Single RyR2 Research Articles

Phosphatidylinositol 3,5 bisphosphate increases contractility and intracellular calcium in cardiac muscle by directly activating the cardiac ryanodine receptor.

We have previously reported that treatment of left ventricular cardiac muscle strips with phosphatidylinositol 3,5 bisphosphate (PI(3,5)P2) during field stimulation enhanced the magnitude of isometric force, the rate of force development, and the area associated with the contractile waveforms. In addition, PI(3,5)P2 caused an increase in intracellular calcium (Ca) in cardiac myocytes. These data suggested that PI(3,5)P2 increased contractile force by promoting extracellular Ca entry or by facilitating intracellular Ca release. To elucidate the mechanism responsible for the effects of PI(3,5) on intracellular Ca, we have performed ratiometric Ca imaging in isolated adult mouse cardiac myocytes. We have found that PI(3,5)P2 induced a dramatic increase in intracellular Ca levels, which was inhibited by preincubation with ryanodine. To confirm that PI(3,5)P2 was acting on RyR2 we performed electrophysiological and biochemical studies. PI(3,5)P2 increased [3H]ryanodine binding and this effect was prevented by preincubation with an antibody against PI(3,5)P2. Furthermore, single RyR2 channels reconstituted in lipid bilayers were activated by PI(3,5)P2 in a dose‐dependent manner, indicating that PI(3,5)P2 binds directly to RyR2. We provide inaugural evidence that PI(3,5)P2 regulates cardiac contractility via modulation of intracellular Ca by direct binding and activation of the RyR2 channel.

RyR1-mediated Ca2+ Leak and Ca2+ Entry Determine Resting Intracellular Ca2+ in Skeletal Myotubes

The control of resting free Ca(2+) in skeletal muscle is thought to be a balance of channels, pumps, and exchangers in both the sarcolemma and sarcoplasmic reticulum. We explored these mechanisms using pharmacologic and molecular perturbations of genetically engineered (dyspedic) muscle cells that constitutively lack expression of the skeletal muscle sarcoplasmic reticulum Ca(2+) release channels, RyR1 and RyR3. We demonstrate here that expression of RyR1 is responsible for more than half of total resting Ca(2+) concentration ([Ca(2+)](rest)) measured in wild type cells. The elevated [Ca(2+)](rest) in RyR1-expressing cells is not a result of active gating of the RyR1 channel but instead is accounted for by the RyR1 ryanodine-insensitive Ca(2+) leak conformation. In addition, we demonstrate that basal sarcolemmal Ca(2+) influx is also governed by RyR1 expression and contributes in the regulation of [Ca(2+)](rest) in skeletal myotubes.

Identification of Changes in the Functional Profile of the Cardiac Ryanodine Receptor Caused by the Coupled Gating Phenomenon

The objective of this work was to identify and further characterize potential changes in the functional profile of the cardiac ryanodine receptor (RyR2) channel caused by the coupled gating phenomenon. By reconstituting an ion channel into a planar lipid membrane, we showed that coupled RyR2 channels were activated by cytosolic Ca(2+) with similar efficacy and potency as reported for the single RyR2 channel. In contrast, all examined parameters of gating kinetics were affected by the functional interaction between channels. Ignoring brief closings during main open events, the average open and closed times were considerably prolonged and the frequency of opening was reduced. Interestingly, when luminal Ca(2+) was used as a charge carrier, Ca(2+)-activated coupled RyR2 channels did not exhibit a sudden switch from slow to fast gating kinetics at an open probability of 0.5 as reported for the single RyR2 channel. Regarding flicker gating, the average closed time was significantly shorter and the frequency of closing was greatly enhanced. Furthermore, in contrast to the single RyR2 channel, both parameters for coupled channels were independent of cytosolic Ca(2+). Selected permeation properties of coupled RyR2 channels were comparable to those found for the single RyR2 channel. The Ca(2+) current amplitude-luminal Ca(2+) relationship displayed a simple saturation and the channel selectivity for Ba(2+) and Ca(2+) ions was similar. Our results suggest that the major targets influenced by coupled gating are likely the gates of individual RyR2 channels recruited into a functional complex, thus ensuring the correlation of Ca(2+) fluxes.

