The effects of high myoplasmic l-lactate concentrations (20–40 mM) at constant pH (7.1) were investigated on contractile protein function, voltage-dependent Ca2+ release, and passive Ca2+ leak from the sarcoplasmic reticulum (SR) in mechanically skinned fast-twitch (extensor digitorum longus; EDL) and slow-twitch (soleus) fibers of the rat. l-Lactate (20 mM) significantly reduced maximum Ca2+-activated force by 4 ± 0.5% (n = 5, P < 0.05) and 5 ± 0.4% (n = 6, P < 0.05) for EDL and soleus, respectively. The Ca2+ sensitivity was also significantly decreased by 0.06 ± 0.002 (n = 5,P < 0.05) and 0.13 ± 0.01 (n = 6, P < 0.001) pCa units, respectively. Exposure tol-lactate (20 mM) for 30 s reduced depolarization-induced force responses by ChCl substitution by 7 ± 3% (n = 17, P < 0.05). This inhibition was not obviously affected by the presence of the lactate transport blocker quercetin (10 μM), or the chloride channel blocker anthracene-9-carboxylic acid (100 μM). l-Lactate (20 mM) increased passive Ca2+ leak from the SR in EDL fibers (the integral of the response to caffeine was reduced by 16 ± 5%,n = 9, P < 0.05) with no apparent effect in soleus fibers (100 ± 2%, n = 3). These results indicate that the l-lactate ion per se has negligible effects on either voltage-dependent Ca2+ release or SR Ca2+ handling and exerts only a modest inhibitory effect on muscle contractility at the level of the contractile proteins.
- muscle fatigue
- excitation-contraction coupling
- contractile apparatus
- calcium release
in most instances, muscle fatigue is associated with prolonged periods of intermittent contractions that typically give rise to alterations in the level of myoplasmic metabolites [see review by Fitts (12) and Allen et al. (1)]. Lactic acidosis is thought to be a major factor in such metabolic fatigue. Acidosis of the myoplasm per se has been shown to affect such parameters as maximum Ca2+-activated force, Ca2+ sensitivity of the myofilaments, and Ca2+ release from ryanodine receptors [see review by Allen et al. (1)]. However, in mammalian muscle, the effects of acidosis on these parameters are small (20, 25, 35), indicating that other metabolic changes are largely responsible for the decline in force in mammalian muscle.
Accompanying the increase in the proton concentration is a concomitant increase in the myoplasmic l-lactate ion concentration, which has been reported to reach as high as 40–55 mmol/l cell water at exhaustion [see review by Juel (15)]. Elevated myoplasmic concentrations of l-lactate per se (independent of pH) have been reported to have small, variable effects on the contractile apparatus and predominantly strong inhibitory effects on Ca2+ release from the sarcoplasmic reticulum (SR) in SR vesicle preparations and skinned fibers. Maximum Ca2+-activated force in chemically skinned fast-twitch fibers has been reported to be either slightly reduced (2) or increased (9) (∼5%). In intact fibers, force response has also been reported to be unaffected (34) or decreased (14, 30), particularly at physiological temperatures (30). Such typically small variations in force may be due to differences between the methods employed.
However, the reported inhibitory effects of l-lactate ion per se on both Ca2+ uptake and Ca2+ release from the SR have been more pronounced. In studies using SR vesicle preparations, Ca2+ release activated by either caffeine or Ag+ was reduced by more than 30% in the presence of high concentrations of l-lactate ion (10,11, 30). Similarly, in single-channel recordings of ryanodine receptors, l-lactate also reduced the mean open time by as much as 75% (10,11). In contrast, it was recently reported thatl-lactate reduced Ca2+ uptake and increased Ca2+-induced Ca2+ release (4). These reports suggest that the l-lactate ion per se, independent of pH, may greatly contribute to skeletal muscle fatigue, particularly late fatigue, in which Ca2+ release from the SR is significantly reduced.
Although l-lactate has been shown to significantly affect Ca2+ release under the above conditions, it cannot be determined from such experiments to what extentl-lactate will affect Ca2+ release from SR Ca2+-release channels in which Ca2+ release is coupled to active voltage sensors. Furthermore, although intact fibers possess functional excitation-contraction (E-C) coupling, it is difficult to eliminate other sites of action or effects, such as any metabolic effects associated with the mitochondria, and to control myoplasmic conditions. Therefore, a more systematic study of the effects of l-lactate per se on E-C coupling, Ca2+ release, and mechanical force under identical conditions would provide further insight into the physiological role ofl-lactate in fatigue.
Using the mechanically skinned fiber technique, we could systematically examine the effects of the l-lactate ion per se, independent of pH, on force, Ca2+ release, and E-C coupling under controlled conditions in the same preparation. We sought to test the hypothesis that l-lactate inhibition of normal voltage-dependent Ca2+ release will result in the reduction of force. Under these conditions, we demonstrated that thel-lactate ion per se only slightly reduced depolarization-induced force responses; this effect was largely attributed to a reduction in the maximum Ca2+-activated force and not to a significant reduction in Ca2+ release from the SR. We conclude that normal E-C coupling and Ca2+release are unaffected by high myoplasmic concentrations ofl-lactate ion. Preliminary results have been reported previously in abstract form (27).
Isolation of skinned fibers.
