Skeletal muscle satellite cells (SC) are considered to play a crucial role in muscular adaption to various forms of training in muscle fiber maintenance and repair (1). In human skeletal muscle, changes in SC number were observed in the first days after an intense single bout of resistance exercise in several investigations. The increase in SC number, mostly identified by the expression of Pax7, was most robust when the exercise was performed as exhaustive eccentric exercise (2–10). These findings supported the hypothesis that repeated high mechanical force during eccentric contraction causes myofiber damage, which triggers the release of growth factors from within the myofiber and initiates regeneration and remodeling processes (11).
Regeneration and remodeling processes are mediated by proliferation, activation and differentiation of SCs orchestrated by the expression of different myogenic regulatory factors (MRF, Myf 5, MyoD, myogenin, MRF4) within the SC (12). To our best knowledge, in human skeletal muscle, a significant increase of MyoD-positive satellite cells, indicative for increased SC activation, was only reported after exhaustive eccentric resistance exercise of the knee extensor muscles with a high number of 196.1 ± 37.2 (13) and 300 repetitions (8), respectively. In two studies (14,15), the expression of myogenin, indicating SC differentiation, was found 7 d after 210 forced eccentric contractions of the quadriceps muscle induced by electrical stimulation and could not be induced by volitional eccentric muscle contractions. In contrast, McKay et al. (6) reported a significant increase in myogenin-positive cells 24, 72, and 96 h after 30 sets of 10 maximal volitional lengthening contractions of the quadriceps muscle.
In common strength training programs, far less than 200 repetitions are performed, usually as combined concentric and eccentric actions. Recently, some investigations focused on modifications of the conventional concentric/eccentric strength training, which aim to enhance muscular adaptation. Accentuated eccentric loading was discussed as a superior method by which to enhance strength and power performance (16,17). In two of our previous studies, there were indications for the development of a stronger and faster muscle phenotype after several weeks of concentric/eccentric strength training with eccentric overload (CON/ECC+) compared with equivalent conventional concentric/eccentric strength training (CON/ECC). Furthermore, we also found some signs for enhanced SC activation and muscle regeneration. After 6 wk of intense strength training, steady-state mRNA-expression of MRF4 and of neonatal myosin heavy chain (MHCneo) was only increased if the strength training was performed with eccentric overload (CON/ECC+) in young strength-trained men (18). In a study on quadriceps, muscle regeneration after anterior cruciate ligament reconstruction, a significant increase in myofibers expressing MHCneo was only found after CON/ECC+ (19). The findings of these studies suggest that SC might play a crucial role in the enhanced muscular adaption to CON/ECC+.
We hypothesized that the combination of the metabolic stress during concentric muscle contractions with damaging eccentric overload during an exhaustive CON/ECC+ exercise bout might provide an effective stimulus for SC activation and differentiation as observed after electrically stimulated eccentric muscle contractions. Therefore, 30 male recreational athletes were randomly assigned to one bout of exhaustive CON/ECC+ or CON/ECC leg extension exercise. Biopsies from the vastus lateralis muscle were obtained 1 wk before and 1 wk after the exercise bout (contralateral leg) to investigate SC content, activated and differentiating SC as well as the number of MHCneo expressing myofibers. As indicators for myofiber damage, plasma creatine kinase (CK) activity and myoglobin concentration were determined. All variables were also measured in 10 nonexercising male control subjects at the same time points.
METHODS
Subjects
Forty recreationally active male subjects volunteered to participate in the study. Thirty of them were randomly assigned to one bout of exhaustive leg extension exercise on a conventional leg extension device (Core Health & Fitness, Nautilus, Vancouver, WA) (CON/ECC; n = 15; age, 23.3 ± 3.5 yr; height, 180.5 ± 5.4 cm; weight, 74.6 ± 7.6 kg) or on a computer-driven isokinetic device with eccentric overload (IsoMed 2000; D&R Ferstl, Hemau, Germany) (CON/ECC+; n = 15; age, 24.7 ± 3.3 yr; height, 181.6 ± 6.1 cm; weight, 77.4 ± 8.9 kg). Ten subjects formed the control group (CG) (n = 10; age: 24.1 ± 3.6 yr; height, 179.6 ± 5.9 cm; weight, 79.8 ± 11.1 kg) without any exercise. Inclusion criteria were recreational activity with an average of three workouts per week, not more than one session of leg strength training and the absence of any health problem in a physical examination performed before enrolment. Throughout the study, the subjects were not allowed to perform any other exercises besides the tests and the training session of the study. Furthermore, they had to refrain from protein or creatine supplements.
The study was approved by the local ethics committee and conducted in accordance with the Declaration of Helsinki. Each subject provided written informed consent to participate.
