Lactate and force production in skeletal muscle

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1 J Physiol (2005) pp Lactate and force production in skeletal muscle Michael Kristensen, Janni Albertsen, Maria Rentsch and Carsten Juel Copenhagen Muscle Research Centre, University of Copenhagen, Denmark Lactic acid accumulation is generally believed to be involved in muscle fatigue. However, one study reported that in rat soleus muscle (in vitro), with force depressed by high external K + concentrations a subsequent incubation with lactic acid restores force and thereby protects against fatigue. However, incubation with 20 mm lactic acid reduces the ph gradient across the sarcolemma, whereas the gradient is increased during muscle activity. Furthermore, unlike active muscle the Na + K + pump is not activated. We therefore hypothesized that lactic acid does not protect against fatigue in active muscle. Three incubation solutions were used: 20 mm Na-lactate (which acidifies internal ph), 12 mm Na-lactate +8 mm lactic acid (which mimics the ph changes during muscle activity), and 20 mm lactic acid (which acidifies external ph more than internal ph). All three solutions improved force in K + -depressed rat soleus muscle. The ph regulation associated with lactate incubation accelerated the Na + K + pump. To study whether the protective effect of lactate/lactic acid is a general mechanism, we stimulated muscles to fatigue with and without pre-incubation. None of the incubation solutions improved force development in repetitively stimulated muscle (Na-lactate had a negative effect). It is concluded that although lactate/lactic acid incubation regains force in K + -depressed resting muscle, a similar incubation has no or a negative effect on force development in active muscle. It is suggested that the difference between the two situations is that lactate/lactic acid removes the negative consequences of an unusual large depolarization in the K + -treated passive muscle, whereas the depolarization is less pronounced in active muscle. (Resubmitted 25 October 2004; accepted 11 November 2004; first published online 18 November 2004) Corresponding author C. Juel: Copenhagen Muscle Research Centre, August Krogh Institute, Universitetsparken 13, DK-2100 Copenhagen, Denmark. cjuel@aki.ku.dk The exercise-induced accumulation of lactic acid in skeletal muscle and the resulting decrease in cellular ph have been widely considered to contribute to fatigue (for review see Westerblad et al. 1991; Fitts, 1994). However, newer studies have pointed out that acidification has only a minor negative effect on force production at body temperature (Pate et al. 1995, Westerblad et al. 1997). It was a surprise when Nielsen et al. (2001) demonstrated that lactic acid incubation resulted in regained force in K + -depressed muscle. The authors concluded that lactic acid has a protective role on force production in muscle (Nielsen et al. 2001), which was confirmed in a succeeding paper (Pedersen et al. 2003). The underlying mechanism has been described in a recent paper (Pedersen et al. 2004). It was shown that intracellular acidosis decreases chloride permeability in the t-tubules, which allows action potentials to be propagated despite muscle depolarization. In the study by Nielsen et al. (2001), resting soleus muscle was incubated in high potassium concentrations to mimic the concentrations reported for interstitial potassium during high-intensity exercise (Nielsen et al. 2004). This treatment does not activate the Na + K + pump, and therefore leads to a larger depolarization than obtained if potassium accumulation was the result of muscle activity, which also activates the pump. Furthermore, the K + -treated muscles were incubated with lactic acid, which lowered the extracellular ph more than the intracellular ph; thus reducing the ph gradient across sarcolemma. In some experiments the ph gradient was essentially abolished (in fact a small gradient in the opposite direction was obtained). In contrast, when muscles are stimulated to fatigue, lactate and H + accumulate intracellularly resulting in a large internal ph decrease (Juel et al. 2004), whereas the external (interstitial) ph is affected to a lesser degree (Street et al. 2001). Consequently, during normal muscle activity, the ph gradient across the sarcolemma is increased. It is therefore uncertain whether the protective effect of lactic acid is a general mechanism that is also active during normal muscle activity. Regulation of cellular ph in skeletal muscle is dependent on the activity of the Na + /H + exchanger and bicarbonate-dependent transport systems (Juel, 1998). DOI: /jphysiol

