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REVIEWS
NOVEL THERAPEUTIC APPROACHES
FOR HEART FAILURE BY
NORMALIZING CALCIUM CYCLING
Xander H. T. Wehrens and Andrew R. Marks*
Congestive heart failure is the leading cause of death in the Western world. Abnormal
intracellular calcium (Ca2+) handling is central to the pathogenesis of heart failure because it
contributes to a decrease in ventricular contractile function. Chronic hyperactivity of the
sympathetic nervous system causes increased phosphorylation of the ryanodine receptor
intracellular Ca2+-release channel, a key Ca2+-handling protein in the heart, by protein kinase A.
Alteration of the structure and function of ryanodine receptors contributes to defective
intracellular Ca2+ handling and an increased propensity for cardiac arrhythmias in failing hearts.
Novel therapeutic strategies are now being evaluated to specifically correct defective
Ca2+-handling in heart failure.
MYOCYTES
Muscle cells that contract
rhythmically in the heart.
SYSTOLE
The phase of the cardiac cycle
during which the ventricles
contract.
DIASTOLE
The phase of the cardiac cycle
during which the ventricles
are relaxed.
Department of Physiology
and Cellular Biophysics,
Center for Molecular
Cardiology,
Department of Medicine,
Columbia University College
of Physicians and Surgeons,
630W 168th Street,
P&S 9-401, New York,
New York 10032, USA.
Correspondence to A.R.M.
e-mail:
[email protected]
doi:10.1038/nrd1440
Heart failure (HF) is the leading cause of death in the
Ca2+ cycling in the normal heart
The term excitation–contraction (EC) coupling
United States. This syndrome affects about 2–3% of
the total population, with a prevalence of 5 million
describes the process of converting electrical depolariindividuals1. The most common cause of HF is corozation of the plasma membrane to contraction of the
nary artery disease, followed by hypertension and
cardiomyocyte3. The release of Ca2+ from the sarcoplasmic reticulum (SR) in the cardiomyocyte initiates
valvular pathologies. Following an initial cardiac insult
contraction of the heart during SYSTOLE. The subseaccompanied by impaired contractility, ventricular
quent reuptake of Ca2+ into the SR or extrusion from
remodelling (changes in wall thickness and/or volume)
the cytoplasm enables relaxation of the heart during
and activation of the sympathetic nervous system
DIASTOLE (FIG. 1).
activity occur as compensatory responses to maintain
cardiac output. Although these responses initially supRelease of intracellular Ca2+ during systole. The contracport cardiovascular homeostasis, they eventually lead to
tile force in cardiomyocytes is determined by the
increases in oxygen and energy consumption, structural
amplitude of the Ca2+ transient generated by SR Ca2+
abnormalities (for example, chamber dilatation and
release via intracellular Ca2+ release channels/ryanodine
fibrosis) and functional abnormalities (for example,
receptor-2 (RyR2)4,5. Depolarization of the plasma
reduced contractility), which ultimately impair the ability
membrane during the cardiac action potential causes
to pump blood and can deteriorate into overt HF.
the activation of voltage-gated L-type Ca2+ channels
During HF, cardiac contractility is impaired by
(LTCC, or dihydropyridine receptors) in the sarcoabnormalities in the structure and function of molelemmal membrane encompassing the TRANSVERSE (T)
cules responsible for the rhythmic release and reup2+
2+
TUBULES. Additional Ca can enter via the T-type Ca
take of calcium (Ca2+) ions within the MYOCYTES2. In the
6
+
2+
channels (TTCC) or the Na /Ca exchanger (NCX) in
past five years, important new insights have been
its reverse mode7. The ensuing Ca2+ influx then triggers
obtained into the molecular basis of defective Ca2+
a much greater Ca2+ release from the SR via RyR2
cycling in failing hearts. Attractive new therapeutic
targets have emerged.
through a process called Ca2+-induced Ca2+ release
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T-tubule
Plasma membrane
Actin
Myosin
LTCC
NCX
N
N
C
Ca2+
Ca2+
Ca2+
C
RyR2
Calstabin2
Ca2+
SERCA2a
PLB
Sarcoplasmic reticulum
Figure 1 | Excitation–contraction coupling in the heart. Excitation–contraction (EC)
coupling in the heart involves depolarization of the transverse tubule (T-tubule), which
activates voltage-gated L-type Ca2+ channels (LTCC). The small amount of Ca2+ influx through
LTCC triggers a large-scale Ca2+ release from the sarcoplasmic reticulum (SR) through
ryanodine receptors (RyR2). The increase in cytoplasmic Ca2+ concentration will induce
muscle contraction. To enable relaxation, intracellular Ca2+ is pumped back into the SR via SR
Ca2+-ATPase (SERCA2a), which is regulated by phospholamban (PLB), or extruded from the cell
via the Na+/Ca2+-exchanger (NCX).
TRANSVERSE (T) TUBULE
An invagination of the plasma
membrane that contains ion
channels and ion transporters
that are in a close spatial
relationship with ion channels
on the sarcoplasmic reticulum
(to enable efficient excitation–
contraction coupling).
CATECHOLAMINES
Hormones (for example,
adrenaline and noradrenaline)
that affect the sympathetic
nervous system, produced in
the medulla of the adrenal
gland. Catecholamines are
derivatives of the steroid
catechol, which is derived from
the amino acid tyrosine.
EC COUPLING GAIN
The ability of Ca2+ influx through
voltage-gated L-type Ca2+
channels to trigger Ca2+ release
from the sarcoplasmic reticulum.
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(CICR)8. The tenfold increase in cytoplasmic Ca2+ concentrations during systole results in actin–myosin crossbridge formation that is activated by Ca2+ binding to
troponin C. This results in displacement of tropomyosin,
myofilament movement and contraction of the myocyte.
Removal of cytoplasmic Ca2+ during diastole. Myocardial relaxation during diastole is initiated by the
removal of Ca2+ from the cytoplasm, which results in
deactivation of the contractile machinery. Cytosolic
Ca2+ is pumped back into the SR by sarcoplasmic reticulum ATP-ase (SERCA2a)9. The activity of this enzyme is
regulated by the binding of phospholamban (PLB)10.
In its non-phosphorylated form, PLB inhibits SERCA2a
activity10, whereas phosphorylation of PLB reverses the
inhibition. Cytosolic Ca2+ can also be expelled from
the cardiomyocyte via the sarcolemmal NCX11.
Phosphorylation of the Ca2+ channels and pumps by
PKA is the downstream event in a signalling cascade
that begins with activation of β-adrenoceptors on the
plasma membrane by CATECHOLAMINES. This allows for
the activation of adenylate cyclase by specific G proteins,
leading to increased cytosolic levels of cAMP and activation of PKA15. Once activated, PKA directly phosphorylates important Ca2+-cycling proteins, including LTCCs,
RyR2 and PLB. Activation of the β-adrenoceptor signalling pathway increases EC COUPLING GAIN, thereby
increasing the amount of Ca2+ released by RyR2 per
amount of trigger Ca2+ entering the cell through LTCC
(FIG. 2)16. This signalling pathway, also known as the FIGHTOR-FLIGHT RESPONSE, is highly conserved in evolution and
allows for the rapid enhancement of cardiac contractility
during exercise or stress5.
There is ample evidence that CaMKII is important in
regulating EC coupling in the heart13,17–19. An increase
in heart rate increases CaMKII activity in the heart, which
can result in phosphorylation of LTCCs17,20, RyR213,19
and PLB by CaMKII13,21–23. The functional effects of phosphorylation of these key Ca2+-handling proteins by
CaMKII includes an increase in contractile force (for
example, a positive force–frequency relationship)13. More
rapid release and reuptake of Ca2+ provides more time
for diastolic filling of the ventricles at higher heart rates18.
