When muscle fibers are repeatedly stimulated, they may become depolarized and force output decline. Excitation of the transverse tubular system (T-system) is critical for activation, but its role in muscle fatigue is poorly understood. Here, mechanically skinned fibers from rat fast-twitch muscle were used, because the sarcolemma is absent but the T-system retains normal excitability and its properties can be studied in isolation. The T-system membrane was fully polarized by bathing the skinned fiber in an internal solution with 126 mM K+ (control solution) or set at partially depolarized levels (approximately −63 and −58 mV) in solutions with 66 or 55 mM K+, respectively, and action potentials (APs) were triggered in the sealed T-system by field stimulation. Prolonged depolarization of the T-system reduced tetanic force proportionately more than twitch force, with greater effect at higher stimulation frequency (responses at 20 and 100 Hz reduced to 71 and 62% in 66 mM K+ and to 54 and 35% in 55 mM K+, respectively). Double-pulse stimulation showed that depolarization increased the repriming period (estimated minimum time before a second AP can be produced) from ∼4 ms to ∼7.5 and 15 ms in the 66 and 55 mM K+ solutions, respectively. These results demonstrate that T-system depolarization reduces tetanic force by impairing AP repriming, rather than by preventing AP generation per se or by inactivating the T-system voltage sensors. The findings also explain why it is advantageous to reduce the rate of motoneuron stimulation to muscles during repeated or prolonged periods of activity.
- muscle fatigue
- excitation-contraction coupling
action potentials (APs) trigger contraction in vertebrate skeletal muscle by an intricate series of events known as excitation-contraction (EC) coupling (27). An early critical step in this sequence is the spread or propagation of APs along the sarcolemma and then down into a complex network of invaginations known as the transverse tubular system (T-system). The APs then activate the voltage sensor molecules (also known as dihydropyridine receptors), which in turn trigger Ca2+ release through the adjacent ryanodine receptor-Ca2+ release channels in the sarcoplasmic reticulum (SR). The consequent rise in myoplasmic Ca2+ concentration ([Ca2+]) leads to the generation of tetanic force by the contractile apparatus. Repeated activation of muscle, however, leads to a reduction in force output, commonly referred to as muscle fatigue. Many factors may cause muscle fatigue, with the relative contribution of the various factors depending on muscle fiber type and the level and duration of the activity (2, 20, 39). With repeated or high rates of AP stimulation, there can be a net loss of K+ ions from the myoplasm and concomitant accumulation of K+ ions in the interstitium (∼9 mM or higher) (28, 37), and muscle fibers can become depolarized to approximately −60 mV (3, 22, 26, 42). Depolarization to such levels for seconds to minutes might adversely affect EC coupling by both 1) interfering with the size and time course or propagation of APs over the sarcolemma and/or T-system (4, 10, 11, 34), owing to fast and slow inactivation of Na+ channels (19, 36) and other membrane permeability changes (38), and 2) inactivating some of the voltage sensors in the T-system and thereby reducing SR Ca2+ release (12, 27).
Although the T-system is often regarded as a likely site of EC coupling failure, at least for the case of continuous high-frequency stimulation (6, 18, 26, 43), the exact mechanisms involved have not been identified, largely because of the great difficulty in measuring, let alone controlling, the electrical events occurring in the T-system itself (1, 38). Here, we identify which specific events in the T-system are adversely affected by sustained depolarization by utilizing a mechanically skinned fiber preparation where sarcolemmal events are completely eliminated and the T-system membrane potential can be directly controlled by appropriate choice of bathing solution, and with the normal mechanism of AP-induced Ca2+ release retained and fully functional (25, 31, 33). In this preparation, the T-system seals off upon skinning (23) and can be polarized at different levels according to the [K+] and [Cl−] in the solution bathing the myoplasmic space (13, 24), and APs are elicited in the T-system by applying brief electric field stimulation.
