Abstract
In skeletal muscle, the release of calcium (Ca2+) by ryanodine sensitive sarcoplasmic reticulum (SR) Ca2+ release channels (i.e., ryanodine receptors; RyR1s) is the primary determinant of contractile filament activation. Much attention has been focused on calsequestrin (CASQ1) and its role in SR Ca2+ buffering as well as its potential for modulating RyR1, the L-type Ca2+ channel (dihydropyridine receptor, DHPR) and other sarcolemmal channels through sensing luminal [Ca2+]. The genetic ablation of CASQ1 expression results in significant alterations in SR Ca2+ content and SR Ca2+ release especially during prolonged activation. While these findings predict a significant loss-of-function phenotype in vivo, little information on functional status of CASQ1 null mice is available. We examined fast muscle in vivo and in vitro and identified significant deficits in functional performance that indicate an inability to sustain contractile activation. In single CASQ1 null skeletal myofibers we demonstrate a decrease in voltage dependent RyR Ca2+ release with single action potentials and a collapse of the Ca2+ release with repetitive trains. Under voltage clamp, SR Ca2+ release flux and total SR Ca2+ release are significantly reduced in CASQ1 null myofibers. The decrease in peak Ca2+ release flux appears to be solely due to elimination of the slowly decaying component of SR Ca2+ release, whereas the rapidly decaying component of SR Ca2+ release is not altered in either amplitude or time course in CASQ1 null fibers. Finally, intra-SR [Ca2+] during ligand and voltage activation of RyR1 revealed a significant decrease in the SR[Ca2+]free in intact CASQ1 null fibers and a increase in the release and uptake kinetics consistent with a depletion of intra-SR Ca2+ buffering capacity. Taken together we have revealed that the genetic ablation of CASQ1 expression results in significant functional deficits consistent with a decrease in the slowly decaying component of SR Ca2+ release.
Highlights
Specialized systems have evolved for the robust and rapid release of calcium (Ca2+) ions from the sarcoplasmic reticulum (SR) in skeletal and cardiac muscle
Recent studies in muscle fibers with varied levels of CASQ1 [43] as well is in muscle fibers genetically depleted of CASQ1 and or CASQ1 and 2 [24,27,28,44] have demonstrated that CASQ is not required for EC coupling, but have reaffirmed its importance as a Ca2+ buffer in skeletal muscle SR
Our main findings are: (A.) Both voluntary and involuntary exercise capacity was greatly reduced in the CASQ1 null mice. (B.) While a substantial deficit in peak force produced by the CASQ1 null extensor digitorum longus (EDL) muscles could be almost fully accounted for by a decrease in mass these muscles exhibited a dramatic frequency dependent inability to sustain tension during a pulse train. (C.) With electric field stimulation of intact flexor digitorum brevis (FDB) fibers there is a significant decrease in the Ca2+ transients in CASQ1 null myofibers for single action potentials, and a collapse of the Ca2+ transient with repetitive trains of pulses. (D) Under voltage clamp, the peak SR Ca2+ release flux, as well as total SR Ca2+ release is significantly reduced in CASQ1 null fibers
Summary
Specialized systems have evolved for the robust and rapid release of calcium (Ca2+) ions from the sarcoplasmic reticulum (SR) in skeletal and cardiac muscle. A single action potential initiates a release of ,200 mM of Ca2+ (per liter of fiber volume) from the SR which causes a ‘‘twitch’’ contraction. Repetitive trains of action potentials are a hallmark of skeletal muscle activation. These contractions are sustained by the reserve of releasable Ca2+ in the basal state and a rapid restoration of SR Ca2+ content due to increased sarcoendoplasmic reticulum ATPase activity (SERCA) and dynamic refilling of the SR through sarcolemmal Ca2+ influx (i.e., excitation-coupled calcium entry; ECCE [5,6,7]) during repetitive stimuli. Recent work to elucidate the control of this dynamic regulation of SR Ca2+ has focused on calsequestrin (CASQ) and its binding partners in the SR membrane as potential modulators of the RyR1, the DHPR and other sarcolemmal channels through sensing luminal [Ca2+] [8,9,10]
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