Enhanced Beta-Adrenergic Response, Spontaneous Calcium Release, and Arrhythmogenic Propensity in Mice with a Phosphomimetic Mutation of a PKA Site (S2030D) in the Cardiac Ryanodine Receptor

The phosphorylation of the cardiac calcium release channel/ryanodine receptor (RyR2) by protein kinase A (PKA) has been extensively investigated, but its functional consequences remain poorly understood and controversial. We have previously shown that S2030 is a major PKA site in RyR2 responding to beta-adrenergic stimulation, and that phosphomimetic mutation of this PKA site (S2030D) enhances luminal calcium activation of single RyR2 channels and increases the propensity for spontaneous calcium release in HEK293 cells during store calcium overload. To further investigate the physiological and pathophysiological significance of PKA phosphorylation of S2030, we generated a knock-in mouse model harboring the phosphomimetic S2030D mutation in RyR2. We found that S2030D mutant mice displayed higher isoproterenol-induced heart rate increase than wt mice. ECG recordings revealed that S2030D mutant mice were more susceptible to bidirectional ventricular tachycardia induced by the injection of epinephrine plus caffeine as compared to wt mice. To determine the impact of the S2030D mutation on sarcoplasmic reticulum (SR) calcium handling, ventricular myocytes isolated from wt and mutant mice were loaded with fluo-4 and field-stimulated. Calcium imaging showed that S2030D mutant cells exhibited enhanced spontaneous calcium release activity in the presence of isoproterenol. These observations indicate that the S2030D mutation in RyR2 enhances the beta-adrenergic response, and suggest that excessive PKA phosphorylation of S2030 in RyR2 may increase the propensity for stress-induced spontaneous SR calcium release and ventricular arrhythmias (supported by CIHR).

Incorporation of RyR2 and Other Ion Channels into Nanopore Based Planar Lipid Bilayers for Low Noise Single Channel Recordings

Measurement of ion channel activity at the single molecule level in isolated planar lipid bilayers (PLB) is a critical biophysical tool for understanding the function of such proteins. However, current PLB techniques involving bilayers painted across apertures >100 μm suffer from high noise arising from the capacitance of the bilayer and is severely limited in bandwidth. Therefore, it is difficult to investigate the fast gating properties of certain channels e.g the Ryanodine Receptor (RyR2). RyR2 channels, which play a key role in the intracellular Ca2+ induced calcium release mechanism, demonstrate a complex gating characterized by bursts of very fast open-close transitions that cannot be resolved by conventional PLB apparatus. Furthermore, the low current amplitude these channels produce in presence of the physiological ion Ca2+, complicates the analysis.We have developed a robust platform based on glass or quartz nanopore membranes (GNMs, 200-3000 nm radius pore size), for performing high bandwidth, low noise measurements of such ion channels in lipid bilayers. Previously, we have demonstrated incorporation of bacterial toxins [1] and porins in these small bilayers. Here we report the successful incorporation and measurement of RyR2 activity in such a system. Vesicles prepared from sarcoplasmic reticulum enriched in RyR2 channels were fused through osmotic swelling to PLBs formed on a GNM with a 3000 nm radius orifice, allowing unprecedented resolution of single RyR2 channel events at 10 kHz.In addition, we have fused vesicles containing nystatin and ergosterol to small PLBs (< 1000nm radius) successfully. The latter method has the potential to provide a general technique for incorporation of a variety of ion channels in small GNM bilayers.[1] White et al J. Am. Chem. Soc., 129, 11766-11775, 2007

