Mutation-linked defective interdomain interactions within ryanodine receptor cause aberrant Ca²âºrelease leading to catecholaminergic polymorphic ventricular tachycardia.

BACKGROUND: The molecular mechanism by which catecholaminergic polymorphic ventricular tachycardia is induced by single amino acid mutations within the cardiac ryanodine receptor (RyR2) remains elusive. In the present study, we investigated mutation-induced conformational defects of RyR2 using a knockin mouse model expressing the human catecholaminergic polymorphic ventricular tachycardia-associated RyR2 mutant (S2246L; serine to leucine mutation at the residue 2246). METHODS AND RESULTS: All knockin mice we examined produced ventricular tachycardia after exercise on a treadmill. cAMP-dependent increase in the frequency of Ca²âº sparks was more pronounced in saponin-permeabilized knockin cardiomyocytes than in wild-type cardiomyocytes. Site-directed fluorescent labeling and quartz microbalance assays of the specific binding of DP2246 (a peptide corresponding to the 2232 to 2266 region: the 2246 domain) showed that DP2246 binds with the K201-binding sequence of RyR2 (1741 to 2270). Introduction of S2246L mutation into the DP2246 increased the affinity of peptide binding. Fluorescence quench assays of interdomain interactions within RyR2 showed that tight interaction of the 2246 domain/K201-binding domain is coupled with domain unzipping of the N-terminal (1 to 600)/central (2000 to 2500) domain pair in an allosteric manner. Dantrolene corrected the mutation-caused domain unzipping of the domain switch and stopped the exercise-induced ventricular tachycardia. CONCLUSIONS: The catecholaminergic polymorphic ventricular tachycardia-linked mutation of RyR2, S2246L, causes an abnormally tight local subdomain-subdomain interaction within the central domain involving the mutation site, which induces defective interaction between the N-terminal and central domains. This results in an erroneous activation of Ca²âº channel in a diastolic state reflecting on the increased Ca²âº spark frequency, which then leads to lethal arrhythmia.

Dynamic, inter-subunit interactions between the N-terminal and central mutation regions of cardiac ryanodine receptor.

Naturally occurring mutations in the cardiac ryanodine receptor (RyR2) have been linked to certain types of cardiac arrhythmias and sudden death. Two mutation hotspots that lie in the N-terminal and central regions of RyR2 are predicted to interact with one another and to form an important channel regulator switch. To monitor the conformational dynamics involving these regions, we generated a fluorescence resonance energy transfer (FRET) pair. A yellow fluorescent protein (YFP) was inserted into RyR2 after residue Ser437 in the N-terminal region, and a cyan fluorescent protein (CFP) was inserted after residue Ser2367 in the central region, to form a dual YFP- and CFP-labeled RyR2 (RyR2(S437-YFP/S2367-CFP)). We transfected HEK293 cells with RyR2(S437-YFP/S2367-CFP) cDNAs, and then examined them by using confocal microscopy and by measuring the FRET signal in live cells. The FRET signals are influenced by modulators of RyR2, by domain peptides that mimic the effects of disease causing RyR2 mutations, and by various drugs. Importantly, FRET signals were also readily detected in cells co-transfected with single CFP (RyR2(S437-YFP)) and single YFP (RyR2(S2367-CFP)) labeled RyR2, indicating that the interaction between the N-terminal and central mutation regions is an inter-subunit interaction. Our studies demonstrate that FRET analyses of this CFP- and YFP-labeled RyR2 can be used not only for investigating the conformational dynamics associated with RyR2 channel gating, but potentially, also for identifying drugs that are capable of stabilizing the conformations of RyR2.

Catecholaminergic polymorphic ventricular tachycardia is caused by mutation-linked defective conformational regulation of the ryanodine receptor.

