Correction of Defective Interdomain Interaction Within...

Correction of Defective Interdomain Interaction WithinRyanodine Receptor by Antioxidant Is a New Therapeutic

Strategy Against Heart FailureMasafumi Yano, MD, PhD; Shinichi Okuda, MD, PhD; Tetsuro Oda, MD; Takahiro Tokuhisa, MD;

Hiroki Tateishi, MD; Mamoru Mochizuki, MD; Toshiyuki Noma, BS; Masahiro Doi, MD, PhD;Shigeki Kobayashi, MD, PhD; Takeshi Yamamoto, MD, PhD; Yasuhiro Ikeda, MD, PhD;

Tomoko Ohkusa, MD, PhD; Noriaki Ikemoto, PhD; Masunori Matsuzaki, MD, PhD

Background—Defective interdomain interaction within the ryanodine receptor (RyR2) seems to play a key role in thepathogenesis of heart failure, as shown in recent studies. In the present study we investigated the effect of oxidativestress on the interdomain interaction, its outcome in the cardiac function in heart failure, and the possibility of preventingthe problem with antioxidants.

Methods and Results—Sarcoplasmic reticulum (SR) vesicles were isolated from dog left ventricular (LV) muscle (normalor 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 SRvesicles isolated from the EV�, oxidative stress of the RyR2 (reduction in the number of free thiols) was severe, butit 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 anormal amount in EV�. The NO donor 3-morpholinosydnonimine (SIN-1) reproduced, in normal SR, several abnormalfeatures seen in failing SR, such as defective interdomain interaction and abnormal Ca2� leak. Both cell shortening andCa2� transients were impaired by SIN-1 in isolated normal myocytes, mimicking the pathophysiological conditions infailing 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 theRyR2. This prevented Ca2� leak and LV remodeling, leading to an improvement of cardiac function and an attenuationof LV remodeling. (Circulation. 2005;112:3633-3643.)

Key Words: calcium � free radicals � heart failure � sarcoplasmic reticulum

Aconsiderable body of evidence suggests that an abnormalregulation of the intracellular Ca2� by the sarcoplasmic

reticulum (SR) is the chief pathogenic mechanism for varioustypes of dysfunctions seen in heart failure. In our recent studiesof the canine model of pacing-induced heart failure, we havedemonstrated that the Ca2�-release function of the ryanodinereceptor (RyR2) is defective in heart failure, presumably becauseof a partial loss of FKBP12.6 from the RyR2, and this defectiveregulation of the RyR2 causes an abnormal Ca2� leak bymediation of a conformational change in the RyR2.1 Marx et al2

demonstrated that protein kinase A (PKA)–mediated hyperphos-phorylation of the RyR2 causes dissociation of FKBP12.6 fromthe RyR2, resulting in a defective channel function due to anincreased sensitivity to Ca2�-induced activation. In failing hearts,

reduced levels of PP1 and PP2A in the RyR2 macromolecularcomplex rather than increased PKA activity appear to beresponsible for the RyR2 hyperphosphorylation and the forma-tion of “leaky” channels.2 We found that both �-blockers3 and anew cardioprotective agent, JTV519,4,5 correct the defectiveFKBP12.6-mediated control of the RyR2, improving cardiacfunction during the development of heart failure. Wehrens et al6

also demonstrated that JTV519 increased the binding affinity ofFKBP12.6 to the RyR2, which stabilized the closed state of theRyR2 channels and prevented the abnormal Ca2� leak thatwould otherwise have triggered ventricular arrhythmias. Thesestudies suggest that securing the FKBP12.6-mediated stabiliza-tion of the RyR2 may be a new therapeutic strategy against heartfailure.

Received April 13, 2005; revision received September 13, 2005; accepted September 19, 2005.From the Department of Medical Bioregulation, Division of Cardiovascular Medicine, Yamaguchi University School of Medicine, Yamaguchi, Japan

(M.Y., S.O., T. Oda, T.T., H.T., M. Mochizuki, T.N., M.D., S.K., T.Y., Y.I., T. Ohkusa, M. Matsuzaki); Boston Biomedical Research Institute,Watertown, Mass (N.I.); and Department of Neurology, Harvard Medical School, Boston, Mass (N.I.).

Correspondence to Masafumi Yano, MD, PhD, Department of Medical Bioregulation, Division of Cardiovascular Medicine, Yamaguchi UniversitySchool of Medicine, 1-1-1 Minamikogushi, Ube, Yamaguchi 755-8505, Japan. E-mail [email protected]

© 2005 American Heart Association, Inc.

