| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
MUSCLE CELL BIOLOGY AND CELL MOTILITY
Department of Zoology, La Trobe University, Bundoora Campus, Melbourne, Victoria, Australia
Submitted 20 September 2005 ; accepted in final form 17 November 2005
 |
ABSTRACT |
|---|
 TOP ABSTRACT METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
|---|
skeletal muscle; low-frequency fatigue; ryanodine receptor; skinned fiber
20 µM [Ca2+]i irreversibly reduced Ca2+ release and force production by interrupting the coupling between the T-system and the SR Ca2+ release channels. A comparable Ca2+-mediated reduction in Ca2+ release has also been demonstrated in intact fibers with repeated tetanic contractions, and a correlation with the total time during which [Ca2+]i was above resting levels was shown (3, 4). Unclear, however, is which aspect of the repeated [Ca2+] transients causes the disruption of the E-C coupling, in particular whether it depends primarily on peak [Ca2+] reached locally at particular sites or on the temporal summation of the increased [Ca2+] in the cytoplasm as a whole. Such information is important for the identification of the biochemical mechanism responsible for disrupting E-C coupling. In the present study, in which we used mechanically skinned mammalian skeletal muscle fibers in which the normal E-C coupling mechanism remained fully functional (13, 15), we were able to manipulate [Ca2+]i in various ways and to determine whether it affected E-C coupling. In this preparation, we were able to make direct comparisons between the effect of a uniform rise in [Ca2+] throughout the fiber and the effect of triggering normal Ca2+ release, which raises the [Ca2+] proportionately more near the release channels than in the cytoplasm as a whole. We examined the effect of prolonged exposure to relatively low levels of [Ca2+] and showed that when the [Ca2+] is maintained for 1–3 min at a constant level in the physiological range throughout the cytoplasm, it caused a partial disruption of depolarization-induced Ca2+ release, regardless of whether the release was elicited by direct depolarization of the T-system by solution change or by AP stimulation (21). Because the [Ca2+] applied to the fiber was precisely known, this allowed comparison of the [Ca2+]- and time-dependent characteristics of E-C uncoupling with those of biochemical reactions that putatively might underlie the process. We further showed that Ca2+ released by tetanic stimulation also causes uncoupling if the release is potentiated by the presence of caffeine and that this effect is stopped by buffering the released Ca2+ with BAPTA. Elevating [Ca2+]i via the endogenous release system caused greater disruption of E-C coupling than could be explained on the basis of the accompanying rise in bulk [Ca2+]. Thus these experiments have demonstrated that it is the local peak Ca2+ release during tetanus, not the time integral of the bulk [Ca2+] rise, that is the primary factor determining the extent of uncoupling. Our results suggest that during prolonged vigorous activity, the many repeated episodes of relatively high triadic [Ca2+] may disrupt E-C coupling and lead to long-lasting fatigue. The likely biochemical basis of this uncoupling mechanism and its possible role as a protective mechanism in muscle are discussed below.
|
METHODS |
|---|
|
TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
|---|
Male Long-Evans hooded rats were killed with an overdose of inhaled halothane (Fluothane; 2% vol/vol). All procedures were performed with the approval of the La Trobe University Animal Ethics Committee. The extensor digitorum longus (EDL) muscle was removed, pinned at resting length under paraffin oil, and kept on ice (15). As previously described (21), muscles were mechanically skinned, mounted between forceps, and connected to a force transducer (model AME801; SensoNor, Horten, Norway) with a resonance frequency >2 kHz, stretched to 1.2 times resting length, and transferred to a bath containing 2 ml of the appropriate K+-based solution according to the type of experiment involved (see below). Force responses were amplified and recorded on a chart recorder and/or on a computer equipped with PowerLab hardware (400 series; ADInstruments, Sydney, Australia) and Chart software (version 5.2; ADInstruments). All experiments were performed at room temperature (24 ± 2°C).
Solutions
All chemicals were obtained from Sigma (St. Louis, MO) unless stated otherwise. All solutions had pH 7.10 ± 0.01 and osmolality 295 ± 5 mosmol/kgH2O, and free Mg2+ concentration ([Mg2+]) was 1 mM, except where specified otherwise. Free [Ca2+] at 
0.1 µM was verified using a Ca2+-sensitive electrode (Orion, Cambridge, MA).
Solutions used for depolarization-induced force responses. The K+-HDTA solution used to polarize the T-system contained (in mM) 126 K+, 37 Na+, 50 hexamethylethylene-diamine-tetraacetic acid (HDTA2–; Fluka, Buchs, Switzerland), 8 total ATP, 8.6 total Mg2+, 10 creatine phosphate (CrP), 0.05 total EGTA, and 90 HEPES, pCa 6.9 (0.12 µM free [Ca2+]). When depolarizing the T-system by Na+/K+ substitution, a Na+-HDTA solution similar to the K+-HDTA solution was used, but with all of the K+ replaced by Na+.