Recent work by Molkentin et al.14 has identified
PKC-α as a fundamental regulator of cardiac contractility
and Ca2+ handling in myocytes. The Ca2+/phospholipiddependent PKC exists as a family of at least 12 distinct
isoforms24. The conventional PKC isoforms (α, βΙ, βΙΙ
and γ) are activated by Ca2+ and lipids, whereas the novel
(δ, ε, η and θ) and atypical (ζ, ι, υ and λ) PKC isoforms
do not require Ca2+ for maximal activation. Activation
of angiotensin-II (AngII) receptors, α1-adrenoceptors
and endothelin-1 receptors (ET-1R) has been shown
to stimulate PKC via Gq-coupled phospholipase C
(PLC)25,26. PKC-α is the predominant PKC isoenzyme
expressed in the heart27. PKC-α can directly phosphorylate protein phosphatase inhibitor-1 (I-1), augmenting
the activity of protein phosphatase-1 (PP1) and causing
hypophosphorylation of PLB14. Decreased PLB phosphorylation could result in inhibition of SERCA2a and
impaired Ca2+ reuptake into the SR (FIG. 2).
Structure and function of RyR2
RyR2 is the predominant isoform in the heart and has
an essential function in EC coupling28. RyRs are homotetrameric channels located on the SR membrane29.
Each RyR monomer comprises a 560-kDa molecule,
characterized by an enormous N-terminal domain protruding into the cytosol, and a smaller C-terminal
domain containing the transmembrane segments (FIG. 3).
Regulation of Ca2+ cycling in the heart
The N-terminal domain serves as a scaffold for channel
The magnitude and timing of the Ca2+ transient, which
modulators, which regulate the function of the C-terminal
determines the strength of contraction, is dynamically
Ca2+-conducting pore region. An array of channel modu2+
regulated by phosphorylation of both Ca -handling
lators are bound to the cytoplasmic scaffold domain,
pumps and ion channels. Several kinases, including proincluding calmodulin (CaM)30, the channel-stabilizing
protein calstabin2 (the 12.6-kDa, also known as
tein kinase A (PKA)12, Ca2+/calmodulin-dependent
protein kinase (CaMKII)13 and protein kinase C (PKC)14
FKBP12.6)31, PKA12, PP1 and PP2A (and their targetcontribute to these effects (FIG. 2).
ing proteins 32), CaMKII 13,33 and sorcin (FIG. 3)34.
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REVIEWS
β-AR N
Plasma membrane
β
C
γ
α
AC
T-tubule
Ca2+
cAMP
N
AT-IIR
LTCC
CaMKII
C
N
C
AKAP
CaM
PKCα
PKA
PLC
Gαq
N
α-AR
Ca2+
N
mAKAP
PKA
C
NCX
PKA
Ca
C
A
PK
P
KA
mA
MK
II
Calstabin2 CaMKII
I-1
RyR2
SERCA2a
SERCA2a
PLB
PLB
PPI
Sarcoplasmic reticulum
FIGHT-OR-FLIGHT RESPONSE
An evolutionarily conserved
mechanism which allows for the
rapid enhancement of cardiac
contractility and cardiac output
during exercise or sudden
stress. This stress response is
mediated by the activation of
the sympathetic nervous
system, which leads to
phosphorylation of an array of
intracellular proteins in the
heart, including ryanodine
receptor-2, by protein kinase A.
CA2+-RELEASE UNITS
Structures containing two
proteins essential to EC
coupling: the L-type Ca2+
channels (LTCC) on the plasma
membrane and the ryanodine
receptors (RyR2) on the
sarcoplasmic reticulum. The
LTCC–RyR2 complexes are
organized in to lattices which
allow a large population of
receptors to be simultaneously
switched on, or off, by a very
small change in ligand
concentration.
Figure 2 | Regulation of intracellular Ca2+ signalling in the heart. Several intracellular signalling pathways can increase the
gain of the excitation–contraction coupling system. Agonist-activation of the β-adrenoceptor allows for activation of adenylate
cyclase (AC) via specific G proteins. The subsequent generation of cAMP activates protein kinase A (PKA), which can be
targeted to L-type Ca2+ channel (LTCC) via A-kinase anchoring protein (AKAP), and ryanodine receptor-2 (RyR2) and Na+/Ca2+
exchanger (NCX) via muscle AKAP (mAKAP), respectively. Faster heart rates increase the average cytosolic Ca2+ concentration,
which activates Ca2+/calmodulin-dependent protein kinase (CaMKII). CaMKII can phosphorylate LTCC, RyR2 (to which CaMKII
is directly targeted) and phospholamban (PLB). Activation of the angiotensin-II receptor (AT-IIR), α-adrenoceptor or endothelin-1
receptor (ET-1R) activates phospholipase C (PLC) via specific G proteins, which in turn activate protein kinase C (PKC-α). PKC-α
can directly phosphorylate protein phosphatase inhibitor-1 (I-1), augmenting the activity of the protein phosphatase-1 (PP1) and
causing hypophosphorylation of PLB. SERCA2a, sarcoplasmic reticulum ATP-ase.
Highly conserved leucine/isoleucine zipper (LIZ)
This phenomenon, called ‘coupled gating’, enables
CA -RELEASE UNITS consisting of groups of RyR2 to open
motifs in RyR2 form binding sites for similar LIZs in
and close simultaneously.
targeting proteins for kinases (for example, PKA) and
phosphatases (for example, PP1 and PP2A) that reguRegulation of ryanodine receptors. A variety of cellular
late RyR function (FIG. 3)32. RyR also binds proteins at the
luminal SR surface (for example, triadin, junctin and
mediators and modifications can modulate the activity
calsequestrin (CSQ)). Junctin35 and triadin36 are presumof RyR2 in the heart, including Ca2+, Mg2+, ATP, phosably involved in anchoring RyR, whereas CSQ provides a
phorylation, oxidation and so on (for a more extensive
high-capacity intracellular Ca2+ buffer37,38.
review on RyR modulation see REF. 4). Phosphorylation
Calstabin2 is a peptidyl-prolyl cis–trans isomerase
by PKA increases the probability that the RyR2 channel
that binds to RyR2 with a stoichiometry of one calstaadopts an open conformation by increasing the sensibin2 bound to each RyR2 monomer. Binding of
tivity of RyR2 to Ca2+-dependent activation12,39,42,43.
Phosphorylation of serine 2809 on RyR2 results in the
calstabin2 stabilizes the channel in the closed state during
dissociation of the channel-stabilizing protein calstabin2
the resting phase of the heart (diastole)31,39. In addition
to stabilizing individual RyR2 channels, calstabin2
from the channel complex, which increases the sensitivity
functionally couples groups of RyR2 channels to
of the channel to Ca2+-dependent activation13. Recently,
40,41
enable synchronous opening during EC coupling .
the CaMKII phosphorylation site on RyR2 (serine 2815)
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VOLUME 3 | JULY 2004 | 3
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the sensitivity to Ca2+-dependent activation. In contrast
to phosphorylation by PKA, phosphorylation by CaMKII
does not induce the dissociation of calstabin2 from the
RyR2 channel13.
Activity of RyR2 is also regulated by protein phosphatases targeted to the macromolecular channel complex via specific adaptor proteins (FIG. 2)32,44. We have
previously demonstrated that both PP1 and PP2A are
bound to RyR2 via the adaptor proteins spinophilin
and PR130, respectively 32. It is believed that PP1
reduces RyR2 activity45, although contrary results have
been reported46.