Circulation is available at http://www.circulationaha.org DOI: 10.1161/CIRCULATIONAHA.105.555623

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Recently, it has been demonstrated that the mode ofinterdomain interaction of 2 specific domains within theRyR2 (N-terminal and central domains), which contain sev-eral mutations reported in arrhythmogenic right ventricularcardiomyopathy type 2 (ARVD/C2) and polymorphic ven-tricular tachycardia,7–9 plays a key role in Ca2� channelregulation and in the pathogenesis of heart failure. In normaloperation, the interaction of these domains is tight (zipped),stabilizing the closed state of the channel; on stimulation ofthe RyR2, the interdomain interaction becomes loose (un-zipped), and the channel opens.10 In diseased conditions, amutation in either of these domains weakens the interdomaininteraction even in resting or nonactivating conditions, anddiseased channels remain partially open. As shown in ourrecent study of the pacing-induced heart failure model,11

abnormal interdomain interaction as well as dissociation ofthe RyR2-bound FKBP12.6 destabilizes the channel gating ofthe RyR2 and then produces Ca2� leak, suggesting that theweakened interdomain interaction is one of the key mecha-nisms underlying the pathogenesis of ARVD/C2, polymor-phic ventricular tachycardia, and heart failure.

It has been suggested that oxygen free radicals, producedby the reduction of oxygen during many cellular reactions,are involved in the pathogenesis of a variety of cardiovasculardiseases, including heart failure.12 Highly reactive free radi-cals have a severe effect on many cellular structures and on avariety of important cellular functions. Recently, it has beensuggested that RyR channel gating is regulated by the redoxstate and that oxidation or nitrosylation of the cysteineresidues in the RyR produces considerable changes in chan-nel function.13

Edaravone (3-methyl-1-phenyl-2-pyrazolin-5-one; MCI-186), a synthetic antioxidant, is a ubiquitously acting directfree radical scavenger. Because of its highly efficient functionof detoxifying the devastatingly reactive ·OH, edaravonedirectly neutralizes peroxyl radicals (LOO·).14 We hypothe-sized that in heart failure the production of oxygen freeradicals may affect the channel regulation of the RyR2 andcause abnormal contractile and relaxation function. If this isthe case, edaravone, because of its antioxidant effect, mayrestore normal channel gating in the failing RyR2, therebypreventing the development of heart failure.

In the present study we tested this hypothesis by investi-gating the effects of oxidative stress on several key factorsthat are likely involved in the pathogenesis, such as theinterdomain interaction and the RyR2-associated FKBP12.6,as described above. As shown here, the NO donor3-morpholinosydnonimine (SIN-1) produced, in the normalSR, the same type of dysfunctions as seen in the SR from thefailing heart (pacing-induced heart failure), such as destabi-lization of interdomain interaction, partial dissociation ofFKBP12.6, and Ca2� leak. Interestingly, the antioxidantedaravone reversed all 3 of these problems in pacing-inducedheart failure.

MethodsMaterialsFK506, edaravone, SEA0400, and JTV519 were provided by Fuji-sawa Pharmaceutical Co Ltd, Mitsubishi Pharma Corporation Co

Ltd, Taisho Pharmaceutical Co, Ltd, and Aetas Pharmaceutical CoLtd, respectively.

Animal PreparationIn beagle dogs weighing 10 to 13 kg, heart failure was induced by 28days of rapid ventricular (RV) pacing at 250 bpm (referred to as 4Wpacing) with the use of an externally programmable miniaturepacemaker (Medtronic Inc), and both left ventricular (LV) pressureand 2-dimensional echocardiograms were measured in the consciousstate, as described elsewhere.1,3,4 The long-term administration of theantioxidant edaravone (0.6 mg/kg per day IV) was immediatelyfollowed by the initiation of RV pacing. The care of the animals andthe protocols used were in accordance with guidelines of the AnimalEthics Committee of Yamaguchi University School of Medicine.

Preparation of SR VesiclesSR vesicles were prepared from dog LV, as described elsewhere.1,3,4

Ca2� Uptake and Leak AssaysCa2� uptake and Ca2� leak assays were performed as describedpreviously.1,3,4

[3H]Dihydro-FK506 and [3H]RyanodineBinding Assays[3H]Dihydro-FK506 and [3H]ryanodine (Dupont NEN) binding as-says were performed as described elsewhere.1,3,4

Peptides Used and Peptide SynthesisWe used a synthetic peptide corresponding to residues 2460 to 2495of the RyR2, DPc10, that includes 1 residue mutable in polymorphicventricular tachycardia, as described previously11,15: 2459GFCP-DHKAAMVLFLDRVYGIEVQDFLLHLLEVGFLP2494.

The peptide was synthesized on an Applied Biosystems model 431Asynthesizer with the use of Fmoc [N-(9-fluorenyl)methoxycarbonyl] asthe �-amino protecting group, as described previously.15

Site-Directed Fluorescence Labeling of the RyR2Specific fluorescence labeling of the RyR2 moiety of the SR wasperformed with the use of the cleavable hetero-bifunctional cross-linking reagent sulfosuccinimidyl 3-([2-(7-azido-4-methylcoumarin-3-acetamido)ethyl]dithio)propionate (SAED) from PIERCE, withDPc10 as a site-specific carrier, as described previously.11,16

Fluorescence Quenching of the MethylcoumarinAcetate Probe Attached to the DPc10 Binding SiteThe zipped and unzipped states of the interacting domains of theRyR2 were assessed by the fluorescence quench technique describedpreviously.11,16 The principle of the fluorescence quench assay ofdomain unzipping is that a large-size quencher BSA-QSY is inac-cessible to the attached methylcoumarin acetate in the zipped state,whereas it becomes accessible to the methylcoumarin acetate site inthe unzipped state. To form the quencher, QSY 7 carboxylic acid wasconjugated with BSA by incubating 5 mmol/L QSY 7 carboxylicacid with 0.5 mmol/L BSA in 20 mmol/L HEPES (pH 7.5) for 60minutes at 22°C in the dark. Unreacted QSY 7 carboxylic acid wasremoved by means of Sephadex G50 gel filtration. Fluorescencequenching by both QSY 7 BSA conjugate (a large-size quencher)and acrylamide (a small-size quencher) was performed by measuringsteady state fluorescence of labeled methylcoumarin acetate (exci-tation at 368 nm, emission at 455 nm) in the presence or absence ofthe quencher. The data were analyzed with the use of the Stern-Volmer equation.

