SERCA in genesis of arrhythmias: what we already know and what is new?

ABSTRACT

This review mainly focuses on the structure, function of the sarco(endo)plasmic reticulum calcium pump (SERCA) and its role in genesis of arrhythmias. SERCA is a membrane protein that belongs to the family of P-type ion translocating ATPases and pumps free cytosolic calcium into intracellular stores. Active transport of [Ca.sup.2+] is achieved, according to the E1-E2 model, changing of SERCA structure by [Ca.sup.2+]. The affinity of [Ca.sup.2+] -binding sites varies from high (E1) to low (E2). Three different SERCA genes were identified-SERCA1, SERCA2, and SERCA3. SERCA is mainly represented by the SERCA2a isoform in the heart. In heart muscle, during systole, depolarization triggers the release of [Ca.sup.2+] from the sarcoplasmic reticulum (SR) and starts contraction. During diastole, muscle relaxation occurs as [Ca.sup.2+] is again removed from cytosol, predominantly by accumulation into SR via the action of SERCA2a. The main regulator of SERCA2a is phospholamban and another regulator proteolipid of SERCA is sarcolipin. There are a lot of studies on the effect of decreased and/or increased SERCA activity in genesis of arrhythmia. Actually both decrease and increase of SERCA activity in the heart result in some pathological mechanisms such as heartfailure and arrhythmia.

Keywords: sarco(endo)plasmic reticulum, SERCA, arrhythmia, calcium channels

Introduction

Cardiac physiology is a major area of research in basic and clinical medicine. Studying the molecular determinants of cardiac disorders becomes more important since a major portion of human ailments comprises cardiac diseases. In particular, disorders of the heart that derive from altered calcium homeostasis, such as, cytoplasmic calcium overload caused by abnormal calcium signaling is thought to be a common mechanism underlying some of these abnormalities (1).

In cardiac muscle, the sarcoplasmic reticulum (SR) plays a central role in the contraction and relaxation cycle by regulating the intracellular [Ca.sup.2+]. levels (2, 3). As shown in Figure 1 multitude channels are involved in intracellular [Ca.sup.2+] regulation mechanism. The surface membrane (sarcolemma) is shown invaginating into the transverse tubule system which contains the junctions with the SR and dihydropyridine receptor (DHPR) L-type channels (4). The main role of [Ca.sup.2+] ions entering the myocyte through the DHPR is activation of the ryanodine receptor (RYR) (4). In heart muscle, during systole, depolarization triggers [Ca.sup.2+] entry into the cell via (DHPR) L-type [Ca.sup.2+] channels. And the L-type [Ca.sup.2+] channels triggers the release of [Ca.sup.2+] from the sarcoplasmic reticulum via the ryanodine receptor channels (RYR2). This process, known as [Ca.sup.2+] induced [Ca.sup.2+] release, causes an increase in cytosolic contraction. Subsequently, [Ca.sup.2+] binds to troponin C (TnC), which contains the binding sites for the [Ca.sup.2+] and starts the cross-bridge movement of myofibrils resulting in force development and contraction (5). During diastole, muscle relaxation occurs as [Ca.sup.2+] is again removed from cytosol, predominantly by accumulation into sarcoplasmic reticulum via the action of sarco(endo)plasmic reticulum [Ca.sup.2+] ATPase (SERCA). The SERCA uses hydrolysis of ATP as a source of energy for [Ca.sup.2+] transport from the cytosol into the lumen of SR (1, 6). In the SR, [Ca.sup.2+] becomes bound to calsequestrin (CSQ), which is the major calcium binding protein in the sarcoplasmic reticulum (3). The relaxation is facilitated by 1 ) the SR [Ca.sup.2+] ATPase (SERCA2), which pumps [Ca.sup.2+] back into the SR and is primarily responsible for the myocardial relaxation (2, 5) 2) the [Na.sup.+]/[Ca.sup.2+] exchanger (NCX), which uses the energy of the [Na.sup.+] and [Ca.sup.2+] gradients across the plasma membrane to exchange 3 extracellular [Na.sup.+] ions for 1 intracellular [Ca.sup.2+] ion (28%) and 3) the plasma membrane [Ca.sup.2+] ATPase (PMCA), which presses out [Ca.sup.2+] from the cell using the energy liberated by ATP hydrolysis (5).

