© 1998 American Heart Association, Inc.
Editorial |
Calcium Pump Overexpression and Myocardial Function
Implications for Gene Therapy of Myocardial Failure
Correspondence to Gerd Hasenfuss, MD, Universität Göttingen, Zentrum Innere Medizin, Abteilung Kardiologie und Pneumologie, Robert-Koch-Strasse 40, 37075 Göttingen, Germany. E-mail hasenfus{at}med.uni-goettingen.de
Key Words: Ca2+-ATPase overexpression • myocardium • gene therapy • heart failure
The contraction-relaxation cycle of the heart is
controlled by the sequential rise and fall of the cytosolic calcium
concentration. Calcium entry through L-type calcium channels during the
action potential serves to trigger calcium release from the
sarcoplasmic reticulum (SR) leading to activation of contractile
proteins and force generation (for review, see Reference 11 ). In
addition, there is calcium influx by reverse-mode sodium-calcium
exchange. The contribution of calcium influx through L-type calcium
channels, or sodium calcium exchange, and of SR calcium release to
systolic calcium transients differs from species to
species.2 The global increase in calcium is
immediately followed by calcium removal, resulting in subsequent
deactivation of the contractile machinery and myocardial relaxation.
Calcium removal from the cytosol occurs by the activity of the SR pump,
by exchange of calcium for sodium via the sarcolemmal sodium-calcium
exchanger (Na+-Ca2+
exchanger), by mitochondrial calcium uptake, and by calcium extrusion
via the sarcolemmal calcium pump.1 Again, the
contribution of these systems removing calcium from the cytosol to the
decay of the calcium transient and to subsequent relaxation varies in a
species-dependent manner. Using the rapid-cooling contracture
technique, Bers et al suggested that in rat ventricular
myocardium, 
92% of calcium removal occurs by SR calcium
uptake and only 7% by
Na+-Ca2+
exchange.3 4 In other species (human, rabbit,
ferret, cat, and guinea pig) the balance is more in the range of 70%
to 75% SR calcium uptake and 25% to 30%
Na+-Ca2+
exchange.3 4 According to these studies, calcium
uptake by mitochondria and transsarcolemmal calcium extrusion by the
sarcolemmal calcium pump were suggested to be of minor importance.
The relevance of calcium extrusion by the sarcolemmal calcium pump is again evaluated in the study by Hammes et al5 in this issue of Circulation Research. The plasma membrane calmodulin-dependent calcium ATPase (PMCA) is a ubiquitous calcium transporting enzyme that plays a dominant role in nonexcitable cell types.6 7 So far, 4 different isoforms have been cloned, and 3 different isoforms seem to be expressed in myocardial tissue.8 9 PMCA is not homologous with SR Ca2+-ATPase and has a different ATP to calcium stoichiometry.10 One calcium ion seems to be transported per ATP hydrolyzed.10 11 Hammes et al5 established transgenic rat lines carrying the human PMCA 4 cDNA under control of the ventricle-specific myosin light chain-2 promoter. Although transgenes exhibited a 2.5-fold PMCA overexpression in neonatal and a 1.6-fold overexpression in adult cardiomyocytes, no significant differences in hemodynamic parameters between control and transgenic animals were observed in anesthetized rats. Accordingly, no relevant differences in fura-2 calcium transients and L-type calcium currents were observed. These data confirm elegantly that PMCA does not significantly contribute to calcium elimination in cardiac myocytes. However, because the rate of protein synthesis was increased in neonatal myocytes from transgenic compared with wild-type animals in the presence of 2% FCS, as well as on incubation with phenylephrine or isoproterenol, the authors suggest that PMCA may influence growth regulation.5 Moreover, because the authors were able to show that PMCA is at least partially localized in caveolae, they speculate that PMCA may modulate caveolar signal transduction and thereby influence growth regulation. This is an interesting hypothesis that is in line with previous studies suggesting that PMCA isoform 1a overexpression is involved in regulation of vascular smooth muscle cell growth.12 However, additional research is warranted to study the role of PMCA in myocardial growth regulation and the influence of altered expression or functional modulation of this calcium pump on myocardial development, hypertrophy, or failure.
