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The role of protein S-acylation in cardiac function

by Dr O. Robertson-Gray and Dr W. Fuller

Cardiovascular disease (CVD) is the umbrella term for conditions affecting the heart or blood vessels and remains the leading cause of death worldwide. New therapeutic strategies to aid in the prevention and treatment of CVD are therefore necessary. This article discusses the potential for S-acylation, a post-translational protein modification, to be used as a therapeutic target for CVD primarily affecting cardiac function.

Protein S-acylation and zDHHC-containing protein acyltransferases

Protein S-acylation is the reversible post-translational attachment of a 16-carbon fatty acid, typically palmitate, to cysteine thiols via a thioester bond and is commonly referred to as ‘palmitoylation’. Catalysed by zinc finger and DHHC-motif-containing protein acyltransferases (zDHHC-PATs), the addition of palmitate increases protein hydrophobicity and alters many key features such as protein structure, assembly and, importantly, function. Recent investigations have determined that at least 454 cardiac proteins covering numerous cellular functions are palmitoylated [1]. Twenty-three human zDHHC-PAT isoforms are expressed throughout the secretory pathway; however, across species the expression of just 11 have been detected within the heart (zDHHCs 1, 2, 4, 5, 7, 8, 9, 17, 18, 20 & 21; [1]).

The reversal of palmitoylation (depalmitoylation) occurs via the activity of acyl-protein thioesterases (APTs), APT1 and APT2 [2, 3]. For many years these enzymes were considered the only two capable of catalysing depalmitoylation; however, it has since been determined that ABHD17A, ABHD17B and ABHD17C are also protein depalmitoylases [4] and the future discovery of additional enzymes capable of depalmitoylation can be anticipated.

Palmitoylation and cardiac excitation–contraction coupling

Excitation–contraction coupling (E–C coupling) is the process which instigates cardiac muscle contraction through numerous intracellular events that are prompted by the electrical excitation of cardiomyocytes (Fig. 1). This process is precisely controlled via the tightly-regulated activity of an array of ion channels and transporters, several of which are established drug targets for the management of cardiovascular disease (CVD).

An action potential triggers Ca2+ movement across the cell membrane via opening of L-Type Ca2+ channels (LTCCs). This in turn activates ryanodine receptors (RyR2s) located on the sarcoplasmic reticulum (SR) prompting Ca2+-induced Ca2+ release (CICR) from the SR store. This newly released Ca2+ instigates contraction by binding to the myofilament protein troponin C, in turn causing cross bridging of actin and myosin filaments. For the cell to relax, the sarcoplasmic/ endoplasmic reticulum calcium-adenosine triphosphatase 2a (SERCA2a) drives Ca2+ from the cytosol of the cell into the SR. To further reduce the cytosolic Ca2+ concentration, the sodiumcalcium exchanger (NCX1) at the sarcolemmal membrane works in forward mode to extrude one Ca2+ ion in exchange for three Na+ ions. Ca2+ consequently becomes unbound from troponin C resulting in relaxation. Although a plethora of E–C coupling proteins are known to be palmitoylated, only a limited number can be discussed within the scope of this article.

Nav1.5 voltage-gated sodium channel

Nav1.5 is the molecular target of class 1 antiarrhythmic drugs and is the 2016-amino-acid pore-forming α-subunit of the voltage-gated sodium channel. The α-subunit generates sodium currents (INa) by permitting the flow of sodium ions through the membrane and plays a pivotal role in the cardiac conduction system by generating the fast depolarization at the start of the cardiac action potential (phase 0). Mutations in the gene encoding Nav1.5 (SCN5A) are associated with an assortment of cardiac diseases including Brugada and long QT syndrome (type 3) emphasizing the importance of maintaining Nav1.5 function.

Although Nav1.5 palmitoylation sites are yet to be experimentally verified, bioinformatics software CSS-Palm 3.0 identified four potential palmitoylation sites located in the cytoplasmic linker between the second and third domain of the channel (C981, C1176, C1178 and C1179). Interestingly, one of these, C981, is the site of a missense mutation implicated in familial long QT syndrome [6]. To elucidate the role of Nav1.5 palmitoylation in the heart, a variety of methods have been utilized including the mutation of all four identified cysteines to alanine via site-directed mutagenesis, in addition to electrophysiological and biochemical assays. Such studies have determined that both channel availability and ‘late’ sodium current is enhanced by palmitoylation, which in turn increases cardiac excitability and prolongs the cardiac action potential. Conversely, inhibiting palmitoylation enhances closed-state channel inactivation and reduces myocyte excitability [7]. Ultimately then, Nav1.5 palmitoylation controls channel properties of profound importance for the heart. Studies are now required to identify the cysteine or combination of cysteines responsible for the electrophysiological effects elicited by Nav1.5 palmitoylation, and to understand the contribution of Nav1.5 mis-palmitoylation to action potential abnormalities in CVD.

