Ryanodine receptor = in heart failure: potential therapeutic sites Guy Vassort

From: Subject: Ryanodine receptor in heart failure: potential therapeutic sites Guy Vassort Date: Thu, 3 Nov 2011 10:29:36 -0600 MIME-Version: 1.0 Content-Type: text/html; charset="iso-8859-1" Content-Transfer-Encoding: quoted-printable Content-Location: http://bvs.sld.cu/revistas/car/vol16_1_10/car05110.htm X-MimeOLE: Produced By Microsoft MimeOLE V6.00.2900.2180 Ryanodine receptor = in heart failure: potential therapeutic sites Guy Vassort

Rev Cubana Cardiol Cir Cardiovasc 2010;16(1):25-40=20

INSUFICIENCIA CARD=CDACA

Ryanodine receptor in heart failure: potential therapeutic = sites=20

Guy = Vassort=20

Laboratoire de=20 Physiopathologie Cardiovasculaire, INSERM U-637, CHU Arnaud de = Villeneuve,=20 Montpellier, France

ABSTRACT=20

Heart = failure (HF)=20 is one of the most important contributors to morbidity and mortality = from=20 cardiac diseases. Ca²⁺ handling abnormalities play an = important role=20 in the development of HF. The ryanodine receptor, RyR2 is a large = tetrameric=20 protein associated with FK506-binding immunophilin protein, FKBP12.6 or=20 calstabin2 and many kinases and phosphatases to form a huge complex. = FKBP12.6 is=20 thought to maintain the RyR2 in a closed state. Hyperphosphorylated = state of=20 RyR2 and FKBP12.6 dissociation occur in heart failure. RyR2 leakage may = not=20 necessarily depend upon phosphorylation. Other mechanisms include = unzipping of=20 the N-terminal and central domains of RyR2, and abnormal sensing of the=20 sarcoplasmic reticulum (SR) Ca²⁺ load. The = 1,4-benzothiazepine=20 derivative JTV519/K201 has significant beneficial contractile effect in = HF.=20 Several models are proposed to account for its restoring effects. Heart = failure=20 is also accompanied by increased oxidative stress and NOsynthase = activity that=20 both alter RyR2 properties and lead to RyR2 leak. SR-Ca²⁺ = release can=20 be antagonized by reducing agents and by S107, a K201-analog. Abnormal=20 SR-Ca²⁺ release during diastole also results in changes in = membrane=20 potential and the triggering of ventricular arrhythmia. Besides the = above=20 alterations in RyR2 properties, multiple mutations occurring in RyR2 = protein are=20 linked to catecholaminergic polymorphic ventricular tachycardia. The=20 anti-arrhythmic properties of K201 and its mechanisms of action remain=20 controversial. Controlling the interactions RyR2 - FKBP12.6 could be an=20 important candidate target for pharmaceutical management of cardiac=20 insufficiency and ventricular arrhythmia during HF.

Key words: Heart failure, = ryanodine=20 receptor.

RESUMEN=20

La = insuficiencia=20 card=EDaca (IC) contribuye de manera importante a la morbilidad y = mortalidad por=20 enfermedades card=EDacas. Las anomal=EDas en el manejo del = Ca²⁺ tienen un=20 papel preponderante en el desarrollo de la IC. El receptor a la = ryanodina, RyR2=20 es una prote=EDna tetram=E9rica grande que se asocia a una prote=EDna = inmunofilina que=20 une al FK506, la FKBP12.6 o calstabina2, as=ED como a muchas kinasas y = fosfatasas=20 para formar un gigantesco complejo. Existe la idea de que la FKBP12.6 = mantiene=20 al RyR2 en un estado cerrado. El estado de hiperfosforilaci=F3n del RyR2 = y la=20 disociaci=F3n de la FKBP12.6 de este, ocurren en la insuficiencia = card=EDaca. La=20 fuga (de Ca²⁺) a trav=E9s del RyR2 no depende necesariamente = de la=20 fosforilaci=F3n. Otros mecanismos incluyen el despliegue del extremo = N-terminal y=20 los dominios centrales del RyR2 y sensaje anormal de la carga de = Ca²⁺=20 del ret=EDculo sarcoplasm=E1tico (RS). El derivado de benzotiazepina = JTV519/K201=20 tiene efectos contr=E1ctiles beneficiosos en la IC. Se han propuesto = varios=20 modelos para dar cuenta de sus efectos restauradores. La insuficiencia = card=EDaca=20 se acompa=F1a tambi=E9n de un incremento en el stress oxidativo y = de la=20 actividad de la NOS-sintetasa, ambos alteran las propiedades del RyR2 y = llevan a=20 una p=E9rdida (de Ca²⁺) desde el RyR2. La liberaci=F3n de = Ca²⁺=20 del RS puede ser antagonizada por agentes reductores y por el S107, un = an=E1logo=20 del K201. La liberaci=F3n an=F3mala de Ca²⁺ del RS durante la = di=E1 puede=20 resultar tambi=E9n en cambios en el potencial de membrana y disparar = arritmias=20 ventriculares. Adem=E1s de las alteraciones antes descritas en las = propiedades del=20 RyR2, existen m=FAltiples mutaciones que ocurren en la prote=EDna del = RyR2 y est=E1n=20 vinculadas a la taquicardia ventricular polim=F3rfica = catecolamin=E9rgica. Las=20 propiedades antiarr=EDtmicas del K201 y sus mecanismos de acci=F3n son = tema de=20 controversias. El control de las interacciones RyR2 - FKBP12.6 puede ser = candidato a un sitio blanco de importancia para el tratamiento = farmacol=F3gico de=20 la insuficiencia card=EDaca y de las arritmias ventriculares durante la = IC.=20

Palabras clave: Insuficiencia = card=EDaca,=20 receptor a la ryanodina.