Rigor solutions with set [Ca2+].
Solutions with zero ATP (rigor solutions) were used to set a constant [Ca2+] throughout the cytoplasm. The control rigor solution was similar to K+-HDTA solution, but with all ATP and CrP replaced isosmotically with additional HDTA (68 mM total), total Mg2+ reduced to 1.5 mM to keep free [Mg2+] at 1 mM, and 0.5 mM free EGTA (pCa 8). This solution was used for the control treatment as well as the wash solution after Ca2+ treatment. The Ca2+ rigor solutions were similar to the control rigor solution, but with the free [Ca2+] set at values in the range of 1–20 µM. The solutions contained a total of 0.1–0.25 mM EGTA, and the HDTA also acted as a weak but appreciable Ca2+ buffer (20).
Other solutions.
The load solution was the K+-HDTA solution with 1 mM total EGTA at pCa 6.7 (0.2 µM free [Ca2+]). The low-[Mg2+] solution used to directly activate the SR Ca2+-release channels was similar to the K+-HDTA solution, but with free [Mg2+] of only 15 µM (1.0 mM total Mg2+). The maximum activation solution was similar to the K+-HDTA solution, but with all HDTA replaced by 49.5 mM Ca2+-EGTA and 0.5 mM free EGTA to heavily buffer the free [Ca2+] at 20 µM (pCa 4.7). This maximum activation solution was also mixed at various proportions with a matching relaxing solution containing 50 mM free EGTA to obtain solutions with strongly buffered [Ca2+] between 0.1 and 20 µM, which were used to assess the Ca2+ sensitivity of the contractile proteins. In the solutions used for caffeine tetani, 5 mM caffeine and 0.2 mM free BAPTA (from a 50 mM BAPTA stock solution matching the K+-HDTA solution) were added to the K+-HDTA solution as required.
Experiments
In all experiments, each skinned fiber was initially equilibrated in K+-HDTA solution for 2 min before stimulation and treatment. The T-system was depolarized either by substituting all K+ in the solution with Na+ (Na+ depolarizations) or by APs evoked by electrical field stimulation. At the end of most experiments, the fiber was transferred to the low-[Mg2+] solution to demonstrate that the SR release channels were still functioning and that the SR was not depleted of Ca2+. In this solution, free [Mg2+] was only 15 µM instead of 1 mM and rapid lowering of [Mg2+]i directly activated the Ca2+ release channels (15). In freshly skinned fibers, when the SR was loaded at the endogenous Ca2+ content level, exposure to this low-[Mg2+] solution induced a force response reaching 74 ± 1% (n = 4) of maximum Ca2+-activated force (defined below), taking 6.6 ± 0.9 s from the time of solution application until 80% of peak force was reached. When the SR was loaded at greater levels, the force rose faster and higher, with experiments with added Ca2+ (0–0.5 mM) showing that the maximum force level obtainable in the low-[Mg2+] solution was only 85 ± 1% (n = 4) of the maximum Ca2+-activated force measured under standard conditions (1 mM free Mg2+ and 7 mM MgATP) by applying the maximum activation solution (pCa 4.7).
Na+ depolarizations.
When required, a skinned fiber was depolarized by substituting the Na+-HDTA solution for 2–3 s, which typically resulted in Ca2+ release and a substantial force response. This procedure was repeated every 1 min until the peak force response reached a stable level (typically 80–100% of maximum Ca2+-activated force), with the fiber repolarized for 1 min in K+-HDTA solution between Na+ depolarizations. Usually three to five Na+ depolarizations were required for the force response to become stable (<5% different from previous response). After exposure to elevated [Ca2+], the Na+ depolarizations were repeated (for an example of a protocol, see Fig. 1) and the peak force was compared with that before Ca2+ exposure. Only fibers producing a pretreatment force response of >80% maximum Ca2+-activated force were used in the analysis.
|
Elevating [Ca2+]i by exposure to solution with set [Ca2+]. After the pretreatment depolarizations, the fiber was transferred to the control rigor solution (pCa 8) for 1 min to remove all ATP from the fiber, which induced a state of rigor in the fiber. The removal of all ATP prevented any uptake of Ca2+ by the SR when [Ca2+] was subsequently raised and prevented any Ca2+ release at a rate greater than the Ca2+ buffering could counteract effectively (19). This allowed the fiber to be exposed to a set level of [Ca2+] that was homogeneous throughout the fiber. After rigor was induced, the fiber was exposed to elevated [Ca2+] by moving the fiber to a Ca2+ rigor solution for 3 min (or other time as specified) with [Ca2+] in the range of 1–20 µM. The Ca2+ was then washed out by a 30-s exposure to control rigor solution before being returned to the K+-HDTA solution. In control experiments, the fibers were left in control rigor solution for an equivalent time.