Na+ pump (Na+/K+ ATPase)

The activity of the Na+ pump – a sarcolemmal active transporter responsible for the coupled exchange of two K+ ions for three Na+ ions – is essential to every eukaryotic cell. The pump itself consists of a catalytic α subunit (the molecular target of cardiotonic steroids such as digoxin), and regulatory β and γ subunits. In the heart, the Na+ pump establishes the ion gradients that support the electrical excitability of the tissue.

The Na+ pump is one of several E–C coupling proteins targeted by a novel endocytosis pathway known as massive endocytosis (MEND). MEND is activated after reoxygenation of anoxic cardiac muscle (an experimental model of myocardial infarction [8]) and causes the rapid internalization of up to 70% of the cell surface membrane. A number of ion channels and transporters, including the Na+ pump, are preferentially internalized by the MEND pathway. To date, the mechanisms underlying this pathway remain poorly understood, but it has been shown to involve palmitoylation of surface membrane proteins by the palmitoylating enzyme zDHHC5, which leads to their clustering and internalization. Deletion of zDHHC5 protects cardiac muscle from MEND and also protects against experimental myocardial infarction, positioning zDHHC5 as a drug target for the treatment of a heart attack.

Phospholemman (PLM)

PLM is a sarcolemmal phosphoprotein consisting of 72 amino acids which regulates the Na+ pump in the heart. Palmitoylation of PLM occurs at Cys40 and Cys42 which reside close to the pump’s α subunit [9, 10]; however, Cys40 is the main palmitoylation site. Palmitoylation of PLM at Cys40 by zDHHC5 inhibits Na+ pump activity [10], and therefore targeting PLM palmitoylation by zDHHC5 may serve as a method to restore pump activity in cardiac disease. The first steps towards therapeutic targeting of PLM palmitoylation were recently reported by our research group. We identified the mechanism by which zDHHC5 recruits its substrate PLM and designed a disruptor peptide to prevent the zDHHC5–PLM interaction that selectively reduced PLM palmitoylation in cardiac myocytes [11]. This important proof of principle experiment demonstrates selective targeting of zDHHC-PATs could be achieved therapeutically if we can identify small molecules that will target zDHHC-PAT interactions with their substrates.

Phospholamban (PLB)

PLB resides in the SR membrane and is composed of just 52 amino acids. Its interaction with SERCA2a inhibits the pump’s ability to transport Ca2+ into the SR. However, this inhibitory action is repressed when PLB is phosphorylated at serine 16 by protein kinase A (PKA) during β-adrenergic signalling. In such instances, the rate at which SERCA2a transports Ca2+ across the SR membrane is greatly enhanced, thus making PLB a key regulator of cardiac function.

PLB is palmitoylated at Cys36 by zDHHC16. PLB palmitoylation promotes its phosphorylation by PKA and consequently increases cardiac output. Knocking out zDHHC16 in mice results in a diminished cardiac output as a consequence of bradycardia and a reduced stroke volume. zDHHC16 knockout (KO) mice also exhibit cardiomyopathy [12], indicating that palmitoylation of PLB and possibly other cardiac proteins by zDHHC16 plays a significant role in maintaining cardiac structure and function.

Ryanodine receptor (RyR2)

Ryanodine receptors are enormous tetrameric proteins composed of approximately 20¦000 amino acids that reside within the membrane of the SR. RyR2, the cardiac isoform, releases Ca2+ from the SR store in response to Ca2+ influx, usually via the opening of adjacent LTCCs. Mutations in the gene encoding the RyR2 receptor result in conditions such as familial polymorphic ventricular tachycardia [13], which can result in sudden cardiac death.