RATIONALE: Catecholaminergic polymorphic ventricular tachycardia (CPVT) is caused by a single point mutation in a well-defined region of the cardiac type 2 ryanodine receptor (RyR)2. However, the underlying mechanism by which a single mutation in such a large molecule produces drastic effects on channel function remains unresolved. OBJECTIVE: Using a knock-in (KI) mouse model with a human CPVT-associated RyR2 mutation (R2474S), we investigated the molecular mechanism by which CPVT is induced by a single point mutation within the RyR2. METHODS AND RESULTS: The R2474S/+ KI mice showed no apparent structural or histological abnormalities in the heart, but they showed clear indications of other abnormalities. Bidirectional or polymorphic ventricular tachycardia was induced after exercise on a treadmill. The interaction between the N-terminal (amino acids 1 to 600) and central (amino acids 2000 to 2500) domains of the RyR2 (an intrinsic mechanism to close Ca(2+) channels) was weakened (domain unzipping). On protein kinase A-mediated phosphorylation of the RyR2, this domain unzipping further increased, resulting in a significant increase in the frequency of spontaneous Ca(2+) transients. cAMP-induced aberrant Ca(2+) release events (Ca(2+) sparks/waves) occurred at much lower sarcoplasmic reticulum Ca(2+) content as compared to the wild type. Addition of a domain-unzipping peptide, DPc10 (amino acids 2460 to 2495), to the wild type reproduced the aforementioned abnormalities that are characteristic of the R2474S/+ KI mice. Addition of DPc10 to the (cAMP-treated) KI cardiomyocytes produced no further effect. CONCLUSIONS: A single point mutation within the RyR2 sensitizes the channel to agonists and reduces the threshold of luminal [Ca(2+)] for activation, primarily mediated by defective interdomain interaction within the RyR2.

Aberrant interaction of calmodulin with the ryanodine receptor develops hypertrophy in the neonatal cardiomyocyte.

We have shown previously that the inter-domain interaction between the two domains of RyR (ryanodine receptor), CaMBD [CaM (calmodulin)-binding domain] and CaMLD (CaM-like domain), activates the Ca(2+) channel, and this process is called activation-link formation [Gangopadhyay and Ikemoto (2008) Biochem. J. 411, 415-423]. Thus CaM that is bound to CaMBD is expected to interfere the activation-link formation, thereby stabilizing the closed state of the channel under normal conditions. In the present paper, we report that, upon stimulation of neonatal cardiomyocytes with the pro-hypertrophy agonist ET-1 (endothelin-1), CaM dissociates from the RyR, which induces a series of intracellular events: increased frequency of Ca(2+) transients, translocation of the signalling molecules CaM, CaMKII (CaM kinase II) and the transcription factor NFAT (nuclear factor of activated T-cells) to the nucleus. These events then lead to the development of hypertrophy. Importantly, an anti-CaMBD antibody that interferes with activation-link formation prevented all of these intracellular events triggered by ET-1 and prevented the development of hypertrophy. These results indicate that the aberrant formation of the activation link between CaMBD and CaMLD of RyR is a key step in the development of hypertrophy in cultured cardiomyocytes.

Effects of conformational peptide probe DP4 on bidirectional signaling between DHPR and RyR1 calcium channels in voltage-clamped skeletal muscle fibers.

In skeletal muscle, excitation-contraction coupling involves the activation of dihydropyridine receptors (DHPR) and type-1 ryanodine receptors (RyR1) to produce depolarization-dependent sarcoplasmic reticulum Ca²âº release via orthograde signaling. Another form of DHPR-RyR1 communication is retrograde signaling, in which RyRs modulate the gating of DHPR. DP4 (domain peptide 4), is a peptide corresponding to residues Leu²44²-Pro²477 of the central domain of the RyR1 that produces RyR1 channel destabilization. Here we explore the effects of DP4 on orthograde excitation-contraction coupling and retrograde RyR1-DHPR signaling in isolated murine muscle fibers. Intracellular dialysis of DP4 increased the peak amplitude of Ca²âº release during step depolarizations by 64% without affecting its voltage-dependence or kinetics, and also caused a similar increase in Ca²âº release during an action potential waveform. DP4 did not modify either the amplitude or the voltage-dependence of the intramembrane charge movement. However, DP4 augmented DHPR Ca²âº current density without affecting its voltage-dependence. Our results demonstrate that the conformational changes induced by DP4 regulate both orthograde E-C coupling and retrograde RyR1-DHPR signaling.

Intracellular translocation of calmodulin and Ca2+/calmodulin-dependent protein kinase II during the development of hypertrophy in neonatal cardiomyocytes.

