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J Appl Physiol 94: 2475-2482, 2003. First published February 14, 2003; doi:10.1152/japplphysiol.01128.2002
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Vol. 94, Issue 6, 2475-2482, June 2003

Intracellular sodium in mammalian muscle fibers after eccentric contractions

Ella W. Yeung1, Heather J. Ballard2, J.-P. Bourreau2, and David G. Allen3

1 Department of Rehabilitation Sciences, Hong Kong Polytechnic University, Hung Hom, Kowloon; 2 Department of Physiology, University of Hong Kong, Hong Kong, China; and 3 Institute for Biomedical Research and Department of Physiology, University of Sydney F13, Sydney NSW 2006, Australia


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The effect of eccentric contractions on intracellular Na+ concentration ([Na+]i) and its distribution were examined in isolated rat and mouse muscle fiber bundles. [Na+]i was measured with either Na+-binding benzofuran isophthalate or sodium green. Ten isometric contractions had no significant effect on force (measured after 5 min of recovery) and caused no significant change in the resting [Na+]i (7.2 ± 0.5 mM). In contrast 10 eccentric contractions (40% stretch at 4 muscle lengths/s) reduced developed force at 100 Hz to 45 ± 3% of control and increased [Na+]i to 16.3 ± 1.6 mM (n = 6; P < 0.001). The rise of [Na+]i occurred over 1-2 min and showed only minimal recovery after 30 min. Confocal images of the distribution of [Na+]i showed a spatially uniform distribution both at rest and after eccentric contractions. Gd3+ (20 µM) had no effect on resting [Na+]i or control tetanic force but prevented the rise of [Na+]i and reduced the force deficit after eccentric damage. These data suggest that Na+ entry after eccentric contractions may occur principally through stretch-sensitive channels.

muscle; eccentric damage; intracellular sodium; gadolinium; stretch-sensitive channels


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

MUSCLE DAMAGE IS A common consequence of intense muscular activity and is more severe when the activity involves stretch of contracting muscles (eccentric contraction). After repeated eccentric contractions, particularly by untrained subjects, the muscles exhibit an immediate muscle weakness and over the subsequent days they remain weak but also become tender, painful, and stiff (33).

The cellular mechanisms that underlie the immediate weakness and the subsequent muscle damage after eccentric contractions have been investigated intensively in recent years. An early finding was that the sarcomere structure is disturbed with overstretched sarcomeres and wavy Z-lines distributed randomly throughout the affected fibers (15). A theoretical basis for these structural abnormalities was proposed by Morgan (31) who pointed out that sarcomeres are unstable on the descending limb of the length-tension curve, particularly when undergoing stretch. The resulting "popping sarcomere" theory can help to explain the development of the structural abnormalities, but it is probable that sarcomere disorganisation is not the sole explanation for the muscle weakness (32, 37).

There is good evidence that changes in excitation-contraction coupling also contribute to the muscle weakness caused by eccentric contractions. This was first demonstrated by Warren et al. (38), who showed that, after eccentric contractions, the reduction of the caffeine-induced force was less than the reduction in the stimulated force. Because caffeine bypasses the normal mechanism of Ca2+ release, this suggests that damage to excitation-contraction coupling is an important component of the weakness. This finding was confirmed by measurements of myoplasmic Ca2+ ([Ca2+]i), which showed that the stimulation-induced rise in [Ca2+]i was reduced after eccentric contractions (2, 20). However, the mechanism of the disturbance to excitation-contraction coupling remains uncertain.

Another component of eccentric damage is an increase in membrane permeability. For example, serum albumin was shown to enter 20% of fibers fixed after a period of eccentric contractions (30). Fibers damaged by eccentric contractions can also be identified by the uptake of fluorescent dyes such as orange procion (9). Resting [Ca2+]i increases within 10 min of eccentric contractions, and, although the mechanism has not been established, this might also be a consequence of increased membrane permeability (2, 20). In addition, large rises in mitochondrial Ca2+ have been observed 1-2 days after eccentric exercise (11). The rise in resting [Ca2+]i might initiate the impairment of excitation-contraction coupling by activating proteases that damage the sarcoplasmic reticulum (SR) Ca2+-release channel (5, 7, 26). It is also proposed that Ca2+-activated proteases contribute to the cell damage that occurs several days after the eccentric activity (4).

