Lactate and force production in skeletal muscle

J Physiol 562.2 (2005) pp 521–526 521

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, onestudy 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 protectsagainst fatigue. However, incubation with 20 mM lactic acid reduces the pH gradient acrossthe sarcolemma, whereas the gradient is increased during muscle activity. Furthermore, unlikeactive muscle the Na+–K+ pump is not activated. We therefore hypothesized that lactic aciddoes 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 thepH changes during muscle activity), and 20 mM lactic acid (which acidifies external pH morethan internal pH). All three solutions improved force in K+-depressed rat soleus muscle. The pHregulation associated with lactate incubation accelerated the Na+–K+ pump. To study whetherthe protective effect of lactate/lactic acid is a general mechanism, we stimulated muscles tofatigue with and without pre-incubation. None of the incubation solutions improved forcedevelopment in repetitively stimulated muscle (Na-lactate had a negative effect). It is concludedthat although lactate/lactic acid incubation regains force in K+-depressed resting muscle, a similarincubation has no or a negative effect on force development in active muscle. It is suggestedthat the difference between the two situations is that lactate/lactic acid removes the negativeconsequences of an unusual large depolarization in the K+-treated passive muscle, whereas thedepolarization 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. Email: [email protected]

The exercise-induced accumulation of lactic acid inskeletal muscle and the resulting decrease in cellular pHhave been widely considered to contribute to fatigue (forreview see Westerblad et al. 1991; Fitts, 1994). However,newer studies have pointed out that acidification hasonly a minor negative effect on force production at bodytemperature (Pate et al. 1995, Westerblad et al. 1997). Itwas a surprise when Nielsen et al. (2001) demonstratedthat lactic acid incubation resulted in regained force inK+-depressed muscle. The authors concluded that lacticacid has a protective role on force production in muscle(Nielsen et al. 2001), which was confirmed in a succeedingpaper (Pedersen et al. 2003). The underlying mechanismhas been described in a recent paper (Pedersen et al.2004). It was shown that intracellular acidosis decreaseschloride permeability in the t-tubules, which allowsaction potentials to be propagated despite muscledepolarization.

In the study by Nielsen et al. (2001), resting soleusmuscle was incubated in high potassium concentrationsto mimic the concentrations reported for interstitialpotassium during high-intensity exercise (Nielsen et al.

2004). This treatment does not activate the Na+–K+

pump, and therefore leads to a larger depolarization thanobtained if potassium accumulation was the resultof muscle activity, which also activates the pump.Furthermore, the K+-treated muscles were incubatedwith lactic acid, which lowered the extracellular pHmore than the intracellular pH; thus reducing the pHgradient across sarcolemma. In some experiments thepH gradient was essentially abolished (in fact a smallgradient in the opposite direction was obtained). Incontrast, when muscles are stimulated to fatigue, lactateand H+ accumulate intracellularly resulting in a largeinternal 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, thepH gradient across the sarcolemma is increased. It istherefore uncertain whether the protective effect of lacticacid is a general mechanism that is also active duringnormal muscle activity.

Regulation of cellular pH in skeletal muscle isdependent on the activity of the Na+/H+ exchanger andbicarbonate-dependent transport systems (Juel, 1998).

C© The Physiological Society 2004 DOI: 10.1113/jphysiol.2004.078014

522 M. Kristensen and others J Physiol 562.2

Both Na+/H+ exchange and Na+/bicarbonate cotransportmediate an influx of Na+. Since the Na+–K+ pump isstimulated by Na+ influx (Buchanan et al. 2002), it is apossibility that the influx of Na+ during pH regulation issufficient to stimulate the Na+–K+ pump. Furthermore,since there is a close relation between pump activity andmembrane potential (Overgaard & Nielsen, 2001), thismay in turn affect the membrane potential and muscleexcitability.

The present study investigated the effect of lactic acid,Na-lactate and a combination of lactic acid and Na-lactateon muscle force. Since an activation of the Na+–K+

pump may take place during some of the experimentalconditions, we have measured potassium uptake duringsimilar conditions. In addition, in order to evaluatethe effect of internal lactic acid on muscle fatigue wecompared force production during fatiguing stimulationin control muscle and muscles pre-incubated withNa-lactate, lactic acid or a combination of lactic acid andNa-lactate.

We hypothesized that pH regulation stimulates theNa+–K+ pump, which in turn may influence musclefunction. Furthermore, lactic acid accumulation in activemuscle is not expected to protect the muscle fromdeveloping fatigue. In contrast, if lactic acid accumulationhas any influence on muscle force the effect is expected tobe negative.

