Review
Cardiovascular Research (2008) 77, 293–301
doi:10.1093/cvr/cvm004
Disruption of calcium homeostasis and arrhythmogenesis
induced by mutations in the cardiac ryanodine
receptor and calsequestrin
Nian Liu1 and Silvia G. Priori1,2*
1
Molecular Cardiology, Fondazione Salvatore Maugeri, Via Maugeri 10/1-A, 27100 Pavia Italy; and 2Department of Cardiology,
University of Pavia, Pavia, Italy
Received 7 May 2007; received in revised form 13 July 2007; accepted 17 July 2007; online publish-ahead-of-print 14 August 2007
Time for primary review: 21 days
KEYWORDS
Ventricular arrhythmias;
SR (function);
Sudden death;
E-c coupling;
Calcium (cellular)
Development of cardiac arrhythmias in several degenerative cardiac disorders such as heart failure is
precipitated by abnormalities in intracellular calcium regulation. Recently, the identification of
mutations in proteins responsible for the control of intracellular calcium has been associated with an
inherited arrhythmogenic syndrome called catecholaminergic polymorphic ventricular tachycardia
(CPVT). Here, we review the current knowledge about the molecular pathophysiology of CPVT and we
discuss some potentially innovative strategies for controlling calcium-handling abnormalities in CPVT
that may provide novel therapeutic options for affected patients.
1. Introduction
A great body of evidence has demonstrated that calciumhandling abnormalities play an important role in the
pathophysiology of heart diseases, such as heart failure
and cardiomyopathies.1 As a consequence, the concept
that a new strategy for the control of cardiac rhythm
abnormalities could be developed through the modulation
of the key proteins that control intracellular calcium, has
raised substantial interest in the field.2,3 In order to move
forward in this direction, it is necessary to make advances
in the understanding of calcium homeostasis under normal
settings as well as in disease states. Recently, the
mutations identified in CPVT patients in the cardiac
ryanodine receptor’s gene (RyR2) and in the cardiac
calsequestrin’s gene (CASQ2)4,5 have provided an ideal
substrate to test novel approaches to modulate calcium
homeostasis for therapeutic purposes. In this review, we
outline the current knowledge about the mechanisms by
which mutations in RyR2 and in CASQ2 facilitate the
development of ventricular tachyarrhythmias and cardiac
arrest and we speculate on the chances of developing innovative curative strategy for calcium-associated inherited
arrhythmogenic syndromes.
* Corresponding author. Tel: þ39 0382 592051; fax: þ39 0382 592059.
E-mail address: [email protected]
2. Intracellular calcium cycling in
cardiac myocytes
The delicate balance that regulates calcium fluxes between
the intracellular compartment and the extracellular space
in cardiac myocytes is critical to ensure cellular viability,
to preserve normal contractile function and to provide a
stable heart rhythm.
During the plateau phase of the cardiac action potential, a
small amount of calcium enters the cardiac myocytes
through the voltage-dependent L-type calcium channels,
causing calcium release into the cytosol through the RyR2
channel that is located in the membrane of the sarcoplasmic
reticulum (SR). This process, called calcium-induced calcium
release (CICR),6 is the basis of cardiac excitation–contraction
(E–C) coupling: it increases intracellular calcium from 100 to
about 1 mM and activates the contractile apparatus.
The calcium release termination mechanisms are complex
and rather controversial, there is evidence for several mechanisms: RyR adaptation, RyR inactivation, SR depletion, and
luminal regulation.7 The attainment of higher concentrations of calcium allows the activation of the contractile
elements of the cardiac sarcomere and subsequently
reuptake of calcium into the SR ensures relaxation. To preserve the proper function of the contractile apparatus cytosolic calcium levels are lowered by two systems: the first is
represented by the calcium-pump ATPase (SERCA2) that is
responsible for reuptake approximately 75% of calcium
into the SR; the second mechanism is represented by the
Published on behalf of the European Society of Cardiology. All rights reserved. & The Author 2007.