Although there is unequivocal evidence for increased membrane permeability after eccentric contractions, the pathway involved is less clear. Membrane tears are often proposed and are consistent with the movement of large molecules such as serum albumin into the cell. If membrane tears were present, one might expect to see localized elevations of [Ca2+]i around the site of the tear. However, attempts to observe such localized elevations have been unsuccessful (3, 20). Another possible contributor to the increased membrane permeability is the involvement of stretch-sensitive channels. Muscles have been shown to possess stretch-sensitive channels, and these are usually nonspecific cation channels opened by stretch (14, 16). Opening of such a channel after eccentric contractions would be expected to cause a depolarization; such a depolarization has been observed and was eliminated by both gadolinium and streptomycin, which are blockers of stretch-sensitive channels (29, 40).

Our laboratory recently showed (41) that, after eccentric contractions, muscles developed vacuoles that filled with an extracellular marker (sulphorhodamine B), suggesting that they were attached to the T-system. Such vacuoles had previously been observed under a range of situations in which a muscle was eliminating an osmotic load (24, 27). We proposed that eccentric damage produced T-tubular tears that allowed the myoplasmic Na+ ([Na+]i) to rise and that vacuoles were a consequence of the osmotic load caused by the Na+ pump extruding the excess Na+ along with osmotically equivalent water.

In the present study, we sought to test two assumptions of the above proposal. One is that [Na+]i should rise after eccentric contractions, and the second was that there might be localized regions of elevated [Na+]i close to the putative membrane tears. We found that [Na+]i increased after eccentric contractions, as expected, but we could find no evidence of localized increases in [Na+]i suggestive of membrane tears. We therefore used gadolinium to test whether the Na+ entry might occur through stretch-sensitive channels.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The experiments were in two parts. The first set of experiments was performed to determine the spatially averaged changes of [Na+]i after eccentric contractions by using the fluorescent indicator Na-binding benzofuran isophthalate (SBFI). The second set of experiments examined the spatial distribution of [Na+]i after eccentric contractions by using confocal microscopy and sodium green.

Contraction protocols. Details of the experimental protocol have been described previously (41, 42). Briefly, tetani were produced by 0.5-ms stimuli (1.2 × threshold) at 100 Hz and lasted for 400 ms. In this protocol, 10 tetani were given at 4-s intervals with the muscle at the muscle length that gave optimal tetanic tension (Lo). The control series consisted of 10 isometric tetani. In the eccentric series, the fibers were stretched from Lo to Lo + 40% over 100 ms (4 muscle lengths/s) starting 200 ms after the start of the tetanus and returning to the original length after the muscle had relaxed.

To determine the effect of each protocol on force production, tetani at 10, 20, 30, 50, 70, and 100 Hz were recorded before and 5-10 min after the contraction protocol.

Experiments were performed in the following solution (in mM): 121 NaCl, 5 KCl, 1.8 CaCl2, 0.5 MgCl2, 0.4 NaH2PO4, 24 NaHCO3, 5.5 glucose, and fetal calf serum (0.2%; Gibco). The solution was bubbled with 95% O2-5% CO2, pH 7.3. All experiments were at room temperature (22 ± 1°C). In a few experiments, a low-Na+ solution (0.4 mM Na+ concentration) was made in which N-methyl-D-glucamine (NMG) replaced most Na+. This solution contained (in mM) 121 NMG Cl, 5 KCl, 1.8 CaCl2, 0.5 MgCl2, 0.4 NaH2PO4, 24 NMG HCO3, 5.5 glucose, and was bubbled with 95% O2-5% CO2, pH 7.3.

SBFI experiments. Sprague-Dawley rats (200-250 g) were anaesthetized with intraperitoneal pentobarbital Na, and muscle bundles were dissected from the extensor digitorum longus (EDL) muscle. The procedures complied with protocols approved by the Committee on the Use of Live Animals in Research at the University of Hong Kong. EDL muscle bundles consisted of three to five cells (for details see Ref. 42). The muscle bundle was mounted between the arm of a motor and the force transducer so that known length changes could be imposed on the fiber while measuring force production.

