Inna Györke

Regulation of the Cardiac Ryanodine Receptor Channel by Luminal Ca2+ Involves Luminal Ca2+ Sensing Sites

The mechanism of activation of the cardiac calcium release channel/ryanodine receptor (RyR) by luminal Ca2+ was investigated in native canine cardiac RyRs incorporated into lipid bilayers in the presence of 0.01 microM to 2 mM Ca2+ (free) and 3 mM ATP (total) on the cytosolic (cis) side and 20 microM to 20 mM Ca2+ on the luminal (trans) side of the channel and with Cs+ as the charge carrier. Under conditions of low trans Ca2+ (20 microM), increasing cis Ca2+ from 0.1 to 10 microM caused a gradual increase in channel open probability (Po). Elevating cis Ca2+ above 100 microM resulted in a gradual decrease in Po. Elevating trans [Ca2+] enhanced channel activity (EC50 approximately 2.5 mM at 1 microM cis Ca2+) primarily by increasing the frequency of channel openings. The dependency of Po on trans [Ca2+] was similar at negative and positive holding potentials and was not influenced by high cytosolic concentrations of the fast Ca2+ chelator, 1,2-bis(2-aminophenoxy)ethane-N,N,N, N-tetraacetic acid. Elevated luminal Ca2+ enhanced the sensitivity of the channel to activating cytosolic Ca2+, and it essentially reversed the inhibition of the channel by high cytosolic Ca2+. Potentiation of Po by increased luminal Ca2+ occurred irrespective of whether the electrochemical gradient for Ca2+ supported a cytosolic-to-luminal or a luminal-to-cytosolic flow of Ca2+ through the channel. These results rule out the possibility that under our experimental conditions, luminal Ca2+ acts by interacting with the cytosolic activation site of the channel and suggest that the effects of luminal Ca2+ are mediated by distinct Ca2+-sensitive site(s) at the luminal face of the channel or associated protein.

The Role of Calsequestrin, Triadin, and Junctin in Conferring Cardiac Ryanodine Receptor Responsiveness to Luminal Calcium

The level of Ca inside the sarcoplasmic reticulum (SR) is an important determinant of functional activity of the Ca release channel/ryanodine receptor (RyR) in cardiac muscle. However, the molecular basis of RyR regulation by luminal Ca remains largely unknown. In the present study, we investigated the potential role of the cardiac SR luminal auxiliary proteins calsequestrin (CSQ), triadin 1, and junctin in forming the luminal calcium sensor for the cardiac RyR. Recordings of single RyR channels incorporated into lipid bilayers, from either SR vesicle or purified RyR preparations, were performed in the presence of MgATP using Cs+ as the charge carrier. Raising luminal [Ca] from 20 microM to 5 mM increased the open channel probability (Po) of native RyRs in SR vesicles, but not of purified RyRs. Adding CSQ to the luminal side of the purified channels produced no significant changes in Po, nor did it restore the ability of RyRs to respond to luminal Ca. When triadin 1 and junctin were added to the luminal side of purified channels, RyR Po increased significantly; however, the channels still remained unresponsive to changes in luminal [Ca]. In RyRs reassociated with triadin 1 and junctin, adding luminal CSQ produced a significant decrease in activity. After reassociation with all three proteins, RyRs responded to rises of luminal [Ca] by increasing their Po. These results suggest that a complex of CSQ, triadin 1, and junctin confer RyR luminal Ca sensitivity. CSQ apparently serves as a luminal Ca sensor that inhibits the channel at low luminal [Ca], whereas triadin 1 and/or junctin may be required to mediate interactions of CSQ with RyR.

Redox modification of ryanodine receptors contributes to sarcoplasmic reticulum Ca2+ 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 ([Ca2+]c) and sarcoplasmic reticulum (SR) luminal Ca2+ ([Ca2+]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 Ca2+ leak was significantly enhanced in permeabilized myocytes, resulting in reduced [Ca2+]SR in HF compared to control cells. Both SR Ca2+ leak and [Ca2+]SR were partially normalized by treating HF myocytes with reducing agents. Conversely, oxidizing agents accelerated SR Ca2+ leak and decreased [Ca2+]SR in cells from normal hearts. Moreover, exposure to antioxidants significantly improved intracellular Ca2+-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 Ca2+ and was partially normalized by reducing agents through restoring luminal Ca2+ sensitivity oxidation of control RyR2s enhanced their luminal Ca2+ 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.

