Ventricular Tachycardia from
Bedside to Bench and Beyond
Guy Katz, BA, Michael Arad, MD, and
Michael Eldar, MD
Abstract: Catecholaminergic polymorphic ventricular
tachycardia (CPVT) is a primary electrical myocardial
disease characterized by exercise- and stress-related
ventricular tachycardia manifested as syncope and
sudden death. The disease has a heterogeneous genetic
basis, with mutations in the cardiac Ryanodine Recep-
tor channel (RyR2) gene accounting for an autosomal-
dominant form (CPVT1) in approximately 50% and
mutations in the cardiac calsequestrin gene (CASQ2)
accounting for an autosomal-recessive form (CPVT2)
in up to 2% of CPVT cases. Both RyR2 and calseques-
trin are important participants in the cardiac cellular
We review the physiology of the cardiac calcium
homeostasis, including the cardiac excitation contrac-
tion coupling and myocyte calcium cycling. The patho-
physiology of cardiac arrhythmias related to myocyte
calcium handling and the effects of different modula-
tors are discussed.
The putative derangements in myocyte calcium ho-
meostasis responsible for CPVT, as well as the clinical
manifestations and therapeutic options available, are
described. (Curr Probl Cardiol 2009;34:9-43.)
normal hearts.1The most prevalent and researched of this group of
atecholaminergic polymorphic ventricular tachycardia (CPVT)
belongs to a family of primary electrical heart diseases character-
ized by severe arrhythmias in young patients with apparently
Curr Probl Cardiol 2009;34:9-43.
0146-2806/$ – see front matter
Curr Probl Cardiol, January 20099
diseases is the congenital long QT syndrome (LQTS), which also includes
the Brugada syndrome, the short-coupled variant of torsade de pointes,
the recently described short QT syndrome, and idiopathic ventricular
CPVT was first described in a case report in 1975.3The late Philippe
Coumel described four children with catecholamine-induced syncopal
ventricular tachycardia (VT) in 1978.4In 1995, a comprehensive study of
CPVT by Leenhardt et al from the Coumel group5described 21 children
suffering from stress- or emotion-induced syncope (single or recurrent),
with no evidence of structural heart disease and normal QT interval (see
below). There was a family history of syncope or sudden death in seven
patients, suggesting that CPVT has a genetic origin. However, no genetic
analysis was reported.
Our understanding of the genetic basis of this disease began with a
report by Swan et al6who described two unrelated Finish families with a
typical presentation of CPVT segregating in an autosomal-dominant
mode. Symptoms in these families appeared at an average age of 21 years
with a high mortality rate among affected individuals (31% by the age of
30 years). Using linkage analysis, they mapped the disease to chromo-
some 1q42-43. Priori et al7and shortly afterwards Laitinen et al8
identified mutations in the cardiac ryanodine receptor gene (RyR2) in
families suffering from this type of CPVT, now termed CPVT1. A
recessive form of the disease has been described by Lahat et al from our
group.9We studied members of seven nuclear families belonging to a
Bedouin tribe from the north of Israel with a history typical of CPVT. By
means of genome-wide linkage analysis the disease was mapped to
chromosome 1p13-21,9and later, a missense mutation in a highly
conserved region of the cardiac calsequestrin gene (CASQ2) was identi-
fied as the potential cause of this form of CPVT,10now termed CPVT2.
As previously stated, the first comprehensive description of CPVT was
provided in 1995 by Leenhardt et al,5who described 21 patients (12
males), 20 of whom had suffered from syncopal episode(s). Almost half
of the patients had been diagnosed as suffering from epilepsy prior to
being correctly diagnosed with CPVT. Their age at first syncope ranged
between 3 and 16 years (mean, 7.8 ? 4) and they had a slow resting heart
rate of 60 ? 9 bpm. Physical examination, baseline electrocardiograms,
and cardiac echocardiographic examinations were normal. Programmed
electrical stimulation in six patients did not elicit significant arrhythmias.
The hallmark of the disease was a reproducible form of polymorphic
10 Curr Probl Cardiol, January 2009
ventricular tachycardia (PVT), which developed in a typical manner
during exercise or emotion (Fig 1A and B). First, ventricular premature
complexes (VPC) appeared at rates between 120 and 130 bpm (mean,
122 ? 13), sometimes with concurrent development of junctional rhythm.
With continued catecholaminergic drive, the VPCs increased in number
and PVT appeared. Fifteen patients developed salvos of bidirectional VT.
In many patients a pattern of right bundle branch block with alternating
left and right axis deviation was noted. On continued effort, very fast PVT
followed, resulting in VF-like arrhythmia at the time of syncope.
M. M. Scheinman and J. N. Weiss: Other patients with genetic cardiac
rhythm disorders may have exercise-induced provocation of serious ventric-
ular arrhythmias. This is especially true for patients with the long QT
syndrome (ie, LTQ1, LTQ4, and the Andersen–Tawil syndrome). The differ-
entiation of these patients from those with CPVT relies on the facts that CPVT
patients have normal QT (or QTc) intervals and because of the very stereo-
typic response to exercise as nicely summarized in the Leenhardt study.
Treatment with amiodarone and type I antiarrhythmic drugs was ineffec-
tive. Beta-receptor blockers (mostly nadolol) were very effective in
preventing the PVTs. During a mean follow-up of 7 years, two patients
died suddenly (during treatment with beta-receptor blockers) and two had
FIG 1. Catecholamine-dependent ventricular tachycardia in men and mice. (A) Bidirectional
ventricular tachycardia in a nongenotyped patient. (Courtesy of Dr. Samuel Viskin).
(B) Exercise-induced polymorphic ventricular tachycardia in a patient homozygous for D307H
CASQ2 mutation. (C) Bidirectional ventricular tachycardia recorded at rest in a mouse
homozygous for CASQ2 knockout. (D) Polymorphic ventricular tachycardia recorded during
treadmill exercise in a mouse homozygous for D307H CASQ2 mutation. (Color version of
figure is available online.)
Curr Probl Cardiol, January 200911
syncope (one when skipping or delaying a beta-receptor blocker drug, and
the other with documented VF while treated with amiodarone). Seven
patients (30%) had a family history of syncope or sudden death and
autosomal-dominant heredity was suspected.
A subsequent significant contribution was made by Priori et al in
2002,11who described 30 probands referred because of exercise- or
emotion-related VT (either bidirectional or polymorphic) or VF. RyR2
mutations were found in 14 (47%) of them. During exercise test, 24
(80%) patients had VT. Clinical and genetic investigation of 118 family
members revealed an additional cohort of 13 CPVT patients, of whom 9
had VT of VF during exercise test and 9 carried RyR2 mutations. In the
23 RyR2 mutation carriers (CPVT1 patients consisting of 14 probands
and 9 cohort patients), first syncope occurred during childhood (mean
age, 8 ? 2 years). Syncopal episodes were much more prevalent among
the males (11 of 13) than among the females (2 of 10). The 20
nongenotyped subjects differed from the CPVT1 patients in that they
were mostly females (18/20) and their first syncope occurred at the
relatively late age of 20 ? 12 years. Programmed electrical stimulation
was performed in 21 patients but had a negligible contribution to the
diagnosis of CPVT.
All 39 clinically affected patients were treated with beta-receptor
blockers and followed for about 4 years. Eighteen (46%) patients had VT
or VF while treated. An implantable cardioverter defibrillator (ICD) was
implanted in 12 patients, of whom 6 received appropriate shocks during
a 2-year follow-up. There was a high juvenile sudden cardiac death rate
among CPVT patients (19 of 148 subjects) with no difference between
genotyped and nongenotyped subjects.