We have recently shown that stimulation of cultured neonatal cardiomyocytes with endothelin-1 (ET-1) first produces conformational disorder within the ryanodine receptor (RyR2) and diastolic Ca(2+) leak from the sarcoplasmic reticulum (SR), then develops hypertrophy (HT) in the cardiomyocytes (Hamada et al., 2009 [3]). The present paper addresses the following question. By what mechanism does crosstalk between defective operation of RyR2 and activation of the HT gene program occur? Here we show that the immuno-stain of calmodulin (CaM) is localized chiefly in the cytoplasmic area in the control cells; whereas, in the ET-1-treated/hypertrophied cells, major immuno-staining is localized in the nuclear region. In addition, fluorescently labeled CaM that has been introduced into the cardiomyocytes using the BioPORTER system moves from the cytoplasm to the nucleus with the development of HT. The immuno-confocal imaging of Ca(2+)/CaM-dependent protein kinase II (CaMKII) also shows cytoplasm-to-nucleus shift of the immuno-staining pattern in the hypertrophied cells. In an early phase of hypertrophic growth, the frequency of spontaneous Ca(2+) transients increases, which accompanies with cytoplasm-to-nucleus translocation of CaM. In a later phase of hypertrophic growth, further increase in the frequency of spontaneous Ca(2+) transients results in the appearance of trains of Ca(2+) spikes, which accompanies with nuclear translocation of CaMKII. The cardio-protective reagent dantrolene (the reagent that corrects the de-stabilized inter-domain interaction within the RyR2 to a normal mode) ameliorates aberrant intracellular Ca(2+) events and prevents nuclear translocation of both CaM and CaMKII, then prevents the development of HT. These results suggest that translocation of CaM and CaMKII from the cytoplasm to the nucleus serves as messengers to transmit the pathogenic signal elicited in the surface membrane and in the RyR2 to the nuclear transcriptional sites to activate HT program.

Defective calmodulin binding to the cardiac ryanodine receptor plays a key role in CPVT-associated channel dysfunction.

Calmodulin (CaM), one of the accessory proteins of the cardiac ryanodine receptor (RyR2), is known to play a significant role in the channel regulation of the RyR2. However, the possible involvement of calmodulin in the pathogenic process of catecholaminergic polymorphic ventricular tachycardia (CPVT) has not been investigated. In this study, we investigated the state of RyR2-bound CaM and channel dysfunctions using a knock-in (KI) mouse model with CPVT-linked RyR2 mutation (R2474S). Without added effectors, the affinity of CaM binding to the RyR2 was indistinguishable between KI and WT hearts. In response to cAMP (1 micromol/L), the RyR2 phosphorylation at Ser2808 increased in both WT and KI hearts to the same extent. However, cAMP caused a significant decrease of the CaM-binding affinity in KI hearts, but the affinity was unchanged in WT. Dantrolene restored a normal level of CaM-binding affinity in the cAMP-treated KI hearts, suggesting that defective inter-domain interaction between the N-terminal domain and the central domain of the RyR2 (the target of therapeutic effect of dantrolene) is involved in the cAMP-induced reduction of the CaM-binding affinity. In saponin-permeabilized cardiomyocytes, the addition of cAMP increased the frequency of spontaneous Ca(2+) sparks to a significantly larger extent in KI cardiomyocytes than in WT cardiomyocytes, whereas the addition of a high concentration of CaM attenuated the aberrant increase of Ca(2+) sparks. In conclusion, CPVT mutation causes defective inter-domain interaction, significant reduction in the ability of CaM binding to the RyR2, spontaneous Ca(2+) leak, and then lethal arrhythmia.

Identification of target domains of the cardiac ryanodine receptor to correct channel disorder in failing hearts.

BACKGROUND: We previously demonstrated that defective interdomain interaction between N-terminal (0 to 600) and central regions (2000 to 2500) of ryanodine receptor 2 (RyR2) induces Ca2+ leak in failing hearts and that K201 (JTV519) inhibits the Ca2+ leak by correcting the defective interdomain interaction. In the present report, we identified the K201-binding domain and characterized the role of this novel domain in the regulation of the RyR2 channel. METHODS AND RESULTS: An assay using a quartz-crystal microbalance technique (a very sensitive mass-measuring technique) revealed that K201 specifically bound to recombinant RyR2 fragments 1741 to 2270 and 1981 to 2520 but not to other RyR2 fragments from the 1 to 2750 region (1 to 610, 494 to 1000, 741 to 1260, 985 to 1503, 1245 to 1768, 2234 to 2750). By further analysis of the fragment(1741-2270), K201 was found to specifically bind to its subfragment(2114-2149). With the use of the peptide matching this subfragment (DP(2114-2149)) as a carrier, the RyR2 was fluorescently labeled with methylcoumarin acetate (MCA) in a site-directed manner. After tryptic digestion, the major MCA-labeled fragment of RyR2 (155 kDa) was detected by an antibody raised against the central region (Ab(2132)). Moreover, of several recombinant RyR2 fragments, only fragment(2234-2750) was specifically MCA labeled; this suggests that the K201-binding domain(2114-2149) binds with domain(2234-2750). Addition of DP(2114-2149) to the MCA-labeled sarcoplasmic reticulum interfered with the interaction between domain(2114-2149) and domain(2234-2750), causing domain unzipping, as evidenced by an increased accessibility of the bound MCA to a large-size fluorescence quencher. In failing cardiomyocytes, the frequency of spontaneous Ca2+ spark was markedly increased compared with normal cardiomyocytes, whereas incorporation of DP(2114-2149) markedly decreased the frequency of spontaneous Ca2+ spark. CONCLUSIONS: We first identified the K201-binding site as domain(2114-2149) of RyR2. Interruption of the interdomain interaction between the domain(2114-2149) and central domain(2234-2750) seems to mediate stabilization of RyR2 in failing hearts, which may lead to a novel therapeutic strategy against heart failure and perhaps lethal arrhythmia.