Luminal Regulation of Single RyR2 Channels by Cardiac Calsequestrin

We examined the hypothesis that calsequestrin (CSQ2) can regulate the RyR2 activity using a combination of single channel electrophysiology and lanthanide resonance energy transfer (LRET). Under steady-state, open probability (Po) of RyR2 of cardiac SR fractions from dog is modulated by luminal [Ca2+]. Po increased when luminal [Ca2+] was increased from 6 μM to 2 mM at a fixed cytosolic [Ca2+] of 2 μM. This effect on RyR2 appears to be mediated by luminal sites. Interestingly, RyR2 Po also increases when 2 mM Mg2+ was added to the luminal side. To gain mechanistic insights on the Casq2-mediated luminal regulation, we used the binding of Casq2 to Tb+3 as a functional assay. Luminescence produced by LRET between the tryptophan's of purified dog Casq2 and the lanthanide increased as function of the [Tb+3]. This fluorescence was reduced as [Ca2+] increased suggesting that Ca2+ binds to purified Casq2 by displacing tightly bound Tb+3 from a common binding site. To further explore the specificity of this regulation, we expressed recombinant dog Casq2 in E coli. The specificity of this interaction was assessed by LRET lifetime measurements. Interestingly, we found that the displacement of Tb3+ by Ca2+ was not significantly different than the displacement of Tb3+ by Mg2+ (∼ 2.5 mM) suggesting that Ca2+ and Mg2+ share a common binding site. Finally, we explore the hypothesis that Tb3+ bound to Casq2 was able to modulate the RR2 activity. At fixed cytosolic [Ca2+] of 2 μM, Po of single RyR2 increased as a function of luminal [Tb+3 (KD ∼ 500 nM, Hill coeff. ∼4). These results are consistent with the idea that a multimeric form of Casq2 acts as luminal divalent cation sensor and translates it into changes in RyR2 gating. Supported by NIH R01-HL-084487 to AE.

In Vitro Interactions Between the Beta1a Subunit of the Skeletal Muscle Dhpr and RyR1

The beta1a subunit of the skeletal muscle DHPR plays two important roles in excitation-contraction (EC) coupling in skeletal muscle. Beta1a was originally found to target the alpha1S subunit of the DHPR to sarcolemmal tetrads which oppose skeletal ryanodine receptor (RyR1) channels in the sarcoplasmic reticulum. It was later found to also aid in transmission of EC coupling between the alpha1S subunit and RyR1 (1,2,3), through its C-terminal tail residues (1,2). This presumably depends on an interaction between the C-terminal tail of beta1a and RyR1. The full beta1a subunit binds to RyR1 in affinity chromatography experiments (4), but direct binding of the C-tail to RyR1 has not been reported, nor have direct functional interactions between the proteins. We show here that a peptide corresponding to the native sequence of residues in the C-terminus of beta1a, but not a scrambled sequence, bind to RyR1. In addition the peptide and the full β1a subunit significantly increase both [3H]ryanodine binding and single RyR1 channel activity at concentrations of 0.1 to 1nM, with maximum 3- to 5-fold activation at a concentration of ∼10nM. The increase in RyR1 channel activity with both constructs is essentially irreversible within the lifetime of the bilayer, indicating tight binding. These results show that the C-terminal tail of the beta1a subunit is capable of directly binding to and activating RyR1 and hence suggest that beta1a may enhance EC coupling by virtue of its ability to activate RyR1.1. Beurg M et al. Biophys J1999 77, 2953-2967.2. Zhou W et al. Cell Calcium 2006 39, 227-236.3. Schredelseker J et al. J Biol Chem 2009 284, 1242-51.4. Cheng W et al. Proc Natl Acad Sci 2005 102, 19225-19230.