[FIGURE 1 OMITTED]

SERCA is a membrane protein that pumps free cytosolic calcium into intracellular stores, has been implicated in several cardiac disorders (7). SERCA, mainly represented by the SERCA2a isoform in the heart, an facilitates the storage and distribution of [Ca.sup.2+] ions in the SR (8). Decreased sarco(endo)plasmic reticulum (SR) [Ca.sup.2+]-uptake and decreased expression of the SR [Ca.sup.2+]-ATPase, SERCA2a, are key features of cardiac myocyte dysfunction in both experimental and human heartfailure (8,9). In recentyears, some authors used a genetic strategy to modify cellular calcium handling by overexpressing sarcoplasmic reticulum ATPase via an adenovirus vector similar to pharmacologic strategies for reducing cytosolic free calcium, such as calcium channel blockers and beta-blockers (10). Excessive [Ca.sup.2+] delivery by SERCA overexpression carries the potential risk of cardiac arrhythmias, as well (7).

In this brief review, we aimed to give an overview of recent advances in the rapidly growing field of factors modulating SERCA activity and arrhythmia.

The SERCA Pump Molecular structures of SERCA

The SERCA pump is an ~110-kDa transmembrane protein and belongs to the family of P-type ion translocating ATPases, which includes [Na.sup.+]-[K.sup.+]-ATPase and gastric [H.sup.+]-[K.sup.1]-ATPase among others, and are fundamental in establishing ion gradients by pumping ions across biological membranes. SERCA pumps calcium ion from the cytoplasm into the SR against a large concentration gradient (11-15). The SERCA [Ca.sup.2+] pump protein consists of a single polypeptide chain folded into four major domains. The structure of SR [Ca.sup.2+]-ATPase with two bound [Ca.sup.2+] in the transmembrane (M) region, which consists of ten helices. The cytoplasmic part of [Ca.sup.2+]-ATPase consists of three domains (A, actuator or anchor; N, nucleotide; and P, phosphorylation), well separated in this [Ca.sup.2+]-bound form (13). Molecular cloning analyses identified three different SERCA genes, SERCA1, SERCA2, and SERCA3, which encode at least five [Ca.sup.2+] pump isoforms (11, 15-17). SERCA1 and SERCA2 genes are different in their C-terminate (18). The SERCA1 gene encodes two alternatively spliced transcripts, SERCA1a and SERCA1b, which are exclusively expressed in skeletal muscle. SERCA1a is predominantly expressed in the adult stages; SERCA1 b is mainly expressed in the fetal/neonatal stages. The SERCA2 gene encodes SERCA2a, SERCA2b and SERCA2c isoforms. The SERCA2a isoform is identical to SERCA2b except for its carboxyl terminate. The cardiac isoform of sarcoplasmic reticulum calcium-ATPase (SERCA2a) is the main regulator of cytosolic calcium, which is not only determining the electrical, but also the contractile, properties of myocardium (18). SERCA2b isoform is commonly expressed but is found at high levels in smooth muscle tissues (11, 16,17). Quite recently, a new SERCA2c mRNA was described, and it is mainly expressed in cardiac and skeletal muscle, which exhibits functional similarities, but also functional differences. Relative to SERCA2a and SERCA2b, SERCA2c protein presents a distinct localization in left ventricle of normal hearts (17). The third gene, SERCA3, is expressed in a limited set of non-muscle cells and encoded as SERCA3b-SERCA3f in human, SERCA3b-SERCA3c in mice, and SERCA3b-SERCA3c in rat proteins. The SERCA3 isoforms are differently co-expressed in a variety of cells and tissues including muscle and non-muscle tissues (17).

SERCA related [Ca.sup.2+] transport cycle

Active transport of [Ca.sup.2+] is achieved, according to the E1-E2 model, by changing the affinity of [Ca.sup.2+] -binding sites from high (E1) to low (E2) (13).

As shown in Figure 2, the conformational change of SERCA by [Ca.sup.2+] provides us a hint for the understanding of how the E2 form changes to the E1-[Ca.sup.2+]. form by [Ca.sup.2+], and the transport of [Ca.sup.2+] into the lumen through the SR membrane (12).

[FIGURE 2 OMITTED]

In the E1 conformation, the two [Ca.sup.2+] -binding sites are of high affinity and are facing the cytoplasm. In the E2 state the [Ca.sup.2+] binding sites are of low affinity and are facing the luminal side. Either cytosolic ATP or [Ca.sup.2+] can bind first to the E1 conformation. The 2[Ca.sup.2+]-E1-ATP form undergoes phosphorylation to form 2[Ca.sup.2+]-E1-P, the high energy phosphointermediate, in which the bound [Ca.sup.2+] ions become occluded. This intermediate is also called the ADP-sensitive form, because in the presence of ADP the backward reaction occurs with release of the bound [Ca.sup.2+] and synthesis of ATP. Conversion to the low energy intermediate is accompanied by a major conformational change to 2[Ca.sup.2+]-E2-P (ADP intensive form), whereby the [Ca.sup.2+] binding sites are converted to a low affinity state and reorient towards the luminal face. The cycle ends with the sequential release of [Ca.sup.2+] and phosphate and a major conformational change from the E2 to the E1 state (19).