The muscle fiber was loaded with the membrane permeable AM form of SBFI (Molecular Probes). Stock solution was prepared by dissolving SBFI-AM in dimethyl sulphoxide (DMSO) to a concentration of 2 mM. The loading solution was made by adding 2 µl of 25% wt/wt Pluronic F-127 (Molecular Probes) in DMSO and 32 µl of fetal bovine serum to 3 µl of SBFI-AM stock solution. The vial was sonicated and then diluted in 1 ml of standard solution. The final concentration of SBFI-AM in the solution was 6 µM. Loading was carried out for 60-90 min at room temperature. Measurement of [Na+]i was performed by using the dual-excitation ratiometric method, employing the fluorescence imaging system (DeltaScan 4000, Photon Technology International). The loaded muscle fiber was excited at 340 and 380 nm wavelengths, and emitted light was recorded at 530 nm.

An in vivo calibration of SBFI fluorescence was obtained by exposing the muscle fiber to the calibration solution to create conditions that allow equilibration of [Na+]i with extracellular Na+ concentration. The solution contained 10 mM HEPES, 1 mM EGTA, 10 mM glucose, 0.2 µg/ml gramicidin D, 100 µM strophanthidin, and 40 µM monesin, with pH adjusted to 7.3 (36). Different proportions of Na+ (0, 10, 20, 40, 70, and 140 mM) and K+ solutions were made up to keep the same total cation concentration. The resulting calibration curve was used to convert SBFI fluorescence ratios to [Na+]i.

Sodium green experiments. Adult, male mice were killed by rapid neck disarticulation and a single muscle fiber was dissected from the flexor digitorum brevis muscle (for details, see Ref. 41). The experiments were approved by the Animal Ethical Committee of the University of Sydney. The fiber was mounted in a chamber that allowed length changes and simultaneous measurements of force and fluorescence signals.

[Na+]i was measured with the nonratiometric indicator sodium green (Molecular Probes). The fiber was examined with laser scanning confocal microscopy (Leica TCS SL, Heidelberg, Germany) by using a ×63, numerical aperture 1.2 water immersion objective. Sodium green was excited by using the 488-nm line at ~25% of maximum strength, and fluorescence emission from 510- to 560-nm wavelength was collected. The working distance of the above objective (220 µm above the coverslip) was sufficient to focus on a fiber attached to a force transducer and the muscle lever. Images were routinely collected every 2 or 5 min, and under these circumstances we observed no obvious bleaching or dye loss over the time course of experiments lasting 1 h. Sodium green fluorescence was quantitified by using NIH Image (Scion) by measuring the fluorescence intensity over a large region of the fiber (~20 × 20 µm). This was expressed as a percentage of the initial signal.

In preliminary experiments, we varied the loading conditions to achieve optimal fluorescence images. Loading with 10 µM sodium green AM for >30 min resulted in fluorescent images that showed a nonuniform distribution of fluorescence with lines of increased intensity across the fiber repeated every sarcomere. To determine whether this fluorescence represented a nonuniform distribution of sodium green or arose from gradients of [Na+]i, we exposed two fibers to a low-Na+ solution. The dominant effect was a decline in fluorescence signal presumably caused by a fall in [Na+]i, but the characteristic periodic elevations were still present. This suggests that the periodic staining is associated with organelle uptake or binding to proteins as noted with many other indicators (21, 23). In subsequent experiments the fiber was incubated with 5 µM sodium green AM for 15-20 min at room temperature, and under these conditions this type of nonuniformity was almost absent.

Drugs. SBFI-AM, sodium green AM (Molecular Probes), and ouabain (Sigma-Aldrich) were prepared as stock solutions in DMSO. The DMSO concentration did not exceed 0.2% in the final solution. Gadolinium (III) chloride, hexahydrate (Sigma-Aldrich) was prepared as stock solution (1 M in water), and the Gd3+ was added to the experimental solution immediately before the experiments.