Defective regulation of the ryanodine receptor induces hypertrophy in cardiomyocytes.

Recent studies on cardiac hypertrophy animal model suggest that inter-domain interactions within the ryanodine receptor (RyR2) become defective concomitant with the development of hypertrophy (e.g. de-stabilization of the interaction between N-terminal and central domains of RyR2; T. Oda, M. Yano, T. Yamamoto, T. Tokuhisa, S. Okuda, M. Doi, T. Ohkusa, Y. Ikeda, S. Kobayashi, N. Ikemoto, M. Matsuzaki, Defective regulation of inter-domain interactions within the ryanodine receptor plays a key role in the pathogenesis of heart failure, Circulation 111 (2005) 3400-3410). To determine if de-stabilization of the inter-domain interaction in fact causes hypertrophy, we introduced DPc10 (a peptide corresponding to the G(2460)-P(2495) region of RyR2, which is known to de-stabilize the N-terminal/central domain interaction) into rat neonatal cardiomyocytes by mediation of peptide carrier BioPORTER. After incubation for 24h the peptide induced hypertrophy, as evidenced by significant increase in cell size and [(3)H]leucine uptake. K201 or dantrolene, the reagents known to correct the de-stabilized inter-domain interaction to a normal mode, prevented the DPc10-induced hypertrophy. These results suggest that disruption of the normal N-terminal/central inter-domain interaction within the RyR2 is a causative mechanism of cardiomyocyte hypertrophy.

Interaction of the Lys(3614)-Asn(3643) calmodulin-binding domain with the Cys(4114)-Asn(4142) region of the type 1 ryanodine receptor is involved in the mechanism of Ca2+/agonist-induced channel activation.

In the present study we show that the interaction of the CaM (calmodulin)-binding domain (Lys(3614)-Asn(3643)) with the Cys(4114)-Asn(4142) region (a region included in the CaM-like domain) serves as an intrinsic regulator of the RyR1 (type-1 ryanodine receptor). We tested the effects of antibodies raised against the two putative key regions of RyR1 [anti-(Lys(3614)-Asn(3643)) and anti-(Cys(4114)-Asn(4142)) antibodies]. Both antibodies produced significant inhibition of [3H]ryanodine-binding activity of RyR1. This suggests that the inter-domain interaction between the two domains, Lys(3614)-Asn(3643) and Cys(4114)-Asn(4142), activates the channel, and that the binding of antibody to either side of the interacting domain pair interfered with the formation of a 'channel-activation link' between the two regions. In order to spectroscopically monitor the mode of interaction of these domains, the site of inter-domain interaction was fluorescently labelled with MCA [(7-methoxycoumarin-4-yl)acetyl] in a site-directed manner. The accessibility of the bound MCA to a large molecular mass fluorescence quencher, BSA-QSY (namely, the size of a gap between the interacting domains) decreased with an increase of [Ca2+] in a range of 0.03-2.0 microM, as determined by Stern-Volmer fluorescence quenching analysis. The Ca2+-dependent decrease in the quencher accessibility was more pronounced in the presence of 150 microM 4-CmC (4-chlorometacresol), and was reversed by 1 mM Mg2+ (a well-known inhibitor of Ca2+/agonist-induced channel activation). These results suggest that the Lys(3614)-Asn(3643) and Cys(4114)-Asn(4142) regions of RyR1 interact with each other in a Ca2+- and agonist-dependent manner, and this serves as a mechanism of Ca2+- and agonist-dependent activation of the RyR1 Ca2+ channel.