Ca2+
C
RII
PP2A
LIZ2
PP1
LIZ1
C
RII
RyR2
LIZ3
Serine 2809 S
CaMKII
S Serine 2815
CaM
Sorcin
Triadin
Junctin
Defective Ca2+ handling in failing hearts
Sarcoplasmic reticulum
CSQ
Calstabin2
Spinophilin
mAKAP
PR130
CSQ
Figure 3 | Ryanodine receptor-2 is a macromolecular complex. The ryanodine receptor-2
(RyR2) macromolecular complex includes four identical RyR2 subunits, each of which binds
one calstabin2, as well as the phosphatases and kinases shown in figure 2 (for reasons of
clarity, only one protein kinase A (PKA) is shown). Leucine/isoleucine zippers (LIZ) on ryanodine
receptor-2 (RyR2) mediate binding of adaptor proteins that target protein phosphatases PP1
and PP2A, and protein kinase A (PKA) to the channel complex. PKA consists of two regulatory
(RII) and two catalytic (C) subunits. Calstabin2, calmodulin (CaM), CaMKII and sorcin also bind
to the cytoplasmic surface of RyR2. The encircled ‘S’ residues indicate the PKA (serine 2809)
and CaMKII (serine 2815) phosphorylation sites within the RyR2 protein. Triadin and junctin
have transmembrane domains and bind to the sarcoplasmin reticulum (SR) side of RyR2.
Calsequestrin (CSQ) binds and unbinds to the triadin–junctin–RyR2 complex, depending on
the SR Ca2+ concentration during the excitation– contraction coupling cycle.
was identified using site-directed mutagenesis and
phospho-epitope-specific antibodies13. Phosphorylation
of RyR2 at serine 2815 increases the probability that the
channel adopts an open conformation by augmenting
Defective intracellular Ca2+ handling is central to the
depressed contractility and diminished contractile reserve
observed in heart failure2. Cardiomyocytes isolated from
failing hearts are characterized by a reduction in the
systolic Ca2+ transient amplitude, an increase in diastolic
Ca2+ concentrations and a slowed rate of diastolic Ca2+transient decay47,48. These changes result in a decreased
SR Ca2+ content and a lower EC coupling gain2,16.
Chronically elevated plasma concentrations of catecholamines contribute to the alterations in intracellular
Ca2+ handling in patients with HF49,50. Chronic activation
of the β-adrenoceptor pathway results in maladaptive
changes in the heart, including a decrease in expression
and coupling of β-adrenoceptors51,52, an increase in
expression of the inhibitory G protein Gi, a rise in the
expression of the β-adrenoceptor kinase (which phosphorylates and desensitizes β-adrenoceptors)53, and a
decrease in expression and function of adenylate
cyclase15. Downstream effects of increased intracellular
kinase activity include PKA-mediated hyperphosphorylation of LTCC54, NCX55 and RyR212. Defective Ca2+
cycling in failing hearts can also result from the altered
expression and function of proteins required for Ca2+
homeostasis. In failing hearts, the expression of SERCA2a
and RyR2 are downregulated, and NCX expression is
generally upregulated56–58.
Box 1 | Mutations in the cardiac RyR2 linked to exercise-induced sudden cardiac death
Sudden cardiac death is associated with common cardiac diseases and conditions, most notably heart failure (roughly
50% of heart failure patients die from fatal ventricular arrhythmias). However, fatal arrhythmias can also occur in young
and otherwise healthy individuals without known structural heart disease101,102. Catecholaminergic polymorphic
ventricular tachycardia (CPVT) is an arrhythmogenic disorder of the heart characterized by stress- or exercise-induced
ventricular tachycardias that lead to sudden cardiac death103–105. Affected individuals typically present during
adolescence with repetitive exercise-triggered syncopal events leading to sudden cardiac death in 30–50% by 30 years of
age106,107. Genetic linkage studies and DNA sequencing have identified mutations in the cardiac ryanodine receptor gene
(hRyR2) in individuals with CPVT104,105.
The analysis of the biophysical properties of CPVT-mutant RyR2 channels has provided new insights into the
molecular basis for the triggers that initiate arrhythmias39. Under non-stimulated, resting conditions, CPVT-mutant
RyR2 channels are indistinguishable from normal (wild-type) channels39,108, consistent with the clinical observation that
CPVT patients do not develop arrhythmias at rest107. However, CPVT-linked mutant RyR2 channels display abnormal
single-channel function following phosphorylation by protein kinase A (for example, to mimic exercise or stress)39,108,109.
Mutant RyR2 channels also have a decreased affinity for the channel-stabilizing molecule calstabin2, compared with
wild-type channels. These findings indicate that during exercise, calstabin2-depleted CPVT-mutant RyR2 channels
might trigger sarcoplasmic reticulum Ca2+ leak, which could initiate ventricular arrhythmias39. Consistent with this
model is the finding that calstabin2-deficient mice consistently develop ventricular arrhythmias and sudden cardiac
death during exercise-stress testing39.
© 2004 Nature Publishing Group
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Table 1 | Therapeutic strategies for preventing abnormal Ca2+ release
Strategy
Drug
References
Enhancing calstabin2 binding to RyR2
Increasing RyR2 binding-affinity for calstabin2
JTV519
Overexpression of calstabin2 in myocytes
(Adenovirus)
75,77
96
Normalizing β-adrenoceptor signalling
Reducing PKA phosphorylation of RyR2
Reducing PKA activity
Beta-blockers
64–66
Enhancing PP1/PP2A activity
Beta-blockers
64
PKA, protein kinase A; PP, protein phosphatase; RyR2, ryanodine receptor-2.
Ryanodine receptor dysfunction in heart failure. Chronic
hyperactivity of the β-adrenoceptor signalling pathway
in patients with HF leads to hyperphosphorylation of
RyR2 by PKA12,59,60. Reduced levels of PP1 and PP2A in
the RyR2 macromolecular complex might contribute
to the maintenance of long-term hyperphosphorylation
of RyR2 by PKA12,32. Hyperphosphorylation of serine
2809 on RyR2 results in the dissociation of the channelstabilizing protein calstabin2, which causes a leftward
shift in the Ca2+-sensitivity of the channel (for example,
RyR2 is more easily activated at the same Ca2+ concentrations)12. In addition, RyR2 can open aberrantly during
diastole, leading to SR Ca2+ ‘leak’, which can result in
resetting the SR Ca2+ content to a lower level. Decreased
SR Ca2+ loading, in turn, reduces EC coupling gain and
contributes to impaired systolic contractility12. Increased
SR Ca2+ leak due to impaired binding of calstabin2 to
RyR2 can also lead to ventricular arrhythmias and sudden
cardiac death in patients with heart failure or inherited
exercise-induced ventricular arrhythmias (BOX 1)39.
Novel therapeutic strategies for HF
A better understanding of intracellular signalling cascades involved in Ca2+ cycling in cardiomyocytes has led
to the identification of several new therapeutic targets to
improve systolic and diastolic function in the failing
Table 2 | Therapeutic strategies for enhancing SR Ca2+ loading
Strategy
myocardium (TABLES 1,2,3). Some treatments aimed at
normalizing intracellular Ca2+ handling are already
being used for the treatment of HF patients, whereas
others are presently being evaluated in preclinical trials
or animal models.
Drug
References
Increasing SERCA activity
Stimulating SERCA2a activity
Gingerol analogues
Overexpressing SERCA2a
(Adenovirus)
78,80,97
81,82
Overexpressing SERCA1a
(Adenovirus)
98,99
Increasing PLB phosphorylation
N/A
84,85
Antisense gene therapy
(Antisense)
86
Gene therapy with rAAV-PLB
(AAV-virus)
84,85
Inhibiting PKC-α activity
N/A
Decreasing PLB activity
14
Decreasing NCX activity
NCX antagonist (reverse-mode)
KB-R7943/ SEA0400
NCX antagonist (forward-mode)
SEA0400
β-adrenoceptor blockers (beta-blockers) are one of the
few classes of drugs that improve cardiac contractile
function and reduce mortality rates in patients with
congestive heart failure61–63. β-adrenoceptor blockers
are known to depress cardiac function in healthy
hearts, and so it might seem rather counter-intuitive to
use this class of drugs for the treatment of HF. Recent
studies, however, have provided more insight into the
molecular mechanisms by which β-adrenoceptor blockers
improve cardiac contractility in patients with HF58,64.