M. M. Scheinman and J. N. Weiss: Of great concern is the possibility of
“resistance” to beta-blocker therapy. In younger patients, patient compliance
is a great issue even when dealing with potentially life-threatening arrhyth-
mias. This has certainly been our experience with children of the long QT
As in several other primary electrical diseases, the penetrance
of CPVT1 was not complete. In the report by Priori et al,114 (17%) of the
CPVT1 subjects were silent gene carriers. Incomplete penetrance in
CPVT1 patients was later confirmed by others.12
Thus, the Priori et al report11added several important clinical observa-
tions to those of Leenhardt et al5: (1) about one-half of the symptomatic
autosomal-dominant CPVTs are due to RyR2 mutations, while the genetic
12 Curr Probl Cardiol, January 2009
basis for the other half is currently unknown; (2) syncope in CPVT may
be due to VF (in addition to bidirectional and polymorphic VT); (3) the
first syncope may occur relatively late in the third decade of life; (4) males
are at an increased risk for syncope; (5) beta-blockers are only partially
effective; and (6) a significant minority requires ICDs.
Relatively little is known about the nongenotyped patients. Interest-
ingly, most of them were female but the reason for this gender
inequality is not known.11There is a definite genetic heterogeneity in
CPVT1. In a study by Tester et al13of 11 patients who were referred
for genetic testing with a clinical diagnosis of CPVT, 3 had mutations
in the KCNJ2 gene (associated with LQT7), a gene not tested in the
Sinus Bradycardia in CPVT
Sinus bradycardia was reported by Leenhardt et al in 19955but was not
mentioned by Priori et al.11This feature was later described in 29
nongenotyped Japanese CPVT patients14and in a European series of 12
families carrying 13 different RyR2 missense mutations.12Mutation
carriers in these series had a resting heart rate that was 20 beats/min
lower than that of age- and gender-matched controls, and 12 beats/min
lower than that of their nonaffected family members. The heart rate of
affected males was significantly lower than that of females (compared
to matched controls). The reason for bradycardia is unknown. Postma
et al12hypothesize that it may result from impaired Ca2?handling by
mutated RyR2 channels in sinoatrial node cells or be mediated by a
Several atypical clinical features of CPVT1 were detected in a family
described by Allouis et al,15in which 11 subjects carried the G14876A
mutation in the RyR2 gene, 10 of whom were symptomatic. Several
unusual characteristics were noted, as follows: (1) while four patients on
exercise had bidirectional or polymorphic VT, five patients had mostly
monomorphic VPCs or short VTs (up to quadruplets); (2) while symp-
toms (syncope and sudden death) occurred mostly during exercise or
emotion, two young patients died suddenly during sleep. One of these
patients had never had exercise-related symptoms, while the other (the
proband) had experienced several emotion-related syncopal episodes.
Based on these findings, the authors recommend a full-scale family
screening of each case of sudden cardiac death in a young patient, in order
to detect a typical catecholamine-related presentation to facilitate a
Curr Probl Cardiol, January 200913
M. M. Scheinman and J. N. Weiss: Ackerman and colleagues have
emphasized the importance of looking for RyR2 mutations in patients with
sudden death (forensic autopsy) and in survivors of sudden cardiac death.
CPVT and Congenital LQTS
It is obvious that phenotypical CPVT is quite similar to congenital
LQTS.16In fact, 9 of the 30 probands in the Priori series11were
misdiagnosed as “LQTS with normal QT interval”16because syncope
occurred during emotion and exercise. Tester et al17found RyR2
mutations in 17 of 269 (6%) unrelated, genotype-negative patients
referred for LQTS genetic testing. Interestingly, the presentation was
near-drowning in five and excitement- or exertion-related syncope in
three patients. Six patients had VPCs up to nonsustained VT on exercise
Moreover, it is well known that congenital LQT1 syndrome (due to
mutations in the KCNQ1 gene) is characterized by swimming-related
symptoms. Among 43 patients with swimming-related syncope referred
for genetic testing, 28 (65.1%) had LQT1 syndrome and 9 (20.1%) had
RyR2 mutation.18All subjects with the LQT1 syndrome had a high
clinical probability, while all those with the CPVT1 genotype had a low
clinical probability of LQTS. Molecular autopsy revealed mutations in the
RyR2 gene in two other cases of unexplained drowning.12Catechol-
amine-related syncope may also occur in the setting of other primary
electrical diseases. These include some patients with the congenital LQT2
syndrome, LQT4,19LQT515(generally due to polymorphic VT), and
LQT7 (due to bidirectional VT).
The clinical manifestations of CPVT2 are similar to those of CPVT1.
The largest series to date includes 13 patients from seven nuclear families
belonging to a Bedouin tribe, all harboring a homozygous D307H
mutation in the CASQ2 gene. Syncope occurred in 12 of them, and an
asymptomatic 7-year-old boy developed PVT on the treadmill. Symptoms
began at a mean age of 6 ? 3 years (range, 3-12) and were always
emotion- or exercise-related. The resting heart rate was quite slow, 64 ?
13 beats/min. The penetrance was complete, as is usually the case in
homozygous monogenic diseases. Lethality in untreated subjects is high,
and nine children in these seven families died suddenly during the decade
before CPVT was diagnosed.
At the last follow-up (personal communication, Dr. Asad Khoury,
October 2006), 12 affected families were identified in the tribe, encom-
passing 25 CPVT2 patients. The follow-up period ranged between 2 and
14 Curr Probl Cardiol, January 2009
11 years (mean, 5.2). To date, electrocardiograms and echocardiograms in
all patients, and cardiac magnetic resonance imaging in six of them, are
normal. Syncope occurred in 13 and seizures in 7 patients, while PVT
developed during exercise tests in all 25 patients. All are being treated
with beta-receptor blockers (mostly propranolol, 4.8 mg/kg/day). During
the follow-up period, 17 (68%) patients remained asymptomatic, includ-
ing 12 patients who had been symptomatic previously. ICDs were
implanted in six patients (the four with syncope and two following the
sudden death of a sibling). Four (16%) patients had at least one syncope
episode and four (16%) patients died suddenly. Some of these patients
are known to have poor compliance, and two of the four who died
suddenly had refused ICD implantation.
Molecular Basis of Calcium-Mediated Arrhythmia
The following sections review the physiology of excitation-contraction
coupling, providing the background for discussing the pathogenesis of
calcium-mediated arrhythmia in general and genetically determined
CPVT in particular.
Physiology of Myocardial Calcium Cycling:
Excitation-Contraction Coupling and Calcium-Induced
The process coupling cardiac electrical activity to mechanical activity is
referred to as excitation-contraction (E-C) coupling (Fig 2). Calcium
(Ca2?) is an essential ion largely responsible for the E-C coupling
mediating cardiac contraction and relaxation.20-22The diastolic-free Ca2?
concentrations in the extracellular space [Ca2?]e, in the cytosolic com-
partment [Ca2?]i, and in the sarcoplasmic reticulum [Ca2?]SRare
approximately 1, 100, and 1-3 mM, respectively. Therefore, a gradient
of ?104magnitude exists across the sarcolemmal and sarcoplasmic
reticulum (SR) membranes, which is of crucial importance for cardiomy-
ocyte contraction and relaxation.23During systole, [Ca2?]irapidly rises
from ?100 nM to ?1 ?M, allowing Ca2?binding to troponin C and
triggering the cascade of conformational changes leading to sarcomere
contraction. Given the enormous buffering capacity (??100) of the
cytosolic compartment, as a result of numerous Ca2?binding proteins
such as troponin C, SR Ca2?pump, myosin, etc, a large amount of Ca2?
(?100 ?mol Ca2?per liter) has to enter the cytosol to allow for the
increase in [Ca2?]i. There is a small influx of Ca2?(ICa) resulting from
the opening of the voltage-dependent Ca2?channel during cardiac action
potential, which triggers a massive release from the SR24by a mechanism
Curr Probl Cardiol, January 2009 15
called “Ca2?-induced Ca2?release” (CICR).25,26To accomplish effec-
tive CICR, voltage-dependent calcium channels are concentrated in
membrane folds (transverse tubules, “T tubules”) in close proximity to
clusters of calcium release channels (also called “the ryanodine recep-
tors”) at the terminal cisternae of the SR. Local increase in [Ca2?]ileads
to the opening of adjacent SR channels, releasing more Ca2?from the SR
to facilitate contraction.
