RyR Opening Research Articles

A systematic approach for assessing Ca²⁺ handling in cardiac myocytes.

In cardiac myocytes, Ca(2+) release from the sarcoplasmic reticulum (SR) Ca(2+) store through the opening of ryanodine receptors (RyRs) is the major source of Ca(2+) for activation of myofilaments and contraction. Over the past 20 years, tools have become available to study this release process in detail, allowing new insights into the regulation of SR Ca(2+) release and RyR function. To assess these processes, we recommend and here review a systematic approach that evaluates the essential transport mechanisms and Ca(2+) fluxes in isolated single cardiac myocytes by using fluorescent Ca(2+) indicators and whole-cell recording of membrane voltage and ionic currents under voltage clamp. The approach includes an assessment of the L-type Ca(2+) current as a trigger for opening of RyRs and release of SR Ca(2+), of the SR Ca(2+) content, of intrinsic properties of RyRs, and of Ca(2+)-removal systems.

The Ryanodine Receptor Patchwork: Knitting Calcium Spark Dynamics

The Ryanodine Receptor Patchwork: Knitting Calcium Spark Dynamics

Activation and propagation of Ca2+ release from inside the sarcoplasmic reticulum network of mammalian skeletal muscle.

Skeletal muscle fibres are large and highly elongated cells specialized for producing the force required for posture and movement. The process of controlling the production of force within the muscle, known as excitation-contraction coupling, requires virtually simultaneous release of large amounts of Ca(2+) from the sarcoplasmic reticulum (SR) at the level of every sarcomere within the muscle fibre. Here we imaged Ca(2+) movements within the SR, tubular (t-) system and in the cytoplasm to observe that the SR of skeletal muscle is a connected network capable of allowing diffusion of Ca(2+) within its lumen to promote the propagation of Ca(2+) release throughout the fibre under conditions where inhibition of SR ryanodine receptors (RyRs) was reduced. Reduction of cytoplasmic [Mg(2+)] ([Mg(2+)]cyto) induced a leak of Ca(2+) through RyRs, causing a reduction in SR Ca(2+) buffering power argued to be due to a breakdown of SR calsequestrin polymers, leading to a local elevation of [Ca(2+)]SR. The local rise in [Ca(2+)]SR, an intra-SR Ca(2+) transient, induced a local diffusely rising [Ca(2+)]cyto. A prolonged Ca(2+) wave lasting tens of seconds or more was generated from these events. Ca(2+) waves were dependent on the diffusion of Ca(2+) within the lumen of the SR and ended as [Ca(2+)]SR dropped to low levels to inactivate RyRs. Inactivation of RyRs allowed re-accumulation of [Ca(2+)]SR and the activation of secondary Ca(2+) waves in the persistent presence of low [Mg(2+)]cyto if the threshold [Ca(2+)]SR for RyR opening could be reached. Secondary Ca(2+) waves occurred without an abrupt reduction in SR Ca(2+) buffering power. Ca(2+) release and wave propagation occurred in the absence of Ca(2+)-induced Ca(2+) release. These observations are consistent with the activation of Ca(2+) release through RyRs of lowered cytoplasmic inhibition by [Ca(2+)]SR or store overload-induced Ca(2+) release. Restitution of SR Ca(2+) buffering power to its initially high value required imposing normal resting ionic conditions in the cytoplasm, which re-imposed the normal resting inhibition on the RyRs, allowing [Ca(2+)]SR to return to endogenous levels without activation of store overload-induced Ca(2+) release. These results are discussed in the context of how pathophysiological Ca(2+) release such as that occurring in malignant hyperthermia can be generated.

Spontaneous, Pro-Arrhythmic Calcium Signals Disrupt Electrical Pacing in Mouse Pulmonary Vein Sleeve Cells