INTRODUCTION

Heart = failure (HF)=20 is a leading cause of cardiovascular morbidity and mortality with about = half of=20 the deaths occurring suddenly, often following ventricular arrhythmia. = HF is=20 characterized by a disturbance of the normal pumping of blood to the = peripheral=20 organs to meet the metabolic demands of the body that is accompanied by=20 activation of neurohumoral factors.¹ Regardless of the = initial cause=20 of the myocardial damage (cardiomyopathy, myocardial ischemia, = hypertension...),=20 HF eventually develops when such damage persists for a prolonged period. = There=20 is a strong correlation between chronic activation of the sympathetic = nervous=20 system and poor prognosis of HF².

A great = body of=20 evidence has accumulated that indicated calcium-handling abnormalities = play an=20 important role in the development of HF. In many cases, altered = Ca²⁺=20 cycling precedes the observed depression of mechanical = performance.³=20 Because intracellular Ca²⁺concentration, = [Ca²⁺]_i=20directly regulates contractility of cardiomyocytes, a reduced=20 [Ca²⁺]_itransient amplitude in HF results in = decreased=20 force development. Furthermore, both the prolonged decay of the=20 [Ca²⁺]_itransient and the increased diastolic=20 [Ca²⁺]_i may contribute to slow relaxation and = reduce=20 refilling. Several defects in the [Ca²⁺]_{i =}homeostasis have=20 been reported including depressed Ca²⁺uptake, storage and = /or=20 release of Ca²⁺from the sarcoplasmic reticulum, SR. In = particular,=20 the abnormal Ca²⁺- release from the SR has been shown to = play a=20 critical role both in the weakened pump activity and in the triggering = of=20 ventricular arrhythmia. In this review, I shall focus on the underlying=20 mechanisms of disease-linked channel disorders of the ryanodine = receptor, RyR in=20 the pathogenesis of HF and on the possibility of developing new = therapeutic=20 strategies against HF using RyR as the target.

In the = normal=20 heart, intracellular Ca²⁺ movements critically regulate = mechanical=20 contractions. During the early plateau phase of the cardiac action = potential, a=20 small amount of Ca²⁺ first enters through the L-type = Ca²⁺=20 channel or dihydropyridine receptor, DHPR. This Ca²⁺ influx = triggers=20 a large-scale Ca²⁺ release from the SR through the = Ca²⁺=20 release channel, RyR. Close spatial coupling between DHPR and RyR = clusters and=20 the relative insensitivity of RyR to be triggered by Ca^{2+ =}together=20 ensure the stability of this positive-feedback system of Ca²⁺⁼²⁰amplification. This process referred as cardiac = excitation-contraction,=20 E-C coupling is characterized by a transient increase in = [Ca²⁺]_i=20from 100 nM to about 1 =ECM. For termination of SR-Ca²⁺ = release,=20 RyR adaptation, RyR inactivation, and SR-Ca²⁺ depletion may = play=20 important roles by acting in a synergistic manner. The released = Ca²⁺=20 then binds to the troponin C, which induces activation of the = myofilaments and a=20 consequent muscle contraction. Relaxation is initiated by dissociation = of=20 Ca²⁺ from troponin C, followed by its reuptake into the SR = through=20 Ca²⁺-ATPase, SERCA2a and subsequent trans-sarcolemmal = Ca²⁺=20 removal through the Na⁺/Ca²⁺ exchanger, NCX = operating in=20 its forward mode.⁴

The RyR = is a huge=20 tetrameric protein, and each monomer is constituted of ~5000 amino acids = and has=20 a molecular weight of 565 kDa. About 90 % of the RyR polypeptide = chain=20 forms a bulky cytoplasmic domain that modulates the channel function. = The=20 remaining 10 % of the RyR sequence (C-terminal region) forms = transmembrane and=20 channel pore regions. Three mammalian isoforms of RyR have been = identified. Of=20 these, RyR1 is found in skeletal muscle⁵ while RyR2 is = predominantly=20 expressed in cardiac muscle.⁶ RyR3 is ubiquitously expressed = at low=20 levels. RyR exists as a scaffolding protein bound with many accessory = proteins,=20 producing a huge macromolecular complex (Figure = 1). It=20 associates with FK506-binding immunophilin protein (FKBP; indeed = FKBP12.6, or=20 calstabin2 in heart), protein kinase A (PKA), protein phosphatases 1 = (PP1) and=20 2A (PP2A), calmodulin, calmodulin kinase II, CaMKII, and the = phosphodiesterase=20 PDE4D3. Both junctin and triadin, which anchor calsequestrin, bind to = the=20 luminal site of the RyR2. The RyRs are = closely=20 associated with DHPR and this spatial association of the two channels = forms a=20 key functional unit in cardiac E-C coupling.^{7, 8} In addition = to=20 Ca²⁺, endogenous effectors such as Mg²⁺, ATP, = reactive=20 oxygen, and nitrogen molecules regulate RyR2.

