Calsequestrin content and SERCA determine normal and maximal Ca²⁺ storage levels in sarcoplasmic reticulum of fast‐ and slow‐twitch fibres of rat

Abstract

Whilst calsequestrin (CSQ) is widely recognized as the primary Ca2+ buffer in the sarcoplasmic reticulum (SR) in skeletal muscle fibres, its total buffering capacity and importance have come into question. This study quantified the absolute amount of CSQ isoform 1 (CSQ1, the primary isoform) present in rat extensor digitorum longus (EDL) and soleus fibres, and related this to their endogenous and maximal SR Ca 2+ content. Using Western blotting, the entire constituents of minute samples of muscle homogenates or segments of individual muscle fibres were compared with known amounts of purified CSQ1. The fidelity of the analysis was proven by examining the relative signal intensity when mixing muscle samples and purified CSQ1. The CSQ1 contents of EDL fibres, almost exclusively type II fibres, and soleus type I fibres [SOL (I)] were, respectively, 36 ± 2 and 10 ± 1 μmol (l fibre volume) −1 , quantitatively accounting for the maximal SR Ca 2+ content of each. Soleus type II [SOL (II)] fibres (∼20% of soleus fibres) had an intermediate amount of CSQ1. Every SOL (I) fibre examined also contained some CSQ isoform 2 (CSQ2), which was absent in every EDL and other type II fibre except for trace amounts in one case. Every EDL and other type II fibre had a high density of SERCA1, the fast-twitch muscle sarco(endo)plasmic reticulum Ca 2+ -ATPase isoform, whereas there was virtually no SERCA1 in any SOL (I) fibre. Maximal SR Ca 2+ content measured in skinned fibres increased with CSQ1 content, and the ratio of endogenous to maximal Ca 2+ content was inversely correlated with CSQ1 content. The relative SR Ca 2+ content that could be maintained in resting cytoplasmic conditions was found to be much lower in EDL fibres than in SOL (I) fibres (∼20 versus >60%). Leakage of Ca2+ from the SR in EDL fibres could be substantially reduced with a SR Ca 2+ pump blocker and increased by adding creatine to buffer cytoplasmic [ADP] at a higher level, both results indicating that at least part of the Ca 2+ leakage occurred through SERCA. It is concluded that CSQ1 plays an important role in EDL muscle fibres by providing a large total pool of releasable Ca 2+ in the SR whilst maintaining free [Ca 2+ ] in the SR at sufficiently low levels that Ca 2+ leakage through the high density of SERCA1 pumps does not metabolically compromise muscle function.

Enhanced Ca2+ transport and muscle relaxation in skeletal muscle from sarcolipin-null mice

“…It is well accepted that SERCA1a is expressed in fast-twitch skeletal muscle fibers, whereas SERCA2a is expressed in slow-twitch fibers in rats (23,36). Here, we show that the different SERCA isoforms are, generally, also confined to entirely different muscle fibers in mouse skeletal muscle with SERCA1a expressed in fast-twitch (type II) fibers and SERCA2a expressed in slow-twitch (type I) fibers.…”

Calsequestrin depolymerizes when calcium is depleted in the sarcoplasmic reticulum of working muscle

“…S4, it increased markedly in the first bleached region of on Casq-null mice. Most, however, were done on WT mice; therefore, the exogenous Casq1 (which was expressed at 2-5 μmol/L cytosol) was less than 10% of the total [estimated at 50 μM or greater (25,26)]. The evolution of f ðtÞ in a representative case is shown in Fig.…”

Fiber Types in Mammalian Skeletal Muscles

“…Both isoforms can be found in slow fibers, whereas only CASQ1 is expressed in fast fibers (177,680). Calsequestrin content is greater in fast than in slow fibers (466) and a quantitative analysis on single murine fibers (554) points to a concentration of 36 M in fast fibers (only CASQ1) and 11 M in slow fibers (CASQ1 and CASQ2). CASQ plays two important roles: 1) calcium buffer since, due to the large number of acidic residues, each CASQ1 molecule binds up 80 calcium ions and each CSAQ2 up to 60 calcium ions, and 2) modulator of calcium release due to its interaction with RyR (64).…”

“…As mentioned above, CASQ is the most important calcium binding protein inside SR. Due to the larger SR volume (202) and the greater abundance of CASQ (466,554), the SR of fast fibers is only filled with calcium for 35% of its capacity at resting concentrations of cytosolic free calcium: thus any increase of cytosolic free calcium will be followed by a fast and effective resequestration into the SR with little increase of intrareticular free calcium concentration (128,242). In contrast, in slow fibers, the SR is completely saturated with calcium at resting concentrations of cytosolic free calcium (242); thus during the reuptake phase of a cytosolic calcium transient the saturation of SR will slow down the removal of calcium from cytosol by backinhibition of the Ca 2ϩ pump (367).…”