Report

Intracellular Acidosis Enhances the Excitability of Working Muscle

See allHide authors and affiliations

Science  20 Aug 2004:
Vol. 305, Issue 5687, pp. 1144-1147
DOI: 10.1126/science.1101141

Abstract

Intracellular acidification of skeletal muscles is commonly thought to contribute to muscle fatigue. However, intracellular acidosis also acts to preserve muscle excitability when muscles become depolarized, which occurs with working muscles. Here, we show that this process may be mediated by decreased chloride permeability, which enables action potentials to still be propagated along the internal network of tubules in a muscle fiber (the T system) despite muscle depolarization. These results implicate chloride ion channels in muscle function and emphasize that intracellular acidosis of muscle has protective effects during muscle fatigue.

Contraction in a twitch skeletal muscle fiber in response to a nerve impulse is the result of a complex series of events known as excitation-contraction-coupling (1) (ECC). ECC consists of (Fig. 1) (i) initiation and propagation of an action potential (AP) along the surface membrane and into the T system, (ii) activation of the voltage sensors (VSs) in the tubular wall, (iii) signal transmission to the sarcoplasmic reticulum (SR) from which the activator ion Ca2+ is released, and (iv) activation by Ca2+ of the Ca2+-regulatory system associated with the contractile apparatus. Intense muscle activity leads to a decline in mechanical performance (power output, force, and velocity of shortening), which is generally known as muscle fatigue (14). Intracellular acidification of the working muscle associated with the production of lactic acid has been said to contribute to muscle fatigue (2). This is because intracellular acidification reduces the sensitivity of the contractile apparatus to Ca2+ and, under some circumstances, the maximum Ca2+-activated force that is generated (2). However, the adverse actions of intracellular pH are not as great as originally thought (5, 6). Another factor in muscle fatigue is reduced ability of the T system to conduct APs as a result of excitation-induced accumulation of K+ in the T system (3, 4). The accumulation of K+ causes depolarization, which inactivates the Na+ channels responsible for the generation and propagation of APs (3, 7). Unexpectedly, the loss of muscle excitability and force seen in depolarized muscles is greatly counteracted by intracellular acidification (8), although the underlying mechanism is not understood.

Fig. 1.

Modes of activation of mechanically skinned muscle fibers. The force responses (right) were all obtained with the same preparation. Calibration bars for all force responses: vertical, 0.3 mN, and horizontal, 5 s. Upward-pointing arrows indicate time of activation and downward-pointing arrows indicate subsequent relaxation in a heavily buffered EGTA solution ([Ca2+] < 1 nM). (A) Electrical stimulation initiates APs in the sealed T system. Shown is a tetanic contraction at 25 Hz stimulation with square pulses of 2-ms duration for 1s and field strength of 70 V/cm in a standard K-hexamethylene-diamine-tetraacetate (KHDTA) solution with Cl (10, 11, 21). (B) Depolarization of the T system (by replacing all K+ in the solution with Na+) activates VSs independently of APs in the T system. The force response resulted from transfer of the preparation from a standard K-HDTA solution to depolarizing Na-HDTA solution (5). (C) Direct activation of SR Ca2+-release channels, causing Ca2+ release from the SR, and force production when free [Mg2+] in the solutions was lowered from 1 mM to 0.015 mM (5). (D) Direct activation of the contractile apparatus in Ca2+-buffered solutions (5, 10, 11, 21). The preparation was transferred from the standard K-HDTA solution ([Ca2+] = 100 nM) to heavily buffered Ca-EGTA solution ([Ca2+] = 30 μM).

In this study, we used a muscle fiber preparation from the extensor digitorum longus (EDL) muscle of the rat in which the surface membrane is removed by microdissection, causing the T system to seal off and all steps in ECC to be maintained (5, 911). This preparation allows direct access to the intracellular environment, thus permitting separate control of the intracellular pH and T system membrane potential. Such mechanically skinned muscle fibers can be activated at any individual step in the ECC process (i) by electrical stimulation that triggers APs in the T system (10, 11), (ii) by direct activation of the VSs in the T system (5), (iii) by directly releasing Ca2+ from the SR (5), or (iv) by directly activating the contractile apparatus in Ca2+-buffered solutions (5, 10) (Fig. 1). Thus, it is possible to determine the separate and the combined effects of T system depolarization and intracellular acidification on T system excitability (that is, generation and propagation of APs in the T system) and to assess likely effects on muscle fatigue.