Phosphorylation of RYR2 by cAMP-dependent protein kinase and by = calmodulin kinase II

The = RyR2 release=20 channel is also regulated by evolutionarily highly conserved signaling = pathways=20 that control the E-C coupling in the heart. Phosphorylations of = RyR₂=20 at Ser2030 by cAMP-dependent protein kinase, PKA and at Ser2809 = (corresponding=20 to Ser2808 in human) by PKA and CaMKII have been described. According to = Marks's=20 group, RyR₂ phosphorylation at Ser2809 plays a key role in = regulating=20 the channel in response to stress following activation of the = sympathetic=20 nervous system, the "fly-or-fight"response.⁹However = phosphorylation=20 of RyR₂ may not correlate directly with cellular cAMP levels = since=20 the RyR2 is involved in a large macromolecular complex including PKA and = phosphatases. PKA phosphorylation potently modulates RyR2 function and = is=20 physiologically regulated in vivo. PKA hyperphosphorylation = caused by HF=20 after activation of the b-adrenergic cascade driven by the nervous = system=20 initially results in a compensatory response aimed at enhancing cardiac=20 contractility. Maladaptative response occurs along with sustained = activation.=20 Chronic activation desensitizes the cardiac b-adrenergic signaling = system, which=20 further contributes to reduced inotropic reserve and worsens remodeling = in HF as=20 part of the maladaptative response that ultimately fails to protect the = heart=20 and preserves cardiac mechanical function.

Dysregulation of=20 the RyR2 by hyperphosphorylation is characterized by improper control of = SR-Ca²⁺ release and diastolic Ca²⁺ leak. = Hyperactivation=20 of PKA, by causing an increased phosphorylation at Ser2809 of the RyR2, = induces=20 dissociation of the FKBP12.6 subunit from RyR2. This accessory protein, = FKBP12.6=20 is suggested to stabilize a closed state of the TyR2 channel and to = reduce RyR2=20 sensitivity to Ca²⁺. FKBP12.6 binds to RyR2with a = stoichiometric ratio of 1 FKBP12.6 to 1 RyR2 monomer, or 4 FKBP12.6 to = the RyR2=20 tetramer. Studies from both Marks's and Yano's groups^{9, 10}=20 demonstrated that altered stoichiometry between RyR2 and FKBP12.6 leads = to SR-=20 Ca²⁺leak and cardiac contractile dysfunction. In HF = long-term=20 hyperphosphorylation of RyR2 can be maintained through a reduction in = the=20 protein abundance of PP1 and PP2A, contributing to an increased S2808=20 phosphorylation state. Furthermore, a splice variant of the PDE4 family, = PDE4D3=20 containing an N-terminal targeting motif for mAKAP forms a = PKA-mAKAP-PDE4D3=20 signaling molecule with PDE4D3 contributing to the RyR2 complex. In = human HF,=20 PDE4D3 levels in the RyR2 complex were reduced by 43 %.¹¹ = Owing to a=20 partial loss of FKBP12.6 from RyR₂, in a lipid-bilayer = experiment=20 single-channel activity was found to be hypersensitized to = Ca²⁺, thus=20 expectedly causing in HF a diastolic Ca²⁺ leak at a = concentration of=20 Ca²⁺ (approximately 100 nM) at which no significant = Ca²⁺=20 release is induced in normal heart.⁹ The dissociation of = FKBP12.6=20 from RyR2 also functionally uncouples multiple RyR2 and disturbs both = the=20 simultaneous opening of RyR2 during systole and their simultaneous = closing=20 during diastole. Indeed in vivo, a diastolic Ca²⁺ leak = has=20 been reported in a rabbit model of myocardial infarction.¹² =

However = using the=20 same pacing-induced HF dog model, Jiang et al. obtained = conflicting=20 results with no change in RyR2/FKBP12.6 association or single channel=20 behavior.¹³ They attributed the depressed and slow Ca²⁺⁼²⁰transient characteristic of HF to abnormal Ca²⁺uptake = by a=20 depressed SERCA2a, more than to SR-Ca²⁺release. Several = other groups=20 have challenged the role of FKBP12.6 as a channel stabilizer in various=20 experimental conditions. Phosphorylation at Ser2808 did not dissociate = FKBP12.6=20 from RyR2,¹⁴ and the constitutive phosphorylation of Ser2808 = by=20 mutations (S2808D) failed to disrupt the FKBP12.6_RyR2 = interaction.¹⁵=20 Prolonged RyR2 phosphorylation via inhibition of protein phosphatase 1 = appears=20 cardioprotective.¹⁶ The significant role of PKA = phosphorylation of=20 another site (Ser-2030) on hypersensitized channel to activation by = luminal=20 Ca²⁺ in disease-linked RyR2 channel disorder has also been = stressed=20 out.¹⁷ As well it was pointed out the importance of the = extent of=20 RyR2 phosphorylation by PKA since 100 % RyR2 phosphorylation, but not 75 = %, is=20 required to increase the probability of RyR2 channel to be in the open=20 state.¹⁸ Besides Li et al.¹⁹ found = that the=20 onset of SR-Ca²⁺ leak by PKA phosphorylation is fully = dependent on=20 phospholamban phosphorylation and the subsequent increase in = SR-Ca²⁺=20 content rather than on RyR2 phosphorylation.

