion, this study demonstrates that muscle twitches evoked by TMS or MNS provide similar descriptions of fatigue during intermittent submaximal contractions compared with maximum voluntary efforts. This evidence supports the use of a twitch-based approach to the assessment of dynamic response characteristics of muscle fatigue during submaximal exercise. Acknowledgments The authors thank Professors Simon Gandevia and Janet Taylor from Neuroscience Research Australia for their help and advice in the early phase of this project.
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