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J Mol Cell Cardiol 33, 1053–1063 (2001) doi:10.1006/jmcc.2001.1366, available online at http://www.idealibrary.com on Review Article SERCA Pump Level is a Critical Determinant of Ca2+ Homeostasis and Cardiac Contractility Muthu Periasamy1 and Sabine Huke Division of Cardiology, University of Cincinnati, College of Medicine, 231 Albert Sabin Way, Cincinnati, Ohio 45267-0542, USA (Received 7 February 2001, accepted 7 February 2001) M. P S. H. SERCA Pump Level is a Critical Determinant of Ca2+ Homeostasis and Cardiac Contractility. Journal of Molecular and Cellular Cardiology (2001) 33, 1053–1063. The control of intracellular calcium is central to regulation of cardiac contractility. A defect in SR Ca2+ transport and SR Ca2+ ATPase pump activity and expression level has been implicated as a major player in cardiac dysfunction. However, a precise cause–effect relationship between alterations in SERCA pump level and cardiac contractility could not be established from these studies. Progress in transgenic mouse technology and adenoviral gene transfer has provided new tools to investigate the role of SERCA pump level in the heart. This review focuses on how alterations in SERCA level affect Ca2+ homeostasis and cardiac contractility. It discusses the consequences of altered SERCA pump levels for the expression and activity of other Ca2+ handling proteins. Furthermore, the use of SERCA pump as a therapeutic target for gene therapy of heart failure is evaluated. 2001 Academic Press K W: SR Ca2+ ATPase; Contractility; Transgenic; Gene therapy. Introduction The study of sarcoplasmic reticulum (SR) gene expression and SR calcium transport function has been an active area of research for decades because of its importance to cardiac function and disease. A regulated release and uptake of intracellular Ca2+ between SR and cytoplasm tightly controls the contraction–relaxation cycle of the heart. Muscle contraction is initiated when Ca2+ enters the cell via L-type Ca2+ channels in the sarcolemma and, as a consequence, triggers the release of a much larger amount of Ca2+ from the SR via SR Ca2+ release channels (ryanodine receptor, RyR).1,2 The free cytosolic Ca2+ concentration determines the extent of muscle activation and therefore regulates force development. The SR Ca2+ ATPase (SERCA) pumps the Ca2+ back into the SR and is therefore responsible for muscle relaxation and for replenishing Ca2+ stores needed for the next 1 contraction.3 SERCA pump activity is regulated by the small 52-aminoacid phosphoprotein phospholamban (PLB), which in its unphosphorylated state lowers the affinity of SERCA for Ca2+.4 Moreover, Ca2+ removal by the Na+–Ca2+ exchanger in the sarcolemma contributes to maintenance of intracellular Ca2+ homeostasis.5 A number of studies, conducted on animal models of heart failure and human failing hearts, suggest that alterations in SR Ca2+ handling are a critical feature of the hypertrophied or failing myocardium. Alterations in the expression of different SR proteins and its associated Ca2+ transport abnormalities in cardiac hypertrophy and heart failure have been recently reviewed by Houser et al.6 The purpose of this review is to highlight the role of SERCA in intracellular Ca2+ homeostasis and cardiac contractility as learned from in vivo and in vitro genetic manipulations of the SERCA pump. This review focuses on the following major questions: (i) Is E-mail: [email protected] 0022–2828/01/061053+11 $35.00/0 2001 Academic Press 1054 M. Periasamy and S. Huke the protein level of SERCA pump critical for Ca2+ homeostasis and cardiac function? (ii) How do alterations in SERCA pump level affect other Ca2+ handling proteins? (iii) Can SERCA pump serve as a therapeutic target to restore cardiac function in failing myocardium? The SR Ca2+ ATPase Gene Family The SERCA pump is a transmembrane protein of >110 kDa and belongs to a family of highly conserved proteins. Molecular cloning analyses have identified a family of SERCA pumps encoded by three highly homologous genes (SERCA1, SERCA2 and SERCA3) (reviewed in Arai et al.7). The SERCA1 gene encodes two alternatively spliced transcripts, SERCA1a and SERCA1b, which differ only by a few amino acids in their carboxyl terminus.8 SERCA1b is expressed in the fetal/neonatal stages of fast twitch skeletal muscle development and is gradually replaced by the SERCA1a isoform in the adult fast twitch skeletal muscle.9 The SERCA2 gene is also alternately spliced and encodes SERCA2a and SERCA2b isoforms.10 SERCA2a protein terminates with four unique amino acids (Ala-ILe-Leu-Glu), whereas SERCA2b has an extended hydrophobic sequence of 49 amino acids at its carboxyl terminus.11,12 The SERCA2b isoform is expressed ubiquitously and is found to be associated with IP3 gated Ca2+ stores,11,13 whereas SERCA2a is the primary isoform expressed in the heart.10,14,15 SERCA2a Pump Expression Level in the Heart The SERCA2a isoform plays a central role in SR Ca2+ handling required for excitation–contraction coupling in the heart. Although SERCA2a isoform is expressed at