Calsequestrin determines the functional size and stability of cardiac intracellular calcium stores: Mechanism for hereditary arrhythmia.

Calsequestrin is a high-capacity Ca-binding protein expressed inside the sarcoplasmic reticulum (SR), an intracellular Ca release and storage organelle in muscle. Mutations in the cardiac calsequestrin gene (CSQ2) have been linked to arrhythmias and sudden death. We have used Ca-imaging and patch-clamp methods in combination with adenoviral gene transfer strategies to explore the function of CSQ2 in adult rat heart cells. By increasing or decreasing CSQ2 levels, we showed that CSQ2 not only determines the Ca storage capacity of the SR but also positively controls the amount of Ca released from this organelle during excitation–contraction coupling. CSQ2 controls Ca release by prolonging the duration of Ca fluxes through the SR Ca-release sites. In addition, the dynamics of functional restitution of Ca-release sites after Ca discharge were prolonged when CSQ2 levels were elevated and accelerated in the presence of lowered CSQ2 protein levels. Furthermore, profound disturbances in rhythmic Ca transients in myocytes undergoing periodic electrical stimulation were observed when CSQ2 levels were reduced. We conclude that CSQ2 is a key determinant of the functional size and stability of SR Ca stores in cardiac muscle. CSQ2 appears to exert its effects by influencing the local luminal Ca concentration-dependent gating of the Ca-release channels and by acting as both a reservoir and a sink for Ca in SR. The abnormal restitution of Ca-release channels in the presence of reduced CSQ2 levels provides a plausible explanation for ventricular arrhythmia associated with mutations of CSQ2.

Regulation of calcium release by calcium inside the sarcoplasmic reticulum in ventricular myocytes

To study the effects of changes in sarcoplasmic reticulum (SR) intraluminal Ca2+ on the Ca2+ release mechanism, we correlated the activity of single cardiac ryanodine receptor (RyR) channels, monitored in planar bilayers, with the properties of spontaneous elementary Ca2+ release events (sparks) in intact ventricular myocytes, monitored by scanning confocal microfluorimetry. Under both normal conditions and Ca2+ overload, induced by elevation of extracellular [Ca2+], Ca2+ sparks represented single populations of events. During Ca2+ overload, the frequency of sparks increased from 0.8 to 3.1 events per second per 100 μm line scanned, and their amplitude increased from 100 nM to 400 nM. The duration of the Ca2+ sparks, however, was not altered. Changes in the properties of Ca2+ sparks were accompanied by only an ≈ 30% increase in the SR Ca2+ content, as determined by emptying the intracellular Ca2+ stores using caffeine. When single Ca2+ release channels were incorporated into lipid bilayers and activated by cytoplasmic Ca2+ (≈ 100 nM) and ATP (3 mM), elevation of Ca2+ on the luminal side from 20 μM to 0.2–20 mM resulted in a 1.2-fold to 7-fold increase, respectively, in open probability (Po). This potentiation of Po was due to an increase in mean open time and frequency of events. The relative effect of luminal Ca2+ was greater at low levels of cytoplasmic [Ca2+] than at high levels of cytoplasmic [Ca2+], and no effect of luminal Ca2+ was observed to occur in channels activated by 0.5–50 μM cytoplasmic Ca2+ in the absence of ATP. Our results suggest that SR Ca2+ release channels are modulated by SR intraluminal Ca2+. These alterations in properties of release channels may account for, or contribute to, the mechanism of spontaneous Ca2+ release in cardiac myocytes

Abnormal Interactions of Calsequestrin With the Ryanodine Receptor Calcium Release Channel Complex Linked to Exercise-Induced Sudden Cardiac Death