Several = investigators soon proposed that CaMKII- rather than PKA-dependent=20 phosphorylation was critical for RyR2-FKBP12.6 dissociation and the = consequent=20 diastolic Ca²⁺leak. Notably besides the originally reported=20 Ser2808/2809 (shared amino acids with a putative PKA site), CaMKII can=20 phosphorylate several other sites in RyR2 including Ser2815, resulting = in more=20 active RyR2 channels.^20,21 Thus it is proposed that CaMKII, = activated=20 by b-adrenergic stimulation is responsible for the SR-Ca^{2+ =}leak in=20 rabbit heart, instead of PKA activation. This is supported by the = increased=20 spark frequency and low diastolic Ca²⁺content observed in=20 CaMKII-transgenic mice.²² However, in the presence of = elevated=20 Ca²⁺, the multimeric CaMKII are autophosphorylated to become=20 constitutively active and exhibit multiple cellular phosphorylation = sites=20 including DHPR, phospholamban, nuclear sites, besides 8 potential sites = on RyR2.=20 This might account for the diversity of the reported effects. Thus = recently, it=20 was shown that over-expression of CaMKII suppresses Ca^{2+ =}sparks and=20 Ca²⁺waves in cultured rat cardiomyocytes thereby affording a = negative feedback that stabilizes local and global Ca^{2+ =}-induced=20 Ca²⁺release in the heart.²³

JTV519, = to-day=20 known as K201, is a 1,4-benzothiazepine derivative which has been found = to be=20 more effective than other Ca²⁺antagonist channels at = reducing=20 Ca²⁺- nduced myocardial damage. It was shown to improve=20 contractility and prevent the development of heart failure in paced dog = heart=20 probably by preventing dissociation of FKBP12.6 from RyR2.²⁴ = Thus,=20 JTV519 treatment increased calstabin binding to PKA-hyperphosphorylated = RyR2 and=20 cardiac function in dogs with pacing-induced HF²⁴ and as well = in=20 calstabin2^-/-- and PDE4^-/-- deficient = mice.^24,25=20However JTV519 exhibits multiple cellular effects including = inhibition of=20 SR-Ca²⁺ uptake^{26, 27} while during ischemia, the=20 JTV519-dependent attenuation of the large decrease in ATP content was = prevented=20 by NOsynthase inhibitors.²⁸

Regulation of the interdomain interaction within = RyR2

The = concept that=20 interactions between the N-terminal domainand the central = domain of=20 RyR1 are involved in Ca²⁺ channelregulation has = emerged=20 from recent domain peptide probestudies.^29,30=20 Accordingto this concept, in the resting or nonactivated = state, the=20 N-terminaland central domains make close contact at several=20 subdomains(domain zipping). The conformational constraints = imparted=20 bythe zipped configuration of these two domains stabilize = and=20 maintainthe closed state of Ca²⁺ channel. Some=20 mutationsof RyR2 reported in cardiac disease patients are = located=20 inthe regions corresponding to the skeletal N-terminal and=20 centraldomains harboring most of the malignant hyperthermia=20 mutationsthat cause an increased Ca²⁺ leak. = Knowing that=20 RyR1 and RyR2 shares about 60 % similarities, this suggests that RyR2=20 sharesa common domain-mediated channel regulation mechanism = with=20 RyR1and that the increased Ca²⁺ leak of diseased = RyR2=20 channels maybe explained by the altered mode of interdomain=20 interactions.

