Effect of low cytoplasmic [ATP] on excitation-contraction coupling in fast-twitch muscle fibres of the rat

Abstract

In this study we investigated the roles of cytoplasmic ATP as both an energy source and a regulatory molecule in various steps of the excitation-contraction (E-C) coupling process in fast-twitch skeletal muscle fibres of the rat. Using mechanically skinned fibres with functional E-C coupling, it was possible to independently alter cytoplasmic [ATP] and free [Mg2+]. Electrical field stimulation was used to elicit action potentials (APs) within the sealed transverse tubular (T-) system, producing either twitch or tetanic (50 Hz) force responses. Measurements were also made of the amount of Ca2+ released by an AP in different cytoplasmic conditions. The rate of force development and relaxation of the contractile apparatus was measured using rapid step changes in [Ca2+]. Twitch force decreased substantially (approximately 30%) at 2 mm ATP compared to the level at 8 mm ATP, whereas peak tetanic force only declined by approximately 10% at 0.5 mm ATP. The rate of force development of the twitch and tetanus was slowed only slightly at [ATP] > or = 0.5 mm, but was slowed greatly (> 6-fold) at 0.1 mm ATP, the latter being due primarily to slowing of force development by the contractile apparatus. AP-induced Ca2+ release was decreased by approximately 10 and 20% at 1 and 0.5 mm ATP, respectively, and by approximately 40% by raising the [Mg2+] to 3 mm. Adenosine inhibited Ca2+ release and twitch responses in a manner consistent with its action as a competitive weak agonist for the ATP regulatory site on the ryanodine receptor (RyR). These findings show that (a) ATP is a limiting factor for normal voltage-sensor activation of the RyRs, and (b) large reductions in cytoplasmic [ATP], and concomitant elevation of [Mg2+], substantially inhibit E-C coupling and possibly contribute to muscle fatigue in fast-twitch fibres in some circumstances.

Figure 1. Low cytoplasmic [ATP] decreases and slows the twitch response

A–D, representative examples of twitch responses at various low [ATP]; each panel shows data from a different skinned fibre. Unless indicated otherwise, the skinned fibre was bathed in the control conditions (8 mm ATP and 1 mm free Mg²⁺). Several twitches were elicited under each condition with similar results. Timing of stimulus (2 ms pulse, 70 V cm⁻¹) is indicated by a vertical bar below each force recording. See Table 1 for mean data.

Figure 2. Effect of low cytoplasmic [ATP] on tetanic responses

A–D, representative examples of tetanic responses at various low [ATP]; each panel shows data from a different skinned fibre. A period of 2 min was allowed between successive tetani. Period of stimulation (50 Hz) is indicated by the rectangle below each force recording; the duration of the stimulation was longer in low [ATP] to ensure that peak force was obtained. The rates of rise and decay of the tetani were slowed as [ATP] was decreased, and peak tetanic force was reduced at [ATP] ≤ 1 mm (see Table 1 for mean data).

Figure 3. Force development in response to a rapid rise in [Ca²⁺] at various [ATP]

A, the [Ca²⁺] within a skinned fibre was rapidly raised by transferring the fibre from a weakly Ca²⁺-buffered solution (100 μm EGTA) at pCa 7.0 to a strongly buffered solution (∼50 mm CaEGTA) at pCa 4.7. The procedure was repeated with [ATP] set at each of the levels indicated. The SR and other membranous compartments were removed beforehand by treating the fibre with TritonX-100. All solutions had 30 units ml⁻¹ exogenous CPK added. B, superimposed and expanded records of the force responses from A. The unevenness in the trace for 100 μm ATP was caused by movement during the solution exchange procedure. See Table 2 for mean data.