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a familial arrhythmogenic disorder associated with mutations in the cardiac ryanodine receptor (RyR2) and cardiac calsequestrin (CASQ2) genes. Previous in vitro studies suggested that RyR2 and CASQ2 interact as parts of a multimolecular Ca2+-signaling complex; however, direct evidence for such interactions and their potential significance to myocardial function remain to be determined. We identified a novel CASQ2 mutation in a young female with a structurally normal heart and unexplained syncopal episodes. This mutation results in the nonconservative substitution of glutamine for arginine at amino acid 33 of CASQ2 (R33Q). Adenoviral-mediated expression of CASQ2R33Q in adult rat myocytes led to an increase in excitation–contraction coupling gain and to more frequent occurrences of spontaneous propagating (Ca2+ waves) and local Ca2+ signals (sparks) with respect to control cells expressing wild-type CASQ2 (CASQ2WT). As revealed by a Ca2+ indicator entrapped inside the sarcoplasmic reticulum (SR) of permeabilized myocytes, the increased occurrence of spontaneous Ca2+ sparks and waves was associated with a dramatic decrease in intra-SR [Ca2+]. Recombinant CASQ2WT and CASQ2R33Q exhibited similar Ca2+-binding capacities in vitro; however, the mutant protein lacked the ability of its WT counterpart to inhibit RyR2 activity at low luminal [Ca2+] in planar lipid bilayers. We conclude that the R33Q mutation disrupts interactions of CASQ2 with the RyR2 channel complex and impairs regulation of RyR2 by luminal Ca2+. These results show that intracellular Ca2+ cycling in normal heart relies on an intricate interplay of CASQ2 with the proteins of the RyR2 channel complex and that disruption of these interactions can lead to cardiac arrhythmia.

Abnormal Calcium Signaling and Sudden Cardiac Death Associated With Mutation of Calsequestrin

Abstract— Mutations in human cardiac calsequestrin (CASQ2), a high-capacity calcium-binding protein located in the sarcoplasmic reticulum (SR), have recently been linked to effort-induced ventricular arrhythmia and sudden death (catecholaminergic polymorphic ventricular tachycardia). However, the precise mechanisms through which these mutations affect SR function and lead to arrhythmia are presently unknown. In this study, we explored the effect of adenoviral-directed expression of a canine CASQ2 protein carrying the catecholaminergic polymorphic ventricular tachycardia–linked mutation D307H (CASQ2D307H) on Ca2+ signaling in adult rat myocytes. Total CASQ2 protein levels were consistently elevated ≈4-fold in cells infected with adenoviruses expressing either wild-type CASQ2 (CASQ2WT) or CASQ2D307H. Expression of CASQ2D307H reduced the Ca2+ storing capacity of the SR. In addition, the amplitude, duration, and rise time of macroscopic ICa-induced Ca2+ transients and of spontaneous Ca2+ sparks were reduced significantly in myocytes expressing CASQ2D307H. Myocytes expressing CASQ2D307H also displayed drastic disturbances of rhythmic oscillations in [Ca2+]i and membrane potential, with signs of delayed afterdepolarizations when undergoing periodic pacing and exposed to isoproterenol. Importantly, normal rhythmic activity was restored by loading the SR with the low-affinity Ca2+ buffer, citrate. Our data suggest that the arrhythmogenic CASQ2D307H mutation impairs SR Ca2+ storing and release functions and destabilizes the Ca2+-induced Ca2+ release mechanism by reducing the effective Ca2+ buffering inside the SR and/or by altering the responsiveness of the Ca2+ release channel complex to luminal Ca2+. These results establish at the cellular level the pathological link between CASQ2 mutations and the predisposition to adrenergically mediated arrhythmias observed in patients carrying CASQ2 defects.

Dual effects of tetracaine on spontaneous calcium release in rat ventricular myocytes.

