The effect of sports specialization on musculus quadriceps function after exercise-induced muscle damage

1 The effect of sports specialization on musculus quadriceps function after exercise-induced muscle damage Albertas Skurvydas, Marius Brazaitis, Tomas Venckūnas, Sigitas Kamandulis, Aleksas Stanislovaitis, and Aurelijus Zuoza Abstract: The primary aim of the present study was to examine the effect of eccentric exercise-induced (100 submaximal eccentric contractions at an angular velocity of 60 s 1, with 20-s rest intervals) muscle damage on peripheral and central fatigue of quadriceps muscle in well-trained long-distance runners, sprint runners, volleyball players, and untrained subjects. We found that (i) indirect symptoms of exercise-induced muscle damage (prolonged decrease in maximal voluntary contraction, isokinetic concentric torque, and electrically induced (20 Hz) torque) were most evident in untrained subjects, while there were no significant differences in changes of muscle soreness and plasma creatine kinase 48 h after eccentric exercise between athletes and untrained subjects; (ii) low-frequency fatigue was greater in untrained subjects and volleyball players than in sprint runners and long-distance runners; (iii) in all subjects, electrically induced (100 Hz) torque decreased significantly by about 20%, while central activation ratio decreased significantly by about 8% in untrained subjects and sprint runners, and by about 3% 5% in long-distance runners and volleyball players. Thus, trained subjects showed greater resistance to exercise-induced muscle damage for most markers, and long-distance runners had no advantage over sprint runners or volleyball players. Key words: eccentric exercise, peripheral and central fatigue, well-trained athletes, electrical stimulation, creatine kinase activity, muscle soreness. Résumé : Cette étude se propose principalement d analyser l effet d une lésion musculaire suscitée par une séance d exercices pliométriques (100 exercices pliométriques d intensité sous-maximale à une vélocité de 60 s 1 avec des intervalles de repos d une durée de 20 s) sur la fatigue d origine centrale et périphérique du muscle quadriceps de sujets entraînés (coureurs de longue distance, sprinteurs à la course, joueurs de volleyball) et de sujets non entraînés. Nos observations sont les suivantes : (i) les symptômes indirects de lésion musculaire suscitée par l exercice physique (diminution prolongée de la tension isométrique maximale volontaire, du moment de force isokinétique miométrique au cours d un effort maximal et du moment de force consécutif à une stimulation électrique (20 Hz)) sont plus manifestes chez les sujets non entraînés; il n y a cependant pas de différences significatives entre les athlètes et les sujets sédentaires en ce qui concerne la douleur musculaire et l activité de la créatine kinase 48 h après la fin de la séance d exercices pliométriques (ii) la fatigue à basse fréquence est plus grande chez les sujets non entraînés et les joueurs de volleyball que chez les sprinteurs à la course et les coureurs de longue distance, (iii) chez tous les sujets, le moment de force produit par la stimulation électrique (100 Hz) diminue significativement d environ 20 %; le ratio d activation centrale diminue significativement d environ 8 % chez les sujets non entraînés et les sprinteurs à la course et d environ 3 à 5 % chez les coureurs de longue distance et les joueurs de volleyball. En conclusion, tel que le révèle la plupart des marqueurs, les sujets entraînés présentent une plus grande résistance aux lésions musculaires suscitées par l exercice physique, mais les coureurs de longue distance ne sont pas plus avantagés que les sprinteurs à la course et les joueurs de volleyball. Mots clés : exercice pliométrique, fatigue centrale et périphérique, athlètes bien entraînés, stimulation électrique, activité de la créatine kinase, douleur musculaire. [Traduit par la Rédaction] Introduction Exercise-induced fatigue occurs because of limitations in skeletal muscles or in the nervous system (Gandevia 2001; Bilodeau 2006; Streckis et al. 2007). The terms peripheral fatigue and central fatigue have been used to denote these 2 possible sites of muscle fatigue (Gandevia 2001). The peripheral mechanisms include metabolic fatigue (Allen et al. 2008), exercise-induced muscle damage (Fridén and Lieber 2001; Clarkson and Hubal 2002; Byrne et al. 2004; Skurvy- Received 1 March 2011. Accepted 8 August 2011. Published at www.nrcresearchpress.com/apnm on. A. Skurvydas, M. Brazaitis, T. Venckūnas, S. Kamandulis, A. Stanislovaitis, and A. Zuoza. Sports and Movement Science Centre, Lithuanian Academy of Physical Education, Sporto 6, LT 44221, Kaunas, Lithuania. Corresponding author: Marius Brazaitis (e mail: marius_brazaitis@yahoo.com). Appl. Physiol. Nutr. Metab. 36: 1 8 (2011) doi:10.1139/h11-112