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294
N. Liu and S.G. Priori
Figure 1 Intracellular signalling pathway associated with calcium cycling in cardiomyocytes. NCX, sodium calcium exchanger; b-R, b adrenergic receptor; LTCC,
L-type calcium channel; PKA, Protein Kinase A; P, phosphorylation sites; CaMKII, calmodulin-dependent protein kinase II; FKBP12.6, FK506-binding Protein 12.6;
RyR2, ryanodine receptor 2; PLN, phospholamban; SERCA2, sarcoplasmic reticulum calcium-pump ATPase.
sodium–calcium exchanger (NCX) that extrudes the remaining portion of cytosolic calcium from the cytoplasm. Overall,
the gating of RyR2 and its multiple accessory proteins exquisitely control cardiac E–C coupling.8
RyR2 activity is highly regulated by signalling pathways
as shown in Figure 1. Under adrenergic stimulation,
beta-adrenergic receptors activate a GTP-binding protein,
which stimulates adenylyl cyclase to produce cAMP, which
in turn activates Protein Kinase A (PKA). This kinase phosphorylates RyR2 and other central proteins related to E–C
coupling, such as phospholamban and the L-type calcium
channels, thus causing gain of function of calcium cycling
in cardiomyocytes in response to adrenergic activation. A
cogent body of evidence has supported the view that a regulatory protein called FKBP12.6 is critical in modulating the
response of RyR2 to PKA phosphorylation.9–11 Data collected
from single channel gating studies of RyR2 reconstituted in
lipid bilayers and from biochemical studies suggested that
FKBP12.6 stabilizes RyR2 in the closed state and that hyperphosphorylation of RyR2 at Serine 2809 by PKA causes
FKBP12.6 dissociation from RyR2 increasing the open probability of RyR2. It has been suggested that enhanced
FKBP12.6 dissociation from RyR2 is a central event in the
pathophysiology of heart failure. Albeit attractive, this
hypothesis is still highly controversial.12,13
More recently, several authors have stressed the importance
of Ca2þ/calmodulin-dependent protein kinase II (CaMKII)
phosphorylation that also regulates RyR2 activity.14,15 CaMKII
can phosphorylate RyR2 at Serine 2815 which activates RyR2
without interfering with FKBP12.6 binding. Recently, evidence
from a phospholamban knock-out mouse model showed that
both CaMKII and PKA are able to phosphorylate RyR2 with
possibly distinctly different functional consequences. CaMKII
appears to be able to increase calcium spark frequency
directly via phosphorylation of RyR2 in the absence of
increased SR calcium load while PKA-mediated effects on
sparks frequency rely predominantly on elevation of SR
calcium load.16–18 It is fair to point out however that some
studies showed that CaMKII mediated phosphorylation inhibits
RyR2,19,20 the reason for discrepancies is unclear and it has
been suggested it may be related to the cellular model used.20
3. Catecholaminergic polymorphic
ventricular tachycardia
Catecholaminergic polymorphic ventricular tachycardia
(CPVT) is an inherited disease characterized by adrenergically
mediated ventricular tachyarrhythmias leading to syncope and
sudden cardiac death. The clinical presentation encompasses
exercise- or emotion-induced syncopal events and a distinctive
pattern of reproducible, stress-related, bidirectional VT in the
absence of either structural heart disease and of prolonged QT
interval (Figure 2).21 Since 2001, molecular genetic studies
have unveiled that CPVT results from inherited defects of
intracellular calcium handling in cardiac myocytes. Two
genetic variants of CPVT have been identified, one transmitted
as an autosomal dominant trait caused by mutations in the
gene encoding the RyR25 and one recessive form caused by
mutations in the cardiac-specific isoform of the CASQ2.4
4. Triggered activity is the electrophysiological
mechanism for catecholaminergic polymorphic
ventricular tachycardia
Even before the identification of the gene responsible for
CPVT, the hypothesis that arrhythmias in CPVT were likely
Disruption of calcium homeostasis and arrhythmogenesis
295
observation is important as it may explain why the treatment with beta-blockers affords only an incomplete protection from life-threatening arrhythmias in CPVT patients.