Statistics. Data are expressed as means ± SE. Statistical significance was tested with paired or unpaired Student's t-test, as appropriate, and P < 0.05 was taken to be significant.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Effects of eccentric contraction on force and [Na+]i. The eccentric protocol reduced developed force in the rat EDL muscle bundles to 45 ± 3% of control and shifted the Lo by 14 ± 4%. [Na+]i was measured continuously by using SBFI in EDL muscle bundles subjected to the isometric and eccentric contraction protocols (Fig. 1). The resting level of [Na+]i for the isometric series (7.2 ± 0.5 mM; n = 6) was not significantly different from that of the eccentric series (7.7 ± 0.6 mM; n = 6). Figure 1A shows the record of [Na+]i before and after the isometric protocol. After the isometric tetani, there was no significant change in [Na+]i (7.3 ± 0.2 mM; n = 6). In contrast, after eccentric contractions, the [Na+]i level increased markedly, reaching a peak of 16.3 ± 1.6 mM (P < 0.001; n = 6) after 10 min (Fig. 1B). The rise of [Na+]i did not occur immediately but increased to a steady level over several minutes. The time to 50% rise measured from the end of the last tetanus was 2.2 ± 0.4 min (n = 6).


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  		Fig. 1.   Intracellular Na+ concentration ([Na+]i) records during isometric protocol (A) and eccentric (Ecc) protocol (B). Records of [Na+]i were obtained from Na-binding benzofuran isophthalate fluorescence in rat extensor digitorum longus muscle bundles. Timing of the isometric contractions in A and the eccentric contractions in B are indicated by the bars. Note that the large increase in [Na+]i only occurred after the Ecc protocol. Isom, isometric contraction.

Ouabain experiments. The increase in [Na+]i after eccentric contractions could arise because Na+ efflux pathways were inhibited or because Na+ influx was increased. To distinguish between these possibilities, we used ouabain to block the Na+ pump, which is the main efflux pathway.

Figure 2A illustrates the [Na+]i tracing of a representative EDL muscle bundle in which 0.5 mM ouabain was applied for 10 min starting 1 min before the isometric contractions. At the end of this 10 min, [Na+]i had increased to 9.9 ± 1.5 mM (n = 5). In similar experiments but with eccentric contractions, the [Na+]i increased to 26.4 ± 3.0 mM (n = 6), which was significantly greater than the isometric tetani series (P < 0.001). If the rise in [Na+]i after eccentric contractions was caused principally by inhibition of the Na+ pump, then addition of ouabain might have had little effect on the rise of [Na+]i. Instead, the peak [Na+]i was significantly increased from 16.3 ± 1.6 mM in the absence of ouabain to 26.4 ± 3.0 mM in its presence (P < 0.01; unpaired t-test). This result suggests that inhibition of the Na+ pump does not contribute greatly to the rise of [Na+]i after eccentric contractions.


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  		Fig. 2.   Effect of ouabain on the increase in [Na+]i caused by Isom (A) and Ecc (B) protocols. The muscle bundle was exposed to 0.5 mM ouabain for 10 min, as indicated by the open bars. Closed bars indicate Isom or Ecc contractions. Note that the increase in [Na+]i after Ecc contractions is enhanced by ouabain.

Confocal images of the distribution of [Na+]i in single mouse fibers. A key issue was to determine the location and distribution of the rise in [Na+]i after eccentric damage. Figure 3A is an image of a single mouse fiber under ordinary light transmission that shows regular alignment of the sarcomeres. Confocal images of the sodium green fluorescence from the same fiber are shown from before the eccentric series (Fig. 3B) and then at ~1 min after the eccentric contraction series (Fig. 3C). Under control conditions, distribution of fluorescence intensity appears quite uniform. After eccentric contractions, the fluorescence signal was increased substantially but remained spatially uniform. Each fiber was carefully scanned along its length and at multiple depths, and no obvious regions of focal increase in fluorescence intensity were observed.


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  		Fig. 3.   Confocal images of a mouse flexor digitorum brevis (FDB) fiber loaded with sodium green. A: image of a fiber under ordinary light transmission. B and C: confocal images of the same fiber showing the distribution of sodium green fluorescence. The control fiber (B) shows a uniform fluorescence distribution. After Ecc contractions, the same fiber shows a significant increase in fluorescence signal without obvious localized elevations (C). Fluorescence intensity is color coded, with black, dark red, yellow, and white indicating progressive increases in fluorescence intensiy. Scale bars, 25 µm.