We hypothesized that maintaining the T-system partially depolarized at approximately −60 mV would have comparatively little effect on the ability of single AP stimulation to activate the voltage sensors and trigger Ca2+ release, but would markedly slow the restoration of the membrane electrical properties following each AP, thus greatly interfering with the ability of the T-system to propagate closely spaced APs and hindering tetanic force production. We investigated this by setting the T-system potential in a skinned muscle fiber at various levels and monitoring the force responses to stimulation with single pulses or pairs or trains of closely spaced pulses. This indicated that the reduction in tetanic force occurring with prolonged T-system depolarization is indeed primarily caused by a reduction in the ability of the T-system to propagate closely spaced APs. This readily explains earlier findings on the relative extent of reduction in twitch and tetanic responses in fatigued muscle fibers in vitro (29, 40) and also importantly highlights one major reason why it is beneficial for the rate of neural stimulation to muscles in vivo to decline during periods of prolonged or repeated activity (7, 21).
With the approval of the La Trobe University Animal Ethics Committee, male Long-Evans hooded rats (∼6 mo old) were anesthetized with Fluothane (2% vol/vol) in a glass chamber and killed by asphyxiation. Both extensor digitorum longus (EDL) muscles were rapidly excised and immediately pinned at resting length under paraffin oil in a petri dish lined with Sylgard 184 (Dow Corning, Midland, MI) and kept cool (∼10°C) on an ice pack. Individual fibers were mechanically skinned by rolling back the sarcolemma by microdissection with fine forceps. A segment of the skinned fiber was then clamped at one end with fixed forceps and secured at the other end with fine silk thread to a force transducer (AME801, resonance frequency >2 kHz; SensoNor, Horten, Norway) and set at 120% of resting length. The mounted skinned fiber segment (∼2-mm long, diameter 30–50 μm) was then transferred to a small Perspex well containing 2 ml of the standard K+-hexamethylene-diamine-tetraacetate (K-HDTA) solution (see below) for 2 min to replace all of the in vivo diffusible myoplasmic constituents with the experimental bathing solution. All experiments were conducted at ∼24°C. Data are expressed as means ± SE, with the number of fibers studied denoted as “n.” Student's t-test (paired or unpaired as appropriate) was used to determine statistical significance (probability value, P < 0.05).
All chemicals were purchased from Sigma-Aldrich (St. Louis, MO) unless otherwise specified. The standard K-HDTA solution (control solution) contained the following (in mM): HDTA2− (Fluka, Buchs, Switzerland), 48.5; total ATP, 8; creatine phosphate (CrP), 10; Na+, 36; K+, 126; Cl−, 3; total Mg2+, 8.5; total EGTA, 0.075; HEPES, 90; pH 7.1 and pCa (−log10[Ca2+]) 6.9, except where stated. Similar solutions with total [K+] of 66 and 55 mM were made using NH4+ for equimolar replacement of K+ while keeping the other constituents (e.g., Na+, ATP, etc.) unchanged, except that the total [Cl−] was increased to 6 and 7 mM, respectively (with HDTA2− reduced by 1.5 and 2 mM to compensate for the additional 3 and 4 mM Cl−), so as to keep the [K]·[Cl] product approximately unchanged. Substitution of NH4+ for K+ has been shown previously (30, 31) to be an effective way of reducing [K+] in a solution with little direct effect on the contractile apparatus or SR properties. All solutions had an osmolality of 295 ± 5 mosmol/kgH2O and a free [Mg2+] of 1 mM based on apparent Mg2+ affinity constants of 6.9 × 103 M−1 for ATP, 8 M−1 for HDTA, and 15 M−1 for CrP (25). The pCa of solutions (for pCa <7.2) was measured with a Ca2+-sensitive electrode (Orion Research, Cambridge, MA). In some experiments, 5 mM anhydrous caffeine was dissolved directly into the test solution. This slight increase in osmolality would have had negligible effects on the properties of the contractile apparatus.