Blockade of β-adrenoceptors reduces intracellular
cAMP levels and decreases the activity of PKA. The
downstream effects of reduced PKA activity include a
reversal of the hyperphosphorylation of RyR2 by
PKA64–66. This restores normal stoichiometry of the
RyR2 macromolecular complex by increasing the binding of calstabin2 to RyR2 (FIG. 4)12,64,66. Increased binding
of calstabin2 normalizes the function of RyR2 channels
in failing hearts64. The restoration of normal RyR2
structure and function might in part explain the improved contractility observed in HF patients treated with
β-adrenoceptor blockers. In addition, β-adrenoceptor
blockers might improve the energy balance in failing
hearts, which are typically energy starved and depleted of
high-energy phosphate. Finally, β-adrenoceptor blockers have been shown to reverse HF-specific alterations
in cardiac gene expression, which might be involved in
progression of the disease58.
The pro-drug Nolomirole (Chiesi Farmaceutici),
when converted into its active metabolite, acts as a selective presynaptic agonist for dopaminergic (DA2) receptors and α2-adrenoceptors. Stimulation of these receptors
reduces noradrenaline release67. Nolomirole is presently
being compared with placebo in the Echocardiography
and Heart Outcome Study (ECHOS)68.
Increased activity of G-protein-coupled receptor
kinase (GRK, or β-ARK) contributes to desensitization of
β-adrenoceptors in failing hearts15,69. These observations
led to the hypothesis that cardiac function might be
restored by selectively inhibiting GRK70. Although no
pharmacological GRK-inhibitors have so far been identified that would allow validation of this hypothesis, several experimental studies using the C terminus of GRK2
(also known as β-ARKct) as a dominant-negative
approach seem to support this theory70,71. The β-ARKct
peptide inhibits GRK-mediated β-adrenoceptor phosphorylation, as well as generalized Gβγ signalling, and
overexpression of GRK2 has led to improved contractility
in animal models of HF15,71.
93
100
Normalizing intracellular Ca2+ release
Treatment with β-adrenoceptor blockers can improve
cardiac function by reversing hyperphosphorylation
of RyR2 by PKA, which allows for increased binding of
© 2004 Nature Publishing Group
N/A, not available; NCX, Na+/Ca2+ exchanger; PKC-α, protein kinase C-α; PLB, phospholamban;
rAAV, recombinant adeno-associated virus; SERCA, sarcoplasmic reticulum ATP-ase; SR, sarcoplasmic reticulum.
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Table 3 | Therapeutic strategies for normalizing β-adrenoceptor signalling
Strategy
Drug
Reducing norepinephrine excretion
Nolomirole
References
67
Blocking β-adrenoceptors
Beta-blockers
Inhibiting G-protein-coupled receptor kinase
N/A
61,63
15
N/A, not available.
calstabin2 and normalization of Ca2+ release64,66. Potential
side effects, however, include depressed cardiac function
and reduced exercise tolerance72,73.
We have recently shown that a genetically altered
calstabin2 protein can bind to PKA-phosphorylated
RyR239, indicating that stabilizing the RyR2 channel
complex might be a promising therapeutic strategy for
the treatment of HF74. The 1,4-benzothiazepine derivative JTV519 also effectively enhances calstabin2 binding
to PKA-phosphorylated RyR2 (FIG. 4; BOX 2)75,76. By inducing a conformational change in RyR2 that allows calstabin2 to rebind to the channel, the drug inhibited
diastolic Ca 2+ leakage from the SR, and improved
a Heart failure
contractile performance in a canine experimental
model of HF77. Moreover, we demonstrated in a
genetic mouse model that JTV519 can very effectively
suppress ventricular arrhythmias by rebinding calstabin2
to RyR275, indicating that JTV519 and its derivatives
could constitute a novel class of drugs for the treatment of heart failure and cardiac arrhythmias39,74,75,
although it is not known whether JTV519 directly
binds RyR2 or calstabin2.
Enhancing SR Ca2+ loading
Increasing the reuptake of Ca2+ into the SR by stimulating SERCA2a has been proposed as an attractive
approach to improving systolic and diastolic function
in the failing myocardium78–80. Proof-of-principle
experiments have been performed using adenovirusmediated gene transfer of SERCA2a, which normalized
Ca2+ handling and cardiac contractility in a rat model
of heart failure78. Pharmacological stimulation of the
pump itself is also possible, with several compounds
showing SERCA2a agonist activity in vitro81,82. The
development of such pump-stimulating drugs is still
b β-adrenoceptor blocker
P S
S P
S
S
PKA
P
S
PKA
P
S
S
RyR2
S
RyR2
Sarcoplasmic
reticulum
c JTV519
P S
P
JTV519
S
P
S
PKA
S
P
P
S
S
Secondary
to treatment
Calstabin2-binding site
JTV519
Calstabin2
S
Serine 2809
P
RyR2
Phosphate group
Figure 4 | Action of β-adrenoceptor blockers and JTV519 on ryanodine receptor-2 in heart failure. a | Chronic hyperactivity
of the β-adrenoceptor signalling pathway leads to hyperphosphorylation (P) of serine 2809 on RyR2 by PKA. Phosphorylation of
serine 2809 leads to the dissociation of calstabin2 from the phosphorylated RyR2 monomer. b | β-adrenoceptor blockers
antagonize the β-adrenoceptor signalling pathway and decrease PKA activity in the heart, which reduces the number of serine
2809 residues phosphorylated on RyR2. Reduced phosphorylation of RyR2 by PKA will allow for increased binding of calstabin2.
c | The drug JTV519 increases the binding affinity of calstabin2 for RyR2 (presumably by inducing a conformational change that
allows calstabin2 to bind to PKA-phosphorylated RyR2; see insert). Increased calstabin2 binding and improved contractility might
indirectly lead to decreased β-adrenoceptor signalling in the JTV519-treated failing heart.
© 2004 Nature Publishing Group
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Box 2 | JTV519
The 1,4-benzothiazepine derivative JTV519 was initially developed by Kaneko et al. to prevent cardiac cell damage
due to Ca2+ overload110,111. The drug was found to bind allosterically to annexin V and inhibit annexin-V-dependent
Ca2+ influx110. In addition, JTV519 has been reported to protect the heart against ischaemia/reperfusion-induced Ca2+
overload and myocardial stunning112–115.
JTV519 could also represent a very promising new drug for the treatment of heart failure76,77, as well as ventricular75
and supraventricular arrhythmias116,117. Recent studies have convincingly demonstrated that treatment with JTV519
corrects the defective interaction between calstabin2 and RyR2 in a canine model of heart failure77. Increased binding
of calstabin2 to RyR2 prevents diastolic Ca2+ leak from the sarcoplasmic reticulum (SR), which is believed to underlie
decreased cardiac contractility in heart failure118.
Using a genetic mouse model of exercise-induced cardiac arrhythmias, JTV519 was shown to very effectively prevent
ventricular tachycardias and sudden cardiac death in calstabin2-haploinsufficient mice75. The finding that JTV519 did
not prevent arrhythmias in calstabin2-deficient mice proves that the presence of calstabin2 in the heart is required for
the therapeutic effects of JTV519 (REF. 75). Indeed, the mechanism of action is that JTV519 increases the binding
affinity of RyR2 for calstabin2, and thereby prevents diastolic SR Ca2+ leaks that trigger arrhythmias and contractile
dysfunction in heart failure12,75.
confined to the laboratory and no specific agents have
been put forward for clinical testing. However, there are
also potential inherent risks of SR Ca2+ overload due to
SERCA2a stimulation, which could result in cardiac
arrhythmias83.
