RyR2 Open Probability Research Articles

Iron (II) Modulation of the Cardiac Ryanodine Receptor (RyR2)

Cardiomyopathies and arrhythmias are major causes of mortality in chronic iron overload. There is evidence that iron overload impairs cardiomyocytes Ca2+ homeostasis (Baptista-Hon et al, 2005). However its molecular substrates remain unknown. Cardiac ryanodine receptors (RyR2) dysfunction is implicated in several diseases where Ca2+ homeostasis is lost. We therefore wanted to investigate RyR2 role as a potential target for iron-induced cardiomyopathies.We isolated heavy sarcoplasmic reticulum (HSR) vesicles containing RyR2 from sheep hearts. RyR2 were reconstituted into L-α-phosphatidylethanolamine (PE) bilayers. Unitary currents were measured under voltage-clamp with 250mM Cs+ as the charge carrier and 10μM activating Ca2+. High affinity [3H]ryanodine binding of the native vesicles was detected by liquid scintillation counting. Non-specific binding determined by incubations with 100x cold ryanodine.Fe2+ reduced RyR2 open probability and conductance in a dose dependent manner. Lifetime analysis revealed 5 shut times components and 3 open times components in control. Fe2+ caused an extra-shut component. Furthermore, there was a dose dependent shift in the open time constants towards the faster components. The binding assays revealed a [Fe2+] dependent, co-operative reduction in [3H]ryanodine binding to HSR vesicles. Preliminary data of [3H]ryanodine binding in increasing [Ca2+] showed a rightward shift in the presence of Fe2+.The results presented here show for the first time that Fe2+ is a potent inhibitor of RyR2. The mechanism of this inhibition may be due to competition with Ca2+ for RyR2 activation sites. Suppression of RyR2 activity by Fe2+ may therefore be one of the mechanisms involved in iron-induced cardiomyopathies.ReferencesBaptista-Hon, D, Diaz, M. E., and Elliot, A. C. Acute exposure to iron (II) alters calcium handling in isolated rat ventricular myocytes. Journal of Molecular and Cellular Cardiology 39, 179. 2005.

The Ryanodine Receptor Pore Blocker Neomycin also Inhibits Channel Activity via a Previously Undescribed High-Affinity Ca2+ Binding Site

In this study, we present evidence for the mechanism of neomycin inhibition of skeletal ryanodine receptors (RyRs). In single-channel recordings, neomycin produced monophasic inhibition of RyR open probability and biphasic inhibition of [(3)H]ryanodine binding. The half-maximal inhibitory concentration (IC(50)) for channel blockade by neomycin was dependent on membrane potential and cytoplasmic [Ca(2+)], suggesting that neomycin acts both as a pore plug and as a competitive antagonist at a cytoplasmic Ca(2+) binding site that causes allosteric inhibition. This novel Ca(2+)/neomycin binding site had a neomycin affinity of 100 nM: and a Ca(2+) affinity of 35 nM,: which is 30-fold higher than that of the well-described cytoplasmic Ca(2+) activation site. Therefore, a new high-affinity class of Ca(2+) binding site(s) on the RyR exists that mediates neomycin inhibition. Neomycin plugging of the channel pore induced brief (1-2 ms) conductance substates at 30% of the fully open conductance, whereas allosteric inhibition caused complete channel closure with durations that depended on the neomycin concentration. We quantitatively account for these results using a dual inhibition model for neomycin that incorporates voltage-dependent pore plugging and Ca(2+)-dependent allosteric inhibition.

Nitroxyl: A positive inotrope enhancing SR CA2+ cycling in isolated myocytes

Objectives. Nitroxyl (HNO) is a positive inotrope in vivo. Here we aim to elucidate the mechanisms for HNO-induced inotropy. We demonstrate that HNO enhances sarcoplasmic reticular (SR) Ca2+ cycling, in a thiol-sensitive manner. Methods and results. In murine isolated myocytes, the HNO donor Angeli's Salt (AS) increased sarcomere shortening in a dose-dependent manner (for instance, the increase with 1 mM AS was 117±25%; p<.01 vs. base). This effect was not mimicked by the NO donor DEA/NO. Preincubation with reduced glutathione (GSH, 4 mM, 3 h) incremented intracellular thiol content by 6% (p<.05), and AS/HNO response was markedly blunted in these cells: 58±19% (p=.05 vs. AS 1 mM alone). AS/HNO activated cardiac ryanodine receptor (RyR2). In reconstituted RyR2, HNO increased RyR2 open probability (Po) without altering the unitary conductance: Po increased from 0.16±0.03 to 0.25±0.05, 0.46±0.07 and 0.69±0.11 after 0.1, 0.3, and 1 mM AS, respectively. These changes were fully reverted by addition of the reducing agent DTT (2 mM) to the cis side (0.11±0.04). In addition, HNO dose-dependently increased Ca2+ sparks frequency (CSF): with 0.5 mM AS/HNO CSF was 26±3 vs. 4±1 sparks/100 μm/s (p<.01). Again, pretreatment with cell-permeable GSH abrogated HNO-induced increase in CSF. Finally, to assess whether AS also affects SERCA2a function, AS (250 μM) was added to isolated cardiac mouse SR vesicles. HNO markedly increased the rate of SR Ca2+ uptake. Conclusions. Thus, HNO increases and sustain inotropy by enhancing SR Ca2+ cycling, without altering diastolic Ca2+ levels and/or SR Ca2+ load.

