Inactivation Of Sarcoplasmic Reticulum Ca2 Research Articles

Sarcoplasmic Reticulum (SR) Ca2+ Depletion Predominantly Contributes to the Decline of SR Ca2+ Release during Long-Lasting Depolarizations in Skeletal Muscle Fibers

High amplitude depolarizations of skeletal muscle fibers induce massive SR Ca2+ release that progressively declines with time and completely annihilates if depolarization is maintained during several tens of seconds. Several processes may be involved in the decline of SR Ca2+ release: cytosolic Ca2+-dependent closure of SR Ca2+ release channels, voltage-dependent inactivation of SR Ca2+ release channels and SR Ca2+ depletion. This study aimed to determine how these processes are involved during long-lasting depolarizations of isolated mouse muscle fibers under voltage control by measuring cytosolic Ca2+ changes using fura-2 or luminal SR Ca2+ changes using fluo5N in the presence of 50 mM internal EGTA. Decays of cytosolic Ca2+ signals elicited by 50-s duration depolarizations became more marked and faster with depolarization amplitude. Pre-depolarizations of 2-min duration and of increasing amplitude induced a reduction of voltage-activated cytosolic Ca2+ signals with a mean voltage of −50 mV inducing half-maximum reduction. A comparable protocol applied to fibers loaded with fluo5N showed that low voltage depolarizing prepulses induced a marked SR Ca2+ depletion that contributed to reduce a subsequent voltage-activated SR Ca2+ change with a mean voltage of −50 mV inducing half-maximum reduction. Measuring SR Ca2+ changes in response to long-lasting depolarizations indicated that SR Ca2+ release channels inactivated in response to much higher depolarizations with a mean half-maximum inactivation voltage of −20 mV. Finally, trains of action potential of 50 s duration produced cytosolic Ca2+ signals that decayed with time, whereas SR Ca2+ changes did not display any sign of inactivation. These results indicate that the decline in SR Ca2+ release during long-lasting depolarizations mainly results from SR Ca2+ depletion. The work was supported by AFM, CNRS and University Lyon 1.

Modulation of sarcoplasmic reticulum Ca2+ release in skeletal muscle expressing ryanodine receptor impaired in regulation by calmodulin and S100A1.

In vitro, calmodulin (CaM) and S100A1 activate the skeletal muscle ryanodine receptor ion channel (RyR1) at submicromolar Ca(2+) concentrations, whereas at micromolar Ca(2+) concentrations, CaM inhibits RyR1. One amino acid substitution (RyR1-L3625D) has previously been demonstrated to impair CaM binding and regulation of RyR1. Here we show that the RyR1-L3625D substitution also abolishes S100A1 binding. To determine the physiological relevance of these findings, mutant mice were generated with the RyR1-L3625D substitution in exon 74, which encodes the CaM and S100A1 binding domain of RyR1. Homozygous mutant mice (Ryr1(D/D)) were viable and appeared normal. However, single RyR1 channel recordings from Ryr1(D/D) mice exhibited impaired activation by CaM and S100A1 and impaired CaCaM inhibition. Isolated flexor digitorum brevis muscle fibers from Ryr1(D/D) mice had depressed Ca(2+) transients when stimulated by a single action potential. However, during repetitive stimulation, the mutant fibers demonstrated greater relative summation of the Ca(2+) transients. Consistently, in vivo stimulation of tibialis anterior muscles in Ryr1(D/D) mice demonstrated reduced twitch force in response to a single action potential, but greater summation of force during high-frequency stimulation. During repetitive stimulation, Ryr1(D/D) fibers exhibited slowed inactivation of sarcoplasmic reticulum Ca(2+) release flux, consistent with increased summation of the Ca(2+) transient and contractile force. Peak Ca(2+) release flux was suppressed at all voltages in voltage-clamped Ryr1(D/D) fibers. The results suggest that the RyR1-L3625D mutation removes both an early activating effect of S100A1 and CaM and delayed suppressing effect of CaCaM on RyR1 Ca(2+) release, providing new insights into CaM and S100A1 regulation of skeletal muscle excitation-contraction coupling.