Elevating [Ca2+]i with Ca2+ released via the endogenous system. In these experiments, three to five 50-Hz tetani were elicited in the presence of 5 mM caffeine with or without 0.2 mM free BAPTA. The skinned fiber was transferred to the K+-HDTA solution containing 5 mM caffeine (with or without BAPTA) at least 10 s before the first tetanus and remained in this solution until the last caffeine-tetanus. The force responses to twitch, 5-, 10-, and 50-Hz stimulation or just to 50-Hz stimulation were examined in normal K+-HDTA solution before and after application of caffeine tetani. Control fibers were stimulated with the same total number of tetani without caffeine or BAPTA present.
Ca2+ sensitivity of contractile apparatus after exposure to elevated [Ca2+].
In a separate set of experiments, the force-[Ca2+] relationship of fibers was examined before and after exposure to elevated [Ca2+]. The contractile apparatus was directly activated by transferring the skinned fiber to strongly buffered Ca2+-EGTA solutions, increasing [Ca2+] stepwise in the range from 0.3 to 5 or 20 µM (pCa 6.5–5.3 or 4.7), exposing the fiber to each solution for the shortest possible time but just long enough to reach a plateau in force (usually <5 s). In each fiber, the [Ca2+] staircase was repeated at least twice before Ca2+ rigor treatment was begun and twice afterward. During rigor treatment, [Ca2+] was elevated to 8 µM for 3 min or to 100 µM as indicated. In each fiber, the force at each pCa in a given staircase was expressed as a percentage of the respective maximum Ca2+-activated force and Hill curve fit to the force-pCa data. The maximum Ca2+-activated force and the pCa50 (i.e., pCa at half-maximum) of each staircase were compared before and after treatment in each fiber.
|
RESULTS |
|---|
|
TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
|---|
Exposing a skinned EDL fiber for 3 min to a set [Ca2+]i level between 1 and 20 µM caused an irreversible decrease in the force response to depolarization (Fig. 1 and 2). The desired [Ca2+] was applied homogeneously throughout the cytoplasm by applying buffered solutions and preventing any Ca2+ uptake or appreciable rate of Ca2+ efflux by removal of all ATP (see METHODS). The control treatment, which involved exposing a skinned fiber to zero ATP conditions for the same length of time while keeping the [Ca2+] low (pCa 8), caused only a small decline in the force response (e.g., Fig. 1) (postcontrol rigor response 88.6 ± 4.2% of pretreatment values, n = 12; P < 0.05). The effect of elevated [Ca2+] was clearly concentration dependent as shown in Fig. 2, which presents the data for fibers that underwent only a single treatment (either the control treatment or a raised [Ca2+] treatment). A second treatment of the same fiber produced comparable results (see, e.g., Fig. 1). The depolarization-induced force response was abolished completely after 3 min at 20 µM [Ca2+] in every fiber examined. The pCa50 (i.e., pCa at 50% force) of the best fit Hill curve indicated that a 50% reduction in the depolarization-induced force response occurred with 3-min exposure to 5.4 µM [Ca2+]. The reduction in depolarization-induced force was evidently not due to depletion of SR Ca2+. A 15- to 20-s loading period was used for each fiber to ensure full restoration of any Ca2+ lost from the SR during the procedure, and we directly verified in every case that the SR was loaded at or above the normal endogenous level (10) by subsequently exposing each fiber to a solution with low free [Mg2+]. This procedure invariably induced the release of a large amount of Ca2+ from the SR, with the force rising rapidly (typically <2 s) (see, e.g., Fig. 1) to close to the maximum possible in the low-[Mg2+] solution (see METHODS). The response to the low-[Mg2+] solution also demonstrated that the SR Ca2+ release channels still functioned when directly stimulated. Within the time scale of these experiments, the reduction in depolarization-induced force responses after Ca2+ treatment was irreversible. The response did not show any appreciable recovery from the level attained after the initial load period when examined during an additional 10–15 min and, in two cases, during an additional 30-min period. Applying a second 15-s Ca2+ load also had no significant effect on the size of the depolarization-induced response. In the three fibers examined in which the 5 or 8 µM [Ca2+] exposure reduced the response from 44% to 66% of the pretreatment level, the response after the second load differed by only 3 ± 2%, and in two fibers in which 20 µM [Ca2+] abolished the response completely, there was still no response after such additional loading.
|
100 µM [Ca2+] for 3 min also had no significant effect on either maximum Ca2+-activated force or Ca2+ sensitivity (n = 8; data not shown).