Experimental Design
Exercise groups
The timeline of the study protocol is illustrated in Figure 1. All subjects of the exercise groups performed a standardized familiarization training. On the same occasion, they were also familiarized with the isokinetic testing. The first muscle biopsy was taken after 7 without any exercise (t0). One week later (t1), subjects performed one bout of exhaustive leg extension resistance exercise, either in the conventional concentric/eccentric mode (CON/ECC) or as concentric/eccentric exercise with eccentric overload (CON/ECC+). The second muscle biopsy was obtained 1 wk after the exhaustive leg extension exercise from the contralateral leg (t8).
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FIGURE 1: Timeline of the experimental protocol.
Isokinetic strength tests of the quadriceps femoris muscle group of both legs were conducted immediately before (t1a) as well as immediately (t1b), 24 (t2), 48 (t3), 72 h (t4), and 7 d (t8) after the exhaustive training bout. Before all strength tests, venous blood was drawn for determination of CK and myoglobin.
Control group
Muscle biopsies were performed at the same time intervals as in the exercise groups. Venous blood was drawn immediately before both muscle biopsies for determination of CK and myoglobin.
Exhaustive Leg Extension Exercise
After a standardized warm-up procedure on a stationary bike (10 min at 1.5 W·kg−1 and 60–80 rpm; Ergoline, Bitz, Germany), the subjects performed one bout of exhaustive one-legged concentric/eccentric extension resistance exercise supervised by professional strength training instructors. Both legs were exercised, and six sets of eight repetitions were performed with each leg. The starting leg was randomly determined. On both strength training devices, subjects were seated upright with a hip angle of approximately 80°. The motion ranged from 100° to 10° (10°, full leg extension). The axis of rotation was set in the middle of the lateral femoral condyle.
Conventional leg extension exercise
The subjects sat upright leaning against the seatback while holding the installed handles. First, the one repetition maximum (1RM) was determined for the first leg. After 5 min of rest, the subjects performed six sets of eight repetitions of leg extension exercise with a load equivalent to 80% of 1RM. The series of eight repetitions were separated by 1 min of rest. The load was the same for the concentric and eccentric phase. In case of fatigue symptoms, the load was reduced by 2.5% to ensure completion of six sets with eight repetitions. After 5 min of rest, 1RM of the contralateral leg was measured, and the leg was exercised as described. Training load was 47.2 ± 8.8 kg (right leg) and 47.6 ± 6.7 kg (left leg), respectively.
Computer-guided leg extension exercise with eccentric overload
The subjects sat upright leaning against the seatback with their arms folded across their chest. They were secured with shoulder pads and with a belt across the hip; the thigh of the exercising leg was fixed with a belt as well. The load was controlled by velocity with 60°·s−1 for the concentric and 180°·s−1 for the eccentric phase using the active-assistive mode in the computer-guided isokinetic device (IsoMed 2000; D&R Ferstl, Hemau, Germany). In each of the six sets, the subjects were instructed to develop maximal force onto the lever arm during the eight repetitions in the concentric, as well as in the eccentric phase, which resulted in an eccentric load that was approximately 1.3-fold higher than the concentric load.
Isokinetic Muscle Strength Testing
After a standardized warm-up procedure on a bicycle ergometer (10 min at 1.5 W·kg−1; body mass, 60–70 rpm), maximum strength was assessed using the leg extension module of the IsoMed 2000 (D&R Ferstl). For familiarization, subjects completed a five repetitions warm-up of leg extension in concentric mode with increasing, but submaximal intensity. After 1 min rest, peak torque was determined out of three repetitions at an angular velocity of 60°·s−1 and out of five repetitions at 180°·s−1 between 10° and 100° of knee-flexion for both legs separately. Subjects were seated upright with 80° of hip flexion and with their arms folded across the chest. They were secured with shoulder pads and with a belt across the hip; the thigh of the tested leg was fixed with a belt as well.
Determination of CK and Myoglobin in Venous Blood
In supine position, blood samples (7.5 mL) were drawn from an antecubital vein into heparin tubes (Monovette-S Lithium-Heparin, catalog no. 04.1936, Sarstedt AG & Co. KG, Nümbrecht, Germany). Creatine kinase and myoglobin were measured on the same day in the laboratory of the university hospital. Creatine kinase was analyzed from plasma by an enzymatic kinetic essay with NAC (N-acetyl cysteine) method at 340 nm (EC 2.7.3.2) using an ADVIA Chemistry XPT System (Siemens Healthcare, Erlangen, Germany). The content of myoglobin in plasma was determined by a chemiluminescent two-way immunoassay (Sandwich, ADVIA Centaur XPT Immunoassay-System; Siemens Healthcare). In the university hospital, plasma CK activity and myoglobin concentration are routinely measured every day. The intra-assay coefficients of variation are 1.2 for CK activity and vary between 4.9 and 2.6 for values between 70 and 220 μg·L−1 for myoglobin concentration. The interassay coefficients vary between 3.3 and 1.1 (CK activity between 80 and 615 U·L−1) and between 7.4 and 4.5 (myoglobin concentration between 75 and 260 μg·L−1, respectively.