The pulmonary vein, which returns oxygenated blood to the left atrium, is ensheathed by a population of unique, myocyte-like cells called pulmonary vein sleeve cells (PVCs). These cells autonomously generate action potentials that propagate into the left atrial chamber and cause arrhythmias resulting in atrial fibrillation; the most common, often sustained, form of cardiac arrhythmia. In mice, PVCs extend along the pulmonary vein into the lungs, and are accessible in a lung slice preparation. We exploited this model to study how aberrant Ca2+ signaling alters the ability of PVC networks to follow electrical pacing. Cellular responses were investigated using real-time 2-photon imaging of lung slices loaded with a Ca2+-sensitive fluorescent indicator (Ca2+ measurements) and phase contrast microscopy (contraction measurements). PVCs displayed global Ca2+ signals and coordinated contraction in response to electrical field stimulation (EFS). The effects of EFS relied on both Ca2+ influx and Ca2+ release, and could be inhibited by nifedipine, ryanodine or caffeine. Moreover, PVCs had a high propensity to show spontaneous Ca2+ signals that arose via stochastic activation of ryanodine receptors (RyRs). The ability of electrical pacing to entrain Ca2+ signals and contractile responses was dramatically influenced by inherent spontaneous Ca2+ activity. In PVCs with relatively low spontaneous Ca2+ activity (<1 Hz), entrainment with electrical pacing was good. However, in PVCs with higher frequencies of spontaneous Ca2+ activity (>1.5 Hz), electrical pacing was less effective; PVCs became unpaced, only partially-paced or displayed alternans. Because spontaneous Ca2+ activity varied between cells, neighboring PVCs often had different responses to electrical pacing. Our data indicate that the ability of PVCs to respond to electrical stimulation depends on their intrinsic Ca2+ cycling properties. Heterogeneous spontaneous Ca2+ activity arising from stochastic RyR opening can disengage them from sinus rhythm and lead to autonomous, pro-arrhythmic activity.

Dynamics of Calcium Sparks and SR Calcium Leak During Excitation-Contraction Coupling in Mouse Heart Cells

Stable calcium-induced calcium release (CICR) is critical for maintaining normal cell contraction during cardiac excitation-contraction (EC) coupling in heart cells. The fundamental element of CICR in heart is the calcium (Ca2+) spark which arises from the regenerative release of Ca2+ from a cluster of ryanodine receptors (RyR2) in the junctional sarcoplasmic reticulum membrane. We have shown how stochastic gating of RyR2s could produce an SR Ca2+ leak capable of balancing SR Ca2+-ATPase (SERCA2a) activity under quiescent conditions (Williams et al. BJ 2011). This investigation suggested the surprising finding that a single, or even multiple, RyR2 openings could fail probabilistically to trigger a Ca2+ spark. To further investigate this effect, we expanded upon that formulation to create a detailed, local control model of EC coupling in mouse heart. A number of features were added or modified, including the addition of a novel seven-state Markov chain model of the sarcolemmal L-type Ca2+ channel (LCC). This model features dynamic action-potential (AP) generation, robust Ca2+ spark initiation and termination, realistic [Ca2+]i transients, and true SR Ca2+ pump/leak balance. The model suggests that numerous LCC openings are often required to trigger a single Ca2+ spark. This is consistent with our prior work where multiple RyR2 openings were needed to trigger a spontaneous Ca2+ spark during quiescent conditions. We also investigated how RyR2 mutations associated with catecholaminergic polymorphic ventricular tachycardia (CPVT) influence the dynamics of Ca2+ sparks, “invisible” non-spark Ca2+ leak, [Ca2+]i transients, and SR Ca2+ content. We observe that CPVT mutations can lead to unstable Ca2+ spark dynamics, altering SR Ca2+ content and promoting [Ca2+]i signaling instability. Our new model provides significant insights into the dynamics of local control of CICR under physiological and pathological conditions.

Modification of Cardiac Ryanodine Receptor Gating by a Peptide from the Central Domain of the RyR2

The effect of a domain peptide DPCPVTc from the central region of the RYR2 on ryanodine receptors isolated from rat heart was examined in planar lipid bilayers. At a zero holding potential and at 100 nM cytosolic and 8 mM luminal Ca2+ concentration, DPCPVTc induced concentration-dependent activation of the ryanodine receptor that led up to 20-fold increase of open probability at saturating DPCPVTc concentrations. The effect of the peptide appeared within 30 s after addition to the experimental chamber. At all DPCPVTc concentrations RyR2 channels displayed large variability in open probability, open time and opening frequency. DPCPVTc prolonged RyR2 openings up to 8× and increased RyR2 opening frequency by up to 100%. With increasing DPCPVTc concentration, the fraction of high open probability records increased up to 5×, and their open time increased up to 4×. The closed times did not depend on DPCPVTc concentration either in low- or high-open probability records. The DPCPVTc concentration dependence of all gating parameters had EC50 of 20 µM and a Hill slope of 2. Comparison of the effects of DPCPVTc with those of ATP [1] and cytosolic Ca2+ [2] suggests that activation does not involve luminal feed-through and is not caused by modulation of the cytosolic activation A-site. The data suggest that although “domain unzipping” by DPCPVTc occurs in both modes of RyR activity, it affects RyR gating only when the channel resides in the H-mode of activity.Supported in part by the European Union Contract No. LSHM-CT-2005-018802/CONTICA and by grants APVV-LPP-0441-09, APVV-0628-10 and VEGA 2/0197/11.[1] Tencerova B, Zahradnikova A, Gaburjakova J, Gaburjakova M. J Gen Physiol 140: 93, 2012.[2] Gaburjakova J, Gaburjakova M. J Membr Biol 212: 17, 2006.