For examination of contractile apparatus properties, solutions similar to the standard K-HDTA solution were made in which all HDTA was replaced with EGTA or CaEGTA for very strong Ca2+ buffering. The maximum Ca2+-activating solution, “max,” contained 50 mM CaEGTA and had a pCa ∼4.7, and the “relaxing” solution contained 50 mM free EGTA and had a pCa >10, with total Mg2+ adjusted to maintain 1 mM free. These two solutions were mixed in the appropriate ratio to produce solutions with pCa in the range of 6.7–4.7, and then these were added in a 1:9 ratio to each of the 126 mM K+, 66 mM K+, and 55 mM K+ solutions to buffer the [Ca2+] at the desired level (with 5 mM total EGTA-CaEGTA) with only relatively minor changes to the other constituents.
T-system AP generation by transverse electric field stimulation.
The skinned fiber segment was positioned parallel to, and midway between, two platinum electrodes in a stimulating chamber containing 130 μl of the standard K-HDTA solution. An in-house stimulator was used to apply electric field pulses (duration, 1 ms; 75 V/cm) to generate APs in the sealed T-system synchronously along the whole length of the fiber segment (41). The electric field pulse strength was ∼2.5-fold greater than required to elicit maximum twitch force (i.e., supramaximal). Twitch and tetanic force responses were elicited with single-pulse and 20-, 25-, 50-, and 100-Hz stimulation, respectively. When the frequency was altered from 50 to 100 Hz, the number of pulses per burst was also altered so that the duration of the burst was maintained at 400 ms (i.e., 20 pulses at 50 Hz or 40 pulses at 100 Hz). When lower frequencies were used (i.e., 20 and 25 Hz), the number of pulses was kept the same (at 20) but the burst duration increased to ensure that peak tetanic force was always reached well before the end of the burst. Whenever a fiber was transferred from one condition to another, it was equilibrated for >20 s before stimulation.
To quantify the ability of the T-system to propagate two successive closely spaced APs, pairs of pulses (duration, 1 ms; 75 V/cm) were applied with the interpulse interval ranging between 1 and 50 ms. If the second pulse in a pair was applied too soon for the T-system to sustain another AP, the twitch response would not be much different than that for single-pulse stimulation (see Refs. 17, 32, 41). This would indicate that the T-system membrane was still refractory at that time. If, however, the second pulse did elicit an AP, then the twitch response, a sensitive indicator of the amount of Ca2+ released, should increase substantially in size. In this study, the “repriming period” was defined as the minimum time needed between two pulses (in nearest whole ms) for the twitch force to display >50% of the maximum incremental increase in force obtainable with two successive pulses.
Contractile apparatus experiments.
The direct effect of the depolarizing solutions (55 and 66 mM K+ solutions) on the contractile apparatus was examined by activating the contractile apparatus in the given conditions with the free [Ca2+] heavily buffered (5 mM total EGTA-CaEGTA) at various levels, as described previously (e.g., see Ref. 16). Briefly, the skinned fiber segment was first treated in Triton X-100 (1% vol/vol) in relaxing solution for 5 min to completely lyse all membranous compartments that might otherwise affect the free [Ca2+] within the fiber and then was washed (two 1-min washes in relaxing solution) to remove all Triton X-100. The fiber was then activated by exposure to sequences of solutions with progressively higher free [Ca2+] (pCa 9 to pCa 4.7), first under control conditions (i.e., standard K-HDTA solution), then in one of the test conditions (i.e., randomly either “55 mM K+ solution” or “66 mM K+ solution”), in control conditions again, in the other test condition, and finally in control conditions once again. Force increased in a staircase-like manner to each solution sequence, and each sequence was repeated twice 1 min apart, which always verified a high level of reproducibility. Force elicited at each pCa in a sequence was expressed as a percentage of the corresponding maximum Ca2+-activated force, plotted against pCa and fit with a Hill curve using GraphPad Prism 3 (GraphPad Software, San Diego, CA), to determine the pCa producing 50% of maximum Ca2+-activated force (pCa50) and the Hill coefficient. Values obtained on the two repetitions of a given condition were first averaged. The values so obtained for a given test condition (i.e., 55 or 66 mM K+ solution) were then expressed relative to the average of the bracketing control solution values to eliminate any changes associated with repeated activation or time.
Effect of 55 and 66 mM K+ solutions on contractile apparatus properties.