β-Adrenergic signaling accelerates and synchronizes cardiac ryanodine receptor response to a single L-type Ca 2+ channel

As the most prototypical G protein-coupled receptor, beta-adrenergic receptor (betaAR) regulates the pace and strength of heart beating by enhancing and synchronizing L-type channel (LCC) Ca(2+) influx, which in turn elicits greater sarcoplasmic reticulum (SR) Ca(2+) release flux via ryanodine receptors (RyRs). However, whether and how betaAR-protein kinase A (PKA) signaling directly modulates RyR function remains elusive and highly controversial. By using unique single-channel Ca(2+) imaging technology, we measured the response of a single RyR Ca(2+) release unit, in the form of a Ca(2+) spark, to its native trigger, the Ca(2+) sparklet from a single LCC. We found that acute application of the selective betaAR agonist isoproterenol (1 microM, < or = 20 min) increased triggered spark amplitude in an LCC unitary current-independent manner. The increased ratio of Ca(2+) release flux underlying a Ca(2+) spark to SR Ca(2+) content indicated that betaAR stimulation helps to recruit additional RyRs in synchrony. Quantification of sparklet-spark kinetics showed that betaAR stimulation synchronized the stochastic latency and increased the fidelity (i.e., chance of hit) of LCC-RyR intermolecular signaling. The RyR modulation was independent of the increased SR Ca(2+) content. The PKA antagonists Rp-8-CPT-cAMP (100 microM) and H89 (10 microM) both eliminated these effects, indicating that betaAR acutely modulates RyR activation via the PKA pathway. These results demonstrate unequivocally that RyR activation by a single LCC is accelerated and synchronized during betaAR stimulation. This molecular mechanism of sympathetic regulation will permit more fundamental studies of altered betaAR effects in cardiovascular diseases.

Ryanodine Receptor Luminal Ca 2+ Regulation: Swapping Calsequestrin and Channel Isoforms

Sarcoplasmic reticulum (SR) Ca 2+ release in striated muscle is mediated by a multiprotein complex that includes the ryanodine receptor (RyR) Ca 2+ channel and the intra-SR Ca 2+ buffering protein calsequestrin (CSQ). Besides its buffering role, CSQ is thought to regulate RyR channel function. Here, CSQ-dependent luminal Ca 2+ regulation of skeletal (RyR1) and cardiac (RyR2) channels is explored. Skeletal (CSQ1) or cardiac (CSQ2) calsequestrin were systematically added to the luminal side of single RyR1 or RyR2 channels. The luminal Ca 2+ dependence of open probability ( Po) over the physiologically relevant range (0.05–1 mM Ca 2+) was defined for each of the four RyR/CSQ isoform pairings. We found that the luminal Ca 2+ sensitivity of single RyR2 channels was substantial when either CSQ isoform was present. In contrast, no significant luminal Ca 2+ sensitivity of single RyR1 channels was detected in the presence of either CSQ isoform. We conclude that CSQ-dependent luminal Ca 2+ regulation of single RyR2 channels lacks CSQ isoform specificity, and that CSQ-dependent luminal Ca 2+ regulation in skeletal muscle likely plays a relatively minor (if any) role in regulating the RyR1 channel activity, indicating that the chief role of CSQ1 in this tissue is as an intra-SR Ca 2+ buffer.

Ca 2+-Calmodulin Increases RyR2 Open Probability yet Reduces Ryanoid Association with RyR2

We have shown that physiological levels of Ca 2+-calmodulin (Ca 2+CaM; 50–100 nM) activate cardiac ryanodine receptors (RyR2) incorporated into bilayers and increase the frequency of Ca 2+ sparks and waves in cardiac cells. In contrast, it is well known that Ca 2+CaM inhibits [ 3H]ryanodine binding to cardiac sarcoplasmic reticulum. Since the [ 3H]ryanodine binding technique does not reflect the effects of Ca 2+CaM on RyR2 open probability ( Po), we have investigated, using the reversible ryanoid, ryanodol, whether Ca 2+CaM can directly influence the binding of ryanoids to single RyR2 channels independently of Po. We demonstrate that Ca 2+CaM reduces the rate of ryanodol association to RyR2 without affecting the rate of dissociation. We also find that ryanodol-bound channels fluctuate between at least two distinct subconductance states, M 1 and M 2, in a voltage-dependent manner. Ca 2+CaM significantly alters the equilibrium between these two states. The results suggest that Ca 2+CaM binding to RyR2 causes a conformation change to regions of the channel that include the ryanoid binding site, thereby leading to a decrease in ryanoid association rate and modulation of gating within the ryanoid/RyR2 bound state. Our data provide a possible explanation for why the effects of Ca 2+CaM at the single-channel level are not mirrored by [ 3H]ryanodine binding studies.