Modification of cardiac RYR2 gating by a peptide from the central domain of the RYR2

AbstractThe effect of a domain peptide DPCPVTc from the central region of the RYR2 on ryanodine receptors from rat heart has been examined in planar lipid bilayers. At a zero holding potential and at 8 mmol L−1 luminal Ca2+ concentration, DPCPVTc induced concentrationdependent activation of the ryanodine receptor that led up to 20-fold increase of PO at saturating DPCPVTc concentrations. DPCPVTc prolonged RyR2 openings and increased RyR2 opening frequency. At all peptide concentrations the channels displayed large variability in open probability, open time and frequency of openings. With increasing peptide concentration, the fraction of high open probability records increased together with their open time. The closed times of neither low- nor high-open probability records depended on peptide concentration. The concentration dependence of all gating parameters had EC50 of 20 μmol L−1 and a Hill slope of 2. Comparison of the effects of DPCPVTc with the effects of ATP and cytosolic Ca2+ suggests that activation does not involve luminal feed-through and is not caused by modulation of the cytosolic activation A-site. The data suggest that although “domain unzipping” by DPCPVTc occurs in both modes of RyR activity, it affects RyR gating only when the channel resides in the H-mode of activity.

A proteolytic cleavage to separate the sarcolemma/T‐tubule from the sarcoplasmic reticulum

A proteolytic cleavage to separate the sarcolemma/T‐tubule from the sarcoplasmic reticulum

Modeling the Effect of Unitary Calcium Current on Neighboring Ryanodine Receptors during Calcium Induced Calcium Release

In resting cardiac cells, the open probability (Po) of single ryanodine receptors (RyRs) is very low and consequently little Ca2+ is released from the sarcoplasmic reticulum (SR). However, a stochastic RyR opening will cause diastolic local Ca2+ release from the SR that can activate neighboring closed RyRs. This inter-RyR Ca2+-induced Ca2+ release (CICR) may generate diastolic Ca2+ sparks. It is known that elevating SR Ca2+ load above normal levels dramatically increases spark frequency and increases the unitary RyR Ca2+ current. It is this current that acts on neighboring RyRs through CICR. We have developed a simple model based on experimental single-channel RyR Ca2+ sensitivity to understand how unitary RyR Ca2+ current may control CICR within a group of neighboring RyRs (a Ca2+ release unit, CRU). The model predicts how the current carried by an open RyR influences the activity of neighboring RyRs in a CRU. These predictions match published experimental single and clustered RyR channel results obtained in bilayer studies.

In Cardiomyocytes, Binding of Unzipping Peptide Activates Ryanodine Receptor 2 and Reciprocally Inhibits Calmodulin Binding

One hypothesis for elevated Ca(2+) leak through cardiac ryanodine receptors (ryanodine receptor 2 [RyR2]) in heart failure is interdomain unzipping that can enhance aberrant channel activation. A peptide (domain peptide corresponding to RyR2 residues 2460-2495 [DPc10]) corresponding to RyR2 central domain residues 2460-2495 recapitulates this arrhythmogenic RyR2 leakiness by unzipping N-terminal and central domains. Calmodulin (CaM) and FK506-binding protein (FKBP12.6) bind to RyR2 and stabilize the closed channel. Little is known about DPc10 binding to the RyR2 and how that may interact with binding (and effects) of CaM and FKBP12.6 to RyR2. To measure, directly in cardiac myocytes, the kinetics and binding affinity of DPc10 to RyR2 and how that affects RyR2 interaction with FKBP12.6 and CaM. We used permeabilized rat ventricular myocytes and fluorescently labeled DPc10, FKBP12.6, and CaM. DPc10 access to its binding site is extremely slow in resting RyR2 but is accelerated by promoting RyR opening or unzipping (by unlabeled DPc10). RyR2-bound CaM (but not FKBP12.6) drastically slowed DPc10 binding. Conversely, DPc10 binding significantly reduced CaM (but not FKBP12.6) binding to the RyR2. Fluorescence resonance energy transfer measurements indicate that DPc10-binding and CaM-binding sites are separate and allow triangulation of the structural DPc10 binding locus on RyR2 vs FKBP12.6-binding and CaM-binding sites. DPc10-RyR2 binding is sterically limited by the resting zipped RyR2 state. CaM binding to RyR2 stabilizes this zipped state, whereas RyR2 activation or prebound DPc10 enhances DPc10 access. DPc10-binding and CaM-binding sites are distinct but are allosterically interacting RyR2 sites. Neither DPc10 nor FKBP12.6 influences RyR2 binding of the other.