Figure 4 shows the time course of sodium green fluorescence intensity under resting conditions and after isometric and eccentric tetani. In the muscle fibers examined (n = 10), the resting fibers showed no significant changes of fluorescence intensity over 10 min (see also Fig. 5). After the 10 isometric tetani, there was a suggestion of a small rise of [Na+]i, but this did not reach statistical significance. After the eccentric tetani, [Na+]i intensity was monitored every 5 min over 30 min. The sodium green fluorescence was significantly greater in the first image after the eccentric contractions (collected at ~1 min) and reached a peak of 124 ± 9% at 10 min. The fluorescence remained significantly elevated for 25 min (P < 0.05) before it started to decline.


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  		Fig. 4.   Effect of isometric and Ecc contractions on sodium fluorescence. Comparison of fluorescence intensity normalized to initial control after isometric contractions and Ecc contractions (n = 10) in mouse FDB fibers is shown. Isometric contractions did not lead to a significant change in sodium fluorescence, whereas after Ecc contractions fluorescence was significantly elevated above control level. Values are means ± SE. * Significantly different from control situations (P < 0.05).



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  		Fig. 5.   Effect of Gd3+ on sodium fluorescence in control conditions and after Ecc contractions. Graph shows sodium green fluorescence normalized to initial control in mouse FDB fibers. The fiber was exposed to 20 µM Gd3+ for 10 min under control conditions, and the Gd3+ was then washed off. There was no significant change in fluorescence. The Ecc protocol was then instituted, and 20 µM Gd3+ was applied immediately afterward. Note that the increase in sodium fluorescence seen after Ecc contractions (Fig. 4) was eliminated in the presence of Gd3+. Values are means ± SE.

We also checked in these experiments whether the source of elevated [Na+]i arose from influx from the extracellular space. In three experiments, the extracellular solution was changed to a low-Na+ solution immediately after the end of a series of eccentric contractions. In these experiments, the normalized sodium green fluorescence 5 min after the eccentric contractions declined to 86 ± 10% of the original control. This value is significantly less (P < 0.05; unpaired t-test) than that observed after eccentric contraction in normal solution (123 ± 8%). These data are consistent with the earlier ouabain data and suggest that the elevated [Na+]i arises from increased influx from the extracellular space.

Gadolinium experiments. The sodium green data indicate an increased [Na+]i after eccentric contractions consistent with the SBFI results but showed no detectable localized elevations of [Na+]i suggestive of tears in the membrane. We therefore used gadolinium, an established blocker of stretch-sensitive channels, to see whether the Na+ influx might be through this class of channels. The effect of Gd3+ on [Na+]i at baseline and after eccentric contractions was studied (n = 6). Gd3+ was applied for 10 min under control conditions and did not produce a significant change in sodium green fluorescence. However, after the eccentric contractions, the previously observed increase in sodium green fluorescence was eliminated and there was no significant change in fluorescence (Fig. 5). On removal of Gd3+ 10 min after eccentric contractions, the fluorescence signal did not change significantly. Thus the rise of [Na+]i was abolished by Gd3+, suggesting that the stretch-activated channels provide the main entry pathway for Na+.

Effect of inhibition of stretch-activated channels on force. The force deficit provides an indication of the extent of muscle damage, and, given that Gd3+ prevented the rise in [Na+]i after eccentric contractions, it was of interest to examine whether the force deficit was affected by Gd3+. Figure 6 shows force-frequency curves normalized to the control 100-Hz stimulation. To determine whether Gd3+ had any effect on force under normal conditions, we assessed the force-frequency relations (n = 8) both in standard solution and in the presence of Gd3+. There was no significant difference at any frequency of stimulation. The eccentric contraction protocol (n = 9) produced a large and significant reduction in force at all stimulation frequencies. The addition of Gd3+ (20 µM) for 10 min starting immediately after the end of the eccentric contractions (n = 6) led to a small improvement of force that was significant at 70 and 100 Hz (P < 0.05). For instance at 100-Hz stimulation, tetanic force after eccentric contractions was 36 ± 5%, and after the Gd3+ treatment it was 49 ± 4% of the initial control. In some Gd3+ experiments (n = 4), after a further 20 min of recovery in the absence of Gd3+, force was remeasured, and this improvement was maintained.