The direct effects of the 55 and 66 mM K+ solutions on the properties of the force-generating machinery (contractile apparatus) were examined first, because force was used as a measure of Ca2+ release, and hence any alterations in the properties of the contractile apparatus would affect the interpretation of the AP-mediated Ca2+ release experiments. The force-pCa relationship for each solution condition was determined by exposing skinned EDL fibers to sequences of solutions with the free [Ca2+] set at various levels (see methods), and each relationship was then fit with a Hill curve (e.g., Fig. 1). Mean values for pCa50 and Hill coefficients (h) in each test condition, and in control conditions before and afterward, are given in Table 1. In the four fibers examined this way, the maximum Ca2+-activated force was slightly reduced by ∼4 and 5% in the 66 and 55 mM K+ solutions, respectively (mean maximum 95.5 ± 0.5% and 94.8 ± 0.8% of control level, P < 0.05). The 66 mM K+ and 55 mM K+ solutions also caused a moderate reduction in the Ca2+ sensitivity of the contractile apparatus (mean change, ΔpCa50, was −0.07 ± 0.01 in both the 66 and 55 mM K+ solutions, P < 0.05). The Hill coefficient (a measure of the steepness in the relationship between [Ca2+] and force) was not significantly altered. Overall, the effects of the 66 mM K+ and 55 mM K+ solutions on the contractile apparatus were almost identical. These changes in contractile apparatus properties by themselves should result in only a moderate reduction in twitch force and a very small reduction in tetanic force if the amount of Ca2+ released per AP remains unchanged (see discussion). Thus any substantial reduction in twitch or tetanic force (e.g., >10%) occurring when fibers are in the 66 mM K+ or 55 mM K+ solutions would be indicative of reduced Ca2+ release.
Effect of chronic partial depolarization of T-system on twitch responses.
As described previously (15, 31, 33), when mechanically skinned fibers from rat EDL muscle are bathed in the control solution conditions (126 mM K+), the sealed T-system becomes polarized to approximately −80 mV, and electrical stimulation can be used to trigger APs in the T-system and consequent twitch and tetanic force responses (e.g., Fig. 2). The T-system of a skinned fiber can be chronically depolarized to various levels by appropriate reduction of the [K+] in the bathing solution (24, 31). The solutions used here had a constant [K]·[Cl] product, and the T-system potential is estimated to be approximately −63 mV in the 66 mM K+ solution and approximately −58 mV in the 55 mM K+ solution (see Ref. 31). When the T-system potential was maintained at these depolarized levels, twitch and tetanic force responses were reduced (e.g., Fig. 2A), with the relative effect on tetanic force being substantially greater. These effects were readily reversible by repolarizing the T-system by bathing the skinned fiber in the control (126 mM K+) solution again for 30 s or more. On average, when a fiber was bathed in the 66 mM K+ and 55 mM K+ solutions, the twitch force to a single AP stimulus (i.e., to a single applied pulse) was reduced to 77 and 65% of the control level, respectively, with the extent of the reduction differing considerably between different fibers (e.g., Fig. 2B).
Effect of chronic partial depolarization on AP repriming in the T-system.
Prolonged depolarization of muscle fibers to potentials of approximately −60 to −55 mV is known to cause “slow inactivation” of a large proportion of the Na+ channels and can result in complete AP failure (34, 36). If an AP is generated, the membrane becomes unable to support another AP shortly thereafter (refractory period), because too few of the available Na+ channels have recovered from fast inactivation to generate a sufficiently large Na+ current to overcome the leak currents through K+ and Cl− channels (19, 38). The specific ability of the T-system to support closely spaced successive APs, when polarized at particular levels, was examined by applying pairs of identical pulses with various times between the two pulses (“interpulse interval,” 1–50 ms) and recording the subsequent effect on peak twitch force. Twitch force is substantially increased if the second pulse in a pair is also able to elicit an AP in the T-system, because the additional Ca2+ release triggered by this second AP augments that released by the first AP, leading to a higher level of activation of the contractile apparatus (32, 41). The twitch response is a sensitive indicator of small changes in Ca2+ release because it produces submaximal force (typically ∼20–40% of maximum) (32, 33). If the T-system was well polarized in the 126 mM K+ control solution (approximately −80 mV or more negative), twitch size remained relatively unchanged when the interpulse intervals were 1–3 ms (Fig. 2). However, when the interpulse interval was ≥4 ms, there was a sudden jump in twitch size to a relatively constant level, indicating that, at ≥4 ms, a second AP propagated in the T-system and elicited a second release of Ca2+ from the SR (indicating a repriming period of ∼4 ms; see methods for definition). In contrast, when the T-system was chronically partially depolarized in the 66 mM K+ and 55 mM K+ solutions, there was a less vivid rise in twitch force, and the AP repriming period increased on average to ∼7.5 and ∼15 ms, respectively (Figs. 2 and 3 and Table 2). Thus, if the T-system is maintained partially depolarized, its ability to propagate a second closely spaced AP is greatly impaired.
