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Slowed relaxation in fatigued skeletal muscle fibers of Xenopus and Mouse. Contribution of [Ca2+]i and cross-bridges.
Westerblad H
,
Lännergren J
,
Allen DG
.
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Slowing of relaxation is an important characteristic of skeletal muscle fatigue. The aim of the present study was to quantify the relative contribution of altered Ca2+ handling (calcium component) and factors downstream to Ca2+ (cross-bridge component) to the slowing of relaxation in fatigued fibers of Xenopus and mouse. Two types of Xenopus fibers were used: easily fatigued, type 1 fibers and fatigue resistant, type 2 fibers. In these Xenopus fibers the free myoplasmic [Ca2+] ([Ca2+]i) was measured with indo-1, and the relaxation of Ca2(+)-derived force, constructed from tetanic [Ca2+]i records and in vivo [Ca2+]i-force curves, was analyzed. An alternative method was used in both Xenopus and mouse fibers: fibers were rapidly shortened during the initial phase of relaxation, and the time to the peak of force redevelopment was measured. These two methods gave similar results and showed proportional slowing of the calcium and cross-bridge components of relaxation in both fatigued type 1 and type 2 Xenopus fibers, whereas only the cross-bridge component was slowed in fatigued mouse fibers. Ca2+ removal from the myoplasm during relaxation was markedly less effective in Xenopus fibers as compared to mouse fibers. Fatigued Xenopus fibers displayed a reduced rate of sarcoplasmic reticulum Ca2+ uptake and increased sarcoplasmic reticulum Ca2+ leak. Some fibers were stretched at various times during relaxation. The resistance to these stretches was increased during fatigue, especially in Xenopus fibers, which indicates that longitudinal movements during relaxation had become less pronounced and this might contribute to the increased cross-bridge component of relaxation in fatigue. In conclusion, slowing of relaxation in fatigued Xenopus fibers is caused by impaired Ca2+ handling and altered cross-bridge kinetics, whereas the slowing in mouse fibers is only due to altered cross-bridge kinetics.
Figure 6. Releases during relaxation in a Xenopus fiber (A and B) and in a mouse fiber (D and E). Records were obtained in control (A and D) and at the end of phase 2 (B and E). Force records with full lines were obtained with the release and records with dashed lines in a preceding tetanus without release. Upward arrows show time of releases; the amplitude of releases was 200 μm for the Xenopus fiber and 70 μm for the mouse fiber. Measurements were performed from the end of stimulation (dotted lines) to the time of peak force after the release (t1) and then to the time when the same relative force was produced in the preceding tetanus without release (t2). Note that in the mouse fiber the shortening step resulted in a reduction of the resting force, and measurements were therefore performed at somewhat different absolute force levels. Mean data of the time of real force relaxation (t1 + t2, •) and the time to peak force after the release (t1, ▾) obtained in Xenopus fibers (C; n = 6) and mouse fibers (F ; n = 4). The relaxation time of real force in control was in each experiment set to 100%.
Figure 1. Tetanic force and [Ca2+]i during fatiguing stimulation in type 1 and type 2 fibers. Mean values (±SEM) of tetanic force (A) and tetanic [Ca2+]i (B) during fatigue in type 1 fibers (○; n = 3) and type 2 fibers (•; n = 4). Data obtained in the first tetanus, the tenth tetanus, the 20th tetanus, at the end of phase 2, and at the end of fatiguing stimulation. [Ca2+]i signals were corrected for changes in pH (see materials and methods) at the end of phase 2 and at the end of fatiguing stimulation.
Figure 3. [Ca2+]i-force relations of a type 1 fiber in control and during fatigue. (A) Examples of tetanic [Ca2+]i and force records used to construct [Ca2+]i-force curves. Dashed lines indicate how measurements were performed. The “Control” tetanus was produced at a reduced stimulation frequency (15 Hz) before fatiguing stimulation, while the “Control (recovery)” tetanus was produced at 20 min after the end of fatiguing stimulation. Periods of stimulation are indicated below force records. Curves in B represent least square fits of Eq. 1 to data-points obtained in control (○, reduced stimulation frequency; ▿, during recovery after fatiguing stimulation) and during fatigue (•), respectively. Arrows indicate data-points from the tetani shown above.
Figure 4. Real and Ca2+-derived force of Xenopus fibers in control and at the end of phase 2. Records obtained from a type 1 fiber in control (A) and at the end of phase 2 (B). Records with higher noise level represent Ca2+-derived force. Dashed lines indicate end of tetanic stimulation. Measurements were performed at 70% of the tetanic force (horizontal lines); t1 represents the calcium component, and t2 represents the cross-bridge component of relaxation. C shows collected data (mean ± SEM; n = 7) of the relative time of real force relaxation (t1 + t2, •) and relaxation of the calcium component (t1, ▾). The relaxation time of real force in control was in each experiment set to 100%.
Figure 5. Analysis of the function of SR Ca2+ pumps in control and at the end of phase 2. (A) Average records from seven fibers of [Ca2+]i after tetanic stimulation in control and at the end of phase 2. Dashed curves obtained by a least square fit of the sum of two exponentials to the records. Tetanic stimulation ended at 0 s. B shows plots of the rate of [Ca2+]i decline (d([Ca2+]i)/dt) vs. [Ca2+]i in control and at the end of phase 2. Data-points for control (○) and end of phase 2 (•) were obtained from fitted curves in A. Curves in B were produced by fitting Eq. 2 to the data-points.
Figure 7. Releases during relaxation in a Xenopus fiber where the SR Ca2+ uptake was inhibited by tBuHQ. Force records with (full lines) and without (dashed lines) releases before (A) and after 10-min exposure to 500 nM tBuHQ (B). Dotted lines show end of tetanic stimulation; upward arrows indicate the time when a 250 μm release was performed. Measurements indicated by horizontal lines; observe that the time to peak force after the release (i.e., the calcium component) increased in tBuHQ, whereas the time from the peak to the same real force level (i.e., the cross-bridge component) remained constant. C shows mean data (n = 6) of the relative relaxation time of real force (•) and the calcium component (▾) from control and tBuHQ.
Figure 8. Ramp stretches at constant velocity performed during relaxation in a Xenopus fiber (A and B) and a mouse fiber (C and D). Dashed lines show force of tetani produced immediately before the tetani with stretches. Dotted lines mark the beginning and end of stretches; the amplitude of the stretch was 100 μm for the Xenopus fiber and 40 μm for the mouse fiber. Periods of stimulation are given below each force record. Observe that the stretch in the Xenopus fiber was produced later into relaxation at the end of phase 2 than in control. Note also the different time scales for Xenopus and mouse.
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