Regulation of SERCA

The key role played by calcium pumps in controlling of cytoplasmic calcium ions, regulation of calcium pump activity will have profound effects on calcium signaling (12). The need for an accurate regulation of SERCA2's [Ca.sup.2+] affinity is underscored by the existence of two membrane inserted regulator proteins phospholamban (PLB) and sarcolipin (SLN) (5).

The main regulator of SERCA2a is phospholamban, which has been accepted as a key regulator of SERCA2a and cardiac contractility. It interacts with SERCA1a, SERCA2a and SERCA2b but not SERCA3. It is a 52-amino acid transmembrane protein and is expressed predominantly in cardiac muscle. Phospholamban monomers interact with SERCA2a and reversibly inhibit the [Ca.sup.2+]. transport activity of the pump. It interacts with SERCA molecules to lower the apparent affinity of SERCA2a for [Ca.sup.2+] without altering its maximal pumping rate (12, 15, 20). At non resting [Ca.sup.2+] concentrations, the binding of [Ca.sup.2+] to the pump promotes the dissociation of the PLB/SERCA2a complex (8,12, 15, 20).

Another proteolipid, which appears to be involved in the regulation of SERCA1 activity is sarcolipin. This 31-amino acid peptide co-localizes with SERCA1 and is most abundant in fast twitch muscle (12). Indeed SLN and PLB are homologous proteins and members of the same gene family. They appear to bind to the same regulatory site in SERCA. Sarcolipin is superinhibitory compared with co-expression of SERCA1a and PLB (20, 21). Like PLB, SLN interacts with and inhibits SERCA by lowering its apparent [Ca.sup.2+] affinity without pronounced effects on the maximal pumping rate (5). Sarcolipin has the ability to interact not only with SERCA1 a but also with SERCA2a affecting their [Ca.sup.2+]affinity to a similar extent and, also its shown that SLN expression may be prominent in the heart. As reported by Vangheluve et al, previously, SLN was considered to be the regulator of SERCA1a and hence the fast skeletal muscle counterpart of the SERCA2a inhibitor PLB in the heart. In humans, SLN mRNA is also found in the heart (5).

In heartfailure, PLB and SERCA2a are both down-regulated so that [Ca.sup.2+] stores are less effectively filled through the action of SERCA2a. As the [Ca.sup.2+] store is depleted, the force of contraction is diminished. Since SLN might be involved in such pathological conditions, it is important to understand any potential that exists for the involvement of SLN in heart diseases (20).

The [Ca.sup.2+]/calmodulin dependent protein kinase II (CaMKII) also is accepted as a mediator of SERCA2a (8). It is shown that phosphorylation of SERCA2a by CaMKII modulates the maximal activity of the SERCA2a without changing the apparent affinity of the pump. Hawkins et al (22) and Frank (8) reported that this phosphorylation is selective and comes into being in the cardiac and smooth muscle SR while not in the skeletal muscle.

Phosphorylation of PLB by protein kinase A and/or [Ca.sup.2+]/calmodulin kinase II relieves the inhibition of the pump and stimulates [Ca.sup.2+] uptake activity (5, 15).

SERCA and cardiac arrhythmias

It is reported that, 50% and 80% of deaths in patients suffering from various kinds of heart diseases are caused by cardiac arrhythmias (23). Cardiac arrhythmias mostly occur in diseased hearts as a result of an abnormality in ion channels. Over the past three decades, the researchers have been focused on the role of abnormal calcium signaling in the genesis of cardiac arrhythmias (24). Insufficient calcium delivery to the myofilaments causes a weak contraction, while excessive calcium delivery carries the risk of activation of proteases and other maladaptive calcium-sensitive pathways that lead to cell death, and can resultwith the generation of pathological membrane currents. In many studies conducted on human and animal models, altered [Ca.sup.2+] homeostasis in cardiac cells has been evaluated as a common finding in heart diseases. Decreased SERCA uptake and decreased expression of the SERCA2a, cause cardiac myocyte dysfunction (9). Among the most documented alterations in [Ca.sup.2+] homeostasis is a decrease in SERCA function, caused by a decrease in SERCA protein and/or activity resulting from a relative increase of phospholamban. These changes bring the alterations in intracellular [Ca.sup.2+] cycling and damaged cardiac function (7).