Inspection of the data from the individual fibers shown in Fig. 2B indicates a number of things. First, with depolarization in the 55 mM K+ solution, there was a considerable range in the extent of impairment in different fibers, which is not surprising given that AP failure occurs with a steep voltage dependence with depolarization near −60 mV (34). Second, fibers in which the twitch response to a single AP was reduced to a greater degree (see values for “0”-ms interval in Fig. 2B) in general showed greater impairment of the response to the second stimulus in a pair, in particular displaying a greater increase in repriming time. This is further analyzed in Fig. 4, which plots the relative twitch size to a single AP stimulus vs. the observed repriming period required to elicit a second AP when individual fibers were depolarized in either the 66 mM K+ or 55 mM K+ solution. The repriming period was ∼4 ms in fully polarized fibers (not shown), whereas in the depolarized fibers, it ranged from 5 ms up to 20 ms or greater. Figure 4 shows that depolarization had a proportionately greater effect on the repriming time needed to elicit a second T-system AP than it had on the twitch response to a single AP stimulus, with repriming time increasing ∼5-fold when twitch size decreased ∼30%. Furthermore, a substantial part of the observed reduction in twitch force to a single AP was attributable purely to direct effects of the depolarizing solutions of the contractile apparatus (likely reducing the force response to ∼90% of control level) (see above and discussion). Thus it was apparent that T-system depolarization interfered to a greater extent with the generation and propagation of a second AP in a pair than it did with the first AP.
Effect of chronic partial depolarization on tetanic force responses elicited at different frequencies.
Given the above, it was important to examine what happened when multiple successive stimuli were applied, as occurs during normal tetanic stimulation. Tetanic force responses were elicited at various stimulation frequencies (20, 25, 50, or 100 Hz, in pseudo-random order) in both the control solution and the 55 mM K+ and 66 mM K+ depolarizing solutions. To enable comparison between different fibers, tetanic forces measured in each fiber were normalized to that produced at 50 Hz in control conditions (126 mM K+); such stimulation produced 97.5 ± 0.8% (n = 27) of the maximum Ca2+-activated force measured in the same fibers (see methods). At every stimulation frequency examined, there was a reduction in peak tetanic force when the fiber was chronically partially depolarized with either the 55 or 66 mM K+ solution (Fig. 5). When a number of stimulation frequencies were examined in the same fiber in such depolarizing conditions, it was found that peak tetanic force was progressively smaller the higher the stimulation frequency used (in the range 20–100 Hz, irrespective of stimulus order) (Fig. 5, A and C). This was in accord with the data for paired pulse stimulation (Figs. 2 and 3), where it was found that force responses in depolarized fibers typically increased substantially when the interpulse interval was increased over the range of 10–50 ms, which corresponds to the spacing between successive stimuli for 100- and 20-Hz stimulation, respectively.
Relationship between repriming period and peak tetanic force.