The mechanically skinned fiber preparation described previously (17, 28) was used. Briefly, male Wistar rats (3–5 mo) were anesthetized and killed by halothane overdose, and both the extensor digitorum longus (EDL) and the soleus muscles were removed. Whole muscles were immediately placed under paraffin oil after dissection, and single fibers were mechanically skinned, leaving ∼70–90% of the fiber bulk. A segment of the skinned fiber was attached to a force transducer (KG3, Scientific Instruments) to monitor isometric force. The resting length of the fiber was measured, and the fiber was then stretched 20%, after which the fiber diameter was measured. The skinned fiber was then bathed in a 2-ml bath containing a potassium hexamethylenediamine tetraacetate (K-HDTA) solution (see below) for 2 min before being stimulated by rapid substitution of an appropriate solution. Fibers were used with their resting steady-state total SR Ca2+ content at pCa 7.1, which has previously been shown to be equivalent to the normal endogenous SR Ca2+content of a fiber (13). Consequently, fibers were not additionally loaded unless otherwise stated. All experiments were performed at 24 ± 1°C unless stated otherwise. The number of fibers used in any given experiment when statistical comparisons were made ranged between 3 and 17. Fibers were taken from at least two different rats.
Solutions for skinned fibers.
All chemicals were obtained from Sigma unless specified otherwise. The HDTA2− was obtained from Fluka, Buchs, Switzerland. The composition of standard solutions used in skinned fiber experiments is summarized in Table 1.
The K-HDTA solutions containing either 0.1 or 0.05 mM free Mg2+ were made by mixing appropriate volumes of a standard K-HDTA solution (1 mM free Mg2+ and K-HDTA without Mg2+). Confirmation of fiber types (i.e., fast- and slow-twitch) was achieved by exploiting the differences in the strontium (Sr2+) activation characteristics of fast- and slow-twitch fibers. The Sr2+ solution was mixed with an appropriate volume of the relaxing solution to achieve a free Sr2+ concentration ([Sr2+]) of 4 μM (pSr: −log10 [Sr2+] of 5.4). This [Sr2+] triggered near-maximum force in slow-twitch fibers, whereas no force response was produced in fast-twitch fibers because of an ∼10-fold difference in the Sr2+ sensitivity (22). In all solutions containing 40 mM l- ord-lactate and 20 mM pyruvate, the free acid replaced either an equimolar amount of HEPES buffer or HEPES and HDTA2− to maintain similar osmolality and ionic strength. Solutions containing 20 mM l-lactate were made by mixing appropriate amounts of the standard K-HDTA and the equivalent 40-mM l-lactate solutions together. Unless otherwise stated, all solutions had a free Mg2+ concentration ([Mg2+]) of 1 mM, pH of 7.10, and osmolality of 295 ± 5 mosmol/kg.
Determination of the force-Ca2+ relationship in skinned fibers.
To determine the relationship between force and Ca2+ in both EDL and soleus fibers, mechanically skinned fibers were treated with 2% (vol/vol) Triton X-100 in the relaxing solution for 5 min to destroy all membranes. Fibers were then washed for a further 2 min in a relaxing solution. Submaximal force responses were then elicited by exposure to a series of heavily buffered Ca-EGTA solutions with increasing free Ca2+ concentration ([Ca2+]) until maximum force was achieved in either the presence or absence of l-lactate (or pyruvate). Solutions with varying amounts of free Ca2+ were made by mixing specific volumes of the appropriate maximal solution and the relaxing solutions together. The protocol was repeated twice under each of the conditions (i.e., before and during exposure to lactate and pyruvate and after washout) and averaged. Between each run, fibers were returned to an appropriate relaxing solution. The free [Ca2+] for each solution was later determined by using a potentiometric method previously described (32). The Ca2+ sensitivity of the contractile apparatus was determined by normalizing submaximal force responses to the maximum Ca2+-activated force response obtained under the same conditions. The effects of l-lactate (or pyruvate) on the maximum Ca2+-activated force were determined by comparing the average of two maximum force responses obtained in the presence ofl-lactate (or pyruvate) to the average maximum force responses obtained before and after treatment. This eliminated any error caused by the gradual loss of force with repeated activation. Maximum force values in mechanically skinned fibers ranged between 30 and 40 N/cm2 in both EDL and soleus fibers and were consistent with previously published skinned-fiber data.
Depolarization-induced force responses.
As described previously (16, 17), the transverse tubular system (T system) can be rapidly depolarized by substituting a solution in which all the K+ is replaced with either Na+ or choline chloride (ChCl), and this depolarization initiates the normal sequence of E-C coupling events that leads to the production of a transient force response (see Fig.1 A). Typically, between 15 and 30 such depolarization-induced force responses can be elicited in the same fiber, provided that the fiber is returned to the high K+ concentration (for at least 20–30 s between successive depolarizations) to repolarize the T system. Skinned fibers were not additionally loaded with Ca2+, because they retained their preskinned “endogenous” level of SR Ca2+ after skinning.
In this study, both ChCl and Na+ were used as depolarizing stimuli. Depolarization of the T system with ChCl has been shown to be more effective than depolarization with Na+ because of the simultaneous increase in the myoplasmic Cl− concentration, which overcomes any polarizing effects of transverse tubular Cl− (7). Thus the effects ofl-lactate on both a weak (Na+) and strong (ChCl) depolarizing stimulus were examined to maximize the sensitivity of detection of any l-lactate effect on depolarization-induced responses. Depolarization-induced responses were only examined in fast-twitch (EDL) fibers in this study. Previous attempts by others (31) and us (data not shown) have revealed that depolarization of skinned slow-twitch fibers produces only small force responses (∼10% of the maximum Ca2+-activated force). It is thought that this reflects a lower density of voltage sensor-ryanodine receptor couplings in slow- compared with fast-twitch fibers (31).