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  		Fig. 6.   Effect of Gd3+ on force-frequency data before and after Ecc damage. Four force-frequency curves are shown that were determined under the following conditions for mouse FDB fibers: open circle , control; , control conditions plus 20 µM Gd3+; triangle , 5 min after the Ecc protocol; black-triangle, 10 min after the Ecc protocol with Gd3+ present from the end of the Ecc protocol. All data points were normalized to the initial control force at 100 Hz. Values are means ± SE. * Significant difference between Ecc and Ecc + Gd3+.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Muscle bundles exposed to the eccentric protocol used in this study exhibit the following features: 1) reduced force at 100-Hz stimulation and 2) an increase in the length at which maximum force is developed (41, 42). Previously, we have also shown that single fibers show the characteristic disorganization of sarcomere structure (3) and that these changes were not produced either by an equivalent number of isometric tetani or by stretching relaxed fibers (2, 3, 41, 42). All these features have been described in intact humans or animal muscles subject to eccentric exercise (for review, see Ref. 32) and suggest that isolated muscle bundles can provide a realistic model of eccentric damage.

We previously showed that after eccentric contractions single fibers can develop vacuoles attached to the T-tubules and suggested that these arose when the Na+ pump extruded excess Na+ and water that had accumulated in the fiber through tears in the T-tubular system. In this study, we used two different Na+-sensitive fluorescent indicators to examine the changes in [Na+]i after isometric and eccentric contractions. Each action potential in a frog muscle has been estimated to cause the entry of 7 µmol Na+/l fiber water (18) so that after 10 × 0.4-s tetani at 100 Hz we might expect a total influx of Na+ equivalent to 400 × 7 µM = 2.8 mM. The rise of [Na+]i will be smaller to the extent that the Na+ pump is activated during the period of activity. Although we did not observe a significant increase after isometric tetani when all experiments were considered, in 2 of 6 fibers small increases in [Na+]i (1-2 mM) were observed after the isometric tetani. This suggests that the expected increase in [Na+]i associated with action potentials was below the resolution of our methods.

In contrast, a clear and statistically significant increase in [Na+]i after eccentric contractions was observed both in the rat EDL by using SBFI and in the mouse flexor digitorum brevis by using sodium green. SBFI was calibrated by using standard techniques, and the results suggest that [Na+]i rose from a resting level of 7.7 mM to a peak of ~16.3 mM (an increase of 112%). In contrast, the sodium green fluorescence increased by only 24%. In the present study, we did not attempt to calibrate the sodium green fluorescence. However, when the resting [Na+]i is assumed to be 7.5 and the data from the Molecular Probes catalog (where Na+ + K+ = 135 mM) is used, a 24% increase in fluorescence suggests a rise in [Na+]i to 14 mM, which is similar to the SBFI data. Typically, the rise of [Na+]i takes 1-3 min to reach a peak and declines only slowly after 25 min.

Route of entry of Na+ after eccentric contractions. The fact that the rise of [Na+]i was increased after blocking the Na+ pump suggests that the rise of [Na+]i is not caused by reduced activity of the Na+ pump. Furthermore, the rise of [Na+]i was eliminated in low-Na+ solution, confirming that it arises from increased Na+ influx from the extracellular space. In our laboratory's previous study (41), we suggested that the increased Na+ entry might arise through tears in the T-tubules caused by relative movement of the sarcomeres. If this were the case, one might expect to see elevations of [Na+]i localized around the sites of the tears. To test this possibility, we imaged the [Na+]i in resting fibers and after eccentric contractions when [Na+]i was elevated. We never observed localized elevations of [Na+]i as predicted. This might be because the tears are small and occur at multiple sites, and the Na+ rapidly becomes uniformly distributed by diffusion. Interestingly, the same lack of localized elevations was observed when [Ca2+]i was imaged (3, 20), and a possible explanation for that result was that the Ca2+ that leaked into the fiber was rapidly taken up by the SR. Thus one reason for imaging [Na+]i was that it is not rapidly sequestered in the cell and therefore localized elevations might be more easily detected. Although it remains possible that the elevations of [Na+]i were too small for us to detect, our present conclusion is that it is unlikely that tears in the membrane are the main cause of the early Na+ influx.

