INTRODUCTION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

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 that L-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 extent L-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 of L-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 the L-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 of L-lactate ion. Preliminary results have been reported previously in abstract form (27).

	METHODS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

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.

2

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 of L-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. 1A). 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.

View larger version (28K): [in this window] [in a new window]   Fig. 1.   Effect of continuous exposure to 20 mM L-lactate on depolarization-induced force responses in single mechanically skinned extensor digitorum longus (EDL) fibers. Single EDL fibers were bathed in a K+ solution containing 20 mM L-lactate immediately after they were skinned, and depolarization-induced force responses were elicited by either Na+ (A and B) or ChCl (C) substitution. A: time scale is 2 s during depolarizations by Na+ and 30 s elsewhere. max, Maximum Ca2+-activated force at ~20 µM free Ca2+ concentration. Summarized data (B and C) show the mean amplitude of depolarization-induced force responses (expressed as percent of the largest response obtained in each fiber) in control (B, n = 8; C, n = 6) and lactate-treated (B, n = 9; C, n = 6) fibers. The number of fibers contributing to the mean value diminishes over time. Data points that bear no standard error bar represent single fibers that produced more than 24 responses before rundown. * A single fiber that produced more than 32 responses before rundown.

Effects of L-lactate on depolarization-induced force responses. A number of experiments were used to examine the effects of L-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 in RESULTS.

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 of L-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 the L-lactate solution containing the same concentration of inhibitor. After three responses and 1.5 min of exposure to L-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 higher L-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. 2C and Table 3).

View larger version (16K): [in this window] [in a new window]   Fig. 2.   Effect of intermittent exposure of 20 mM L-lactate on depolarization-induced force responses in EDL fibers. Depolarization-induced force responses were elicited by either ChCl (A) or Na+ (B) substitution in the presence and absence of 20 mM L-lactate. Time scale, 2 s during depolarizations by ChCl and 30 s elsewhere. C: mean amplitude (± SE) of the force response to depolarization by either choline chloride (ChCl; n = 17) or Na+ (n = 9) substitution in EDL fibers after application and subsequent washout of 20 mM L-lactate. Response in each fiber was expressed as a percentage of that obtained before addition of L-lactate.

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 with L-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.

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

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).

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 1A 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 of L-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.

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 and B, shows examples of the effects of 20-mM L-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. 2A) and 4% (e.g., Fig. 2B), respectively. Typically, the first response to depolarization by ChCl substitution was significantly reduced by about 7% of the control response before exposure to 20 mM L-lactate (see Fig. 2C and Table 3). 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. 2C 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).

View larger version (11K): [in this window] [in a new window]   Fig. 3.   Reverse protocol of intermittent L-lactate exposure. In this instance, an EDL fiber was initially exposed to 20 mM L-lactate immediately after skinning and was then washed to remove L-lactate. The protocol was then repeated again for comparison. This experiment showed that the reduction in force in the presence of L-lactate was not due simply to the natural decline in force with repeated activation (i.e., rundown). Note that the subsequent washout after readdition of L-lactate resulted in the same initial reduction in force described in Fig. 2, A and B. Time scale, 2 s during depolarizations by ChCl and 30 s elsewhere.

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 (see METHODS).

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.

View larger version (18K): [in this window] [in a new window]   Fig. 4.   Effect of 20 mM L-lactate on the repriming period of depolarization-induced responses. Skinned EDL fibers were repolarized in a K+ solution with or without 20 mM L-lactate for a given period of time ranging between 6 s and 1 min. Fibers were then fully depolarized with ChCl in the presence or absence of L-lactate. In each fiber, the control and L-lactate values were obtained. Responses were normalized to the maximum response in the absence or presence of L-lactate obtained after 1 min repriming. Values are means ± SE from 7 fibers.

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. 2C 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 to L-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 with L-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 the L-lactate solution back to the control solution.

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).

View larger version (9K): [in this window] [in a new window]   Fig. 5.   Effect of L-lactate on net Ca2+ leak from the SR in a mechanically skinned EDL fiber. The fiber was subjected to a series of load-and-deplete cycles in the presence and absence of 20 mM L-lactate and was always loaded for a constant period of 30 s (see METHODS). Note a reduction in the area of the caffeine response in the presence of L-lactate. Bars under the caffeine responses represent a faster chart speed of 2 s.

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⁺] (see METHODS). 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; see METHODS), 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.

View larger version (8K): [in this window] [in a new window]   Fig. 6.   Effect of 20 mM L-lactate on Ca2+ release induced by lowering the free Mg2+ concentration in a single soleus fiber. Lowering the free Mg2+ concentration bathing the muscle fiber from 1 mM to 0.1 mM induces massive Ca2+ release, resulting in a large, rapid force response (see first response). See METHODS for the protocol. In the presence of L-lactate, the force responses elicited were reduced by ~5%. Force responses were recorded at the fast time speed of 2 s.

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

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 mM L-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 that L-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 that L-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. 1C). 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. 1B). 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 mM L-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 extracellular L-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 by L-lactate, although tetanic force was depressed. The slight reduction in fatigability seen by Westerblad and Allen (34) may reflect similar mechanisms whereby L-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 of L-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 that L-lactate does not appear to reduce force output by reducing the ability of the voltage sensors to evoke Ca²⁺ release.

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.