REVIEWS
PHOSPHOLAMBAN: A CRUCIAL REGULATOR OF CARDIAC CONTRACTILITY David H. MacLennan* and Evangelia G. Kranias ‡ Heart failure is a major cause of death and disability. Impairments in blood circulation that accompany heart failure can be traced, in part, to alterations in the activity of the sarcoplasmic reticulum Ca2+ pump that are induced by its interactions with phospholamban, a reversible inhibitor. If phospholamban becomes superinhibitory or chronically inhibitory, contractility is diminished, inducing dilated cardiomyopathy in mice and humans. In mice, phospholamban seems to encumber an otherwise healthy heart, but humans with a phospholamban-null genotype develop early-onset dilated cardiomyopathy. C A LC I U M CARDIAC RESERVE
The maximum percentage that the cardiac output can increase above normal — the ability of the heart to adjust rapidly to demands placed on it. β-AGONIST
A molecule that activates β-adrenergic receptors.
*Banting and Best Department of Medical Research, University of Toronto, Charles H. Best Institute, 112 College Street, Toronto, Ontario M5G 1L6, Canada. ‡ Department of Pharmacology and Cell Biophysics, University of Cincinnati College of Medicine, Cincinnati, Ohio 45267, USA. Correspondence to D.H.M. e-mail: david.maclennan@ utoronto.ca doi:10.1038/nrm1151
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Cardiac function is regulated on a beat-to-beat basis through the sympathetic nervous system. When the demand arises, the heart can respond to stress and increase blood flow to peripheral tissues within seconds. This is thanks to the large CARDIAC RESERVE in humans; the slow heart beat rate and submaximal contractility at rest are increased markedly after the release of adrenaline into the blood1. Adrenaline and other β-AGONISTS initiate an important signal-transduction pathway in the heart by binding to and activating β-ADRENERGIC RECEPTORS in the cell membrane (FIG. 1). The signal proceeds through Gs proteins to stimulate the formation of cyclic AMP by adenylate cyclase2. Elevations in cAMP concentration activate cAMP-dependent protein kinase (PKA), which then phosphorylates and alters the function of a few cardiac proteins that have key effects on the overall cardiac function. Prominent among these proteins is phospholamban (PLN), a small, reversibly phosphorylated, transmembrane protein that is located in the cardiac SARCOPLASMIC RETICULUM (SR), which, depending on its phosphorylation state, binds to and regulates the activity of a Ca2+ pump, the SARCO(ENDO)PLASMIC RETICULUM Ca -ATPASE SERCA2a. The trigger for cardiac contraction is the elevation of the Ca2+ concentration in the cytoplasm of the muscle cell (BOX 1), which is mediated by Ca2+-release channels 2+
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(RYANODINE RECEPTORS; RyRs) that tap the Ca2+ store in the lumen of the SR, or plasma-membrane Ca 2+ channels (DIHYDROPYRIDINE RECEPTORS; DHPRs) that tap the high concentrations of Ca2+ in the extracellular space. The trigger for relaxation is the lowering of the cytosolic Ca2+ concentration by the combined activity of SERCA2a, PLASMA-MEMBRANE Ca -ATPASES (PMCAs) and Na /Ca EXCHANGERS (NCXs) that replenish the SR and extracellular Ca2+ stores3. In humans, the activity of SERCA2a determines the rate of removal of >70% of cytosolic Ca 2+, thereby determining the rate of relaxation of the heart, and influences cardiac contractility by determining the size of the lumenal Ca2+ store that is available for release in the next beat. In its dephosphorylated state, PLN binds to SERCA2a at resting Ca2+ concentrations and inhibits Ca2+ pump activity: phosphorylation of PLN alters the PLN–SERCA2a interaction, relieving Ca2+-pump inhibition and enhancing relaxation rates (LUSITROPIC effects) and contractility (INOTROPIC effects)4. In this review, we discuss advances in the understanding of the molecular aspects of the interaction between PLN and SERCA2a that have been derived from extensive mutagenesis and structural modelling studies5. We describe gain- and loss-of-inhibitoryfunction PLN mutants, which led to the investigation 2+
+
2+
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REVIEWS β-adrenergic receptor
β-ADRENERGIC STIMULATION
The ligand- or agonistdependent activation of β-adrenergic receptors and subsequent signalling events.