Stimulation via=20 E-Ccoupling or pharmacological agents weakens these critical = interdomaincontacts, resulting in loss of conformational = constraints=20 (domainunzipping) and thus lowering of the energy barrier = for=20 Ca²⁺channel opening. Weakening of these interdomain=20 interactionsmay also occur via mutation or with the use of = synthetic=20 domainpeptides. Thus, a cardiac domain peptide (DPc10)=20 correspondingto the Gly2460-Pro2495 region of the rabbit = RyR2=20 (equivalentto the Gly2459-Pro2494 region of the human RyR2) = was=20 found toproduce significant activation of the RyR2 channel,=20 especiallyat low Ca²⁺ concentrations (0.1 to 0.3 = =B5M). The=20 addition of DPc10 induced an unzipped state of the interacting = N-terminal and=20 central domains, as evidenced by the increase in accessibility of = labeled RyR2=20 to a fluorescence quencher.³¹ A singleArg-to-Ser = mutation=20 made in DPc10, mimicking the A2474Shuman mutation, abolished = all of=20 the effects that would havebeen produced by DPc10. These = data=20 presented strong evidencethat synthetic domain peptides=20 corresponding to key subdomainsof RyR2 are capable of = mimicking=20 diseased conditions of theRyR2 channel by interfering with = the=20 interdomain interaction.Interestingly, the binding region of = FKBP12.6 to RyR2, the residues sequence 2361 to 2496⁹ is = included in=20 the sequence of DPc10 (2460 to 2495). Thissuggests that = there is a=20 close mechanistic relationshipbetween the PKA-mediated = FKBP12.6=20 dissociation and abnormaldomain-domain interactions such as = that=20 seen in the DPc10-mediatedchannel hypersensitization. Thus = when=20 DPc10 was introduced, the Ca²⁺ transient decreased and its = time decay=20 was prolonged. Domain unzipping facilitated PKA phosphorylation and the=20 resultant FKBP12.6 dissociation in normal SR. In the absence of cAMP, = DPc10=20 caused domain unzipping and Ca²⁺leak in DPc10-introduced=20 cardiomyocytes without causing any changes in the level of = phosphorylation and=20 in the amount of the RyR2-FKBP12.6 complex. In pacing-induced dog = failing heart,=20 the domain unzipping has already occurred, together with FKBP12.6 = dissociation=20 and Ca²⁺ leak. K201 was proposed to reverse domain unzipping = and=20 reduce Ca²⁺leak.³¹ The K201-binding site was = recently=20 identified at domain 2114-2149 of RyR2 (Figure = 2).=20 K201 reverses the mode of interdomain interaction from defective = unzipped to=20 normal zipped configuration and stops Ca²⁺leak without = requiring=20 rebinding of FKBP12.6 to RyR2 in failing HF.³² The authors = also=20 demonstrated that the domain peptide, DP2114-2149, after intracellular=20 incorporation, was able to improved Ca²⁺ transient and cell=20 shortening in FK506-treated cardiomyocytes as well in cardiomyocytes = isolated=20 from paced-induced failing hearts. Thus in destabilized RyR2, K201 like=20 DP2114-2149 are able to bring back a normal zipped state. The authors = further=20 suggest that K201 primarily modifies domain-domain interaction and by=20 establishing a stable conformation it then favors FKBP12.6 binding to = RyR2=20 (Figure 2).

Oxidation and nitrosylation of RyR2

Besides = several=20 potential phosphorylation sites, the tetrameric RyR2 channel contains=20 approximately 84 free thiols and is S-nitrosylated in vivo.=20 S-Nitrosylation of up to 12 sites (3 per subunit) led to progressive = channel=20 activation that was reversed by denitrosylation.³³Thus = alterations=20 in phosphorylation status is not the only biochemical modification that = could=20 alter RyR2 in HF. HF is also accompanied by an increased oxidative = stress=20 resulting from formation of reactive oxygen species, ROS.³⁴ = Using=20 co-immunoprecipitation assays of solubilized native RyR2 from cardiac = muscle SR=20 with recombinant [³⁵S]FKBP12.6, it was found that the=20 sulfydryl-oxidizing agents, H₂O₂ and diamide, = result in=20 diminished RyR2-FKBP12.6 binding. Furthermore, a cysteine-null mutant = FKBP12.6=20 retained redox-sensitive interaction with RyR2, suggesting that the = effect of=20 the redox reagents is exclusively via sites on the ryanodine receptor. = In this=20 study, K201 did not restore normal FKBP binding under oxidizing=20 conditions.³⁵ Scavenging free radicals by low dose of = carvedilol, a=20 non-selective b-blocking agent with antioxidant properties, prevented=20 SR-Ca²⁺ leak and demonstrated in vivo beneficial = effects of=20 the cardiac function.³⁶ This could account for the beneficial = effects=20 of carvedilol over metoprolol in failing patients.³⁷

In a = canine model=20 of HF, the ratio of reduced to oxidized glutathione, as well as the = level of=20 free thiols on RyR2 decreased markedly compared to controls consistent = with the=20 increase in oxidized stress in HF. Consecutively, RyR2-mediated = Ca²⁺⁼²⁰leak, without significant changes in the SERCA-mediated = SR-Ca²⁺⁼²⁰uptake, was significantly enhanced resulting in reduced = SR-Ca²⁺⁼²⁰load. Importantly SR-Ca²⁺leak and SR-Ca^{2+ =}load were=20 both partially normalized by treating failing cardiomyocytes with = reducing=20 agents while oxidizing agents applied on control cells caused a rapid = decline in=20 SR-Ca²⁺load and increased SR-Ca^{2+ =}leak.³⁸ In=20 this work, the increase in RyR2 single channel activity in HF vs = control=20 attributed to increased sensitivity to activation by luminal = Ca²⁺ was=20 always restored by reducing agents.