5. Origin of bidirectional ventricular
tachycardia in catecholaminergic
polymorphic ventricular tachycardia
Figure 2 Exercise stress test in a patient with polymorphic VT and carrier of
a RyR2 mutation. Ventricular arrhythmias are observed with a progressive
worsening during exercise. Typical bidirectional VT develops after 1 min of
exercise at a sinus heart rate of approximately 120–130 b.p.m. Arrhythmias
rapidly recede during recovery.
to be initiated by delayed afterdepolarizations (DADs) and
triggered activity had been advanced based on the observation that the bidirectional VT observed in CPVT patients
closely resembles digitalis-induced arrhythmias.21 Accordingly, it is well appreciated that digitalis-induced intracellular calcium overload leads to activation of NCX that extrudes
Ca2þ in exchange for Naþ with a stoichiometry of 1:3 that
generates a net inward current (the so-called transient
inward current). The transient inward current induces
DADs during the diastolic interval that may reach threshold
and trigger premature ventricular beats and ventricular
tachycardias by a mechanism called triggered activity.
We recently developed a conditional knock-in mouse
model carrier of the RyR2R4496C mutation presenting a phenotype that reproduces the typical bidirectional VT observed
in patients (Figure 3).22 This model has allowed to gain
important insights in the pathophysiology of CPVT. In ventricular myocytes isolated from the heart of RyR2R4496Cþ/2
mice, we were able to perform action potential recording
and to prove that DADs develop in paced RyR2R4496Cþ/2 myocytes even in the absence of adrenergic stimulation: such
a behaviour is never observed in WT myocytes. Upon
beta-adrenergic stimulation, we observed further enhancement of triggered action potentials arising from DADs
(Figure 4)23 suggesting that triggered activity is the likely
mechanism for CPVT. The role of triggered activity in the
genesis of arrhythmias in CPVT has further been highlighted
by observations made in other animal models of the disease
such as the R176Qþ/2 knock-in mice and Casq22/2
knock-out mice.24,25 Recently, Paavola et al.26 for the first
time demonstrated DADs in monophasic action potential
recordings in CPVT patients with RyR2 mutations. Interestingly, all the data from transgenic mice and CPVT patients
support the evidence that RyR2 mutations increase susceptibility to adrenergic stimulation but they also induce abnormal electrical behaviour in resting conditions. Such an
The most typical VT observed in CPVT patients, the so-called
bidirectional VT, derives its name from the fact that on
surface ECG it presents an alternating QRS axis morphology
with a rotation of 180 degrees of the QRS on a beat-to-beat
basis. Several investigators have been attracted by the
unusual morphology of the tachycardia and have investigated the propagation pattern of excitation in models of
bidirectional VT in vitro. Nam et al.27 used caffeine and isoproterenol to mimic the defective calcium homeostasis in
canine wedge preparation, they observed that DAD-induced
triggered activity arises most frequently in the epicardium
and bidirectional VT develops as a consequence of alternation in the origin of ectopic activity between endocardial
and epicardial regions.
Different results were presented by Katra et al.28 using a
canine wedge model of enhanced calcium entry: these
authors demonstrated that the spontaneous calcium
release originating from the endocardium has the largest
amplitude and the earliest onset and is, thereby, more
likely to initiate DAD-mediated triggered activity. Since it
is well known that Purkinje fibres are more sensitive to
calcium overload than ventricular myocytes,29 we were
intrigued by the hypothesis that bidirectional VT could be
triggered by DADs originating from right and left branches
of the Purkinje fibres in an alternate fashion. Recently, in
collaboration with the group of Jalife, it has been possible
to support this evidence using epicardial optical mapping
and volume-conducted ECGs in the isolated and perfused
heart of our RyR2R4496Cþ/2 knock-in mouse model. Activation maps showed that characteristic alternating-QRS
ECG pattern of bidirectional VT corresponded to epicardial
breakthroughs whose origin alternated, on a beat-to-beat
basis, between right and left ventricle.30
6. Molecular mechanism in catecholaminergic
polymorphic ventricular tachycardia
In vitro studies (performed in lipid bilayers, HEK293 cells,
HL1-cardiomyocytes) suggested that the RyR2 mutations
produce a ‘gain of function’ and cause diastolic Ca2þ
‘leakage’ from the SR at conditions of sympathetic activation12,31,32 leading to intracellular calcium overload that
is responsible for the development of DADs and triggered
activity. Despite intensive investigations, the molecular
mechanisms by which RyR2 mutations alter the physiological
properties of RyR2 in CPVT to initiate such arrhythmogenic
cascade remain highly controversial.12,23,33
In the following sections, we will review the three leading
mechanisms proposed to explain how mutations identified in
CPVT patients may alter the function of RyR2.