Given the failure to detect evidence for localized Na+ entry, we explored the possibility that Na+ might enter through a source that was widely distributed across the surface membrane and would produce a generalized [Na+]i elevation. As noted in the introduction, McBride et al. (29) observed a membrane depolarization after eccentric contractions that was inhibited by Gd3+, a blocker of stretch-sensitive channels. We therefore tested Gd3+ and found that 20 µM Gd3+ completely prevented the rise of [Na+]i after eccentric contractions while having no effect on the resting [Na+]i. Gd3+ blocks stretch-sensitive channels at ~5-10 µM (40) but also blocks some other channels (see below). Gd3+ had no detectable effect on contraction, suggesting that the channels involved in excitation and Ca2+ release were little affected. Instead, these results suggest that a stretch-activated Na+-permeable channel is opened after eccentric contractions. The rise in [Na+]i lasts at least 20 min, suggesting that the channels opened by stretch remain open for many minutes. Alternatively, the sarcomere disorganization, which is a key feature of eccentric damage, might cause T-tubules or elements of the surface membrane to be distorted and thereby stretch nearby channels for many minutes after eccentric exercise.

If the early increase in membrane permeability arises from opening of stretch-sensitive channels, what then causes the permeability to large molecules such as albumin and procion orange (9, 30)? We speculate that this increase in permeability to large molecules is a secondary consequence of the early ionic changes. For instance, as suggested below, the rise in [Ca2+]i may activate proteases and cause damage to membranes sufficient to allow these large molecules to enter.

Stretch-sensitive channels in muscle. Addition of 20 µM Gd3+ abolished the [Na+]i rise after eccentric contractions, but the channel and the molecular species of Gd3+ involved are uncertain. Gd3+ is an established blocker of stretch-sensitive channels but has been reported to block a range of other channels (6) including L-type Ca2+ channels (25), Na+ and K+ channels (12), and store-operated channels (13).

Gd3+ has very high binding constants for carbonate, phosphate (PO<UP><SUB>4</SUB><SUP>3−</SUP></UP>) and proteins; the equilibrium binding constant for Gd3+ and PO<UP><SUB>4</SUB><SUP>3−</SUP></UP> is 1033 M (28). Calculation suggests that in the presence of 25 mM HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> and 1 mM H2PO<UP><SUB>4</SUB><SUP>−</SUP></UP> (pH 7.4) the steady-state free Gd3+ would be extremely low (10-20 M) (6). Many studies have been performed in HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>, H2PO<UP><SUB>4</SUB><SUP>−</SUP></UP>, and protein-free solutions to minimize these problems, and in such solutions Gd3+ effectively blocks stretch-sensitive channels at ~5-10 µM. However, the dilemma is that other studies performed in the presence of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>, H2PO<UP><SUB>4</SUB><SUP>−</SUP></UP>, and protein appear to show that Gd3+ has roughly similar potency even though the calculated steady-state free Gd3+ concentration should be negligible (17, 34). Two possible resolutions to this dilemma are that 1) the binding of Gd3+ to carbonate or phosphate is quite slow so that appreciable amounts of free Gd3+ are present for some minutes after addition of Gd3+ or 2) the active species for channel blocking is not Gd3+ but Gd2(CO3)3 or some other bound species. One reason for suggesting this is that even in the nominal absence of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>, solutions that are equilibrated with air contain atmospheric CO2 (0.05%), and this leads to the presence of about 260 µM HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> at pH 7.4 (10). Because the binding of Gd3+ to carbonate is so strong, even this small amount of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> will lead to binding of most of the Gd3+ as Gd2(CO3)3. This argument suggests that even in nominally CO2- or HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>-free solutions, most of the Gd3+ will be in the form Gd2(CO3)3, which could therefore be the active species for channel blocking.

