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Sphingosylphosphocholine modulates the ryanodine receptor/calcium-release channel of cardiac sarcoplasmic reticulum membranes.

ABSTRACT: Sphingosylphosphocholine (SPC) modulates Ca2+ release from isolated cardiac sarcoplasmic reticulum membranes; 50 microM SPC induces the release of 70 80% of the accumulated calcium. SPC release calcium from cardiac sarcoplasmic reticulum through the ryanodine receptor, since the release is inhibited by the ryanodine receptor channel antagonists ryanodine. Ruthenium Red and sphingosine. In intact cardiac myocytes, even in the absence of extracellular calcium. SPC causes a rise in diastolic Ca2+, which is greatly reduced when the sarcoplasmic reticulum is depleted of Ca2+ by prior thapsigargin treatment. SPC action on the ryanodine receptor is Ca(2+)-dependent. SPC shifts to the left the Ca(2+)-dependence of [3H]ryanodine binding, but only at high pCa values, suggesting that SPC might increase the sensitivity to calcium of the Ca(2+)-induced Ca(2+)-release mechanism. At high calcium concentrations (pCa 4.0 or lower), where [3H]ryanodine binding is maximally stimulated, no effect of SPC is observed. We conclude that SPC releases calcium from cardiac sarcoplasmic reticulum membranes by activating the ryanodine receptor and possibly another intracellular Ca(2+)-release channel, the sphingolipid Ca(2+)-release-mediating protein of endoplasmic reticulum (SCaMPER) [Mao, Kim, Almenoff, Rudner, Kearney and Kindman (1996) Proc.Natl.Acad.Sci. U.S.A 93, 1993-1996], which we have identified for the first time in cardiac tissue.

SUBMITTER: Betto R

PROVIDER: S-EPMC1218195 | biostudies-other | 1997 Feb

REPOSITORIES: biostudies-other

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Recovery of cardiac calcium release is controlled by sarcoplasmic reticulum refilling and ryanodine receptor sensitivity.

Project description:AimsIn heart cells, the mechanisms underlying refractoriness of the elementary units of sarcoplasmic reticulum (SR) Ca(2+) release, Ca(2+) sparks, remain unclear. We investigated local recovery of SR Ca(2+) release using experimental measurements and mathematical modelling.Methods and resultsRepeated Ca(2+) sparks were induced from individual clusters of ryanodine receptors (RyRs) in quiescent rat ventricular myocytes, and we examined how changes in RyR gating influenced the time-dependent recovery of Ca(2+) spark amplitude and triggering probability. Repeated Ca(2+) sparks from individual sites were analysed in the presence of 50 nM ryanodine with: (i) no additional agents (control); (ii) 50 µM caffeine to sensitize RyRs; (iii) 50 µM tetracaine to inhibit RyRs; or (iv) 100 nM isoproterenol to activate β-adrenergic receptors. Sensitization and inhibition of RyR clusters shortened and lengthened, respectively, the median interval between consecutive Ca(2+) sparks (caffeine 239 ms; control 280 ms; tetracaine 453 ms). Recovery of Ca(2+) spark amplitude, however, was exponential with a time constant of ∼100 ms in all cases. Isoproterenol both accelerated the recovery of Ca(2+) spark amplitude (τ = 58 ms) and shortened the median interval between Ca(2+) sparks (192 ms). The results were recapitulated by a mathematical model in which SR [Ca(2+)] depletion terminates Ca(2+) sparks, but not by an alternative model based on limited depletion and Ca(2+)-dependent inactivation of RyRs.ConclusionTogether, the results strongly suggest that: (i) local SR refilling controls Ca(2+) spark amplitude recovery; (ii) Ca(2+) spark triggering depends on both refilling and RyR sensitivity; and (iii) β-adrenergic stimulation influences both processes.

| S-EPMC3156908 | biostudies-literature

Size Matters: Ryanodine Receptor Cluster Size Affects Arrhythmogenic Sarcoplasmic Reticulum Calcium Release.