Termination of Ca2?release and reduction of cytosolic calcium [Ca2?]i
to the basal level allow Ca2?dissociation from the myofilaments and
mechanical relaxation.20,26Ca2?is transported out of the cytosol by four
different pathways, which differ between species in their relative contri-
bution. In the rabbit, cat, dog, guinea pig, and humans most of the Ca2?
(?70%) is recycled into the SR via SR Ca2?-ATPase (SERCA), while
?28% is extruded from the cell through Na?/Ca2?exchanger (NCX),
about 1% is removed by the sarcolemmal Ca2-ATPase, and an additional
1% is transported into the mitochondria.25The balance in the mouse and
rat is different: SERCA removes up to 92% of cytosolic calcium, while
FIG 2. Ca2?transport in ventricular myocytes. NCX-Na?/Ca2?exchange, ATP-Ca2?ATPase,
RyR-ryanodine receptor channel. SR, sarcoplasmic reticulum; PLB, phospholamban; Ica, Ca2?
current through l-type Ca2?channel. Insert: the time course of action potential (AP), Ca2?
transient and contraction at 37°C in a rabbit ventricular myocyte. (Adopted with permission
from Bers DM. Cardiac excitation-contraction coupling. Nature 2002;415:198-205.20) (Color
version of figure is available online.)
16Curr Probl Cardiol, January 2009
NCX Ca2?accounts for only ?7%.27The remaining 1% is split between
the sarcolemmal Ca2?-ATPase and the mitochondrial uniporter. The
principal regulators of Ca2?influx and efflux are therefore the L-type
calcium channel (LTCC), the SR calcium release channel, NCX, and
SERCA. The amount of Ca2?entering the cell during systole as well as
the amount of Ca2?extruded during diastole is smaller in rodents, a point
to be considered when studying human disease in rodent models.20
Voltage-Dependent Calcium Channels
Two kinds of voltage-dependent Ca2?channels (L- and T-type) open in
response to membrane depolarization to mediate calcium current (ICa)
where Ca2?enters the cytosol along with the electrochemical gradient.
T-type Ca2?channels are functionally important in pacemaker tissue,
while they are less abundant in the ventricle. The LTCC (also called
dihydropyridine receptors) open at higher membrane potential (?30 mV
versus ?60 mV), have larger conductance and slower inactivation
compared to T-type Ca2?channels, and account for the majority of ICa
during action potential.28,29Inactivation of LTCC primarily occurs by a
rise in intracellular Ca2?and also by a voltage-dependent mechanism.
The ?1-subunit of LTCC possesses the main channel regulatory charac-
teristics: voltage dependency, sites for ligand binding, and phosphoryla-
tion. The Ca2?-dependent inactivation site of the LTCC is located in the
carboxyl tail of the ?1-subunit next to the channel pore and is a target for
drugs designed to block channel activity, such as phenylalkylamines
(verapamil) and dihydropyridines (nifedipine). During ?-adrenergic stim-
ulation, protein kinase A (PKA) phosphorylates LTCC, decreases the
threshold potential, and increases ICaby two- to fourfold. Calmodulin-
dependent protein kinase (CaMK2) also phosphorylates LTCC on the
?1-subunit and ?2a-subunit, increasing the opening probability.30
The NCX is an electrogenic counter-transporter trading three Na?ions
for one Ca2?ion, which can operate in two modes (forward Ca2?
extrusion and reverse Ca2?entry), thereby generating two kinds of
currents. The INa/Cadirection and current intensity are determined by the
transmembrane concentrations of Ca2?and Na?, and by membrane
potential (Em). During the cardiac cycle, the INa/Cacurrent direction
changes twice. The first change occurs quickly after the upstroke of action
potential when NCX transports Ca2?into the cell, creating a net
Curr Probl Cardiol, January 2009 17
repolarizing current. The second change occurs after repolarization when
NCX removes Ca2?out of the cytosol, generating a net depolarizing
The contribution of NCX activity to CICR during cardiac systole is
disputable.31Functioning in reverse mode, NCX increases [Ca2?]iand
could help trigger CICR. Ca2?entry through NCX was shown to cause
SR Ca2?release at very high membrane potentials (eg, 70 mV). This
mechanism may even allow SR Ca2?release and contraction, while ICa
through LTCC is blocked.32However, CICR induced by NCX-mediated
Ca2?influx occurs with a 60- to 120-ms delay after phase 0 of the action
potential compared to ?10 ms when Ca2?influx occurs through the
LTCC. Under prevailing physiological conditions, given a lower mem-
brane potential and lack of confinement of NCX to the transverse tubular
system, the contribution of NCX-mediated Ca2?entry to CICR appears to
be quite small.33,34
NCX does serve as the major Ca2?transporter out of the cell and hence
plays a key role in the relaxation along with SERCA. NCX working in
forward mode causes membrane depolarization. Increased NCX activity
is a major contributor to diastolic afterdepolarizations and triggered
arrhythmia during pathological conditions associated with calcium over-
load. Overexpression of NCX leads to depletion of SR Ca2?stores and
contractile failure. In this model, adrenergic stimuli generate a large
inward current during diastole, which is mediated by NCX functioning in
“forward mode” and causes a delayed afterdepolarization (DAD). Such
potential may reach the threshold and evoke premature action potential,
an electrical event which may propagate into cardiac arrhythmia.25,35
M. M. Scheinman and J. N. Weiss: The similarity between afterdepolariza-
tion triggered ventricular arrhythmias observed with digitalis toxicity as well
as in patients with CPVT led Priori and colleagues to suspect abnormalities in
Ca2?metabolism in the genesis of arrhythmias in CPVT. This led in part to the
successful discovery of RyR2 as a candidate gene in these patients. Digitalis
leads to characteristic arrhythmias due to Na?/K? ATPase inhibition and
genesis of the Ca2?overload state. This is similar to subjects with impaired
Ca2?handling due to RyR2 or CASQ2 mutations.
The cardiac SR is an intracellular Ca2?storage organelle that provides
most of the Ca2?needed for contraction (Fig 3). SR surrounds each
myofibril and is divided into segments containing a longitudinal part and
18Curr Probl Cardiol, January 2009
terminal cisternae. The junction between the transverse tubule and two
cisternae (one on each side) is called a triad.
FIG 3. Schematic illustration of the conformational changes occurring in calsequestrin (CASQ) in the
course of Ca2?binding and the resulting modulation of ryanodine channel (RyR) activity. (A)
CASQ molecule undergoes stabilization, shrinkage, and polymerization. These changes affect the
interaction with RyR, increasing the channel open probability. With even higher SR Ca2?load,
CASQ dissociates from the membrane, disinhibiting the RyR channel. (B) A close-up on calseques-
trin. Three-dimensional model of CASQ molecule according to Wang et al.41The molecule
aspartic and glutamic amino acids. Aspartate 307 substituted to histidine in a human CPVT causing
mutation is shown in red. Mutation is assumed to impair the conformational changes occurring in
CASQ2 with Ca2?binding and the ability to regulate RyR2 channel. (C) A close-up on SR Ca2?
channel complex. The channel is composed of four RyR2 polypeptide chains and is associated on
the luminal side with triadin (T), junctin (J), and calsequestrin (CASQ2). On the cytosolic side the
channel is located in close proximity to L-type Ca2?channels on T-tubules (not shown), and each chain
is capable of binding FKBP12.6, calmodulin (CaM), and being phosphorylated by protein kinase A
(PKA) and Ca2?/calmodulin-dependent protein kinase 2 (CaMK2). In addition, the cytosolic domains
version of figure is available online.)
Curr Probl Cardiol, January 2009 19
The cardiac couplon (or junction) is a SR calcium release unit that
comprises 10-25 LTCCs organized as a circular cluster around ?100 SR
release channels. Each triad has a couplon on each side of the transverse
tubule.36About 10,000 couplons are activated during cardiac contraction
per ventricular myocyte.24
The SR calcium release channel and the calcium pump (SERCA) are the
principal SR membrane proteins (Fig 3). The reuptake of Ca2?back to
the SR is performed by SERCA, which is the most abundant protein in the
SR membrane, accounting for ?35-40% of total protein. The process of
Ca2?pumping against a 1,000- to 10,000-fold concentration gradient
involves ATP hydrolysis and exchange of H?per Ca2?ion. SERCA
Km(Ca2?) is ?300 nM. Therefore, under physiological conditions, the
pump reaches its maximal output at systolic [Ca2?]iof ?1 ?M.