|
Electrical field stimulation in the skinned fibers resulted in AP-induced depolarization of the T-system and consequent twitch and tetanic force responses (e.g., Fig. 4). Because the normal cytoplasmic Ca2+ buffer, parvalbumin, had been washed from the skinned fibers, the force response of the twitch was relatively large compared with that typically observed in intact fibers (46.6 ± 2.3% of tetanic force; n = 16) and the force response fused at a relatively low frequency. Maximal tetanic force was reached at a stimulation frequency of 50 Hz, and the force response at 100 Hz was not significantly different (96.6 ± 1.3% of 50-Hz response; n = 5). As was true with regard to the Na+ depolarization-induced force responses, the force responses to AP stimulation were irreversibly decreased after exposure to elevated [Ca2+]i (Figs. 4 and 5). The 50-Hz tetanic force response was reduced by 1-min exposure to 2 µM [Ca2+] to 76.4 ± 6.2% (n = 7; P < 0.005) of its pretreatment level, and with 5 and 8 µM [Ca2+] to 52.5 ± 18.5% (n = 4; P < 0.001) and 39.0 ± 16.0% (n = 5; P < 0.001), respectively (each fiber treated only once). The force response at 100 Hz was decreased similarly (posttreatment 100-Hz force response 93.0 ± 9.5% of corresponding response at 50 Hz, n = 5; P > 0.2). The reduction in the force response was not due to depletion of SR Ca2+; each fiber underwent a 5-s period of loading after the rigor period (enough to load an additional 25–35% of normal endogenous SR Ca2+ content; Refs. 9, 10) before the response to AP stimulation was ascertained, and there was no significant increase in the response when fibers underwent an additional 5-s Ca2+ load (change in 50-Hz tetanic response after second load, –2.0 ± 2.9%, n = 4; P > 0.5). The control rigor treatment (pCa 8) for the same total time (2.5 min) had no significant effect on the AP-induced force responses (n = 4; P > 0.5) (Fig. 5B).
|
|
The rate of force development during 50-Hz tetanic stimulation, expressed as the average rate of rise of tetanic force to the same absolute force level (90% of post-Ca2+ rigor peak force response), slowed after exposure to elevated [Ca2+] (see, e.g., Fig. 4E and averaged data in Fig. 5C). This finding is further evidence of reduced Ca2+ release in response to AP stimulation. The rigor period in itself had no negative effect on the rate of force development (n = 4; P > 0.5).
The reduction in force responses after the exposure to elevated [Ca2+]i is not due to damage to the T-system, which remains tightly sealed after exposure to high [Ca2+] (11, 16) and does not seem to be due to AP failure in the T-system. The latter possibility was investigated by applying pairs of electrical pulses with the interpulse interval between the first and second pulses ranging from 3 to 20 ms. These pulse pairs were applied before and after exposure to elevated [Ca2+] sufficient to substantially reduce, but not to abolish completely, the twitch response to AP stimulation (1 min at 5 or 8 µM [Ca2+]). This procedure tests the time required for Na+ channels in the T-system to recover from the first AP and generate a second propagated AP, which leads to a further brief but large release of Ca2+ from the SR and a substantial increase in the peak size of the resulting force response. The two APs occurred so closely in time that only a single, larger, twitchlike force response was observed (see Fig. 3 in Ref. 19). As shown in Fig. 6, after Ca2+ treatment, the recovery period required to generate a second AP remained unchanged at a value in the range of 4–5 ms. This finding contrasts greatly with what was observed previously when reductions in AP-induced responses were caused by chronic partial depolarization of the T-system and consequent AP failure (18), in which the repriming time required for the second of the pulses to lead to an appreciably greater force response increased to 20 ms or more.
|
Finally and importantly, we investigated whether excessive Ca2+ release from the SR could also lead to the disruption of E-C coupling. When 12 successive tetani were elicited in a skinned fiber in the standard K+-HDTA solution (200-ms periods of 50-Hz stimulation 1 min apart), there was only a slight reduction in peak force during the sequence of stimuli (–3.6 ± 1.2%, n = 4; P < 0.05). When 5 mM caffeine was present in the solution, the tetanic force was initially increased slightly and substantially prolonged (e.g., Fig. 7, A and E), and after raising [Ca2+]i in this manner for only three to five tetani, peak tetanic force measured under the original control conditions was substantially reduced relative to its pretreatment level (P < 0.05) (Figs. 7 and 8). This reduction was not due to depletion of the SR Ca2+ stores, because a 5-s load did not increase tetanic force (Figs. 7A and 8A), and direct stimulation of the SR by lowering [Mg2+]i at the end of the experiment showed that the SR was loaded at or above endogenous levels (data not shown). Similar effects were observed in all 10 fibers in which [Ca2+]i was raised in this way, with peak tetanic force after the caffeine-tetani being, on average, 79.4 ± 4.4% of pretreatment values (P < 0.01). Figure 8A shows mean data for the four fibers in which caffeine-tetani were applied and the stimulation protocol maintained for a total of 12 or 13 tetani, as conducted with matching control fibers shown in the figure. After caffeine-tetanic treatment, twitch force was significantly more depressed than tetanic force, being reduced to 62.0 ± 6.1% of the pretreatment level (n = 10; P < 0.005). Force production was also depressed at the other two intermediate stimulation frequencies (all 4 frequencies tested in 4 fibers; see mean data shown in Fig. 8B).