JTV519 (K201) reduces sarcoplasmic reticulum Ca2+ leak and improves diastolic function in vitro in murine and human non‐failing myocardium

Ca²⁺ leak from the sarcoplasmic reticulum (SR) via ryanodine receptors (RyR2s) contributes to cardiomyocyte dysfunction. RyR2 Ca²⁺ leak has been related to RyR2 phosphorylation. In these conditions, JTV519 (K201), a 1,4-benzothiazepine derivative and multi-channel blocker, stabilizes RyR2s and decrease SR Ca²⁺ leak. We investigated whether JTV519 stabilizes RyR2s without increasing RyR2 phosphorylation in mice and in non-failing human myocardium and explored underlying mechanisms. SR Ca²⁺ leak was induced by ouabain in murine cardiomyocytes. [Ca²⁺]-transients, SR Ca²⁺ load and RyR2-mediated Ca²⁺ leak (sparks/waves) were quantified, with or without JTV519 (1 µmol·L⁻¹). Contribution of Ca²⁺ -/calmodulin-dependent kinase II (CaMKII) was assessed by KN-93 and Western blot (RyR2-Ser(2814) phosphorylation). Effects of JTV519 on contractile force were investigated in non-failing human ventricular trabeculae. Ouabain increased systolic and diastolic cytosolic [Ca²⁺](i) , SR [Ca²⁺], and SR Ca²⁺ leak (Ca²⁺ spark (SparkF) and Ca²⁺ wave frequency), independently of CaMKII and RyR-Ser(2814) phosphorylation. JTV519 decreased SparkF but also SR Ca²⁺ load. At matched SR [Ca²⁺], Ca²⁺ leak was significantly reduced by JTV519, but it had no effect on fractional Ca²⁺ release or Ca²⁺ wave propagation velocity. In human muscle, JTV519 was negatively inotropic at baseline but significantly enhanced ouabain-induced force and reduced its deleterious effects on diastolic function. JTV519 was effective in reducing SR Ca²⁺ leak by specifically regulating RyR2 opening at diastolic [Ca²⁺](i) in the absence of increased RyR2 phosphorylation at Ser(2814) , extending the potential use of JTV519 to conditions of acute cellular Ca²⁺ overload.

PKA phosphorylation of cardiac ryanodine receptor modulates SR luminal Ca2+ sensitivity

During physical exercise and stress, the sympathetic system stimulates cardiac contractility via β-adrenergic receptor activation, resulting in protein kinase A (PKA)-mediated phosphorylation of the cardiac ryanodine receptor, RyR2, at Ser2808. Hyperphosphorylation of RyR2-S2808 has been proposed as a mechanism contributing to arrhythmogenesis and heart failure. However, the role of RyR2 phosphorylation during β-adrenergic stimulation remains controversial. We examined the contribution of RyR2-S2808 phosphorylation to altered excitation-contraction coupling and Ca(2+) signaling using an experimental approach at the interface of molecular and cellular levels and a transgenic mouse with ablation of the RyR2-S2808 phosphorylation site (RyR2-S2808A). Experimentally challenging the communication between L-type Ca(2+) channels and RyR2 led to a spatiotemporal de-synchronization of RyR2 openings, as visualized using confocal Ca(2+) imaging. β-Adrenergic stimulation re-synchronized RyR2s, but less efficiently in RyR2-S2808A than in control cardiomyocytes, as indicated by comprehensive analysis of RyR2 activation. In addition, spontaneous Ca(2+) waves in RyR2-S2808A myocytes showed significantly slowed propagation and complete absence of acceleration during β-adrenergic stress, unlike wild type cells. Single channel recordings revealed an attenuation of luminal Ca(2+) sensitivity in RyR2-S2808A channels upon addition of PKA. This suggests that phosphorylation of RyR2-S2808 may be involved in RyR2 modulation by luminal (intra-SR) Ca(2+) ([Ca(2+)](SR)). We show here by three independent experimental approaches that PKA-dependent RyR2-S2808 phosphorylation plays significant functional roles at the subcellular level, namely, Ca(2+) release synchronization, Ca(2+) wave propagation and functional adaptation of RyR2 to variable [Ca(2+)](SR). These results indicate a direct mechanistic link between RyR2 phosphorylation and SR luminal Ca(2+) sensing.