2 Appl. Physiol. Nutr. Metab. Vol. 36, 2011 das et al. 2006, 2008, 2010), and low-frequency fatigue (LFF) (Skurvydas et al. 2006, 2008; Dundon et al. 2008). Most frequently, the differences in muscle fatigue between power and endurance athletes are explained by different muscle metabolic capacities (Häkkinen and Myllylä 1990). It has been shown that an exercise-induced drop in maximal muscle force during metabolic fatigue was significantly smaller in endurance athletes than in power and strength athletes and that recovery tended to be quicker in endurance athletes (Häkkinen and Myllylä 1990). However, other types of fatigue (i.e., non-metabolic fatigue, or fatigue that occurs in the absence of significant metabolic disturbance) have also been documented (Allen et al. 2008). It is well established that unaccustomed eccentric exercise brings on the symptoms of exercise-induced muscle damage (Warren et al. 1999; Fridén and Lieber 2001; Clarkson and Hubal 2002; Byrne et al. 2004). These include the prolonged impairment of muscle function (ranging from 30 60 min to 2 4 weeks after exercise) (Warren et al. 1999; Byrne et al. 2004; Hubal et al. 2007; Black and McCully 2008; Chapman et al. 2008; Skurvydas et al. 2006, 2008); the disruption of the intracellular muscle structure, sarcolemma, and extracellular matrix (Fridén and Lieber 2001; Clarkson and Hubal 2002); remodeling of myofiber structure (Yu et al. 2002); leakage from injured muscle fibers; delayed-onset muscle soreness, stiffness, and swelling (Clarkson and Hubal 2002); and induces a rightward shift in the optimum joint angle for voluntary isometric strength (Proske and Morgan 2001; McHugh and Tetro 2003; Philippou et al. 2004; Chen et al. 2007) and concentric strength (Yeung and Yeung 2008; Skurvydas et al. 2010). Muscle-damaging exercise also impairs voluntary activation of muscle (Prasartwuth et al. 2006). There is a paucity of literature describing how resistance (Newton et al. 2008), speed-, and endurance-trained (Skurvydas et al. 2002) individuals respond to maximal eccentric or stretch-shortening exercise. Previous research indicates that chronically resistance-trained men experience smaller changes in muscle function, limb circumference, and creatine kinase (CK) leakage after maximal eccentric exercise than untrained males (Newton et al. 2008). Patel et al. (1998) documented that increasing muscle oxidative capacity in response to isometric electrical stimulation training did not protect muscles against eccentric contraction-induced damage. However, no previous studies have examined differences between specific athletic populations. One can find contradictory data as to resistance of the fast type and slow type muscle fibres to eccentric contraction-induced damage. It has been reported, for instance, that when performing eccentric exercise type IIb muscle fibres seem to be preferentially damaged (Macpherson et al. 1996; Fridén and Lieber 2001). Also, it has been shown that endurance training has a protective effect on eccentric contraction-induced muscle damage (Evans et al. 1986). It would seem, therefore, that muscles of well-trained long-distance runners should be more resistant than those of untrained subjects, sprinters, and volleyball players to eccentric exercise-induced muscle damage since they are not only well trained in terms of muscular endurance but also characterized by a predominance of oxidative, type I muscle fibres (Saltin et al. 1977). Therefore, the purpose of this study was to compare the changes in indirect markers of exercise-induced muscle damage among well-trained long-distance runners, sprint runners, volleyball players, and untrained subjects in response to damaging eccentric exercise. In addition, the central and peripheral fatigue differences between athletes and untrained subjects were examined. Materials and methods Subjects Healthy untrained (UT) men (mean ± SD; n = 10, age 22.4 ± 1.7 years, weight 74.9 ± 8.9 kg), long-distance