Mechanisms of Abnormal Calcium Homeostasis in Mutations Responsible for Catecholaminergic Polymorphic Ventricular Tachycardia

Catecholaminergic polymorphic ventricular tachycardia is a heritable arrhythmia unmasked by exertion or stress and is characterized by triggered activity and sudden cardiac death. In this study, we simulated mutations in 2 genes linked to catecholaminergic polymorphic ventricular tachycardia, the first located in calsequestrin (CSQN2) and the second in the ryanodine receptor (RyR2). The aim of the study was to investigate the mechanistic basis for spontaneous Ca2+ release events that lead to delayed afterdepolarizations in affected patients. Sarcoplasmic reticulum (SR) luminal Ca2+ sensing was incorporated into a model of the human ventricular myocyte, and CSQN2 mutations were modeled by simulating disrupted RyR2 luminal Ca2+ sensing. In voltage-clamp mode, the mutant CSQN2 model recapitulated the smaller calcium transients, smaller time to peak calcium transient, and accelerated recovery from inactivation seen in experiments. In current clamp mode, in the presence of beta stimulation, we observed delayed afterdepolarizations, suggesting that accelerated recovery of RyR2 induced by impaired luminal Ca2+ sensing underlies the triggered activity observed in mutant CSQN2-expressing myocytes. In current-clamp mode, in a model of mutant RyR2 that is characterized by reduced FKBP12.6 binding to the RyR2 on beta stimulation, the impaired coupled gating characteristic of these mutations was modeled by reducing cooperativity of RyR2 activation. In current-clamp mode, the mutant RyR2 model exhibited increased diastolic RyR2 open probability that resulted in formation of delayed afterdepolarizations. In conclusion, these minimal order models of mutant CSQN2 and RyR2 provide plausible mechanisms by which defects in RyR2 gating may lead to the cellular triggers for arrhythmia, with implications for the development of targeted therapy.

Is the Ryanodine Receptor a Target for Antiarrhythmic Therapy?

See related article, pages 1299–1305 In cardiac myocytes, Ca2+ influx and efflux must be in balance to ensure cellular viability, normal contractile function, and a stable heart rhythm. Therefore Ca2+ fluxes between the major cellular compartments and the extracellular space have to adapt to a wide range of changing conditions. Failure to do so can result in Ca2+ overload of the sarcoplasmic reticulum (SR), leading to arrhythmogenic spontaneous release of SR Ca2+ by ryanodine receptors (RyRs). Recently, it was shown that suppressing RyR open probability (Po) was protective in a mouse model of a congenital arrhythmia caused by increased Ca2+ leak from RyRs. It was suggested that such a strategy could be applied more widely to treat patients with common ventricular arrhythmias.1 Is it possible to suppress SR Ca2+ release without jeopardizing contractile function and aggravating Ca2+ overload? In this issue of Circulation Research , Venetucci et al2 answer this question by using the analytic techniques they have used so successfully in the past to examine Ca2+ fluxes and autoregulation in normal cells.3 Their surprising finding is that reducing RyR Po in Ca2+-overloaded myocytes not only suppresses arrhythmogenic spontaneous Ca2+ release, but also increases the amplitude of the Ca2+ transient while maintaining Ca2+ homeostasis. To fully appreciate this finding, it is essential to review the profile of Ca2+ fluxes under both physiological conditions and during arrhythmogenic events. Under normal conditions, Ca2+ enters the cardiomyocyte at the beginning of each contractile cycle through L-type Ca2+ channels (LCCs) and minimally raises the cytoplasmic Ca2+ concentration. This “trigger calcium” binds to RyRs and induces an even greater release of stored Ca2+ from the SR into the cytoplasm, which causes myofilament …

