Mice Null for Calsequestrin 1 Exhibit Deficits in Functional Performance and Sarcoplasmic Reticulum Calcium Handling

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

Non-thiol reagents regulate ryanodine receptor function by redox interactions that modify reactive thiols

The Ca(2+) release channel (CRC) from sarcoplasmic reticulum (SR) is rich in thiol groups, and their oxidation/- reduction by thiol reagents activates/inhibits the CRC. Most channel regulators are not thiol reagents, and the mechanism of their action is illusive. Here the authors show that many channel activators act as electron acceptors, while many channel inhibitors act as electron donors in free radical reactions. The channel activator, caffeine, and the CRC inhibitor, tetracaine, are shown to interact competitively, which suggests that there exists a common site(s) on the CRC, that integrates the donor/acceptor effects of ligands. Moreover, channel activators shift the redox potential of reactive thiols on the ryanodinereceptor (RyR) to more negative values and decrease the number of reactive thiols, while channel inhibitors shift the redox potential to more positive values and increase the number of reactive thiols. These observations suggest that thenon-thiol channel modulators shift the thiol-disulfide balance within CRC by transiently exchanging electrons with the Ca(2+) release protein

β1a490–508, a 19-Residue Peptide from C-Terminal Tail of Cav1.1 β1a Subunit, Potentiates Voltage-Dependent Calcium Release in Adult Skeletal Muscle Fibers

The α1 and β1a subunits of the skeletal muscle calcium channel, Cav1.1, as well as the Ca(2+) release channel, ryanodine receptor (RyR1), are essential for excitation-contraction coupling. RyR1 channel activity is modulated by the β1a subunit and this effect can be mimicked by a peptide (β1a490-524) corresponding to the 35-residue C-terminal tail of the β1a subunit. Protein-protein interaction assays confirmed a high-affinity interaction between the C-terminal tail of the β1a and RyR1. Based on previous results using overlapping peptides tested on isolated RyR1, we hypothesized that a 19-amino-acid residue peptide (β1a490-508) is sufficient to reproduce activating effects of β1a490-524. Here we examined the effects of β1a490-508 on Ca(2+) release and Ca(2+) currents in adult skeletal muscle fibers subjected to voltage-clamp and on RyR1 channel activity after incorporating sarcoplasmic reticulum vesicles into lipid bilayers. β1a490-508 (25 nM) increased the peak Ca(2+) release flux by 49% in muscle fibers. Considerably fewer activating effects were observed using 6.25, 100, and 400 nM of β1a490-508 in fibers. β1a490-508 also increased RyR1 channel activity in bilayers and Cav1.1 currents in fibers. A scrambled form of β1a490-508 peptide was used as negative control and produced negligible effects on Ca(2+) release flux and RyR1 activity. Our results show that the β1a490-508 peptide contains molecular components sufficient to modulate excitation-contraction coupling in adult muscle fibers.Research reported in this publication was supported by the National Institute of Arthritis and Musculoskeletal and Skin Diseases of the National Institutes of Health, Bethesda, MD under award No. R37-AR055099 (to M.F.S.) and by the Australian National Health and Medical Research Council Project Grant No. APP1020589 (to A.F.D.). R.O.O. was supported by the National Institute of Arthritis and Musculoskeletal and Skin Diseases of the National Institutes of Health training grant No. T32 AR007592 to the Interdisciplinary Program in Muscle Biology, University of Maryland School of Medicine

Fluo-4 fluorescence transients are altered in CASQ1 null FDB myofibers.

<p>Free [Ca²⁺] and Ca²⁺ release flux from WT (left) and CASQ1 null fibers (right). Average (n = 4) time course of fluo-4 F/F₀ records expanded in time elicited at different voltages are displayed for WT fibers (<b>A</b>) and for CASQ1 null fibers (<b>B</b>). (<b>C</b>) and (<b>D</b>) are free [Ca²⁺] waveforms derived from <i>A</i> and <i>B</i> while (<b>E</b>) and (<b>F</b>) are Ca²⁺ release flux calculated from <i>C</i> and <i>D</i>. Comparison of the two sets of data show significant suppression of the amplitude of F/F₀, free Ca²⁺ and peak and maintained Ca²⁺ release flux in the CASQ1 null fibers.</p

CASQ1 null fibers lack the slowly decaying component of release.