Ca2+‐calmodulin can activate and inactivate cardiac ryanodine receptors

Ca(2+)-calmodulin (Ca(2+)CaM) is widely accepted as an inhibitor of cardiac ryanodine receptors (RyR2); however, the effects of physiologically relevant CaM concentrations have not been fully investigated. We investigated the effects of low concentrations of Ca(2+)CaM (50-100 nmol.L(-1)) on the gating of native sheep RyR2, reconstituted into bilayers. Suramin displaces CaM from RyR2 and we have used a gel-shift assay to provide evidence of the mechanism underlying this effect. Finally, using suramin to displace endogenous CaM from RyR2 in permeabilized cardiac cells, we have investigated the effects of 50 nmol.L(-1) CaM on sarcoplasmic reticulum (SR) Ca(2+)-release. Ca(2+)CaM activated or inhibited single RyR2, but activation was much more likely at low (50-100 nmol.L(-1)) concentrations. Also, suramin displaced CaM from a peptide of the CaM binding domain of RyR2, indicating that, like the skeletal isoform (RyR1), suramin directly competes with CaM for its binding site on the channel. Pre-treatment of rat permeabilized ventricular myocytes with suramin to displace CaM, followed by addition of 50 nmol x L(-1) CaM to the mock cytoplasmic solution caused an increase in the frequency of spontaneous Ca(2+)-release events. Application of caffeine demonstrated that 50 nmol x L(-1) CaM reduced SR Ca(2+) content. We describe for the first time how Ca(2+)CaM is capable, not only of inactivating, but also of activating RyR2 channels in bilayers in a CaM kinase II-independent manner. Similarly, in cardiac cells, CaM stimulates SR Ca(2+)-release and the use of caffeine suggests that this is a RyR2-mediated effect.

Ca2+-calmodulin Increases RyR2 Open Probability Yet Reduces Ryanoid Association With RyR2

We have previously demonstrated that low, physiological concentrations of Ca2+-calmodulin (Ca2+CaM; 50–100 nM) activate RyR2 channels incorporated into bilayers (Sigalas & Sitsapesan (2007) Biophys.J., 88a). In contrast, we find that Ca2+CaM dose-dependently inhibits [3H]ryanodine binding to cardiac HSR. Since it appears that the [3H]ryanodine binding assay is not accurately reporting the effects of Ca2+CaM on RyR2 Po we have investigated, using the reversible ryanoid, ryanodol, whether Ca2+CaM can directly influence the binding of ryanoid compounds to RyR2 independently of Po. Purified single sheep RyR2 channels were incorporated into artificial membranes under voltage-clamp conditions. In symmetrical solutions containing 250 mM K+, 100 μM free Ca2+, pH 7.2, and 20 mM caffeine to clamp Po to high levels, 1 μM CaM had no significant effect on Po. This agrees with our previous work on native RyR2 channels demonstrating that high levels of CaM can reverse the activating effects of low [Ca2+CaM] but do not significantly lower Po below control values. We find that Ca2+CaM significantly reduces the rate of association of ryanodol to RyR2 but does not affect the rate of dissociation. Ca2+CaM also significantly alters channel behaviour within the ryanodol-modified conductance state. Our results lead to two important conclusions: 1. A Ca2+CaM-induced decrease in the rate of association of ryanoids to RyR2 is consistent with the reduction in [3H]ryanodine binding caused by Ca2+CaM, providing an explanation why the functional effects of Ca2+CaM at the single-channel level are not faithfully mirrored in the [3H]ryanodine binding studies. 2. The important, wider implications of this study are that [3H]ryanodine binding assays, in the absence of other supporting data, may not provide reliable information about the Po of RyR channel populations. Supported by the BHF.

