Reducing chloride conductance prevents hyperkalaemia-induced loss of twitch force in rat slow-twitch muscle

Abstract

Exercise-induced loss of skeletal muscle K(+) can seriously impede muscle performance through membrane depolarization. Thus far, it has been assumed that the negative equilibrium potential and large membrane conductance of Cl(-) attenuate the loss of force during hyperkalaemia. We questioned this idea because there is some evidence that Cl(-) itself can exert a depolarizing influence on membrane potential (V(m)). With this study we tried to identify the possible roles played by Cl(-) during hyperkalaemia. Isolated rat soleus muscles were kept at 25 degrees C and twitch contractions were evoked by current pulses. Reducing [Cl(-)](o) to 5 mM, prior to introducing 12.5 mM K(o), prevented the otherwise occurring loss of force. Reversing the order of introducing these two solutions revealed an additional effect, i.e. the ongoing hyperkalaemia-related loss of force was sped up tenfold after reducing [Cl(-)](o). However, hereafter twitch force recovered completely. The recovery of force was absent at [K(+)](o) exceeding 14 mM. In addition, reducing [Cl(-)](o) increased membrane excitability by 24%, as shown by a shift in the relationship between force and current level. Measurements of V(m) indicated that the antagonistic effect of reducing [Cl(-)](o) on hyperkalaemia-induced loss of force was due to low-Cl(-)-induced membrane hyperpolarization. The involvement of specific Cl(-) conductance was established with 9-anthracene carboxylic acid (9-AC). At 100 microm, 9-AC reduced the loss of force due to hyperkalaemia, while at 200 microm, 9-AC completely prevented loss of force. To study the role of the Na(+)-K(+)-2Cl(-) cotransporter (NKCC1) in this matter, we added 400 microm of the NKCC inhibitor bumetanide to the incubation medium. This did not affect the hyperkalaemia-induced loss of force. We conclude that Cl(-) exerts a permanent depolarizing influence on V(m). This influence of Cl(-) on V(m), in combination with a large membrane conductance, can apparently have two different effects on hyperkalaemia-induced loss of force. It might exert a stabilizing influence on force production during short periods of hyperkalaemia, but it can add to the loss of force during prolonged periods of hyperkalaemia.

Figure 1. Effect of lowering [Cl⁻]_o on twitch-force development during hyperkalaemia

A, twitch force recorded from soleus muscle is shown once every 4 min, although measurements were actually made once every minute. After 55 min, the modified KR solution (○; n = 10, +s.e.m.) was replaced by the high-K⁺_o solution (▽; n = 10). This 12.5 mm K⁺_o solution was maintained in two preparations (▽; n = 2, +s.d.), while the other eight preparations were introduced to the high-K⁺_o/low-Cl⁻_o solution at t = 105 min (▾; n = 8, +s.e.m.). B, isometric twitch-force–stimulation-strength relationships. These three twitch curves were recorded during the experiment depicted in A. Twice during the presence of the control solution, at time points t = 0 and t = 50 min (○; n = 6, −s.e.m.), and once during the presence of the high-K⁺_o/low-Cl⁻_o solution at t = 200 min (▾; n = 6, +s.e.m.).

Figure 2. Lowering [Cl⁻]_o prevents hyperkalaemia-induced loss of twitch force and affects muscle excitability

A, twitch force recorded from soleus muscle is shown once every 4 min. After 5 min, the modified Krebs–Ringer solution (○; n = 11, +s.e.m.) was replaced by the low-Cl⁻_o solution (•); at t = 55 min the low-Cl⁻_o solution was replaced by the high-K⁺_o/low-Cl⁻_o solution (▾); the modified Krebs–Ringer solution was reintroduced at t = 105 min (○). B, isometric twitch-force–stimulation-strength relationships. These four twitch curves were recorded during the experiment depicted in A. Twice during the presence of the control solution, at time points t = 0 (○) and t = 150 min (○), once during the presence of the low-Cl⁻_o solution at t = 50 min (•), and once during the presence of the high-K⁺_o/low-Cl⁻_o solution at t = 100 min (▾).