Phospholamban is a SR transmembrane protein that normally inhibits
SERCA by increasing Km(Ca2?). Phospholamban phosphorylation by
PKA and CaMK2 relieves inhibition, which in turn increases SERCA
activity and SR Ca2?uptake and enhances relaxation.37The SR contains
proteins that are essential for SR Ca2?storage and the regulation of its
release. Calsequestrin is the most abundant SR luminal Ca2?binding
protein, which serves as a Ca2?ion buffer as well as its sensor to regulate
the release through the ryanodine receptor channel.38The buffering
capacity of calsequestrin allows the SR to store up to 20 mM Ca2?,
leaving the free Ca2?concentration at 1 mM.
Calsequestrin is predominantly located within the terminal cisternae.39
The cardiac isoform, CASQ2, is a 399 amino acid protein, which is
encoded by a gene of 11 exons located in chromosome 1 (1p11-p13.3).
The skeletal and cardiac muscle isoforms (CASQ1, CASQ2, respectively)
are encoded by two different genes. CASQ2 can be found in both cardiac
and slow-twitch skeletal muscle, whereas CASQ1 is rather restricted to
the fast-twitch skeletal muscle. There is a high degree of homology
between the different isoforms and between different species, implying
that studies on one animal or tissue may be pertinent to others.40Crystal
structure of rabbit skeletal calsequestrin revealed three thioreduxin-like
domains and an acidic core, which are presumably stabilized upon Ca2?
binding (Fig 3B41). The fact that calsequestrin contains an excess of
60-70 negatively charged amino acid residues is responsible for its
remarkable Ca2?storage capacity, enabling it to bind ?40 Ca2?
ions.42,43CASQ2 interacts with SR membrane proteins, triadin, and
junctin through its C-terminal and with other CASQ2 molecules through
its N-terminal. Upon increase of [Ca2?]SRand Ca2?binding, CASQ2
undergoes a conformational change, condenses, and polymerizes (Fig
20Curr Probl Cardiol, January 2009
3A). At [Ca2?]SRof ?1 mM the CASQ2 polymer is stable and anchored
to the SR membrane through triadin and junctin. Higher [Ca2?]SR
concentrations (5-10 mM) cause CASQ2 to dissociate from the SR
membrane.41,44,45Luminal Mg2?competes with Ca2?(with lower
affinity) on binding to CASQ2. Hydrogen ions also bind to the acidic
residues. During systole, protons counterflow into the SR to compensate
for Ca2?efflux, reduce luminal pH, and decrease the Ca2?binding
affinity through conformational change and unfolding of the CASQ
In addition, CASQ2 may undergo glycosylation and phosphorylation,
either by casein kinase II present in the SR lumen or by autocatalytic
activity in the presence of MgATP. The physiologic role of CASQ2
phosphorylation as well as its ability to phosphorylate other SR proteins
is currently unknown. However, it was demonstrated that dephosphory-
lated CASQ2 may bind to purified ryanosine receptor channels and
increase their channel activity.40,46
Other Ca2?binding proteins have been identified. A histidine-rich Ca2?
binding protein and sarcalumenin are distributed through SR and may
modulate RyR activity in a phosphorylation-dependent manner. Calreti-
culin is a ?46 kDa protein found in the smooth muscle and endoplasmic
reticulum, where it functions as a chaperone helping in the folding of
proteins and glycoproteins. It is highly expressed in the heart during
embryonic development but is replaced by calsequestrin after birth.47,48
The Ryanodine Receptor
The ryanodine receptor traverses the SR membrane from the SR lumen
to the cytosol and comprises the core of a Ca2?release channel (Fig 3C).
It normally opens to release Ca2?from the SR in response to an increase
in the cytosolic Ca2?in the heart or membrane potential in skeletal
muscle. Ryanodine, a plant alkaloid, is a specific ligand having a biphasic
effect on the channel: it increases Ca2?release at low concentration
(?0.01-10 ?M), whereas higher concentrations (200 ?M) have an
There are three mammalian isoforms of ryanodine receptor (RyR1 to
RyR3) expressed in different tissues and encoded by three different genes
having ?70% homology. RyR1 is mostly expressed in the skeletal
muscle. RyR3 is associated mainly with the brain but can also be found
in other tissues like abdominal organs, skeletal, and smooth muscle. RyR2
is considered to be a cardiac isoform but is expressed in many other cell
types, in particular, the brain and the kidney.24,51The skeletal and cardiac
isoforms differ in their opening mechanism. RyR1 opening is voltage
Curr Probl Cardiol, January 200921
dependent and presumably involves a physical interaction between RyR1
and LTCC but does not require Ca2?influx to occur. In contrast, Ca2?
entry through LTCC is necessary for activating RyR2 through the CICR
mechanism in the heart.52
The cardiac SR Ca2?release channel is a huge structure comprising
four RyR2 units of 565 kDa each. RyR2 monomer is encoded by a gene
located on chromosome 1 (1q42-q43), comprising two principle parts. A
large (?4500 amino acids) N-terminal cytosolic part has multiple binding
sites and serves to regulate channel activity (Fig 3C). A smaller
C-terminal part (?500 amino acids) appears to contain the transmem-
brane domains (most probably six). Each monomer contributes two of
these domains to create the channel pore located between the parallel
domains of all four RyR2 molecules. The C-terminal part also contains
sequences for linking between the RyR2 monomers, and for interaction
with CASQ2, triadin, and junctin proteins, responsible for the SR luminal
regulation of Ca2?release.44,53
RyR2 channel activity is tightly linked to cytosolic calcium. The gating
model suggests three functional states. At rest, the channel is closed and
can be opened by binding Ca2?to the low affinity activation site. As
[Ca2?]irises further, Ca2?binds with higher affinity to an inactivation
site, switching the channel to a closed mode. Once [Ca2?]idecreases,
Ca2?first dissociates from the low affinity site and only later from the
high affinity site, switching the channel from the closed-inactive state to
the resting state. Another model of regulation involves channel adapta-
tion: after rapid activation following [Ca2?]ielevation, the open proba-
bility is lowered, presumably to protect from inappropriate release. This
partial refractoriness can be overcome by a further increase in [Ca2?]i
Luminal Ca2?affects RyR2 opening by direct and indirect means and
thus modulates the amount of Ca2?released from the SR. At low
[Ca2?]SR, the capacity of ICato generate CICR is low. Increasing
[Ca2?]SRraises the RyR2 open probability and affects RyR2 sensitivity
Calsequestrin and transmembrane proteins triadin and junctin partici-
pate in Ca2?sensing and RyR2 regulation. Without CASQ2, triadine and
junctin stimulate the channel. CASQ2 serves as a SR-calcium sensor and
regulates RyR2 through its interaction with triadin and junctin. At low
[Ca2?]SRCASQ2 inhibits the channel but, as [Ca2?]SRrises, CASQ2
undergoes conformational changes, causing up-regulation of channel
activity. At high [Ca2?]SR(which may be beyond the physiological
22Curr Probl Cardiol, January 2009
range) CASQ2 polymerizes and dissociates from the complex, further
increasing channel activity (Fig. 344,58).