|
|
7.5 with 0.2 mM BAPTA added). Importantly, when each of these fibers was returned to the standard solution and briefly reloaded (5 s), peak tetanic force recovered fully to pretreatment level (n = 4; P > 0.5) (Fig. 8A), demonstrating that the treatment had not caused any detectable disruption of E-C coupling. |
DISCUSSION |
|---|
|
TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
|---|
The finding reported herein that elevated [Ca2+]i reduced the force response to AP stimulation in rat EDL fibers across the full force-frequency range, with proportionately greatest effect occurring at low frequencies (see Fig. 5, A and B, and Fig. 8B), is similar to that found previously when intact fibers of mouse fast-twitch muscle were subjected to repeated tetani with caffeine present to raise [Ca2+]i (3). This strongly suggests that the same uncoupling mechanism underlies both sets of findings. In the intact fiber studies of Chin and colleagues (3, 4), which were performed at room temperature as were the experiments described herein, the disruption in E-C coupling remained unchanged for 1 h and was clearly distinct from a faster-recovering component of fatigue that was evidently dependent on the metabolic status of the fiber (4). The long-duration deficit in force production observed with prolonged or vigorous stimulation in both human and nonhuman muscle is sometimes called low-frequency fatigue because it is more prominent at low stimulation frequencies, although perhaps it is better described as long-duration fatigue because the persistent nature of the deficit is probably the most important feature, one that shows that it is distinct from metabolic fatigue (1, 6, 7) and is likely caused by structural and/or protein changes in the muscle fiber. The fact that the force response is reduced proportionately more at low-stimulation frequencies than at high-stimulation frequencies most likely simply reflects the sigmoid relationship between [Ca2+]i and force (e.g., Fig. 3), as a given reduction in [Ca2+]i would cause a bigger drop in force when the [Ca2+]i initially produces submaximal force rather than near-maximal force. Thus it is not the absolute frequency of stimulation but rather the relative level of force produced by the stimulus that is important in this regard. In the present experiments, in which parvalbumin was washed from the fiber after skinning, there was less total Ca2+ buffering in the cytoplasm when Ca2+ was released in response to AP stimulation, and consequently the twitch force was a relatively large proportion of peak tetanic force and tetani also became fused and produced maximum force at lower-stimulation frequencies than observed in intact fibers. When this effect is taken into account, the force-frequency deficit observed in the skinned fibers after exposure to elevated [Ca2+] is highly comparable to that observed in intact fibers (3, 4).
Key Determinants and Site of Ca2+-Dependent Uncoupling
The experiments of Chin and colleagues (3, 4) strongly suggested that long-duration fatigue results from increases in [Ca2+]i, but inherent in the use of intact fibers is that those experiments did not identify clearly whether the phenomenon was dependent primarily on peak [Ca2+] reached during tetanus, on the associated rise in resting [Ca2+], or on the temporal sum or any other aspect related to the [Ca2+] changes. It also is not clear in their studies whether the effect was dependent on [Ca2+] in the cytoplasm as a whole or on [Ca2+] in a particular region. Furthermore, a large disparity in the measured values of peak tetanic [Ca2+] in intact, fast-twitch murine fibers has been reported by different investigators [1–2 µM reported using indo-1, Ref. 3; 20 µM reported using furaptra, also known as mag-fura-2; Ref. 2]. In the present study using skinned fibers in which it is possible to apply a known [Ca2+]i throughout the fiber, we were able to determine accurately the Ca2+ and time dependence of the uncoupling effect, allowing quantitative comparison with the properties of biochemical processes that putatively might underlie this uncoupling. Also, we were able to demonstrate that it is the peak local [Ca2+] that is of primary importance.
First, it is apparent that E-C uncoupling does not depend simply on the total time integral of the [Ca2+]i but instead proceeds disproportionately faster at higher [Ca2+]i. When examining coupling with Na+ depolarizations in rat EDL fibers, 180-s exposure to 5.4 µM [Ca2+]i is required to cause 50% uncoupling (Fig. 2) (time integral, 970 µM·s), whereas at 23 µM [Ca2+]i, only 10-s exposure is needed (11) (time integral, 230 µM·s). When [Ca2+]i was kept at 1 or 2 µM for 180 s, little if any reduction in the force response to Na+ depolarization was observed, even though the [Ca2+]i time integral (180 and 360 µM·s, respectively) (Fig. 2) was similar to or exceeded the value associated with 50% uncoupling at 23 µM [Ca2+]i.