To further explore the route of Na+ entry following eccentric damage we also used another chemically unrelated blocker of stretch-sensitive channels streptomycin. In two experiments streptomycin (100 µM) increased the normalized fluorescence in resting fibres by + 11% and the normalized fluorescence 10 min after eccentric contractions was reduced to 82% of the initial control. Thus streptomycin, like Gd3+, had little effect on resting [Na+]i but prevented the rise after eccentric contractions.

We conclude that the Na+ enters through a channel that is blocked by Gd3+. The channel may well be activated directly by stretch, but other mechanisms are possible, e.g., stretch might activate a second messenger that then activates the channel.

Mechanism of force reduction after eccentric contractions. As noted in the introduction, the force deficit after eccentric contractions is known to have at least two causes (for review, see Refs. 1, 32, 37). One component is caused by the sarcomeres inhomogeneity, which is characteristic of eccentric damage (15). Because the longer sarcomeres act as additional compliance, this component of the force deficit can be quantified by the increase in developed force when the muscle is restretched to the new Lo (39, 41). We have previously shown that, in a mammalian muscle preparation subject to the present protocol, this requires a stretch of ~15% and the force recovers from ~43 to 78% of the original control (42). The force deficit that remains after restretching to the new Lo arises partly from damage to the processes of excitation-contraction coupling as shown by the fact that the tetanic Ca2+ signal is reduced after eccentric contractions and that this reduction can be overcome by caffeine (2, 38). Thus it is of interest in the present experiments that not only did Gd3+ prevent the increase in [Na+]i after eccentric contractions but it also prevented some of the force deficit. Given that the stretch-sensitive channels described in muscles are nonspecific cation channels (14, 16), it is tempting to speculate that the rise of resting [Ca2+]i observed after eccentric damage (2, 20) also occurs by Ca2+ entry through the same stretch-activated channels.

Elevation of [Na+]i after eccentric contractions might have various effects on muscle function. The electrochemical gradient for Na+ influx will be reduced by the increased [Na+]i, and this will reduce the amplitude of the action potential and potentially reduce SR Ca2+ release, which depends on the magnitude of the depolarization (19) . However, this effect is likely to be quite small because it is generally thought that there is a large safety margin in the relation between action potential amplitude and Ca2+ release (for discussion, see Ref. 35). The reduced inward Na+ gradient will also reduce the rate of extrusion of protons on the Na+/H+ exchanger (22), and this may partly explain the reduced operation of the Na+/H+ exchanger observed after eccentric contractions (42).

It has often been noted that muscles swell after eccentric contractions (8). This is visible in our single fibers that showed substantial increases in diameter after eccentric contractions (compare Fig. 3, B and C). This swelling will be partly explained by the increase in [Na+]i and associated H2O; in addition, the extracellular vacuoles will also contribute to the increase in diameter (41).

When it is assumed that Ca2+ ions also enter through the stretch-activated channels, this pathway could contribute to the elevated resting [Ca2+]i (3, 20) and to the increases in mitochondrial Ca2+ (11) observed after eccentric contractions. In skinned fibers, it has been shown that a period of elevated [Ca2+]i reduces subsequent SR Ca2+ release, possibly by activating proteases that damage the ryanodine receptor (26). In intact fibers, it has also been shown that maneuvers that elevate resting [Ca2+]i cause a subsequent reduction in the amplitude of the Ca2+ transients (5, 7). Thus it is possible that both the elevated resting [Ca2+]i and the reduced Ca2+ transients seen after eccentric contraction are a consequence of Ca2+ entry through stretch-activated channels. Thus our observation that Gd3+ prevents one component of eccentric damage may also arise through blocking ion entry through stretch-sensitive channels.


    ACKNOWLEDGEMENTS

This work will be submitted to the University of Hong Kong by Ella W. Yeung as part of her doctoral thesis.


    FOOTNOTES

Address for reprint requests and other correspondence: D. G. Allen, Univ. of Sydney, Sydney NSW 2006, Australia (E-mail: davida{at}physiol.usyd.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.

First published February 14, 2003;10.1152/japplphysiol.01128.2002

Received 6 December 2002; accepted in final form 7 February 2003.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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J APPL PHYSIOL 94(6):2475-2482
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