If the potential across the T system is reduced for a sustained period, a proportion of the VSs become dysfunctional (inactivated). This was achieved here by equilibrating the skinned fibers in solutions with decreased myoplasmic (intracellular) K+ concentration ([K+]i) at constant [K+]i[Cl]i product ([Cl]i is the concentration of Cl) (12). The remaining (noninactivated) VSs were then activated by fully depolarizing the T system with application of a solution with all K+ removed (Fig. 2A). The force responses to full depolarization were then plotted against the [K+]i in the various equilibration solutions to show the inactivation behavior of the VSs. Because the inactivation curves of the VSs (Fig. 2B) at pH = 6.6 and pH = 7.1 were virtually the same, we conclude that the ECC steps starting from VS activation up to the activation of the contractile apparatus (Fig. 1) are largely unaffected by intracellular acidification. This is consistent with inactivation of VSs being unaffected by intracellular acidosis (13). The overlap of the two curves in Fig. 2B also confirms that the membrane potential across the T system at any given [K+]i was essentially the same at pH values of 7.1 and 6.6. The values of membrane potential for the various [K+]i were calculated from the Goldman-Hodgkin-Katz equation (14), assuming 4 mM for K+ and 150 mM for Na+ in the lumen of the sealed T system, relative permeability for K+ as compared to Na+ of 100:1, and passive distribution of Cl across the T system. In contrast, force responses to APs in the T system elicited by electrical stimulation (10, 11) did display pH dependence (Fig. 2, C and D) (preparations were tetanically stimulated at 25 Hz at pH = 7.1 and at pH = 6.6). When the T system was moderately depolarized at [K+]i values of 75 and 60 mM, there was a significantly greater loss of tetanic force at pH = 7.1 than at pH = 6.6 (P < 0.05, n = 10). The loss in force response with AP stimulation occurred at more negative potentials than did VS inactivation (compare Fig. 2D with Fig. 2B), showing that the force loss was due predominantly to AP failure rather than inadequate VS activation. These results indicate that intracellular acidosis protects against the loss of force caused by depolarization when activation is initiated by APs, as occurs in vivo, and indicate that this protective effect may be due to enhanced excitability of the T system.

Fig. 2.

Effect of pH and T system depolarization on force responses induced by direct VS activation (A and B) or tetanic stimulation at 25 Hz (C and D) in the presence of Cl in mechanically skinned fibers at 25° ± 2°C. EDL muscles of rats [Long Evans Hooded, 6-month-old males, killed by halothane overdose (5)] were placed in paraffin oil and skinned fibers were prepared as described (5, 10, 11, 21). Fibers were attached to a sensitive force transducer and bathed in solutions mimicking the myoplasmic environment (5, 10, 11, 21). The K-HDTA standard solution at pH = 7.10 contained 126 mM K+, 17 mM methylsulfate, 3 mM Cl, 40 mM Na+, 40 mM HDTA2–, 1 mM Mg2+ (free), 8 mM adenosine triphosphate, 10 mM creatine phosphate, 90 mM Hepes buffer, 0.05 mM Bapta [(1,2-bis0-aminophenoxy)ethane-N,N,N′,N-tetraacetic acid], and 100 nM Ca2+. The K-HDTA standard solution at pH = 6.60 was identical to the K-HDTA solution at pH = 7.10 with respect to all ions except that it contained 9 mM Pipes and Hepes was reduced to 80 mM to maintain osmotic balance (295 ± 2 mmol/kg). In solutions with decreased [K+]i, K+ was replaced with NH4+ and methylsulfate with Cl to maintain constant [K+]i [Cl]i product. (A and C) Representative force traces from two individual fibers. Bars under traces represent the duration of stimulation [ion substitution for (A) and 25-Hz tetanic stimulation for (C)]. The voltage sensor–activated force response at pH = 6.6 (75 mM K+) is only slightly smaller than the corresponding response at pH = 7.1, with the difference entirely due to the decrease in maximum Ca2+-activated force at pH = 6.6, which was smaller by 19 ± 9% (n = 49) compared with that at pH = 7.1. The horizontal lines in (A) and (C) indicate the maximum Ca2+-activated force generated at pH = 7.1 and pH = 6.6 in buffered Ca-EGTA solutions ([Ca2+] = 30 μM) in which HDTA2– was replaced by Ca-EGTA2–. Calibration bars: vertical, 0.1 mN, and horizontal, 5 s. Membrane potentialin the various [K+]i solutions was calculated from the Goldman-Hodgkin-Katz equation (14). For each data point n was from 5 to 8.