RyR2 is palmitoylated in the heart [1], but the functional consequences of this are not yet known. Interestingly RyR1, the isoform present in skeletal muscle, is palmitoylated at 18 cysteine residues, which reduces its activity and reduces stimulus-coupled Ca2+ release [14]. Based on this, palmitoylation of the cardiac isoform is likely to play a key regulatory role in RyR2 receptormediated Ca2+ release from the SR. If the functional impact of RyR2 palmitoylation is anything like that of RyR1, mutations at or near the palmitoylation sites or mis-regulation of the enzymes responsible for controlling RyR2 palmitoylation would contribute to cardiac arrhythmias and therefore represent a novel therapeutic target in cardiac arrhythmogenesis.

Na+/Ca2+ Exchanger (NCX1)

NCX1 is a sarcolemmal ion transporter which mediates the bidirectional movement of calcium ions across the cell membrane. In forward mode, NCX1 extrudes one Na+ ion from the cell in exchange for three Ca2+ ions, and therefore controls cardiac relaxation. Because NCX1 is a passive exchanger, the mode in which it operates (forward = Ca2+ extrusion, reverse = Ca2+ influx) is dependent on multiple variables: the sodium gradient, the membrane potential and the subsarcolemmal free calcium concentration.

Several in vivo studies have advanced the understanding of NCX1’s contribution to cardiac function. Global KO of NCX1 in mice is embryonically lethal [15], but cardiac-specific NCX1 re-expression is insufficient for embryonic survival [16], therefore indicating that NCX1 expression out-with the embryonic heart is critical to development. Remarkably, a cardiac-specific NCX1 KO model demonstrated a very modest 20–30% reduction in cardiac contractility due to adaptive responses elsewhere in the myocardium [17]. Increased NCX1 activity is implicated in numerous pathological conditions including heart failure [18], myocardial ischemia/reperfusion injury [19] and arrhythmogenesis [20]. It may therefore be possible to target NCX1 for therapeutic benefit in cardiac disease.

NCX1.1 (the predominant splice variant in cardiac muscle) is palmitoylated at Cys739 within the intracellular loop. NCX1.1 activity is controlled by the availability of phosphatidylinositol 4,5-bisphosphate (PIP2); in the absence of PIP2, NCX1 inactivates. Palmitoylation of NCX1 is not only required for its inactivation but hastens the speed at which the exchanger inactivates [21]. Approximately 60% of NCX1 is palmitoylated in ventricular muscle [21], suggesting that palmitoylation creates functionally different subpopulations of NCX1 in the heart. Palmitoylation does not control NCX1 trafficking to the cell membrane, nor does it have a profound effect on either the forward and reverse modes [21]. However, by changing the sensitivity of NCX1 to inactivation, palmitoylation does ultimately change NCX1-mediated calcium fluxes in the cell [22]. The role of NCX1 palmitoylation in cardiac function remains unreported; however, our research group is currently investigating this role by conducting a range of biochemical and physiological studies using a novel transgenic mouse model expressing a form of NCX1 that cannot be palmitoylated in the heart.

Conclusions and future perspectives

The list of cardiac proteins now known to be palmitoylated reads like a ‘who’s who’ of E–C coupling and established cardiac drug targets. It is evident from published data and ongoing studies that palmitoylation plays a pivotal role in regulating ion transport in cardiomyocytes, but to date we know relatively little about whether abnormal palmitoylation of cardiac proteins directly contributes to CVD. Novel therapies designed to augment or inhibit the palmitoylation of specific E–C coupling proteins or therapies designed to target the zDHHC-PATs responsible for their palmitoylation may prove beneficial in the treatment of cardiac arrythmias and the contractile dysfunction associated with calcium mishandling in cardiomyocytes. Additional studies are now required to further elucidate the role of palmitoylation in cardiac function and to identify any mutations in the genes encoding the zDHHC-PATs which may contribute to impaired contractility and arrhythmogenesis within the heart. In the fullness of time we anticipate both diagnostic and therapeutic monitoring of protein palmitoylation in the heart (or of genetic or epigenetic modifiers of the palmitoylating and depalmitoylating enzymes) could aid the clinical management of CVD. Of particular interest in this regard is the concept that the synthesis, dietary availability and cellular uptake of the fatty acid palmitate is an important factor that influences zDHHC-PAT activity. Serum palmitate and the amount of saturated fat in the diet may ultimately then give us insight into nanoscale cellular events controlling gross cardiac function.


We acknowledge financial support for our research programme from the British Heart Foundation.

The authors
Olivia Robertson-Gray PhD, William Fuller* PhD
Institute of Cardiovascular & Medical Sciences, University of Glasgow, UK

*Corresponding author


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