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MUSCLE CELL BIOLOGY AND CELL MOTILITY

Transverse tubular system depolarization reduces tetanic force in rat skeletal muscle fibers by impairing action potential repriming

Department of Zoology, La Trobe University, Melbourne, Victoria, Australia

Submitted 5 January 2007 ; accepted in final form 16 February 2007

	ABSTRACT

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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.

T-system; muscle fatigue; excitation-contraction coupling

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).

	METHODS

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Preparations. 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).

Solutions. 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): HDTA^2– (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 HDTA^2– 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 x 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.

	RESULTS

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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, pCa₅₀, 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.

View larger version (8K): [in this window] [in a new window]   Fig. 1. Effect of 55 and 66 mM K+ solutions on Ca2+ sensitivity of the contractile apparatus. The force-pCa relationship in a mechanically skinned extensor digitorum longus (EDL) fiber was obtained in control solution (, solid line) before, between, and after exposure to the 55 mM K+ solution (, solid line) and the 66 mM K+ solution (, dashed line) (see METHODS). The force elicited at each pCa was normalized to the maximum Ca2+-activated force produced at pCa 4.7 under the same condition. Mean values and changes in the pCa producing 50% of maximum Ca2+-activated force (pCa50) and Hill coefficient (h) for 4 fibers are shown in Table 1.

View larger version (20K): [in this window] [in a new window]   Fig. 2. Effect of chronic partial depolarization on action potential (AP) repriming in the transverse tubular system (T-system). A: representative example of twitch responses elicited in a skinned EDL fiber by single supramaximal electric field pulse or by pairs of identical pulses separated by 1–50 ms (interpulse interval, shown at top of each response) when the T-system was either normally polarized (control conditions, 126 mM K+ solution) or partially depolarized (in 55 mM K+ solution). "0-ms" interpulse spacing indicates application of a single pulse alone. In control conditions, there was a large increase in twitch force to a pulse pair if the interpulse spacing was 4 ms, indicating that the second pulse of the pair had also succeeded in eliciting an AP in the T-system, hence causing additional Ca2+ release and force. In the 55 mM K+ solution, there was no increment in twitch force unless the pulses in a pair were 10 ms apart; tetanic stimulation (Tet 50 Hz) under control conditions produced 100% of maximum Ca2+-activated force (latter not shown). B: twitch force vs. interpulse spacing in control solution () and in 55 mM K+ solution () in all 9 fibers studied in both conditions. All values were normalized to twitch size to a single pulse in control conditions in same fiber. Mean repriming period values are shown in Table 2.

View larger version (19K): [in this window] [in a new window]   Fig. 3. Mean data of AP repriming at different T-system potentials. Mean ± SE twitch force elicited by pairs of supramaximal field pulses (with varying interpulse spacings, 1–50 ms as in Fig. 2) when the T-system was well polarized (in 126 mM K+ control solution; ) and chronically depolarized in the solution with 66 mM K+ () and 55 mM K+ () (membrane potential estimated as approximately –63 and –58 mV, respectively). When the T-system was more depolarized, the interpulse interval had to be increased to longer times in order for the second pulse to trigger an AP and produce an increased twitch response; 0-ms interpulse interval indicates that only a single pulse was applied. Mean repriming period values are shown in Table 2. Nos. of fibers examined at each interpulse interval were as follows: control solution, n = 19–23 for 0- to 20-ms and 4 for 30- to 50-ms spacings; 66 mM K+ solution, n = 10–14 for 0–20 ms and 3 for 30–50 ms; 55 mM K+ solution, n = 7–9 for 0–20 ms and 4 for 30–50 ms.

View larger version (7K): [in this window] [in a new window]   Fig. 4. Relationship between twitch size to a single AP and AP repriming period. Data for individual fibers depolarized in 66 mM K+ solution (, n = 13) or in 55 mM K+ solution (, n = 8) showing relative twitch size to a single AP stimulus vs. the repriming time observed with paired pulses. Experiments were conducted as in Fig. 2. Twitch size is expressed as a percentage of that measured when the T-system was fully polarized in the control solution (126 mM K+). Linear regression analysis correlation index, r2 = 0.2825; P < 0.05. One of the nine fibers shown in Fig. 2B had to be omitted because it did not show any detectable repriming within the range of the pulse spacings studied.