RyR is = also=20 regulated by S-nitrosylation which increases RyR open probability in = both=20 skeletal and cardiac muscles.³⁹ The neuronal isoform of = NOsynthase,=20 nNOS orNOS1 is also found to be constitutively expressed in = cardiomyocytes where=20 it localizes to the SR and co-immunoprecipitates with RyR2 under = physiological=20 conditions. In HF there is an increase in NOS1 that translocates from = the SR to=20 the plasma membrane.⁴⁰ NOS1-knockout mice have a reduced RyR2 = S-nitrosylation with no change in the stoichiometry and binding of = FKBP12.6 and=20 RyR2 phosphorylation. Diastolic Ca²⁺levels are elevated in=20 NOS1-knockout mice and this is accompanied by a proarrhythmic phenotype. = Mice=20 deficient in the endothelial NOsynthase isoform, eNOS, or NOS3 did not = exhibit=20 alterations in any of these parameters.⁴¹ The inducible = isoform,=20 iNOS, or NOS2 is expressed under pathophysiological conditions such as=20 ischemia/reperfusion, ageing and HF. It is known to target RyR2. NOS2-NO = depresses b-adrenergic stimulated RyR function through a = cGMP-independent=20 pathway (eg, NO- and/or peroxynitrite-dependent redox modification) = although the=20 detailed mechanism was not more clarified.⁴²

Cardiac = contractility is also reduced in diabetes. In this pathological = condition,=20 alterations in the Ca²⁺ transient were also related to=20 hyperphosphorylated RyR2 and depressed FKBP12.6.⁴³ Of note = all these=20 effects significantly recovered after angiotensin II application which = reduced=20 the oxidized protein thiol level in membrane fraction.⁴⁴ = Exercise=20 training has clear beneficial effects over various physiopathological = states.=20 Exercise training during diabetes normalizes RyR2 function and = Ca²⁺=20 release from the SR while PKA activity was reduced by 75 %, but CaMKII = activity=20 was increased by 50 %.⁴⁵

In=20 dystrophic-deficient muscle nNOS is reduced by up to 80 % and has been=20 implicated in the pathology of the muscular dystrophy. RyR1 isolated = from=20 mdx skeletal muscle showed an increased S-nitrosylation = accompanied by=20 FKBP12 (or calstabin-1) depletion. S107, a novel RyR2-specific = benzothiazepine=20 derivative compound that binds to RyR1 and enhances the binding of = calstabin-1=20 to the nitrosylated channel inhibits SR-Ca²⁺leak, improves = muscle=20 function and increases exercise performance.⁴⁶ Similar = beneficial=20 effects are currently observed in the mouse dystrophic heart (Matecki S. = Lacampagne A. et al., unpublished).

Abnormal sensing of cytoplasmic and luminal Ca²⁺ by=20 RyR2

The = RyR2=20 macromolecular complex resides at the interface between plasma membrane, = cytoplasmic and SR luminal environments, thus its ability to decode and = modulate=20 Ca²⁺fluxes. Indeed, a steep dependence of Ca²⁺ = release on=20 the SR-Ca²⁺ content identifying a threshold for spontaneous=20 Ca²⁺ release and implicating spontaneous Ca²⁺ = release=20 occurs when SR-Ca²⁺ content reaches a critical level = independent of=20 membrane depolarization.⁴⁷ Thus an increase in luminal=20 Ca²⁺ concentration may increase the open probability of the = RyR2=20 channel, while reducing the RyR2 threshold such as after adding caffeine = would=20 facilitate the spontaneous SR-Ca²⁺ release. Functional = analyses=20 revealed that RyR2 mutations sensitized the channel to activation by = cytosolic=20 Ca²⁺ and that abnormal prolongation of Ca²⁺ = release=20 occurred via reduced Ca²⁺- dependent channel inhibition. = This=20 mechanism has not yet been clearly implicated in the origin of depressed = contractility during HF (but see below in Arrhythmia). It has been = recently=20 reported that the antagonistic effect of K201 on the spontaneous=20 SR-Ca²⁺ release induced by SR-Ca²⁺overload does = not=20 require the presence of calstabin2 in rat ventricular myocytes and = HEK-293 cells=20 expressing RyR2.⁴⁸

RyR2 in arrhythmogenesis

Besides = alterations=20 in muscle contraction, aberrant Ca²⁺release from the SR = during=20 diastole through leaky cardiac RyR2 can also result in diastolic changes = in=20 membrane potential (eg delayed after-depolarisation, DAD) and is a = trigger for=20 ventricular arrhythmia. Such a diastolic leak might occur as a = consequence of=20 anomalous RyR2 behavior as described above due to hyperphosphorylation, = altered=20 interdomain interaction, SR-Ca²⁺overload. It was as well = observed=20 from experimentally induced FKBP12.6 deficiency such as in calstabin2+/- = mice.²¹Furthermore a mouse model with conditional = cardiac-specific=20 overexpression of FKBP12.6 demonstrates that an increased FKBP12.6 = binding to=20 RyR2 prevents triggered VT in normal hearts in stress conditions, = probably by=20 reducing diastolic SR- Ca²⁺ leak. Moreover during = b-adrenergic=20 stimulation, the increased phosphorylation level of RyR2 is accompanied = by an=20 increased FKBP12.6 binding, at odds with the original Marks's group=20 proposal.⁴⁹

