Subject: Ryanodine receptor in heart failure: potential therapeutic sites Guy Vassort
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Date: Thu, 3 Nov 2011 10:29:36 -0600

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

INSUFICIENCIA CARDÍACA

Ryanodine receptor in heart failure: potential therapeutic sites

Guy Vassort

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

ABSTRACT

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

Key words: Heart failure, ryanodine
receptor.

RESUMEN

La insuficiencia
cardíaca (IC) contribuye de manera importante a la morbilidad y mortalidad por
enfermedades cardíacas. Las anomalías en el manejo del Ca2+ tienen un
papel preponderante en el desarrollo de la IC. El receptor a la ryanodina, RyR2
es una proteína tetramérica grande que se asocia a una proteína inmunofilina que
une al FK506, la FKBP12.6 o calstabina2, así como a muchas kinasas y fosfatasas
para formar un gigantesco complejo. Existe la idea de que la FKBP12.6 mantiene
al RyR2 en un estado cerrado. El estado de hiperfosforilación del RyR2 y la
disociación de la FKBP12.6 de este, ocurren en la insuficiencia cardíaca. La
fuga (de Ca2+) a través del RyR2 no depende necesariamente de la
fosforilación. Otros mecanismos incluyen el despliegue del extremo N-terminal y
los dominios centrales del RyR2 y sensaje anormal de la carga de Ca2+
del retículo sarcoplasmático (RS). El derivado de benzotiazepina JTV519/K201
tiene efectos contráctiles beneficiosos en la IC. Se han propuesto varios
modelos para dar cuenta de sus efectos restauradores. La insuficiencia cardíaca
se acompaña también de un incremento en el stress oxidativo y de la
actividad de la NOS-sintetasa, ambos alteran las propiedades del RyR2 y llevan a
una pérdida (de Ca2+) desde el RyR2. La liberación de Ca2+
del RS puede ser antagonizada por agentes reductores y por el S107, un análogo
del K201. La liberación anómala de Ca2+ del RS durante la diá puede
resultar también en cambios en el potencial de membrana y disparar arritmias
ventriculares. Además de las alteraciones antes descritas en las propiedades del
RyR2, existen múltiples mutaciones que ocurren en la proteína del RyR2 y están
vinculadas a la taquicardia ventricular polimórfica catecolaminérgica. Las
propiedades antiarrítmicas del K201 y sus mecanismos de acción son tema de
controversias. El control de las interacciones RyR2 - FKBP12.6 puede ser
candidato a un sitio blanco de importancia para el tratamiento farmacológico de
la insuficiencia cardíaca y de las arritmias ventriculares durante la IC.

Palabras clave: Insuficiencia cardíaca,
receptor a la ryanodina.

INTRODUCTION

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

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

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

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

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

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

Dysregulation of
the RyR2 by hyperphosphorylation is characterized by improper control of
SR-Ca2+ release and diastolic Ca2+ leak. Hyperactivation
of PKA, by causing an increased phosphorylation at Ser2809 of the RyR2, induces
dissociation of the FKBP12.6 subunit from RyR2. This accessory protein, FKBP12.6
is suggested to stabilize a closed state of the TyR2 channel and to reduce RyR2
sensitivity to Ca2+. FKBP12.6 binds to RyR2 with a
stoichiometric ratio of 1 FKBP12.6 to 1 RyR2 monomer, or 4 FKBP12.6 to the RyR2
tetramer. Studies from both Marks's and Yano's groups9, 10
demonstrated that altered stoichiometry between RyR2 and FKBP12.6 leads to SR-
Ca2+ leak and cardiac contractile dysfunction. In HF long-term
hyperphosphorylation of RyR2 can be maintained through a reduction in the
protein abundance of PP1 and PP2A, contributing to an increased S2808
phosphorylation state. Furthermore, a splice variant of the PDE4 family, PDE4D3
containing an N-terminal targeting motif for mAKAP forms a PKA-mAKAP-PDE4D3
signaling molecule with PDE4D3 contributing to the RyR2 complex. In human HF,
PDE4D3 levels in the RyR2 complex were reduced by 43 %.11 Owing to a
partial loss of FKBP12.6 from RyR2, in a lipid-bilayer experiment
single-channel activity was found to be hypersensitized to Ca2+, thus
expectedly causing in HF a diastolic Ca2+ leak at a concentration of
Ca2+ (approximately 100 nM) at which no significant Ca2+
release is induced in normal heart.9 The dissociation of FKBP12.6
from RyR2 also functionally uncouples multiple RyR2 and disturbs both the
simultaneous opening of RyR2 during systole and their simultaneous closing
during diastole. Indeed in vivo, a diastolic Ca2+ leak has
been reported in a rabbit model of myocardial infarction.12