Methods

The handling of animals was in accordance with Danishanimal welfare regulations. The experiments were carriedout using soleus muscles from male Wistar rats (bodyweight 60–70 g) killed by decapitation.

Electrical stimulation

Muscles were isolated and placed in a muscle chamber withan isometric force transducer (Danish Myo Technology).Muscles were stimulated with Ag/AgCl electrodes and theforce development digitalized and computer recorded.The standard incubation media was (mm) 122 NaCl, 25NaHCO3, 2.8 KCl, 1.2 KH2PO4, 1.2 MgSO4, 1.3 CaCl2,5 d-glucose bubbled with 5% CO2–95% O2 at 30◦C(pH 7.4). In experiments with a high K+ concentration, anequivalent amount of Na+ was omitted. One experimentwas carried out in a bicarbonate-free Tris buffer (mm):147 NaCl, 2.8 KCl, 1.2 KH2PO4, 1.2 MgSO4, 1.3 CaCl2,10 Tris buffer, and pH adjusted (to 7.4 and 6.8) withHCl. In the experiments with Na-lactate, a similar amountof NaCl was omitted. In the main experiment, a shorttetanic contraction was obtained every 10 min using 30 Hzpulse trains of 1.5 s duration and supramaximal voltage.A 30 Hz stimulation was chosen because this frequencyfalls within the middle of the discharge rate–frequencycurve for soleus (Henning & Lomo, 1985). In the fatigue

experiments, muscles were stimulated every 3 s with a33 Hz pulse-train lasting 1 s. One soleus muscle from eachrat was incubated for 15 min in the test solution, the othersoleus muscle served as control.

Activity of the Na+–K+ pump

The activity of the Na+–K+ pump was quantifiedaccording to the method of Buchanan et al. (2002). Inshort, after equilibration for 15 min, the resting musclewas incubated for 10 min in the standard incubationmedia containing 0.5 µCi ml−1 86Rb+. After incubationthe resting muscle was washed twice for 30 min in a largevolume of ice-cold sucrose buffer containing (mm) 263sucrose, 10 Tris-HCl, 2.8 KCl, 1.3 CaCl2, 1.2 MgSO4, 1.2KH2PO4 (pH 7.4). The washed muscle was blotted onpaper, weighed and the 86Rb+ activity was determinedin a β-counter. The 86Rb+ uptake was converted toK+ uptake by using the 86Rb+ activity and the K+

concentration in the incubation medium.

Statistics

Data are expressed as means ± s.e.m. In the fatigueexperiments the force development in control muscleand muscle incubated with lactic acid or Na-lactate werecompared using two-way ANOVA for repeated measures.A t test was used to identify the points of difference withsignificance set at P < 0.05.

Results

The combined effect of pH and elevated K+

An increase in the external K+ concentration from 4 to10 mm reduced the tetanic force to 20–25% of the controlforce obtained at 4 mm K+. The effect evolved slowlyand a force plateau was seen after approximately 100 minincubation. A subsequent addition of 20 mm lactic acid(with 10 mm K+ still present) led to force recovery. As amean, 40 min incubation with 20 mm lactic acid increasedthe force to 80% of the initial force (Fig. 1).

The addition of 20 mm Na-lactate to muscle in whichforce had been depressed by 10 mm K+, induced a partialand slowly evolving recovery in force. Tris-buffer (pH 6.8)had no effect on force in K+-depressed muscle (Fig. 1).Incubation of K+-treated muscle with a mixture of 12 mmNa-lactate + 8 mm lactic acid (pH 7.18) led to a nearlycomplete force recovery (to 80% of initial force).

The cost of pH regulation

Since pH regulation is associated with an influx ofNa+ (Juel, 1998), which may stimulate the Na+–K+

pump, we measured the potassium uptake rate duringthe experimental conditions used in the previousexperiments. The effect of ouabain (10−3 m) demonstratedthat the main fraction of the potassium uptake in resting

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J Physiol 562.2 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 rateof K+ uptake compared to control (n = 8, P < 0.05).The increase in pump activity was approximately 43% ifonly the ouabain-suppressible component is taken intoconsideration. The pump activity was lower (P < 0.05)with 12 mm Na-lactate + 8 mm lactic acid compared to20 mm Na-lactate, and even lower (P < 0.05) if muscleswere incubated with 20 mm lactic acid.