In every fiber where measurements were made of both 1) the repriming period of double-pulse stimuli and 2) the tetanic responses at the corresponding stimulation frequencies, there was a clear correlation between the response to a pair of stimuli and the corresponding tetanic force response (Fig. 6). Figure 6A shows a typical example. In control conditions (126 mM K+), the twitch response to a pair of pulses was virtually identical for interpulse intervals of 10 and 20 ms, and, similarly, the peak tetanic force at the corresponding frequencies (100 and 50 Hz, respectively) was also virtually the same. When the same fiber was partially depolarized in 55 mM K+ solution, the twitch elicited by the pair of pulses 10 ms apart was substantially smaller than that for the pair 20 ms apart, and the 100-Hz tetanus was much smaller than the 50-Hz tetanus. In fact, in this case, the twitch response to the pulse pair 10 ms apart was not much different than that to a single pulse alone, and, most significantly, the response to a train of 40 similarly spaced stimuli (i.e., the 100-Hz tetanic response) was only marginally larger and was actually smaller than the response to a single pair of pulses 20 ms apart. Mean data for six fibers examined in this way are given in Table 3. These data illustrate how repeatedly stimulating a fiber at too high a frequency not only may be of little use, but in fact may actually adversely affect the response. Figure 6, B and C, shows the correlation between relative twitch size for pulse pairs 10 and 20 ms apart and relative tetanic response at 100 and 50 Hz. In every case where chronic depolarization (55 or 66 mM K+ solution) caused a relative decrease in the twitch response to a pulse pair 10 ms apart compared with that for pulses 20 ms apart, the tetanic force response to 100-Hz stimulation decreased relative to that to 50-Hz stimulation. On average, the relative reduction in the response for the pulse pair 10 ms apart was very similar to that for the tetanic response at 100 Hz (see regression lines in Fig. 6, B and C). Evidently, the ability of the T-system to reprime sufficiently to allow a second closely spaced AP not only affects the response to pairs of pulses but also greatly influences the tetanic force produced by a train of similarly spaced stimuli.
Effect of caffeine on twitch and tetanic force responses during chronic partial depolarization.
Finally, to verify that the reduction in peak tetanic force caused by chronic partial depolarization was indeed due to reduced AP-mediated SR Ca2+ release, 5 mM caffeine was added to the 66 mM K+ solution (see Fig. 7). If the reduction was due to less Ca2+ being released by the SR, addition of caffeine (a known stimulator of the ryanodine receptors) should potentiate Ca2+ release and, hence, markedly increase the amount of tetanic force. In four fibers examined this way (e.g., Fig. 7), the twitch and tetanic (50 Hz) responses elicited in 66 mM K+ solution without caffeine were reduced to 71.8 ± 7.7% and 57.0 ± 9.3% of the respective bracketing control levels (P < 0.05), and when 5 mM caffeine was present in the 66 mM K+ solution, both the twitch and the tetanic force responses were substantially potentiated (to 127.3 ± 10.2% and 81.0 ± 7.8% of the respective bracketing control levels, P < 0.05, Table 2). In the same four fibers, the repriming period in control solutions was 4.0 ± 0.1 ms, and this extended out to 7.8 ± 0.3 ms in the 66 mM K+ solution and was unchanged on addition of 5 mM caffeine (7.8 ± 1.2 ms, Table 2). Thus the increase in tetanic force with caffeine was not due to better T-system AP repriming but was instead primarily due to greater Ca2+ release per AP (see discussion).
This study directly demonstrates that chronic partial depolarization of the T-system in rat skeletal muscle fibers causes a large reduction in tetanic force and, by examining the force responses to single and paired stimulation pulses under the same conditions, establishes the mechanistic basis of this reduction.
Effect of depolarizing solutions on contractile apparatus properties and twitch response to a single AP.
Because this study used force measurements to characterize SR Ca2+ release and excitability, it was first necessary to quantify the effects of the various conditions on the force-pCa relationship of the contractile apparatus. It was found that the effects of the 55 and 66 mM K+ solutions on the contractile apparatus were almost identical (see Fig. 1 and Table 1) and relatively small (∼5% reduction in maximum Ca2+-activated force and a 0.07 pCa unit decrease in the Ca2+ sensitivity). Furthermore, previous findings (16) showed that changes in steady-state Ca2+ sensitivity of the contractile apparatus have substantially less of an effect on the size of the twitch response than might be inferred simply from the difference in force levels on the two force-pCa curves at some fixed pCa value. This is not surprising, because the total amount of Ca2+ released by a single AP is ∼100-fold greater than the rise in free [Ca2+] (i.e., ∼230 vs. ∼2 μM) (5, 32), and most of the released Ca2+ binds to troponin-C, eliciting the force response, and consequently a small change in the affinity of the troponin-C sites would only substantially affect the total amount bound to them if there were a concomitant large increase in the amount of Ca2+ binding to other sites in the fiber over that same small pCa range.