Effects of l-lactate on depolarization-induced force responses.
A number of experiments were used to examine the effects ofl-lactate on depolarization-induced force responses. The effect of l-lactate on depolarization-induced force responses was examined in fibers by 1) exposing fibers continuously to l-lactate (immediately after skinning);2) intermittent exposure in which the fiber serves as its own control; and 3) a reverse protocol of intermittent exposure to rule out any natural decline in force with repeated activation. The details of these experiments are described inresults.
Effects of blockers of l-lactate transport and diffusion.
The effect of a specific blocker of l-lactate transport, quercetin [half-maximum inhibition of 3 μM (15)] was examined to eliminate any active accumulation of l-lactate into the T system. In addition, any passive diffusion ofl-lactate may involve movement through anion channels such as Cl− channels. Thus the effects of a Cl−channel blocker, anthracene-9-carboxylic acid (9-AC), on the ability of 20 mM l-lactate to inhibit E-C coupling was also examined. Fibers were first exposed to a solution containing the required concentration of inhibitor. When the amplitude of the force response reached a steady state, fibers were then exposed to thel-lactate solution containing the same concentration of inhibitor. After three responses and 1.5 min of exposure tol-lactate, fibers were then washed in the continued presence of the inhibitor. Both quercetin and 9-AC were dissolved in ethanol as a stock solution and diluted 1,000-fold in appropriate solutions with matching control solutions containing an equivalent amount of ethanol.
Effects of d-lactate and pyruvate on depolarization-induced responses.
We sought to determine whether structurally similar compounds produce similar effects on E-C coupling. In addition, the effects of higherl-lactate concentrations were also examined. The effects of 40 mM l-lactate, 20 mM d-lactate, and the structurally similar carboxylate, pyruvate (20 mM), were examined by treating fibers with each drug for 30 s and examining the first response to depolarization. Fibers were then washed for 30 s, and the subsequent response was measured. All responses were compared with the response to depolarization before treatment (as described above). Fibers were depolarized primarily by ChCl substitution, because the effects of either ChCl or Na+ were identical after a 30-s exposure to l-lactate (see Fig.2 C and Table 3).
Determination of net Ca2+ leak from the SR.
The methods employed in this study have been previously described in detail (5, 8, 28). Briefly, the SR of a skinned fiber was first depleted of virtually all the total SR Ca2+ content with a K-HDTA solution containing 0.05 mM free [Mg2+], 30 mM caffeine, and 0.5 mM total EGTA (termed the caffeine solution). Most of the SR Ca2+ content is released with the caffeine solution (13). Fibers were then washed in a buffered K-HDTA solution (with 0.5 mM total EGTA) for 1 min, Ca2+ loaded in a heavily buffered load solution (with 1 mM Ca-EGTA, pCa 6.7) for 30 s, and then briefly exposed to a buffered K-HDTA solution (with 1 mM EGTA) for 6 s to quench Ca2+ loading. Fibers were then equilibrated in a weakly buffered K-HDTA solution (with 150 μM total EGTA) for 30 s with or without 20 mM l-lactate before being equilibrated in an equivalent but more heavily buffered solution (with 0.5 mM total EGTA) for 30 s with or without l-lactate. This latter solution was termed the leak solution (because any Ca2+uptake was eliminated by the presence of 0.5 mM EGTA) and could be used to estimate the rate of Ca2+ leak from the SR. The fiber was then briefly washed in a weakly buffered K-HDTA solution for 6 s to predominantly wash l-lactate from the fiber, and then the Ca2+ remaining in the SR after this time was released by use of the caffeine solution. The area under the curve (integral) of the force response after treatment withl-lactate was then compared with the average integral of control responses before and after treatment. The integral of the caffeine response has been previously shown to be approximately linearly proportional to the length of time the fiber is loaded (5, 8, 28). Thus the integral of the force response provides a good indication of the amount of releasable Ca2+ in the SR. In this study, we were simply interested in whether l-lactate affected Ca2+leak, which would be reflected as either an increase or decrease in the integral of the response to caffeine.
Determination of the effect of l-lactate on Ca2+ release induced by lowering the free myoplasmic [Mg2+].
In both EDL and soleus fibers, Ca2+ release could also be elicited in endogenously loaded fibers by substituting a K+solution containing 0.1 mM free [Mg2+] for a K+ solution with 1 mM free [Mg2+]. This method differs from caffeine in its action in that it involves a removal of channel inhibition rather than direct channel activation (18, 19). This caused a rapid, large submaximal force response that would gradually terminate as the SR resequestered Ca2+. However, in this instance, Ca2+ release was terminated by simply reexposing the fiber to control solution containing 1 mM free [Mg2+] (e.g., Fig. 6). Provided that fibers were returned to the K+ solution with 1 mM free [Mg2+] for 2 min between responses (to restore any Ca2+ that may have been lost during the release), subsequent identical force responses could be elicited. In this way, fibers did not need to be additionally loaded with Ca2+ between responses. This procedure was repeated in the presence of 20 mM l-lactate after a 30-s preequilibration in a standard K-HDTA solution (1 mM Mg2+) containing 20 mM l-lactate. The force response obtained in the presence of l-lactate was then compared with the average responses before and after treatment. It should be noted that lowering the free [Mg2+] to 0.1 mM in these fibers did induce a large, although submaximal, amount of Ca2+ release and that the SR Ca2+ content was not finely controlled in these experiments (unlike the Ca2+-leak experiments described above).