Immunoblot AnalysisImmunoblot analyses for FKBP12.6, SR Ca2�-ATPase, and phos-pholamban (PLB) were performed as described elsewhere.1,3,4 Byemploying the method of Marx et al,2 we achieved coimmunopre-cipitation of FKBP12.6 from SR using anti-RyR2 antibody (Onco-gene Research Products) followed by immunoblotting with anti-

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FKBP12 (C-19) antibody (Santa Cruz Biotechnology). The relativephosphorylation level of RyR was determined by immunoblottingwith anti-phosphoRyR2 (P2809), which was kindly provided by DrAndrew R. Marks (Columbia University). Specific antibodies againstphosphoserine 16-phospholamban (PLB; Upstate Biotech) and anepitope common to all PLB forms (PLB; Upstate Biotech) were alsoused.

Oxidative Stress Level in LV Muscle and RyR2To determine the oxidative stress level in the hearts, immunohisto-chemical analysis was performed with the use of mouse monoclonalanti–4-hydroxy-2-nonenal (HNE)–modified protein antibody (1:50dilution, NOF Medical Department), according to the method ofNakamura et al.17

The content of free thiols (the number of reduced cysteines) in thecanine RyR2 was determined with the use of the monobromobimane(mBB, Calbiochem) fluorescence technique.18,19 SR vesicles wereincubated with an excess concentration (250 �mol/L) of lipophilic,thiol-specific agent mBB for 1 hour in the dark at 24°C. Thefluorescence emission intensity of the RyR2-bound mBB (ie, thethiol content) was then measured at 482 nm by exciting at 382 nm

(Perkin-Elmer luminescence spectrophotometer LS50B). As a refer-ence, matched experiments were performed in the presence of 100�mol/L dithiothreitol (DTT).19 The mBB fluorescence in the RyR2,expressed as a percentage of the reference, was defined as therelative content of free thiols in the RyR2.

In canine cardiac myocytes, a fluorescent probe, 2�,7�-dichlorofluorescin diacetate (DCFH-DA; Molecular Probes), wasused for the assessment of intracellular reactive oxygen species(ROS) formation.20 This assay is widely used as a reliable methodfor the measurement of intracellular ROS such as hydrogen peroxide(H2O2), hydroxyl radicals (OH·), and hydroperoxides (ROOH).Fluorescence images (excitation at 490 nm and emission at 530 nm)were acquired with a microscope (LSM 510 Carl Zeiss).

Ca2� Transient and Cell Shortening inDog MyocytesMyocyte shortening and intracellular calcium were measured asdescribed previously.11 In brief, myocytes were incubated with 1�mol/L fura 2-AM, 0.0045% Pluronic F-127 (Sigma), and 0.1%dimethyl sulfoxide for 30 minutes, then washed twice with HEPES

Figure 1. Effects of edaravone on car-diac function and neurohormonal factors.A, Representative M-mode echocardio-gram. Note that both LV end-diastolicand LV end-systolic diameters weresmaller in edaravone-treated dog than inedaravone-untreated dog. B, Plasmanorepinephrine, angiotensin II, and atrialnatriuretic peptide (ANP) levels beforeand after RV pacing for 4 weeks with orwithout edaravone treatment. EV(�) indi-cates edaravone-untreated 4-weekpaced group; EV(�), edaravone-treated4-week paced group.

TABLE 1. Hemodynamic Data

HR,bpm

LVPSP,mm Hg

LVEDP,mm Hg

�dP/dt,mm Hg/s

Tau,ms

LVEDD,mm

LVESD,mm

LVFS,%

Edaravone-untreated (n�7 )

Prepacing 122�9 124�16 8.5�2.5 3008�190 18.2�3.8 30.8�1.2 19.2�1.1 38.0�2.1

4w pacing 118�15 116�22 35.2�2.8* 1358�214* 39.9�8.6* 40.9�2.0* 36.8�2.8* 12.2�3.3*

Edaravone-treated (n�5)

Prepacing 132�16 124�15 7.4�2.1 3256�262 17.6�4.3 30.4�1.4 19.3�1.6 38.2�3.3

4w pacing 130�10 112�16 20.2�6.0*† 1825�215*† 26.2�3.5*‡ 35.4�1.5*‡ 27.1�1.5*‡ 20.2�2.5*‡

HR indicates heart rate; LVPSP, LV peak-systolic pressure; LVEDP, LV end-diastolic pressure; �dP/dt, peak �dP/dt of LV pressure; Tau, time constant of LVpressure decay during isovolumic relaxation period; LVEDD, LVend-diastolic diameter; LVESD, LV end-systolic diameter, and FS, fractional shortening:(LVEDD�LVESD)/LVEDD�100. Data are mean�SD.