On the other hand, Chen et al. (9) had shown that transgenic SERCA2a overexpression increased the risk of acute arrhythmias and sudden death in rats (Fig. 3). Some of suggested consequences during overexpression of SERCA are as following: i) SERCA2a overexpression may improve [Ca.sup.2+] handling but also induce arrhythmias due to immediate [Ca.sup.2+] reuptake before troponin C binding can occur (25). ii) Overexpressing SERCA dramatically alters the balance between the major calcium- handling proteins. Like digoxin, SERCA overexpression favors sequestration of calcium by the SR instead of being extruded by the NCX (1). A larger SR store will initially lead to an increase in the calcium transient, autoregulation is ensured by (a) more rapid inactivation of subsequent calcium currents and, therefore, (b) reduced calcium entry through L-type calcium channels. The net effect is to reduce transsarcolemmal calcium flux while maintaining a normal systolic transient (26). iii) Increases in SERCA protein abundance result in an increased SERCA2a load. The SERCA [Ca.sup.2+] overload may produce spontaneous [Ca.sup.2+] releases and thereby lead to ectopic activity. iv) Elevated intracellular [Ca.sup.2+] may also close gap junctions, decreasing cell-to-cell coupling, and thereby decreasing action potential conduction directly provoking arrhythmias (27).

[FIGURE 3 OMITTED]

Conclusion

Actually both decrease and increase of SERCA activity in the heart raised many interesting questions resulting in a variety of pathological manifestations including contractile dysfunction and electrical instability. Nevertheless, we can view this as yet another motivation for us to return to the fundamental mechanism of heart failure and arrhythmia in search for better therapeutic approaches.

References

(1.) Wang Y, Goldhaber J. Return of calcium: manipulating intracellular calcium to prevent cardiac physiologies. Proc Natl Acad Sci USA 2004;101: 5697-8.

(2.) Aoyagi T, Yonekura K, Eto Y, Matsumoto A, Yokoyama I, Sugiura S, et al. The sarcoplasmic reticulum [Ca.sup.2+]-ATPase (SERCA2) gene promoter activity is decreased in response to severe left ventricular pressure overload hypertrophy in rat hearts. J Mol Cell Cardiol 1999; 31: 919-26.

(3.) Vangheluwe P, Louch WE, Ver Heyen M, Sipido K, Raeymaekers L, Wuytack F. [Ca.sup.2+] transport ATPase isoforms SERCA2a and SERCA2b are targeted to the same sites in the murine heart. Cell Calcium 2003; 34: 457-64.

(4.) Dulhunty AF, Beard N, Pouliquin P, Casarotto MG. Agonist and antagonists of the cardiac ryanodine receptor: Potential therapeutic agents? Pharmacol Ther 2007;113: 247-63.

(5.) Vangheluwe P, Sipido KR, Raeymaekers L, Wuytack. New perspectives on the role of SERCA2's [Ca.sup.2+] affinity in cardiac function. Biochem Biophys Acta 2006;1763:1216-28.

(6.) Asahi M, Nakayama H, Tada M, Otsu K. Regulation of sarco (endo)plasmic reticulum [Ca.sup.2+] adenosine triphosphatase by phospholamban and sarcolipin: implication for cardiac hypertrophy and failure. Trends Cardiovasc Med 2003;13:152-7.

(7.) Rubio M, Bodi I, Fuller-Bicer GA, Hahn HS, Periasam M, Schwartz A. Sarcoplasmic reticulum adenosine triphosphatase overexpression in the L-type [Ca.sup.2+] channel mouse results in cardiomyopathy and [Ca.sup.2+] -induced arrhythmogenesis. J Cardiovasc Pharmacol Ther 2005;10: 235-49.

(8.) Frank KF, Bolck B, Erdmann E, Schwinger RH. Sarcoplasmic reticulum [Ca.sup.2+]-ATPase contraction and relaxation. Cardiovasc Res 2003; 57: 20-7.

(9.) Chen Y, Escoubet B, Prunier F, Amour J, Simonides WS, Vivien B, et al. Constitutive cardiac overexpression of sarcoplasmic/ endoplasmic reticulum [Ca.sup.2+]-ATPase delays myocardial failure after myocardial infarction in rats at a cost of increased acute arrhythmias. Circulation 2004;109: 1898-903.

(10.) Del Monte F, Lebeche D, Guerrero JL, Tsuji T, Doye AA, Gwathmey JK, et al. Abrogation of ventricular arrhythmias in a model of ischemia and reperfusion by targeting myocardial calcium cycling. Proc Natl Acad Sci USA 2004;101: 5622-7.

