Depletion of Ca2+ in the sarcoplasmic reticulum stimulates Ca2+ entry into mouse skeletal muscle fibres

Abstract

To examine whether a capacitative Ca2+ entry pathway is present in skeletal muscle, thin muscle fibre bundles were isolated from extensor digitorum longus (EDL) muscle of adult mice, and isometric tension and fura-2 signals were simultaneously measured. The sarcoplasmic reticulum (SR) in the muscle fibres was successfully depleted of Ca2+ by repetitive treatments with high-K+ solutions, initially in the absence and then in the presence of a sarcoplasmic/endoplasmic reticulum Ca2+-ATPase (SERCA) inhibitor. Depletion of the SR of Ca2+ enabled us for the first time to show convincingly that the vast majority of the voltage-sensitive Ca2+ store overlaps the caffeine-sensitive Ca2+ store in intact fibres from mouse EDL muscle. This conclusion was based on the observation that both high-K+ solution and caffeine failed to cause a contracture in the depleted muscle fibres. The existence of a Ca2+ influx pathway active enough to refill the depleted SR within several minutes was shown in skeletal muscle fibres. Ca2+ entry was sensitive to Ni2+, but resistant to nifedipine and was suppressed by plasma membrane depolarisation. Evidence for store-operated Ca2+ entry was provided by measurements of Mn2+ entry. Significant acceleration of Mn2+ entry was observed only when the SR was severely depleted of Ca2+. The Mn2+ influx, which was blocked by Ni2+ but not by nifedipine, was inwardly rectifying, as is the case with the Ca2+ entry. These results indicate that the store-operated Ca2+ entry is similar to the Ca2+ release-activated Ca2+ channel (CRAC) current described in other preparations.

Figure 1. Depletion of the Ca²⁺ store in mouse EDL muscle fibres and Ca²⁺ entry concomitant with refilling of the SR

Initially, fibres were incubated in 2Ca-5K solution and treated with 2Ca-100K solution for 30 s to observe high-K⁺ contracture (lower left-hand trace). The incubation solution was then changed to 0Ca-5K solution and stimulation with 0Ca-100K solution (30 s) was repeated every 10 min. The time-integrated high-K⁺ contracture gradually decreased with each stimulation, reaching steady state after the 7th-10th treatment, at which time it had diminished to an average of about 12 % of the initial magnitude (partial depletion). Then 10 μm CPA was added to the solution (prior to the 9th treatment in this experiment), and high-K⁺ stimuli were further repeated every 10 min until fibres did not develop contractures (18th, severe depletion). The fibres were also challenged with 25 mm caffeine (25Caf), resulting in only slight tension development. After washout of CPA with 0Ca-5K solution, the fibres were incubated in 2Ca-5K solution for 2 min, followed by washing with 0Ca-5K solution, and were subsequently challenged with 0Ca-100K solution and 25 mm caffeine to determine the extent of refilling of the SR with Ca²⁺. Ordinal numbers indicate the total number of high-K⁺ treatments in Ca²⁺-free solution. The horizontal line indicates the extracellular Ca²⁺ concentration. CPA was present in the media during the period indicated by the thick line. The time scale can be applied to the tension traces but not to the intervals between. The fluorescence ratio signals corresponding to the tension responses below are also shown (upper traces); note that the Ca²⁺ signal at the 9th high-K⁺ stimulation suffered from large movement artifact, when the fibres moved out of the field, distorting the ratio signal.

Figure 9. Ca²⁺ depletion and Mn²⁺ influx in the presence of thapsigargin

SR was depleted by the procedure described in Fig. 1 with 10 μm thapsigargin (TG) used instead of CPA. Fluorescence signals are also shown together with tension signals. Mn²⁺ influx was determined before and after Ca²⁺ depletion and after refilling of Ca²⁺.

Figure 7. Effect of Ca²⁺ depletion on rate of Mn²⁺ influx

Mn²⁺ influx was determined in the same muscle fibres under four SR loading states as follows: (1) after the first high-K⁺ stimulus in Ca²⁺-free solution where the SR was in the fully loaded state; (2) after partial depletion of Ca²⁺ in the absence of CPA where the fibres developed one-fifth of the initial tension; (3) after severe depletion of Ca²⁺ in the presence of CPA where no high-K⁺ contracture was observed; (4) after refilling of Ca²⁺ by incubation with 2 mm Ca²⁺ for 2 min when a considerable high-K⁺ contracture was observed. Fluorescence signals of fura-2 are also shown, together with high-K⁺ contractures.