DHPR
NCX
PMCA Ca2+
1 Ca2+
Adenylate cyclase
SARCOPLASMIC RETICULUM
(SR). An organellar membrane system that encases each myofibril within a muscle cell. Its essential components are a Ca2+-ATPase (Ca2+ pump), lumenal Ca2+-sequestering proteins and a Ca2+-release channel.
Gsα 3 Na+
Ca2+ Ca2+
Myofibril
β-adrenergic receptor kinase
+ + + +
RyR Junctin/triadin
SARCO(ENDO)PLASMIC RETICULUM Ca2+-ATPASE
(SERCA). A pump that is located in sarcoplasmic or endoplasmic reticulum membranes that couples ATP hydrolysis to the transport of Ca2+ from cytosolic to lumenal spaces.
P
C-protein
P
Troponin I
cAMP
DIHYDROPYRIDINE RECEPTOR
(DHPR). A slow, or L-type, voltage-dependent Ca2+-entry channel that is located in the plasma membrane. DHPRs require a membrane potential that is greater than –30 mV for activation, and they are commonly found in neurons, neuroendocrine cells and muscle cells. PLASMA-MEMBRANE Ca2+-ATPASE
(PMCA). A plasma-membrane pump that couples ATP hydrolysis to the transport of Ca2+ from cytosolic to extracellular spaces.
Ca2+
Ca2+
ATP
SERCA2a
RYANODINE RECEPTOR
(RyR). A Ca2+-release channel that is located in the membrane of the sarcoplasmic and the endoplasmic reticulum that is regulated by protein–protein interactions with the dihydropyridine receptor and by a series of ligands, including Ca2+ itself.
Ca2+–calsequestrin
PKA
Ca2+
PLN P
PKA [Ca2+] Systolic 1 µM Diastolic 0.1 µM SR 500 µM Extracellular 2–3 mM
AKAP PP1
RgI SR
Mitochondria
Figure 1 | Interactions between cardiac signalling pathways. The heart provides an example of how two signalling pathways that are involved in elevating the levels of two intracellular second messengers, cyclic AMP (cAMP) and Ca2+, can interact physiologically (the two pathways are shown by the blue and red arrows, respectively). In response to depolarization, Ca2+ enters the cytoplasm through Ca2+ channels in the plasma membrane (dihydropyridine receptors; DHPRs). This ‘trigger’ Ca2+ then binds to the Ca2+-release channels (ryanodine receptors; RyRs) to stimulate Ca2+ release from the sarcoplasmic reticulum (SR). After activating muscle contraction by binding to troponin C in the thin filament, Ca2+ is removed from the myoplasm by plasma-membrane Ca2+-ATPases (PMCAs) or Na+/Ca2+ exchangers (NCXs), which are located in the plasma membrane, or by the sarco(endo)plasmic reticulum Ca2+-ATPase SERCA2a, which is located in the SR. As SERCA2a activity accounts for the removal of >70% of myoplasmic Ca2+ in humans, it determines both the rate of Ca2+ removal (and, consequently, the rate of cardiac muscle relaxation) and the size of the Ca2+ store (which affects cardiac contractility in the subsequent beat). SERCA2a activity is regulated by its interaction with phospholamban (PLN), which is a target for phosphorylation by protein kinase A (PKA) through the second signalling pathway — the β-adrenergic-receptor pathway (see main text). In its dephosphorylated form, PLN is an inhibitor of SERCA2a, but, when phosphorylated by PKA (or Ca2+/CaM kinase), PLN dissociates from SERCA2a, activating this Ca2+ pump. As a result, the rate of cardiac relaxation is increased and, on subsequent beats, contractility is increased in proportion to the elevation in the size of the SR Ca2+ store and the resulting increase in Ca2+ release from the SR. PLN is dephosphorylated by a protein phosphatase (PP1), which terminates the stimulation phase. AKAP, A-kinase anchoring protein; [Ca2+], Ca2+ concentration; Rgl, regulatory binding subunit A.