2 522 M. Kristensen and others J Physiol Both Na + /H + exchange and Na + /bicarbonate cotransport mediate an influx of Na +.Since the Na + K + pump is stimulated by Na + influx (Buchanan et al. 2002), it is a possibility that the influx of Na + during ph regulation is sufficient to stimulate the Na + K + pump. Furthermore, since there is a close relation between pump activity and membrane potential (Overgaard & Nielsen, 2001), this may in turn affect the membrane potential and muscle excitability. The present study investigated the effect of lactic acid, Na-lactate and a combination of lactic acid and Na-lactate on muscle force. Since an activation of the Na + K + pump may take place during some of the experimental conditions, we have measured potassium uptake during similar conditions. In addition, in order to evaluate the effect of internal lactic acid on muscle fatigue we compared force production during fatiguing stimulation in control muscle and muscles pre-incubated with Na-lactate, lactic acid or a combination of lactic acid and Na-lactate. We hypothesized that ph regulation stimulates the Na + K + pump, which in turn may influence muscle function. Furthermore, lactic acid accumulation in active muscle is not expected to protect the muscle from developing fatigue. In contrast, if lactic acid accumulation has any influence on muscle force the effect is expected to be negative. Methods The handling of animals was in accordance with Danish animal welfare regulations. The experiments were carried out using soleus muscles from male Wistar rats (body weight g) killed by decapitation. Electrical stimulation Muscles were isolated and placed in a muscle chamber with an isometric force transducer (Danish Myo Technology). Muscles were stimulated with Ag/AgCl electrodes and the force development digitalized and computer recorded. The standard incubation media was (mm) 122 NaCl, 25 NaHCO 3, 2.8 KCl, 1.2 KH 2 PO 4, 1.2 MgSO 4, 1.3 CaCl 2, 5 d-glucose bubbled with 5% CO 2 95% O 2 at 30 C (ph 7.4). In experiments with a high K + concentration, an equivalent amount of Na + was omitted. One experiment was carried out in a bicarbonate-free Tris buffer (mm): 147 NaCl, 2.8 KCl, 1.2 KH 2 PO 4, 1.2 MgSO 4, 1.3 CaCl 2, 10 Tris buffer, and ph adjusted (to 7.4 and 6.8) with HCl. In the experiments with Na-lactate, a similar amount of NaCl was omitted. In the main experiment, a short tetanic contraction was obtained every 10 min using 30 Hz pulse trains of 1.5 s duration and supramaximal voltage. A 30 Hz stimulation was chosen because this frequency falls within the middle of the discharge rate frequency curve for soleus (Henning & Lomo, 1985). In the fatigue experiments, muscles were stimulated every 3 s with a 33 Hz pulse-train lasting 1 s. One soleus muscle from each rat was incubated for 15 min in the test solution, the other soleus muscle served as control. Activity of the Na + K + pump The activity of the Na + K + pump was quantified according to the method of Buchanan et al. (2002). In short, after equilibration for 15 min, the resting muscle was incubated for 10 min in the standard incubation media containing 0.5 µci ml 1 86 Rb +.After incubation the resting muscle was washed twice for 30 min in a large volume of ice-cold sucrose buffer containing (mm) 263 sucrose, 10 Tris-HCl, 2.8 KCl, 1.3 CaCl 2, 1.2 MgSO 4, 1.2 KH 2 PO 4 (ph 7.4). The washed muscle was blotted on paper, weighed and the 86 Rb + activity was determined in a β-counter. The 86 Rb + uptake was converted to K + uptake by using the 86 Rb + activity and the K + concentration in the incubation medium. Statistics Data are expressed as means ± s.e.m. In the fatigue experiments the force development in control muscle and muscle incubated with lactic acid or Na-lactate were compared using two-way ANOVA for repeated measures. A t test was used to identify the points of difference with significance set at P < Results The combined effect of ph and elevated K + An increase in the external K + concentration from 4to 10 mm reduced the tetanic force to 20 25% of the control force obtained