Second, the experiments described herein (Fig. 5B) have shown that when applying Ca2+ homogeneously through the intracellular space, the [Ca2+] must be maintained continuously at 2 µM for 60 s to cause a decrease in the AP-induced force responses comparable to those observed in intact murine fibers when subjecting them to repeated tetani in the presence of 10 mM caffeine (3). In those intact fiber experiments, [Ca2+] repeatedly rose to high levels locally and then declined, and the associated [Ca2+] time integral on the basis of the Ca2+ signal measured over the whole fiber was estimated to be 25 µM·s. In the present study, the peak tetanic force was reduced by only 20% after [Ca2+] was kept constant for 60 s at a level sufficient to produce near-maximal force (i.e., 2 µM) (see Fig. 3), and the [Ca2+] time integral in this case was 120 µM·s. This shows that [Ca2+]i must be elevated to relatively high levels far above the resting level for substantial uncoupling to occur in a matter of minutes. Furthermore, we found that a similar degree of uncoupling (20% reduction in peak tetanic force) occurred in the skinned fibers when [Ca2+] was only briefly elevated by applying just three to five tetani in the presence of caffeine (see Fig. 8 and RESULTS). The uncoupling in the latter case was clearly the result of the high [Ca2+] during stimulation and was not due to the caffeine or AP stimulation per se, because it did not occur if 0.2 mM BAPTA was present to buffer the released Ca2+ (Fig. 8). In the skinned fibers, the cytoplasm is open to the buffered bathing solution and resting [Ca2+] would have remained at 0.12 µM for most of the period between tetani even in the continued presence of caffeine. Thus it is apparent that the uncoupling was caused predominantly by the very high [Ca2+] reached during the tetanic stimulation in caffeine, not by any rise in [Ca2+]i between stimuli, particularly given that raising the [Ca2+]i to a steady level of <1 µM for 60 s evidently has little effect on E-C coupling (Figs. 2 and 5).
It is further apparent that the extent of uncoupling observed in the caffeine tetani case must have been caused by extremely high [Ca2+], most likely that occurring close to the Ca2+ release channels. Because the force response in these skinned fibers is able to follow the [Ca2+] prevailing in the cytoplasmic space with a time delay of only 100–200 ms (5), the steady-state force [Ca2+]-dependence measured in these fibers (e.g., Fig. 3) can be used to calculate an upper limit for the total [Ca2+] time integral during the three to five tetanic force responses in caffeine (see, e.g., Fig. 7E), except for the part contributed by the 200- to 400-ms peak Ca2+ release during each tetanus, in which the force response certainly failed to track the rapid, large changes in free [Ca2+]. We found that for the fibers treated with caffeine-tetani, [Ca2+] was clearly <2 µM for the great majority of the time and that the total [Ca2+] time integral for the whole treatment, other than during the three to five peak release periods, was <25 µM·s. Thus the disparity between this case and the case in which a constant level of 2 µM [Ca2+] was applied ([Ca2+] time integral 120 µM·s) is difficult to explain unless the free [Ca2+] causing the uncoupling in the caffeine-tetani case reaches >>10 µM during the brief periods of peak Ca2+ release (total release period 1–1.5 s). When it is further noted that the presence of 200 µM free BAPTA, a fast Ca2+ buffer, throughout the fiber during release only reduced the tetanic force response in caffeine to close to the control tetanic level (see Fig. 7F), it is apparent that the rapid uncoupling in the caffeine-tetani case must have been driven by an extremely high local free [Ca2+], possibly on the order of 100 µM or more. The absence of parvalbumin in the skinned fibers could make them somewhat more susceptible than intact fibers to damage by peak Ca2+ release flux, although this effect is likely relatively small because most of the parvalbumin in vivo acts as a slow rather than a fast Ca2+ buffer as it already has Mg2+ bound. The fact that skinned fibers do have differences from intact fibers, however minor, means one can never be certain that the results are fully applicable to muscle in vivo. Nevertheless, the present experiments clearly demonstrate that it is peak local [Ca2+], not the temporal sum of [Ca2+] in the bulk cytoplasm, that is predominantly responsible for uncoupling.
In summary, it appears that any Ca2+-dependent uncoupling occurring in a working fiber is caused primarily by high local [Ca2+] reached in the vicinity of the triad junction, not by any rise in resting [Ca2+] near the triads or by peak [Ca2+] reached during stimulation elsewhere in the bulk of the cytoplasm, even though the latter may be sufficient to produce maximal force production by the contractile apparatus. Uncoupling appears to occur to a marked extent only if 1) [Ca2+] is elevated for a comparatively long continuous period (1–3 min) to a level producing near-maximal force (>2 µM Ca2+), which fast-twitch fibers do not normally experience, or 2) peak [Ca2+] near the triad junction is potentiated above the level it normally reaches. It also appears that the uncoupling effect is primarily a Ca2+- and time-dependent phenomenon and is not noticeably sensitized or dependent on activation of the coupling mechanism between voltage sensors and the Ca2+ release channels, because 1) high levels of applied Ca2+ cause uncoupling in unstimulated fibers and 2) the coupling mechanism can be activated 15 times or more by Na+ depolarizations, lasting 2–3 s each and releasing sufficient Ca2+ for near-maximal force without causing much Ca2+-dependent uncoupling (see, e.g., Refs. 14, 15). The considerable [Ca2+]-time integral that prevails in the latter case indicates that the uncoupling mechanism could not be sensitized appreciably by voltage sensor or release channel activation per se. Furthermore, the lack of appreciable uncoupling occurring with repeated Na+ depolarizations, in which the peak rate of Ca2+ release is much smaller than with AP stimulation, is fully consistent with uncoupling being caused primarily by very high triadic [Ca2+].