Alternatively, SR Ca2+ reuptake might also be induced by increasing levels of PLB phosphorylation84,85,
because PLB inhibits SERCA2a function in its unphosphorylated form. Other studies indicate that antisense
PLB gene transfer into cardiomyocytes isolated from
failing human hearts could normalize contractile
function86. However, augmentation of Ca2+ cycling by
decreasing PLB function might only be beneficial in
certain forms of HF, because two inactivating mutations of PLB seem to be deleterious in humans with
inherited forms of HF characterized by putative inactive
PLB mutants87.
Inhibition of PKC-α has also been proposed as a
pharmacological target for treating HF, because PKC-α
activity is increased in failing hearts14,88,89. Antagonizing
PKC-α is expected to enhance Ca2+ reuptake, and
therefore increase the force of contraction of the
myocardium90. PKC-α inhibitors would inhibit the signalling cascade downstream from the action of classic
inotropic agents, so there might be fewer side effects
compared with β-adrenoceptor blockers or phosphodiesterase inhibitors. Pharmacological tools to inhibit
1.
2.
3.
4.
5.
6.
Hunt, S. A. et al. ACC/AHA guidelines for the evaluation
and management of chronic heart failure in the adult:
executive summary. J. Am. Coll. Cardiol. 38, 2101–2113
(2001).
Pieske, B., Maier, L. S., Bers, D. M. & Hasenfuss, G. Ca2+
handling and sarcoplasmic reticulum Ca2+ content in
isolated failing and nonfailing human myocardium. Circ. Res.
85, 38–46 (1999).
Bers, D. M. Cardiac excitation–contraction coupling. Nature
415, 198–205 (2002).
Fill, M. & Copello, J. A. Ryanodine receptor calcium release
channels. Physiol. Rev. 82, 893–922 (2002).
Wehrens, X. H. & Marks, A. R. Altered function and
regulation of cardiac ryanodine receptors in cardiac disease.
Trends Biochem. Sci. 28, 671–678 (2003).
Sipido, K. R., Carmeliet, E. & Van de Werf, F. T-type Ca2+
current as a trigger for Ca2+ release from the sarcoplasmic
reticulum in guinea-pig ventricular myocytes. J. Physiol. 508,
439–451 (1998).
NATURE REVIEWS | DRUG DISCOVERY
7.
PKC are, with very few exceptions, not isoform specific, and specific inhibitors of PKC-α have not been
identified yet88,91.
Because NCX translocates ions in either direction
across the plasma membrane, mode-specific inhibitors
of NCX could have interesting therapeutic potential. For
example, specific blockade of excessive Ca2+ influx via
reverse-mode NCX activity might reduce Ca2+ overload
and cardiac arrhythmias (TABLES 1,2,3)92,93. On the other
hand, blockade of the forward mode might decrease
excessive Ca2+ efflux from the cytoplasm, and improve
systolic function by increasing SR Ca2+ loading94,95.
Conclusion
A better understanding of the mechanisms underlying
HF has led to the identification of novel therapeutic
targets. Because beta-blockers have been shown to reduce
mortality, patients with HF should receive a β-adrenoceptor blocker unless contraindicated. One reason why
beta-blockers might be beneficial in HF is because they
restore the normal structure and function of the RyR2
channel complex. Reduced PKA hyperphosphorylation
of RyR2 results in rebinding of the channel-stabilizing
protein calstabin2. The new drug JTV519 could also
normalize intracellular Ca2+ release by increasing the
binding of calstabin2 to RyR2. New therapies will aim to
restore normal Ca2+ cycling in the failing heart.
Sipido, K. R., Maes, M. & Van de Werf, F. Low efficiency of
Ca2+ entry through the Na+-Ca2+ exchanger as trigger for
Ca2+ release from the sarcoplasmic reticulum. A comparison
between L-type Ca2+ current and reverse-mode Na+–Ca2+
exchange. Circ. Res. 81, 1034–1044 (1997).
8. Fabiato, A. Calcium-induced release of calcium from the
cardiac sarcoplasmic reticulum. Am. J. Physiol. 245,
C1–C14 (1983).
A landmark paper that provided the first description
and characterization of the excitation–contraction
coupling process in cardiac myocytes.
9. Koss, K. L., Grupp, I. L. & Kranias, E. G. The relative
phospholamban and SERCA2 ratio: a critical determinant
of myocardial contractility. Basic Res. Cardiol. 92 (Suppl. 1),
17–24 (1997).
10. Jones, L. R., Simmerman, H. K., Wilson, W. W., Gurd, F. R.
& Wegener, A. D. Purification and characterization of
phospholamban from canine cardiac sarcoplasmic
reticulum. J. Biol. Chem. 260, 7721–7730 (1985).
11. Bers, D. M. & Bridge, J. H. Relaxation of rabbit ventricular
muscle by Na–Ca exchange and sarcoplasmic reticulum
calcium pump. Ryanodine and voltage sensitivity. Circ. Res.
65, 334–342 (1989).
12. Marx, S. O. et al. PKA phosphorylation dissociates
FKBP12.6 from the calcium release channel (ryanodine
receptor): defective regulation in failing hearts. Cell 101,
365–376 (2000).
The first report on PKA-hyperphosphorylation of the
cardiac ryanodine receptors (RyR2) as a cause of
abnormal Ca2+ cycling and impaired contractility in
human heart failure.
13. Wehrens, X. H., Lehnart, S. E., Reiken, S. R. & Marks, A. R.
Ca2+/calmodulin-dependent protein kinase II
phosphorylation regulates the cardiac ryanodine receptor.
Circ. Res. 94, e61–e70 (2004).
14. Braz, J. C. et al. PKC-α regulates cardiac contractility and
propensity toward heart failure. Nature Med. 10, 248–254
(2004).
© 2004 Nature Publishing Group
VOLUME 3 | JULY 2004 | 7
REVIEWS
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
26.
27.
28.
29.
30.
31.
32.
33.
34.
A demonstration of the role of PKC-α as a nodal
integrator of cardiac contractility by sensing
intracellular Ca2+ and signal transduction events.
Altered PKC-α signalling can profoundly affect the
propensity toward heart failure.
Rockman, H. A., Koch, W. J. & Lefkowitz, R. J. Seventransmembrane-spanning receptors and heart function.
Nature 415, 206–212 (2002).
Gomez, A. M. et al. Defective excitation–contraction
coupling in experimental cardiac hypertrophy and heart
failure. Science 276, 800–806 (1997).
Dzhura, I., Wu, Y., Colbran, R. J., Balser, J. R. & Anderson,
M. E. Calmodulin kinase determines calcium-dependent
facilitation of L-type calcium channels. Nature Cell Biol. 2,
173–177 (2000).
DeSantiago, J., Maier, L. S. & Bers, D. M. Frequencydependent acceleration of relaxation in the heart depends
on CaMKII, but not phospholamban. J. Mol. Cell. Cardiol.
34, 975–984 (2002).
Maier, L. S. et al. Transgenic CaMKIIδC overexpression
uniquely alters cardiac myocyte Ca2+ handling: reduced SR
Ca2+ load and activated SR Ca2+ release. Circ. Res. 92,
904–911 (2003).
Wu, Y., MacMillan, L. B., McNeill, R. B., Colbran, R. J. &
Anderson, M. E. CaM kinase augments cardiac L-type Ca2+
current: a cellular mechanism for long Q-T arrhythmias. Am.
J. Physiol. 276, H2168–H2178 (1999).
Napolitano, R., Vittone, L., Mundina, C., Chiappe de
Cingolani, G. & Mattiazzi, A. Phosphorylation of
phospholamban in the intact heart. A study on the
physiological role of the Ca2+-calmodulin-dependent
protein kinase system. J. Mol. Cell. Cardiol. 24, 387–396
(1992).
Hagemann, D. et al. Frequency-encoding Thr17
phospholamban phosphorylation is independent of Ser16
phosphorylation in cardiac myocytes. J. Biol. Chem. 275,
22532–22536 (2000).
Hagemann, D. & Xiao, R. P. Dual site phospholamban
phosphorylation and its physiological relevance in the heart.