Quantification of the CICR Response to Artificial Ca Sparks in Striated Muscle

In cardiac muscle physiologic Ca2+ release is CICR, but in skeletal muscle the role of CICR remains unsettled. “Artificial Ca2+ sparks” of up to 8 μM were produced by 2-photon irradiation of Ca2+ cages, applied in the near vicinity of membrane-permeabilized skeletal fibers from frogs and mice, and rabbit atrial and ventricular myocytes, while confocally imaging their Ca2+ response with Fluo-4FF. In response to transients as low as 0.28 μM, frog fibers produced waves (n=16) that satisfied the definition of CICR (Endo, PhRev 2009), including inhibition by Mg2+. Cardiomyocytes responded similarly, with waves that could propagate fully or partially through the cells. Responses were absent in mouse skeletal fibers (n=51) unless RyR opening was promoted pharmacologically (n=24). The underlying Ca2+ flux was calculated from fluorescence images by generalizing the “backward” method (Rios et al, JGP 1999). In frog fibers, fluxes were found to reach 55 mM/s, which is close to values measured under voltage clamp depolarization. A positive three-way correlation was found among flux, speed and extent of propagation. Accordingly, the responses of cardiomyocytes and pharmacologically conditioned mammalian fibers, both of which had smaller flux, were of lower velocity and sometimes limited propagation range. The quantitative properties of the propagating wave, in all cases, were well described by a simple continuum model of sources and sinks, with a common threshold [Ca2+] for channel opening (Kupferman et al, BJ 1997). This description suggests a fundamental commonality of mechanisms. The lower flux and higher threshold [Ca2+] in mice are consistent with the notion that CICR is supported by isoform 3 or beta (in the frog), and that isoform 1 in situ is less susceptible to CICR. Supported by NIAMS and NHLBI (NIH).

Refractoriness of Calcium Release Units in Rat Cardiac Myocytes

Calcium release units (CRUs) in cardiac myocytes contain a large number of ryanodine receptors (RyRs), but it seems that only a small fraction of RyRs is being activated during a single calcium release event. Repetitive activity of CRUs is not well understood from the point of refractoriness. We used direct measurement of Ca-spikes activated by calcium current (ICa) to unveil the refractoriness of CRU firing from the properties and occurrence of calcium spikes under control conditions and in the presence of the DHPR activator FPL.ICa was activated by voltage pulses from 50 to 0 mV under whole-cell patch-clamp. Ca-spikes were measured by confocal microscopy using Ca2+ indicators (Fluo-3 and Oregon Green BAPTA-5N) in the presence of EGTA. A large majority of CRUs responded to stimulation by a single Ca-spike, while 13.4 % produced two subsequent (twin) Ca-spikes. Early Ca-spikes (single spikes and the first of twin Ca-spikes) had similar latencies, while the second Ca-spikes were significantly delayed. Amplitude distribution of the early Ca-spikes consisted of four quantal levels with equal amplitudes and binomially distributed frequency of occurrence, supporting the hypothesis that different Ca-spike amplitudes are due to a different number of independent quantal levels, likely individual RYR openings. The probability of occurrence of second Ca-spikes was inversely proportional to the quantal size of the early Ca-spike. In the presence of FPL, the probability of occurrence of twin spikes was decreased relative to control, despite persistent activation of DHPR receptors.We conclude that refractoriness of local calcium release does not result from Ca-dependent DHPR inactivation, but rather it is caused by inactivation of the release unit due to local depletion of the SR.Supported by APVV-0721-10, APVV-0441-09, VEGA 2/0203/11, VEGA 2/0917/11, and VEGA 2/0190/10.