Muscle Biopsy Sampling
Biopsies from the vastus lateralis muscle were obtained in the rested condition 7 d before (t0) and 7 d after the exhaustive bout of leg extension resistance exercise (t8) under local anesthesia, using the Bergström technique (20). The muscle tissue was immediately freed from visible connective tissue, rapidly frozen in isopentane cooled by liquid nitrogen and subsequently stored at −80°C. To avoid any residual effects from the preceding biopsy, the postexercise biopsy was taken from the contralateral leg.
Immunohistochemistry
All biopsies were assigned a random unique identification number, thereby blinding the investigator to subject identity and time point. However, simultaneous analysis of the biopsies obtained from each subject before and after the exhaustive leg extension exercise bout in the same staining procedure was ensured. Immunofluorescent analyses were performed on serial sections for SC number, activated SC, differentiating SC, fiber type distribution, and myofibers expressing neonatal myosin (MHCneo). For all analyses, transverse sections (10 μm) were cut in a cryostat (−20°C) mounted on Super Frost Plus slides (ThermoFisher Scientific, Waltham, MA) and allowed to dry for 20 min. Sections were then fixed in 4% paraformaldehyde (PFA, Roti-Histofix 4%, catalog no. P087; Carl Roth, Karlsruhe, Germany) for 10 min. For all further procedures, sections were then washed three times for 5 min in phosphate-buffered saline (PBS) and blocked for 10 min at room temperature (RT) in PBS containing 0.5% Triton (Triton X-100, catalog no. 108643; Merck Millipore, Billerica, MA) and 1% bovine serum albumin (Albumin Bovine Fraction V, catalog no. 11922; Serva Electrophoresis, Heidelberg, Germany).
SC number
For determination of the SC number, staining of Pax7, laminin, type II myosin, and nuclei was done on the same section. Sections were incubated overnight at RT with the primary antibodies anti-Pax7 (1:500; catalog no. MAB1675; Bio-Techne [R&D Systems], Minneapolis, MN), anti-laminin (1:1000, catalog no. ab11575, Abcam, Cambridge, UK), and anti-Fast Myosin Skeletal Heavy Chain (1:1000, catalog no. ab91506; Abcam). The next day, the sections were washed in PBS followed by incubation in appropriate secondary antibodies: Alexa-Fluor 488 goat antimouse (Pax7; 1:1000, catalog no. A.11029, ThermoFisher [Invitrogen]) and Alexa-Fluor 568 goat anti-rabbit (Laminin and MHC II; 1:1000, catalog no. A.11036; ThermoFisher [Invitrogen]) for 2 h at RT. Then, the sections were washed in PBS and allowed to dry completely in the dark. Finally, sections were incubated for 5 min in medium containing 4′,6-diamidino-2-phenolindole (DAPI) to visualize nuclei (Fluoroshield Mounting Medium With DAPI, ab 104139; Abcam) and fixed at 4°C for 24 h. Staining specificity was confirmed using appropriate negative controls.
Activated SC
For the analysis of myogenic activation, sections were incubated overnight at RT with primary antibodies against Pax7 (1:500; catalog no. MAB1675; Bio-Techne [R&D Systems]) and against MyoD (1:500; catalog no. ab133627; Abcam). The secondary antibodies Alexa-Fluor 488 goat antimouse and Alexa-Fluor 568 goat antirabbit were used for visualization of Pax7 and for MyoD, respectively, and the staining procedure was finished as described previously.
Differentiating SC
For determination of the number of differentiating SC, sections were incubated overnight at RT with primary antibodies against myogenin (1:100, clone F5D; Developmental Studies Hybridoma Bank, The University of Iowa, Iowa City, IA) and against laminin (1:1000, catalog no. ab11575, Abcam). The secondary antibodies Alexa-Fluor 488 goat antimouse and Alexa-Fluor 568 goat antirabbit were used for visualization of myogenin and laminin, respectively, and the staining procedure was finished as described previously.
Myofibers expressing neonatal myosin
Sections were incubated overnight with the primary antibodies anti-MHCneo (1:20, NCL-MHCn; Leica Biosystems, Wetzlar, Germany), anti-Laminin (1:1000, catalog no. ab11575, Abcam) and anti-Fast Myosin Skeletal Heavy Chain (1:1000, catalog no. ab91506, Abcam). The secondary antibody Alexa-Fluor 488 goat antimouse was used for visualization of MHCneo and Alexa-Fluor 568 goat antirabbit for laminin as well as for MHC II. The staining procedure was finished as described above.