Trends Cardiovasc. Med. 12, 51–56 (2002).
Dempsey, E. C. et al. Protein kinase C isozymes and the
regulation of diverse cell responses. Am. J. Physiol. Lung
Cell. Mol. Physiol. 279, L429–L438 (2000).
Wang, J., Liu, X., Arneja, A. S. & Dhalla, N. S. Alterations in
protein kinase A and protein kinase C levels in heart failure
due to genetic cardiomyopathy. Can. J. Cardiol. 15,
683–690 (1999).
Sugden, P. H. & Bogoyevitch, M. A. Intracellular signalling
through protein kinases in the heart. Cardiovasc. Res. 30,
478–492 (1995).
Pass, J. M. et al. Enhanced PKCβ II translocation and PKCβ II-RACK1 interactions in PKCε-induced heart failure: a role
for RACK1. Am. J. Physiol. Heart Circ. Physiol. 281,
H2500–H2510 (2001).
Tunwell, R. E. et al. The human cardiac muscle ryanodine
receptor-calcium release channel: identification, primary
structure and topological analysis. Biochem. J. 318,
477–487 (1996).
Otsu, K. et al. Molecular cloning of cDNA encoding the Ca2+
release channel (ryanodine receptor) of rabbit cardiac
muscle sarcoplasmic reticulum. J. Biol. Chem. 265,
13472–13483 (1990).
A report of the molecular cloning of the cardiac
ryanodine receptor from rabbit heart, showing that
the RyR2 isoform contains almost 5,000 amino acids
and has 67% sequence similarity to the skeletal
muscle isoform RyR1.
Chu, A., Sumbilla, C., Inesi, G., Jay, S. D. & Campbell, K. P.
Specific association of calmodulin-dependent protein kinase
and related substrates with the junctional sarcoplasmic
reticulum of skeletal muscle. Biochemistry 29, 5899–5905
(1990).
Brillantes, A.–M. B. et al. FKBP12 Optimizes function of the
cloned expressed calcium release channel (ryanodine
receptor). Biophys. J. 66, A19 (1994).
Marx, S. O. et al. Phosphorylation-dependent regulation of
ryanodine receptors. A novel role for leucine/isoleucine
zippers. J. Cell. Biol. 153, 699–708 (2001).
Demonstration that the ryanodine receptor is a
macromolecular complex, and that protein kinase A
and protein phosphatases PP1 and PP2 are targeted
to RyR2 via specific adaptor proteins through leucine/
isoleucine zipper motifs.
Currie, S., Loughrey, C. M., Craig, M. A. & Smith, G. L.
Calcium/calmodulin-dependent protein kinase IIδ
associates with the ryanodine receptor complex and
regulates channel function in rabbit heart. Biochem. J. 377,
357–366 (2004).
Meyers, M. B. et al. Association of sorcin with the cardiac
ryanodine receptor. J. Biol. Chem. 270, 26411–26418 (1995).
35. Zhang, L., Kelley, J., Schmeisser, G., Kobayashi, Y. M. &
Jones, L. R. Complex formation between junctin, triadin,
calsequestrin, and the ryanodine receptor. Proteins of the
cardiac junctional sarcoplasmic reticulum membrane. J.
Biol. Chem. 272, 23389–23397 (1997).
Study establishing the binding of several proteins in
the sarcoplasmic reticulum to the luminal side of the
ryanodine receptor.
36. Flucher, B. E. et al. Triad formation: organization and
function of the sarcoplasmic reticulum calcium release
channel and triadin in normal and dysgenic muscle in vitro.
J. Cell. Biol. 123, 1161–1174 (1993).
37. Collins, J., Tarcsafalvi, A. & Ikemoto, N. Identification of a
region of calsequestrin that binds to the junctional face
membrane of sarcoplasmic reticulum. Biochem. Biophys.
Res. Commun. 167, 189–193 (1990).
38. Viatchenko-Karpinski, S. et al. Abnormal calcium signaling
and sudden cardiac death associated with mutation of
calsequestrin. Circ. Res. 94, 471–477 (2004).
39. Wehrens, X. H. et al. FKBP12.6 deficiency and defective
calcium release channel (ryanodine receptor) function linked
to exercise-induced sudden cardiac death. Cell 113,
829–840 (2003).
40. Marx, S. O., Ondrias, K. & Marks, A. R. Coupled gating
between individual skeletal muscle Ca2+ release channels
(ryanodine receptors). Science 281, 818–821 (1998).
41. Gaburjakova, M. et al. FKBP12 binding modulates
ryanodine receptor channel gating. J. Biol. Chem. 276,
16931–16935 (2001).
42. Hain, J., Onoue, H., Mayrleitner, M., Fleischer, S. &
Schindler, H. Phosphorylation modulates the function of the
calcium release channel of sarcoplasmic reticulum from
cardiac muscle. J. Biol. Chem. 270, 2074–2081 (1995).
43. Valdivia, H. H., Kaplan, J. H., Ellis-Davies, G. C. & Lederer,
W. J. Rapid adaptation of cardiac ryanodine receptors:
modulation by Mg2+ and phosphorylation. Science 267,
1997–2000 (1995).
44. Lokuta, A. J., Rogers, T. B., Lederer, W. J. & Valdivia, H. H.
Modulation of cardiac ryanodine receptors of swine and
rabbit by a phosphorylation-dephosphorylation
mechanism. J. Phys. 487, 609–622 (1995).
45. Sonnleitner, A., Fleischer, S. & Schindler, H. Gating of the
skeletal calcium release channel by ATP is inhibited by
protein phosphatase 1 but not by Mg2+. Cell Calcium 21,
283–290 (1997).
46. Terentyev, D., Viatchenko-Karpinski, S., Gyorke, I., Terentyeva,
R. & Gyorke, S. Protein phosphatases decrease sarcoplasmic
reticulum calcium content by stimulating calcium release in
cardiac myocytes. J. Physiol. 552, 109–118 (2003).
47. Beuckelmann, D., Nabauer, M. & Erdmann, E. Intracellular
calcium handling in isolated ventricular myocytes from
patients with terminal heart failure. Circulation 85,
1046–1055 (1992).
An important paper providing more insight into
abnormalities in Ca2+ cycling in myocytes isolated
from patients with heart failure.
48. Beuckelmann, D. J., Nabauer, M., Kruger, C. & Erdmann, E.
Altered diastolic Ca handling in human ventricular myocytes
from patients with terminal heart failure. Am. Heart J. 129,
684–689 (1995).
49. Kluger, J., Cody, R. J. & Laragh, J. H. The contributions of
sympathetic tone and the renin–angiotensin system to
severe chronic congestive heart failure: response to specific
inhibitors (prazosin and captopril). Am. J. Cardiol. 49,
1667–1674 (1982).
50. Cohn, J. N. et al. Plasma norepinephrine as a guide to
prognosis in patients with chronic congestive heart failure.
N. Engl. J. Med. 311, 819–823 (1984).
51. Bristow, M. R. et al. Reduced β1 receptor messenger RNA
abundance in the failing human heart. J. Clin. Invest. 92,
2737–2745 (1993).
52. Daaka, Y., Luttrell, L. M. & Lefkowitz, R. J. Switching of the
coupling of the β2-adrenergic receptor to different G
proteins by protein kinase A. Nature 390, 88–91 (1997).
53. Ungerer, M., Bohm, M., Elce, J. S., Erdmann, E. &
Lohse, M. J. Altered expression of β-adrenergic receptor
kinase and β1-adrenergic receptors in failing human heart.
Circulation 87, 454–463 (1993).
54. Chen, X. et al. L-type Ca2+ channel density and regulation
are altered in failing human ventricular myocytes and
recover after support with mechanical assist devices. Circ.
Res. 91, 517–524 (2002).
55. Wei, S. K. et al. Protein kinase A hyperphosphorylation
increases basal current but decreases β-adrenergic
responsiveness of the sarcolemmal Na+–Ca2+ exchanger in
failing pig myocytes. Circ. Res. 92, 897–903 (2003).