296
Figure 3
N. Liu and S.G. Priori
Bidirectional ventricular tachycardia induced by administration of epinephrine (2 mg/kg) and caffeine (120 mg/kg) in a RyR2R4496Cþ/2 knock-in mouse.
Figure 4 Action potential recording from a WT myocyte (A) and a RyR2R4496Cþ/2 myocyte (B) in the absence (top panel) and in the presence (bottom panel) of
isoproterenol (30 nM). Arrows indicate the last five paced action potentials.
6.1. Reduced binding affinity of FKBP12.6
to CPVT-associated RyR2 mutations
Over the years, Marks and collaborators have supported the
view that an abnormal Ca2þ leakage through the RyR2 is
present in heart failure, and it is caused by an enhanced dissociation of FKBP12.6 from RyR2.34,35 More recently, the
same group extended this hypothesis to account for the
RyR2 dysfunction observed in CPVT.10,32 Their studies
showed that CPVT-associated RyR2 mutations (S2246L,
R2474S, and R4497C) exhibited reduced binding affinity to
FKBP12.6 under basal conditions and that this defect is
further exaggerated after PKA phosphorylation. Subsequently,
this group generated a mutant form of FKBP12.6 with serine
residue 37 substituted for aspartate acid (FKBP12.6-D37S)
that can bind to PKA phosphorylated RyR2 and the RyR2S2809D mutant.10 Intriguingly, binding of FKBP12.6-D37S to
RyR2-R2474S channel rescued normal channel gating and
decreased open probability. These data proposed that
RyR2 mutants reduce the binding affinity of RyR2 for the
regulatory protein FKBP12.6 and that this defective behaviour is further aggravated when PKA phosphorylates RyR2
in response to adrenergic stimulation causing further
Disruption of calcium homeostasis and arrhythmogenesis
dissociation of FKBP12.6 and promoting Ca2þ leakage from
the SR.
Albeit this hypothesis is attractive and provides a reasonable explanation for arrhythmogenesis related to RyR2
mutations, other investigators challenged it and failed to
provide supportive data from different experimental protocols. George et al.12 found that CPVT-associated RyR2
mutant channels (S2246L, N4104K, and R4497C) have normal
RyR2/FKBP12.6 interaction but an abnormally augmented
response to isoproterenol and forskolin. Jiang et al.36
revealed that phosphorylation of RyR2 by PKA does not
dissociate FKBP12.6 from RyR2 mutant channels [Q4201R,
I4867M, S2246L, R2474S, R176Q(T2504M), and L433P].
Recently, we compared the RyR2–FKBP12.6 association
in WT and in RyR2R4496Cþ/2 mice: Western blot analysis
indicated that the relative amounts of FKBP12.6 to RyR2
found in heavy SR of the stimulated hearts was similar
to that found in unstimulated controls, for both WT
and RyR2R4496Cþ/2 mice.23 These observations indicate
normal RyR2–FKBP12.6 interaction in the heart from both
WT and RyR2R4496Cþ/2 animals, and further suggest that
RyR2–FKBP12.6 binding is not altered following treatment
with caffeine and epinephrine. Interestingly, the FKBP12.6–
RyR2 ratio in the RyR2R4496Cþ/2 mice was higher compared
with WT, suggesting higher FKBP12.6-binding affinity for
RyR2R4496C compared with WT RyR2. Alternatively, in the
mutant mice, there were altered cellular FKBP12.6 levels
and consequently SR levels, which could be part of a compensatory mechanism for abnormalities of mutant RyR2
channels.