Force traces and analysis.
The mechanically skinned muscle fiber was bathed in the standard K-HDTA solution (1 mM free Mg2+, 50 μM EGTA, pCa 7.0) in all force traces unless otherwise indicated. Data are reported in the text as means ± SE. Appropriate statistical analysis was carried out by either ANOVA or paired t-tests where appropriate, and results were considered significant if P < 0.05. Hill curves were fitted to the indicated data using Graphpad Prism software (v2.01, San Diego, CA). Parameters such as the Hill coefficient and 10 and 50% maximal Ca2+-activated force were derived from the individual curves and averaged.
Effects of l-lactate on the contractile apparatus.
Only mechanically skinned fibers were used in this study and will be simply referred to as “skinned fibers” from here on unless otherwise stated. l-Lactate (20 mM) significantly reduced the Ca2+ sensitivity in both EDL and soleus fibers, reducing the pCa required to produce 50% maximal Ca2+-activated force in both, with the largest effects observed in soleus. Table 2 shows the summarized mean data. In both EDL and soleus fibers, there was no change in the Hill coefficient, whereas the maximum Ca2+-activated force was reduced by ∼5%, respectively (see Table 2).
Examination of 20 mM pyruvate, a structurally similar carboxylate, produced similar effects on all parameters measured (see Table 2), with significant decreases in maximum Ca2+-activated force and larger inhibitory effects on the Ca2+ sensitivity in soleus than EDL. The effect of higher concentrations of l-lactate on force was not examined because the majority of subsequent experiments were carried out in the presence of 20 mMl-lactate.
Effect of continuous exposure to l-lactate on depolarization-induced responses.
As mentioned in methods, between 15 and 30 depolarization-induced force responses can be elicited in a single skinned fiber before force falls below 50% of the peak response in that fiber. This phenomenon is termed rundown and reflects a gradual loss of E-C coupling (17). It stands to reason that factors that inhibit E-C coupling will increase the overall rate of rundown in a fiber, particularly if submaximal force responses are more greatly affected. Figure 1 A illustrates a typical example of the effects of 20 mM l-lactate on depolarization-induced force responses in a single EDL fiber. In this experiment, the response to depolarization varies over time from near maximal to submaximal responses as rundown occurs. In this way we can examine the effects ofl-lactate on both maximum and submaximal force responses. Immediately after skinning, a fiber was exposed to a K-HDTA solution containing 20 mM l-lactate and was repeatedly depolarized with a Na+ solution until rundown had occurred, some 23 responses later. It can be seen that depolarization-induced responses could be elicited with no sudden sharp fall in the response to depolarization as responses became more submaximal as the fiber ran down.
In Fig. 1, B and C, the mean amplitudes of depolarization-induced force responses to ChCl and Na+substitution in control and l-lactate-treated fibers are shown. In each fiber, the amplitude of each force response to depolarization was normalized to the largest force response obtained in that fiber. Statistical analysis revealed no significant difference in the mean peak force response over time between control andl-lactate-treated fibers depolarized by ChCl (Fig.1 B). However, there was a significant difference in the mean peak force responses elicited by Na+ depolarization, which can be observed as a slight transient potentiation of the response to depolarization in l-lactate-treated fibers from between 8.5 and 9.5 min (Fig. 1 C).
Nevertheless, in control fibers, the average number of responses elicited by either ChCl or Na+ was not significantly different, with a mean of 20 ± 2 responses (n = 8) and 20 ± 3 responses (n = 6), respectively. The number of depolarization-induced responses in fibers treated with 20 mM l-lactate was also not statistically different from that in controls; mean of 20 ± 2 responses (n = 9) by ChCl substitution and 25 ± 6 responses (n = 6) by Na+ substitution.
Effect of intermittent exposure to l-lactate on depolarization-induced responses.
It is possible that fatigue concentrations of l-lactate (20 mM) may have more subtle effects on E-C coupling that were not revealed using previous protocols. To examine this, skinned fibers were exposed to l-lactate intermittently. Figure 2, A andB, shows examples of the effects of 20-mMl-lactate exposure on the response to depolarization by ChCl and Na+ substitution, respectively. After 30-s exposure to 20 mM l-lactate, the first response to depolarization was reduced by 15% (e.g., Fig. 2 A) and 4% (e.g., Fig. 2 B), respectively. Typically, the first response to depolarization by ChCl substitution was significantly reduced by about 7% of the control response before exposure to 20 mMl-lactate (see Fig. 2 C and Table3). In fibers depolarized by Na+ substitution, there was a similarly small reduction in the first response to depolarization by 20 mM l-lactate (see Fig. 2 C and Table 3). In fibers treated for 30 s in 40 mM l-lactate, depolarization-induced responses by either ChCl or Na+ substitution were also similarly reduced (see Table 3).
Longer exposure to 20 mM l-lactate did not affect subsequent depolarization-induced responses by ChCl substitution (e.g., Fig. 2 A). By 1.5 min of exposure and three responses, the mean response to depolarization was ∼90% of the control response before treatment (see Fig. 2 C and Table 3). In ChCl-stimulated fibers, washout of l-lactate after a 1.5-min exposure typically resulted in a small fall in the subsequent response to depolarization (see Fig. 2 A and Table 3). Additional washout did not cause any further recovery (e.g., Fig. 2,A and C).
