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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 © 2004 Nature Publishing Group NATURE REVIEWS | DRUG DISCOVERY VOLUME 3 | JULY 2004 | 1 REVIEWS 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. 2 | JULY 2004 | VOLUME 3 (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. © 2004 Nature Publishing Group www.nature.com/reviews/drugdisc 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) © 2004 Nature Publishing Group NATURE REVIEWS | DRUG DISCOVERY 2+ VOLUME 3 | JULY 2004 | 3 REVIEWS 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 4 | JULY 2004 | VOLUME 3 www.nature.com/reviews/drugdisc REVIEWS 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. NATURE REVIEWS | DRUG DISCOVERY VOLUME 3 | JULY 2004 | 5 REVIEWS 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 6 | JULY 2004 | VOLUME 3 www.nature.com/reviews/drugdisc REVIEWS 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). 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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 NATURE REVIEWS | DRUG DISCOVERY VOLUME 3 | JULY 2004 | 9