Figure 4. Relaxation upon rapidly lowering [Ca²⁺] is slowed at low [ATP]

Maximum force (at various [ATP]) was elicited by exposing the Triton X-100-treated fibre to a solution with moderate Ca²⁺ buffering (1 mm CaEGTA) at pCa 4.7. Force was then rapidly abolished by transferring the fibre (at vertical dotted line) to a very strongly buffered relaxing solution (50 mm free EGTA) at pCa > 10, with the [ATP] unchanged. The horizontal bar indicates the maximum Ca²⁺-activated force level in each condition. The unevenness in force traces was caused by the solution exchange procedure. The time for the response to fall from 90 to 10% of maximum (FT_90–10) is shown under each trace. All solutions contained 30 units ml⁻¹ exogenous CPK. See Table 3 for mean data.

Figure 5. Assay of amount of Ca²⁺ released by AP-stimulation at different [ATP]

Twitch and tetanic (50 Hz) force responses were first elicited under control conditions (8 mm ATP) as in Figs 1 and 2. In the presence of 0.5 mm ATP, the twitch response (to a single pulse) reached only ∼40% of the control twitch, and a double pulse stimulus (D10; two pulses 10 ms apart to elicit two successive APs) produced a peak force that was ∼95% of that produced by a single pulse in 8 mm ATP. The same fibre was then transferred to corresponding solutions with 50 μm TBQ and 160 μm BAPTA added, and stimulated by single pulses at 15 s intervals. In TBQ-BAPTA the force response declined very slowly (∼1 s) because all Ca²⁺ re-uptake by the SR was blocked. The peak of the force response was indicative of the amount of Ca²⁺ released by the stimulus; this amount could be calculated from the size of the force response and the known amount and Ca²⁺-binding properties of the BAPTA and TnC (see Methods). The single AP stimuli elicited the release of ∼206 μm Ca²⁺ on each of the first two trials in 8 mm ATP, only ∼141 μm Ca²⁺ in the two trials in 0.5 mm ATP, and then ∼174–183 μm Ca²⁺ after returning to 8 mm ATP. Note that the presence of BAPTA meant that force was detected only when the Ca²⁺ release exceeded a certain threshold level, and also that the SR became progressively depleted of Ca²⁺ during the successive stimuli in TBQ-BAPTA. The 50 Hz tetanic response reached the maximum Ca²⁺-activated force level (latter not shown).

Figure 6. Effect of adenosine on the twitch response at various [ATP]

A–D, twitch responses were elicited by single 2 ms pulses as in Fig. 1. Different fibres shown in each panel. Control responses (8 mm ATP) were always produced before and after exposure to the different ratios of ATP:adenosine (ADEN). Several twitches were elicited under each condition (not shown). A, ratio 4:1 (i.e. 8 mm ATP and 2 mm ADEN). B, ratio 1:1 (2 mm ATP and 2 mm ADEN). C, ratio 1:2 (1 mm ATP and 2 mm ADEN). D, ratio 1:4 (1 mm ATP and 4 mm ADEN). Clearly, as the ratio ATP:ADEN was decreased so too was the relative peak of the twitch response.

Figure 7. Relative amount of Ca²⁺ released by AP stimulation in different conditions

The bars show the relative amount of Ca²⁺ released by a single AP (open bars) or a pair of APs (hatched bar) in the indicated conditions, expressed as a percentage of that released by a single AP in the control conditions with 8 mm total ATP and 1 mm free Mg²⁺. Data obtained by measurements in TBQ-BAPTA as shown in Fig. 5.

Figure 8. Effect of 3 mm Mg²⁺ on twitch and tetanic responses at high and low [ATP]

Twitch and tetanic force responses recorded in the presence of 3 mm Mg²⁺ and 8 mm ATP or 0.5 mm ATP, with bracketing responses under control conditions (8 mm ATP and 1 mm Mg²⁺). Raising the [Mg²⁺] to 3 mm at constant 8 mm ATP reduced the twitch response by ∼6-fold and slowed and reduced the tetanic response by ∼2-fold. There was a greater reduction in both the twitch and tetanic response when the rise in [Mg²⁺] was accompanied by a decrease in [ATP] from 8 mm to 0.5 mm (see text for mean data in five fibres). Twitches elicited by single stimuli and tetani by 50 Hz stimulation (respectively indicated by ticks and rectangles under traces).