*P�0.01 vs prepacing group; †P�0.05, ‡P�0.01 vs edaravone-untreated group.

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buffer containing (in mmol/L) NaCl 126, KCl 4.4, MgCl2 1.0, CaCl2

1.08, HEPES 24, glucose 11, NaOH 13, and probenecid 0.5 (pH 7.4).Cells were stimulated by a field electric stimulator (IonOptix) at 1.0Hz. A dual-excitation spectrofluorometer was used to record fluo-rescence emissions (505 nm) elicited from exciting wavelengths at340 and 380 nm. The intracellular calcium was monitored as the ratioof fluorescence of the cell at 360 and 380 nm excitation.

Statistical AnalysisPaired t tests were performed for before and after comparisons ofhemodynamic data. ANOVA was used to compare data groups.When we identified a significant trend by the F test, we used theScheffé post hoc test to compare the data. For comparison ofmultiple measurements, repeated-measures analysis was used. Dataare expressed as mean�SD. We accepted a probability value of�0.05 as statistically significant.

ResultsDetermination of an Appropriate Concentration ofEdaravone for Long-term AdministrationTo determine appropriate concentrations of edaravone forlong-term administration, we examined the concentration-dependent effect of edaravone on hemodynamic parametersin normal dogs. Various amounts of edaravone were admin-istered intravenously for 3 days for each dose, starting at 0.1mg/kg/day with gradual increments to 1.2 mg/kg/day. At adose of �0.9 mg/kg/day, both LV pressure and the peak�dP/dt of LV pressure tended to decrease, while the dogswere kept in a conscious state. Therefore, we used a dose of0.6 mg/kg/day for long-term administration of edaravone inthis study to circumvent the acute hemodynamic effect ofedaravone.

Hemodynamic and Humoral DataIn the edaravone-treated (EV�) dogs with long-term RVpacing, both systolic and diastolic functions were largelypreserved, and none of these dogs developed heart failure(Table 1, Figure 1A). Plasma norepinephrine, angiotensin II,and atrial natriuretic peptide were all higher in the edaravone-untreated paced (EV�) dogs than in normal dogs (beforepacing). Long-term administration of edaravone significantlyreduced the levels of all of these neurohormonal factors in thepaced dogs (Figure 1B). These data indicate that in theedaravone-treated dogs, there were no signs of heart failureafter long-term RV pacing.

Levels of Oxidative Stress Are Elevated inFailing HeartsTo examine the extent of oxidative stress in the pacing-induced failing hearts, we determined the levels of HNE-modified protein in cardiac tissue. Positive immunohisto-chemical staining for HNE-modified protein was distinct inthe failing cardiac myocytes (Figure 2A, HNE�, EV�).However, in the edaravone-treated paced hearts, positiveimmunohistochemical staining for HNE-modified proteinwas absent (Figure 2A, HNE�, EV�). The relative contentof free thiols in the RyR2 (for definition, see Methods) wasconsiderably reduced in the untreated failing hearts (EV�)but was normal in EV� hearts (Figure 2B), indicating that theoxidation of the RyR2 is in fact involved in the oxidativestress in failing hearts. After treatment with the NO donor

SIN-1, which generates the NO-related species peroxynitrite(OONO�),19 approximately half of the free thiols remainedunreactive (not oxidized) in all groups, consistent with thefinding seen in the RyR1.21

Effects of Edaravone on SR Ca2� Leak and DefectiveFKBP12.6-RyR2 Interaction in Failing HeartsAddition of 0.3 �mol/L thapsigargin to normal SR vesiclesproduced little Ca2� leak, whereas addition of 30 �mol/LFK506 together with 1 �mol/L thapsigargin produced apronounced leak (Figure 3A). In contrast, in the failing(EV�) SR vesicles, the addition of thapsigargin alone pro-duced a prominent Ca2� leak, but the addition of FK506produced no further increase (Figure 3A). In the EV� SRvesicles, spontaneous Ca2� leak was not observed, and FK506increased Ca2� leak as in the normal SR (Figure 3A).

Figure 2. A, Immunohistochemical examination of HNE-modifiedproteins in normal and failing hearts. There is no positive stain-ing in normal myocytes, but positive (brown) staining seen infailing hearts. B, mBB fluorescence intensity of the RyR2 (corre-sponding to content of free thiols) in presence or absence of300 �mol/L SIN-1. Relative content of free thiols in RyR2expressed as percentage of fluorescence intensity of mBBreacted with 100 �mol/L DTT (bottom).




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In the EV� SR vesicles, the RyR2 was PKA-hyperphos-phorylated, but edaravone treatment reduced the channelphosphorylation to a level seen in the normal hearts (Figure3B). The amount of the RyR2-associated FKBP12.6 wasdecreased by long-term RV pacing, but the decrease wasprevented by edaravone treatment (Figure 3C).