Critical Roles of Hydrophobicity and Orientation of Side Chains for Inactivation of Sarcoplasmic Reticulum Ca2+-ATPase with Thapsigargin and Thapsigargin Analogs

Thapsigargin (Tg), a specific inhibitor of sarco/endoplasmic Ca(2+)-ATPases (SERCA), binds with high affinity to the E2 conformation of these ATPases. SERCA inhibition leads to elevated calcium levels in the cytoplasm, which in turn induces apoptosis. We present x-ray crystallographic and intrinsic fluorescence data to show how Tg and chemical analogs of the compound with modified or removed side chains bind to isolated SERCA 1a membranes. This occurs by uptake via the membrane lipid followed by insertion into a resident intramembranous binding site with few adaptative changes. Our binding data indicate that a balanced hydrophobicity and accurate positioning of the side chains, provided by the central guaianolide ring structure, defines a pharmacophore of Tg that governs both high affinity and access to the protein-binding site. Tg analogs substituted with long linkers at O-8 extend from the binding site between transmembrane segments to the putative N-terminal Ca(2+) entry pathway. The long chain analogs provide a rational basis for the localization of the linker, the presence of which is necessary for enabling prostate-specific antigen to cleave peptide-conjugated prodrugs targeting SERCA of cancer cells (Denmeade, S. R., Jakobsen, C. M., Janssen, S., Khan, S. R., Garrett, E. S., Lilja, H., Christensen, S. B., and Isaacs, J. T. (2003) J. Natl. Cancer Inst. 95, 990-1000). Our study demonstrates the usefulness of a simple in vitro system to test and direct development toward the formulation of new Tg derivatives with improved properties for SERCA targeting. Finally, we propose that the Tg binding pocket may be a regulatory site that, for example, is sensitive to cholesterol.

Negative Relationship Between Fractional SR Ca2+ Release and Stimulation Rate

Typically, contraction amplitude in rodent myocardium is inversely related to stimulation rate. We calculated Ca2+ fluxes across the sarcolemma and the sarcoplasmic reticulum (SR) membrane, and SR Ca2+ content to estimate the fraction of the SR Ca2+ load released at a twitch (FR) during steady-state (SS) stimulation at 0.2-2 Hz in intact rat cardiomyocytes. Cytosolic [Ca2+] ([Ca2+]i) was measured with indo-1. SR Ca2+ content was determined from the transient in response to 10 mM caffeine in Na+,Ca2+-free medium. From Ca2+ buffer parameters, and [Ca2+]i values and decline kinetics during transients in which SR Ca2+-ATPase and/or Na+-Ca2+ exchanger (NCX) were inhibited, SR- and NCX-mediated Ca2+ fluxes at each rate were estimated and integrated up to attainment of SS diastolic [Ca2+]i. Assuming equal inward and outward Ca2+ fluxes at SS, Ca2+ influx was considered as the NCX-mediated flux, whereas SR Ca2+ release and uptake fluxes were considered equivalent. FR was taken as the ratio of the integrated SR-mediated Ca2+ flux and SR Ca2+ content. Increasing rate did not affect significantly SR Ca2+ content (128±4 and 133±4 μM at 0.2 and 2 Hz, respectively), but decreased (p<0.01) both Ca2+ influx (16.9±1.4 vs. 6.7±0.3 μM at 0.2 and 2 Hz) and SR-dependent Ca2+ flux (93±4 vs. 77±3 μM at 0.2 and 2 Hz). Estimated FR showed a negative relationship with the stimulation rate (0.73±0.03 vs. 0.58±0.02, p<0.01), possibly due to decreased Ca2+ influx, although action potential duration was increased (APD90= 76±9 vs. 130±11 ms, p<0.01). Alternatively, incomplete time-dependent recovery from inactivation of SR Ca2+ channels might contribute to depress FR. These results support the proposal that changes in FR underlie the negative force-frequency relationship in rodents. (FAPESP, CNPq, FAEPEX).

Single-Channel Characterization of the Rabbit Recombinant RyR2 Reveals a Novel Inactivation Property of Physiological Concentrations of ATP

Ryanodine receptor 2 (RyR2) cDNA has been available for more than 15 years; however, due to the complex nature of ligand gating in this channel, many aspects of recombinant RyR2 function have been unresearched. We established a stable, inducible HEK 293 cell line expressing full-length rabbit RyR2 cDNA and assessed the single-channel properties of the recombinant RyR2, with particular reference to ligand regulation with Ca2+ as the permeant ion. We found that the single-channel conductances of recombinant RyR2 and RyR2 isolated from cardiac muscle are essentially identical, as is irreversible modification by ryanodine. Although it is known that RyR2 expressed in HEK 293 cells is not associated with FKBP12.6, we demonstrate that these channels do not exhibit any discernable disorganized gating characteristics or subconductance states. We also show that the gating of recombinant RyR2 is indistinguishable from that of channels isolated from cardiac muscle when activated by cytosolic Ca2+, caffeine or suramin. The mechanisms underlying ATP activation are also similar; however, the experiments highlighted a novel effect of ATP at physiologically relevant concentrations of 5-10 mM. With Ca2+ as permeant ion, 5-10 mM ATP consistently inactivated recombinant channels (15/16 experiments). Such inactivation was rarely observed with native RyR2 isolated from cardiac muscle (1 in 16 experiments). However, if the channels were purified, inactivation by ATP was then revealed in all experiments. This action of ATP may be relevant for inactivation of sarcoplasmic reticulum Ca2+ release during cardiac excitation-contraction coupling or may represent unnatural behavior that is revealed when RyR2 is purified or expressed in noncardiac systems.