Given that the altered solution conditions were found to have almost identical effects on the contractile apparatus in every fiber (see small SEs for ΔpCa50 and Δh data in Table 1), it can be inferred from the twitch size data in Fig. 4 that the direct effects of the depolarizing solutions on the contractile apparatus are likely responsible for reducing the twitch response to ∼85–90% of the control level, and that the larger reduction seen in some fibers must have been due to a reduction in the amount of Ca2+ released from the SR. This indicates that reductions in Ca2+ release in the 66 mM K+ and 55 mM K+ solutions on average caused ∼15 and 30% reductions in twitch force, respectively (Fig. 4). Our previous studies have shown that twitch force is a sensitive measure of the amount of Ca2+ released from the SR and that reducing the amount of Ca2+ release by ∼20 and 40% results in twitch force decreasing by ∼50 and 80%, respectively (15, 17); conversely, increasing the amount of Ca2+ release by ∼25% by eliciting a second AP in a well-polarized fiber (32) increases twitch force by ∼60% (e.g., Fig. 3). Thus it is evident that chronically depolarizing the T-system to approximately −63 mV and −58 mV in the 66 mM and 55 mM K+ solutions, respectively, likely caused only ∼8 and 15% reductions in the amount of Ca2+ released in response to single AP stimulation. This clearly shows that, when the T-system was depolarized to such levels, it was still capable of propagating single APs and that the combined effects of any changes in the T-system AP characteristics (e.g., size and duration) and any steady-state inactivation of the voltage sensors caused only comparatively minor reductions in the amount of Ca2+ release.
Effect of depolarization on T-system AP repriming.
In contrast, the ability of the T-system to generate a second AP soon after was greatly impaired when the T-system was partially depolarized. This was demonstrated by applying pairs of pulses with various interpulse spacings (see Figs. 2–4 and Table 2). When the fiber was well polarized, an interpulse interval of ≥4 ms was sufficient to allow a second AP to propagate through the T-system and trigger Ca2+ release (defined as the repriming period; see methods and results). However, when the fiber was chronically partially depolarized in the 66 mM K+ or 55 mM K+ solution, the repriming period increased approximately two- to fourfold, even though there were only relatively minor reductions in the twitch response to single AP stimulation (see Fig. 4). When the resting potential in the T-system was approximately −63 to −58 mV, some of the voltage-dependent Na+ channels likely became dysfunctional because of the phenomenon of slow inactivation (35, 36), but evidently enough must have remained operable to support the generation and propagation of a single AP. However, the occurrence of that AP evidently altered the membrane properties, preventing propagation of another AP for a period of time afterward, ∼4 ms when the T-system was fully polarized and ∼6–20 ms when it was partially depolarized. This is likely to be predominantly due to the fact that all of the pool of available Na+ channels would have undergone “fast inactivation” during the course of the first AP and that these channels only recover relatively slowly (i.e., over many ms) and incompletely from such inactivation if the membrane is kept partially depolarized (i.e., at approximately −60 mV) (19). It is possible too that the slowing of AP repriming occurring with T-system depolarization was also due partly to increases in the leak conductances to K+ and Cl− (9, 13, 38), which would have meant that more of the Na+ channels would have had to recover in order for an AP to propagate successfully. In any case, the summed effect is that, when the T-system is partially depolarized at approximately −63 to −58 mV, it can facilitate propagation of individual APs but not of subsequent closely spaced APs. If, however, the T-system is depolarized much beyond this level (e.g., approximately −50 mV), there is complete failure of all APs and evoked force responses in this skinned fiber preparation (31), as occurs in similar intact fiber preparations (11, 34).