Figure 2. Response of Ca²⁺-depleted fibres to electrical stimulation

SR of the fibres was depleted of Ca²⁺ and refilled as described in Fig. 1. During the procedure, twitch stimuli were applied at 0.33 Hz. Tetanus stimuli at 100 Hz for 0.2 s were applied after depletion and re-introduction of Ca²⁺ where indicated.

Figure 3. Time course of refilling of SR in severely depleted muscle fibres

A, the protocol for experiments to determine the rate of refilling. SR in muscle fibres was completely depleted of Ca²⁺ in the presence of 10 μm CPA. After washout of CPA, the fibres were then incubated with 2Ca-5K solution for a specified period (t min), washed with 0 Ca-5K solution, and challenged with 0Ca-100K solution to elicit a high-K⁺ contracture as a measure of the extent of refilling of the SR. The SR was then depleted again and the same protocol was repeated. B, high-K⁺ contractures after various periods of incubation in 2Ca-5K solution.

Figure 4. Effect of Ca²⁺ channel blockers on refilling of the SR

A, effect of Ni²⁺. SR in muscle fibres was completely depleted of Ca²⁺ in the presence of CPA, and the fibres were then incubated with 2Ca-5K solution containing 0 (left) or 5 mm (right) Ni²⁺ for 2 min in the absence of CPA. After washout of Ca²⁺ (and Ni²⁺), recovered tension of high-K⁺ contracture was determined. Note that recovery of the high-K⁺ contracture was much smaller in the presence of Ni²⁺ than that in its absence. B, effect of nifedipine. The SR was so severely depleted as described above that a high-K⁺ contracture failed to develop. The caffeine contracture was also marginal. The fibres were then incubated in 0Ca-5K for 10 min and in 2Ca-5K solution for 2 min in the presence of 10 μm nifedipine. After washing out of Ca²⁺ and nifedipine, fibres were successively stimulated with high K⁺ and caffeine. The high-K⁺ contracture was only small, but caffeine caused considerable tension. Vertical bars indicate 1 mN.

Figure 5. Effect of membrane depolarisation on refilling of the SR

A, Ca²⁺ entry in the presence of nifedipine. Nifedipine (10 μm) was applied after the SR had been severely depleted in the presence of CPA. After washout of CPA, the fibres were incubated with 2Ca-150K solution for 3 min, washed with 0Ca-5K solution and challenged with 25 mm caffeine (a). After washing out the caffeine, the fibres were incubated with 2Ca-5K solution for 3 min to determine Ca²⁺ influx after repolarisation (b). B, Ca²⁺ entry in the absence of nifedipine. Fibres had been depleted of Ca²⁺ from the store. After incubation with 2Ca-150K for 3 min (a), 2Ca-5K for 3 min (b), 2Ca-150K for 0.5 min (c) and 2Ca-150K for 3 min (d), the fibres were challenged with 25 mm caffeine in 0Ca-5K solution. After the caffeine treatment in b, fibres were also challenged with 0Ca-150K solution to confirm the response to membrane depolarisation. Vertical bars indicate 1 mN.

Figure 6. Time course of Mn²⁺ influx into mouse skeletal muscle cells

Mn²⁺ quenching of the fura-2 fluorescence signal (F₃₈₀; cps, counts s⁻¹) was monitored in muscle fibres that were Ca²⁺ depleted in the presence of CPA. Downward arrows indicate addition of 0.5 mm Mn²⁺ to the 0Ca-5K solution while upward arrows indicate washout of Mn²⁺. The final steady fluorescence level was almost the same as the intrinsic fluorescence level.

Figure 8. Effects of Ni²⁺ and membrane depolarisation on Mn²⁺ influx

Mn²⁺ influx was determined in muscle fibres with severely depleted SR. A, effect of Ni²⁺ on Mn²⁺ influx. Mn²⁺ (0.5 mm) was applied to Ca²⁺-depleted muscle fibres and after 30 s, NiCl₂ (2 mm) was added. Note that the decrease in fluorescence intensity suddenly stopped after the application of Ni²⁺. B, effect of depolarisation. Measurements were carried out in the presence of 10 μm nifedipine. The fibres were successively incubated with 0Ca-5K solution for 30 s, 0Ca-150K solution for 30 s and 0Ca-5K solution for 40 s; all the solutions contained 0.5 mm MnCl₂.

Figure 10. Ca²⁺ depletion in the presence of BHQ

SR was depleted by the procedure described in Fig. 1 with 10 μm BHQ used instead of CPA. Note that a considerable caffeine contracture was still observed whereas the high-K⁺ contracture became too small to be detected in the presence of BHQ. See also Table 2 for effect of BHQ on Mn²⁺ influx.