runners (LDR) (n = 10, 5 km personal best ranging 14 14.5 min, training experience 9.9 ± 2.3 years, age 24.4 ± 2.5 years, weight 66.8 ± 2.6 kg), sprint runners (S) (n = 10, 100 m personal best ranging 10.5 11 s, training experience 7.1 ± 2.0 years, age 23.4 ± 2.7 years, weight 78.1 ± 6.8 kg), and volleyball players (VP) (n = 10, training experience 8.4 ± 2.8 years, age 22.4 ± 3.7 years, weight 82.5 ± 8.3 kg). All athletes were national level competitors and were considered well-trained. The UT subjects were physically active but none of them took part in any formal physical exercise. The experiments were performed during the period of winter training when LDR and S trained regularly 5 7 times per week, whereas VP were in competition period. In the training of sprinters, exercises developing strength and power were predominant while training developing aerobic endurance was emphasized in the LDR. The athletes stopped training 2 days before the experiment and did not train during the experiment. The volleyball players were tested in between competition periods during the championship. During the last month before testing, the athletes did not have any resistance training for lower limbs. Each subject read and signed a written informed consent form consistent with the principles outlined in the Declaration of Helsinki. The ethics committee of Kaunas University of Medicine approved this study. Muscle-damaging eccentric exercise The subjects performed eccentric knee extension with the dominant leg. Ten series of 10 eccentric repetitions were performed at an angular velocity of 60 s 1 in the passive mode. The contractions were separated by 20 s, and the series were separated by 1-min intervals of rest. The subjects were asked to resist throughout the range of motion and to reach 90% of peak concentric torque. The output from voluntary performance was displayed on a voltmeter located in front of the subject, and participants were asked to match the target torque as accurately as possible. The knee angle range was 30 110 (full knee extension = 0 ). The average peak eccentric torque and the work done were calculated for each contraction. The coefficient of variation (CV) of 100 eccentric repetitions was used as an index of intraindividual variability in the eccentric peak torque and the work done, and were calculated by applying the following formula: CV ¼ mean SD 100% Isometric torque and electrical stimulation The isometric torque of the knee extensor muscles was measured using an isokinetic dynamometer (System 3; Biodex Medical Systems, Shirley, N.Y., USA). The subjects sat

Skurvydas et al. 3 upright in the dynamometer chair with the knee joint positioned at an angle of 60 (full knee extension = 0 ). Shank, trunk, and shoulders were stabilized by belts. The equipment and procedure for electrical stimulation were essentially the same as previously described (Skurvydas et al. 2006, 2010; Streckis et al. 2007). Direct muscle stimulation was applied using 2 carbonized rubber electrodes covered with a thin layer of electrode gel (ECG EEG Gel; Medigel, Modi in, Israel). One of the electrodes (6 11 cm) was placed transversely across the width of the proximal portion of the quadriceps femoris muscle next to the inguinal ligament. Another electrode (6 20 cm) covered the distal portion of the muscle above the patella. A standard electrical stimulator (MG 440; Medicor, Budapest, Hungary) was used. The electrical stimulation was delivered in square-wave pulses, each 1 ms in duration. The tolerance of volunteers to electrical stimulation was assessed on a separate occasion before the actual testing. All participants in this study showed good compliance with the procedure and were recruited for the study. The intensity of electrical stimulation was determined on an individual basis by applying tetanus stimuli to the muscle. During this procedure, the voltage was increased until no increment in torque response was elicited by an additional 10% voltage increase. Quadriceps torques evoked by 1-s trains of electrical stimulation at 20 Hz (P20) and 100 Hz (P100) that were separated by a 5-s rest interval were measured. Maximal voluntary isometric contraction torque (MVC) separated by a 2-min rest interval was measured twice. The ratio of torque production at 20 and 100 Hz stimulation was calculated and analyzed, since eccentric contractions are known to depress force evoked