The reasons for the discrepancies in the FKBP12.6-binding
affinity for CPVT-associated RyR2 mutant channels observed
by different groups are not clear, it is possible that they
depend on differences in experimental conditions.36,37
6.2. Defective intermolecular domain interactions
in catecholaminergic polymorphic ventricular
tachycardia-associated RyR2 mutations
Intermolecular interaction between discrete RyR2 domains
is necessary for the proper folding of the RyR2 channel.38
In the non-activated state, the N-terminal and central
domains make close contact with several subdomains
(zipping domains) that concur to stabilize and maintain
the closed state of the channel. It has been proposed that
RyR2 mutations may cause unzipping of these regions
leading to RyR2 hyperactivation or hypersensitization that
may result in Ca2þ leak from the SR.39 A similar mechanism
has been demonstrated to underlie malignant hyperthermia
and central core disease that are caused by mutations in the
RyR1 isoform that is expressed in skeletal muscles. Since
skeletal RyR1 and cardiac RyR2 have a high level of homology,40 it appears plausible that domain unzipping may
also occur in the cardiac Ca2þ-release channel RyR2.41,42
Further data in support of the critical role of domain–
domain interaction in the regulation of RyR2 function
came from Oda et al.,41 who designed a synthetic peptide
DPc10 corresponding to Gly2460-Pro2495 of RyR2 (located
in the central domains of RyR2) as a site-directing carrier.
DPc10 could induced an unzipped state of the interacting
N-terminal and central domains that resulted in Ca2þ leak
through the RyR2 and facilitated cAMP-dependent hyperphosphorylation of RyR2 and FKBP12.6 dissociation from RyR2.
297
More interestingly, a single Arg-to-Ser mutation made in
DPc10, mimicking the RyR2 mutation A2474S, abolished all
of the effects that would have been produced by DPc10
(Figure 5). Since this mutation seems to be located in the
binding region of FKBP12.6 to RyR2, it is suggested that
there might be 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.
Recently, George et al.43 using high-resolution confocal
microscopy and fluorescence resonance energy transfer
analysis, provided the first cell-based evidence to support
the hypothesis that SCD-linked mutations occurring in the
central domain (S2246L) and the C-terminal domain
(N4104K and R4497C) directly cause RyR2 channel instability
via defective interdomain interaction, resulting in Ca2þ
release dysfunction. The same authors, using noise analysis,
a powerful tool to elucidate mechanistically relevant information in the amplitude patterning of experimental traces,
demonstrated that there are differences in the precise mode
of Ca2þ release dysfunction and conformational instability
arising from central or C-terminal domain mutations. According to this hypothesis, C-terminal domain mutations exhibit
postactivation channel instability that is not present in the
mutations located in the central domain of RyR2. These findings support the view that abnormal interdomain interaction
is a fundamental event in RyR2-mediated arrhythmogenesis
and suggest that the mutation-site may be an important
determinant of RyR2 channel dysfunction. In addition, this
new evidence introduces the view that FKBP12.6 dissociation
from RyR2 may be a consequence rather than the cause of
RyR2 instability.
6.3 Enhanced store overload-induced calcium
release in catecholaminergic polymorphic
ventricular tachycardia-associated RyR2
mutations
Diaz et al.44 observed the steep dependence of Ca2þ release
on the SR Ca2þ content in rat ventricular myocytes, which
identifies a threshold for spontaneous Ca2þ release and
implicates that spontaneous SR Ca2þ release occurs in the
absence of membrane depolarization when SR Ca2þ content
reaches a critical level. This behaviour may result from the
fact that an increase of Ca2þ concentration inside the SR
increases the open probability of the RyR2. It is well known
that reducing the RyR2 threshold would facilitate the spontaneous SR Ca2þ release, such as in presence of caffeine,45
so that spontaneous release may be considered the result of
an increase in SR Ca2þ content and/or of a change in the
Ca2þ release threshold. Jiang et al.46 first demonstrated the
link between defective luminal Ca2þ activation of RyR2 and
CPVT, and referred to this process with the term ‘enhanced
store overload-induced calcium release’ (SOICR). These
authors used HEK293 cell lines stably expressing the CPVT
RyR2 mutants, N4104K, R4496C, and N4895D and demonstrated that they exhibit a reduced threshold for SOICR.
They also showed that these RyR2 mutations increase the sensitivity of single RyR2 channels to luminal calcium dependent
activation and enhanced the basal level of [3H]ryanodine
binding. Subsequently, this group confirmed and extended
this hypothesis to six other RyR2 mutations located in different regions of the channel [Q4201R, I4867M, S2246L, R2474,
298
N. Liu and S.G. Priori
Figure 5 The proposed interaction between the N-terminal and central domains (left). The postulated domain unzipping is caused either by the R2474S
mutation or a competitive interaction with DPc10 (right). Reproduced from Ref.,42 with the permission of the publisher.