Image Analyses of Immunohistochemistry
Images were obtained at ×20 magnification using the fluorescence microscope Axio Observer and the appropriate software program Zen (Carl Zeiss, Oberkochen, Germany). On average, 579 ± 266 myofibers per section were analyzed before and 454 ± 208 myofibers per section after the exhaustive leg extension exercise. For determination of total SC number, the criterion for SC identification was simultaneous Pax7 and myonucleus (DAPI) staining within the myofiber (sublaminar). The number of Pax7-positive (Pax7+) cells (SCs) associated with MHC I+ (type I) or MHC II+ (type II) fibers was quantified and the number of SC divided by the total number of myofibers was categorized as SC per fiber. An activated SC was characterized by simultaneous MyoD, Pax7, and DAPI staining (MyoD+/Pax7+/DAPI). The number of activated SC was expressed relative to total SC number. Simultaneous myogenin and DAPI staining in sublaminar cells (myogenin+/DAPI) was regarded as indicating differentiating SC (SCdiff). The number of SCdiff was related to total number of myofibers per section. The number of MHCneo-positive fibers was related to total fiber number from the section.
Statistical Analyses
The effects of one bout of exhaustive resistance exercise on the (maximum values of) dependent variables (satellite cells, quadriceps strength, and blood values) were assessed using ANCOVA with training group (CON/ECC+ vs CON/ECC/CG) and baseline value as covariates. First, the normal distribution of all data was checked using the Kolmogorov–Smirnov test. This test failed for all data except for quadriceps strength. These data were log-transformed to normal distribution for further analysis except for differentiating SC, which could not be transformed to normal distribution. For analyzing the corresponding time effects (baseline vs 7 d after the training bout or peak value [strength tests, CK, and myoglobin]), the paired t test was applied. To evaluate the changes of differentiating SC, the Wilcoxon signed-rank test was performed. In addition, effect sizes were determined by partial eta-squared (ηp2). To investigate the relationship between selected variables, Pearson product moment correlations or Spearman’s rank-order correlations were calculated, respectively. For all calculations, a value of P ≤ 0.05 was considered statistically significant. All data are presented as mean ± SD. All statistical tests were processed using SPSS 26.0 software for Windows (SPSS Inc., Chicago, IL). The figures were created using GraphPad Prism 5.0 (GraphPad Software Inc., La Jolla, CA).
RESULTS
Effects of One Bout of Exhaustive Leg Extension Exercise on Muscle Strength and Integrity
Quadriceps strength
No significant differences were detected between CON/ECC+ and CON/ECC for the changes in the maximum values of peak torque at both angular velocities after the exhaustive bout of one-legged leg extension resistance exercise for both legs.
At an angular velocity of 60°·s−1, a significant increase in peak torque was found in both exercise groups. The maximum was observed 7 d after the exhaustive exercise bout CON/ECC+: increase from 2.5 ± 0.6 to 2.8 ± 0.4 N·m·kg−1 (right leg, P = 0.007), from 2.3 ± 0.4 to 2.8 ± 0.4 N·m·kg−1 (left leg, P < 0.001), CON/ECC: increase from 2.4 ± 0.5 to 2.8 ± 0.5 N·m·kg−1 (right leg, P < 0.001), from 2.2 ± 0.5 to 2.7 ± 0.5 N·m·kg−1 (left leg, P < 0.001). Also, at an angular velocity of 180°·s−1, there was a significant increase in peak torque in both exercise groups with a maximum value 7 d after the exhaustive exercise bout CON/ECC+: increase from 1.8 ± 0.3 to 2.1 ± 0.3 N·m·kg−1 (right leg, P < 0.001), from 1.8 ± 0.3 to 2.1 ± 0.3 N·m·kg−1 (left leg, P < 0.001), CON/ECC: increase from 1.9 ± 0.3 to 2.1 ± 0.3 N·m·kg−1 (right leg, P = 0.001), from 1.7 ± 0.3 to 2.0 ± 0.3 N·m·kg−1 (left leg, P < 0.001) (Fig. 2).
FIGURE 2: Effects of one exhaustive bout of CON/ECC+ or CON/ECC leg extension exercise on quadriceps strength determined as peak torque in isokinetic leg extension tests performed at angular velocities of 60°·s−1 (A: right leg, B: left leg) and 180°·s−1 (C: right leg, D: left leg). **P < 0.01, ***P ≤ 0.001 compared with the respective value before exercise (t1a).
Blood variables indicative for myofiber damage
Significant group effects were observed for the change in CK and myoglobin between CON/ECC+ and CG (CK: P < 0.001; F(1.22) = 35.576; ηp2 = 0.618; myoglobin: P < 0.001; F(1.21) = 17,464; ηp2 = 0.454) as well as between CON/ECC and CG (CK: P < 0.001; F(1.22) = 31.739; ηp2 = 0.591; myoglobin: P = 0.001; F(1.21) = 14.901; ηp2 = 0.415).