Expression of the SR Ca2+-ATPase and other calcium regulatory proteins has been extensively studied in various animal models of heart failure as well as in the failing human heart (for review, see Reference 1313 ). There is accumulating evidence that reduced expression and/or function of the SR Ca2+-ATPase as well as increased expression and function of the Na+-Ca2+ exchanger are key changes contributing to altered calcium homeostasis in the failing human heart.13 Altered calcium homeostasis is obvious from studies showing a reduced total amount of calcium cycling in isolated muscle strip preparations,14 reduced systolic calcium transients and increased diastolic calcium levels in isolated myocytes,15 as well as reduced rate of calcium removal in both muscle strips and myocytes from failing human hearts.14 15 Furthermore, disturbed calcium cycling underlies blunting of the frequency potentiation of contractile force, which is an important defect in the failing human heart.16 SR Ca2+-ATPase and the Na+-Ca2+ exchanger work in concert to eliminate calcium from the cytosol, facilitating dissociation of calcium from myofilament regulatory proteins and subsequent relaxation.1 17 However, the Na+-Ca2+ exchanger in its forward mode is a competitor of SR Ca2+-ATPase with respect to SR calcium accumulation and availability for systolic activation of contractile proteins. The relevance of calcium influx by the Na+-Ca2+ exchanger to calcium transients is not clear.18 On the basis of these considerations, inhibition of the Na+-Ca2+ exchanger or stimulation of calcium transport by SR Ca2+-ATPase should increase SR calcium load and thus improve systolic myocardial function. However, whereas the former possibility would likely result in cytosolic calcium overload, only the latter would improve both systolic and diastolic performance of the myocardium.
Stimulation of SR calcium pump activity, therefore, may be an attractive approach to improve systolic and diastolic function of the failing human heart. SR Ca2+-ATPase is inhibited by phospholamban, and this inhibition is relieved on phosphorylation of phospholamban by protein kinase A or calcium calmodulin-dependent kinase.13 19 Therefore, interventions that specifically result in increased phospholamban phosphorylation, or in decreased phospholamban protein levels, or interventions that inhibit SR Ca2+-ATPase to phospholamban interaction may be promising therapeutic possibilities to treat heart failure. In this regard, it was shown that overexpression of phospholamban in transgenic animals decreases cardiac contractility whereas phospholamban knockout increases myocardial performance.20 21
Alternatively, function of the failing myocardium may be improved by overexpression of the SR calcium pump. Sarcoendoplasmic reticulum Ca2+-ATPases are encoded by 3 genes, and 5 different isoforms are expressed: the adult fast-twitch skeletal muscle isoform (SERCA1a), its alternatively spliced neonatal isoform (SERCA1b), the cardiac/slow twitch skeletal muscle isoform (SERCA2a), its alternatively spliced smooth muscle/nonmuscle isoform (SERCA2b), and an isoform expressed in a broad variety of muscle and nonmuscle tissues (SERCA3) (for review, see Reference 2222 ). The strategy to improve contractility by overexpressing SERCA is supported by data from He et al,23 who generated transgenic mice overexpressing SERCA2a. In a heterozygous transgenic mouse line, they observed a 1.2-fold increase in SERCA2a protein levels, which was associated with a moderately increased rate of cardiac contraction and relaxation in intact animals as well as in isolated myocytes. Furthermore, adenovirus-mediated gene transfer of SERCA2a in neonatal rat cardiac myocytes resulted in an increased peak and an abbreviation of the calcium transients and an enhanced contraction.24 In addition, Giordano et al25 showed that adenovirus-mediated expression of SERCA2a in neonatal myocytes with decreased SERCA2a protein levels due to downregulation by phorbol ester can reconstitute depressed SERCA levels and improve calcium cycling. Of note, increases in SERCA2a levels and associated changes in function were rather small in these studies.
The article by Loukianov et al26 published in
this issue of Circulation Research elegantly confirms that
overexpression of SERCA can increase myocardial
contractility. Most interestingly, the authors
generated transgenic mice that specifically overexpress the fast-twitch
skeletal muscle isoform of the SR calcium pump termed "SERCA1a,"
driven by a cardiac-specific 
-myosin heavy chain promoter. One line
that expressed 9 copies of the transgene exhibited a 2.5-fold increase
in total SR calcium pump protein levels, resulting in a 1.7-fold
increase in the maximal velocity of oxalate-facilitated calcium uptake.