Figure 3. Lowering [Cl⁻]_o affects the response of membrane potential (V_m) to changes in [K⁺]_o

Resting membrane potential was recorded from rat soleus muscle for a range of K⁺_o concentrations (5, 8, 11, 12.5, 14 and 17 mm). The solutions contained either 130.2 mm Cl⁻_o (○, control) or 5.2 mm Cl⁻_o (•). Extracellular Cl⁻ was replaced by equimolar amounts of isethionate. Muscles incubated at either 5 or 130 mm Cl⁻ were subjected to the full range of K⁺_o concentrations, starting at 5 mm. Each point is the mean V_m value of four muscles under steady-state conditions. Error bars represent s.e.m. There is a significant interaction between the effects of [Cl⁻]_o and [K⁺]_o on V_m (P < 0.001, linear mixed-effect model); the regression lines at 130 and 5 mm Cl⁻_o are V_m=−85.1 + 27.3 log [K⁺]_o and V_m=−111.2 + 48.8 log[K⁺]_o, respectively. These lines intersect at a [K⁺]_o of 16.4 mm.

Figure 4. No influence of 9-anthracene carboxylic acid (9-AC) on hyperkalaemia-induced loss of twitch force

Twitch force recorded from soleus muscle is shown once every 4 min. At t = 5 min, the modified Krebs–Ringer (KR) solution (○; n = 6, +s.e.m.) was replaced by modified KR containing 0.5% v/v ethanol (○). At t = 55 min this solution was replaced by the high-K⁺_o solution, again containing 0.5% v/v ethanol (•). This latter fluid was replaced by the high-K⁺_o solution containing 100 μm 9-AC and 0.5% v/v ethanol at t = 105 min (▾).

Figure 5. Effect of pre-incubating soleus muscle with 9-AC on hyperkalaemia-induced loss of twitch force

The effect of hyperkalaemia on twitch force in the absence (open symbols) and in the presence of 100 or 200 μm 9-AC (filled symbols, A and B, respectively). Four animals were used in the experiment depicted in A, and each animal contributed to both groups (with and without 9-AC). Muscles from the right and left legs were evenly distributed between the two groups. The same holds for the experiment depicted in B. The 9-AC groups were exposed to 100 or 200 μm 9-AC at t = 5 min (•; n = 4, ±s.e.m.), while the control groups received only the solvent (○; n = 4, ±s.e.m.). In the experiment with 100 μm 9-AC, the solutions contained 0.5% v/v ethanol. In the experiment with 200 μm 9-AC, the solutions contained 0.05% v/v DMSO. In addition, all groups were exposed to 12.5 mm K⁺_o at t = 55 min (▾,▽). After the measurement of t = 105 min fluids were replaced by modified Krebs–Ringer solution, still containing the solvent (○).

Figure 6. No effect of pre-incubating soleus muscle with bumetanide on hyperkalaemia-induced loss of twitch force

A, the effect of hyperkalaemia on twitch force in the absence (open symbols) and in the presence of 400 μm bumetanide (filled symbols). The bumetanide group was exposed to 400 μm bumetanide at t = 5 min (•; n = 4, +s.e.m.), while the control group only received the solvent methanol (0.4% v/v) (○; n = 4, −s.e.m.). In addition, both groups were exposed to 12.5 mm K⁺_o at t = 55 min (▾,▽). After the measurement of t = 105 min, fluids were replaced by modified Krebs–Ringer solution, still containing 0.4% v/v methanol (○). B, isometric twitch-force–stimulation-strength relationships from the bumetanide group. These three twitch curves were recorded during the experiment depicted in A at time points t = 0 (○, lower curve; modified Krebs–Ringer solution), t = 50 (•; modified Krebs–Ringer solution containing 400 μm bumetanide and 0.4% v/v methanol) and t = 150 min (○, upper curve; modified Krebs–Ringer solution containing 0.4% v/v methanol).