In Na+ depolarized fibers, the second and third response in the presence of 20 mM l-lactate continued to fall, reaching a greater level of inhibition (e.g., Figs. 2 B and2 C). By 1.5 min of exposure and three depolarizations, the response to depolarization significantly fell by 18% (see Table 3) of the control response before treatment. This additional reduction in force may be attributed to the weaker ability of Na+ to depolarize the T system. After washout of l-lactate, the subsequent response to depolarization showed a significantly larger reduction in amplitude compared with the corresponding response to ChCl (e.g., Fig. 2, A–C). On average, the first response after washout fell by 36% of control responses beforel-lactate exposure (see Fig. 2 C, Table 3). Further washout produced some additional recovery in amplitude but responses never fully recovered back to initial control levels.
The relatively small effects of l-lactate on E-C coupling described above may reflect, in part, some degree of rundown in the fiber; that is, the gradual loss of force over time, irrespective ofl-lactate treatment. To eliminate this possibility, EDL fibers were exposed to a reverse of the protocol above, whereby fibers were exposed to 20 mM l-lactate immediately after skinning and depolarization-induced responses to ChCl were elicited (e.g., Fig.3.). Once the amplitude of the force response attained a steady level, fibers were then washed, and further depolarization-induced responses were elicited in the absence ofl-lactate until steady-state force was achieved.
In Fig. 3, it can be seen that the response to depolarization recovered by ∼16% after washout of l-lactate. The first response to depolarization after 30-s washout of l-lactate significantly recovered in amplitude by an average of 11 ± 3% in all five fibers examined. The increase in force was similar to the reduction in force seen when l-lactate was applied for either 30 s or 1.5 min after a control response (see Fig.2 C and Table 3). Interestingly, the response to depolarization after washout of l-lactate could apparently fully recover in these fibers (see Fig. 3). In contrast, the results described in the previous section show a reduction in the first response to depolarization after washout of l-lactate (see Fig. 2 C and Table 3).
When the fiber in Fig. 3 was returned to the l-lactate solution, the response to depolarization fell by an equivalent 16%, but this time the subsequent response after washout ofl-lactate also fell before recovering in a similar manner described earlier (see Fig. 2 C). This was observed in all five fibers. These results could be explained by an effect ofl-lactate on either the resting membrane potential or the osmolality of the T system and/or SR after l-lactate was reapplied and then removed and was most noticeable when fibers were first exposed to control solutions before l-lactate exposure.
Inhibitors of l-lactate transport do not alter the effect of l-lactate on E-C coupling.
The effect of l-lactate on depolarization-induced responses described above, particularly after washout, may be due to the movement of l-lactate into the sealed T tubules and/or SR lumen. To eliminate this possibility, inhibitors of the lactate transporter (quercetin) and Cl− channels (9-AC) were examined (seemethods).
Table 3 shows the summarized data. Neither 10 μM quercetin nor 100 μM 9-AC prevented the small reduction in the response to depolarization in the presence of 20 mM l-lactate. After both 30-s and 1.5-min exposures to l-lactate and inhibitor, the results were similar to those obtained in the absence of the inhibitors (i.e., Table 3). However, washout of l-lactate for 30 s resulted in the complete recovery of the response to depolarization in fibers treated with 100 μM 9-AC.
Effect of l-lactate on the repriming rate of depolarization-induced responses in EDL.
As mentioned in methods, provided that the fiber is returned to a K+ solution for 30 s to repolarize the T system, subsequent depolarization-induced responses of equivalent amplitude can still be elicited. The length of time that a fiber is required to repolarize between successive responses is determined by the rate at which the voltage sensors revert from their inactivated state back to their resting state. This time is termed the repriming period. If l-lactate were to enter the T system and dissociate, some effect on the resting membrane potential would be expected, given that l-lactate is largely charged at pH 7.1. An effect on the membrane potential would be expected to alter the repriming period in skinned fibers.
Figure 4 shows the mean force response to depolarization achieved after a given repriming period in the standard K+ solution. Submaximal depolarization-induced responses were normalized to the maximum response obtained after a 1-min repriming period in the presence or absence of 20 mMl-lactate. It can be seen that even after just a 6-s exposure to the K+ solution, depolarization could still evoke a large force response. In the absence of l-lactate, the response to depolarization after a 6-s repriming was 47 ± 7% (n = 7) of the response obtained after 1 min. In the presence of l-lactate, the response to depolarization after the same repriming period was not significantly different (mean of 47 ± 14%, n = 7). Clearly, the presence ofl-lactate did not alter the repriming rate. Importantly, these data additionally show that submaximal force responses were not affected by the presence of l-lactate.
Effects of d-lactate and pyruvate on depolarization-induced responses.
We next sought to determine whether structurally similar compounds produce similar effects on E-C coupling. The data are summarized in Table 3. Exposure to both d-lactate and pyruvate (20 mM) for 30 s significantly reduced the response to depolarization. After a 30-s washout, fibers completely recovered in each instance (e.g., Table 3). This data contrasts with the results obtained in fibers treated with l-lactate for up to 1.5 min (e.g., Fig.2 C and Table 3). However, fibers in this instance were only exposed to either compound for just 30 s. When seven other fibers were exposed to either 20 or 40 mM l-lactate for just 30 s, the response to depolarization after washout also fully recovered (see Table 3). Hence, it appears that longer exposure tol-lactate (1.5 min compared with 30 s) impairs the recovery of depolarization-induced responses after washout, and this was only obvious in fibers treated intermittently withl-lactate. This again suggests that l-lactate may be exerting osmotic effects within the T system and/or the SR lumen but that these changes are most apparent after the transition from thel-lactate solution back to the control solution.