at 4 mm K +. The effect evolved slowly and a force plateau was seen after approximately 100 min incubation. A subsequent addition of 20 mm lactic acid (with 10 mm K + still present) led to force recovery. As a mean, 40 min incubation with 20 mm lactic acid increased the force to 80% of the initial force (Fig. 1). The addition of 20 mm Na-lactate to muscle in which force had been depressed by 10 mm K +, induced a partial and slowly evolving recovery in force. Tris-buffer (ph 6.8) had no effectonforceink + -depressed muscle (Fig. 1). Incubation of K + -treated muscle with a mixture of 12 mm Na-lactate + 8mm lactic acid (ph 7.18) led to a nearly complete force recovery (to 80% of initial force). The cost of ph regulation Since ph regulation is associated with an influx of Na + (Juel, 1998), which may stimulate the Na + K + pump, we measured the potassium uptake rate during the experimental conditions used in the previous experiments. The effect of ouabain (10 3 m) demonstrated that the main fraction of the potassium uptake in resting

3 J Physiol Lactate and force production in skeletal muscle 523 soleus muscle is mediated by the Na + K + pump (Fig. 2). Adding 20 mm Na-lactate significantly increased the rate of K + uptake compared to control (n = 8, P < 0.05). The increase in pump activity was approximately 43% if only the ouabain-suppressible component is taken into consideration. The pump activity was lower (P < 0.05) with 12 mm Na-lactate + 8mm lactic acid compared to 20 mm Na-lactate, and even lower (P < 0.05) if muscles were incubated with 20 mm lactic acid. Incubation for 15 min with 0.5 mm DIDS (inhibitor of Na-bicarbonate cotransport) mm amiloride (inhibitor of Na + /H + exchange) before the addition of 20 mm Na-lactate, reduced (P < 0.05) the potassium uptake to a value lower than control, but similar to the rate obtained with 20 mm lactic acid. Effect of lactate on fatigue development The three lactate-containing test solutions (Na-lactate, Na-lactate/lactic acid, and lactic acid) all improved the force development in K + -depressed muscle (Fig. 1). We therefore tested the three solutions in association with continuous muscle activation. The force production during repetitive stimulation was gradually reduced and was approximately 15% of the initial force after 5 min stimulation. The fatigue development in muscle pre-incubated for 15 min with 20 mm Na-lactate was compared to control (Fig. 3A). An analysis of variance including all data points revealed a strong tendency for a lower force development in muscle incubated with 20 mm Na-lactate (P = 0.056, n = 16). If the data points for the first 2 min of the stimulation period were tested separately, a significantly lower force was obtained in the muscles incubated with 20 mm Na-lactate (P < 0.05). Another series of muscle was incubated with 12 mm Na-lactate + 8mm lactic acid (Fig. 3B). This treatment had no significant effect on force development. Finally, a series of muscles were incubated with 20 mm lactic acid (Fig. 3C). For some data points force development was lower in the lactic acid-incubated muscle. Overall, the stimulation experiments did not support any protective effectof lactate/lactic acid on force development in active muscle. Discussion Muscle force in soleus muscle recovered if K + -depressed muscle was incubated with 20 mm lactic acid (Fig. 1). We were therefore able to reproduce the experiments by Nielsen et al. (2001). In that study, incubation with 20 mm lactic acid reduced extracellular ph by 0.64 units (from 7.44 to 6.80), whereas intracellular ph was reduced by 0.39 units (from 7.28 to 6.89). Thus, the transmembrane ph gradient was essentially abolished (in fact a minor gradient in the opposite direction was induced). The interstitial ph in human skeletal muscle has been measured with the microdialysis technique (Street et al. Figure 1. Effect of 20 mm lactic acid or 20 mm Na-lactate or HCl titration on tetanic force in muscle pre-incubated with 10 mm K +,effect of 20 mm lactic acid on force development in muscle pretreated with 10 mm K + (n = 8)., effect of 20 mm Na-lactate (n = 14). muscles were pre-incubated with 10 mm K + in Tris-buffer ph 7.4 and subsequently incubated with Tris-buffer ph 6.8 titrated with HCl (no lactate present) (n = 3)., Effect of a mixture of 12 mm Na-lactate and 8 mm lactic acid (n = 4). Error bars show S.E.