The latter clearly indicates that the overexpressed SERCA1a pumps are
functional. Furthermore, isolated cardiomyocytes displayed
increased rates of contraction and relaxation and abbreviations of the
calcium transients, indicating increased rates of SR calcium uptake and
altered kinetics of calcium release. In addition, isolated
work-performing heart preparations from transgenic mice revealed
significantly higher systolic and lower diastolic
pressures as well as increased rates of pressure rise and fall.
Interestingly, despite a 2.5-fold increase in total SERCA protein
levels, endogenous SERCA2a expression was reduced by 50%
in transgenic animals. This suggests that exogenous SERCA1a can replace
the endogenous SERCA2a protein.26
Unfortunately, no data are available regarding the influence of the
transgene on SERCA2a transcription.
In close agreement with this study is an article recently published by Inesi et al.27 They demonstrated highly efficient adenovirus-mediated expression of SERCA1a, driven by the cell-specific cardiac troponin T promoter in cultured chick embryo cardiac myocytes. By using specific antibodies to SERCA1a and SERCA2a, the authors showed that both endogenous and transgenic ATPases are prevalently associated with the microsomal fraction (ie, sarcoplasmic reticulum). Calcium uptake measurements suggested that a 4-fold increase in uptake rates may reflect an upper limit for the ability of these myocytes to express functional protein. Similar to the finding of Loukianov et al,26 SERCA2a appeared to be reduced by 30% to 60% in transgenic myocytes expressing SERCA1a.
The structural and functional differences of the respective SERCA isoforms are not completely understood. It has been shown that the primary structure of SERCA1a and SERCA2a protein is more than 80% identical.28 Furthermore, expression of the different pump isoforms in COS-1 cells revealed that SERCA1a and SERCA2a isoforms are similar regarding calcium transport capacity and apparent affinity for calcium.29 However, those in vitro studies are limited by absence of phospholamban and other native regulators. This may explain why the calcium transporting capacity of cardiac microsomes was found to be much lower than that in fast skeletal muscle SR, although this may partly be explained by differences in SERCA pump density as well.30
From the studies by Loukianov et al26 and Inesi et al,27 no information on phospholamban regulation of SERCA1a can be derived. However, it was shown that phospholamban can regulate SERCA1a as well as SERCA2a activity.31 32 Furthermore, additional studies showing that apparent calcium affinity of SERCA is similar in wild-type and SERCA1a transgenic animals indicate that SERCA1a and SERCA2a are similarly regulated by phospholamban (M. Periasamy, personal communication, July 1998).
It is important to keep in mind that mouse myocardium and rat myocardium are considerably different from human myocardium regarding excitation-contraction coupling and contractile protein isoforms (for review, see Reference 3333 ). In particular, mouse myocardium appears to be much more tolerant of calcium overload than other species.34 Therefore, what is true in the mouse and rat cannot be readily extrapolated to other animal species and humans.
In summary, the studies presented indicate that overexpression of SERCA2a as well as transgenic expression of SERCA1a and most likely other genetically engineered calcium pump proteins can influence myocardial calcium handling and function substantially. Because disturbed calcium handling seems to play a significant role in the pathophysiology of heart failure, the relevance of overexpression of SERCA to rescue disturbed calcium cycling and myocardial function of the failing heart is of particular importance. Moreover, overexpression of SERCA as an inotropic intervention may be attractive from the point of view of energy economy. Although SERCA overexpression may result in increased ATP hydrolysis related to SR calcium transport, unlike cAMP-mediated inotropic interventions, it should not decrease myofilament calcium sensitivity or economy of force generation at the level of the contractile machinery.35 Therefore, from a pathophysiological as well as an energy economy point of view, overexpression of SERCA seems to be a challenging approach to correct altered calcium cycling in heart failure. SERCA2a but also SERCA 1a and other modified SERCA molecules should be evaluated, and the functional differences between those molecules must be clarified. Most importantly, studies in human myocardium or in animal models of human heart failure are needed to support the hypothesis that stimulation of SR calcium transport can rescue altered excitation-contraction coupling in the failing myocardium.
Footnotes
The opinions expressed in this editorial are not necessarily those of the editors or of the American Heart Association.
References
1.
Barry WH, Bridge JHB. Intracellular calcium
homeostasis in cardiac myocytes. Circulation. 1993;87:1806–1815.
2. Bers DM. Possible sources and sinks of activator calcium. In: Bers DM, ed. Excitation-Contraction Coupling and Cardiac Contractile Force. Norwell, Mass: Kluwer Academic Publishers; 1991:33–48.