Possible Role and Basis of Uncoupling
On the basis of the present findings, one can speculate about the possible biochemical basis and role of Ca2+-dependent uncoupling. The properties of Ca2+-dependent uncoupling indicate that it involves interruption of communication between voltage sensors in the T-system and the Ca2+ release channels in the SR at the triad junction. We previously documented that elevated [Ca2+] appears to cause physical alterations of the triad junction (11). Such alterations could be caused by activation of the Ca2+-dependent proteases calpain-3 and µ-calpain (also known as calpain-1), which we recently showed (17) are present in rat EDL muscle and are activated to a small extent by 1-min exposure to 2 µM [Ca2+] and considerably more by higher [Ca2+], fully consistent with the present findings. Furthermore, calpain-3 and at least some of the µ-calpain are bound in mammalian fibers at or near the N2A line on titin (8, 22), which is at the A-I boundary close or adjacent to the location of the triad junction. The Ca2+ dependence of the uncoupling phenomenon appears to be well attuned to normal muscle function because it does not appear to be activated appreciably during normal Ca2+ release and because muscle fibers are not affected deleteriously by normal levels of activity (observed in control tetani shown in Fig. 8A), but only when the Ca2+ release is excessive or prolonged (3, 4). Thus the Ca2+-dependent uncoupling mechanism may have an important protective role in muscle, because this mechanism seems well designed to interrupt the coupling and reduce Ca2+ release at its source whenever the release exceeds the requirements for normal muscle function, so the uncoupling acts to prevent the widespread activation of Ca2+-dependent proteases and consequent generalized muscle damage that would otherwise follow.
|
GRANTS |
|---|
|
TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
|---|
|
ACKNOWLEDGMENTS |
|---|
|
FOOTNOTES |
|---|
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
|
REFERENCES |
|---|
|
TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
|---|
2. Baylor SM and Hollingworth S. Sarcoplasmic reticulum calcium release compared in slow-twitch and fast-twitch fibres of mouse muscle. J Physiol 551: 125–138, 2003.
3. Chin ER and Allen DG. The role of elevations in intracellular [Ca2+] in the development of low frequency fatigue in mouse single muscle fibres. J Physiol 491: 813–824, 1996.[ISI][Medline]
4. Chin ER, Balnave CD, and Allen DG. Role of intracellular calcium and metabolites in low-frequency fatigue of mouse skeletal muscle. Am J Physiol Cell Physiol 272: C550–C559, 1997.
5. Dutka TL and Lamb GD. Effect of low cytoplasmic [ATP] on excitation-contraction coupling in fast-twitch muscle fibres of the rat. J Physiol 560: 451–468, 2004.
6. Edwards RH, Hill DK, Jones DA, and Merton PA. Fatigue of long duration in human skeletal muscle after exercise. J Physiol 272: 769–778, 1977.
7. Jones DA. High- and low-frequency fatigue revised. Acta Physiol Scand 156: 265–270, 1996.[CrossRef][ISI][Medline]
8. Keira Y, Noguchi S, Minami N, Hayashi YK, and Nishino I. Localization of calpain 3 in human skeletal muscle and its alteration in limb-girdle muscular dystrophy 2A muscle. J Biochem (Tokyo) 133: 659–664, 2003.
9. Lamb GD and Cellini MA. High intracellular [Ca2+] alters sarcoplasmic reticulum function in skinned skeletal muscle fibres of the rat. J Physiol 3: 815–827, 1999.
10. Lamb GD, Cellini MA, and Stephenson DG. Different Ca2+ releasing action of caffeine and depolarisation in skeletal muscle fibres of the rat. J Physiol 531: 715–728, 2001.
11. Lamb GD, Junankar PR, and Stephenson DG. Raised intracellular [Ca2+] abolishes excitation-contraction coupling in skeletal muscle fibres of rat and toad. J Physiol 489: 349–362, 1995.[ISI][Medline]
12. Lamb GD and Posterino GS. Effects of oxidation and reduction on contractile function in skeletal muscle fibres of the rat. J Physiol 546: 149–163, 2003.
13. Lamb GD and Stephenson DG. Calcium release in skinned muscle fibres of the toad by transverse tubule depolarization or by direct stimulation. J Physiol 423: 495–517, 1990.