56. Brillantes, A., Allen, P. & Marks, A. Molecular cloning of the
human cardiac calcium release channel cDNA: expression
studies in end-stage human heart Failure. Circulation 84
(Suppl. II), 442 (1991).
57. Dipla, K., Mattiello, J. A., Margulies, K. B., Jeevanandam, V.
& Houser, S. R. The sarcoplasmic reticulum and the
Na+/Ca2+ exchanger both contribute to the Ca2+ transient of
failing human ventricular myocytes. Circ. Res. 84, 435–444
(1999).
58. Lowes, B. D. et al. Myocardial gene expression in dilated
cardiomyopathy treated with β-blocking agents. N. Engl. J.
Med. 346, 1357–1365 (2002).
59. Reiken, S. et al. Protein kinase A phosphorylation of the
cardiac calcium release channel (ryanodine receptor) in
normal and failing hearts. Role of phosphatases and response
to isoproterenol. J. Biol. Chem. 278, 444–453 (2003).
60. Yano, M. et al. Altered stoichiometry of FKBP12.6 versus
ryanodine receptor as a cause of abnormal Ca2+ leak
through ryanodine receptor in heart failure. Circulation 102,
2131–2136 (2000).
61. MERIT-HF. Effect of metoprolol CR/XL in chronic heart
failure: Metoprolol CR/XL Randomised Intervention Trial in
Congestive Heart Failure (MERIT-HF). Lancet 353,
2001–2007 (1999).
62. Packer, M. et al. The effect of carvedilol on morbidity and
mortality in patients with chronic heart failure. U. S.
Carvedilol Heart Failure Study Group. N. Engl. J. Med. 334,
1349–1355 (1996).
Landmark randomized controlled clinical trail
showing that β-adrenoceptor blockers decrease
mortality in patients with heart failure.
63. CIBIS-II. The Cardiac Insufficiency Bisoprolol Study II (CIBISII): a randomised trial. Lancet 353, 9–13 (1999).
64. Reiken, S. et al. Beta-blockers restore calcium release
channel function and improve cardiac muscle performance
in human heart failure. Circulation 107, 2459–2466 (2003).
65. Reiken, S. et al. β-adrenergic receptor blockers restore
cardiac calcium release channel (ryanodine receptor)
structure and function in heart failure. Circulation 104,
2843–2848 (2001).
66. Doi, M. et al. Propranolol prevents the development of heart
failure by restoring FKBP12.6-mediated stabilization of
ryanodine receptor. Circulation 105, 1374–1379 (2002).
67. Masson, S., Chimenti, S. & Salio, M. CHF-1024, a DA2/α2
agonist, blunts norepinephrine excretion and cardiac fibrosis
in pressure overload. Cardiovasc. Drug Ther. 15, 131–138
(2001).
68. Bayes, M., Rabasseda, X. & Prous, J. R. Gateways to
clinical trials. Methods Find. Exp. Clin. Pharmacol. 25,
565–597 (2003).
69. Freedman, N. J. et al. Phosphorylation and desensitization
of the human β1-adrenergic receptor. Involvement of Gprotein-coupled receptor kinases and cAMP-dependent
protein kinase. J. Biol. Chem. 270, 17953–17961 (1995).
70. Harding, V. B., Jones, L. R., Lefkowitz, R. J., Koch, W. J. &
Rockman, H. A. Cardiac β-ARK1 inhibition prolongs survival
and augments beta-blocker therapy in a mouse model of
severe heart failure. Proc. Natl Acad. Sci. USA 98,
5809–5814 (2001).
71. Rockman, H. A. et al. Expression of a β-adrenergic receptor
kinase 1 inhibitor prevents the development of myocardial
failure in gene-targeted mice. Proc. Natl Acad. Sci. USA 95,
7000–7005 (1998).
Experimental study demonstrating the important role
of β-adrenoceptor kinase inhibitors as modifiers of βadrenoceptor signalling in the development of heart
failure.
72. Gullestad, L. et al. Effect of metoprolol CR/XL on exercise
tolerance in chronic heart failure — a substudy to the
MERIT-HF trial. Eur. J. Heart Fail. 3, 463–468 (2001).
73. White, M. et al. Role of β-adrenergic receptor
downregulation in the peak exercise response in patients
with heart failure due to idiopathic dilated cardiomyopathy.
Am. J. Cardiol. 76, 1271–1276 (1995).
74. Most, P. & Koch, W. J. Sealing the leak, healing the heart.
Nature Med. 9, 993–934 (2003).
75. Wehrens, X. H. et al. Protection from cardiac arrhythmia
through ryanodine receptor-stabilizing protein calstabin2.
Science 304, 292–296 (2004).
76. Kohno, M. et al. A new cardioprotective agent, JTV519,
improves defective channel gating of ryanodine receptor in
heart failure. Am. J. Physiol. Heart Circ. Physiol. 284,
H1035–H1042 (2003).
77. Yano, M. et al. FKBP12.6-mediated stabilization of calciumrelease channel (ryanodine receptor) as a novel therapeutic
strategy against heart failure. Circulation 107, 477–484
(2003).
78. Hajjar, R. J. et al. Modulation of ventricular function through
gene transfer in vivo. Proc. Natl Acad. Sci. USA 95,
5251–5256 (1998).
79. Minamisawa, S. et al. Chronic phospholamban–sarcoplasmic reticulum calcium ATPase interaction is the critical
calcium cycling defect in dilated cardiomyopathy. Cell 99,
313–322 (1999).
© 2004 Nature Publishing Group
8
| JULY 2004 | VOLUME 3
www.nature.com/reviews/drugdisc
REVIEWS
80. Meyer, M. & Dillmann, W. H. Sarcoplasmic reticulum Ca2+ATPase overexpression by adenovirus mediated gene
transfer and in transgenic mice. Cardiovasc. Res. 37,
360–366 (1998).
81. Ohizumi, Y., Sasaki, N. & Shibusawa, K. Stimulation of
sarcoplasmic reticulum Ca2+-ATPase by gingerol analogous.
Biol. Pharm. Bull. 19, 1377–1379 (1996).
82. Berrebi-Bertrand, I., Lahouratete, P. & Lahouratete, V.
Mechanisms of action of sarcoplasmic reticulum calciumuptake activators: discrimination between sarcoplasmic
reticulum Ca2+-ATPase and phospholamban interaction. Eur.
J. Biochem. 247, 801–809 (1997).
83. Volders, P. G. et al. Similarities between early and delayed
afterdepolarizations induced by isoproterenol in canine
ventricular myocytes. Cardiovasc. Res. 34, 348–359 (1997).
84. Hoshijima, M. et al. Chronic suppression of heart-failure
progression by a pseudophosphorylated mutant of
phospholamban via in vivo cardiac rAAV gene delivery.
Nature Med. 8, 864–871 (2002).
85. Iwanaga, Y. et al. Chronic phospholamban inhibition
prevents progressive cardiac dysfunction and pathological
remodeling after infarction in rats. J. Clin. Invest. 113,
727–736 (2004).
86. del Monte, F., Harding, S. E., Dec, G. W., Gwathmey, J. K. &
Hajjar, R. J. Targeting phospholamban by gene transfer in
human heart failure. Circulation 105, 904–907 (2002).
87. Haghighi, K. et al. Human phospholamban null results in
lethal dilated cardiomyopathy revealing a critical difference
between mouse and human. J. Clin. Invest. 111, 869–876
(2003).
88. Bowling, N. et al. Increased protein kinase C activity and
expression of Ca2+-sensitive isoforms in the failing human
heart. Circulation 99, 384–391 (1999).
89. Wang, J., Liu, X., Sentex, E., Takeda, N. & Dhalla, N. S.
Increased expression of protein kinase C isoforms in heart
failure due to myocardial infarction. Am. J. Physiol. Heart
Circ. Physiol. 284, H2277–H2287 (2003).