Identification of a two EF-hand Ca2+ binding domain in lobster skeletal muscle ryanodine receptor/Ca2+ release channel.

The lobster skeletal muscle Ca2+ release channel, known also as the ryanodine receptor, is composed of four polypeptides of approximately 5000 amino acids each, like its mammalian counterparts. Clones encoding the carboxy-terminal region of the lobster ryanodine receptor were isolated from a lobster skeletal muscle cDNA library. Analysis of the deduced 1513 carboxy-terminal amino acid sequence suggests a cytoplasmic Ca2+ binding domain consisting of two EF-hand Ca2+ binding motifs (amino acid residues 594-656). The Ca2+ binding properties of this domain were assessed by preparing bacterial fusion proteins with sequences from the lobster Ca2+ binding domain and the corresponding sequences of the rabbit cardiac and skeletal muscle ryanodine receptors. The lobster skeletal muscle fusion protein bound 45Ca2+ in Ca2+ overlays, and bound two Ca2+ under equilibrium binding conditions with a Hill dissociation constant (KH) of 0.9 mM and coefficient (nH) of 1.4. Rabbit skeletal and cardiac fusion proteins bound two Ca2+ with KHs of 3.7 and 3.8 mM and nHs of 1.1 and 1.3, respectively. Similar to results previously reported for the mammalian RyRs, the lobster RyR was activated by micromolar Ca2+ and inhibited by millimolar Ca2+, as determined in single-channel and [3H]ryanodine binding measurements. These results suggest that the two EF-hand Ca2+ binding domain of the lobster Ca2+ release channel as well as the corresponding regions of the mammalian channels may play a role in Ca2+ inactivation of sarcoplasmic reticulum Ca2+ release.

Ca2+ Modulation of sarcoplasmic reticulum Ca2+ release in rat skeletal muscle fibers

Ca2+ transients and the rate of Ca2+ release (dCaREL/dt) from the sarcoplasmic reticulum (SR) in voltage-clamped, fast-twitch skeletal muscle fibers from the rat were studied with the double Vaseline gap technique and using mag-fura-2 and fura-2 as Ca2+ indicators. Single pulse experiments with different returning potentials showed that Ca2+ removal from the myoplasm is voltage independent. Thus, the myoplasmic Ca2+ removal (dCaREM/dt) was studied by fitting the decaying phase of the Ca2+ transient (Melzer, Ríos & Schneider, 1986) and dCaREL/dt was calculated as the difference between dCa/dt and dCaREM/dt. The fast Ca2+ release decayed as a consequence of Ca2+ inactivation of Ca2+ release. Double pulse experiments showed inactivation of the fast Ca2+ release depending on the prepulse duration. At constant interpulse interval, long prepulses (200 msec) induced greater inactivation of the fast Ca2+ release than shorter depolarizations (20 msec). The correlation (r) between the myoplasmic [Ca2+]i and the inhibited amount of Ca2+ release was 0.98. The [Ca2+]i for 50% inactivation of dCaREL/dt was 0.25 microM, and the minimum number of sites occupied by Ca2+ to inactivate the Ca2+ release channel was 3.0. These data support Ca2+ binding and inactivation of SR Ca2+ release.

Mechanism of the Negative Inotropic Effect of Thiopental in Isolated Ferret Ventricular Myocardium