R176Q(T2504M), and L433P] and demonstrated that HEK cell
lines and HL-1 cardiac cells expressing these CPVT RyR2
mutants all exhibited increased SOICR activity.36 Single
channel analyses revealed that disease-linked RyR2
mutations primarily increase the channel sensitivity to
luminal calcium, but not to cytosolic calcium. These findings
lead to the hypothesis that RyR2 mutations may weaken the
domain–domain interactions that stabilize the channel,
facilitating the conformational changes induced by binding
of calcium to the luminal calcium sensor, enhancing
luminal calcium activation, and reducing the threshold for
SOICR.
7. CASQ2-related catecholaminergic
polymorphic ventricular tachycardia
The CASQ2 protein serves as the major Ca2þ reservoir within
the SR of cardiac myocytes and is part of a protein complex
that contains the ryanodine receptor.47 Given that despite of
50% loss of CASQ2 (i.e. in heterozygous carriers) a normal
clinical phenotype is observed, it is suggested that the
CASQ2-related Ca2þ buffering process can tolerate major
functional impairment. Since the CASQ2-related recessive
variant of CPVT is relatively uncommon,48 only few CASQ2
mutations have been identified and functionally characterized in vitro. These studies have shown that some CASQ2
mutations impair SR Ca2þ storing and reduce the Ca2þ buffering capacity,49,50 whereas others may disrupt CASQ2–
RyR2 interaction and impair the luminal calcium-dependent
RyR2 regulation.51 We recently reported the first CPVT
patient carrier of two distinct CASQ2 mutations thus demonstrating that also compound heterozygous mutations cause
catecholaminergic VT phenotype. More recently, Dirksen
et al.52 developed a cardiac-specific expression of the
CASQ2D307H transgene mouse model (a mutation discovered
in CPVT patients), they demonstrated that CASQ2D307H
mutation impairs myocyte Ca2þ handling and predisposes
the transgenic mice to complex ventricular arrhythmias
under beta-adrenergic stimulation. This transgenic mouse
model for the first time establishes a causal link between
CASQ2 mutation and the CPVT phenotype. These findings
suggest that genetic defects of CASQ2 identified in CPVT
patients can enhance responsiveness of RyR2 to luminal
calcium generating diastolic calcium transients, DADs, and
triggered action potentials. In this respect, both the autosomal dominant RyR2-related and the autosomal recessive
CASQ2-related variants of CPVT not only have a superimposable clinical phenotype but also share a common electrophysiological mechanism represented by DADs mediated
triggered activity.
8. Novel molecular targets for treating
catecholaminergic polymorphic ventricular
tachycardia
The most important result of fundamental research applied
to genetic diseases is that of providing novel insights for the
development of gene specific treatment of the affected
patients. Such an effort has been applied to long QT syndrome and has provided some encouraging results.53,54 It is
expected that in a severe disease such as CPVT, in which
current treatment remains plagued by a high recurrence
rate of life-threatening arrhythmias, novel hope for patients
Disruption of calcium homeostasis and arrhythmogenesis
will come from the growing understanding of the pathophysiology of the disease. Here we will outline some of the
novel therapeutic strategies that may be derived by the
results of in vitro characterization of mutants and by transgenic knock-in mouse models.
9. Preventing triggering factors
The evidence that CPVT is triggered by an increase of the
adrenergic tone and reflexes such as exercise or emotional
stress, provided a clinical rationale for the use of
beta-adrenergic blockers well before the causes of the
disease were known. In the initial clinical observations,
beta-adrenergic blockers appeared to be able to prevent
the occurrence of cardiac events in CPVT patients;21
however, incomplete protection from sudden cardiac death
has been subsequently reported. It is our experience that
over a long follow up at least 30% of patients develop sustained ventricular tachyarrhythmias despite compliance to
therapy.55 It is common practice to consider these individual
candidates for an ICD, however given the young age of the
patients, it would certainly be important to have other
pharmacological options available.