Effect of depolarization on tetanic force responses.
In close accord with the above findings with double-pulse stimulation, tetanic force responses were greatly inhibited at depolarized potentials (Fig. 5), with the severity of the reduction increasing at higher stimulation frequencies. Furthermore, the similarity in the extent of reduction in force responses to paired pulse stimulation and to tetanic stimulation when varying the interpulse interval (Fig. 6, B and C) indicated that the reduction in tetanic force at high frequencies was predominantly due to failure of AP repriming when the fiber was stimulated successively at such close intervals. The fact that this phenomenon is observed even with a pair of pulses demonstrates that it is not due to depolarization caused by stimulation-induced increases in [K+] in the T-system, as this increase would be negligible following a single AP, particularly in the T-system of mammalian muscle fibers where Cl− movements help stabilize the membrane potential (13, 14, 31). It is particularly interesting to note that, when the T-system was depolarized in the 55 mM K+ solution (i.e., to approximately −58 mV), a train of 40 stimuli 10 ms apart (i.e., 100-Hz stimulation) elicited a force response that was only slightly larger than that to a single pulse, and that a comparable response could be elicited with just two pulses, provided they were spaced 20 ms apart (Fig. 6A and Table 3). These data show not only that repeated stimulation at a high frequency can be of little benefit when the T-system is depolarized but also that the high rate of stimulation (100 Hz) can be counterproductive, with there being more Ca2+ released in response to a train of pulses when every second pulse is omitted (i.e., 50 Hz stimulation) and even more released if three of every four pulses are omitted (i.e., 25-Hz stimulation) (see Fig. 5). This is likely because each stimulus activates a proportion of the Na+ channels available for activation at that particular moment, and if this number of channels is insufficient to generate a propagating AP, there is little or no consequent Ca2+ release, and those Na+ channels then would not be available for activation for some time afterward, thereby continually keeping the pool of Na+ channels available for activation at a low level.
Thus the present study demonstrates that the reduction in tetanic force occurring when the T-system is depolarized to near −60 mV is not due to it inhibiting AP conduction per se or causing a marked level of voltage sensor inactivation, but instead primarily to it interfering with the ability of the T-system to support closely spaced successive APs. This is consistent with findings in bundles of intact muscle fibers indicating that membrane potential has to be held more depolarized than this for prolonged periods for there to be large-scale voltage sensor inactivation (12). It is also consistent with the findings that frog muscle fibers became depolarized after vigorous stimulation in vitro (4), and tetanic force was found to decrease to a level that was only slightly larger than the twitch response to single AP stimulation, and also to recover much faster than the twitch response when the muscle was allowed to recover (40). Finally, it accounts for the relatively reduced tetanic force seen at 125 Hz compared with 50 Hz when whole EDL muscles of the mouse were depolarized to approximately −58 mV in an elevated [K+] solution (11).
The present findings also help explain why it is advantageous for an animal to reduce the rate of neural stimulation to muscles in vivo during the course of a prolonged tetanus and after repeated fatiguing stimulation (7, 21). This decline in the neural stimulation rate does not itself reduce force output because accompanying changes in intracellular conditions cause a compensatory decline in the fusion frequency for tetanic force production (4, 8). Certainly, one advantage of such a reduction in stimulation rate is that it reduces the total amount of K+ efflux (and Cl− influx) occurring with repeated stimulation, which ultimately helps minimize the level of fiber depolarization. The results presented here show furthermore that lowering the stimulation rate can also help optimize T-system excitation and consequent force output. This would complement other mechanisms supporting T-system excitability, such as the effect of intracellular acidity in reducing the T-system Cl− conductance (31).
In conclusion, this study demonstrates that T-system depolarization reduces tetanic force production in skeletal muscle fibers by impairing AP repriming in the T-system, and that this constrains the rate of neural stimulation required for optimal activation of the muscles.
This work was supported by the National Health and Medical Research Council of Australia (Grant No. 280623).
We thank Brian Taylor for designing and building the in-house stimulator used here.
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.
- Copyright © 2007 the American Physiological Society