Effects of Oxidative Stress on Ca2� leak,Interdomain Interaction Within RyR2, andFKBP12.6-RyR2 InteractionTo further test the view that oxidative stress of the RyR2 isthe cause of the Ca2� leak, we investigated the effect of SIN-1on the Ca2� flux in normal SR. As shown in Figure 4A, 30�mol/L SIN-1 increased the Ca2� leak, and this Ca2� leak wascompletely inhibited by 1 �mol/L JTV519, which had beenfound to inhibit the Ca2� leak through the stabilization of theRyR23 via restoration of an interdomain interaction within theRyR2.11

To monitor the zipped and unzipped states of the interact-ing domains of the RyR2, we used QSY-BSA as a macro-molecular quencher (Figure 4B). As in the case of our recentstudy with DPc10,11 the slope of the Stern-Volmer plot,which represents KQ or the extent of domain unzipping, wasconsiderably increased by SIN-1 (30 �mol/L), indicating thatSIN-1 in fact induced a sizable opening between the inter-

acting domains (Figure 4B, top). The SIN-1–induced increasein the extent of fluorescence quenching was almost com-pletely reversed by 1 �mol/L JTV519 (Figure 4B, top). Thisis particularly important in view of our recent finding11 thatJTV519 stabilizes the zipped configuration of the interactingdomains and thus prevents Ca2� leak. The extent of fluores-cence quenching (KQ) in the EV� SR vesicles was larger thanin normal SR (compare Reference 11), and the addition ofSIN-1 had no appreciable effect on KQ (Figure 4B, middle).However, JTV519 reduced the KQ in failing SR to a normalvalue even in the presence of SIN-1 (Figure 4B, middle). InEV� vesicles, KQ values were normally restored. Theseresults suggest that domain unzipping had already occurred infailing SR partly by oxidative stress, causing Ca2� leak, andthat JTV519 restored the zipped state and in turn normalchannel gating.

To further resolve the mechanism by which the edara-vone treatment decreased the phosphorylation level of theRyR2 and inhibited FKBP12.6 dissociation from theRyR2, we investigated the effect of oxidative stress on theamount of FKBP12.6 bound with the RyR2. As seen inFigure 4C, addition of SIN-1 had no appreciable effect onthe PKA phosphorylation level of the RyR2 or onFKBP12.6 binding to the RyR2 if cAMP was absent.However, in the presence of SIN-1 (30 �mol/L), the cAMP

Figure 3. A, Representative time coursesof Ca2� uptake and ensuing Ca2� leakfrom SR vesicles (with no cAMP; left).Note in edaravone-treated group, spon-taneous Ca2� leak disappeared andFK506-induced Ca2� leak reappeared asin normal group. Spontaneous andFK506-induced Ca2� leaks in normal,edaravone-untreated, and edaravone-treated SR vesicles are shown (right). B,PKA-mediated phosphorylation of RyR2confirmed by back-phosphorylation.Nonspecific phosphorylation (not inhib-ited by PKA inhibitor [PKI]) was sub-tracted, and resulting value divided byamount of RyR2 protein (determined byimmunoblotting and densitometry) andexpressed as specific PKA-dependent�-32P incorporation �SD. IP indicatesimmunoprecipitation. C, Amount ofRyR2-bound FKBP12.6.




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concentration dependence of both PKA phosphorylation ofthe RyR2 and FKBP12.6 dissociation was shifted towardlower concentrations of cAMP. These results suggest thatdomain unzipping caused by oxidative stress produceslarger extents of PKA phosphorylation of the RyR2 anddissociation of FKBP12.6 from the RyR2 in the presenceof cAMP at micromolar concentrations, which are presum-ably present in physiological conditions.

Figure 4D shows the effect of SIN-1 on the relative contentof free thiols in the RyR2. SIN-1 decreased the number offree thiols of the RyR2 (ie, oxidation). Unlike edaravone,JTV519 did not rescue the oxidative stress of the RyR2induced by SIN-1 (data not shown), indicating that theinhibition of Ca2� leak by JTV519 is mediated by a mecha-nism independent of the RyR2 oxidation.

Effects of Edaravone on the Amount of SRCa2�-ATPase, Rate of Ca2� Uptake, and Ratio ofSer16-Phosphorylated PLB and Total PLB inFailing HeartsAfter rapid RV pacing for 4 weeks, both the SR Ca2� uptakeactivity and the amount of SR Ca2�-ATPase were reduced, andthese changes were not observed in the EV� group (Figure 5A,B). The levels of Ser16-phosphorylated PLB and total PLBamong these groups of SR vesicles are compared in Figure 5C(top: gel picture; bottom: calculated values of relative phosphor-ylation). There was no difference in the level of total PLBamong the 3 groups, but there was a significant decrease in thebasal level of phosphorylated PLB in the failing EV� SRvesicles. In the EV� SR vesicles, the level of phosphorylatedPLB was restored back toward a normal level.