No significant differences were observed between CON/ECC+ and CON/ECC for the changes of plasma CK activity and myoglobin concentration after the exhaustive bout of one-legged leg extension resistance exercise. In the repeated testing during the week after the exhaustive leg extension resistance exercise, significant increases in CK were found from 142 ± 55 U·L−1 to a maximum of 4586 ± 7069 U·L−1 in the CON/ECC+ group (P < 0.001) and from 128 ± 51 U·L−1 to 3942 ± 5422 U·L−1 in the CON/ECC group (P < 0.001).
Significant increases were also found for myoglobin (CON/ECC+: 41 ± 20 μg·L−1 to 720 ± 1335 μg·L−1; P < 0.001; CON/ECC: 34 ± 12 μg·L−1 to 795 ± 1248 μg·L−1; P < 0.001). For both these measures, considerable interindividual variation in the increase was observed.
No significant changes were found in CG (Fig. 3).
FIGURE 3: Effects of one exhaustive bout of CON/ECC+ or CON/ECC leg extension exercise on CK (A) and myoglobin (B) concentration in venous blood. Data are represented as individual values and as group mean.
Exhaustive Strength Training Effects on Satellite Cell Abundance, Activation, and Differentiation
Satellite cell content
No significant differences were observed between CON/ECC+, CON/ECC, and CG for the changes in SC content determined in the muscle biopsy samples obtained 2 wk apart. However, only after the exhaustive leg extension resistance exercise bout with eccentric overload (CON/ECC+), a significant increase in total satellite cell number (P = 0.019) and in satellite cells related to type II fibers (P = 0.011) occurred (Fig. 4). There were no significant differences between CON/ECC+, CON/ECC, and CG in fiber type distribution. Fiber type distribution was similar before and after the exhaustive leg extension exercise.
FIGURE 4: Satellite cell number. A, Representative muscle cross-section stained for MHC II and laminin (red), nuclei (DAPI, blue), and Pax7 (green). Five SC (Pax7/DAPI) are identified, two SC related to a type I fiber (no staining) and three SC related to type II fibers (MHC II, red), Scale bar, 50 μm. B, Satellite cell number before and 7 d after one exhaustive bout of CON/ECC+ or CON/ECC leg extension exercise or after 14 d without exercise (CG). *P ≤ 0.05 compared with the respective value before training.
Activated satellite cells
Significant group effects were observed for the change in the proportion of activated satellite cells (SCact) between CON/ECC+ and CON/ECC (P = 0.036; F(1.27) = 4.869; ηp2 = 0.153) as well as between CON/ECC+ and CG (P = 0.022; F(1.22) = 6.042; ηp2 = 0.215). Seven days after CON/ECC+, the proportion of SCact was significantly increased from 0.132 ± 0.089 to 0.207 ± 0.092 (P = 0.003). No significant changes occurred in the CON/ECC group and in CG (Fig. 5).
FIGURE 5: Activated satellite cells. A, Representative muscle cross-section stained for MyoD (red), nuclei (DAPI, blue) and Pax7 (green). Four SC (Pax7+/DAPI) are identified, one classified as an activated SC (MyoD+/Pax7+/DAPI). Furthermore, two nuclei expressing MyoD (Myo+/Pax7−/DAPI) are shown, Scale bar, 20 μm. B, Proportion of activated SC (MyoD+/Pax7+/DAPI) before and 7 d after one exhaustive bout of CON/ECC+ or CON/ECC leg extension exercise or after 14 d without exercise (CG). **: P ≤ 0.01 compared with the respective baseline value. # P ≤ 0.05 for CON/ECC+ compared with CON/ECC, as well as for CON/ECC+ compared with CG.
Myofibers expressing differentiating satellite cells
No significant changes in the content of SCdiff occurred (Fig. 6).
FIGURE 6: Differentiating satellite cells. A, Representative muscle cross-section stained for laminin (red), nuclei (DAPI, blue) and myogenin (green). One differentiating SC (myogenin/DAPI) is identified, Scale bar, 20 μm. B, Differentiating satellite cells per total myofiber number before and 7 d after one exhaustive bout of CON/ECC+ or CON/ECC leg extension exercise or after 14 d without exercise (CG).
Myofibers expressing neonatal myosin
No significant differences between the groups were observed for the changes in the number of myofibers expressing MHCneo. The number of MHCneo+ myofibers was not significantly changed 7 d after the exhaustive bout of leg extension exercise (CON/ECC+, CON/ECC) or 14 d after the first muscle biopsy, respectively. MHCneo+ myofibers were not visible in the muscle biopsy samples of all subjects. In 10 subjects of CON/ECC+, nine subjects of CON/ECC, five subjects of CG, no MHCneo fibers were found (Fig. 7).