Dynamics of Calcium Sparks and Calcium Leak in the Heart

We present what we believe to be a new mathematical model of Ca2+ leak from the sarcoplasmic reticulum (SR) in the heart. To our knowledge, it is the first to incorporate a realistic number of Ca2+-release units, each containing a cluster of stochastically gating Ca2+ channels (RyRs), whose biophysical properties (e.g., Ca2+ sensitivity and allosteric interactions) are informed by the latest molecular investigations. This realistic model allows for the detailed characterization of RyR Ca2+-release properties, and shows how this balances reuptake by the SR Ca2+ pump. Simulations reveal that SR Ca2+ leak consists of brief but frequent single RyR openings (∼3000 cell−1 s−1) that are likely to be experimentally undetectable, and are, therefore, “invisible”. We also observe that these single RyR openings can recruit additional RyRs to open, due to elevated local (Ca2+), and occasionally lead to the generation of Ca2+ sparks (∼130 cell−1 s−1). Furthermore, this physiological formulation of “invisible” leak allows for the removal of the ad hoc, non-RyR mediated Ca2+ leak terms present in prior models. Finally, our model shows how Ca2+ sparks can be robustly triggered and terminated under both normal and pathological conditions. Together, these discoveries profoundly influence how we interpret and understand diverse experimental and clinical results from both normal and diseased hearts.

Single Ryanodine Receptor Channel Basis of Caffeine's Action on Ca 2+ Sparks

Caffeine (1, 3, 7-trimethylxanthine) is a widely used pharmacological agonist of the cardiac ryanodine receptor (RyR2) Ca 2+ release channel. It is also a well-known stimulant that can produce adverse side effects, including arrhythmias. Here, the action of caffeine on single RyR2 channels in bilayers and Ca 2+ sparks in permeabilized ventricular cardiomyocytes is defined. Single RyR2 caffeine activation depended on the free Ca 2+ level on both sides of the channel. Cytosolic Ca 2+ enhanced RyR2 caffeine affinity, whereas luminal Ca 2+ essentially scaled maximal caffeine activation. Caffeine activated single RyR2 channels in diastolic quasi-cell-like solutions (cytosolic MgATP, pCa 7) with an EC 50 of 9.0 ± 0.4 mM. Low-dose caffeine (0.15 mM) increased Ca 2+ spark frequency ∼75% and single RyR2 opening frequency ∼150%. This implies that not all spontaneous RyR2 openings during diastole are associated with Ca 2+ sparks. Assuming that only the longest openings evoke sparks, our data suggest that a spark may result only when a spontaneous single RyR2 opening lasts >6 ms.

Does Protein Kinase A–Mediated Phosphorylation of the Cardiac Ryanodine Receptor Play Any Role in Adrenergic Regulation of Calcium Handling in Health and Disease?

See related articles, pages 1743–1752 Cardiac myocyte ryanodine receptors (sarcoplasmic reticulum [SR] Ca release channel; cardiac ryanodine receptor [RyR2]) are localized in the junctional SR, in close proximity to L-type Ca channels (LTCCs) embedded in the membranes of the transverse (T)-tubules. This signaling microdomain has been termed the couplon,1 because it is here that excitation–contraction coupling takes place. As the heart fills with blood during diastole, RyR2 is stabilized in a closed state, allowing Ca uptake by the SR Ca ATPase to pump Ca from the cytoplasm into the SR, providing the primary source of Ca to activate the contractile apparatus during the next heart beat (systole). During systole, the cardiac action potential depolarizes the T-tubules and causes the opening of LTCCs. Ca influx through LTCCs elevates the [Ca] within the cytoplasmic space between the T-tubular and SR membrane, which promotes Ca binding to neighboring RyR2s, inducing some to open. Ca then moves out of the SR lumen into the subsarcolemmal, space and the additional elevation of [Ca] induces a regenerative opening of other RyRs in the couplon. The resulting localized Ca signal is called a Ca spark.2 The action potential synchronizes these local Ca release events throughout the cell (termed Ca-induced Ca release) to produce the global cardiac [Ca] transient.3 The stabilized closed state and Ca-dependent opening of RyR2 during diastole and systole are essential for normal cardiac diastolic and systolic function. Destabilizing the behavior of RyR2 would be expected to have a negative …

Impaired Ca2+ Release Synchronization in RyR2-S2808a Mouse Cardiomyocytes During β-Adrenergic Stimulation