Channel sensitivity to activating stimuli is modulated by the energy
state and intracellular Mg2?. ATP activates the channel, and Mg2?
inhibits it. The cytosolic domains interact with various ligands that
regulate its open/close state. Calmodulin has four Ca2?binding sites and
binds to RyR in a Ca2?-dependent fashion. Each calmodulin molecule
binds to a single RyR monomer. Interestingly, while calmodulin de-
creases the RyR2 open probability at all [Ca2?]iconcentrations, its effect
on RyR1 is biphasic: increasing its open probability at basal [Ca2?]ibut
inhibiting it at a concentration higher than 1 ?M CaM.24,50,59
FK binding proteins (FKBPs) bind FK-506 and rapamycin, thus
preventing the mammalian target of rapamycin complex and forestalling
their immunosuppressant and antiproliferative activity. FKBP isoforms,
FKBP12 (calstabin1), and FKBP12.6 (calstabin2) were found to associate
with skeletal and cardiac RyR, respectively. FK-binding proteins bind 1:1
to each RyR monomer, stabilize the channel, and decrease its sensitivity
Overexpression of FKBP12.6 in isolated cardiomyocytes showed a
decrease in Ca2?flux through RyR2 and an increase in [Ca2?]SR,
implying a decrease in Ca2?release through the RyR2. FKBP12.6-
deficient mice suffered from DAD and catecholamine-induced arrhyth-
mia.62Binding of endogenous FKBP12.6 by FK506 or rapamycin leads
to “leaky channels,” an increase in Ca2?sparks and decrease in
[Ca2?]SR.24Cumulatively these data confirm the stabilizing effect of
FKBP12.6 on the RYR channel. Sorcin, a small Ca2?binding protein
localized next to the T-tubules, has an inhibitory effect on RyR2,
resembling FKBP12.6. This effect is attenuated by sorcin phosphoryla-
tion with PKA.63,64
Ryanodine receptor is regulated by phosphorylation and dephosphory-
lation (with kinases and phosphatases, respectively). Marks65showed that
PKA phosphorylation at Ser2809sensitizes the channel to [Ca2?]iby
dissociating FKBP12.6.61Other researchers disputed these findings66and
suggested that PKA effect on single channel activity is dependent on
[Ca2?] concentration.67,68Furthermore, PKA phosphorylates additional
key players in E-C coupling, increasing LTCC current and SERCA
activity, changes which eventually result in greater Ca2?stores and
release from the SR.24
Ca2?/calmodulin-dependent protein kinase 2? (CaMK2?) is the major
cardiac isoform of CaMK, which phosphorylates RyR2, PLB, LTCC, as
well as other sarcolemmal channels. Ca2?binds to CaMK2 and activates
Curr Probl Cardiol, January 200923
kinase activity but the exact location and physiological significance of
CaMK2 phosphorylation of RyR2 are still disputed.30,69,70
Effects of ?-Adrenergic Stimulation
Sympathetic stimulation via ?-adrenergic receptor facilitates contrac-
tion (inotropic effect) and relaxation (lusitropic effect). Ca2?transients
are of relatively short duration and high amplitude with rapid ascent and
decline. The ?-adrenergic receptor receptor activates adenylate cyclase
through Gsprotein, inducing cyclic AMP synthesis. Protein kinase A
(PKA) is then activated to phosphorylate target proteins, mediating the
sympathetic effects on contractility and metabolism. Phosphorylation of
LTCC increases ICa. Phospholamban phosphorylation increases Ca2?
uptake into the SR enhancing relaxation and increasing [Ca2?]SR.RyR2
phosphorylation leads to FKBP12.6 dissociation, to facilitate Ca2?
release. Cumulatively these mechanisms result in increased RyR2 open
probability, thereby explaining increased predisposition to Ca2?-induced
arrhythmia during sympathetic stimulation.54,61
Calcium Sparks, Waves, and Transients
The changes in intracellular calcium during every contraction and
between the contractions can be visualized and measured using fluores-
cence probes. The ascent and descent in [Ca2?]iduring a cardiac cycle is
called Ca2?transient.71An elementary unit of Ca2?release from the SR,
generated by Ca2?influx from a single LTCC and adjacent RyRs, is
regarded as calcium spark.72Ca2?spark may be visualized using a laser
scanning confocal microscope as a single 2- to 4-pA ion current of
?30-ms duration, having an ?1.5 ?m circular radius.73Reasonable
sparks are produced by 10-200 functionally coupled RYR channels. At
negative membrane potentials (? ?40 mV), a spark may be generated by
the opening of a single LTCC, triggering four to six ryanodine receptors.
With the spread of the action potential, a rise in local [Ca2?]iup to 10 ?M
in the vicinity of RyR2 on the terminal cisternae initiates 103-106
simultaneous sparks over the cardiomyocyte, converging into a Ca2?
transient. During diastole, individual sparks are observed at a low rate of
?100 sparks/cell/s (estimated single-channel opening probability of
0.0001/s) and lead to local increases in [Ca2?]iup to ?200 nM but are
stochastic and do not suffice to generate Ca2?transient. In states of
calcium overload the diastolic spark rate and SR channel sensitivity to
cytosolic calcium increase. Some of these events cause sufficient [Ca2?]i
elevation to propagate in cytosol in waves, a pathologic phenomenon
24 Curr Probl Cardiol, January 2009
A subpopulation of RyRs is spread over the SR membrane unrelated to
T-tubules. These “rogue” RyRs can open without anatomic or functional
relationship to the junctional clusters of RyRs. Calcium release from these
channels is uncoordinated and insufficient to generate sparks or signifi-
cantly contribute to CICR. However, it may be involved in diastolic Ca2?
leak, cause SR [Ca2?] depletion, and participate in the pathogenesis of
arrhythmia and contractile dysfunction.68
Triggered Arrhythmia and Delayed Afterdepolarization
Triggered activity is an abnormal depolarization that follows a normal
impulse. Early afterdepolarization occurs during the plateau or late
repolarization phases of an action potential, while DAD is a late event,
initiated after previous action potential has ended.51
Prolongation of action potential duration, as occurs in hypokalemia and
long QT syndrome, can lead to the generation of early afterdepolarization.
Calcium overload increasing the RyR open probability and causing
diastolic calcium waves is the principal cause of DADs.74,75An increase
in diastolic [Ca2?]iis thought to cause DAD by activating a Ca2?-
dependent inward current (Iti), which is heterogeneous. The majority
(?90%) of Iticonsists of INCXin the Ca2?extrusion mode. Ca2?-
activated Cl?current may contribute minimally (?10%). An addi-
tional current may be carried by a Ca2?activated nonselective cation
DAD needs to reach threshold potential to evoke a premature beat to
initiate the arrhythmia. Considering the cytosolic buffering capacity,74it
is estimated that 30-40 ?mol/l of cytosolic calcium or a transient of 424
nM amplitude will be required to evoke an action potential in caffeine-
treated myocytes. In the intact heart, Iticauses a smaller change in
membrane potential due to a higher passive outward current, making it
less susceptible to arrhythmia triggered by calcium overload.75
Inositol Phosphate-Sensitive Channels and Atrial Arrhythmia. Inosi-
tol 1,4,5-trisphosphate receptor (IP3R), a Ca2?-release channel located on
the endoplasmic reticulum, is responsible for Ca2?release from intracel-
lular stores in nonmuscle cells and in smooth muscle.79All three muscle
cell types express IP3R either on the SR or on the perinuclear membrane.
The channel agonist IP3is generated by phospholipase C cleavage of
membrane phosphatidilinositol4,5bisphosphate (PIP2) into IP3and 1,2-
diacylglycerol.80,81IP3R has a homotetrameric structure similar to the
RyR family, and each monomer is ?300 kDa. Three IP3R isoforms are
known: Type 1, which is found in neurons, smooth muscle, and in the
heart conducting system; Type 2, which is 69% identical to type 1, is
Curr Probl Cardiol, January 200925
expressed in the atria and is unique to ventricular myocytes; Type 3,
which is 64% identical to type 1, is selectively expressed in the
conduction system. IP3R ion conductance is lower by ?50% compared to
RyRs. Accordingly, IP3-induced Ca2?sparks have a lower amplitude and
longer rise and descent times.82
In smooth muscle IP3-induced Ca2?release is an important mechanism
contributing to contraction and possibly Ca2?waves and oscillations. The
IP3R blocker, heparin, inhibits IP3-induced contraction. In the heart, IP3R
is expressed in high levels during embryonic life, where it is assumed to
participate in cardiomyocyte differentiation and proliferation but is
decreased after birth. In the ventricular myocyte there are about 50-fold
less IP3Rs than RyR2s, and their location is mainly in the perinuclear
envelope and not in the subsarcolemmal SR. The role of IP3R in E-C
coupling in skeletal and cardiac myocytes appears to be negligible. High
concentrations of IP3can initiate SR Ca2?release and contraction in rat
ventricular myocyte, yet the Ca2?transients are much smaller and slower
compared to those initiated by CICR. It has recently been shown that SR
and nuclear envelope are interconnected, ensuring uniformity of Ca2?
concentration and release.83Therefore, although calcium release through
IP3R does not participate in CICR, it may have a modulator effect on E-C
coupling in ventricular myocytes.