<p>Fluo-4 fluorescence transient measured as ΔF/F0 for WT (left) and CASQ1 null fibers (right). <b>A</b> is the time course of fluo-4 records averaged from 3 WT fibers and <b>B</b> is the time course obtained for CASQ1 null fibers averaged from 3 fibers. 20 mV depolarizing pulses were applied and subsequently terminated at either 20 ms or 80 ms as illustrated on top of figures <b>A</b> and <b>B</b> in order to evaluate the slowly decaying component of release in both WT and CASQ1 null fibers. <b>C</b> and <b>D</b> are Ca²⁺ release flux calculated from <b>A</b> and <b>B</b>. The results showed that CASQ1 buffering triggered a slowly decaying component of release in WT fibers while CASQ1 null fibers lacking this buffering does not exhibit this slowly decaying component of release.</p

Representative raw force records from WT and CASQ1 null mice.

<p><b>A</b> Raw records (top) and same records normalized to peak tension (<b>A</b>, bottom) from the EDL force vs. frequency experiment (WT = black, CASQ1 null = red) with 500 msec trains of pulses of 1, 20, 40, 80, 300 Hz. (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0027036#pone-0027036-g002" target="_blank">Fig. 2A</a> for aggregated data of maximal tension). CASQ1 null EDL muscles exhibited a frequency dependent inability to sustain force during the pulse train. Note that significant difference in maximal force generation normalized when the differences in muscle mass were accounted for (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0027036#pone-0027036-g002" target="_blank">Fig. 2B</a>.). <b>B.</b> Representative normalized force traces from a WT and CASQ1 null muscle in which a 1500 msec train of pulses was delivered at 100 Hz. <b>C.</b> In 4 EDL muscles from each genotype, we demonstrated that while WT muscle showed little change in tension over the 1500 msec, developed tetanic tension in the CASQ1 null muscles fell to 85.2±6.7% (t-test, p<0.05) of the peak value.</p

Kinetics of the rapidly decaying component of the release flux at 20 mV in WT and CASQ1 null fibers.

<p>A decaying exponential fit to the Ca²⁺ release flux at 20 mV was used to determine the amplitude and time constant of the rapidly decaying component of the release flux in both WT and CASQ1 null fibers. The offset of the exponential fit corresponds to an estimate the amplitude of the slow decaying component of the release. <b>A</b> and <b>B</b> independently show comparison of the amplitudes of the fast and of the slow decaying component in both WT and CASQ1 null fibers respectively (n = 7). <b>B</b> shows that the amplitude of the slowly decaying component of release in CASQ1 null fibers was significantly reduced when compared to WT counterparts (* denotes p<0.05) while no significant changes occurred in the amplitude and the decay time constant of the rapidly decaying component of release in CASQ1 null fibers.</p

CASQ1 null myofibers have a reduction in relative SR [Ca²⁺]_free.

<p><b>A.</b> Representative SR Fluo5N fluorescence profile of a WT and CASQ1 null myofibers challenged with 1 mM 4-CmC. This profile was normalized to the maximum 4-CmC depletion (ΔF/F_4-CmC). <b>B.</b> The evaluation of F_initial as a surrogate to SR [Ca²⁺]_free (see results) revealed that CASQ1 null myofibers had a ∼24% reduction in relative SR [Ca²⁺]_free compared to WT myofibers (Mann-Whitney rank-sum test, P<0.05).</p

Kinetic parameters of EDL contractility <i>in vitro</i>.

<p>Like symbols indicate significance p<0.05.</p