Figure 1.
Overview of the three experimental protocols.
Intermittent stimulation at 30 Hz for 1 s every 2 s. A) 20 s exercise (stimulation) followed by 2.5 min rest. B) 100 s exercise followed by 15 min rest. C) 15 min exercise (1st bout) followed by 15 min rest before initiating another 15 min exercise (2nd bout). In all protocols, muscles were harvested (arrows) at start and at end of exercise, as well as after rest. In C, there was an additional harvesting point at 100 s both in the 1st and 2nd bout.
Table 1.
Definitions of calculated contractile parameters.
Figure 2.
Representative tracings of shortening and force in the 15 min exercise protocol (1st bout).
Tracings of shortening (upper panel) and force (lower panel). A) The in vivo prepared muscle was stimulated intermittently with 1s on, 1s off. As stimulation starts, force rises from resting base line tension (Tbl) until it reaches the pre-set afterload (33% of Fmax). From here, the force is maintained while the muscle starts to shorten and reaches the maximal shortening (Smax). When stimulation ceases (after 1 s) the muscle is passively stretched to resting length (L0), and subsequently force declines. B) Representative tracing of a 15 min exercise protocol (1st bout). Note the three phases marked with roman numbers; during the first 20 s exercise there is a prominent fall in Smax (upper panel); at 100 s there is a further reduction in Smax accompanied by a transient rise in Tbl (lower panel); at 15 min there is still reduction in Smax while Tbl is restored to initial values.
Figure 3.
Characteristic fatigue development during the first 100 s of exercise.
Representative tracings of shortening (upper panel) and force (lower panel) assembled from the first 100 s. Tracings at start, 20 s and 100 s exercise are highlighted (black). Note the reduced shortening already at 20 s exercise. At 100 s exercise, there is a further reduction in shortening, and there is also a slowing of the remaining phases of the contraction cycle and especially a significant prolongation of the isometric relaxation phase (lower panel).
Figure 4.
Time course of fatigue and recovery during 20 s and 100 s exercise.
The panels show development of contraction (panels A, B and C) and relaxation (panels D, E and F) parameters during fatigue development (black symbols, solid line) and recovery (white symbols, dotted line). Exercise times are given at bottom x-axis and recovery times at top x-axis. A) Maximal rate of isometric force development, dF/dt. B) Maximal isotonic shortening velocity, dL/dt. C) Maximal shortening, Smax. D) Maximal isotonic relengthening velocity, −dL/dt. E) Maximal isometric relaxation rate, −dF/dt. F) Time to resting length, TTL0 (squares) and tau2 (circles). Symbols are averages ± SEM. * p<0.05 vs. initial value. † p<0.05 vs. 20 s. N start24; 20 s = 12; 100 s = 12; 20 s +2.5 min recovery = 6; 100 s +15 min recovery = 6.
Figure 5.
Expanded view of representative tracings at selected time points in the 1st and 2nd bout.
Shortening (upper panels) and force (lower panels) from a representative tracing at different time points in the 1st (black) and 2nd (grey) exercise bout. A) The initial exercise cycle. B) At 100 s of exercise. C) At 15 min exercise.
Figure 6.
Contractile performance in the 1st bout (open bars) vs. the 2nd bout (black bars).
Contraction (panels A, B and C) and relaxation (panels D, E and F) parameters at start (0 s), at 100 s and at 900 s of shortening contractions. A) Maximal rate of isometric force development, dF/dt. B) Maximal isotonic shortening velocity, dL/dt. C) Maximal shortening, Smax. D) Maximal isotonic relengthening velocity, −dL/dt. E) Maximal isometric relaxation rate, −dF/dt. F) Tau2 values. Bars are averages ± SEM. * p<0.05 vs. initial value. # p<0.05 vs. corresponding 1st bout value. † p<0.05 vs. 100 s. N start 1st = 20; 100 s 1st = 12; 15 min 1st = 6; start 2nd = 12; 100 s 2nd = 20; 15 min 2nd = 6.
Table 2.
Metabolites in soleus muscle at rest (Ctr) and at different exercise and recovery times.
Figure 7.
Isometric relaxation and lactate.
Isometric relaxation rate (−dF/dt) was strongly correlated to muscle lactate throughout the exercise protocols. Data are obtained from all measured time points in the 1st bout (black), after recovery (grey) and in the 2nd bout (white), presented as a linear regression (r2 = 0.81, p<0,01) based on group means ± SEM.
Figure 8.
A) Immunoblot of the extensor digitorum longus (EDL) and soleus (SOL) muscle probed with monoclonal MLC-2 antibody. B) Gel showing myofibrillar proteins from SOL stained with ProQ Diamond for phosphorylated proteins. C) The same gel as in B stained with Sypro Ruby for total proteins. The phosphorylation level of MLC-2s was normalized to protein content of MLC-2s in individual muscles by dividing the staining intensity reflecting phosphoryration level of the MLC-2s (ProQ Diamond) by the staining intensity of the MLC-2s protein band (Sypro Ruby). This normalized phosphorylation level was then calculated relative to the resting, contralateral control muscle. D) Immunoblot of SOL probed with MLC-2 pSer18 confirmed the same phosphorylation pattern as seen with ProQ Diamond gel stain. E) MLC-2s phosphorylation in exercised SOL after different exercise durations (black bars) and after respective recovery periods (grey bars) in the 1st bout, relative to the resting control muscle (100%, white bar). Bars are averages ± SEM. F) MLC-2s phosphorylation plotted against maximal shortening at all measured time points in the 1st bout (black), after recovery (grey) and in the 2nd bout (white). Symbols are group means ± SEM. *p<0.05 vs. control. N, 20 s = 6; 20 s+ recovery = 7; 100 s = 15; 100 s+recovery = 6; 15 min = 8; 15 min+recovery = 6. 100 s 2nd bout = 6; 15 min 2nd bout = 7.
Figure 9.
Effects of afterload and stimulation frequency on dephosphorylation of MLC-2s.
Panel A and B: The effects of altered afterload of the exercising muscle. Stimulation frequency was 30 Hz as in the standard protocols. A) Shortening (Smax) at start was strongly correlated to the pre-set afterload (r2 = 0.99). B) The phosphorylation level of MLC-2s (white bars) relative to resting control (100%) and the corresponding Smax (black bars) at 15 min exercise. Afterload was set to 10, 20 or 33% of Fmax. Panel C and D: The effects of altered muscle stimulation frequency. Afterload was 33% of Fmax as in the standard protocols. C) Smax (black circles) and force development (grey squares) in the unfatigued muscle at various stimulation frequencies. D) The phosphorylation level of MLC-2s (white bars) and Smax (black bars) at 15 min exercise with stimulation frequency 40, 30 or 20 Hz. Symbols are averages ± SEM. *p<0.05 vs. 33% afterload. †p<0.05 vs. 30 Hz. N 10% afterload = 6; 20% afterload = 5; 33% afterload = 8; 20 Hz = 6; 40 Hz = 6.
Figure 10.
A) SR calcium leak after 20 s and 100 s exercise and after recovery from 20 s and 100 s exercise (2.5 min and 15 min recovery, respectively). B) Individual SR calcium leak values from start, 20 s and 100 s time points plotted against corresponding tau2 values (r2 = 0.55, p<0.01). Bars are means ± SEM (N Ctr = 8; 20 s = 4; 20+ rec = 4; 100 s = 6; 100 s+rec = 4).*p<0.05 vs. control.