In rested muscle, Cl is the most membrane-permeant ion (1518). The high membrane permeability to Cl at normal pH stabilizes the resting membrane potential, but the cost of this is that a comparatively large inward Na+ current is needed to sustain a propagating AP (16, 18). Because lowering pH reduces Cl conductance (19, 20), we examined whether Cl plays a role in the protective effect of acidity. When experiments like those in Fig. 2, A to D, were repeated without Cl in the myoplasm and T system, the inactivation curve for tetanic stimulation at pH = 6.6 was no different from that at pH = 7.1 (Fig. 3, C and D). Compared with the inactivation curves in the presence of C1 (indicated with broken lines in Fig. 3D), both inactivation curves were shifted to the right (to lower [K+]i and greater depolarization) but with a greater shift at pH = 7.1 than at pH = 6.6 (Fig. 3D). Thus, the pH effect seen in Fig. 2D is associated with the presence of Cl.

Fig. 3.

Effect of pH and T system depolarization on force responses at 25° ± 2°C induced by direct VS activation (A and B) or tetanic stimulation at 25 Hz (C and D) in the absence of Cl. Before skinning, the muscles were equilibrated for 30 min in Cl-free Ringer's solution containing 65 mM Hepes, 1.2 mM Ca2+, 115 mM sodium methylsulfate, 4 mM K+, 1.2 mM phosphate, and 5 mM glucose. NaOH was used to bring pH to 7.4. All intracellular solutions had the same composition as the solutions described in the legend of Fig. 2 except Cl was replaced with methylsulfate. Membrane potential in the various [K+]i solutions was calculated as described (Fig. 2). (A and C) Representative force traces from two individual fibers. Bars under traces represent the duration of stimulation. (C) The horizontal lines indicate the maximum Ca2+-activated force at pH values of 7.1 and 6.6 in heavily buffered Ca-EGTA solutions (Fig. 2). Calibration bars: vertical 0.1 mN, horizontal 5 s. The broken lines shown in (D) represent the curves from Fig. 2D.

To evaluate whether intracellular acidosis decreases the Cl permeability of the T system, we did an experiment in which preparations were activated by ion substitution after equilibration in a solution with 80 mM K+ and 20 mM Cl, which is about four times the [Cl]i needed to keep the [K+]i[Cl]i constant (Fig. 4). Under these conditions, Cl will tend to depolarize the T system more than the reduction in K+ concentration would, and a high Cl permeability would therefore increase the level of chronic depolarization. Consequently the force response to maximal VS activation was expected to be reduced. The drop in force at 80 mM K+ and 20 mM Cl was larger at pH = 7.1 than at pH = 6.6. From the inactivation curves in Fig. 2B, the membrane potential of the T system at 80 mM K+ and 20 mM Cl was estimated to be –52 mV at pH = 7.1 and –59 mV at pH = 6.6 (n from 7 to 10). On the basis of the Goldman-Hodgkin-Katz equation, this change in membrane potential showed that the permeability of Cl in the T system was reduced by around 74% at pH = 6.6 compared to that at pH = 7.1. The fact that at pH = 6.6 the T system Cl permeability was not completely abolished may explain why the inactivation curve for tetanic stimulation at pH = 6.6 was not shifted as much as the inactivation curves were when Cl was totally removed (Fig. 3D).

Fig. 4.

Effect of pH on Cl permeability in the T system. Representative VS activated force responses in a mechanically skinned fiber equilibrated in solutions of different [K+]i and [Cl]i at pH values of 7.1 and 6.6. Calibration bars: vertical, 0.1 mN; horizontal, 5 s. At constant [K+]i[Cl]i product, the membrane potentials were calculated as described in text, and at 80 K+ and 20 Cl the membrane potential was estimated from Fig. 2B.

We find that, in the presence of Cl, intracellular acidosis increases the excitability of the T system in depolarized muscles fibers, thus counteracting fatigue at a critical step in ECC. In this model of working muscle, acidic pH reduced Cl permeability, thereby reducing the size of the Na+ current needed to generate a propagating AP. Thus, down-regulation of T system Cl permeability by intracellular acidosis is important for preserving a fully operational T system in working muscle.

References and Notes

View Abstract

Stay Connected to Science

Navigate This Article