Lactic acid restores skeletal muscle force in an in vitro fatigue model: are voltage-gated chloride channels involved?

Abstract

High interstitial K(+) concentration ([K(+)]) has been reported to impede normal propagation of electrical impulses along the muscle cell membrane (sarcolemma) and then also into the transverse tubule system; this is one considered underlying mechanism associated with the development of muscle fatigue. Interestingly, the extracellular buildup of lactic acid, once considered an additional cause for muscle fatigue, was recently shown to have force-restoring effects in such conditions. Specifically, it was proposed that elevated lactic acid (and intracellular acidosis) may lead to inhibition of voltage-gated chloride channels, thereby reestablishing better excitability of the muscle cell sarcolemma. In the present study, using an in vitro muscle contractile experimental setup to study functionally viable rectus abdominis muscle preparations obtained from normal swine, we examined the effects of 20 mM lactic acid and 512 μM 9-anthracenecarboxylic acid (9-AC; a voltage-gated chloride channel blocker) on the force recovery of K(+)-depressed (10 mM K(+)) twitch forces. We observed a similar muscle contractile restoration after both treatments. Interestingly, at elevated [K(+)], myotonia (i.e., hyperexcitability or afterdepolarizations), usually present in skeletal muscle with inherent or induced chloride channel dysfunctions, was not observed in the presence of either lactic acid or 9-AC. In part, these data confirm previous studies showing a force-restoring effect of lactic acid in high-[K(+)] conditions. In addition, we observed similar restorative effects of lactic acid and 9-AC, implicating a beneficial mechanism via voltage-gated chloride channel modulation.

Fig. 1.

Lactic acid or the Cl⁻ channel blocker 9-anthracenecarboxylic acid (9-AC) lead to peak force recovery in K⁺-depressed skeletal muscle. A: pH change after addition of 20 mM lactic acid to the tissue baths (n = 8). Data are means ± SE. *P < 0.05, significantly different from normal Krebs solution (Student's t-test). B: evolution of peak force in muscle bundles incubated in 10 mM K⁺ after addition of 20 mM lactic acid (n = 14) or 512 μM 9-AC (n = 15) to the baths. Muscle bundles incubated in either normal Krebs-Ringer solution (n = 13) or in 10 mM K⁺ Krebs-Ringer solution (n = 14) were used as controls. Both treatments (lactic acid and 9-AC) led to increased peak force compared with the K⁺-only group (*P < 0.05 by 2-way ANOVA for repeated measures). Data are means ± SE.

Fig. 2.

Myotonic activity change after normalization of [K⁺] (the same experiments as in Fig. 1). In the experiments with force decline due to elevated [K⁺] (10 mM) in the tissue baths (see Fig. 1), myotonic activity was absent irrespective of the treatment group. On exchange to normal Krebs solution, however, the muscle bundles treated with 512 μM 9-AC showed an abrupt and significant increase in the area under the force curve (AUC) divided by peak force compared with the other groups (induced myotonia) (*P < 0.05 by 2-way ANOVA for repeated measures). Data are means ± SE.

Fig. 3.

Effects of lactic acid or the Cl⁻ channel blocker 9-AC on peak force recovery in K⁺-depressed skeletal muscle: tissue bath temperature set at 30°C. Evolution of peak force in muscle bundles incubated in 10 mM K⁺ after addition of 20 mM lactic acid (n = 6) or 512 μM 9-AC (n = 6) to the baths is shown. Muscle bundles incubated in either normal Krebs-Ringer solution (n = 5) or in 10 mM K⁺ Krebs-Ringer solution (n = 5) were used as controls. Data are means ± SE.

Fig. 4.

Effects of lactic acid or the Cl⁻ channel blocker 9-AC on peak force recovery in K⁺-depressed skeletal muscle: human tissue. Evolution of peak force in muscle bundles incubated in 10 mM K⁺ after addition of 20 mM lactic acid (n = 2) or 512 μM 9-AC (n = 1) to the baths is shown. Muscle bundles incubated in either normal Krebs-Ringer solution (n = 1) or in 10 mM K⁺ Krebs-Ringer solution (n = 1) were used as controls. Data are means ± SE.

Fig. 5.

Effects of lactic acid or the Cl⁻ channel blocker 9-AC on peak force recovery in K⁺-depressed skeletal muscle: tetanic stimulation protocol. Evolution of peak force in muscle bundles incubated in 10 mM K⁺ after addition of 20 mM lactic acid (n = 5) or 512 μM 9-AC (n = 5) to the baths is shown. Muscle bundles incubated in either normal Krebs-Ringer solution (n = 5) or in 10 mM K⁺ Krebs-Ringer solution (n = 5) were used as controls. Data are means ± SE.

Fig. 6.

Myotonic activity after pharmacological blockade of the voltage-gated Cl⁻ channels depends on [K⁺] in the tissue baths. AUC/peak force was increasing in the muscle bundles treated with 9-AC (512 μM). After 30 min, the muscle bundles were exposed to various [K⁺]: 4.6 (n = 4), 6 (n = 4), 8 (n = 4), 10 (n = 4), and 12 mM (n = 4). At a concentration of 10 mM K⁺ in the tissue baths, myotonic activity of the muscle bundles ceased despite continued blockade of the voltage-gated Cl⁻ channels by 9-AC. Data are means ± SE.

Fig. 7.

Effects of epinephrine, ouabain, and the combination of ouabain with either epinephrine or lactic acid on peak force recovery in K⁺-depressed skeletal muscle. Peak forces in the muscle bundles declined roughly 40% after incubation in 10 mM K⁺ in all groups. In groups where 100 μM ouabain was added, peak forces declined steeper over the next 20 min. Epinephrine (1 μM) was then given to baths free of ouabain (n = 6) or in combination with 100 μM ouabain (n = 6). In addition, 20 mM lactic acid was added to ouabain-pretreated baths (n = 5) or ouabain was the sole treatment (n = 4). The peak force in the epinephrine-only group was significantly higher compared with all the groups where ouabain was added to the tissue baths (P < 0.05 by 2-way ANOVA for repeated measures). Data are means ± SE.