Sarcoplasmic reticulum Ca2+ uptake and leak properties and SERCA isoform expression in type I and type II fibres of human skeletal muscle

摘要

The Ca²⁺ uptake properties of the sarcoplasmic reticulum (SR) were compared between type I and type II fibres of vastus lateralis muscle of young healthy adults. Individual mechanically skinned muscle fibres were exposed to solutions with the free [Ca²⁺] heavily buffered in the pCa range (–log10[Ca²⁺]) 7.3–6.0 for set times and the amount of net SR Ca²⁺ accumulation determined from the force response elicited upon emptying the SR of all Ca²⁺. Western blotting was used to determine fibre type and the sarco(endo)plasmic reticulum Ca²⁺-ATPase (SERCA) isoform present in every fibre examined. Type I fibres contained only SERCA2 and displayed half-maximal Ca²⁺ uptake rate at ∼pCa 6.8, whereas type II fibres contained only SERCA1 and displayed half-maximal Ca²⁺ uptake rate at ∼pCa 6.6. Maximal Ca²⁺ uptake rate was ∼0.18 and ∼0.21 mmol Ca²⁺ (l fibre)^–1 s^–1 in type I and type II fibres, respectively, in good accord with previously measured SR ATPase activity. Increasing free [Mg²⁺] from 1 to 3 mm had no significant effect on the net Ca²⁺ uptake rate at pCa 6.0, indicating that there was little or no calcium-induced calcium release occurring through the Ca²⁺ release channels during uptake in either fibre type. Ca²⁺ leakage from the SR at pCa 8.5, which is thought to occur at least in part through the SERCA, was ∼2-fold lower in type II fibres than in type I fibres, and was little affected by the presence of ADP, in marked contrast to the larger SR Ca²⁺ leak observed in rat muscle fibres under the same conditions. The higher affinity of Ca²⁺ uptake in the type I human fibres can account for the higher relative level of SR Ca²⁺ loading observed in type I compared to type II fibres, and the SR Ca²⁺ leakage characteristics of the human fibres suggest that the SERCAs are regulated differently from those in rat and contribute comparatively less to resting metabolic rate.