Thiopental's myocardial depressant effects are well known and most likely involve some alteration in intracellular Ca2+ homeostasis. The aim of this study was to investigate the mechanisms of thiopental's negative inotropic effects and its underlying mechanism in isolated ferret ventricular myocardium (which shows physiologic characteristics similar to human ventricular myocardium), and in frog ventricular myocardium, in which Ca2+ ions for myofibrillar activation are derived almost entirely from transsarcolemmal influx. The authors analyzed the effects of thiopental after beta-adrenoceptor blockade on variables of contractility and relaxation, and on the free intracellular Ca2+ transient detected with the Ca(2+)-regulated photoprotein aequorin. Thiopental's effects also were evaluated in ferret right ventricular papillary muscles in which the sarcoplasmic reticulum (SR) function was impaired by ryanodine and in frog ventricular strips with little or no SR function. At concentration > or = 10(-4) M, which is in the high range of the clinically encountered free plasma thiopental concentrations, thiopental decreased contractility and the amplitude of the intracellular Ca2+ transient. At equal peak force, peak aequorin luminescence in 10(-4) M thiopental and [Ca2+]0 > 2.25 mM was slightly smaller than that in control conditions at [Ca2+]o = 2.25 mM. This indicates that thiopental causes a small increase in myofibrillar Ca2+ sensitivity. After inactivation of sarcoplasmic reticulum Ca2+ release with 10(-6) M ryanodine, a condition in which myofibrillar activation depends almost exclusively on transsarcolemmal Ca2+ influx, thiopental caused a further decrease in contractility and in the amplitude of the intracellular Ca2+ transient, and thiopental's relative negative inotropic effect was not different from that in control muscles not exposed to ryanodine. Thiopental, > or = 10(-4) M, decreased contractility in frog ventricular myocardium. These findings indicate that the direct negative inotropic effect of thiopental results from a decrease in intracellular Ca2+ availability. At least part of thiopental's action is caused by inhibition of transsarcolemmal Ca2+ influx. These effects become apparent at concentrations routinely present during intravenous induction with thiopental.

Mechanism of the Negative Inotropic Effect of Propofol in Isolated Ferret Ventricular Myocardium

The aim of this study was to investigate propofol's effect on myocardial contractility and relaxation and examine its underlying mechanism of action in isolated ferret ventricular myocardium. The effects of propofol on variables of contractility and relaxation and on the free intracellular Ca++ transient detected with the Ca(++)-regulated photoprotein aequorin were analyzed. Propofol's effects were evaluated in a preparation in which the sarcoplasmic reticulum function was impaired by ryanodine. The effects of propofol's solvent, intralipid, on myocardial contractility, relaxation, and the intracellular Ca++ transient also were examined. Propofol, at concentrations of 10 microM or greater, decreased contractility and, at concentrations of 30 of microns or greater, decreased the amplitude of the intracellular Ca++ transient. At equal peak force, control peak aequorin luminescence in [Ca++]o = 2.25 mM and peak aequorin luminescence in 300 microM [Ca++]o = 2.25 mM and peak aequorin luminescence in 300 microM propofol in [Ca++]o > 2.25 mM did not differ, which suggests that propofol does not alter myofibrillar Ca++ sensitivity. After inactivation of sarcoplasmic reticulum Ca++ release with 1 microM ryanodine, a condition in which myofibrillar activation depends almost exclusively on transsarcolemmal Ca++ influx, propofol caused a decrease in contractility and in the amplitude of the intracellular Ca++ transient. Under these conditions, propofol's relative negative inotropic effect did not differ from that in control muscles not exposed to ryanodine. Propofol's solvent, 10% intralipid, exerted a modest positive inotropic effect in this preparation. The intracellular Ca++ transient was unchanged by intralipid. Neither propofol nor intralipid altered the load sensitivity of relaxation. These findings suggest that the negative inotropic effect of propofol results from a decrease in intracellular Ca++ availability with no changes in myofibrillar Ca++ sensitivity. At least part of propofol's action is attributable to inhibition of transsarcolemmal Ca++ influx.

Inactivation of sarcoplasmic-reticulum Ca(2+)-ATPase in low-frequency-stimulated muscle results from a modification of the active site.

Molecular changes underlying the partial inactivation of the sarcoplasmic-reticulum (SR) Ca(2+-) ATPase in low-frequency-stimulated fast-twitch muscle were investigated in the present study. The specific Ca(2+)-ATPase activity, as well as the ATP- and acetyl phosphate-driven Ca2+ uptakes by the SR, were reduced by approx. 30% in 4-day-stimulated muscle. Phosphoprotein formation of the enzyme in the presence of ATP or Pi was also decreased to the same extent. Measurements of ATP binding revealed a 30% decrease in binding to the enzyme. These changes were accompanied by similar decreases in the ligand-induced (ATP, ADP, Pi) intrinsic tryptophan fluorescence. A decreased binding of fluorescein isothiocyanate (FITC) corresponded to the lower ATP binding and phosphorylation of the enzyme. Moreover, Pi-induced changes in fluorescence of the FITC-labelled enzyme did not differ between SR from stimulated and contralateral muscles, indicating that Ca(2+)- ATPase molecules which did not bind FITC were responsible for the decreased Pi-dependent phosphorylation, and therefore represented the inactive form of the enzyme. No differences existed between the Ca(2+)-induced changes in the intrinsic fluorescence of SR from stimulated and contralateral muscles which fit their similar Ca(2+)-binding characteristics. Taking the proposed architecture of the Ca2(+)-ATPase into consideration, our results suggest that the inactivation relates to a circumscribed structural alteration of the enzyme in sections of the active site consisting of the nucleotide-binding and phosphorylation domains.