The amount of IP3R2 is 6-10 times higher in the atria than in the
ventricle. Some of the receptors are located in the SR next to RyR and
adjacent to the IP3generation sites on the sarcolemma. These IP3
receptors can contribute to E-C coupling, cause Ca2?release, and
increase contractility.84Pathological conditions, such as ischemia and
heart failure, are associated with increased IP3generation through
phospholipase C activation. While IP3R activity is apparently not affected
by Ca2?released from the SR via RyR2, the RyR2 is influenced by Ca2?
released via neighboring IP3R (through CICR). It is believed that
IP3-induced Ca2?release in the atria can affect ion channel activity (eg,
LTCC, NCX, and Ca2?-dependent Cl?channel) and participate in
pathogenesis of atrial arrhythmias including fibrillation.
Arrhythmia and Cardiac Glycosides. The enzyme Na,K-ATPase (Na
pump) is an ubiquitous plasma membrane protein that uses energy from
ATP to extrude Na?from and transport K?into the cell. Cardiac
glycosides, such as digoxin and ouabain, belong to the family of
cardiotonic steroids, having in common a high-affinity binding to the
cardiac Na pump. This binding results in partial inhibition of the
enzyme,85,86leading to intracellular accumulation of Na?. The rise in
?initially reduces Ca2?efflux through the NCX, thereby increasing
26Curr Probl Cardiol, January 2009
cytoplasmic and sarcoplasmic Ca2?. This rise in sarcoplasmic Ca2?may
reach a critical level, resulting in secondary spontaneous and synchronous
release of Ca2?from the SR during diastole.75The rise in Ca2?triggers
an Iticurrent responsible for the DADs, which manifest as oscillatory
membrane potentials and can induce an arrhythmia.87In fact, digitalis
intoxication is the most prevalent cause of DAD-induced triggered
activity and typically manifests itself as monomorphic or bidirectional
Arrhythmia in Heart Failure
There is ample evidence for reduced SR Ca2?content in heart failure,
both in animal and in human hearts,88-91ascribed to down-regulation of
SR-Ca2?ATPase (SERCA2a),92,93up-regulation of the inhibitory func-
tion of phospholamban on SERCA2a,94increased expression and func-
tion of NCX,95and enhanced diastolic SR Ca2?leak.96Despite decreased
[Ca2?]SRand RYR2 protein expression, the release channels have an
increased open-probability increasing the spark rate and further depleting
the SR calcium stores.97
Heart failure is associated with action potential prolongation, elevated
cytosolic Na?, and increased calcium entry via NCX, apparently during
systole. The E-C coupling and the calcium transients are reduced in heart
failure models.98The combination of increased NCX and decreased
SERCA2a function leads to greater Ca2?extrusion from the cytosol and
lower SR Ca2?content.95Smaller Ca2?transients contribute to systolic
dysfunction in heart failure. Other changes may be compensatory.
Interestingly, a link appears to exist between a markedly decreased
SERCA2a function and diastolic dysfunction.99
The mechanism of the enhanced diastolic SR Ca2?leak is hotly
debated. According to the hypothesis by Marks et al,51heart failure is a
hyper-adrenergic state resulting in hyper-phosphorylation of RyR2 by
protein kinase A (PKA). This results in partial dissociation of FKBP12.6
from the RyR2, increasing the open probability of the channel and
allowing diastolic Ca2?leak from the SR. Treatment with beta-adrenergic
blockers reduced PKA phosphorylation, restored FKBP12.6 binding, and
normalized channel function in lipid bilayers and myocardial strips from
experimental and human heart failure.94,100Inactivation of phosphodies-
terase 4D3, a component of RYR2 complex, resulted in hyper-phosphor-
ylated channel, dilated cardiomyopathy, and arrhythmia.101Heart failure
and arrhythmia were attenuated in mice by expressing RyR2, which could
not be phosphorylated, due to better FKBP12.6 binding and channel
Curr Probl Cardiol, January 200927
However, other investigators have failed to reproduce several key
aspects of this hypothesis92,103,104and alternative hypotheses have been
Whatever the mechanism, there is no question regarding the role of the
diastolic Ca2?leak in the generation of cardiac arrhythmias in heart
failure patients. In nonischemic heart failure, arrhythmias are mainly due
to a non-reentrant mechanism, particularly DAD-induced triggered activ-
ity.25,107,108The increased inward mode NCX function95,109is coupled
with a significantly reduced inward rectifier current (Ik1),95which
augments Iti-induced membrane depolarization.25Therefore, there is an
increased probability that a resulting DAD will trigger an arrhythmogenic
Catecholamine-Induced Ventricular Tachyarrhythmia
To date, there are 67 known mutations in RYR2 causing CPVT1 and
seven CASQ2 mutations reported to cause CPVT2. All RYR2 mutations,
except one, are localized within four well-defined protein domains (Fig
4A), although other regions may be implicated in RYR1 in association
with malignant hyperthermia and central core disease. As far as one can
extrapolate, currently no specific localization is apparent for CASQ2
mutations, although Arg33 might constitute a hot spot for mutagenesis
FIG 4. CPVT genes. (A) The domains and correspondent amino acids of RyR2 implicated in
CPVT1. Exons where the predominant majority of mutations were found are indicated in red
and define an efficient screening strategy according to ref.134(B) CASQ2 gene structure and
CPVT2 mutations reported so far. Three missense mutations (top) and four mutations predicted
to cause null allele disease in homozygotes or compound heterozygotes. (Color version of
figure is available online.)
28 Curr Probl Cardiol, January 2009
(Fig 4B). The following section discusses how the molecular mechanisms
elucidated through genetic studies contribute towards our understanding
of the pathogenesis and mode of inheritance of CPVT.
The autosomal-dominant form of CPVT is caused by missense muta-
tions in RyR2, which are considered to confer an abnormal “gain of
function.” While the precise molecular mechanism has not been charac-
terized for most of the mutations, they are assumed to cause spontaneous
calcium release due to increased sensitivity to luminal calcium or
increased responsiveness to phosphorylation by sympathetic agonists.
Marks65proposed a unifying model linking the mechanism of genetically
inherited CPVT with that of ventricular tachyarrhythmia in heart failure.
PKA phosphorylation at Ser2809sensitizes the channel to [Ca2?]iby
dissociating FKBP12.6 from the RyR2 complex.61Exercise increases
sympathetic nervous activity, causing PKA phosphorylation, and disso-
ciates FKBP12.6 from RYR2. Single channels from FKBP12.6-deficient
mice demonstrate diastolic Ca2?leak when isolated after exercise but not
at rest.62Programmed electrical stimulation or isoproterenol evokes
monophasic action potential alternans and bidirectional VT in these
mice.62,110Wehrens et al62studied S2246L, R2474S, and R4497C human
CPVT mutations in isolated RYR2 channels to propose an intriguing but
debatable model, suggesting that mutations increase the open probability
of PKA-phosphorylated channels by decreasing their affinity to
FKBP12.6. Similar results were obtained with “Finnish” RYR2 CPVT
mutations: P2328S, Q4201R, V4652F.111
FKBP12.6 capable of binding to either phosphorylated or mutant RYR2
restored normal gating to the mutationally altered channels.62A novel
1-4-benzothiazepine, JTV519 (recently termed K201), was synthesized
and found to increase the binding affinity of FKBP12.6 to RYR2. The
drug stabilized isolated channels expressing CPVT mutations. In
FKBP12.6 knockout model JTV519 completely abolished arrhythmia in
heterozygous but not homozygous mice, suggesting that FKBP12.6
association with RYR2 is essential for normal E-C coupling and that
some FKBP12.6 is necessary for antiarrhythmic drug action.59,110
The role of “FKBP12.6” mechanism in heart failure and CPVT has been
disputed by others.112Jiang et al113found no abnormality in RYR2 and
in particular no change in RyR/FKBP12.6 complexes in canine tachycar-
dia-induced heart failure models and in failing human hearts. HEK293
cells transfected with RYR2R4497Chad abnormal calcium oscillations,
and R4497C mutant channels had increased activity at low cytosolic Ca2?