Calstabin deficiency, ryanodine receptors, and sudden cardiac death

Altered cardiac ryanodine receptor (RyR2) function has an important role in heart failure and genetic forms of arrhythmias. RyR2 constitutes the major intracellular Ca 2+ release channel in the cardiac sarcoplasmic reticulum (SR). The peptidyl–prolyl isomerase calstabin2 (FKBP12.6) is a component of the RyR2 macromolecular signaling complex. Calstabin2 binding to RyR2 is regulated by PKA phosphorylation of Ser 2809 in RyR2. PKA phosphorylation of RyR2 decreases the binding affinity for calstabin2 and increases RyR2 open probability and sensitivity to Ca 2+-dependent activation. In heart failure, a majority of studies have found that RyR2 becomes chronically PKA hyper-phosphorylated which depletes calstabin2 from the channel complex. Calstabin2 dissociation causes a diastolic SR Ca 2+ leak contributing to depressed intracellular Ca 2+ cycling and decreased cardiac contractility. Missense mutations linked to genetic forms of exercise-induced arrhythmias and sudden cardiac death also cause decreased calstabin2-binding affinity and leaky RyR2 channels. We review the importance of calstabin2 for RyR2 function and excitation–contraction coupling, and discuss new observations that implicate dysregulation of calstabin2 binding as a central mechanism for abnormal calcium cycling in heart failure and triggered arrhythmias.

Physiological and pathological modulation of ryanodine receptor function in cardiac muscle

Calcium release from the sarcoplasmic reticulum (SR) in cardiac muscle occurs through a specialised release channel, the ryanodine receptor, RyR, via the process of Ca-induced Ca release (CICR). The open probability of the RyR is increased by elevation of cytoplasmic Ca concentration ([Ca 2+] i). However, in addition to Ca, other modulators affect the RyR open probability. Agents which increase the RyR opening during systole produce a transient increase of systolic [Ca 2+] i followed by a return to the initial level due to a compensating decrease of SR Ca content. Increasing RyR opening during diastole decreases SR Ca content and thereby decreases systolic [Ca 2+] i. We therefore conclude that potentiation of RyR opening will, if anything, decrease systolic [Ca 2+] i. The effects of specific examples of modulators of the RyR, such as phosphorylation, metabolic changes, heart failure and polyunsaturated fatty acids, are discussed.

Removal of clustered positive charge from dihydropyridine receptor II–III loop peptide augments activation of ryanodine receptors

Peptides based on the skeletal muscle DHPR II–III loop have been shown to regulate ryanodine receptor channel activity. The N-terminal region of this cytoplasmic loop is predicted to adopt an α-helical conformation. We have selected a peptide sequence of 26 residues (Ala 667–Asp 692) as the minimum sequence to emulate the helical propensity of the corresponding protein sequence. The interaction of this control peptide with skeletal and cardiac RyR channels in planar lipid bilayers was then assessed and was found to lack isoform specificity. At low concentrations peptide A 667–D 692 increased RyR open probability, whilst at higher concentrations open probability was reduced. By replacing a region of clustered positive charge with a neutral sequence with the same predisposition to helicity, the inhibitory effect was ablated and activation was enhanced. This novel finding demonstrates that activation does not derive from the presence of positively charged residues adjacent in the primary structure and, although it may be mediated by the alignment of basic residues down one face of an amphipathic helix, not all of these residues are essential.

Relative effectiveness of two mathematical models of interpersonal contingency for the prediction of conventional measures of infant development

Calcium (Ca) sparks are the fundamental sarcoplasmic reticulum (SR) Ca release events in cardiac myocytes, and they have a typical duration of 20–40 ms. However, when a fraction of ryanodine receptors (RyRs) are blocked by tetracaine or ruthenium red, Ca sparks lasting hundreds of milliseconds have been observed experimentally. The fundamental mechanism underlying these extremely prolonged Ca sparks is not understood. In this study, we use a physiologically detailed mathematical model of subcellular Ca cycling to examine how Ca spark duration is influenced by the number of functional RyRs in a junctional cluster (which is reduced by tetracaine or ruthenium red) and other SR Ca handling properties. One RyR cluster contains a few to several hundred RyRs, and we use a four-state Markov RyR gating model. Each RyR opens stochastically and is regulated by cytosolic and luminal Ca. We varied the number of functional RyRs in the single cluster, diffusion within the SR network, diffusion between network and junctional SR, cytosolic Ca diffusion, SERCA uptake activity, and RyR open probability. For long-lasting Ca release events, opening events within the cluster must occur continuously because the typical open time of the RyR is only a few milliseconds. We found the following: 1) if the number of RyRs is too small, it is difficult to maintain consecutive openings and stochastic attrition terminates the release; 2) if the number of RyRs is too large, the depletion of Ca from the junctional SR terminates the release; and 3) very long release events require relatively small-sized RyR clusters (reducing flux as seen experimentally with tetracaine) and sufficiently rapid intra-SR Ca diffusion, such that local junctional intra-SR [Ca] can be maintained by intra-SR diffusion and overall SR Ca reuptake.