Changes in Negative Charge at the Luminal Mouth of the Pore Alter Ion Handling and Gating in the Cardiac Ryanodine-Receptor

We have tested the hypothesis that a high density of negative charge at the luminal mouth of the RyR2 pore plays a pivotal role in the high cation conductance and limited selectivity observed in this channel by introducing into each monomer a double point mutation to neutralize acidic residues in this region of the mouse RyR2 channel. The resultant channel, ED4832AA, is capable of functioning as a calcium-release channel in situ. Consistent with our hypothesis, the ED4832AA mutation altered the ion handling characteristics of single RyR2 channels. The mutant channel retains the ability to discriminate between cations and anions but cation conductance is altered significantly. Unitary K + conductance is reduced at low levels of activity but increases dramatically as activity is raised and shows little sign of saturation. ED4832AA no longer discriminates between divalent and monovalent cations. In addition, the gating characteristics of single RyR2 channels are altered markedly by residue neutralization. Open probability in the ED4832AA channel is substantially higher than that of the wild-type channel. Moreover, at holding potentials in excess of ±50 mV several subconductance states become apparent in ED4832AA and are more prevalent at very high holding potentials. These observations are discussed within the structural framework provided by a previously developed model of the RyR2 pore. Our data indicates that neutralization of acidic residues in the luminal mouth of the pore produces wide-ranging changes in the electric field in the pore, the interaction energies of permeant ions in the pore and the stability of the selectivity filter region of the pore, which together contribute to the observed changes ion handling and gating.

Molecular Basis of Luminal Ca2+ Gating of the Cardiac Ryanodine Receptor

It has long been known that Ca2+ release from the sarcoplasmic reticulum (SR) can occur spontaneously when the SR Ca2+ content reaches a critical threshold, and that this spontaneous SR Ca2+ release can lead to lethal cardiac arrhythmias. Although it is clear that spontaneous SR Ca2+ release results from the activation of the cardiac ryanodine receptor (RyR2) by luminal Ca2+, the molecular basis of luminal Ca2+ activation of RyR2 remains undefined and controversial. An increasing body of evidence indicates that the RyR2 channel contains a luminal Ca2+ sensor distinct from its cytosolic Ca2+ sensor. To localize this luminal Ca2+ sensor, we have systematically mutated each of the negatively charged residues in the predicted pore-forming region of RyR2 that is likely to be accessible to luminal Ca2+. We found that alanine-mutations within and near the COOH-terminal end of the TM10 transmembrane helix (the pore inner helix) abolish the luminal, but not the cytosolic, Ca2+ activation of single RyR2 channels. HEK293 cells expressing these alanine-mutants display caffeine-induced Ca2+ release, but show no store-overload-induced Ca2+ release. Interestingly, introducing histidines into this region creates a high affinity metal binding site. The histidine-mutant channels are blocked by Ni2+ (<1 uM), while 100 uM Ni2+ does not block the RyR2 wt channels. Molecular modeling of the RyR2 channel pore based on the three-dimensional structure of the KcsA potassium channel reveals a cluster of negatively charged residues located within the internal pore at the helix bundle crossing, which is thought to form the ion gate of the RyR2 channel. Collectively, our results suggest that luminal Ca2+ opens the RyR2 channel by binding to the intracellular gate.

Dissection of the inhibition of cardiac ryanodine receptors by human glutathione transferase GSTM2-2