and increased sensitivity to caffeine.92In another study, RyR2 mutations
linked to CPVT and sudden death markedly increased the occurrence of
Curr Probl Cardiol, January 200929
store overload-induced calcium release, ie, single RyR2 channels were
more sensitive to activation by luminal Ca2?.114These investigators
studied a series of RyR2 mutations: Q4201R and I4867M from the
C-terminal region, S2246L and R2474S from the central region, and
R176Q(T2504M) and L433P from the N-terminal region, to conclude that
CPVT mutations increase channel sensitivity to luminal, but not to
cytosolic Ca2?activation, and do not affect the interaction with
FKBP12.6.105Studies in transfected cardiomyocytes also support an
FKBP12.6-independent mechanism.106Decreased Mg2?-dependent inhi-
bition, CaMKII-dependent phosphorylation,115conformational instabil-
ity, and altered interdomain interaction116are other possible mechanisms.
It could be proposed that distinct mechanisms may underlie the propen-
sity to aberrant Ca2?release in different sets of ryanodine receptor
mutations to cause triggered activity and ventricular tachyarrhythmia.112
Murine models of human disease are often used to validate mechanisms
emerging from in vitro studies. Mice lacking the ryanodine receptor die
in utero.117Knock-in mice carrying either the R176Q or the R4497C
human mutations recapitulated the CPVT phenotype including polymor-
phic VT in vivo as well as DADs and triggered activity in vitro.118,119
Contrary to humans, murine arrhythmia was poorly responsive to ?-ad-
renergic blockade. Importantly, unlike isolated channels, FKBP12.6
binding to RYR2 from SR membrane was not compromised by the
mutation, and arrhythmia could not be prevented by K201/JTV519.
DADs and triggered activity were abolished by ryanodine.120
The Gyorke group studied the mechanisms of recessively inherited
arrhythmia caused by calsequestrin defects in rat ventricular myocytes.
The levels of calsequestrin expression were manipulated using adenovirus
transfection carrying CASQ2 or antisense CASQ2 transgene. While
cardiomyocytes, partially deficient in CASQ2, had reduced SR Ca2?,
Ca2?transients, and Ca2?sparks, their channels had faster recovery from
inactivation and markedly higher release activity after sympathetic
stimulation.26Arrhythmia was attributed to defective Ca2?-dependent
inactivation of RyR2 channels. Overexpression of CASQ2D307Hprotein
produced effects comparable to CASQ2 deficiency. Cardiomyocytes
displayed diastolic calcium oscillations and delayed afterdepolarizations
when stimulated after exposure to isoproterenol. It was concluded that
decreased SR storage capacity was responsible for disrupted calcium
cycling, which could be restored by a Ca2?buffer-citrate.98
Protein studies suggested a somewhat different mechanism: D307H
mutation compromised CASQ2 ability to undergo conformational change
in response to Ca2?concentration and reduced its binding to triadin and
30 Curr Probl Cardiol, January 2009
junction.121The authors interpreted their findings as reduced ability of
mutant CASQ2 to regulate RyR2 activity. A compatible mechanism was
elucidated in cardiomyocytes transfected with CASQ2 harboring a novel
R33Q mutation, causing recessive CPVT.122Unlike CASQ2D307H,
CASQ2R33Qwas associated with similar Ca2?binding capacity but
lacked the ability to inhibit RYR2 at low luminal [Ca2?]. The mutation
increased the gain of CICR, resulting in leaky SR but with normal
amplitude of cytosolic transients despite reduced SR Ca2?stores.
Truncated calsequestrin protein CASQ2G112?5Xcould not bind cal-
cium, was incapable of conformational changes, and caused severe
depletion of SR calcium and abnormal calcium release when expressed in
rat cardiomyocytes (despite the presence of a normal endogenous pro-
tein). While these findings suggest a dominant negative mechanism, no
clinical phenotype was detected in heterozygous carriers of the mutation.
CASQ2L167Hwas identified alongside CASQ2G112?5Xin another CPVT
patient with compound heterozygosity. This variant had normal Ca2?
binding, slightly depleted SR calcium stores, and reduced spark size but no
spontaneous transients or DADs after isoproterenol.10Collectively, while
implicating either defective Ca2?storage or conformational changes causing
abnormal regulation of SR release, in vitro studies did not identify a unifying
mechanism by which CASQ2 mutations cause disease.
Transgenic mice overexpressing CASQ2D307Hin their hearts had
normal cardiac structure and function but minor ultrastructural alterations
in the morphology of T-tubules and terminal cisternae. While SR Ca2?
stores and ICacurrent were unchanged, transgenic cardiomyocytes had
smaller Ca2?transients and a higher spark rate compared to wild-type
controls. Application of caffeine and isoproterenol resulted in abnormal
calcium release, Ca2?waves, and delayed afterdepolarizations in mutant
CASQ2 overexpressors. ECG recording in intact animals demonstrated
increased prevalence of simple and complex ventricular arrhythmia after
the drug challenge.38
Knollmann et al123were the first to describe mice deficient in cardiac
calsequestrin developed to study CPVT caused by homozygous CASQ2
null-allele mutations. Mice were viable and had lower heart rates but
normal ECG and cardiac contractility. Besides lack of CASQ2 and a
marked decrease in triadin and junctin, there were no changes in protein
levels of RYR2 or SERCA, in NCX function, or LTCC current. There
was modest ventricular hypertrophy (10% increase in heart/body weight
ratio), which was not associated with myofiber disarray or fibrosis but
could be related to expansion of SR visualized on electron microscopy.
Like human patients, homozygous mice suffered from premature beats
Curr Probl Cardiol, January 200931
and polymorphic VT episodes during exercise or isoproterenol infusion.
Catecholamines were less effective in inducing arrhythmia in anesthe-
tized mice. Unlike the results from transfected cardiomyocytes,26calcium
transients, myocyte contractility, and SR calcium stores were surprisingly
well preserved in cells isolated from mutant animals. After isoproterenol
application, cardiomyocytes from mutant mice displayed spontaneous
transient rises in diastolic Ca2?and after-contractions, as well as a 30%
decrease in SR calcium stores, consistent with SR calcium leak. The
authors concluded that calsequestrin is not required for contractile
function but rather to inhibit/control RYR2 channel activity.
Our group engineered murine models of calsequestrin mutations,
expecting to establish an experimental model with maximal genetic
similarity to human recessive CPVT.124Gene-targeted mice with CASQ2
knockout or D307H CASQ2 human mutations were generated. The
human arrhythmic phenotype as well as Ca2?oscillations were recapit-
ulated in homozygous but not heterozygous mice (Fig 1C and D). While
homozygous knockouts had (as expected) neither CASQ2 RNA nor
protein, CASQ2D307H/D307Hhad a normal amount of RNA but a very low
protein level, suggesting protein deficiency as a unifying mechanism of
various CASQ2 mutations causing recessive CPVT. Upregulation of
calreticulin and of the RyR2 protein compensated for calsequestrin
deficiency and allowed mice to survive and have normal cardiac function
at rest. We identified increased expression of RyR2 and loss of channel
inhibition by CASQ2 as mechanisms potentially responsible for calcium
leak and catecholamine-induced arrhythmia. Arrhythmias responded to
Mg, which has RyR2 (as well as LTCC) blocking activity.