4 524 M. Kristensen and others J Physiol ). It was found that interstitial ph was reduced by 0.34 units (from 7.38 to 7.04) during intense exercise, whereas the intracellular ph in fatigued human muscles was reduced by 0.45 units (from 7.14 to 6.69) (Juel et al. 2004). The last ph reduction is probably an underestimation, as ph was measured in muscle homogenates. In any case, there remains no doubt that the ph gradient across sarcolemma in fatigued muscle exceeds the ph gradient at rest. In order to evaluate the effect of external and internal ph changes we used incubations with the Na + salt of lactic acid (Na-lactate) or acombination of Na-lactate and lactic acid. In both cases the inwardly directed lactate gradient stimulates lactate influx mediated by the lactate/h + cotransports and the influx of undissociated lactic acid. Therefore, in both cases H + enters the cell together with lactate ions in a 1 : 1 manner (Juel & Halestrap, 1999). Because of this coupling between lactate and H + transport, both the internal lactate concentration and the H + load will be similar whether the muscle is incubated with lactic acid or Na-lactate (Juel, 1997). In the experiments with Na-lactate, only the internal ph is changed. The treatment with Na-lactate led to a slow and only partial force recovery and there was a large variation between experiments. In order to more closely simulate the ph changes at fatigue we incubated muscles in a mixture of 12 mm Na-lactate + 8mm lactic acid, which acidifies external ph less than internal ph. This treatment Figure 2. The effect of ph regulation on potassium uptake in resting muscle Control: potassium uptake in resting soleus muscle (n = 8). Ouabain: effect of 10 3 M ouabain on potassium uptake (n = 4). Na-lactate: effect of 20 mm Na-lactate on potassium uptake (n = 8). Na-lac/l. acid: effect of 12 mm Na-lactate +8 mm lactic acid (n = 8). L. acid: effect of 20 mm lactic acid (n = 4). DIDS + AMIL: effect of 0.5 mm DIDS and 0.5 mm amiloride on potassium uptake in muscle incubated with 20 mm Na-lactate (n = 4). Significantly different from control; # significantly different from each other. Error bars show S.E. led to a fast force recovery of a magnitude similar to the one obtained with 20 mm lactic acid. If the external ph is changed with HCl titration and no lactate present, the internal ph will only change by approximately 10% of the external ph change (Aickin & Thomas, 1977). In a series of experiments, we used HCl to mainly reduce external ph; this treatment did not induce force recovery. Based on these experiments the underlying mechanism for force recovery in K + -incubated soleus muscle can be discussed. One mechanism could be a stimulation of the Na + K + pump by the Na + influx (Buchanan et al. 2002) during ph regulation, which could lead to a hyperpolarization (Overgaard et al. 1999) and restoration of excitability. Incubation with Na-lactate increased the ouabain-sensitive pump activity by 43% (Fig. 2). The activity was lower with 12 mm Na-lactate + 8mm lactic acid, and even lower with 20 mm lactic acid. Thus, there was a positive correlation between the ph gradient across sarcolemma and the rate of K + uptake (P < 0.05), reflecting that ph regulation is dependent on the existence of a ph gradient. However, stimulation of the pump cannot be the sole explanation for force recovery in K + -depressed muscle; although the pump was stimulated with Na-lactate incubation, lactic acid incubation led to force recovery in K + -depressed muscle without affecting the pump. Also, Nielsen et al. (2001) found that potassium uptake is not increased by lactic acid incubation, and it was argued that the underlying mechanism for the force recovery during lactic acid incubation is related to the changes in ph. The present study demonstrated that a reduction in the ph gradient is not a necessity for force recovery in K + -depressed muscle, although it may contribute. Another important factor could be the acidification during incubation. Although the H + load on the intracellular compartment is the same with the three test solutions, the muscle is able to counteract the acidification during Na-lactate/lactic acid and Na-lactate infusion. Therefore, the resulting acidification is expected to be less in Na-lactate and Na-lactate/lactic acid than in the experiments with lactic acid. The present experiments confirmed that lactate and lactic acid incubation could lead to force recovery in K + -depressed muscle. The question is whether this is a general mechanism active during fatiguing muscle activity, as suggested by Nielsen et al. (2001), or whether the mechanism is only seen in passive muscle pre-incubated with high external K +. In an attempt to solve this problem we stimulated isolated rat soleus muscle to fatigue. In three independent series of experiments we compared control muscle and muscle pre-incubated with either 20 mm Na-lactate, 12 mm Na-lactate + 8mm lactic acid, or 20 mm lactic acid (4 mm K +,ph7.4). In these experiments, the pre-incubation is expected to lead to