1. Confocal microfluorometry was used to study the effects of tetracaine on spontaneous Ca2+ release from the sarcoplasmic reticulum (SR) in isolated rat ventricular myocytes. 2. At low concentrations (0.25‐1.25 mM), tetracaine caused an initial inhibition of spontaneous release events (Ca2+ sparks) and Ca2+ waves, which was followed by a gradual increase in Ca2+ release activity. The frequency and magnitude of sparks were first decreased and then increased with respect to control levels. At high concentrations (> 1.25 mM), tetracaine abolished all forms of spontaneous release. 3. Exposure of the myocytes to tetracaine resulted in a gradual increase in the SR Ca2+ load as indexed by changes in the magnitude of caffeine‐induced Ca2+ transients. 4. In cardiac SR Ca(2+)‐release channels incorporated into lipid bilayers, tetracaine (> 0.25 mM) induced a steady inhibition of channel activity. Addition of millimolar Ca2+ to the luminal side of the channel caused an increase in channel open probability under control conditions as well as in the presence of various concentrations of tetracaine. 5. We conclude that the primary effect of tetracaine on SR Ca(2+)‐release channels is inhibition of channel activity both in vitro and in situ. The ability of tetracaine to reduce spark magnitude suggests that these events are not due to activation of single channels or non‐reducible clusters of channels and, therefore, supports the multichannel origin of sparks. We propose that the paradoxical late potentiation of release by submaximal concentrations of tetracaine is caused by a gradual increase in SR Ca2+ load and subsequent activation of the Ca(2+)‐release channels by Ca2+ inside the SR.

The role of luminal Ca2+ in the generation of Ca2+ waves in rat ventricular myocytes

1 We used confocal Ca2+ imaging and fluo‐3 to investigate the transition of localized Ca2+ releases induced by focal caffeine stimulation into propagating Ca2+ waves in isolated rat ventricular myocytes. 2 Self‐sustaining Ca2+ waves could be initiated when the cellular Ca2+ load was increased by elevating the extracellular [Ca2+] ([Ca2+]o) and they could also be initiated at normal Ca2+ loads when the sensitivity of the release sites to cytosolic Ca2+ was enhanced by low doses of caffeine. When we prevented the accumulation of extra Ca2+ in the luminal compartment of the sarcoplasmic reticulum (SR) with thapsigargin, focal caffeine pulses failed to trigger self‐sustaining Ca2+ waves on elevation of [Ca2+]o. Inhibition of SR Ca2+ uptake by thapsigargin in cells already preloaded with Ca2+ above normal levels did not prevent local Ca2+ elevations from triggering propagating waves. Moreover, wave velocity increased by 20 %. Tetracaine (0·75 mM) caused transient complete inhibition of both local and propagating Ca2+ signals, followed by full recovery of the responses due to increased SR Ca2+ accumulation. 3 Computer simulations using a numerical model with spatially distinct Ca2+ release sites suggested that increased amounts of releasable Ca2+ might not be sufficient to generate self‐sustaining Ca2+ waves under conditions of Ca2+ overload unless the threshold of release site Ca2+ activation was set at relatively low levels (< 1·5 μM). 4 We conclude that the potentiation of SR Ca2+ release channels by luminal Ca2+ is an important factor in Ca2+ wave generation. Wave propagation does not require the translocation of Ca2+ from the spreading wave front into the SR. Instead, it relies on luminal Ca2+ sensitizing Ca2+ release channels to cytosolic Ca2+.

Protein Phosphatases Decrease Sarcoplasmic Reticulum Calcium Content by Stimulating Calcium Release in Cardiac Myocytes

Phosphorylation/dephosphorylation of Ca2+ transport proteins by cellular kinases and phosphatases plays an important role in regulation of cardiac excitation−contraction coupling; furthermore abnormal protein kinase and phosphatase activities have been implicated in heart failure. However, the precise mechanisms of action of these enzymes on intracellular Ca2+ handling in normal and diseased hearts remains poorly understood. We have investigated the effects of protein phosphatases PP1 and PP2A on spontaneous Ca2+ sparks and SR Ca2+ load in myocytes permeabilized with saponin. Exposure of myocytes to PP1 or PP2A caused a dramatic increase in frequency of Ca2+ sparks followed by a nearly complete disappearance of events. These effects were accompanied by depletion of the SR Ca2+ stores, as determined by application of caffeine. These changes in Ca2+ release and SR Ca2+ load could be prevented by the inhibitors of PP1 and PP2A phosphatase activities okadaic acid and calyculin A. At the single channel level, PP1 increased the open probability of RyRs incorporated into lipid bilayers. PP1‐medited RyR dephosphorylation in our permeabilized myocytes preparations was confirmed biochemically by quantitative immunoblotting using a phosphospecific anti‐RyR antibody. Our results suggest that increased intracellular phosphatase activity stimulates RyR‐mediated SR Ca2+ release leading to depleted SR Ca2+ stores in cardiac myocytes.