The muscle specific glutathione transferase GSTM2-2 inhibits the activity of cardiac ryanodine receptor (RyR2) calcium release channels with high affinity and activates skeletal RyR (RyR1) channels with lower affinity. To determine which overall region of the GSTM2-2 molecule supports binding to RyR2, we examined the effects of truncating GSTM2-2 on its ability to alter Ca 2+ release from sarcoplasmic reticulum (SR) vesicles and RyR channel activity. The C-terminal half of GSTM2-2 which lacks the critical GSH binding site supported the inhibition of RyR2, but did not support activation of RyR1. Smaller fragments of GSTM2-2 indicated that the C-terminal helix 6 was crucial for the action of GSTM2-2 on RyR2. Only fragments containing the helix 6 sequence inhibited Ca 2+ release from cardiac SR. Single RyR2 channels were strongly inhibited by constructs containing the helix 6 sequence in combination with adjacent helices (helices 5–8 or 4–6). Fragments containing helices 5–6 or helix 6 sequences alone had less well-defined effects. Chemical cross-linking indicated that C-terminal helices 5–8 bound to RyR2, but not RyR1. Structural analysis with circular dichroism showed that the helical content was greater in the longer helix 6 containing constructs, while the helix 6 sequence alone had minimal helical structure. Therefore the active centre of GSTM2-2 for inhibition of cardiac RyR2 involves the helix 6 sequence and the helical nature of this region is essential for its efficacy. GSTM2-2 helices 5–8 may provide the basis for RyR2-specific compounds for experimental and therapeutic use.

Redox Modification of Ryanodine Receptors Contributes to Sarcoplasmic Reticulum Ca 2+ Leak in Chronic Heart Failure

Abnormal cardiac ryanodine receptor (RyR2) function is recognized as an important factor in the pathogenesis of heart failure (HF). However, the specific molecular causes underlying RyR2 defects in HF remain poorly understood. In the present study, we used a canine model of chronic HF to test the hypothesis that the HF-related alterations in RyR2 function are caused by posttranslational modification by reactive oxygen species generated in the failing heart. Experimental approaches included imaging of cytosolic ([Ca(2+)](c)) and sarcoplasmic reticulum (SR) luminal Ca(2+) ([Ca(2+)]SR) in isolated intact and permeabilized ventricular myocytes and single RyR2 channel recording using the planar lipid bilayer technique. The ratio of reduced to oxidized glutathione, as well as the level of free thiols on RyR2 decreased markedly in failing versus control hearts consistent with increased oxidative stress in HF. RyR2-mediated SR Ca(2+) leak was significantly enhanced in permeabilized myocytes, resulting in reduced [Ca(2+)](SR) in HF compared to control cells. Both SR Ca(2+) leak and [Ca(2+)](SR) were partially normalized by treating HF myocytes with reducing agents. Conversely, oxidizing agents accelerated SR Ca(2+) leak and decreased [Ca(2+)](SR) in cells from normal hearts. Moreover, exposure to antioxidants significantly improved intracellular Ca(2+)-handling parameters in intact HF myocytes. Single RyR2 channel activity was significantly higher in HF versus control because of increased sensitivity to activation by luminal Ca(2+) and was partially normalized by reducing agents through restoring luminal Ca(2+) sensitivity oxidation of control RyR2s enhanced their luminal Ca(2+) sensitivity, thus reproducing the HF phenotype. These findings suggest that redox modification contributes to abnormal function of RyR2s in HF, presenting a potential therapeutic target for treating HF.

Amitriptyline Activates Cardiac Ryanodine Channels and Causes Spontaneous Sarcoplasmic Reticulum Calcium Release

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.

Caffeine induces Ca2+ release by reducing the threshold for luminal Ca2+ activation of the ryanodine receptor

Caffeine has long been used as a pharmacological probe for studying RyR (ryanodine receptor)-mediated Ca(2+) release and cardiac arrhythmias. However, the precise mechanism by which caffeine activates RyRs is elusive. In the present study, we investigated the effects of caffeine on spontaneous Ca(2+) release and on the response of single RyR2 (cardiac RyR) channels to luminal or cytosolic Ca(2+). We found that HEK-293 cells (human embryonic kidney cells) expressing RyR2 displayed partial or 'quantal' Ca(2+) release in response to repetitive additions of submaximal concentrations of caffeine. This quantal Ca(2+) release was abolished by ryanodine. Monitoring of endoplasmic reticulum luminal Ca(2+) revealed that caffeine reduced the luminal Ca(2+) threshold at which spontaneous Ca(2+) release occurs. Interestingly, spontaneous Ca(2+) release in the form of Ca(2+) oscillations persisted in the presence of 10 mM caffeine, and was diminished by ryanodine, demonstrating that unlike ryanodine, caffeine, even at high concentrations, does not hold the channel open. At the single-channel level, caffeine markedly reduced the threshold for luminal Ca(2+) activation, but had little effect on the threshold for cytosolic Ca(2+) activation, indicating that the major action of caffeine is to reduce the luminal, but not the cytosolic, Ca(2+) activation threshold. Furthermore, as with caffeine, the clinically relevant, pro-arrhythmic methylxanthines aminophylline and theophylline potentiated luminal Ca(2+) activation of RyR2, and increased the propensity for spontaneous Ca(2+) release, mimicking the effects of disease-linked RyR2 mutations. Collectively, our results demonstrate that caffeine triggers Ca(2+) release by reducing the threshold for luminal Ca(2+) activation of RyR2, and suggest that disease-linked RyR2 mutations and RyR2-interacting pro-arrhythmic agents may share the same arrhythmogenic mechanism.