Postulated role of interdomain interaction between regions 1 and 2 within type 1 ryanodine receptor in the pathogenesis of porcine malignant hyperthermia.

We have demonstrated recently that CICR (Ca2+-induced Ca2+ release) activity of RyR1 (ryanodine receptor 1) is held to a low level in mammalian skeletal muscle ('suppression' of the channel) and that this is largely caused by the interdomain interaction within RyR1 [Murayama, Oba, Kobayashi, Ikemoto and Ogawa (2005) Am. J. Physiol. Cell Physiol. 288, C1222-C1230]. To test the hypothesis that aberration of this suppression mechanism is involved in the development of channel dysfunctions in MH (malignant hyperthermia), we investigated properties of the RyR1 channels from normal and MHS (MH-susceptible) pig skeletal muscles with an Arg615-->Cys mutation using [3H]ryanodine binding, single-channel recordings and SR (sarcoplasmic reticulum) Ca2+ release. The RyR1 channels from MHS muscle (RyR1MHS) showed enhanced CICR activity compared with those from the normal muscle (RyR1N), although there was little or no difference in the sensitivity to several ligands tested (Ca2+, Mg2+ and adenine nucleotide), nor in the FKBP12 (FK506-binding protein 12) regulation. DP4, a domain peptide matching the Leu2442-Pro2477 region of RyR1 which was reported to activate the Ca2+ channel by weakening the interdomain interaction, activated the RyR1N channel in a concentration-dependent manner, and the highest activity of the affected channel reached a level comparable with that of the RyR1MHS channel with no added peptide. The addition of DP4 to the RyR1MHS channel produced virtually no further effect on the channel activity. These results suggest that stimulation of the RyR1MHS channel caused by affected inter-domain interaction between regions 1 and 2 is an underlying mechanism for dysfunction of Ca2+ homoeostasis seen in the MH phenotype.

Malignant hyperthermia mutation sites in the Leu2442-Pro2477 (DP4) region of RyR1 (ryanodine receptor 1) are clustered in a structurally and functionally definable area.

To explain the mechanism of pathogenesis of channel disorder in MH (malignant hyperthermia), we have proposed a model in which tight interactions between the N-terminal and central domains of RyR1 (ryanodine receptor 1) stabilize the closed state of the channel, but mutation in these domains weakens the interdomain interaction and destabilizes the channel. DP4 (domain peptide 4), a peptide corresponding to residues Leu2442-Pro2477 of the central domain, also weakens the domain interaction and produces MH-like channel destabilization, whereas an MH mutation (R2458C) in DP4 abolishes these effects. Thus DP4 and its mutants serve as excellent tools for structure-function studies. Other MH mutations have been reported in the literature involving three other amino acid residues in the DP4 region (Arg2452, Ile2453 and Arg2454). In the present paper we investigated the activity of several mutants of DP4 at these three residues. The ability to activate ryanodine binding or to effect Ca2+ release was severely diminished for each of the MH mutants. Other substitutions were less effective. Structural studies, using NMR analysis, revealed that the peptide has two a-helical regions. It is apparent that the MH mutations are clustered at the C-terminal end of the first helix. The data in the present paper indicates that mutation of residues in this region disrupts the interdomain interactions that stabilize the closed state of the channel.

Effects of peptide C corresponding to the Glu724-Pro760 region of the II-III loop of the DHP (dihydropyridine) receptor alpha1 subunit on the domain- switch-mediated activation of RyR1 (ryanodine receptor 1) Ca2+ channels.

The Leu720-Leu764 region of the II-III loop of the dihydropyridine receptor is believed to be important for both orthograde and retrograde communications with the RyR (ryanodine receptor), but its actual role has not yet been resolved. Our recent studies suggest that voltage-dependent activation of the RyR channel is mediated by a pair of interacting N-terminal and central domains, designated as the 'domain switch'. To investigate the effect of peptide C (a peptide corresponding to residues Glu724-Pro760) on domain- switch-mediated activation of the RyR, we measured Ca2+ release induced by DP (domain peptide) 1 or DP4 (which activates the RyR by mediation of the domain switch) and followed the Ca2+ release time course using a luminal Ca2+ probe (chlortetracycline) under Ca2+-clamped conditions. Peptide C produced a significant potentiation of the domain-switch-mediated Ca2+ release, provided that the Ca2+ concentration was sufficiently low (e.g. 0.1 microM) and the Ca2+ channel was only partially activated by the domain peptide. However, at micromolar Ca2+ concentrations, peptide C inhibits activation. Covalent cross-linking of fluorescently labelled peptide C to the RyR and screening of the fluorescently labelled tryptic fragments permitted us to localize the peptide-C-binding site to residues 450-1400, which may represent the primary region involved in physical coupling. Based on the above findings, we propose that the physiological role of residues Glu724-Pro760 is to facilitate depolarization-induced and domain-switch-mediated RyR activation at sub- or near-threshold concentrations of cytoplasmic Ca2+ and to suppress activation upon an increase of cytoplasmic Ca2+.