FIGURE 7: Myofibers expressing neonatal myosin. A, Representative muscle cross-section stained for MHC II and laminin (red), nuclei (DAPI, blue), and neonatal myosin (green). Three myofibers expressing MHCneo (green) are identified, all related to type II fibers (red), Scale bar, 50 μm. B, Myofibers expressing MHCneo before and 7 d after one exhaustive bout of CON/ECC+ or CON/ECC leg extension exercise or after 14 d without exercise (CG). Data are represented as individual values and as group mean.
Correlation analyses
Spearman’s rank-order correlation analyses revealed significant correlations between the proportion of activated SC 7 d after the exhaustive bout of resistance exercise (T8) and peak CK values (R = 0.607, P = 0.016), as well as between the proportion of activated SC at that time-point (T8) and peak myoglobin (R = 0.679, P = 0.005). No further significant correlations between the blood variables indicative for myofiber damage and SC content, differentiating SC and MHCneo+ myofibers, respectively, were found.
DISCUSSION
The present study is the first to show that one bout of eccentric-overload strength training, which, as a modification of the conventional concentric/eccentric resistance exercise, is applied in regular strength training programs, induces a significant increase in SC content and in the proportion of activated SC. Seven days (168 h) after a single session of six sets with eight repetitions of CON/ECC+ leg extension exercise, an average 56% increase in the proportion of activated SC was observed whereas the proportion of activated SC remained unchanged after conventional CON/ECC leg extension exercise. The changes in CON/ECC and in the CG were significantly different from the increase in the CON/ECC+ group. To our best knowledge, in human skeletal muscle, activated SC were only observed after high-force eccentric exercise with ~200 to 300 repetitions (4,8,13,21). However, such a high number of eccentric muscle contraction is not part of regular strength training regimens.
There is an ongoing controversial discussion about the singular contribution of eccentric and concentric contractions to muscular hypertrophy, which is the main adaptation to regular strength training. More robust increases in SC number after intense eccentric exercise than after intense concentric exercise bouts are a common finding and have been attributed to myofiber damage caused by active lengthening eccentric contractions. However, increases in SC number have also been observed after nondamaging concentric contractions (11,22). From various animal studies it is well known that SC are essential for myofiber repair and regeneration, comprising complicated biological and molecular processes of SC activation, proliferation, differentiation, and cell fusion (1). In response to myofiber damage, SC start to express MyoD, then start to proliferate to produce myoblasts, committed myoblasts being defined by the expression of myogenin and the loss of Pax7 expression (23).
In the present study, plasma CK activity and myoglobin concentration were repeatedly determined up to day 7 after the exhaustive bout of leg extension exercise as indicators of muscle damage. Surprisingly, despite the enhanced eccentric load, the increases in CK activity and myoglobin concentration were not significantly enhanced after CON/ECC+ compared with CON/ECC leg extension exercise. Impressive interindividual variation was observed for the maximum values of CK activity and of myoglobin concentration ranging from 288 to 26,827 U·L−1 (CK), from 44 to 5360 μg·L−1 (myoglobin) after CON/ECC+ and from 228 to 18,783 U·L−1 (CK), from 46 to 3,932 μg·L−1 (myoglobin) after CON/ECC, respectively. Such interindividual variation has previously been observed (24,25) and might be due to genetic variability emerging in low and high responders with regard to exercise-induced myofiber damage (26). However, it also was questioned if plasma CK activity and myoglobin concentration are reliable indicators of myofiber damage (26). For many years, both measures have been considered as indirect markers of muscle damage, indicating increased muscle membrane permeability. Until now, it has not been proven by histological analyses that the increases in plasma or serum CK activity are caused by significant sarcolemmal disruptions in human muscle after eccentric exercise (26). In some studies, distinct increases in plasma CK activity were not always accompanied by signs of myofiber damage in (immuno-)histochemical analyses of muscle biopsies after high force eccentric muscle contractions (27,28). As an alternative explanation for the increased membrane permeability after eccentric muscle contractions an activation of stretch-activated Na+ and Ca2+ channels has been discussed (26). The lack of a decrease in maximal isokinetic quadriceps strength measured as peak torque values at angular velocities of 60°·s−1 and 180°·s−1 24, 48, 72 and 168 h after exhaustive leg extension exercise is hard to understand. Surprisingly, there even was a successive increase in maximal isokinetic quadriceps strength, even at time points when plasma CK activity was still elevated. In most studies on the effects of damaging eccentric exercise, decreases in maximal isometric voluntary torque were reported (8,14,24,29–31), but also a loss of isokinetic force was observed (13,31,32). In one study, a significant decrease in isokinetic force occurred after an average of 196 repetitions of eccentric muscle contractions, although such loss was not observed after concentric muscle contractions, matched for total work volume (average of 350 concentric contractions) (13). In all the other studies, a nonphysiological amount of eccentric load was applied. In contrast, we investigated resistance exercise, which is used in regular strength training. It might be speculated that the myofiber damage introduced by the CON/ECC+ leg extension exercise of the present study was less severe than after high-force eccentric muscle contractions applied in other investigations. However, inadequate familiarization might also be a cause for the surprising increase in quadriceps strength. Although all subjects of the exercise groups received a familiarization session on the isokinetic testing and training system, the increase in isokinetic force suggests still ongoing familiarization.