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

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

JTV519, to-day
known as K201, is a 1,4-benzothiazepine derivative which has been found to be
more effective than other Ca2+ antagonist channels at reducing
Ca2+ - nduced myocardial damage. It was shown to improve
contractility and prevent the development of heart failure in paced dog heart
probably by preventing dissociation of FKBP12.6 from RyR2.24 Thus,
JTV519 treatment increased calstabin binding to PKA-hyperphosphorylated RyR2 and
cardiac function in dogs with pacing-induced HF24 and as well in
calstabin2-/- - and PDE4-/- - deficient mice.24,25
However JTV519 exhibits multiple cellular effects including inhibition of
SR-Ca2+ uptake26, 27 while during ischemia, the
JTV519-dependent attenuation of the large decrease in ATP content was prevented
by NOsynthase inhibitors.28

Regulation of the interdomain interaction within RyR2

The concept that
interactions between the N-terminal domain and the central domain of
RyR1 are involved in Ca2+ channel regulation has emerged
from recent domain peptide probe studies.29,30
According to this concept, in the resting or nonactivated state, the
N-terminal and central domains make close contact at several
subdomains (domain zipping). The conformational constraints imparted
by the zipped configuration of these two domains stabilize and
maintain the closed state of Ca2+ channel. Some
mutations of RyR2 reported in cardiac disease patients are located
in the regions corresponding to the skeletal N-terminal and
central domains harboring most of the malignant hyperthermia
mutations that cause an increased Ca2+ leak. Knowing that
RyR1 and RyR2 shares about 60 % similarities, this suggests that RyR2
shares a common domain-mediated channel regulation mechanism with
RyR1 and that the increased Ca2+ leak of diseased RyR2
channels may be explained by the altered mode of interdomain
interactions.

Stimulation via
E-C coupling or pharmacological agents weakens these critical
interdomain contacts, resulting in loss of conformational constraints
(domain unzipping) and thus lowering of the energy barrier for
Ca2+ channel opening. Weakening of these interdomain
interactions may also occur via mutation or with the use of synthetic
domain peptides. Thus, a cardiac domain peptide (DPc10)
corresponding to the Gly2460-Pro2495 region of the rabbit RyR2
(equivalent to the Gly2459-Pro2494 region of the human RyR2) was
found to produce significant activation of the RyR2 channel,
especially at low Ca2+ concentrations (0.1 to 0.3 µM). The
addition of DPc10 induced an unzipped state of the interacting N-terminal and
central domains, as evidenced by the increase in accessibility of labeled RyR2
to a fluorescence quencher.31 A single Arg-to-Ser mutation
made in DPc10, mimicking the A2474S human mutation, abolished all of
the effects that would have been produced by DPc10. These data
presented strong evidence that synthetic domain peptides
corresponding to key subdomains of RyR2 are capable of mimicking
diseased conditions of the RyR2 channel by interfering with the
interdomain interaction. Interestingly, the binding region of
FKBP12.6 to RyR2, the residues sequence 2361 to 24969 is included in
the sequence of DPc10 (2460 to 2495). This suggests that there is a
close mechanistic relationship between the PKA-mediated FKBP12.6
dissociation and abnormal domain-domain interactions such as that
seen in the DPc10-mediated channel hypersensitization. Thus when
DPc10 was introduced, the Ca2+ transient decreased and its time decay
was prolonged. Domain unzipping facilitated PKA phosphorylation and the
resultant FKBP12.6 dissociation in normal SR. In the absence of cAMP, DPc10
caused domain unzipping and Ca2+ leak in DPc10-introduced
cardiomyocytes without causing any changes in the level of phosphorylation and
in the amount of the RyR2-FKBP12.6 complex. In pacing-induced dog failing heart,
the domain unzipping has already occurred, together with FKBP12.6 dissociation
and Ca2+ leak. K201 was proposed to reverse domain unzipping and
reduce Ca2+ leak.31 The K201-binding site was recently
identified at domain 2114-2149 of RyR2 (Figure 2).
K201 reverses the mode of interdomain interaction from defective unzipped to
normal zipped configuration and stops Ca2+ leak without requiring
rebinding of FKBP12.6 to RyR2 in failing HF.32 The authors also
demonstrated that the domain peptide, DP2114-2149, after intracellular
incorporation, was able to improved Ca2+ transient and cell
shortening in FK506-treated cardiomyocytes as well in cardiomyocytes isolated
from paced-induced failing hearts. Thus in destabilized RyR2, K201 like
DP2114-2149 are able to bring back a normal zipped state. The authors further
suggest that K201 primarily modifies domain-domain interaction and by
establishing a stable conformation it then favors FKBP12.6 binding to RyR2
(Figure 2).

Oxidation and nitrosylation of RyR2

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

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

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

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

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

Abnormal sensing of cytoplasmic and luminal Ca2+ by
RyR2

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

RyR2 in arrhythmogenesis

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

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

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

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

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

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

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

CONCLUDING REMARKS

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

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

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

Contact: [email-masked]