Figure 4. A, Representative time coursesof Ca2� uptake and Ca2� leak from SRvesicles in presence of 30 �mol/L SIN-1or 1 �mol/L JTV519 together with SIN-1(with no cAMP). B, Stern-Volmer plots offluorescence quenching data with QSY-BSA. Top and middle figures show effectof SIN-1 (30 �mol/L; with no cAMP) inpresence or absence of 1 �mol/LJTV519 in normal and failing SR, respec-tively. Bottom figure shows Stern-Volmerplots of QSY-BSA fluorescence quench-ing data of normal, failing (edaravone-untreated), and edaravone-treatedgroups. C, Effect of cAMP on level PKA-dependent phosphorylation of RyR2 andFKBP12.6 dissociation in presence orabsence of 30 �mol/L SIN-1. Beforeimmunoprecipitation of RyR2, SR vesi-cles were mixed with SIN-1 for 30 minand centrifuged, followed by Westernblotting. D, Fluorescence intensity ofmBB reacted with RyR2 (correspondingto content of free thiols) after treatmentwith various concentrations of SIN-1.Relative content of free thiols in RyR2was expressed as percentage of fluores-cence of mBB reacted with 100 �mol/LDTT (bottom).




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Effects of Edaravone or JTV 519 on Ca2�

Transient and Cell Shortening in Normal andFailing MyocytesWe further assessed whether the adverse effects of oxida-tive stress on SR Ca2� release function and its reversal byJTV519 seen in SR vesicles can be seen in caninemyocytes as well. In normal myocytes, SIN-1 decreasedthe peak of Ca2� transient and prolonged its duration inparallel with a reduced percent cell shortening (Figure 6A).Addition of either JTV519 (1 �mol/L) or edaravone (100�mol/L) resulted in a significant, if not complete, restora-tion of both Ca2� transient and cell shortening to normallevels (Figure 6A). Several reports have suggested thatROS induces intracellular Ca2� overload through activa-tion of a reversed mode of Na�-Ca2� exchange duringreoxygenation.22,23 Therefore, we examined the effect ofthe specific inhibitor of Na�-Ca2� exchange, SEA0400,24

on normal myocyte function. SEA0400 (1 �mol/L) had noappreciable effect on cell shortening and peak Ca2� tran-sient at baseline. In the presence of SEA0400, SIN-1decreased the peak of Ca2� transient and prolonged itsduration in parallel with a reduced percent cell shorteningto an extent similar to those in the absence of SEA0400(Figure 6A). This finding excludes the possibility that theNa�-Ca2� exchange may have been involved in the ob-

served effects of SIN-1 on normal cardiomyocytes, al-though other radicals may influence calcium influxthrough reverse-mode Na�-Ca2� exchange activity. Asshown in Figure 6B, Ca2� transient and cell shortening inthe myocytes isolated from pacing-induced failing doghearts were deteriorated compared with those in normalmyocytes. In the presence of isoproterenol, both Ca2�

transient and cell shortening were partially restored towardnormal by incubation with edaravone (100 �mol/L) for 12hours and also with JTV519 (1 �mol/L), which reversesdomain unzipping.11 Table 2 summarizes the cell shorten-ing and Ca2� transient data in normal and failing myocytes.Thus, all the data obtained from the experiments with SRvesicles and myocytes consistently support the notion thatoxidative stress–induced cardiac dysfunction is induced bydefective interdomain interaction in the RyR2 and that itsreversal either directly by JTV519 or indirectly by edara-vone restores normal cardiac function.

Figure 6C shows fluorescence images after application ofthe fluorescent probe of intracellular ROS, DCFH-DA (1�mol/L), into the normal and failing cardiomyocytes. Innormal cardiomyocytes, fluorescence intensity was markedlyincreased after addition of 100 �mol/L H2O2 or 30 �mol/LSIN-1, whereas it was restored to a normal level in thepresence of 100 �mol/L edaravone. In failing cardiomyo-

Figure 5. A, Representative Western blot of SRCa2�ATPase (top) and densitometric analysis ofWestern blot (bottom). B, Rate of Ca2� uptake. C,Representative Western blot of Ser16-phosphory-lated PLB (p-PLB) and total PLB (t-PLB; top) anddensitometric analysis of Western blot (bottom).p-PLB values (sum of pentamer and monomer, inarbitrary units) normalized with respect to t-PLBvalues (sum of pentamer and monomer, in arbitraryunits). Data presented as mean�SD.




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cytes, fluorescence intensity had already been increased to ahigh level, but a normal level of fluorescence intensity wasrestored by application of 100 �mol/L edaravone (Figure 6C)but not by 1 �mol/L JTV519 (data not shown). Thesefindings indicate that the ROS level is indeed increased infailing myocytes, and it can be reversed by the antioxidantedaravone.