The RyR2 central domain peptide DPc10 lowers the threshold for spontaneous Ca2+ release in permeabilized cardiomyocytes.

OBJECTIVE: In vitro experiments have shown that the ryanodine receptor-2 (RyR2) central domain peptide DPc10 (Gly(2460)-Pro(2495)) mimics channel dysfunction associated with catecholaminergic polymorphic ventricular tachycardia (CPVT) by acting competitively to reduce stabilizing interactions between the N-terminal and central domains. In the present study, DPc10 was used as a tool to establish an adult cell model of the disease and to analyse the underlying mechanisms. METHODS: Rat ventricular myocytes were permeabilized with saponin and perfused with solutions approximating the intracellular milieu containing fluo-3. Sarcoplasmic reticulum (SR) Ca(2+) release was detected using confocal microscopy. DPc10 (10 or 50 microM) was compared with 0.2 mM caffeine, which is known to activate RyR2 and to facilitate Ca(2+)-induced Ca(2+) release (CICR). RESULTS: Introduction of DPc10 induced a transient increase in spark frequency and a sustained rise in resting [Ca(2+)]. Under conditions causing initial Ca(2+) overload of the SR, DPc10 reduced the frequency and amplitude of spontaneous, propagated Ca(2+) release (SPCR). Following equilibration with 10microM DPc10, the cytosolic [Ca(2+)] threshold for SPCR was markedly reduced and the proportion of spontaneously active cells increased. Caffeine induced a similar, transient increase in spark frequency and a reduction in the [Ca(2+)] threshold for SPCR. However, unlike DPc10, caffeine increased SPCR frequency and had no sustained effect on resting [Ca(2+)]. These results suggest that the net effect of DPc10 (and CPVT mutations) on RyR2 function in situ is not only to increase the sensitivity to CICR as caffeine does, but also to potentiate Ca(2+) leakage from the SR. As SPCR can trigger delayed after-depolarisations, the decrease in [Ca(2+)] threshold may contribute to arrhythmias in CPVT patients during exercise or stress.

Correction of defective interdomain interaction within ryanodine receptor by antioxidant is a new therapeutic strategy against heart failure.

BACKGROUND: Defective interdomain interaction within the ryanodine receptor (RyR2) seems to play a key role in the pathogenesis of heart failure, as shown in recent studies. In the present study we investigated the effect of oxidative stress on the interdomain interaction, its outcome in the cardiac function in heart failure, and the possibility of preventing the problem with antioxidants. METHODS AND RESULTS: Sarcoplasmic reticulum (SR) vesicles were isolated from dog left ventricular (LV) muscle (normal or rapid ventricular pacing for 4 weeks with or without the antioxidant edaravone). In the edaravone-treated paced dogs (EV+), but not in the untreated paced dogs (EV-), normal cardiac function was restored almost completely. In the SR vesicles isolated from the EV-, oxidative stress of the RyR2 (reduction in the number of free thiols) was severe, but it was negligible in EV+. The oxidative stress of the RyR2 destabilized interdomain interactions within the RyR2 (EV-), but its effect was reversed in EV+. Abnormal Ca2+ leak through the RyR2 was found in EV- but not in EV+. The amount of the RyR2-bound FKBP12.6 was less in EV- than in normal dogs, whereas it was restored almost to a normal amount in EV+. The NO donor 3-morpholinosydnonimine (SIN-1) reproduced, in normal SR, several abnormal features seen in failing SR, such as defective interdomain interaction and abnormal Ca2+ leak. Both cell shortening and Ca2+ transients were impaired by SIN-1 in isolated normal myocytes, mimicking the pathophysiological conditions in failing myocytes. Incubation of failing myocytes with edaravone restored the normal properties. CONCLUSIONS: During the development of heart failure, edaravone ameliorated the defective interdomain interaction of the RyR2. This prevented Ca2+ leak and LV remodeling, leading to an improvement of cardiac function and an attenuation of LV remodeling.