Na+/Ca2+ EXCHANGER
(NCX). A plasma-membrane enzyme that exchanges three moles of Na+ for one mole of Ca2+ either inward or outward, depending on ionic gradients across the membrane. LUSITROPIC
Affecting cardiac relaxation. INOTROPIC
Affecting the force of cardiac contractions. CARDIOMYOPATHY
A disease of the heart muscle. VMAX
The maximal rate of enzymatic
of mutations in the PLN gene as a potential cause of 6 CARDIOMYOPATHY . We also discuss studies that were carried out in transgenic mice that overexpress superinhibitory forms of PLN and that have confirmed this potential7–9. The creation and analysis of PLN-null mice10 and of mice that overexpress PLN 11 have enabled the elucidation of the remarkable role of PLN in the regulation of the kinetics of cardiac contractility12. The fact that ablation of PLN or suppression of the inhibitory function of PLN can intervene to prevent the progression of dilated cardiomyopathy in wellcharacterized animal models opens the door to the investigation of the diverse pathways that lead to endstage heart failure and to potential therapeutic interventions13–15. Finally, we discuss very recent data,
NATURE REVIEWS | MOLECUL AR CELL BIOLOGY
which show not only that mutations in PLN can cause dilated cardiomyopathy in humans, but also introduce new principles16,17. We learn that humans, unlike mice, require PLN and that dilated cardiomyopathy can result from chronic inhibition of SERCA2a by the prevention of phosphorylation of PLN by PKA. Phospholamban–SERCA interactions
One of the most useful assays for the interaction between PLN and SERCA molecules is the measurement of the Ca2+ dependence of Ca2+ transport in isolated SR vesicles18. By varying the Ca2+ concentration over four orders of magnitude and calculating the rate of ATP-dependent Ca2+ transport relative to the maximal rate observed at 10 µM Ca2+ (V ), the apparent MAX
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REVIEWS
Box 1 | Regulation and dysregulation of the Ca2+ transient During the evolution of phosphate- and organic-acid-based metabolic processes, it became important that cytosolic Ca2+ concentrations be maintained below ~10 µM; and so the need for Ca2+ pumps that lower resting Ca2+ concentrations to 4 at Θ=0.00)16. The effects of the PLN Arg9Cys mutation were characterized by expression in heterologous cell culture, by the creation of a transgenic mouse and by analysis of cardiac tissue obtained from an explant. In all cases, the level of PLN phosphorylation was reduced markedly. In heterologous cell culture, the Arg9Cys mutant PLN had reduced inhibitory properties in the homozygous state, but, when expressed in the heterozygous state, did not function in a dominant fashion to prevent the inhibition of SERCA2a by wild-type PLN. The key effect of the mutation was enhancement of the affinity of Arg9Cys mutant PLN for PKA. In attempting to phosphorylate mutant PLN, PKA becomes trapped in a stabilized mutant PLN–PKA complex and can no longer dissociate to phosphorylate wild-type PLN molecules. The effect seems to be local and restricted to the SR, perhaps because a specific fraction of PKA is associated with
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REVIEWS
Contraction
DHPR
1 Ca2+
RyR
NCX
SR Myofibrils
3 Na+ 1 Ca2+
DHPR
SERCA
NCX
PLN 3 Na+
Mouse Human
Relaxation
Figure 5 | Differences in the regulation of Ca2+ transients between mice and humans. With heart beat rates of up to 800 beats per min, mice are normally at two thirds of the theoretical maximum and the sarcoplasmic reticulum (SR) Ca2+ store is large at normal heart rates. Mice cycle 90% of their cardiac Ca2+ through the SR and only 10% through extracellular spaces in a very short temporal cycle. Humans, with heart beat rates ranging between 60 and 180 beats per min, have a larger cardiac reserve. A major part of this reserve is the potential for a gain in SR Ca2+ content. Normally humans cycle only 70% of their Ca2+ through the SR in a longer temporal cycle. The red arrows show the cycle in humans, including the involvement of the dihydropyridine receptor (DHPR) and Na+/Ca2+ exchanger (NCX), and the blue arrows show the cycle in mice, again including the DHPR and NCX. The red dots represent Ca2+ ions. For a general explanation of cardiac Ca2+ signalling and, specifically, the roles of ryanodine receptors (RyRs), phospholamban (PLN) and sarco(endo)plasmic Ca2+-ATPase (SERCA), see FIG. 1.
the SR through A-kinase anchoring proteins (AKAPs)98. These results explain the dominant effects of the mutation. Affected individuals must go through life with chronically inhibited SERCA2a and can never draw on their full cardiac reserve. The long-term effects of the chronic, specific inhibition of PLN phosphorylation on Ca2+ transients are clearly sufficiently deleterious to cause the onset of dilated cardiomyopathy in humans in their teenage years. However, analysis of the transgenic expression of a PLN Ser16Ala, Thr17Ala double mutant in mice provided different results. This mutant PLN could not be phosphorylated, but produced the same shift in Ca2+ affinity as unphosphorylated, wild-type PLN. The measures of basal contractility and Ca 2+ transients were similar in wild-type and mutant myocytes, but isoproterenol did not increase the rate of Ca2+ removal in the mutant. Increased L-type Ca 2+ current (ICa) density, with unaltered characteristics, was the principal compensation in mutant myocytes. This ICa modulation might compensate in part for the loss in SERCA2a responsiveness and thereby partially normalize β-adrenergic inotropy in Ser16Ala, Thr17Ala double-mutant mice, allowing them to survive without cardiomyopathy73.