DiscussionMany reports suggested that oxidative stress caused byoxygen free radicals is in fact involved in the pathogenesis ofheart failure. Several reports25,26 have shown that there is anincreased oxygen-derived free radical production in heartfailure, and it can be reversed by �-blocker therapy. Recentreports also showed that the RyR is the target of ROS and thatthe channel activity of the RyR is regulated by oxidation ornitrosylation.13 S-Nitrosylation of the RyR2 may have aphysiological significance in the normal heart, as suggestedby several reports.27,28 However, oxidative stress often resultsin a deleterious loss of normal cardiac function, leading toheart failure.13

The most important aspect of this study is the findingthat the pacing-induced heart failure and the oxidativestress–induced contractile dysfunction share at least somecommon causative molecular mechanisms, namely, defec-tive interdomain interaction accompanied by a decrease inthe amount of the RyR2-bound FKBP12.6 and an increasein the extent of phosphorylation. This conclusion is sup-ported by several findings made in the present study. First,administration of the antioxidant edaravone during thecourse of RV pacing prevented the development of heartfailure, as shown consistently in the hemodynamic, hu-moral, and immunohistochemical data. The treatment ofthe SR isolated from normal heart with the oxidant SIN-1resulted in a considerable reduction in the content ofreactive thiols in the RyR2 moiety. In the SR from failinghearts subjected to the RV pacing, the content of reactivethiols was considerably reduced, indicating that oxidativestress has occurred within the RyR2; however, the thiolcontent in the SR from the edaravone-treated hearts wasessentially identical with that of the normal control evenafter RV pacing for 4 weeks. The SIN-1–induced oxidative

Figure 6. Cell shortening and corresponding Ca2� transient in myocytes. A, Dose-dependent effect of SIN-1 in absence or presence ofJTV519 (1 �mol/L), edaravone (100 �mol/L), or SEA0400 (1 �mol/L) in normal myocytes. B, Myocyte response to isoproterenol (50nmol/L) in normal, failing (edaravone-untreated), and edaravone-treated groups. C, DCFH-DA fluorescence images in normal and failingmyocytes. In normal myocytes, H2O2 (100 �mol/L) or SIN-1 (30 �mol/L) was added in presence or absence of edaravone (100 �mol/L).In failing myocytes, edaravone (100 �mol/L) added. Confocal microscopy clearly detects DCFH-DA fluorescence signal, indicated asgreen, both in failing myocytes and in normal myocytes in presence of H2O2 or SIN-1. Cell surface membrane fluorescently labeled asred by wheat germ agglutinin–Alexa Fluor 633 conjugate.




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stress within the RyR2 of normal SR resulted in anincreased extent of domain unzipping (defective interdo-main interaction). Likewise, the RV pacing resulted in thedefective interdomain interaction even without the treat-ment with SIN-1, but edaravone administration during thepacing prevented the defective interdomain interactionfrom occurring. SR Ca2� leak was increased by SIN-1–mediated oxidation of normal SR or RV pacing. The SRCa2� leak produced by RV pacing was prevented byadministration of edaravone during the pacing. Finally,these observations with the isolated SR (SR Ca2� leakinduced by RV pacing or oxidative stress) were reproducedin similar abnormal features of cell shortening and Ca2�

transients of the cardiac myocytes.The next important question concerns the molecular mech-

anism underlying the pathogenesis of the in vivo pacing-induced heart failure and the oxidative stress–induced con-tractile dysfunction in myocytes. As shown in this study, RVpacing as well as oxidation of the RyR2 with SIN-1 weak-ened the interdomain interaction (ie, domain unzipping).Importantly, the RV pacing–induced problem in the interdo-main interaction was corrected not only by JTV519, whichalso corrected SR Ca2� leak in the RV-paced heart, but alsoby edaravone, which corrected the leak problem in the heartsubjected to oxidative stress. Similarly, the SIN-1–inducedproblem in the interdomain interaction was corrected not onlyby the antioxidant edaravone but also by JTV519. Thisclearly indicates that the defect in the mode of interdomaininteraction is the source mechanism of pathogenesis of bothtypes (ie, RV pacing and oxidation) of failing hearts.

According to Baker et al,29 the RyR1 has anoxidoreductase-like domain in the N-terminal region 41 to

420, which may function as a redox sensor. This regioncorresponds to the N-terminal domain, one of the key domainsinvolved in the interdomain interaction. Voss et al30 identifiedseveral thiols of the RyR1 that are highly reactive to thethiol-directed fluorescence reagent 7-diethylamino-3-(4�-maleimidylphenyl)-4-methylcoumarin (hyperreactive thiol[s]).Interestingly, more than half of the corresponding Cys residuesin the RyR2 (2403, 2532, 2573, 2578) are clustered in the regionthat is partially overlapping with the central domain, another keydomain involved in the interdomain interaction. It seems verylikely that the interdomain interaction is under the control ofredox-sensing mechanisms built into these domains. There are anumber of reports of oxidation-induced activation of the RyRCa2� channels with a variety of oxidative reagents.13,19,20,31 Thepresent data suggest that the oxidation-induced domain unzip-ping might be a basic mechanism for the widely observedoxidation-induced SR Ca2� release. Apart from oxidation, spe-cific nitrosylation of Cys-3635, which was found to modifyRyR1 channel activity,32 may also be involved in the defectiveRyR2 channel gating in heart failure.