Reduced Threshold for Luminal Ca2+ Activation of RyR1 Underlies a Causal Mechanism of Porcine Malignant Hyperthermia

Naturally occurring mutations in the skeletal muscle Ca(2+) release channel/ryanodine receptor RyR1 are linked to malignant hyperthermia (MH), a life-threatening complication of general anesthesia. Although it has long been recognized that MH results from uncontrolled or spontaneous Ca(2+) release from the sarcoplasmic reticulum, how MH RyR1 mutations render the sarcoplasmic reticulum susceptible to volatile anesthetic-induced spontaneous Ca(2+) release is unclear. Here we investigated the impact of the porcine MH mutation, R615C, the human equivalent of which also causes MH, on the intrinsic properties of the RyR1 channel and the propensity for spontaneous Ca(2+) release during store Ca(2+) overload, a process we refer to as store overload-induced Ca(2+) release (SOICR). Single channel analyses revealed that the R615C mutation markedly enhanced the luminal Ca(2+) activation of RyR1. Moreover, HEK293 cells expressing the R615C mutant displayed a reduced threshold for SOICR compared with cells expressing wild type RyR1. Furthermore, the MH-triggering agent, halothane, potentiated the response of RyR1 to luminal Ca(2+) and SOICR. Conversely, dantrolene, an effective treatment for MH, suppressed SOICR in HEK293 cells expressing the R615C mutant, but not in cells expressing an RyR2 mutant. These data suggest that the R615C mutation confers MH susceptibility by reducing the threshold for luminal Ca(2+) activation and SOICR, whereas volatile anesthetics trigger MH by further reducing the threshold, and dantrolene suppresses MH by increasing the SOICR threshold. Together, our data support a view in which altered luminal Ca(2+) regulation of RyR1 represents a primary causal mechanism of MH.

Arrhythmogenic right ventricular cardiomyopathy in Boxer dogs is associated with calstabin2 deficiency

Objective To examine the presence and effect of calstabin2-deficiency in Boxer dogs with arrhythmogenic right ventricular cardiomyopathy (ARVC). Animals Thirteen Boxer dogs with ARVC. Materials and methods Tissue samples were collected for histopathology, oligonucleotide microarray, PCR, immunoelectrophoresis, ryanodine channel immunoprecipitation and single-channel recordings, and calstabin2 DNA sequencing. Results In cardiomyopathic Boxer dogs, myocardial calstabin2 mRNA and protein were significantly decreased as compared to healthy control dogs (calstabin2 protein normalized to tetrameric cardiac ryanodine receptor (RyR2) complex: affected, 0.51 ± 0.04; control, 3.81 ± 0.22; P < 0.0001). Calstabin2 deficiency in diseased dog hearts was associated with a significantly increased open probability of single RyR2 channels indicating intracellular Ca 2+ leak. PCR-based sequencing of the promoter, exonic and splice site regions of the canine calstabin2 gene did not identify any causative mutations. Conclusions Calstabin2 deficiency is a potential mechanism of Ca 2+ leak-induced ventricular arrhythmias and heart disease in Boxer dogs with ARVC.