particularly with low stimulation frequencies (low frequency fatigue, LFF) (Skurvydas et al. 2006). Isokinetic concentric torque (IT) measurement The subjects were asked to perform 3 consecutive concentric knee extensions at 30 s 1 with maximal efforts. The angle range was 30 110. Knee flexion was not required, and the shank was passively returned to the starting position. The peak torque attained was recorded as maximal IT. Central activation ratio (CAR) measurements The volunteers were positioned in the dynamometer chair and the stimulating electrodes were placed on the skin above the quadriceps of the dominant leg. After a 5-min rest, 2 MVC efforts (5 s each, separated by a 2-min rest interval) were performed, and torque output traces were recorded. At about 3 s of the MVC, a 250-ms test train of stimuli of 100 Hz (TT100 Hz) was superimposed on the voluntary contraction. CAR was calculated as the ratio of maximal voluntary torque to the peak torque generated when an additional TT100 Hz was superimposed on the MVC (Bilodeau 2006; Streckis et al. 2007). Plasma CK activity Approximately 0.5 ml of mixed capillary blood was drawn from the finger at the baseline and 48 h after the exercise. The samples were immediately centrifuged and analyzed for CK activity using the biochemical analyzer Spotchem EZ SP-4430 (Menarini Diagnostics, UK) with soft reagent strips (ARKRAY Facroty Inc., Shiga, Japan). The normal reference range of plasma CK activity for humans using this method is between 56 and 244 IU L 1 according to the manual provided with the analyzer. Muscle soreness Muscle soreness was reported subjectively using a 10-point visual analogue scale. Each number had a written description for soreness on the scale: 0 (none), 1 (very slight), 2 (slight), 3 (mild), 4 (less than moderate), 5 (moderate), 6 (more than moderate), 7 (intense), 8 (very intense), 9 (barely tolerable), and 10 (intolerably intense). The participants were required to evaluate the severity of soreness in their exercising quadriceps during 2 3 squats at 48 h after the eccentric exercise bout (Skurvydas et al. 2006). Experimental procedure One week before the experiment, each subject was familiarized with the stimulation procedure. During the same visit to the laboratory the subjects learned to perform isometric, isokinetic, and eccentric attempt tests. Upon arrival to the laboratory on the testing day, their blood samples were taken for the analysis of CK activity at baseline. The subjects then completed a warm-up consisting of 5 min of cycling on the cycle ergometer at the intensity that corresponded to the heart rates of 110 130 beats min 1 and were seated into dynamometer chair. Isokinetic concentric and isometric knee extension torques and CAR were assessed at baseline, immediately after (within 2 3 min) and 48 h after the eccentric exercise bout. At each time point, the performance of the knee extensors was tested in the following order: (i) the torque evoked by 20 and 100 Hz electrical stimulation; (ii) the torque produced during MVC (2 trials); (iii) concentric isokinetic torque (3 trials); and (iv) superimposed tetanic train of stimuli (250 ms in duration) at 100 Hz frequency (TT100 Hz) on a 5-s MVC (2 trials). The time interval between each measurement was 2 min in all cases. In addition, muscle soreness and plasma CK activity was determined at 48 h after the eccentric exercise. Statistical analysis The 2-way ANOVA for repeated measures was used to determine the effect of time (i.e., at baseline, immediately after, and 48 h after the eccentric exercise) and group (i.e., UT vs. TD vs. VP vs. S) on markers of exercise-induced muscle damage. If significant effect was found, a post hoc test was performed, applying paired t tests with a Bonferroni correction for time effect and a Tukey post hoc for group effect for multiple comparisons. Descriptive data are presented as means ± SD. The level of significance was set at p < 0.05. Statistical power was calculated for all mechanical markers of muscle damage based on an a level of 0.05, sample size (n = 10), SDs, and averages at baseline and after the eccentric exercise. Statistical power was more than 80% when p < 0.01 and more than 65% in all other cases when p < 0.05. Results Dynamics of eccentric work during exercise The total and average eccentric work done per contraction was significantly (p < 0.05) greater in S and VP than in LDR and UT (Table 1). The changes in peak eccentric torque dur-