90. Hahn, H. S. et al. Protein kinase Cα negatively regulates
systolic and diastolic function in pathological hypertrophy.
Circ. Res. 93, 1111–1119 (2003).
91. Vlahos, C. J., McDowell, S. A. & Clerk, A. Kinases as
therapeutic targets for heart failure. Nature Rev. Drug Discov.
2, 99–113 (2003).
92. Mattiello, J. A., Margulies, K. B., Jeevanandam, V. & Houser,
S. R. Contribution of reverse-mode sodium–calcium
exchange to contractions in failing human left ventricular
myocytes. Cardiovasc. Res. 37, 424–431 (1998).
93. Iwamoto, T., Watano, T. & Shigekawa, M. A novel
isothiourea derivative selectively inhibits the reverse mode of
Na+/Ca2+ exchange in cells expressing NCX1. J. Biol. Chem.
271, 22391–22397 (1996).
94. Hobai, I. A. & O’Rourke, B. Enhanced Ca2+-activated
Na+–Ca2+ exchange activity in canine pacing-induced heart
failure. Circ. Res. 87, 690–698 (2000).
95. Shigekawa, M. & Iwamoto, T. Cardiac Na+–Ca2+ exchange:
molecular and pharmacological aspects. Circ. Res. 88,
864–876 (2001).
96. Prestle, J. et al. Overexpression of FK506-binding protein
FKBP12.6 in cardiomyocytes reduces ryanodine receptormediated Ca2+ leak from the sarcoplasmic reticulum and
increases contractility. Circ. Res. 88, 188–194 (2001).
97. Miyamoto, M. I. et al. Adenoviral gene transfer of SERCA2a
improves left-ventricular function in aortic-banded rats in
transition to heart failure. Proc. Natl Acad. Sci. USA 97,
793–798 (2000).
98. Ennis, I. L., Li, R. A., Murphy, A. M., Marban, E. & Nuss, H. B.
Dual gene therapy with SERCA1 and Kir2.1 abbreviates
excitation without suppressing contractility. J. Clin. Invest.
109, 393–400 (2002).
99. Ji, Y., Loukianov, E., Loukianova, T., Jones, L. R. &
Periasamy, M. SERCA1a can functionally substitute for
SERCA2a in the heart. Am. J. Physiol. 276, H89–H97 (1999).
100. Magee, W. P. et al. Differing cardioprotective efficacy of the
Na+/Ca2+ exchanger inhibitors SEA0400 and KB-R7943.
Am. J. Physiol. Heart Circ. Physiol. 284, H903–H910 (2003).
101. Wehrens, X. H., Vos, M. A., Doevendans, P. A. &
Wellens, H. J. Novel insights in the congenital long QT
syndrome. Ann. Intern. Med. 137, 981–992 (2002).
102. Marks, A. R., Priori, S., Memmi, M., Kontula, K. & Laitinen,
P. J. Involvement of the cardiac ryanodine
receptor/calcium release channel in catecholaminergic
polymorphic ventricular tachycardia. J. Cell. Physiol. 190,
1–6 (2002).
103. Leenhardt, A. et al. Catecholaminergic polymorphic
ventricular tachycardia in children. A 7-year follow-up of 21
patients. Circulation 91, 1512–1519 (1995).
104. Laitinen, P. J. et al. Mutations of the cardiac ryanodine
receptor (RyR2) gene in familial polymorphic ventricular
tachycardia. Circulation 103, 485–490 (2001).
105. Priori, S. G. et al. Mutations in the cardiac ryanodine
receptor gene (hRyR2) underlie catecholaminergic
polymorphic ventricular tachycardia. Circulation 103,
196–200 (2001).
106. Fisher, J. D., Krikler, D. & Hallidie-Smith, K. A. Familial
polymorphic ventricular arrhythmias: a quarter century of
successful medical treatment based on serial exercisepharmacologic testing. J. Am. Coll. Cardiol. 34,
2015–2022 (1999).
107. Swan, H. et al. Arrhythmic disorder mapped to chromosome
1q42–q43 causes malignant polymorphic ventricular
tachycardia in structurally normal hearts. J. Am. Coll.
Cardiol. 34, 2035–2042 (1999).
108. George, C. H., Higgs, G. V. & Lai, F. A. Ryanodine receptor
mutations associated with stress-induced ventricular
tachycardia mediate increased calcium release in stimulated
cardiomyocytes. Circ. Res. 93, 531–540 (2003).
109. Jiang, D., Xiao, B., Zhang, L. & Chen, S. R. Enhanced basal
activity of a cardiac Ca2+ release channel (ryanodine
receptor) mutant associated with ventricular tachycardia and
sudden death. Circ. Res. 91, 218–225 (2002).
110. Kaneko, N. New 1,4-benzothiazepine derivative, K201,
demonstrates cardioprotective effects against sudden
cardiac cell death and intracellular calcium blocking action.
Drug Dev. Res. 33, 429–438 (1994).
111. Kaneko, N., Ago, H., Matsuda, R., Inagaki, E. & Miyano, M.
Crystal structure of annexin V with its ligand K-201 as a
calcium channel activity inhibitor. J. Mol. Biol. 274, 16–20
(1997).
112. Kawabata, H., Ryomoto, T. & Ishikawa, K. Effect of a novel
cardioprotective agent, JTV-519, on metabolism,
contraction and relaxation in the ischemia-reperfused rabbit
heart. Jpn Circ. J. 64, 772–776 (2000).
113. Inagaki, K., Kihara, Y., Izumi, T. & Sasayama, S. The
cardioprotective effects of a new 1,4-benzothiazepine
derivative, JTV519, on ischemia/reperfusion-induced Ca2+
overload in isolated rat hearts. Cardiovasc. Drugs Ther. 14,
489–495 (2000).
114. Inagaki, K. et al. Anti-ischemic effect of a novel
cardioprotective agent, JTV519, is mediated through
specific activation of delta-isoform of protein kinase C in
rat ventricular myocardium. Circulation 101, 797–804
(2000).
115. Ito, K. et al. JTV-519, a novel cardioprotective agent,
improves the contractile recovery after ischaemiareperfusion in coronary perfused guinea-pig ventricular
muscles. Br. J. Pharmacol. 130, 767–76 (2000).
116. Nakaya, H., Furusawa, Y., Ogura, T., Tamagawa, M. &
Uemura, H. Inhibitory effects of JTV-519, a novel
cardioprotective drug, on potassium currents and
experimental atrial fibrillation in guinea-pig hearts. Br. J.
Pharmacol. 131, 1363–1372 (2000).
117. Kumagai, K., Nakashima, H., Gondo, N. & Saku, K.
Antiarrhythmic effects of JTV-519, a novel cardioprotective
drug, on atrial fibrillation/flutter in a canine sterile pericarditis
model. J. Cardiovasc. Electrophysiol. 14, 880–884 (2003).
118. Schlotthauer, K. & Bers, D. M. Sarcoplasmic reticulum Ca2+
release causes myocyte depolarization. Underlying
mechanism and threshold for triggered action potentials.
Circ. Res. 87, 774–780 (2000).
Competing interests statement
The authors declare that they have competing financial interests:
see Web version for details.
Online links
DATABASES
The following terms in this article are linked online to:
Entrez Gene:
http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=gene
Annexin V | calstabin2 | CaMKII | NCX | PKC | PLB | RyR2 |
SERCA2a |
FURTHER INFORMATION
American College of Cardiology/American Heart Association
Guidelines for the Evaluation and Management of Heart
Failure: www.acc.org/clinical/guidelines/failure/hf_index.htm
Andrew R. Marks Lab Home Page:
http://www.cumc.columbia.edu/dept/physio/physio2/marks_new/
Access to this interactive links box is free online.
© 2004 Nature Publishing Group
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