Incubation for 15 min with 0.5 mm DIDS (inhibitorof Na-bicarbonate cotransport) + 0.5 mm amiloride(inhibitor of Na+/H+ exchange) before the addition of20 mm Na-lactate, reduced (P < 0.05) the potassiumuptake to a value lower than control, but similar to therate 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 theforce development in K+-depressed muscle (Fig. 1). Wetherefore tested the three solutions in association withcontinuous muscle activation.

The force production during repetitive stimulation wasgradually reduced and was approximately 15% of theinitial force after 5 min stimulation. The fatiguedevelopment in muscle pre-incubated for 15 min with20 mm Na-lactate was compared to control (Fig. 3A). Ananalysis of variance including all data points revealed

Figure 1. Effect of 20 mM lactic acid or 20 mM Na-lactate or HCl titration on tetanic force in musclepre-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 subsequentlyincubated 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.

a strong tendency for a lower force development inmuscle incubated with 20 mm Na-lactate (P = 0.056,n = 16). If the data points for the first 2 min of thestimulation period were tested separately, a significantlylower force was obtained in the muscles incubated with20 mm Na-lactate (P < 0.05). Another series of musclewas incubated with 12 mm Na-lactate + 8 mm lactic acid(Fig. 3B). This treatment had no significant effect on forcedevelopment. Finally, a series of muscles were incubatedwith 20 mm lactic acid (Fig. 3C). For some data pointsforce development was lower in the lactic acid-incubatedmuscle. Overall, the stimulation experiments did notsupport any protective effect of lactate/lactic acid on forcedevelopment in active muscle.

Discussion

Muscle force in soleus muscle recovered if K+-depressedmuscle was incubated with 20 mm lactic acid (Fig. 1).We were therefore able to reproduce the experiments byNielsen et al. (2001). In that study, incubation with 20 mmlactic acid reduced extracellular pH by 0.64 units (from7.44 to 6.80), whereas intracellular pH was reduced by 0.39units (from 7.28 to 6.89). Thus, the transmembrane pHgradient was essentially abolished (in fact a minor gradientin the opposite direction was induced).

The interstitial pH in human skeletal muscle has beenmeasured with the microdialysis technique (Street et al.



2001). It was found that interstitial pH was reduced by0.34 units (from 7.38 to 7.04) during intense exercise,whereas the intracellular pH in fatigued human muscleswas reduced by 0.45 units (from 7.14 to 6.69) (Juelet al. 2004). The last pH reduction is probably an under-estimation, as pH was measured in muscle homogenates.In any case, there remains no doubt that the pH gradientacross sarcolemma in fatigued muscle exceeds the pHgradient at rest.

In order to evaluate the effect of external and internalpH changes we used incubations with the Na+ salt oflactic acid (Na-lactate) or a combination of Na-lactateand lactic acid. In both cases the inwardly directedlactate gradient stimulates lactate influx mediated by thelactate/H+ cotransports and the influx of undissociatedlactic acid. Therefore, in both cases H+ enters the celltogether with lactate ions in a 1 : 1 manner (Juel &Halestrap, 1999). Because of this coupling between lactateand H+ transport, both the internal lactate concentrationand the H+ load will be similar whether the muscle isincubated with lactic acid or Na-lactate (Juel, 1997).

In the experiments with Na-lactate, only the internalpH is changed. The treatment with Na-lactate led to aslow and only partial force recovery and there was a largevariation between experiments. In order to more closelysimulate the pH changes at fatigue we incubated muscles ina mixture of 12 mm Na-lactate + 8 mm lactic acid, whichacidifies external pH less than internal pH. This treatment

Figure 2. The effect of pH regulation on potassium uptake inresting muscleControl: 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 of20 mM lactic acid (n = 4). DIDS + AMIL: effect of 0.5 mM DIDS and0.5 mM amiloride on potassium uptake in muscle incubated with20 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 theone obtained with 20 mm lactic acid.

If the external pH is changed with HCl titration andno lactate present, the internal pH will only change byapproximately 10% of the external pH change (Aickin &Thomas, 1977). In a series of experiments, we used HCl tomainly reduce external pH; this treatment did not induceforce recovery.