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A second human PLN mutation, Leu39stop, was discovered in two large Greek families17. The heterozygous inheritance of the Leu39stop mutation in one family led to left ventricular hypertrophy in one third of the older affected family members, without diminished contractile performance. However, the inheritance of two copies of the mutant PLN gene led to dilated cardiomyopathy and heart failure in two teenage siblings. In heterologous expression studies, the Leu39stop mutant protein was unstable or misrouted to other membranes and no protein was detected in the endoplasmic reticulum of these cells or in a cardiac explant from one of the affected individuals. As a result, there was no effect of the mutant protein, in either the homozygous or heterozygous state, on the Ca2+ affinity of SERCA2a. Accordingly, these two homozygous mutant individuals can be considered to be equivalent to a PLN-null genotype with a phenotype of dilated cardiomyopathy. So, in contrast to the benefits of PLN ablation in mouse, humans that lack PLN develop lethal cardiomyopathy. A caveat in these studies is that the number of affected individuals is very low and the lod score for linkage of the mutation to the disease is low. So, how can this discrepancy between the cardiac phenotypes in mice and humans be explained? In contrast to humans, mice have relatively little cardiac reserve and differ in the balance of myocyte Ca2+ fluxes (FIG. 5). With heart beat rates ranging up to 800 beats per min, mice are normally at two thirds of the theoretical maximum and the SR Ca2+ store is nearly full at normal heart rates. Humans, with heart beat rates ranging between 60 and 180 beats per min, have a large cardiac reserve1. A major part of this reserve is the potential for a gain in SR Ca2+ content99. If defective PLN regulation limits the normal gain in SR Ca2+ content with increasing heart rate, cardiac reserve will be compromised, as observed in human heart failure99. So, PLN modulation might be of more paramount importance in humans, who have a life span that is 40 times that of a mouse. Conclusion
PLN is an important regulator of the kinetics of cardiac Ca2+ transients and of contractility, and is a key determinant of β-adrenergic stimulation of the heart. Significant advances have been made in understanding the structure and dynamics of the interaction of PLN with SERCA2a. And the generation of mouse models with altered PLN expression levels or activity has allowed a thorough understanding of the ‘physiological brake’ that is provided by PLN in vivo. Although the mouse and human myocardia differ in excitation–contraction coupling and in the isoforms of contractile proteins that they express in different developmental stages, studies of mice form the basis for comparative studies in humans. They also form the basis for potential therapeutic approaches in humans through disruption of PLN inhibition of SERCA2a. Depressed SR Ca 2+ cycling is a common feature of the depressed contractility that is seen in hypertrophied and failing myocardia, so that interference
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REVIEWS with the PLN–SERCA2a interaction through a ‘molecular inotrope’ might rescue contractile dysfunction and prevent transcription-based remodelling of the heart, thereby preventing progression to heart failure. It is evident, however, that elimination of PLN activity might not benefit all forms of heart failure, especially those in which depressed SR Ca2+ cycling is not a primary event.
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Acknowledgements We are grateful to D. Bers, A. Gramolini, K. Haghighi, G. Inesi and C. Toyoshima for their helpful comments on this manuscript. The original studies described in this review were supported by grants to D.H.M. from the Heart and Stroke Foundation of Ontario, the Canadian Institutes for Health Research and the Canadian Genetic Diseases Network of Centres of Excellence, and to E.G.K. by grants from the National Institutes of Health (USA).
Online links DATABASES The following terms in this article are linked online to: LocusLink: http://www.ncbi.nlm.nih.gov/LocusLink/ AKAPs | ATP2A1 | ATP2A2 | Ca2+/CaM kinase | CACNA1S | CASQ2 | PKA | PMCAs | RYR1 | RYR2 Swiss-Prot: http://www.expasy.ch/ calmodulin | calsequestrin | MKP-1 | PLN | SERCA1a | SERCA2a | SLN Access to this interactive links box is free online.
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