Defective regulation of interdomain interactions within the ryanodine receptor plays a key role in the pathogenesis of heart failure.

BACKGROUND: According to our hypothesis, 2 domains within the ryanodine receptor (RyR) of sarcoplasmic reticulum (SR) (N-terminal [0 to 600] and central [2000 to 2500] domains), where many mutations have been found in patients with polymorphic ventricular tachycardia, interact with each other as a regulatory switch for channel gating. Here, we investigated whether the defective FKBP12.6-mediated stabilization of RyR in heart failure is produced by an abnormal interdomain interaction. METHODS AND RESULTS: SR vesicles were isolated from dog left ventricular muscles, and then the RyR moiety of the SR was fluorescently labeled with methylcoumarin acetate (MCA) using DPc10, a synthetic peptide corresponding to Gly2460-Pro2495 of RyR (one of the mutable domains in polymorphic ventricular tachycardia), as a site-directing carrier; the carrier was removed from the RyR after MCA labeling. Addition of DPc10 induced an unzipped state of the interacting N-terminal and central domains, as evidenced by an increase in the accessibility of the RyR-bound MCA fluorescence to a large fluorescence quencher. Domain unzipping resulted in Ca2+ leak through the RyR and facilitated cAMP-dependent hyperphosphorylation of RyR and FKBP12.6 dissociation from RyR. When DPc10 was introduced into the isolated myocytes, the magnitude of intracellular Ca2+ transient decreased, and its decay time was prolonged. In the SR isolated from pacing-induced dog failing hearts, the domain unzipping has already occurred, together with FKBP12.6 dissociation and Ca2+ leak. CONCLUSIONS: The specific domain interaction within the RyR regulates the channel gating property, and the defectiveness in the mode of the interdomain interaction seems to be the initial critical step of the pathogenesis of heart failure.

Abnormal ryanodine receptor function in heart failure.

The abnormally regulated release of Ca2+ from an intracellular Ca2+ store, the sarcoplasmic reticulum (SR), is the mechanism underlying contractile and relaxation dysfunctions in heart failure (HF). According to recent reports, protein kinase A (PKA)-mediated hyperphosphorylation of ryanodine receptor (RyR) in the SR has been shown to cause the dissociation of FK506 binding protein (FKBP) 12.6 from the RyR in heart failure. This causes an abnormal Ca2+ leak through the Ca2+ channel located in the RyR, leading to an increase in the cytosolic Ca2+ during diastole, prolongation of the Ca2+ transient, and delayed/slowed diastolic Ca2+ re-uptake. More recently, a considerable number of disease-linked mutations in the RyR have been reported in patients with catecholaminergic polymorphic ventricular tachycardia (CPVT) or arrhythmogenic right ventricular dysplasia type 2. An analysis of the disposition of these mutation sites within well-defined domains of the RyR polypeptide chain has led to the new concept that interdomain interactions among these domains play a critical role in channel regulation, and an altered domain interaction causes channel dysfunction in the failing heart. The knowledge gained from the recent literature concerning the critical proteins and the changes in their properties under pathological conditions has brought us to a better position to develop new pharmacological or genetic strategies for the treatment of heart failure or cardiac arrhythmia. A considerable body of evidence reviewed here indicates that abnormal RyR function plays an important role in the pathogenesis of heart failure. This review also covers some controversial issues in the literature concerning the involvement of phosphorylation and FKBP12.6.

Probing a putative dantrolene-binding site on the cardiac ryanodine receptor.

Dantrolene is an inhibitor of intracellular Ca2+ release from skeletal muscle SR (sarcoplasmic reticulum). Direct photoaffinity labelling experiments using [3H]azidodantrolene and synthetic domain peptides have demonstrated that this drug targets amino acids 590-609 [termed DP1 (domain peptide 1)] of RyR1 (ryanodine receptor 1), the skeletal muscle RyR isoform. Although the identical sequence exists in the cardiac isoform, RyR2 (residues 601-620), specific labelling of RyR2 by dantrolene has not been demonstrated, even though some functional studies show protective effects of dantrolene on heart function. Here we test whether dantrolene-active domains exist within RyR2 and if so, whether this domain can be modulated. We show that elongated DP1 sequences from RyR1 (DP1-2s; residues 590-628) and RyR2 (DP1-2c; residues 601-639) can be specifically photolabelled by [3H]azidodantrolene. Monoclonal anti-RyR1 antibody, whose epitope is the DP1 region, can recognize RyR1 but not RyR2 in Western blot and immunoprecipitation assays, yet it recognizes both DP1-2c and DP1-2s. This suggests that although the RyR2 sequence has an intrinsic capacity to bind dantrolene in vitro, this site may be poorly accessible in the native channel protein. To examine whether it is possible to modulate this site, we measured binding of [3H]dantrolene to cardiac SR as a function of free Ca2+. We found that > or =10 mM EGTA increased [3H]dantrolene binding to RyR2 by approximately 2-fold. The data suggest that the dantrolene-binding site on RyR2 is conformationally sensitive. This site may be a potential therapeutic target in cardiovascular diseases sensitive to dysfunctional intracellular Ca2+ release.