Ca2+-induced Ca2+ release via ryanodine receptors (RyR2) is crucial for cardiac contractile function. During periods of stress and exercise, the sympathetic nervous system stimulates cardiac contractility. β-Adrenergic receptor activation has been suggested to result in PKA-mediated phosphorylation of RyR2 at Ser2808. Hyperphosphorylation at Ser2808 has also been discussed as possible factor contributing to heart failure. However, the role of RyR2 phosphorylation in inotropic adaptations during β-adrenergic stimulation remains controversial. Previous reports on a mouse model with genetic ablation of this phosphorylation site (S2808A) did not confirm the putative involvement of RyR2 phosphorylation in EC-coupling changes during β-adrenergic stimulation. In the present study, we intensified the search for EC-coupling modifications in S2808A myocytes by challenging EC-coupling near threshold conditions. Single cardiomyocytes were patch-clamped in the whole-cell configuration to measure ICaL, while Ca2+ transients were simultaneously recorded with confocal imaging of fluo-3. The EC-coupling gain, a measure for the effectiveness of ICaL to trigger Ca2+ release from the SR, was determined from control and S2808A cardiomyocytes. Lowering the extracellular Ca2+ concentration, a maneuver often used to unmask latent EC-coupling problems, did not reveal significant differences in the EC-coupling gain in WT and S2808A myocytes before and during β-adrenergic stimulation with isoproterenol. However, comprehensive analysis of subcellular Ca2+ transient kinetics indicated subtle differences in coordination of RyR activation. Uncoupling of the EC-mechanism by reduced [Ca2+]o resulted in a spatiotemporal de-synchronization of RyR openings. β-Adrenergic stimulation re-synchronized RyR openings under the same conditions less effectively in S2808A than in WT cardiomyocytes (time-to-peak of single Ca2+ release sites 181±6 vs. 100±3ms, respectively, P<0.0001). We conclude that although removal of the PKA phosphoepitope at Ser2808 does not critically derange EC-coupling, its ablation may interfere with synchronization of RyR2 activation during β-adrenergic stimulation.

FKBP12.6 Inhibits Resting Ryanodine Receptor Activity but PKA-Dependent Phosphorylation Does Not Alter FKBP12.6-RyR2 Binding in Rat Permeabilized Myocytes

FK506-binding proteins FKBP12.6 and FKBP12 are associated with the cardiac ryanodine receptor (RyR2), and PKA dependent hyperphosphorylation of RyR2 has been proposed to interrupt the FKBP12.6-RyR2 interaction and activate RyR2 opening. However, the physiological function of FKBP12.6/12.0 in cardiac myocytes and the role of PKA-dependent RyR2 phosphorylation are controversial. We used permeabilized rat ventricular myocytes, and fluorescently-labeled FKBP12.6/12.0 to directly measure in situ binding of FKBP12.6/12.0 to RyR2, with simultaneous Ca sparks measurements as an RyR2 functional index.

Trifluoperazine: a rynodine receptor agonist

Trifluoperazine (TFP), a phenothiazine, is a commonly used antipsychotic drug whose therapeutic effects are attributed to its central anti-adrenergic and anti-dopaminergic actions. However, TFP is also a calmodulin (CaM) antagonist and alters the Ca(2+) binding properties of calsequestrin (CSQ). The CaM and CSQ proteins are known modulators of sarcoplasmic reticulum (SR) Ca(2+) release in ventricular myocytes. We explored TFP actions on cardiac SR Ca(2+) release in cells and single type-2 ryanodine receptor (RyR2) channel activity in bilayers. In intact and permeabilized ventricular myocytes, TFP produced an initial activation of RyR2-mediated SR Ca(2+) release and over time depleted SR Ca(2+) content. At the single channel level, TFP or nortryptiline (NRT; a tricyclic antidepressant also known to modify CSQ Ca(2+) binding) increased the open probability (Po) of CSQ-free channels with an EC(50) of 5.2 microM or 8.9 microM (respectively). This Po increase was due to elevated open event frequency at low drug concentrations while longer mean open events sustained Po at higher drug concentrations. Activation of RyR2 by TFP occurred in the presence or absence of CaM. TFP may also inhibit SR Ca uptake as well as increase RyR2 opening. Our results suggest TFP and NRT can alter RyR2 function by interacting with the channel protein directly, independent of its actions on CSQ or CaM. This direct action may contribute to the clinical adverse cardiac side effects associated with these drugs.