Ca²⁺=20 diastolic leak might also be the consequence of RyR2 mutations that are = mostly=20 responsible for the catecholaminergic polymorphic ventricular = tachycardia,=20 CPVT.^{27, 50} CPVT is a life-threatening genetic disease that=20 predisposes young individuals with normal structure to cardiac arrest. = The=20 autosomal dominal form of CVPT is linked to mutations in the cardiac = receptor=20 RyR2 while the autosomal recessive form is linked to cardiac = calsequestrin=20 mutations. Both RyR2 and calsequestrin proteins are fundamentally = involved in=20 the regulation of intracellular Ca²⁺ in cardiomyocytes. To = date, more=20 than 70 RyR2 missense mutations, clustering in 4 short domains, have = been found=20 to belinked with two inherited forms of sudden cardiac = death:=20 catecholaminergicpolymorphic or familial polymorphic = ventricular=20 tachycardia and arrhythmogenic right ventricular cardiomyopathy type 2. = Recent=20 post-mortem analysis has found RyR2 mutations in 14 % of sudden death = victims.=20 All RyR2 mutations, most of them single-base-pair substitutions, cluster = into=20 three regions of the channel thatcorrespond to three = malignant=20 hyperthermia/central core diseasemutation regions = (designated as=20 N-terminal domain, central domain,and channel forming = domain) of=20 RyR1. In 2002 Jiang et al. elucidated the functional properties = of the=20 R4497C mutation identified in humans by expressing in HEK293 = cells.¹³=20 The mutation results in enhanced sensitivity of the channel to the = activation by=20 Ca²⁺ and caffeine, producing a loss of normal function and = causing=20 diastolic leakage. A conditional knock-in mouse model carrier of the = R4496C=20 mutation in RyR2 demonstrates a phenotype that reproduces the typical=20 bi-directional VT observed in patients.⁵¹ Action potential = recordings=20 showed that DAD developed in paced RyR2-R4496C myocytes while triggered = action=20 potentials arising from DAD occurred frequently after b-adrenergic = stimulation.=20 Indeed in vitro studies of several other RyR2 mutations pointed = out that=20 independently from the position of the mutation, the = electrophysiological=20 abnormalities are remarkably similar. The various RyR2 mutations produce = all a=20 gain of function and cause diastolic leak from the SR particularly after = sympathetic stimulation that should lead to intracellular = Ca²⁺=20 overload, responsible for the development of DAD and triggered activity. = But the=20 molecular mechanisms by which RyR2 mutations alter the physiological = properties=20 of the RyR2 channel in CVPT remain controversial.

In line = with the=20 enhanced FKBP12.6-RyR2 dissociation observed in heart failure, = CVPT-associated=20 RyR2 mutations (S2246L, R2447S and R4497C) demonstrate reduced binding = affinity=20 to FKBP12.6 under basal conditions and this defect is amplified after = PKA=20 phosphorylation (Wehrens et al., 2003). This is supported by the fact = that a=20 mutant form of FKBP12.6 (FKBP12.6-D37S) that can bind to = PKA-phosphorylated RyR2=20 and to the RyR2-S2809D mutant demonstrated rescued normal channel gating = and=20 decreased open probability. However these observations were not = confirmed in=20 other works (George, Higgs, & Lai, 2003; D. Jiang et al., 2005). = Furthermore=20 a similar RyR2-FKBP12.6 association was found in control and RyR2-R4496C = +/-=20 mice indicating normal proteins interaction.⁵¹

The = experimental=20 drug JTV519/K201 that has been shown to reduce diastolic SR- Ca²⁺⁼²⁰leak in animal models of heart failure exhibits also strong=20 anti-arrhythmic properties in the calstabin2^{+/ -} -deficient = mouse=20 model under b-adrenergic stimulation. JTV519 was found to normalize = channel=20 gating and increased calstabin2 binding to RyR2 channels. Ventricular=20 tachycardia and sudden cardiac death in calstabin2^+/- were = reversed=20 by treatment with JTV 519.⁵² Recently it was reported that = treatment=20 with S107, known to enhance the binding of calstabin2 to the mutant = RyR2-R2474S=20 channel, inhibits channel leak. Marks's group further proposed that CPVT = is a=20 combined neurocardiac disorder in which leaky RyR2 channels in the brain = cause=20 epilepsy, and the same leaky channels in the heart cause = exercise-induced sudden=20 cardiac death.^53-55However, while the S2246L, R2474S, and = R4497C=20 RyR2-mutated mouse models were reported to demonstrate impaired FKBP12.6 = binding=20 suggested to cause the CVPT phenotype,⁵² K201 failed to show=20 protective effects against caffeine - and catecholamine-induced DAD in = R4496C=20 knock-in mice (equivalent to R4497C mutation in CVPT = patients).⁵¹=20

Unzipping of the=20 N-terminal and central regions leading to RyR2 hyperactivation and=20 Ca²⁺ leak has been involved in HF.³¹ In a more = recent=20 report the same group, using DPc10 and two other synthetic peptides,=20 demonstrated that the three mutable domains of RyR2 could cause = defective=20 interdomain interactions and consequently abnormal Ca²⁺ = cycling=20 leading to heart failure and lethal arrhythmia.⁵⁶