Project description:Ryanodine receptors (RyR) mediate sarcoplasmic reticulum calcium (Ca2+) release and influence myocyte Ca2+ homeostasis and arrhythmias. In cardiac myocytes, RyRs are found in clusters of various sizes and shapes, and RyR cluster size may critically influence normal and arrhythmogenic Ca2+ spark and wave formation. However, the actual RyR cluster sizes at specific Ca2+ spark sites have never been measured in the physiological setting. Here we measured RyR cluster size and Ca2+ sparks simultaneously to assess how RyR cluster size influences Ca2+ sparks and sarcoplasmic reticulum Ca2+ leak. For small RyR cluster sizes (<50), Ca2+ spark frequency is very low but then increases dramatically at larger cluster sizes. In contrast, Ca2+ spark amplitude is nearly maximal even at relatively small RyR cluster size (≈10) and changes little at larger cluster size. These properties agreed with computational simulations of RyR gating within clusters. Our study explains how this combination of properties may limit arrhythmogenic Ca2+ sparks and wave propagation (at many junctions) while preserving the efficacy and spatial synchronization of Ca2+-induced Ca2+-release during normal excitation-contraction coupling. However, variations in RyR cluster size among individual junctions and RyR sensitivity could exacerbate heterogeneity of local sarcoplasmic reticulum Ca2+ release and arrhythmogenesis under pathological conditions.

| S-EPMC6064922 | biostudies-literature

Effect of flecainide derivatives on sarcoplasmic reticulum calcium release suggests a lack of direct action on the cardiac ryanodine receptor.

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| S-EPMC4945764 | biostudies-literature

Dynamic calcium movement inside cardiac sarcoplasmic reticulum during release.

Project description:Intra-sarcoplasmic reticulum (SR) free [Ca] ([Ca](SR)) provides the driving force for SR Ca release and is a key regulator of SR Ca release channel gating during normal SR Ca release or arrhythmogenic spontaneous Ca release events. However, little is known about [Ca](SR) spatiotemporal dynamics.To directly measure local [Ca](SR) with subsarcomeric spatiotemporal resolution during both normal global SR Ca release and spontaneous Ca sparks and to evaluate the quantitative implications of spatial [Ca](SR) gradients.Intact and permeabilized rabbit ventricular myocytes were subjected to direct simultaneous measurement of cytosolic [Ca] and [Ca](SR) and FRAP (fluorescence recovery after photobleach). We found no detectable [Ca](SR) gradients between SR release sites (junctional SR) and Ca uptake sites (free SR) during normal global Ca release, clear spatiotemporal [Ca](SR) gradients during isolated Ca blinks, faster intra-SR diffusion in the longitudinal versus transverse direction, 3- to 4-fold slower diffusion of fluorophores in the SR than in cytosol, and that intra-SR Ca diffusion varies locally, dependent on local SR connectivity. A computational model clarified why spatiotemporal gradients are more detectable in isolated local releases versus global releases and provides a quantitative framework for understanding intra-SR Ca diffusion.Intra-SR Ca diffusion is rapid, limiting spatial [Ca](SR) gradients during excitation-contraction coupling. Spatiotemporal [Ca](SR) gradients are apparent during Ca sparks, and these observations constrain models of dynamic Ca movement inside the SR. This has important implications for myocyte SR Ca handling, synchrony, and potentially arrhythmogenic spontaneous contraction.

| S-EPMC3084972 | biostudies-literature

Amitriptyline activates cardiac ryanodine channels and causes spontaneous sarcoplasmic reticulum calcium release.