4 Appl. Physiol. Nutr. Metab. Vol. 36, 2011 Table 1. Average eccentric torque attained and average eccentric work done in untrained sprint runners, long-distance runners, and volleyball players during eccentric exercise. Subjects Eccentric torque, N m Work done, J Untrained 211.8 (33.7) 199.2 (34.2) Sprint runners 240.7*, (38.1) 228.1*, (23.7) Long-distance runners 191.3 (18.2) 181.1 (15.7) Volleyball players 264.9*, (21.5) 242.4*, (19.1) Note: Values are means (±SD) per repetition. *p < 0.05 compared with untrained. p < 0.05 compared with long-distance runners. Fig. 1. Eccentric torque during eccentric exercise. LDR, long-distance runners; S, sprint runners; UT, untrained subjects; VP, volleyball players. Values are means ± SD. P20, and P100 were significantly (p < 0.05) greater in S and VP than in LDR and UT. There were no significant differences in voluntary and electrically induced knee extension torque between S and VP, while P100 was significantly (p < 0.05) greater in UT compared with LDR. The values of P20/ P100 of UT, S, and LDR were significantly (p < 0.05) greater than those of VP. MVC, IT, P20, and P100 decreased significantly (p < 0.001) in all groups after the 100 eccentric contractions (Fig. 3). P20 and P100 did not recover within 48 h after exercise. In all groups, P20/100 decreased significantly (p < 0.001) after exercise (Fig. 4A). LFF after exercise (i.e., 2 3 min) was significantly (p < 0.05) greater in UT and VP than in S and LDR. MVC, IT, and P20 decreased significantly (p < 0.05) more in UT after exercise than in other groups. There were no significant (p > 0.05) differences in changes of P20, P100, and MVC between S, LDR, and VP. Changes in CAR There was no significant difference in baseline CAR between groups (Fig. 4B). CAR was significantly (p < 0.05) impaired by eccentric exercise in all groups, but remained depressed for 48 h after exercise only in UT. CAR decreased after exercise significantly (p < 0.05) greater in UT and S compared with LDR and VP. Fig. 2. Intraindividual variability (coefficient of variation) of eccentric torque and eccentric work done during eccentric exercise. UT, untrained subjects; S, sprint runners; LDR, long-distance runners; VP, volleyball players. Values are means ± SD. *, p < 0.05, from VP. ing each of 100 contractions are presented in Fig. 1. The intraindividual variability of eccentric work done was significantly (p < 0.05) lower in VP compared with UT, S and LDR, while CV of eccentric torque in UT and LDR was significantly (p < 0.05) greater than in S and VP (Fig. 2). Changes in voluntary and electrically induced knee extension torque The baseline values of voluntary and electrically induced knee extension torque are presented in Table 2. MVC, IT, The changes in optimal knee angle for IT The optimal knee angle for IT was 63.9 ± 5.8, 67.5 ± 4.7, 62.7 ± 5.7 and 63.1 ± 3.9, respectively, in UT, S, LDR, and VP (p > 0.05 between groups) (Fig. 4D). Immediately after exercise the optimal knee angle for IT shifted to significantly (p < 0.05) longer muscle length only in UT and LDR. This shift to longer optimal length in UT subjects did not recover significantly within 48 h after exercise. CK and muscle soreness Plasma CK activity within 48 h after exercise increased from 106.5 ± 64.2 IU L 1, 161.1 ± 107.5 IU L 1, 208.1 ± 169.9 IU L 1, and 200.0 ± 111.7 IU L 1 to 268.4 ± 198.2 IU L 1, 348.4 ± 188.4 IU L 1, 411.9 ± 202.2 IU L 1, 313.9 ± 254.0 IU L 1 in UT, S, LDR, and VP, respectively (p < 0.05, compared with baseline value; p > 0.05, compared among groups). No differences were observed in muscle soreness between groups 48 h after exercise (2.9 ± 1.4, 4.1 ± 2.1, 3.8 ± 1.7, and 2.8 ± 1.4 points in UT, S, LDR, and VP, respectively). Discussion We found that (i) indirect symptoms of exercise-induced muscle damage (prolonged decrease in MVC, IT, and P20) were more evident in UT than in well-trained athletes, while there were no significant differences in changes of muscle soreness and plasma CK activity between athletes and UT; (ii) LFF was greater in UT and VP than in S and LDR; (iii) optimal knee angle for IT shifted significantly by about 5 to the longer muscle length only in UT and LDR after exercise; and (iv) in all groups P100 decreased significantly by about 20%, while CAR decreased significantly by about 8% in UT and S and by approximately 3% 5% in LDR and VP.