5 J Physiol Lactate and force production in skeletal muscle 525 internal lactate and H + accumulation from the start of the stimulation period. In addition, the added external lactate will inhibit the efflux of lactate and H + formed in the muscle fibres during stimulation. In the first two minutes of the stimulation period there was a significant reduction of force development in muscle incubated with 20 mm Na-lactate (Fig. 3A), and a tendency for force reduction with Na-lactate/lactic acid, and lactic acid (Fig. 3B and C). Therefore, this experimental setup could not detect any sign of a protective role of lactate/lactic acid incubation against fatigue development in active muscle. A depressive effect of lactate on tetanic force has been reported for mouse muscles (Spangenburg et al. 1998). The three combinations of lactate/lactic acid restored (more or less) the force development in passive soleus Figure 3. Effect of lactate and lactic acid incubation on force development during fatiguing stimulation From each rat one soleus muscle was incubated with the test solution for 15 min, the other soleus muscle served as control. The values represent the mean force (± S.E.M.) read every 30 s for 5 min., controls., muscles incubated with the test solution. A, muscles were incubated with 20 mm Na-lactate. n = 16 for both series. Control and Na-lactate values significantly different (P < 0.05); #, P = B, muscles incubated with 12 mm Na-lactate + 8 mm lactic acid (n = 8). C, muscles incubated with 20 mm lactic acid (n = 9). Control and values for lactic acid incubation significantly different, P < 0.05

6 526 M. Kristensen and others J Physiol muscle incubated with a high K + concentration, whereas lactate/lactic acid had no protective effect in repetitively stimulated muscle. What is the reason for this difference? The resting muscles were incubated with high K + without activating the Na + K + pump, whereas in active muscle the pump is stimulated by Na + influx during the action potentials and due to Na + influx caused by the ph regulating transport systems (Juel, 1998) as demonstrated in the present study (in vivo the pump is also stimulated by hormones). Therefore, the depolarization in the K + -incubated muscle is expected to be more pronounced than in active muscle. The difference between the two situations could therefore be that lactate/lactic acid removes the negative consequences of an unusually large depolarization in the K + -treated passive muscle. In line with this argument, muscle activity and activation of the pump have been demonstrated to hyperpolarize muscle cells and to restore force in K + -depressed muscle (Overgaard & Nielsen, 2001). In conclusion, we have demonstrated that lactate and lactic acid can lead to force recovery in passive soleus muscle incubated with high K +.However, this is not a general mechanism; lactate/lactic acid incubation has no protective role in active muscle. References Aickin CC & Thomas RC (1977). Micro-electrode measurement of the intracellular ph and buffering power of mouse soleus muscle fibers. JPhysiol 267, Buchanan R, Nielsen OB & Clausen T (2002). Excitation- and β 2 -agonist-induced activation of the Na + K + pump in rat soleus muscle. JPhysiol 545, Fitts RH (1994). Cellular mechanisms of muscle fatigue. Physiol Rev 74, HenningR&LomoT(1985). Firing patterns of motor units in normal rats. Nature 314, Juel C (1997). Lactate-proton transport in skeletal muscle. Physiol Rev 77, Juel C (1998). Muscle ph regulation: role of training. Acta Physiol Scand 162, Juel C & Halestrap AP (1999). Lactate transport in skeletal muscle role and regulation of the monocarboxylate transporter. JPhysiol 517, Juel C, Klarskov C, Nielsen JJ, Krustrup P, Mohr M & Bangsbo J (2004). Effect of high-intensity intermittent training in lactate and H + release from human skeletal muscle. Am JPhysiol Endocrinol Metab 286, E245 E251. Nielsen JJ, Mohr M, Klarskov C, Kristensen M, Krustrup P, Juel C&Bangsbo J (2004). Effect of high-intensity intermittent training on potassium kinetics and performance in human skeletal muscle. JPhysiol 554, Nielsen OB, Paoli F & Overgaard K (2001). Protective effects of lactic acid on force production in rat skeletal muscle. JPhysiol 536, Overgaard K, Nielsen OB, Flatman JA & Clausen T (1999). Relations between excitability and contractility in rat soleus muscle: role of the Na + -K + pump and Na + -K + gradients. JPhysiol 518, Overgaard K & Nielsen OB (2001). Activity-induced recovery of excitability in K (+)-depressed rat soleus muscle. Am J Physiol Regul Integr Comp Physiol 280, R48 R55. Pate E, Bhimani M, Franks-Shiba K & Cooke R (1995). Reduced effect of ph on skinned rabbit psoas muscle mechanics at high temperature: implications for fatigue. JPhysiol 486, Pedersen TH, ClausenT&NielsenOB(2003). Loss of force induced by high extracellular [K + ]inratmuscle: effect of temperature, lactic acid and β 2 -agonist. JPhysiol 551, Pedersen TH, Nielsen OB, Lamb GD & Stephenson DG (2004). Intracellular acidosis enhances the excitability of working muscle. Science 305, Spangenburg EE, Ward CW & Williams JH (1998). Effect of lactate on force production by mouse EDL muscle: implications for the development of fatigue. Can J Physiol Pharmacol 76, Street D, Bangsbo J & Juel C (2001). Interstitial ph in human skeletal muscle during and after dynamic graded exercise. JPhysiol 537, Westerblad H, Bruton JD & Lännergren J (1997). The effect of intracellular ph on contractile function of intact, single fibers of mouse muscle declines with increasing temperature. JPhysiol 500, Westerblad H, Lee JA, Lännergren J & Allen DG (1991). Cellular mechanisms of fatigue in skeletal muscle. Am J Physiol Cell Physiol 261, C195 C209. Acknowledgement This work was supported by The Danish National Research Foundation.