Project description:Patients taking amitriptyline (AMT) have an increased risk of sudden cardiac death, yet the mechanism for AMT's proarrhythmic effects remains incompletely understood. Here, we hypothesize that AMT activates cardiac ryanodine channels (RyR2), causing premature Ca(2+) release from the sarcoplasmic reticulum (SR), a mechanism identified by genetic studies as a cause of ventricular arrhythmias and sudden cardiac death. To test this hypothesis, we measured the effect of AMT on RyR2 channels from mice and sheep and on intact mouse cardiomyocytes loaded with the Ca(2+) fluorescent indicator Fura-2 acetoxymethyl ester. AMT induced trains of long channel openings (bursts) with 60 to 90% of normal conductance in RyR2 channels incorporated in lipid bilayers. The [AMT], voltage, and open probability (P(o)) dependencies of burst frequency and duration indicated that AMT binds primarily to open RyR2 channels. AMT also activated RyR2 channels isolated from transgenic mice lacking cardiac calsequestrin. Reducing RyR2 P(o) by increasing cytoplasmic [Mg(2+)] significantly inhibited the AMT effect on RyR2 channels. Consistent with the single RyR2 channel data, AMT increased the rate of spontaneous Ca(2+) releases and decreased the SR Ca(2+) content in intact cardiomyocytes. Intracellular [AMT] were approximately 5-fold higher than extracellular [AMT], explaining AMT's higher potency in cardiomyocytes at clinically relevant concentrations (0.5-3 muM) compared with its effect in lipid bilayers (5-10 muM). Increasing extracellular [Mg(2+)] attenuated the effect of AMT in intact myocytes. We conclude that the heretofore unrecognized activation of RyR2 channels and increased SR Ca(2+) leak may contribute to AMT's proarrhythmic and cardiotoxic effects, which may be counteracted by interventions that reduce RyR2 channel open probability.

| S-EPMC2685059 | biostudies-other

Identification of the Ca2+-release activity and ryanodine receptor in sarcoplasmic-reticulum membranes during cardiac myogenesis.

Project description:Ca2+-induced Ca2+ release and pH-induced Ca2+ release activities were identified in sarcoplasmic-reticulum (SR) vesicles isolated from adult- and fetal-sheep hearts. Ca2+-induced Ca2+ release and pH-induced Ca2+ release appear to proceed via the same channels, since both phenomena are similarly inhibited by Ruthenium Red. Ca2+ release from fetal SR vesicles is inhibited by higher concentrations of Ruthenium Red than is that from adult membranes. Both fetal and adult SR vesicles bind ryanodine. Fetal SR shows higher ryanodine-binding capacity than adult SR vesicles. Scatchard analysis of ryanodine binding revealed only one high-affinity binding site (Kd 6.7 nM) in fetal SR vesicles compared with two distinct binding sites (Kd 6.6 and 81.5 nM) in the adult SR vesicles. SR vesicles isolated from fetal and adult hearts were separated on discontinuous sucrose gradients into light (free) and heavy (junctional) SR vesicles. Heavy SR vesicles isolated from adult hearts exhibited most of the Ca2+ release activities. In contrast, Ca2+-induced Ca2+ release, pH-induced Ca2+ release and ryanodine receptors were detected in both light and heavy fetal SR. These results suggest that fetal SR may not be morphologically and functionally as well differentiated as that of adult cardiac muscle and that it may contain a greater number of Ca2+-release channels than that present in adult SR membranes.

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Peptide fragments of the dihydropyridine receptor can modulate cardiac ryanodine receptor channel activity and sarcoplasmic reticulum Ca2+ release.

Project description:We show that peptide fragments of the dihydropyridine receptor II-III loop alter cardiac RyR (ryanodine receptor) channel activity in a cytoplasmic Ca2+-dependent manner. The peptides were AC (Thr-793-Ala-812 of the cardiac dihydropyridine receptor), AS (Thr-671-Leu-690 of the skeletal dihydropyridine receptor), and a modified AS peptide [AS(D-R18)], with an extended helical structure. The peptides added to the cytoplasmic side of channels in lipid bilayers at > or = 10 nM activated channels when the cytoplasmic [Ca2+] was 100 nM, but either inhibited or did not affect channel activity when the cytoplasmic [Ca2+] was 10 or 100 microM. Both activation and inhibition were independent of bilayer potential. Activation by AS, but not by AC or AS(D-R18), was reduced at peptide concentrations >1 mM in a voltage-dependent manner (at +40 mV). In control experiments, channels were not activated by the scrambled AS sequence (ASS) or skeletal II-III loop peptide (NB). Resting Ca2+ release from cardiac sarcoplasmic reticulum was not altered by peptide AC, but Ca2+-induced Ca2+ release was depressed. Resting and Ca2+-induced Ca2+ release were enhanced by both the native and modified AS peptides. NMR revealed (i) that the structure of peptide AS(D-R18) is not influenced by [Ca2+] and (ii) that peptide AC adopts a helical structure, particularly in the region containing positively charged residues. This is the first report of specific functional interactions between dihydropyridine receptor A region peptides and cardiac RyR ion channels in lipid bilayers.

| S-EPMC1224045 | biostudies-other

Mitochondrial free calcium regulation during sarcoplasmic reticulum calcium release in rat cardiac myocytes.