A greater damaging effect of one bout of CON/ECC+ compared with CON/ECC leg extension exercise could not be proven in the present study. Nevertheless, significant increases in SC content, SC related to type II myofibers and in the proportion of activated SC were only found when the leg extension exercise was performed with approximately 30% eccentric overload. Furthermore, the proportion of activated SC, determined in the muscle biopsies obtained 7 d after the exhaustive leg-extension exercise, was significantly correlated with the maximal values of CK and of myoglobin in the CON/ECC+ group only. With regard to the observation of Joanisse et al. (33), who found a significant correlation between the expansion of the SC content and CK levels in older adults, it appears that eccentric overload might provide a sufficient stimulus for enhanced activation of SC by inducing some muscle damage. There are only few studies in which an increase in activated SC in human skeletal muscle after a single exercise bout was reported. After an average of 196 eccentric muscle contractions, significant increases in SC content and in MyoD-positive cells per myofiber were observed 24 h after exercise; however, SC numbers related to type I and per type II myofibers did not change significantly (13). Significant increases in SC number related to type II myofibers and in activated SC related to type II myofibers (determined by immunohistochemical DLK1 staining) were described 24 h after about 300 repetitions of eccentric muscle contractions (21). In two further studies, muscle biopsies were repeatedly obtained (4–6, 24, 72 and 96 or 120 h) after 300 maximal eccentric quadriceps contractions and analyzed for SC content (Pax7+/DAPI cells) and activated SC (Pax7+/PCNA+/DAPI or Pax7+/MyoD+ DAPI cells) (4,8). The number of activated SC peaked 72 h (4) or 24 h after the exhaustive eccentric exercise (8) and remained significantly increased 120 and 96 h after the exercise, respectively. In both these studies, SC content peaked 72 h after exercise, declined afterward and was not significantly different from the preexercise value 96 h postexercise in one investigation (8), but still significantly increased 120 h postexercise in the other study (4). In the present investigation, significant increases in SC content and activated SCs were observed 7 d (168 h) after the exhaustive CON/ECC+ leg extension exercise at a time point when biopsies had not been obtained in most other studies. However, after 200 electrically stimulated eccentric contractions, proliferating SC (Ki-67+/Pax7+) peaked on day 7 (168 h) after exercise (30). With regard to the findings in animal studies, MyoD-positive SCs can still be found at that time point (12,23). Cells staining for MyoD+/Pax7−/DAPI might be regarded as differentiating SCs. As described previously in animal studies (34,35), also in the present study, MyoD frequently occurred in Pax7− nuclei, which could not clearly be located within the myofibers and must, at least to a certain extent, be regarded as undefined cells. Because of this observation, we wondered if the Pax7+/MyoD+/DAPI cells represent activated satellite cells. However, in the sections stained with antibodies against Pax7, against laminin and with DAPI, all the Pax7+/DAPI cells were located within the myofiber (sublaminar) and in the serial sections, which were stained with antibodies against Pax7, against MyoD and with DAPI, a similar number of Pax7+/DAPI cells was found. Furthermore, in a study on the satellite cell response to nonhypertrophic exercise stimuli, a similar proportion of Pax7+ cells coexpressing either MyoD or PCNA was observed, PCNA (proliferating nuclear antigen) being marker for cell proliferation. It was concluded that cells expressing both Pax7 and MyoD may be classified as activated SCs (36).
For the evaluation of differentiating SC, simultaneous staining with antibodies against myogenin, against laminin and with DAPI was performed. With this staining procedure, myonuclei (sublaminar nuclei) expressing myogenin could be identified (36). There only was a slight trend toward an increase in myogenin-expressing cells in the CON/ECC+ group, however, with considerable intraindividual variation. Significant increases in myogenin-positive cells were observed in two investigations 168 or 192 h after 200 electrically stimulated lengthening contractions of the quadriceps muscle (14,15). No such increase was found after 200 volitional eccentric contractions (14). Myogenin-expressing cells, representing committed myoblasts, are expected to appear in the middle phases (4–7 d) after muscle damage (23). When McKay et al. (6) observed a significant increase in myogenin-expressing cells as early as 24 h after 300 maximal voluntary eccentric contractions, the authors wondered whether these myogenin-positive cells really represent differentiation. For the formation of new myofibers from myoblast proliferation and differentiation to myotubes, muscle fiber necrosis is essential (37). Although exhaustive 200 volitional eccentric muscle contractions to some extent caused muscle damage with Z-line disruption, electrical stimulation resulted in myonecrosis and subsequent regeneration of muscle tissue (14). With regard to the unchanged number of myofibers expressing MHCneo, it can be speculated that myonecrosis inducing the formation of new myofibers has only occurred in some subjects of the present study. In contrast, a significant increase in MHCneo+ fibers was observed 7 and 30 d after electrically stimulated damaging eccentric muscle contractions (30). The developmental myosin heavy chains are transiently expressed during embryonic and fetal development. Their reexpression in the adult skeletal muscle is regarded as indicative for degeneration/regeneration events in the pathological human skeletal muscle (e.g., in myopathies or polymyositis) and in regenerative processes after severe muscle damage in animal studies (38).