3. Bers DM. Ca transport during contraction and relaxation in mammalian ventricular muscle. In: Hasenfuss G, Just H, eds. Alterations of Excitation-Contraction Coupling in the Failing Human Heart. New York, NY: Springer-Verlag; 1998:1–16.
4.
Bassani JW, Bassani RA, Bers DM. Relaxation in rabbit
and rat cardiac cells: species-dependent differences in cellular
mechanisms. J Physiol (Lond). 1994;476:279–293.
5.
Hammes A, Oberdorf-Maass S, Rother T, Nething K,
Gollnick F, Linz KW, Meyer R, Hu K, Han H, Gaudron P, Ertl G, Hoffmann
S, Ganten U, Vetter R, Schuh K, Benkwitz C, Zimmer HG, Neyses L.
Overexpression of the sarcolemmal calcium pump in the
myocardium of transgenic rats. Circ Res. 1998;83:877–888.
6.
Carafoli E. The Ca2+-pump of the
plasma membrane. J Biol Chem. 1992;267:2115–2118.
7. Carafoli E, Stauffer T. The plasma membrane calcium pump: functional domains, regulation of the activity, and tissue specificity of isoform expression. J Neurobiol. 1994;25:312–324.[Medline] [Order article via Infotrieve]
8.
Stauffer TP, Guerini D, Carafoli E. Tissue
distribution of the four gene products of the plasma membrane
Ca2+ pump. J Biol Chem. 1995;270:12184–12190.
9.
Keeton TP, Burk SE, Shull GE. Alternative splicing of
exons encoding the calmodulin-binding domains and the C
termini of plasma membrane Ca2+-ATPase isoforms
1, 2, 3, and 4. J Biol Chem. 1993;268:2740–2748.
10.
Niggli E, Adunyha ES, Penniston JT, Carafoli E.
Purified (Ca2+-Mg2+)-ATPase
of the erythrocyte membrane. J Biol Chem. 1981;256:395–401.
11. Hasenfuss G, Mulieri LA, Holubarsch C, Pieske B, Just H, Alpert NR. Energetics of calcium cycling in nonfailing and failing human myocardium. In: Holtz J, Drexler H, Just H, eds. Cardiac Adaptation in Heart Failure. New York, NY: Springer-Verlag; 1992:81–92.
12.
Husain M, Jiang L, See V, Bein K, Simons M, Alper SL,
Rosenberg RD. Regulation of vascular smooth muscle cell proliferation
by plasma membrane Ca(2+)-ATPase. Am J
Physiol. 1997;272:C1947–C1959.
13.
Hasenfuss G. Alterations of calcium-regulatory proteins
in heart failure. Cardiovasc Res. 1998;37:279–289.
14.
Hasenfuss G, Mulieri LA, Leavitt JB, Allen PD, Haeberle
JR, Alpert NR. Alteration of contractile function and
excitation-concentration coupling in dilated
cardiomyopathy. Circ Res. 1992;70:1225–1232.
15.
Beuckelmann DJ, Näbauer M, Erdmann E.
Intracellular calcium handling in isolated ventricular
myocardium. Circulation. 1992;85:1046–1055.
16.
Pieske B, Kretschmann B, Meyer M, Holubarsch C, Weirich
J, Posival H, Minami K, Just H, Hasenfuss G. Alterations in
intracellular calcium handling associated with the inverse
force-frequency relation in human dilated
cardiomyopathy. Circulation. 1995;92:1169–1178.
17.
Backx PH, Gao W-D, Azan-Backx MD, Marban E. The
relationship between contractile force and intracellular
[Ca2+] in intact rat cardiac
trabeculae. J Gen Physiol. 1995;105:1–19.
18. Bers DM, Christensen DM, Nguyen TX. Can Ca2+ entry via the Na+-Ca2+-exchanger directly activate cardiac muscle contraction? J Mol Cell Cardiol. 1988;20:405–414.[Medline] [Order article via Infotrieve]
19. James P, Inui M, Tada M, Chiesi M, Carofoli E. Nature and site of phospholamban regulation of the Ca2+ pump of sarcoplasmic reticulum. Nature. 1989;342:90–92.[Medline] [Order article via Infotrieve]
20.
Luo W, Grupp IL, Harrer J, Ponniah S, Grupp G, Duffy
JJ, Doetschman T, Kranias EG. Targeted ablation of the phospholamban
gene is associated with markedly enhanced myocardial
contractility and loss of ß-agonist stimulation.
