Disruption of excitation-contraction coupling and titin by endogenous Ca2+-activated proteases in toad muscle fibres

Abstract

This study investigated the effects of elevated, physiological levels of intracellular free [Ca(2+)] on depolarization-induced force responses, and on passive and active force production by the contractile apparatus in mechanically skinned fibres of toad iliofibularis muscle. Excitation-contraction (EC) coupling was retained after skinning and force responses could be elicited by depolarization of the transverse-tubular (T-) system. Raising the cytoplasmic [Ca(2+)] to approximately 1 microm or above for 3 min caused an irreversible reduction in the depolarization-induced force response by interrupting the coupling between the voltage sensors in the T-system and the Ca(2+) release channels in the sarcoplasmic reticulum. This uncoupling showed a steep [Ca(2+)] dependency, with 50% uncoupling at approximately 1.9 microm Ca(2+). The uncoupling occurring with 2 microm Ca(2+) was largely prevented by the calpain inhibitor leupeptin (1 mm). Raising the cytoplasmic [Ca(2+)] above 1 microm also caused an irreversible decline in passive force production in stretched skinned fibres in a manner graded by [Ca(2+)], though at a much slower relative rate than loss of coupling. The progressive loss of passive force could be rapidly stopped by lowering [Ca(2+)] to 10 nm, and was almost completely inhibited by 1 mm leupeptin but not by 10 microm calpastatin. Muscle homogenates preactivated by Ca(2+) exposure also evidently contained a diffusible factor that caused damage to passive force production in a Ca(2+)-dependent manner. Western blotting showed that: (a) calpain-3 was present in the skinned fibres and was activated by the Ca(2+)exposure, and (b) the Ca(2+) exposure in stretched skinned fibres resulted in proteolysis of titin. We conclude that the disruption of EC coupling occurring at elevated levels of [Ca(2+)] is likely to be caused at least in part by Ca(2+)-activated proteases, most likely by calpain-3, though a role of calpain-1 is not excluded.

Figures

Figure 1. Exposure to physiological levels of intracellular [Ca2+] disrupts excitation–contraction (EC) coupling

Depolarization-induced force responses were elicited in a skinned fibre by substituting all K+ in the solution with Na+ (Depol.). A, when the rigor treatment included a 3 min exposure to 2 μm free Ca2+, it caused an irreversible reduction in the depolarization-induced force response by more than 50% even after additional sarcoplasmic reticulum (SR) loading. B, in another fibre, a 3 min exposure to 3.5 μm free Ca2+ completely abolished the depolarization-induced response. Lowering the free [Mg2+] to 50 μm (low Mg2+), which stimulates the SR Ca2+ release channels directly, indicated that the SR was loaded above the endogenous level and that the Ca2+ release channels were still functional. Maximum Ca2+-activated force was ascertained in a heavily Ca2+-buffered solution (Max, pCa 4.7). Moving the fibre between solutions caused small force artefacts. The time scale was 1 s during the depolarizations and low [Mg2+], and 30 s elsewhere.

Figure 2. Ca2+ dependence of EC uncoupling

Mean size (± s.e.m.) of depolarization-induced force response after a 3 min exposure at the indicated [Ca2+] (in pCa units), expressed as a percentage of the response before treatment. Ca2+ was applied during rigor as in Fig. 1, and n is the number of fibres. The data were fitted with a Hill curve with 50% inhibition at pCa 5.72 (i.e. 1.9 μm Ca2+) and a Hill coefficient of 3.5.

Figure 3. Elevated [Ca2+] treatment during stretch damages passive force development

A, passive force production in a fibre stretched to 2.0 times resting length (in relaxing solution), and then exposed to the control rigor solution (pCa 8). After the fibre was returned to its resting length for 2 min, stretching it again to 2.0 times resting length gave passive force similar to that upon the first stretch. B, when another fibre was treated similarly, except exposed for 3 min to 40 μm Ca2+ during the rigor period, force declined markedly during the Ca2+ exposure, and there was subsequently a great reduction in the amount of passive force produced at 2.0 times resting length. C, in another fibre in which the length was reduced to ∼1.2 times of resting length before applying the rigor and Ca2+ treatment, passive force production (at 2.0 times resting length) was virtually identical before and after the treatment.

Figure 4. Summary of effects of treatment on passive force

Mean (± s.e.m.) of peak of passive force following indicated treatment, expressed as a percentage of that before the treatment. Experiments were performed as shown in Fig. 3, with a 3 min exposure to the [Ca2+] indicated. The number of fibres is shown for each mean. Each fibre was stretched to 2.0 times resting length during the exposure except in case indicated (far right). leu, 1 mm leupeptin. *Significantly less than 100%. **P < 0.01 and ***P < 0.001, mean is significantly less than for the control-rigor case.