Calreticulin is the principal calcium-binding chaperon of endoplasmic
reticulum and smooth muscle cells. In addition to participating in muscle
contraction, calreticulin has multiple actions including gene expression
and protein processing. Cardiac expression during neonatal life is
required for heart development but falls to very low levels after birth.
Calreticulin-deficient animals die in utero because of cardiac defects.
Transgenic mice with cardiac expression during adult life develop dilated
cardiomyopathy and conduction system disease characterized by sinus
node dysfunction and atrioventricular block. Molecular studies identified
decreased expression of connexins 40 and 43 and decreased density of
LTCC.125One would expect that calreticulin expression through adult life
in CASQ2-deficient mice might account for other phenotypic manifesta-
tions (cardiomyopathy, conduction system disease), which were indeed
found in mice48,124and need to be further characterized in humans.
Table 1 provides the mechanisms responsible for Ca2?-mediated
32 Curr Probl Cardiol, January 2009
arrhythmia following digitalis intoxication, heart failure, and CPVT.
Apparently, there are numerous ways to disrupt calcium uptake and
release. Multiple regulatory pathways that evolved to control CICR are
responsible for the remarkable stability of the system and its capacity to
compensate for various perturbations.126It is therefore conceivable that
several stimuli need to coincide to dysregulate the calcium release system.
The clinical presentation of CPVT requires a synergism between a genetic
defect (ie, mutation) and an external stimulus (ie, stress or drug).
Sensitizing the SR release channel in ventricular myocytes by caffeine
increases the spark rate but quickly depletes the SR of calcium and brings
the system to a new steady state with downregulated Ca2?release.
Adding an adrenergic agonist for concomitant increase in SERCA activity
and SR calcium leads to sustained generation of calcium waves.127In a
canine ventricle wedge preparation, a combination of isoproterenol and
caffeine was required to produce monomorphic or bidirectional VT. An
additional extrastimulus was often needed to convert it into polymorphic
VT.128Likewise, human mutations do not suffice to manifest at rest but
create a substrate for arrhythmia following another stressful stimulus. In a
computerized model simulating RYR2 gating, diastolic calcium release,
cytosolic calcium, and DAD evolution, a disrupted RYR2 luminal Ca2?
sensing emerged as a unifying pathway of different RYR2 and CASQ2
M. M. Scheinman and J. N. Weiss: The authors have well described the
normal operation of Ca2?handling during the cardiac cycle. This involves
complex interactions of a number of gene products. While it is clear that
approximately 50% of patients with CPVT have genetic mutations related to
RyR2 or CASQ2 genes, it is clear that multiple other genetic influences are
involved in Ca2?homeostasis. One gets the impression that we are merely
skimming the surface in terms of a complete genetic understanding of CPVT.
This represents a fertile area for future study and discovery.
TABLE 1. Calcium-mediated arrhythmia
(Ca?2overload) Heart failure
RyR2 channelNo change Increased
CalsequestrinNo change Decreased
to luminal Ca?2
Curr Probl Cardiol, January 2009 33
Current and Future Therapies for CPVT
CPVT therapy interferes with E-C coupling and must therefore be
compatible with adequate contractility and physiological electrical activ-
ity. There is general agreement that beta-receptor blockers are the
mainstay of therapy for CPVT. However, inadequate response to beta-
receptor blocker therapy, or an “escape” phenomenon, is not uncommon.
Recurrence and mortality remain high and many patients require an ICD.
High doses of calcium channel blockers showed efficacy in animal
models and were useful in some of the human patients intolerant to
?-blockers. Combining ?-blockers with non-dihydropyridine calcium
channel blockers carries a risk of symptomatic bradycardia and is not
generally recommended. In a recent study in nongenotyped CPVT
patients,130this combination was more effective than either drug alone.
Therefore, cautious administration in highly symptomatic patients, in
particular in the presence of a protective pacemaker-defibrillator, appears
While specifically tailored therapy for a given molecular defect is still
a long way off, agents to stabilize the disinhibited cardiac ryanodine
receptors may target the common pathway underlying CPVT. Although
ineffective in a genetic model of CPVT, JTV519 provided valuable
proof of this concept by suppressing the arrhythmia in FKBP12.6-
deficient mice.62The nonspecific RYR2 blocking effect of Mg2?was
rather remarkable in CASQ2 mice and this electrolyte has a proven
added value in numerous types of ventricular arrhythmia. We suggest
that preserving normal levels of Mg2?and supplementations to
increase intracellular Mg2?should be recommended to CPVT patients
unless otherwise contraindicated. Because much of the proarrhythmic
Iticurrent that underlies the DADs is mediated by the NCX, the
exchanger could also be a potential target for inhibition. Regretfully,
NCX inhibitors are usually more effective for the reverse mode
activity (Ca2?entry), thus carrying a risk of depleting intracellular
Ca2?and suppressing the contractility beyond the potential action
against DADs. It is therefore encouraging that novel substances
emerge that might suppress Itiand attenuate digitalis-induced arrhyth-
mia without compromising contractility.131,132
Exercise is currently prohibited in CPVT patients. The effect of
conditioning, sympathetic denervation, and vagal stimulation should be
explored, because these interventions might also have a profound effect
on susceptibility to arrhythmia and the quality of life of these arrhythmia-
34Curr Probl Cardiol, January 2009
Implications for Genetic Testing
Reaching a genetic diagnosis, ie, identifying a disease-causing muta-
tion, has profound implications on disease management in the affected
individual and his/her family members. Mutations can be identified in
about 50% of clinically diagnosed individuals with CPVT.133RYR2 is
the prevailing candidate gene and should be the first to be screened
unless disease transmission is incompatible with autosomal-dominant
inheritance. Although the value of RYR2 screening in serial victims of
sudden cardiac death (SCD) and negative autopsy is considerably
smaller (?15%),134RYR2 remains the most common single genetic
cause in this population.135The contribution of RYR2 mutation to
swimming-induced arrhythmia, sudden infant death, and LQT-like
presentation is below 5%.18,136
Every family with this malignant disease tends to have its own “private”
mutation, and therefore, examination of the coding sequence appears to
be warranted (NCBI-RYR2 website). Because RYR2 is a huge gene, a
strategy was developed restricting the screening to ?20% of gene length
but covering the regions encompassing ?95% of the mutations described
so far134(Fig 4). Such a selective screening would be the best choice in
light of budget limitations, in particular, when dealing with sporadic cases
and victims of sudden cardiac death.
In a cost-contained environment, screening of CASQ2 is warranted after
excluding RYR2, in particular, when CPVT is familial and the history is
suggestive, or possibly compatible with recessive inheritance (ie, consan-
guinous, similar ethnic background, etc). Screening for LQT genes,
starting with KCNJ2, may be considered in females with bidirectional/
polymorphic VT when negative for RYR2 or CASQ2 mutation.13
Identifying a disease-causing mutation confirms and refines the clinical
diagnosis to facilitate the most appropriate behavioral counseling and
therapy. More importantly, genetic diagnosis creates the possibility of
genotyping and risk-stratifying asymptomatic family members. CPVT is
one of the rare cases when lifestyle modification and even drug therapy
appear to be indicated based on genetic diagnosis in the absence of
clinical phenotype, since sudden death is not uncommonly the first
presentation of this malignant disease.133,137
M. M. Scheinman and J. N. Weiss: We are indeed greatly indebted to the
authors for a superb review of the clinical manifestations underlying genetic
mutations as well as a review of Ca2?homeostasis and how abnormalities in
Ca2?handling lead to life-threatening arrhythmias. This review is up to date
and required reading for clinicians as well as basic scientists interested in
Curr Probl Cardiol, January 2009 35
genetic arrhythmia syndromes. Especially appreciated by clinicians are the
possible innovative future therapeutic options as well as the eminently
practical approach to the genetic study of patients with CPVT.
Acknowledgments We appreciate the help of Dr. Steve de Palma from
Harvard University, Boston and Daniela Tchetchik from Tel Aviv University
in preparing the illustrations. Elaine Finkelstein and Vivienne York provided
invaluable assistance in editing this manuscript. This project is supported by
a grant from the Israel Science Foundation.
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