(11.) Ji Y, Loukianov E, Loukianova T, Jones LR, Periasamy M. SERCA1a can functionally substitute for SERCA2a in the heart. Am J Physiol 1999; 276: H89-97.

(12.) East JM. Sarco(endo)plasmic reticulum calcium pumps: recent advances in our understanding of structure/function and biology. Mol Membr Biol 2000;17: 189-200.

(13.) Toyoshima C, Nomura H. Structural changes in the calcium pump accompanying the dissociation of calcium. Nature 2002; 418: 605-11.

(14.) Sengupta T, Ghoshal S, Sen PC. Stimulation of Mgl. -independent form [Ca.sup.2+] -ATPase by a low molecular mass protein purified from goattestes cytosol. Compar Biochem Physiol 2007;146:131-8.

(15.) Vangheluwe P, Raeymaekers L, Dode L, Wuytack F. Modulating sarco(endo)plasmic reticulum [Ca.sup.2+] ATPase2 (SERCA2) activity: Cell biological implications. Cell Calcium 2005; 38: 291-302.

(16.) Sumbilla C, Cavagna M, Zhong L, Ma H, Lewis D, Farrance I, et al. Comparison of SERCA1 and SERCA2a expressed in COS-1 cells and cardiac myocytes. Am J Physiol 1999; 277: H2381-2391.

(17.) Dally S, Bredoux R, Corvazier E, Andersen JP, Clausen JD, Dode L, et al. [Ca.sup.2+] -ATPases in non-failing and failing heart: evidence for a novel cardiac sarco/endoplasmic reticulum [Ca.sup.2+] -ATPase 2 isoform (SERCA2c). Biochem J 2006; 395: 249-58.

(18.) Pavlovic M, Schaller A, Pfammatter JP, Carrel T, Berdat P, Gallati S. Age-dependent suppression of SERCA2a mRNA in pediatric atrial myocardium. Biochem Biophys Res Comm 2005; 326: 344-8.

(19.) Wuytack F, Raeymaekers L, Missiaen L. Molecular physiology of SERCA and SPCA pumps. Cell Calcium 2002; 32: 279-305.

(20.) Asahi M, Kurdlowski K, Tada M, Mac Lennan DH. Sarcolipin inhibits polymerization of phospholamban to induce superinhibition of sarco(endo)plasmic reticulum Ca.sup.2+]-ATPases (SERCAs). J Biol Chem 2002; 277: 26725-8.

(21.) MacLennan DH, Asahi M, Tupling AR. The regulation of SERCA-type pumps by phospholamban and sarcolipin. Ann N Y Acad Sci 2003; 986: 472-80.

(22.) Hawkins C, Xu A, Narayanan N. Sacroplasmic reticulum calcium pump in cardiac and slow twitch skeletal muscle but not fast twitch skeletal muscle undergoes phosphorylation by endogenous and exogenous [Ca.sup.2+]/calmodulin-dependent protein kinase. Characterization of optimal conditions for calcium pump phosphorylation. J Biol Chem 1994; 269: 31198-26.

(23.) Dai D, Yu F. Ion channelopathy and hyperphosphorylation contributing to cardiac arrhythmias. Acta Pharmacol Sin 2005; 26: 918-25.

(24.) Clusin WT. Calcium and cardiac arrhythmias: DADs, EADs, and alternans. Crit Rev Clin Lab Sci 2003; 40: 337-75.

(25.) Maier LS, Wahl-Schott C, Horn W, Weichert S, Pagel C, Wagner S, et al. Increased SR [Ca.sup.2+] cycling contributes to improved contractile performance in SERCA2a-overexpressiog transgenic rats. Cardiovasc Res 2005; 67: 636-46.

(26.) Eisner DA, Trafford AW, Diaz ME, Overend CL, O'Neill SC. The control of [Ca.sup.2+] release from the cardiac sarcoplasmic reticulum: regulation versus autoregulation. Cardiovasc Res 1998; 38: 589-604.

(27.) Rodenbaugh DW, Collins HL, Nowacek DG, DiCarlo SE. Increased susceptibility to ventricular arrhythmias is associated with changes in [Ca.sup.2+] regulatory proteins in paraplegic rats. Am J Physiol Heart Circ Physiol 2003; 285: H2605-13.

Nilufer Erkasap

Department of Physiology, Medical Faculty, Eskisehir Osmangazi University, Eskisehir, Turkey

Address for Correspondence: Dog. Dr. Nilufer Erkasap, Eskisehir Osmangazi Universitesi Tip Fakultesi, Fizyoloji Anabilim Dali, Eskisehir Turkey E-mail: [e-mail]