14. Lamb GD and Stephenson DG. Effects of FK506 and rapamycin on excitation-contraction coupling in skeletal muscle fibres of the rat. J Physiol 494: 569–576, 1996.[ISI][Medline]
15. Lamb GD and Stephenson DG. Effects of intracellular pH and [Mg2+] on excitation-contraction coupling in skeletal muscle fibres of the rat. J Physiol 478: 331–339, 1994.[ISI][Medline]
16. Launikonis BS, Barnes M, and Stephenson DG. Identification of the coupling between skeletal muscle store-operated Ca2+ entry and the inositol trisphosphate receptor. Proc Natl Acad Sci USA 100: 2941–2944, 2003.
17. Murphy RM, Snow RJ, and Lamb GD. µ-Calpain and calpain-3 are not autolyzed with exhaustive exercise in humans. Am J Physiol Cell Physiol 290: C116–C122, 2006.
18. Nielsen OB, Ortenblad N, Lamb GD, and Stephenson DG. Excitability of the T-tubular system in rat skeletal muscle: roles of K+ and Na+ gradients and Na+-K+ pump activity. J Physiol 557: 133–146, 2004.
19. Posterino GS, Cellini MA, and Lamb GD. Effects of oxidation and cytosolic redox conditions on excitation-contraction coupling in rat skeletal muscle. J Physiol 547: 807–823, 2003.
20. Posterino GS and Lamb GD. Effect of sarcoplasmic reticulum Ca2+ content on action potential-induced Ca2+ release in rat skeletal muscle fibres. J Physiol 551: 219–237, 2003.
21. Posterino GS, Lamb GD, and Stephenson DG. Twitch and tetanic force responses and longitudinal propagation of action potentials in skinned skeletal muscle fibres of the rat. J Physiol 527: 131–137, 2000.
22. Raynaud F, Fernandez E, Coulis G, Aubry L, Vignon X, Bleimling N, Gautel M, Benyamin Y, and Ouali A. Calpain 1-titin interactions concentrate calpain 1 in the Z-band edges and in the N2-line region within the skeletal myofibril. FEBS Lett 272: 2578–2590, 2005.
23. Stephenson DG, Lamb GD, and Stephenson GM. Events of the excitation-contraction-relaxation (E-C-R) cycle in fast- and slow-twitch mammalian muscle fibres relevant to muscle fatigue. Acta Physiol Scand 162: 229–245, 1998.[CrossRef][ISI][Medline]
24. Verburg E, Murphy RM, Stephenson DG, and Lamb GD. Disruption of excitation-contraction coupling and titin by endogenous Ca2+-activated proteases in toad muscle fibres. J Physiol 564: 775–789, 2005.
This article has been cited by other articles:
|
|
 |
|
  R. M. Murphy, T. L. Dutka, and G. D. Lamb Hydroxyl radical and glutathione interactions alter calcium sensitivity and maximum force of the contractile apparatus in rat skeletal muscle fibres J. Physiol., April 15, 2008; 586(8): 2203 - 2216. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. L. Dutka, R. M. Murphy, D. G. Stephenson, and G. D. Lamb Chloride conductance in the transverse tubular system of rat skeletal muscle fibres: importance in excitation-contraction coupling and fatigue J. Physiol., February 1, 2008; 586(3): 875 - 887. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. G. Allen, G. D. Lamb, and H. Westerblad Skeletal Muscle Fatigue: Cellular Mechanisms Physiol Rev, January 1, 2008; 88(1): 287 - 332. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. G. Allen, G. D. Lamb, and H. Westerblad Impaired calcium release during fatigue J Appl Physiol, January 1, 2008; 104(1): 296 - 305. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. L. Dutka and G. D. Lamb Na+-K+ pumps in the transverse tubular system of skeletal muscle fibers preferentially use ATP from glycolysis Am J Physiol Cell Physiol, September 1, 2007; 293(3): C967 - C977. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. L. Dutka and G. D. Lamb Transverse tubular system depolarization reduces tetanic force in rat skeletal muscle fibers by impairing action potential repriming Am J Physiol Cell Physiol, June 1, 2007; 292(6): C2112 - C2121. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. M. Murphy, E. Verburg, and G. D. Lamb Ca2+ activation of diffusible and bound pools of {micro}-calpain in rat skeletal muscle J. Physiol., October 15, 2006; 576(2): 595 - 612. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. D. Lamb and D. G. Stephenson The Last Word: Point:Counterpoint authors respond to commentaries on "Lactic acid accumulation is an advantage/disadvantage during muscle activity" J Appl Physiol, July 1, 2006; 101(1): 369 - 369. [Full Text] [PDF]
|
|
| ||||||
) and after (
) exposure for 3 min to 8 µM free [Ca2+]. Hill plots were fitted to both before and after data. Data shown are from 1 fiber but are representative of all fibers. For all 8 fibers, the average pCa50 was 6.06 ± 0.03 before treatment and 6.06 ± 0.02 after treatment, which did not constitute a significant difference. P > 0.5, paired data.
) and 50-Hz tetanus (
), tetanic force (measured in control conditions) was subsequently reduced to