Taking = into account=20 the steep dependence of Ca²⁺ release on the SR-Ca²⁺⁼²⁰content,⁴⁷ a further deleterious effect may result from = a=20 defective luminal Ca²⁺ activation of RyR2 and CVPT. This has = been=20 referred as "enhanced store overload-induced Ca²⁺ release" = after=20 expressing various CVPT mutants.⁵⁷ In other words, RyR2 = mutations=20 weaken the interdomain interaction facilitating conformational changes = induced=20 by Ca²⁺ binding to the luminal but not cytosolic side, = reducing the=20 threshold for SR-Ca²⁺ release. This is supported by a recent = study of=20 the RYR2-R4496C knock-in mice in which it is demonstrated that Ca^{2+ =}sparks are much more frequent and that permeabilized = cardiomyocytes are=20 much more sensitive to controlled cytosolic Ca²⁺=20 concentration.⁵⁸ This should be related to the previous = observation=20 that PKA-dependent increase in Ca²⁺spark frequency is = entirely=20 attributable to PLB phosphorylation and the resulting SR-Ca²⁺ = load.¹⁹

Interactions=20 between RyR2 and other proteins within the macromolecular complex or its = close=20 environment are also of significant interest. Perturbed interactions = between=20 DHPR and RyR2 even in the absence of channel dysfunction, may lead to = RyR2=20 dysregulation. This functional uncoupling leads to the existence of = "orphaned"=20 RyR2 that underly dyssynchronous Ca²⁺ sparks, a = pro-arrhythmic=20 substrate that would be exacerbated by changes in SR-Ca²⁺ = load during=20 adrenergic stress.⁵⁹ Thus, age-dependent alterations in both = DHPR and=20 RyR2 proteins may contribute to the early onset of CVPT. Furthermore = although=20 purified RyR2 can sense luminal Ca²⁺, it is thought that the = RyR2=20 sensitivity to luminal Ca²⁺ in situ is determined by = its=20 interaction with calsequestrin. Calsequestrin mutations disrupt=20 RyR2-calsequestrin interaction and underlie the autosomal recessive form = of CVPT=20 (CVPT2). An arrhythmic role for mutations in disrupting the organization = of RyR2=20 and the Ca²⁺-sensitive maxi-conductance K+, BK_{Ca =}channels=20 and transient receptor potential, TRP channels is also proposed in = smooth muscle=20 based on the finding that BK_Ca and TRP channels decode = RyR2-dependent=20 Ca²⁺ signals to determine the action potential = duration.⁶⁰=20

CONCLUDING REMARKS

The = fundamental=20 mechanisms of defective RyR2 function in HF and cardiac arrhythmia has = been the=20 matter of numerous studies. A reasonable conclusion seems to be that = several=20 factors produce synergistic effects on Ca²⁺leak in a = cooperative=20 manner after a change in RyR2 protein conformation. They include=20 hyperphosphorylation, domain unzipping, redox modification, = hypernitrosylation=20 and FKBP12.6 dissociation. The later FKBP12.6 dissociation is often = reported to=20 result from the previous factors. It is intriguing to note that the=20 1,4-benzothiazepine derivative drug JTV519/K201 which demonstrates clear = beneficial effects in HF and in most studies on defective RyR2-induced=20 arrhythmia has been used to support each of these hypothetical = mechanisms. For=20 some groups, K201 and the more specific compound S107 prevent calstabin2 = depletion occurring after PKA-, or CaMKII-dependent hyperphosphorylation = and=20 S-hypernitrosylation and reduces Ca²⁺leak. For others, K201 = induces=20 a recovery of the zipped conformation of RyR2 that controls Ca^{2+ =}leak=20 and allows secondarily calstabin2 rebinding. The recent identification = of the=20 K201-binding site might help to identifying new therapeutic agents. In = diabetes,=20 a pathological state that like HF demonstrates hyperphosphorylated RyR2 = and=20 FKBP12 dissociation, exercise training as well as angiotensin II have = been shown=20 to normalize phosphorylated RyR2 status and recover Ca^{2+ =}handling=20 properties of SR. Exercise training has various site effects and is = currently=20 recommended in HF. It is interesting to note that angiotensin II levels = are=20 increased in HF and at first sight this should be beneficial. However in = HF ACE=20 inhibitors, like b-adrenergic inhibitors, demonstrate therapeutic = effects=20 probably by allowing re-sensitization of their respective receptors. = Whatever=20 the precise mechanisms of regulation of activity the RyR2 complex, = controlling=20 the interactions RyR2 - FKBP12.6 is an important candidate target for=20 pharmaceutical management of cardiac insufficiency and ventricular = arrhythmia=20 during HF.

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Recibido: 30 de=20 agosto de 2009
Aprobado: 15 de septiembre de 2009=20

Guy=20 Vassort
Laboratoire de Physiopathologie Cardiovasculaire, INSERM U-637, = CHU=20 Arnaud de Villeneuve, Montpellier, France

Contact: guy.vassort@inserm.fr=20