Disruption of excitation-contraction coupling and titin by endogenous Ca2+-activated proteases in toad muscle fibres
Esther Verburg, Robyn M Murphy, D George Stephenson, Graham D Lamb, Esther Verburg, Robyn M Murphy, D George Stephenson, Graham D Lamb
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…](https://www.ncbi.nlm.nih.gov/pmc/articles/instance/1464466/bin/tjp0564-0775-f1.jpg)
![Figure 2. Ca 2+ dependence of EC…](https://www.ncbi.nlm.nih.gov/pmc/articles/instance/1464466/bin/tjp0564-0775-f2.jpg)
![Figure 3. Elevated [Ca 2+ ] treatment…](https://www.ncbi.nlm.nih.gov/pmc/articles/instance/1464466/bin/tjp0564-0775-f3.jpg)
![Figure 4. Summary of effects of treatment…](https://www.ncbi.nlm.nih.gov/pmc/articles/instance/1464466/bin/tjp0564-0775-f4.jpg)
![Figure 5. Elevated [Ca 2+ ] damages…](https://www.ncbi.nlm.nih.gov/pmc/articles/instance/1464466/bin/tjp0564-0775-f5.jpg)
![Figure 6. Effect of various treatments on…](https://www.ncbi.nlm.nih.gov/pmc/articles/instance/1464466/bin/tjp0564-0775-f6.jpg)
![Figure 7. Leupeptin reduces the initial decay…](https://www.ncbi.nlm.nih.gov/pmc/articles/instance/1464466/bin/tjp0564-0775-f7.jpg)
![Figure 8. Calpain-3 Western blots of muscle…](https://www.ncbi.nlm.nih.gov/pmc/articles/instance/1464466/bin/tjp0564-0775-f8.jpg)
![Figure 9. Silver stained 2.8% SDS-PAGE gel…](https://www.ncbi.nlm.nih.gov/pmc/articles/instance/1464466/bin/tjp0564-0775-f9.jpg)
Source: PubMed