Project description:Cardiac mitochondria can take up Ca(2+), competing with Ca(2+) transporters like the sarcoplasmic reticulum (SR) Ca(2+)-ATPase. Rapid mitochondrial [Ca(2+)] transients have been reported to be synchronized with normal cytosolic [Ca(2+)](i) transients. However, most intra-mitochondrial free [Ca(2+)] ([Ca(2+)](mito)) measurements have been uncalibrated, and potentially contaminated by non-mitochondrial signals. Here we measured calibrated [Ca(2+)](mito) in single rat myocytes using the ratiometric Ca(2+) indicator fura-2 AM and plasmalemmal permeabilization by saponin (to eliminate cytosolic fura-2). The steady-state [Ca(2+)](mito) dependence on [Ca(2+)](i) (with 5 mM EGTA) was sigmoid with [Ca(2+)](mito)<[Ca(2+)](i) for [Ca(2+)](i) below 475 nM. With low [EGTA] (50 microM) and 150 nM [Ca(2+)](i) (+/-15 mM Na(+)) cyclical spontaneous SR Ca(2+) release occurred (5-15/min). Changes in [Ca(2+)](mito) during individual [Ca(2+)](i) transients were small ( approximately 2-10 nM/beat), but integrated gradually to steady-state. Inhibition SR Ca(2+) handling by thapsigargin, 2 mM tetracaine or 10 mM caffeine all stopped the progressive rise in [Ca(2+)](mito) and spontaneous Ca(2+) transients (confirming that SR Ca(2+) releases caused the [Ca(2+)](mito) rise). Confocal imaging of local [Ca(2+)](mito) (using rhod-2) showed that [Ca(2+)](mito) rose rapidly with a delay after SR Ca(2+) release (with amplitude up to 10 nM), but declined much more slowly than [Ca(2+)](i) (time constant 2.8+/-0.7 s vs. 0.19+/-0.06 s). Total Ca(2+) uptake for larger [Ca(2+)](mito) transients was approximately 0.5 micromol/L cytosol (assuming 100:1 mitochondrial Ca(2+) buffering), consistent with prior indirect estimates from [Ca(2+)](i) measurements, and corresponds to approximately 1% of the SR Ca(2+) uptake during a normal Ca(2+) transient. Thus small phasic [Ca(2+)](mito) transients and gradually integrating [Ca(2+)](mito) signals occur during repeating [Ca(2+)](i) transients.

| S-EPMC2683203 | biostudies-literature

Deficient ryanodine receptor S-nitrosylation increases sarcoplasmic reticulum calcium leak and arrhythmogenesis in cardiomyocytes.

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| S-EPMC2154479 | biostudies-literature

Insights into the gating mechanism of the ryanodine-modified human cardiac Ca2+-release channel (ryanodine receptor 2).

Project description:Ryanodine receptors (RyRs) are intracellular membrane channels playing key roles in many Ca(2+) signaling pathways and, as such, are emerging novel therapeutic and insecticidal targets. RyRs are so named because they bind the plant alkaloid ryanodine with high affinity and although it is established that ryanodine produces profound changes in all aspects of function, our understanding of the mechanisms underlying altered gating is minimal. We address this issue using detailed single-channel gating analysis, mathematical modeling, and energetic evaluation of state transitions establishing that, with ryanodine bound, the RyR pore adopts an extremely stable open conformation. We demonstrate that stability of this state is influenced by interaction of divalent cations with both activating and inhibitory cytosolic sites and, in the absence of activating Ca(2+), trans-membrane voltage. Comparison of the conformational stability of ryanodine- and Imperatoxin A-modified channels identifies significant differences in the mechanisms of action of these qualitatively similar ligands.

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