Skurvydas et al. 5 Table 2. Baseline values of voluntary and electrically induced induced quadriceps contraction torque in untrained sprint runners, long-distance runners, and volleyball players. Subjects P20, N m P100, N m P20/P100 MVC, N m IT, N m Untrained 151.1 (38.2) 222.4 (48.7) 0.8 (0.1) 274.8 (52.1) 238.3 (37.8) Sprint runners 202.5*, (40.2) 282.5*, (46.5) 0.81 (0.1) 322.6*, (59.3) 277.6*, (40.1) Long-distance runners 122.1 (23.1) 175.2* (34.2) 0.79 (0.1) 255.6 (30.1) 214.7 (21.1) Volleyball players 170.6*, (38.2) 281.7*, (67.1) 0.7*, (0.1) 357.2*, (39.1) 303.5*, (25.9) Note: Values are means (±SD). P20 and P100, muscle contraction torque induced by stimulating musculus quadriceps femoris at 20 Hz and 100 Hz frequencies; MVC, maximal voluntary contraction torque; IT, maximal knee isokinetic extension torque. *p < 0.05 compared with untrained. p < 0.05 compared with long-distance runners. Fig. 3. Electrically induced muscle contraction torque at 20 Hz (A) and 100 Hz (B), as well maximal voluntary isometric (C) and isokinetic (D) torque in per cent compared with baseline values. UT, untrained subjects; S, sprint runners; LDR, long-distance runners; VP, volleyball players. Values are means ± SD. *, p < 0.05, compared with baseline;, p < 0.05, compared with S, LDR, and VP. Changes in voluntary-induced knee extension torque It is quite understandable why the IT, MVC (Table 1), eccentric torque, and eccentric work done (Table 2) in S and VP were greater than in LDR and UT: one explanation is the prevalence of fast-twitch muscle fibers in the muscles of athletes engaged in speed and power sports while slow-twitch muscle fibres prevail in the muscles of endurance athletes (Saltin et al. 1977; Howald et al. 1985). Also, training modality favours S and VP superiority at the exercise test performed. However, it was unexpected that variability of eccentric torque and variability of work done per repetition was lower in VP compared with UT, S, and LDR, and we do not have a reasonable explanation for the reduction (Fig. 2). Causes of depressed neuromuscular function after eccentric exercise It has been established that decreases in maximal voluntary isometric and isokinetic contraction torque, increases in plasma CK activity and muscle soreness, and a rightward shift in the muscle length-tension relationship are evident for an extended period of time (i.e., 1 7 days after exercise) (Philippou et al. 2004; Prasartwuth et al. 2006; Chen et al. 2007; Hubal et al. 2007; Chapman et al. 2008; Kamandulis et al. 2010). In the present study, for the subjects of all groups the following indirect markers of muscle damage emerged within 48 h of eccentric exercise: muscle soreness; elevated plasma CK activity; decreased P20, P100, MVC, and IT; and increased LFF (Figs. 3 and 4D). CAR decreased after eccentric exercise in all groups and

6 Appl. Physiol. Nutr. Metab. Vol. 36, 2011 Fig. 4. P20/P100 ratio (A), central activation ratio (B) values in per cent, and optimal knee angle in degrees for isokinetic extension torque (C) at baseline and in recovery. UT, untrained subjects; S, sprint runners; LDR, long-distance runners; VP, volleyball players. Values are means ± SD. *, p < 0.05, compared with baseline;, p < 0.05, compared with S, LDR, and VP;, p < 0.05, compared with S and LDR. only in UT did it not fully recover during the next 48 h (Fig. 4B). This is in accord with Prasartwuth et al. (2006), who showed that voluntary activation of muscle decreased after muscle-damaging exercise and remained depressed for 24 h. There is no doubt that in our case the main reasons for the decrease in voluntary and electrically induced quadriceps muscle torque are related to damage of force-bearing structures (Black and McCully 2008, Hubal et al. 2007) and changes in muscle voluntary activation (Prasartwuth et al. 2006). Peripheral fatigue was greater than central fatigue, as the decrease of voluntary and electrically induced torques was greater than the drop of CAR. Furthermore, P20 decreased to a greater extent than P100 after eccentric exercise, indicating that the muscles were subjected to LFF. LFF is characterized by a relatively greater loss of force at low than high stimulation frequencies (Skurvydas et al. 2006; Allen et al. 2008; Dundon et al. 2008), which shows an impairment of the excitation contraction coupling system (Proske and Morgan 2001; Skurvydas et al. 2006, 2008). The decreased force production of eccentric exercise exposed muscle cells can, in principle, be due to (i) reduced Ca 2+ release from the sarcoplasmic reticulum (SR) leading to decreased free myoplasmic [Ca 2+ ] ([Ca 