We also examined the effects of l-lactate on depolarization-induced responses in two EDL fibers at elevated temperature (30°C). The first response to depolarization by ChCl substitution was similarly reduced in peak amplitude in the presence of 20 mM l-lactate by 10 and 5%, respectively, and this was similar to previous observations at room temperature (see Table 3). Subsequent responses were not additionally affected, and washout resulted in the same characteristic failure to recover completely as observed in fibers at room temperature. These data fit within the range of observed values at room temperature (see Table 3).
Effect of l-lactate on net Ca2+ leak from the SR.
The effects of l-lactate on the response to depolarization may be due in part to a reduction in Ca2+ release from the SR. To examine this, we subjected single fibers from both the EDL and the soleus to the leak protocol described in methods to determine the rate of Ca2+ release from the SR under conditions of zero Ca2+ uptake (see 5, 8, 28).
In Fig. 5, a single EDL fiber was subjected to the Ca2+-leak protocol. The first response represents the control response before l-lactate exposure. When the protocol was repeated and the fiber was exposed to 20 mMl-lactate for 30 s, the integral of the subsequent response to caffeine was reduced. Washout of l-lactate and a repeat of the protocol caused recovery of the response to caffeine. In all nine EDL fibers examined, the integral of the response to caffeine was significantly reduced after exposure tol-lactate by an average of 16 ± 5% of the average control response before and after l-lactate exposure. This would suggest that l-lactate actually increased Ca2+ leak to a small extent rather than inhibiting Ca2+ leak. In contrast, the integral of the caffeine response in soleus fibers was not significantly affected after treatment with l-lactate (mean of 100 ± 2%,n = 3).
Effect of l-lactate on low free [Mg2+] induced Ca2+ release from the SR.
To examine the effect of l-lactate on a weaker stimulus of Ca2+ release, Ca2+ release was elicited in skinned fibers from both EDL and soleus by exposure to a K+solution containing 0.1 mM free [Mg2+] (seemethods). By lowering the free [Mg2+], a force transient can be elicited by simple virtue of removing the Mg2+ inhibition of the Ca2+-release channels (18, 19). Unlike the solution typically used to empty the SR of Ca2+ (i.e., the caffeine solution; seemethods), in the absence of caffeine this free [Mg2+] should still exert some inhibition of Ca2+-release channel activity, and, thus, limit the extent of Ca2+ release to some degree.
The response to 0.1 mM free [Mg2+] was clearly reproducible (Fig. 6). After a second response to 0.1 mM free [Mg2+], fibers were exposed to 20 mM l-lactate in the last 30 s of equilibration. Ca2+ release was then elicited by exposure to a K+ solution with 0.1 mM free [Mg2+] and 20 mMl-lactate. In Fig. 6, the response to 0.1 mM free [Mg2+] in the presence of 20 mM l-lactate was only reduced in amplitude by 5%. In four EDL fibers and four soleus fibers examined, the mean response in the presence ofl-lactate was reduced to 97 ± 2% and 96 ± 0.3% of controls before and after treatment. Clearly,l-lactate did not greatly inhibit Ca2+ release. In fact, the small reduction in force was consistent with the reduction in the maximum Ca2+-activated force byl-lactate described earlier.
We sought to test the hypothesis that l-lactate would reduce force output by an inhibition of normal voltage-dependent Ca2+ release. We have demonstrated that high concentrations of l-lactate reduced depolarization-induced responses to a small extent but that this could be largely accounted for by a reduction in the maximum Ca2+-activated force.
Effect of 20 mM l-lactate on the contractile apparatus.
In both EDL and soleus fibers, the maximum Ca2+-activated force was shown to be reduced by ∼5% in the presence of 20 mMl-lactate (Table 2). Previous skinned-fiber studies have reported a similar reduction in maximum Ca2+-activated force in fast-twitch (psoas) and slow-twitch (soleus) fibers from the rabbit (2). In this study, the Ca2+sensitivity of the contractile apparatus was also reduced in both EDL and soleus, with the larger reduction in soleus (Table 2). Pyruvate (a related carboxylate) had similar effects to l-lactate on force, indicating that the effect of l-lactate on force was not unique (see Table 2). These data differed from previous reported effects of l-lactate in skinned fibers (2,9). Results by Andrews et al. (2) showed thatl-lactate (20–25 mM) had no effect on the pCa50 in either fast-twitch (psoas) or slow-twitch (soleus) fibers, although the maximum Ca2+-activated force was reduced in both fiber types, whereas the Hill coefficient was significantly reduced in psoas fibers alone. Chase and Kushmerick (9) showed that 50 mM l-lactate increased the maximum Ca2+-activated force by 5%. Such differences in the effects of l-lactate on the maximum Ca2+-activated force have been attributed to the differences in the major anion substituted for l-lactate in skinned-fiber solutions (2), because different anions have different effects on maximum force production (3). It is invariably difficult to balance ionic strength in solutions with differing anions (i.e., l-lactate) without altering the concentration of one or more of these constituents. Andrews et al. (2) substituted potassium l-lactate for potassium methanesulfonate (K-MeSO3 −) because K-MeSO3 − apparently had the least deleterious effect on maximum force. As with many other anionic salts, such as acetate used by Chase and Kushmerick (9), alteration of the ionic strength with K-MeSO3 − also affects maximum Ca2+-activated force but to a lesser extent than other anions (3). In this study, l-lactate was substituted for either HEPES or a combination of HEPES and HDTA2−. Interestingly, HDTA2− has been shown to have a similarly small effect on maximum Ca2+-activated force as K-MeSO3 − (3), although the effect of HEPES substitution is not known. Thus subtle differences in the effects of l-lactate on various parameters of force measurement between this study and others can be attributed to small variations in the composition of skinned-fiber solutions used. Nevertheless, in both this study and others, it is clear thatl-lactate exerted only a small effect on the contractile apparatus.