Based on these experiments the underlying mechanismfor force recovery in K+-incubated soleus muscle canbe discussed. One mechanism could be a stimulationof the Na+–K+ pump by the Na+ influx (Buchananet al. 2002) during pH regulation, which could lead to ahyperpolarization (Overgaard et al. 1999) and restorationof excitability. Incubation with Na-lactate increasedthe ouabain-sensitive pump activity by 43% (Fig. 2).The activity was lower with 12 mm Na-lactate + 8 mmlactic acid, and even lower with 20 mm lactic acid.Thus, there was a positive correlation between the pHgradient across sarcolemma and the rate of K+ uptake(P < 0.05), reflecting that pH regulation is dependenton the existence of a pH gradient. However, stimulationof the pump cannot be the sole explanation for forcerecovery in K+-depressed muscle; although the pumpwas stimulated with Na-lactate incubation, lactic acidincubation led to force recovery in K+-depressed musclewithout affecting the pump. Also, Nielsen et al. (2001)found that potassium uptake is not increased by lacticacid incubation, and it was argued that the underlyingmechanism for the force recovery during lactic acidincubation is related to the changes in pH. The presentstudy demonstrated that a reduction in the pH gradient isnot a necessity for force recovery in K+-depressed muscle,although it may contribute. Another important factorcould be the acidification during incubation. Althoughthe H+ load on the intracellular compartment is thesame with the three test solutions, the muscle is ableto counteract the acidification during Na-lactate/lacticacid and Na-lactate infusion. Therefore, the resultingacidification is expected to be less in Na-lactate andNa-lactate/lactic acid than in the experiments with lacticacid.

The present experiments confirmed that lactate andlactic acid incubation could lead to force recovery inK+-depressed muscle. The question is whether this is ageneral mechanism active during fatiguing muscle activity,as suggested by Nielsen et al. (2001), or whether themechanism is only seen in passive muscle pre-incubatedwith high external K+. In an attempt to solve thisproblem we stimulated isolated rat soleus muscle tofatigue. In three independent series of experiments wecompared control muscle and muscle pre-incubated witheither 20 mm Na-lactate, 12 mm Na-lactate + 8 mm lacticacid, or 20 mm lactic acid (4 mm K+, pH 7.4). In theseexperiments, the pre-incubation is expected to lead to


J Physiol 562.2 Lactate and force production in skeletal muscle 525

internal lactate and H+ accumulation from the start ofthe stimulation period. In addition, the added externallactate will inhibit the efflux of lactate and H+ formedin the muscle fibres during stimulation. In the first twominutes of the stimulation period there was a significantreduction of force development in muscle incubatedwith 20 mm Na-lactate (Fig. 3A), and a tendency forforce reduction with Na-lactate/lactic acid, and lactic acid

Figure 3. Effect of lactate and lactic acid incubationon force development during fatiguing stimulationFrom each rat one soleus muscle was incubated with thetest solution for 15 min, the other soleus muscle served ascontrol. The values represent the mean force (± S.E.M.)read every 30 s for 5 min. •, controls. �, musclesincubated with the test solution. A, muscles wereincubated with 20 mM Na-lactate. n = 16 for both series.∗Control and Na-lactate values significantly different(P < 0.05); #, P = 0.059. B, muscles incubated with12 mM Na-lactate + 8 mM lactic acid (n = 8). C, musclesincubated with 20 mM lactic acid (n = 9). ∗Control andvalues for lactic acid incubation significantly different,P < 0.05

(Fig. 3B and C). Therefore, this experimental setup couldnot detect any sign of a protective role of lactate/lacticacid incubation against fatigue development in activemuscle. A depressive effect of lactate on tetanic forcehas been reported for mouse muscles (Spangenburget al. 1998).

The three combinations of lactate/lactic acid restored(more or less) the force development in passive soleus



muscle incubated with a high K+ concentration, whereaslactate/lactic acid had no protective effect in repetitivelystimulated muscle. What is the reason for this difference?The resting muscles were incubated with high K+

without activating the Na+–K+ pump, whereas in activemuscle the pump is stimulated by Na+ influx duringthe action potentials and due to Na+ influx causedby the pH regulating transport systems (Juel, 1998) asdemonstrated in the present study (in vivo the pump is alsostimulated by hormones). Therefore, the depolarizationin the K+-incubated muscle is expected to be morepronounced than in active muscle. The difference betweenthe two situations could therefore be that lactate/lacticacid removes the negative consequences of an unusuallylarge depolarization in the K+-treated passive muscle. Inline with this argument, muscle activity and activationof the pump have been demonstrated to hyperpolarizemuscle cells and to restore force in K+-depressed muscle(Overgaard & Nielsen, 2001).

In conclusion, we have demonstrated that lactate andlactic acid can lead to force recovery in passive soleusmuscle incubated with high K+. However, this is not ageneral mechanism; lactate/lactic acid incubation has noprotective role in active muscle.

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Acknowledgement

This work was supported by The Danish National ResearchFoundation.


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Lactate and force production in skeletal muscle