Figure 5. Elevated [Ca2+] damages passive force development

A, complete protocol of the passive force experiment in which a skinned fibre was stretched to ∼2.0 times slack length and then treated. In this example the fibre was stretched to final length in two steps, producing the two peaks in passive force at the beginning. Transferring the fibre to the control-rigor solution (Rig.) initially caused a small increase in force, but then force subsequently declined with approximately its original time course. The fibre was given various treatments, and then rigor was reversed by returning to the control K-HDTA solution (see Methods) with ATP (Post), and the fibre was returned to close to resting length. B, expanded view of period in rigor solutions showing the off-on-off-on effect of elevated [Ca2+] on the rate of decay of passive force. Step reductions in force often occurred when moving the fibre between solutions, most likely because of stretch-induced breaking of the rigor bridges and possibly also titin filaments. C, expanded view of the rigor period in another fibre, showing effect of 1 mm leupeptin (leu). The first two periods are with leupeptin present, in which the rate of decay of passive force in 40 μm Ca2+ is unchanged compared with the preceding control rigor period. The Ca2+ effect could be switched on again by washing out the leupeptin and re-exposing the fibre to 40 μm Ca2+.

Figure 6. Effect of various treatments on the rate of decline of passive force

A, mean (± s.e.m.) rate of decline of passive force for each given condition, expressed relative to the control-rigor period in the same fibre (see Fig. 5). The number of fibres is shown with each mean. 1.2, 8 and 40 μm denote the free [Ca2+] for the 3 min exposure period. leu and calp, the presence of 1 mm leupeptin and 10 μm calpastatin, respectively. 40 μm+ DTDP, pretreatment of the fibre with dithiodipyridine before stretching and treating it with 40 μm Ca2+. Homogenate, exposure to Ca2+-activated homogenate with final [Ca2+] of ∼0.4 μm. B, data in A expressed as absolute values of force decline per minute, with each fibre standardized by the force level immediately prior to the first exposure to rigor solution, and with the paired rate of decline during the control-rigor subtracted from it. In some instances the fibres included in B were not included in the normalized data in A because force decline during the control rigor period was approximately zero. *Significantly steeper decline than paired control-rigor period (P < 0.05). #Significantly smaller rate of decline than with 40 μm Ca2+ without calpastatin (P < 0.05).

Figure 7. Leupeptin reduces the initial decay in maximal active force

A, force responses to first two periods in maximal Ca2+-activation solution (pCa 4.7, 50 mm CaEGTA-EGTA). The skinned fibre was at 120% of resting length, and had not been activated or exposed to any Ca2+ beforehand. The fibre was relaxed in relaxing solution (pCa > 9) and transferred to solution with 0.5 mm EGTA (pCa ∼8) for 30 s before each activation period. Initial peak force defined as 100%. B, similar procedure in another fibre with 0.5 mm leupeptin present in all solutions, showing smaller decline in force during the first activation period. (See text regarding change in baseline force). C, mean relative force (± s.e.m.) reached at the start of each successive 1 min maximal activation period. The force is expressed relative to the initial peak force reached during the first activation period. Open and filled symbols indicate presence and absence of 1 mm leupeptin, respectively (4 fibres each).

Figure 8. Calpain-3 Western blots of muscle homogenates and skinned fibres

A, muscle homogenates from rat muscle under control conditions (Rat CON) and toad muscle under control conditions (Toad CON) or after exposure to 5 mm Ca2+ for 1 min (Toad Ca2+) were analysed for calpain-3. The 94 kDa band disappeared after exposing toad muscle to Ca2+. B, groups of three mechanically skinned single fibres were exposed to 40 μm Ca2+ for 1 min, 8 μm Ca2+ for 3 min and 2 μm Ca2+ for 3 min or to the control-rigor solution (Rig) under paraffin oil. Each fibre had been initially washed in the same droplet of control-rigor solution (wash) for 1–7 min (time matched for all fibre groups); proteins found in this solution were those readily washed out of the 12 fibres. The density of the 94 kDa calpain-3 band in lanes 1–4 was normalized by the 42 kDa protein present (likely to be actin, relative density range 70–100%), and expressed as a percentage of that for the control-rigor treatment was 100, 19, 14 and 0% for control rigor, 2, 8 and 40 μm Ca2+, respectively. Whole, homogenate of toad whole muscle. Note that the sensitive streptavidin method required with the single fibres in B resulted in nonspecific detection of additional protein bands.

Figure 9. Silver stained 2.8% SDS-PAGE gel of skeletal muscle

A, homogenates from rat soleus and toad iliofibularis muscle. Full-length titin (T1, ∼3700 kDa) is similar in size in both samples, whereas nebulin (N) in rat soleus muscle (∼800 kDa) is slightly larger than that in toad iliofibilaris muscle (∼700 kDa). B, when toad muscle was warmed and left at room temperature for a period (RT, see Methods), much of the titin present in control conditions (Con) degraded to titin 2 (T2). C, similar results in rat muscle. D, single skinned fibres from toad iliofibilaris muscle that had been exposed to control-rigor (Rig), 40 μm Ca2+ (Ca2+) or 40 μm Ca2++1 mm leupeptin (Leu) for 3 min whilst stretched on the force transducer at 2.0 resting length (see Methods and Fig. 3). The ratio of T1 to T2 was greatest for the control-rigor case and least for the fibre exposed to 40 μm Ca2+. E, mean densitometry values (± s.e.m.) of the T2 band (as a percentage of T1 + T2) in single skinned fibres (number in parentheses) exposed as in D, with ‘unstretched’ denoting single fibres exposed to Ca2+ as in Fig. 3C. *Significant difference compared to Rig and Leu (one-way analysis of variance, P < 0.05).