Effect of l-lactate on E-C coupling.
The application of l-lactate at fatiguing concentrations (20 and 40 mM) did not markedly inhibit E-C coupling in fast-twitch (EDL) fibers irrespective of whether responses were maximal or submaximal in nature, and in some instances it appeared that lactate actually transiently increased submaximal force during rundown (e.g., Fig. 1 C). The relatively small reduction in depolarization-induced responses (∼7–10%) with intermittent exposure can be accounted for by the reduction in the maximum Ca2+-activated force. Similar observations were made after application of a structurally similar carboxylate, pyruvate (20 mM), which affected both depolarization-induced force responses and maximum Ca2+-activated force to a similar extent. In support of this study, using chemically skinned EDL fibers, Andrews and Nosek (4) reported that l-lactate reduced Ca2+ uptake and increased Ca2+-induced Ca2+ release, which may account for the slight potentiation of submaximal responses near rundown in Na+-stimulated fibers in our study (e.g., Fig. 1 B). In intact muscle, Phillips et al. (26) showed that tetanic force elicited in whole mouse EDL and soleus muscles was unaffected by perfusion of 20 mMl-lactate for 30 min. Westerblad and Allen (34) previously showed that the fatigability of single fast-twitch fibers from the mouse perfused with 20 mM extracellularl-lactate was unaffected or slightly reduced if the extracellular pH was heavily buffered (i.e., with 24 mM NaHCO3). Similar results were observed by Mainwood et al. (23), who showed that twitch tension was potentiated byl-lactate, although tetanic force was depressed. The slight reduction in fatigability seen by Westerblad and Allen (34) may reflect similar mechanisms wherebyl-lactate perfusion actually increased force, as shown by Mainwood et al. (23) and described in soleus muscle by Pannier et al. (24). In the studies by Phillips et al. (26) and Westerblad and Allen (34), the intracellular pH did not change relative to controls on application ofl-lactate in resting (26), activated (26, 34), or fatigued (34) fibers. If we assume that the intracellular l-lactate concentration increased on application, then there is little evidence that the l-lactate ion per se affected E-C coupling under these conditions in these studies. However, l-lactate has also been reported to reduce force output by ∼10–14% after repeated activation in whole dog muscle when infused into the arterial blood supply (final blood lactate concentration 12–15 mM) (14). This reduction in force was similar to the reported effects of l-lactate on maximum Ca2+-activated force in this study and others (2) and could be attributed to such effects. Recently, Spangenburg et al. (30) reported that perfusion of isolated whole EDL muscles with 30–50 mM lactate reduced tetanic force by as much as 38% at 37°C. However, the effects described do not appear to be due to an intracellular effect of l-lactate, because force reduction occurred within the first 60–90 s of perfusing whole EDL muscles. When we examined the effects of 20 mM l-lactate at 30°C (at constant pH) in two fibers, we found that the first response to depolarization was reduced by 10 and 5%, respectively, which fell within the range of observations described at room temperature. Although these experiments were done at 30°C and not at 37°C, the absence of any increased effect observed here, along with the data of Hogan et al. (14), indicates that raised temperature does not obviously alter the effects of l-lactate on E-C coupling and force. The present results indicate thatl-lactate does not appear to reduce force output by reducing the ability of the voltage sensors to evoke Ca2+release.
The apparently larger effect of l-lactate on the response to depolarization in Na+-stimulated fibers could be explained by a comparatively weaker capacity of Na+, compared with ChCl, to depolarize the T system of skinned fibers. This may arise in part from the presence of Cl− within the T system exerting a polarizing effect on the T system membrane (7). Another possibility is that l-lactate affected the resting membrane potential of the T system, which in turn may affect the response obtained by Na+ substitution, given that l-lactate is largely dissociated at pH 7.1 [pK a 3.5; see Juel (15)]. However, it seems unlikely that l-lactate did affect the membrane potential to any large extent. First, the repriming period of depolarization-induced force responses was not significantly affected by 20 mM l-lactate (see Fig. 4). Second, depolarization-induced responses were not greatly inhibited byl-lactate after prolonged or intermittent exposure, irrespective of the concentration used (i.e., 20 or 40 mM; see Fig.2 C and Table 3). Third, quercetin, a known blocker of the lactate-transporter (15), did not prevent the small reduction in the response to depolarization in the presence ofl-lactate (see Table 3), nor did it allow the response to depolarization to recover after washout. This latter point suggests that if l-lactate enters the T system, it is unlikely that it was actively transported.