2+ ] i ), (ii) decreased myofibrillar Ca 2+ sensitivity; and (iii) reduced ability of contractile machinery to produce force (Balnave and Allen 1995). On a simplified model, factors (i) and (ii) would result in a larger force depression at low than high stimulation frequencies because of the sigmoidal shape of the force [Ca 2+ ] i relationship, whereas factor (iii) would give a similar force decrease at all stimulation frequencies. We observed markedly larger force reductions at 20 Hz than 100 Hz stimulation after exercise, which indicate important roles of factors (i) and (ii) in the eccentric exercise-induced force depression. This is in line with the results from single mouse muscle fibres exposed to eccentric contractions where both reduced tetanic [Ca 2+ ] i and decreased myofibrillar Ca 2+ sensitivities were observed (Balnave and Allen 1995). It should be noted, however, that sarcomere instability induced by eccentric contractions can shift the optimal length for active force production to longer lengths, which may exaggerate the force depression at low stimulation frequencies (Parikh et al. 2004). Sarcomere instability may also disrupt the mechanical interaction between the t-tubules and the SR and in this way impair SR Ca 2+ release (Takekura et al. 2001). The results of our study suggest that differences in muscle damage cannot be accounted for by differences only in muscle fiber composition, although it has been established that fast-twitch fibers are more vulnerable (Fridén and Lieber 2001; Guerrero et al. 2008). If muscle damage depended only on muscle composition, then symptoms in LDR would be less pronounced than in S and VP, which was not the case in this study. Besides, it has been postulated that endurance training has a protective effect for eccentric contraction-induced muscle damage (Evans et al. 1986), and muscle fiber oxidative capacity is a determining factor in fiber damage (Fridén and Lieber 2001). Therefore, if this was the case, there should have been a greater decrease of torque in S and VP than in LDR. It has been shown that stretch-induced muscle damage can be dramatically reduced by training (Brown et al. 1997). Normally, S and VP perform more exercise with maximum intensity during their training than LDR; thus S and VP must recruit and train greater proportion of their fast-twitch muscle fibers. Therefore, fast-twitch fibers of S and VP could be more resistant to muscle damage caused by eccentric exercise than fast-twitch fibres of LDR and UT. This may have been one of the causes of a smaller change in optimal angle in S and VP than in LDR and UT (Fig. 4D). This is in accord with the results showing that muscles of power athletes are more resistant to damage when intensive stretch-shortening exercises are performed (Takala et al. 1989; Kyröläinen et al.

Skurvydas et al. 7 1998). Takala et al. (1989) and Kyröläinen et al. (1998) speculated that the differences of muscle protein (CK, myoglobin) levels in blood plasma between the subject groups were because of different muscle fiber composition, motor unit recruitment, and (or) training background. On the other hand, Magal et al. (2010) have recently suggested that plasma CK activity following exercise-induced muscle damage may not be related to muscle fiber composition. However, they found a significant positive correlation between soreness 48 h after exercise and fiber content of IIa and IIb fibres type. Changes in optimal knee angle for IT The rightward shift in the muscle length-tension relationship has been attributed to an increased muscle series compliance due to disrupted sarcomeres (Proske and Morgan 2001) and this shift has been proposed as a reliable indicator of muscle damage (Proske and Morgan 2001; Philippou et al. 2004; Chen et al. 2007). This is in accord with our data that shift to optimal knee angle for IT is clearly evident only in UT and LDR (Fig. 4D). However, it can be concluded that after a series of eccentric muscle contractions, the fall in force is the result of a number of interdependent factors, not all of which are a direct consequence of the damage process (Gregory et al. 2007). For example, the shift to optimal angle may occur because of the increase in muscle-tendon compliance. It has been established that when the number of sarcomeres is added in series during muscle adaptation to eccentric exercise, muscle compliance increases and it results in rightward shift of length-tension properties of muscle where the optimal angle for peak torque occurs at longer muscle lengths (Proske and Morgan 2001). 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