The significant increase in SC content, in the proportion of activated SC, and in the SC number related to type II myofibers after CON/ECC+ provides some evidence for enhanced repair processes because of accentuated eccentric load. It is not very likely that the significant differences in the SC responses were caused by differences in muscle capillarization between the subjects in the respective groups. Nederveen et al. (8) observed an enhanced SC response to one bout of damaging exercise in subjects with greater muscle capillarization who also had a significantly greater proportion of type I fibers than subjects with lower capillarization. Capillarization was not determined in the present study; however, there were no significant differences in the fiber type distribution between the groups of recreationally active subjects, suggesting that the results were not flawed by different training status of the CON/ECC+ and CON/ECC subjects. We and others have shown that chronic CON/ECC+ strength training is superior to conventional CON/ECC strength training with regard to the development of muscle hypertrophy and a fast muscle phenotype (18,39–41). In the present investigation, the significantly increased SC number related to type II myofibers after one bout of CON/ECC+ leg extension exercise was probably caused by increased recruitment of type II myofibers. Also in other studies, SC activity was differently affected by the contraction mode of skeletal muscle, with a greater increase in SC number related to type II fibers after eccentric exercise (13,21). Furthermore, there is some evidence that the accretion of new myonuclei from activated SCs after resistance exercise is of importance for the development of muscle hypertrophy (1). It could be shown that the acute SC response to one bout of conventional CON/ECC resistance exercise is related to the extent of muscle hypertrophy induced by 16 wk of regular CON/ECC training. However, not only the response of SC related to type II fibers but also the response of SC related to type I fibers was relevant for the development of muscle hypertrophy (42). The importance of an elevated SC pool throughout 10 wk of CON/ECC leg extension exercise for the development of muscle hypertrophy was also observed in another investigation (43). However, the increased SC content was not coupled with changes in myonuclear number. Furthermore, the acute SC response to one bout of CON/ECC resistance exercise was no longer significant at the end of the training period probably because of attenuated muscle damage at this time. To our best knowledge, the SC responses during several weeks of CON/ECC+ training have not been investigated so far. Therefore, it is not known, if progressive eccentric-overload training might provide a greater muscle damaging stimulus than conventional CON/ECC training, also after several weeks of regular training still inducing an increase in SC related to type II fibers. It is speculative, but this could be an explanation for the advantages of CON/ECC+ for the induction of muscle hypertrophy and a shift toward a faster muscle phenotype.
Our study has several limitations. No muscle biopsies were obtained during the first 2 d after the exhaustive leg extension exercise and acute effects of the different resistance exercises remain unknown. However, the primary aim of the study was to find out if one bout of exhaustive CON/ECC+ leg extension exercise induces SC differentiation which might be observed 7 d after exercise-induced myofiber damage. To make absolutely sure that exercise effects and no residual effects of preceding muscle biopsies were investigated, we decided against multiple biopsies from one biopsy site and obtained the second muscle biopsy 7 d after the exhaustive leg extension exercise from the vastus lateralis muscle of the contralateral leg. To control for the variability of repeated biopsy sampling in the same individual, muscle biopsies were obtained from a nonexercising CG. Another drawback is that the effects of the different types of resistance exercise could only be tested in different individuals, and this certainly caused some variability because of different training status or to genetic variability in the response to exercise. To test the effects of the different modes of resistance exercise against each other, it would have been advantageous to subject one leg to CON/ECC and the other leg to CON/ECC+ leg extension exercise in the same individual.
CONCLUSIONS
In conclusion, accentuated eccentric load during one bout of concentric/eccentric leg extension exercise induced increases in SC activation, SC content, and in SC number related to type II myofibers. The enhanced and/or prolonged SC response to one bout of CON/ECC+ compared with CON/ECC might be because of the ongoing repair 1 wk after exercise. However, this mode of resistance exercise did not induce SC differentiation and formation of new myofibers as observed after electrically stimulated eccentric muscle contractions. With regard to the discussion of accentuated eccentric loading as a superior method in regular strength training to enhance strength and power performance, it seems interesting to further investigate in the role of SC concerning muscular adaptation to such resistance exercise.
The authors have no conflict of interests to report. The results of the present study do not constitute endorsement by the American College of Sports Medicine and are presented clearly, honestly, and without fabrication, falsification, or inappropriate data manipulation.
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