It was noted that after a 1.5-min exposure to l-lactate, the first response to depolarization after washout typically showed a greater degree of inhibition than the prior response obtained in the presence of l-lactate. The cause of this phenomenon is not known, although it may involve changes in osmotic gradients between the T system (and/or SR) and the myoplasm of the skinned fiber. Even though the solutions used in this study were balanced osmotically, any net passive movement of l-lactate into such compartments would result in an increase in the osmolality with respect to the myoplasm. When fibers were exposed to 9-AC (a Cl− channel blocker) in an attempt to block any passive movements of l-lactate, the response to depolarization in the presence of l-lactate showed the same typical reduction in force (see Table 3). However, the response immediately after washout (30 s) showed complete recovery in all three fibers examined (see Table 3). Such recovery was not typically seen in the absence of 9-AC in fibers exposed tol-lactate for 1.5 min. These data suggested that the reduction in force in the presence of l-lactate was not due to an effect on either E-C coupling or T system membrane potential and, as mentioned above, could be largely accounted for by the effect ofl-lactate on the contractile apparatus. However, possible diffusion of l-lactate into and out of the T system (and/or SR) appears to affect E-C coupling only after l-lactate is washed from the fiber, as this was possibly blocked by 9-AC. Passive movements of l-lactate into and out of such compartments may result in the subsequent swelling and shrinkage of such compartments due to a net diffusion of water. Such osmotic swelling/shrinking has been used as a method of disrupting the T system (termed osmotic shock) and may explain the washout phenomena and lack of complete reversibility after washout of l-lactate. Recently, Lännergren et al. (21) suggested that vacuolation of the T system observed in fatigued frog fibers may arise from the movement of l-lactate into the T system. However, such vacuolation was never observed in mouse fibers, and they suggested that this might be due to the differing morphology of the T system in the two species, resulting in better clearance of lactate in the rat. It is possible that the observed reduction in force after washout of lactate in our study is associated with some form of vacuolation (even though rat skeletal muscle fibers were examined here) because the T system in mechanically skinned fibers is sealed and would prevent any accumulated l-lactate from diffusing out.
Effects of l-lactate on active and passive Ca2+ release from the SR.
Fatigue concentrations of l-lactate (20–30 mM) have previously been shown to inhibit Ca2+ release from SR vesicles and ryanodine receptors incorporated into lipid bilayers (10, 11, 30). Examination of the net Ca2+ leak from the SR under nominal myoplasmic [Ca2+] (pCa < 8.0) in this study actually revealed a small increase in the rate of Ca2+ leak from the SR of EDL fibers in the presence of l-lactate. These data are similar to a recent report by Andrews and Nosek (4) that describes an absence of any effect of l-lactate on Ca2+ leak from the SR, although no numbers were cited.
Using previous estimates of net Ca2+ leak in EDL fibers derived from the area of the caffeine response under the same conditions [∼8–10 μM Ca2+/s (5,28)], the reduction in the area of the caffeine response by 16% in EDL fibers would only amount to an increase in the leakage rate by 1–2 μM Ca2+/s. However, given that the washout period before exposure to the caffeine solution was small (6 s), some, but perhaps not all, of the l-lactate may have been removed from the fiber in this time. Bearing in mind thatl-lactate reduced the Ca2+ sensitivity both in EDL and, to a larger extent, in soleus fibers, the small increase in the Ca2+ leak from EDL fibers would actually be slightly smaller than estimated. Similarly, in soleus fibers, the area of the caffeine response in the presence of l-lactate would also be larger than observed, indicating that the rate of Ca2+leak in soleus may have been reduced by ∼1–2 μM Ca2+/s. Such small effects are relatively insignificant, given that the peak rate of Ca2+ release on voltage-sensor activation is ∼1,000-fold higher (29).
When Ca2+ release was submaximally activated by lowering of the free [Mg2+] to 0.1 mM, the presence ofl-lactate did not affect Ca2+ release (e.g., Fig. 6). Nevertheless, the absence of any obvious effect ofl-lactate on peak Ca2+ release elicited by voltage-sensor activation does not preclude the possibility that the rate of Ca2+ release was unaffected. The small rates of resting Ca2+ leak from the SR were affected by the presence of l-lactate. Rather, these data indicate that the total Ca2+ release from the SR, rather than the Ca2+-release rate, was unaffected. However, even when the stimulus was such that the rate of Ca2+ release was submaximal [i.e., Ca2+ release induced depolarization (Fig. 4) or by 0.1 mM free Mg2+ (Fig. 6)], the effect ofl-lactate was still negligible. Because the rates of Ca2+ release can vary depending on the stimulus used, this suggests that only a very substantial reduction in the rate of Ca2+ release would result in a reduction in the total Ca2+ release, such as that induced either by a depolarization (6) or by lowering the free [Mg2+] as described above. Thus it is likely that the effect of l-lactate on the rate of Ca2+ release is underestimated in this study, because smaller, fast voltage-dependent Ca2+ release (similar to a twitch) cannot be elicited in mechanically skinned fibers. It would be interesting to examine what effect l-lactate had on twitchlike responses in which Ca2+ release is substantially smaller (and faster) than that attained by depolarization here. This may explain the differences in the reported effects of l-lactate described in previous studies in which the rate of Ca2+ release is typically examined (10, 11, 30).
In conclusion, our investigation of the effects ofl-lactate on E-C coupling at constant pH has revealed modest (∼5%) inhibitory effects on the maximum Ca2+-activated force, with little effect on either the voltage-dependent or passive release of Ca2+ from the SR. Thus these data demonstrate that the l-lactate ion per se will not greatly contribute to skeletal muscle fatigue.
This work was supported by the Australian Research Council, the National Health and Medical Research Council, and the Faculty of Medicine, The University of New South Wales, Sydney, Australia.
Address for reprint requests and other correspondence: G. S. Posterino, Dept. of Zoology, La Trobe Univ., Bundoora, Victoria 3083, Australia (E-mail:).
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