View larger version (25K): [in this window] [in a new window]   Fig. 5. Effect of chronic partial depolarization on tetanic force responses. A: representative example of tetanic force responses in one fiber (same fiber as in Fig. 2A). In the 55 mM K+ solution, peak tetanic force decreased substantially as the stimulation frequency was increased (20 to 100 Hz; applied during the period indicated by solid bars), whereas the control tetani (126 mM K+ solution) were close to maximal at 50 and 100 Hz. B. mean ± SE peak tetanic force vs. stimulation frequency, for fibers in 126 mM (control, ), 66 mM (), and 55 mM () K+ solution. Tetanic force was expressed as a percentage of the 50-Hz level in control conditions; n is no. of fibers indicated with each mean value. C: individual fiber data. Lines join values obtained at different stimulation frequencies in same fiber, for fibers in either 66 mM (, n = 3) or 55 mM (, n = 4) K+ solution, together with corresponding data in 126 mM K+ (control, ).

View larger version (25K): [in this window] [in a new window]   Fig. 6. Relationship between responses to paired stimuli and to tetanic stimulation. A: force responses in a fiber to single pulses (marked ‘0’), to pairs of pulses 10 and 20 ms apart (‘10’ and ‘20,’ respectively), and to 50- and 100-Hz tetanic stimulation (Tet) in both control conditions (126 mM K+) and in depolarizing conditions (55 mM K+). Note that 100- and 50-Hz stimulation consisted of trains of 40 pulses 10 and 20 ms apart, respectively. In the 55 mM K+ solution, the twitch response elicited by a pair of pulses 10 ms apart was not much different from that elicited by a single pulse, and the response to 100-Hz stimulation was also only slightly larger. B and C: relative size of twitch response to two pulses 10 ms apart, expressed as a percentage of that to two pulses 20 ms apart, vs. the relative size of the tetanic response to 100-Hz stimulation, expressed as a percentage of that to 50-Hz stimulation. Lines join data for same fiber in control conditions (126 mM K+, ) and when depolarized in 66 mM K+ solution (B; ) or in 55 mM K+ solution (C; ). Thick gray line indicates best fit from linear regression analysis showing a significant correlation between relative twitch size and relative tetanus size (correlation index r2 = 0.436, gradient 0.76, and ordinate intercept 22% for B; and 0.653, 1.14, and –14%, respectively, for C; both P < 0.05). Data indicate that depolarization causes a similar relative reduction in the response to a pair of pulses 10 ms apart and to a train of such pulses (i.e., 100-Hz stimulation).

View larger version (15K): [in this window] [in a new window]   Fig. 7. Effect of 5 mM caffeine on twitch and tetanic force responses in depolarizing conditions. Twitch (tw) and tetanic force (50 Hz) responses were produced in control conditions (126 mM K+) and in 66 mM K+ conditions with and without 5 mM caffeine. Depolarization in the 66 mM K+ solution caused only a small reduction in twitch force but a larger reduction in tetanic force. Addition of 5 mM caffeine to the 66 mM K+ solution greatly increased twitch and tetanic force compared with that observed without caffeine. Time scale: 2 s for twitch and tetani and 5 s for maximum Ca2+-activated force (max).

	DISCUSSION

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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 pCa₅₀ and h 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).

Further implications. 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.

	GRANTS

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This work was supported by the National Health and Medical Research Council of Australia (Grant No. 280623).

	ACKNOWLEDGMENTS

 
We thank Brian Taylor for designing and building the in-house stimulator used here.

	FOOTNOTES

 

Address for reprint requests and other correspondence: T. L. Dutka, Dept. of Zoology, La Trobe Univ., Melbourne 3086, Victoria, Australia (e-mail: t.dutka{at}latrobe.edu.au)

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.

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