Circ Res. 1994;75:401–409.
21. Kadambi VJ, Ponniah S, Harrer JM, Hoit BD, Dorn GW 2nd, Walsh RA, Kranias EG. Cardiac-specific overexpression of phospholamban alters calcium kinetics and resultant cardiomyocyte mechanisms in transgenic mice. J Clin Invest. 1996;97:533–539.[Medline] [Order article via Infotrieve]
22.
Arai M, Hirosuke M, Periasamy M. Sarcoplasmic
reticulum gene expression in cardiac hypertrophy and heart
failure. Circ Res. 1994;74:555–564.
23. He H, Giordano FJ, Hilal-Dandan R, Choi DJ, Rockman HA, McDonough PM, Bluhm WF, Meyer M, Sayen MR, Swanson E, Dillmann WH. Overexpression of the rat sarcoplasmic reticulum Ca2+ ATPase gene in the heart of transgenic mice accelerates calcium transients and cardiac relaxation. J Clin Invest. 1997;100:380–389.[Medline] [Order article via Infotrieve]
24.
Hajjar RJ, Kang JX, Gwathmey JK, Rosenzweig A.
Physiological effects of adenoviral gene transfer
of sarcoplasmic reticulum calcium ATPase in isolated rat myocytes.
Circulation. 1997;95:423–429.
25.
Giordano FJ, He H, McDonough P, Meyer M, Sayen MR,
Dillmann WH. Adenovirus-mediated gene transfer reconstitutes depressed
sarcoplasmic reticulum Ca2+-ATPase levels and
shortens prolonged cardiac myocyte Ca2+
transients. Circulation. 1997;96:400–403.
26.
Loukianov E, Ji Y, Grupp IL, Kirkpatrick DL, Baker DL,
Loukianova T, Grupp G, Lytton J, Walsh RA, Periasamy M. Enhanced
myocardial contractility and increased
Ca2+ transport function in transgenic hearts
expressing the fast-twitch skeletal muscle sarcoplasmic reticulum
Ca2+-ATPase. Circ Res. 1998;83:889–897.
27. Inesi G, Lewis D, Sumbilla C, Nandi A, Strock C, Huff KW, Rogers TB, Johns DC, Kessler PD, Ordahl CP. Cell-specific promoter in adenovirus vector for transgenic expression of SERCA1 ATPase in cardiac myocytes. Am J Physiol. 1998;273:C645–C653.
28. Brandl CJ, Green NM, Korczak B, MacLennan DH. Two Ca2+-ATPase genes: homologies and mechanistic implications of deduced amino acid sequences. Cell. 1986;44:597–607.[Medline] [Order article via Infotrieve]
29.
Lytton J, Westlin M, Hanley MR. Thapsigargin inhibits
the sarcoplasmic or endoplasmic reticulum
Ca2+-ATPase family of calcium pumps. J
Biol Chem. 1991;266:17067–17071.
30.
Shigekawa M, Finegan JAM, Katz AM. Calcium
transport ATPase of canine cardiac sarcoplasmic reticulum. A comparison
with that of rabbit fast skeletal muscle sarcoplasmic reticulum.
J Biol Chem. 1976;251:6894–6900.
31.
Kim HW, Steenaart NA, Ferguson DG, Kranias EG.
Functional reconstitution of the cardiac sarcoplasmic reticulum
Ca2+ ATPase with phospholamban in phospholipid
vesicles. J Biol Chem. 1990;265:1702–1709.
32. Hughes G, Starling AP, Sharma RP, East JM, Lee AG. An investigation of the mechanism of inhibition of the Ca2+-ATPase by phospholamban. Biochem J. 1996;318:973–979.
33.
Hasenfuss G. Animal models of human
cardiovascular disease, heart failure and
hypertrophy. Cardiovasc Res. 1998;39:60–76.
34.
Gao W-D, Perez NG, Marban E. Calcium cycling and
contractile activation in intact mouse cardiac muscle. J
Physiol (Lond). 1998;507:175–184.
35. Hasenfuss G, Mulieri LA, Leavitt BJ, Alpert NR. Influence of isoproterenol on contractile protein function, excitation-contraction coupling, and energy turnover of isolated nonfailing human myocardium. J Mol Cell Cardiol. 1994;26:1461–1469.[Medline] [Order article via Infotrieve]
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