Interdomain interactions within ryanodine receptors regulate Ca2+ spark frequency in skeletal muscle.

DP4 is a 36-residue synthetic peptide that corresponds to the Leu(2442)-Pro(2477) region of RyR1 that contains the reported malignant hyperthermia (MH) mutation site. It has been proposed that DP4 disrupts the normal interdomain interactions that stabilize the closed state of the Ca(2)+ release channel (Yamamoto, T., R. El-Hayek, and N. Ikemoto. 2000. J. Biol. Chem. 275:11618-11625). We have investigated the effects of DP4 on local SR Ca(2)+ release events (Ca(2)+ sparks) in saponin-permeabilized frog skeletal muscle fibers using laser scanning confocal microscopy (line-scan mode, 2 ms/line), as well as the effects of DP4 on frog SR vesicles and frog single RyR Ca(2)+ release channels reconstituted in planar lipid bilayers. DP4 caused a significant increase in Ca(2)+ spark frequency in muscle fibers. However, the mean values of the amplitude, rise time, spatial half width, and temporal half duration of the Ca(2)+ sparks, as well as the distribution of these parameters, remained essentially unchanged in the presence of DP4. Thus, DP4 increased the opening rate, but not the open time of the RyR Ca(2)+ release channel(s) generating the sparks. DP4 also increased [(3)H]ryanodine binding to SR vesicles isolated from frog and mammalian skeletal muscle, and increased the open probability of frog RyR Ca(2)+ release channels reconstituted in bilayers, without changing the amplitude of the current through those channels. However, unlike in Ca(2)+ spark experiments, DP4 produced a pronounced increase in the open time of channels in bilayers. The same peptide with an Arg(17) to Cys(17) replacement (DP4mut), which corresponds to the Arg(2458)-to-Cys(2458) mutation in MH, did not produce a significant effect on RyR activation in muscle fibers, bilayers, or SR vesicles. Mg(2)+ dependence experiments conducted with permeabilized muscle fibers indicate that DP4 preferentially binds to partially Mg(2)+-free RyR(s), thus promoting channel opening and production of Ca(2)+ sparks.

Antibody probe study of Ca2+ channel regulation by interdomain interaction within the ryanodine receptor.

N-terminal and central domains of ryanodine receptor 1 (RyR1), where many reported malignant hyperthermia (MH) mutations are localized, represent putative channel regulatory domains. Recent domain peptide (DP) probe studies led us to the hypothesis that these domains interact to stabilize the closed state of channel (zipping), while weakening of domain-domain interactions (unzipping) by mutation de-stabilizes the channel, making it leaky to Ca2+ or sensitive to the agonists of RyR1. As shown previously, DP1 (N-terminal domain peptide) and DP4 (central domain peptide) produced MH-like channel activation/sensitization effects, presumably by peptide binding to sites critical to stabilizing domain-domain interactions and resultant loss of conformational constraints. Here we report that polyclonal anti-DP1 and anti-DP4 antibodies also produce MH-like channel activation and sensitization effects as evidenced by about 4-fold enhancement of high affinity [3H]ryanodine binding to RyR1 and by a significant left-shift of the concentration-dependence of activation of sarcoplasmic reticulum Ca2+ release by polylysine. Fluorescence quenching experiments demonstrate that the accessibility of a DP4-directed, conformationally sensitive fluorescence probe linked to the RyR1 N-terminal domain is increased in the presence of domain-specific antibodies, consistent with the view that these antibodies produce unzipping of interacting domains that are of hindered accessibility to the surrounding aqueous environment. Our results suggest that domain-specific antibody binding induces a conformational change resulting in channel activation, and are consistent with the hypothesis that interacting N-terminal and central domains are intimately involved in the regulation of RyR1 channel function.