In the dog model of heart failure produced by 4W pacing,the defective interdomain interaction is accompanied by 2other major problems: reduction in the amount of the RyR2-bound FKBP12.6 and an increase in the level of cAMP-dependent phosphorylation of the RyR2.11 It is rather difficultto delineate the sequence of the occurrence of these eventsbecause these 3 problems appear to occur in a coordinatedmanner. The fact that, in the presence of cAMP, SIN-1increased PKA phosphorylation and FKBP12.6 dissociationsuggests that phosphorylation and FKBP12.6 dissociationaccelerate the SIN-1–induced domain unzipping, and domainunzipping accelerates phosphorylation and FKBP12.6 disso-

TABLE 2. In Vivo Cell Shortening and Ca2� Transient in Normal and Failing Myocytes

Normal 4W Pacing

Untreated(n�30)

�JTV519(n�30)

�Edaravone(n�30)

�SEA0400(n�10)

EV�(n�30)

EV�(n�30)

Cell shortening, % decrease from baseline

SIN-1� 8.9�2.1 9.3�2.4 9.2�2.2 9.4�2.5 2.0�0.8§ 4.7�1.1‡

SIN-1 30 �mol/L 5.2�1.8* 8.2�2.5‡ 8.7�1.9‡ 4.4�0.7* � � � � � �

SIN-1 100 �mol/L 2.1�0.9† 6.4�2.2*§ 6.9�2.6*§ 2.6�0.6† � � � � � �

Isoproterenol 50 nmol/L 14.8�4.5† � � � � � � � � � 2.5�1.1§ 8.9�2.6*‡

Peak of Ca2� transient, % increase from baseline

SIN-1� 33.0�3.4 31.3�4.0 33.2�4.1 31.6�2.6 16.7�4.3‡ 25.9�4.6‡

SIN-1 30 �mol/L 26.5�3.1* 28.7�3.8 32.7�4.3 27.6�1.9* � � � � � �

SIN-1 100 �mol/L 14.4�3.7† 25.8�3.1*‡ 27.8�3.9*‡ 14.6�1.7† � � � � � �

Isoproterenol 50 nmol/L 58.4�15.8† � � � � � � � � � 22.8�2.5‡ 39.7�14.1*‡

Time from peak to70% decline, ms

SIN-1� 90.6-�10.6 90.0�22.4 88.9�7.7 92.1�14.5 118.6�13.5† 98.6�8.3

SIN-1 30 �mol/L 109.1�2.1* 97.1�14.5 92.1�6.8 112.0�5.6* � � � � � �

SIN-1 100 �mol/L 115.3�6.4† 108.0�6.4* 93.7�10.3‡ 120.6�4.5† � � � � � �

Isoproterenol 50 nmol/L 56.9�5.7† � � � � � � � � � 110.7�15.8§ 58.2�8.9†

*P�0.05, †P�0.01 vs SIN-1�.‡P�0.05, §P�0.01 vs untreated normal myocytes.




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ciation, letting these 3 events occur in a concerted manner.Thus, like JTV519, the antioxidant edaravone corrected all ofthese problems in a concerted manner.

In addition to the effect of edaravone on the mode ofinterdomain interaction, we should consider various otherpharmacological functions of this drug. Long-term admin-istration of edaravone improved the Ca2� uptake function,SERCA2a expression, and PLB phosphorylation. Theseeffects appear to be similar to that of �-blockers or theangiotensin II receptor antagonist valsartan, both of whichreduce sympathetic activity and thus prevent Ca2� leak.3,33

However, because edaravone does not have a �-blockingeffect by itself, the beneficial effect of edaravone on Ca2�

uptake may be relevant to the inhibitory effect on intra-cellular Ca2� overload. The inhibition of LV dilatation byedaravone may be attributable to other possible mecha-nisms by which antioxidants prevent the progression ofheart failure. Because improvement of LV function afteradministration of edaravone may decrease the sympatheticactivity as a secondary effect, it would be rather difficult toevaluate the specific effect of edaravone as distinct fromthe catecholamine effect.

In conclusion, administration of the antioxidant edaravoneduring RV pacing of the canine heart prevented the develop-ment of heart failure by correcting several problems occur-ring in the RyR2 moiety of the SR of failing hearts, such asdefective interaction of the regulatory domains in the RyR2,partial dissociation of RKBP12.6, PKA hyperphosphoryla-tion, and Ca2� leak. Oxidation of the cysteine residues of theRyR2 with SIN-1 destabilized the interdomain interaction,mimicking the situation in the failing SR, but on treatment ofthe SR with edaravone the normal mode of interdomaininteraction was restored. The present study suggests that theuse of free radical scavengers will be a promising therapeuticstrategy against heart failure.

AcknowledgmentsThis work was supported by grants-in-aid for scientific research fromthe Ministry of Education in Japan (grants 16590689, 16209026,14370228 to Drs Yano and Matsuzaki), by the Takeda ScienceFoundation (to Dr Yano), and by National Institutes of Health grantsHL072841 and AR16922 (to Dr Ikemoto).

DisclosuresThe authors report no conflicts of interest.

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Ikeda, Tomoko Ohkusa, Noriaki Ikemoto and Masunori MatsuzakiMochizuki, Toshiyuki Noma, Masahiro Doi, Shigeki Kobayashi, Takeshi Yamamoto, Yasuhiro Masafumi Yano, Shinichi Okuda, Tetsuro Oda, Takahiro Tokuhisa, Hiroki Tateishi, Mamoru

Antioxidant Is a New Therapeutic Strategy Against Heart FailureCorrection of Defective Interdomain Interaction Within Ryanodine Receptor by

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