10. Restoring the binding affinity of
FKBP12.6 for RyR2
According to the hypothesis proposed by Marks and debated
by other authors,12,23,33 abnormal FKBP12.6–RyR2 interaction may be central in the pathogenesis of CPVT.10,32,56
As discussed in a previous section of this review, Marks’
group proposed the use of a 1,4-benzothiazepine derivative
called JTV 519 (more recently renamed K201) to enhance
the binding of FKBP12.6 to RyR2 and prevent adrenergically
induced arrhythmias and sudden death.9 Although we were
unable to confirm the validity of this approach in our
knock-in animal model,23 the possibility that CPVT associated with other mutations may respond positively to the
drug remains open.
11. Reducing ryanodine receptor
open probability
As discussed earlier, calcium fluxes between cellular compartment and extracellular space must maintain a delicate
balance both at rest and during adrenergic activation. It
appears logical to hypothesize that reducing RyR2 open
probability may be a straightforward strategy to counter
the development of diastolic calcium leak and DADs.
However, it may be complex to achieve this goal without
interfering with normal systolic calcium release. This challenge has been successfully addressed by Venetucci et al.45
who studied rat ventricular myocytes exposed to isoproterenol in which diastolic calcium release was induced. Application of tetracaine (50 mmol/L), a local anaesthetic that
beside blocking INa is also able to reduce RyR2 open probability, abolished the diastolic calcium release. Surprisingly,
this was accompanied by an increase in the amplitude and
duration of the systolic calcium transients. These data
suggest that reducing ryanodine receptor open probability
without altering systolic function may provide a successful
antiarrhythmic strategy in CPVT.
299
Another drug that has been suggested to reduce RyR2
open probability is the calcium channel blocker verapamil:57
its role in the prevention of RyR2 mediated arrhythmias is
unclear. Swan et al.58 reported that verapamil can suppress
premature ventricular complexes and non-sustained ventricular salvoes in the CPVT patients with RyR2 mutation. Our
experience however has been less favourable: although verapamil reduced arrhythmias in CPVT patients during exercise
stress test, it was ineffective in preventing recurrence
of life threatening arrhythmias at follow up. Whether the concentration of the drug administered at therapeutic dosages is
able to affect RyR2 open probability is unknown: systematic
investigations on the role of verapamil in CPVT is warranted.
12. Blocking sodium–calcium exchanger
Intracellular calcium overload can activate NCX that by
exchanging one Ca2þ for three Naþ generating a net
inward current leading to triggered arrhythmias in CPVT: it
may be therefore reasonable to explore NCX as a therapeutic target in CPVT. Since the selective blockade of NCX
may increase calcium load of the cell, it may be important
to consider the use of NCX blockers with a broader action
including blockade of L-type calcium channel.59 No data
are available so far to allow speculations on the efficacy of
this approach in CPVT.
13. Modulation of CAMKII mediated
phosphorylation of RyR2
In our own knock-in mouse model, we demonstrated that
inhibition of CaMKII by KN93 (a selective blocker of CaMKII)
is highly effective in preventing adrenergically mediated
arrhythmias in vivo and the development of DADs in vitro.54
These data suggest that modulation of CaMKII may represent
a novel therapeutic strategy for CPVT.
14. conclusions
In the last few years, investigations directed to the understanding of CPVT, an arrhythmogenic disorder caused by
mutations in the cardiac ryanodine receptor, and in the SR
calcium buffering protein calsequestrin have given momentum to the study of the regulation of intracellular calcium
handling. Albeit several questions on the pathophysiology
of CPVT still remain highly disputed, it is clear that the collaboration between clinical scientists and basic researchers
has provided intriguing speculations about the electrophysiologic mechanisms leading to sudden death in CPVT. Interestingly, the development of knock-in animal models of
CPVT has created a novel substrate suitable to advance
not only the clinical field but also to promote the basic
understanding of arrhythmogenesis related to intracellular
calcium dysregulation. It is expected that these research
effort will concur to the development of new treatment
for the prevention of life-threatening arrhythmias in CPVT
young patients.
Conflict of interest: none declared.
300
Funding
This work was supported by the following grants (to S.G.P.):
Telethon GP0227Y01 and GGP04066, Ricerca Finalizzata
2003/180, Fondo per gli Investimenti della Ricerca di Base
RBNE01XMP4_006, RBCa034X_002, COFIN 2001067817_003,
and European Union 6th Framework for Research and Technological Development (EU FP6) grant STREP LSHM-CT2005-018802.
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