high levels in cardiac myocytes, the expression level of SERCA is not a fixed parameter. There are naturally occurring regional differences, developmental changes and aging-related effects as well as alterations due to variations in thyroid hormone levels. These changes can be drastic, as shown for atrium in comparison to ventricle. The expression level of SERCA in atrium v ventricle is reported to be two-fold in most experimentally used animals and in humans.16,17 With respect to the functional differences, the higher SERCA pump levels may account at least in part for the shorter duration of contraction in atrial v ventricular tissue.16,18 Moreover, it was shown for mouse, rat and rabbit that the expression of SERCA pump gradually increases during development.16,19–22 This increase was again accompanied by a shortening of relaxation time in adult v neonatal ventricle.22 In addition also in adult hearts SERCA levels are not steady, but are influenced by aging and fluctuations in thyroid hormone levels. A decrease in content and activity of SERCA was described in experimental models of senescence and in senescent human myocardium.23,24 This decrease was associated with a prolonged contraction time and depressed myocardial function. Hearts from rats and mice with increased thyroxine levels showed increased SERCA expression and significant increased contractility, whereas hypothyroid hearts displayed decreased SERCA levels together with reduced contractility.25,26 Therefore, several naturally occurring variations in SERCA expression level correlate with the contractile status of the heart. The expression level of SERCA pump protein appears to be a critical determinant of cardiac contractility. In all these studies the alteration in SERCA expression is in consent with the accompanying functional changes, but also changes in other factors might contribute. Several additional tissue-specific (atrium v ventricle), developmental and thyroxine-induced changes may be equally responsible for the observed changes in relaxation time and contractility.27–29 In the past three decades a decrease in SERCA gene expression and activity was observed in a wide variety of pathological conditions. Varying degrees of defects in the SR Ca2+ uptake function have been identified in animal models of heart disease and have been shown to correlate with altered contractile function (reviewed in Arai et al.7). Studies from many laboratories have shown that the expression level of SERCA is significantly decreased in pressure overload (PO)-induced hypertrophy/heart failure.30–34 In these studies decreased SR calcium transport and formation of the phosphoenzyme intermediate, E-P, was observed.7,31–33,35 In addition to studies using animal models of cardiac diseases, there are considerable data indicating that SR Ca2+ transport function is altered in end-stage human heart failure. Intracellular Ca2+ measurements using aequorin and fura-2 showed markedly prolonged Ca2+ transients in both Ca2+ release and uptake phases in muscle samples from failing human hearts.36,37 It was shown that the expression level of SR Ca2+ ATPase was decreased both at the mRNA and protein level in end-stage heart failure.38,39 Some other laboratories could not observe a decrease in SERCA protein expression (for overview see Hasenfuss40). A decrease SERCA Pump and Cardiac Contractility in the level of SR Ca2+ ATPase (mRNA or protein or activity) was closely correlated with decreased myocardial function or altered force–frequency response.38,39,41–43 The studies described above suggest that SERCA pump level is critical for the maintenance of cardiac function, but the true relationship between SERCA levels and muscle function cannot be defined. Too many changes occur in parallel within the heart and myocytes during progression to heart failure. Similarly, physiological differences in SERCA expression (development, aging etc.) are accompanied by simultaneous changes in other excitation– contraction coupling proteins. Studies using these models do not allow us to establish a precise causeeffect relationship between SERCA pump level and cardiac contractility. Therefore, others and we have taken a transgenic approach to specifically manipulate SERCA pump levels in vitro and in vivo. Genetic Manipulation of SERCA Pump Expression To manipulate SERCA pump expression levels several investigators have utilized in vitro-adenoviralmediated gene transfer to produce targeted alterations in protein expression in cardiac myocytes. A number of groups including us have generated genetically altered mouse models to critically assess the functional relevance of changes in SERCA levels for contractile function. The usage of in vivo models allows us to study the long-term changes in cardiovascular function at the levels of both the organ and the whole animal. Transgenic animals with increased SERCA pump levels in the heart (SERCA level ↑) and a mouse model with decreased SERCA2 expression (SERCA level ↓) are powerful tools to define the role of SERCA pump level for Ca2+ homeostasis and cardiac function. In this review we will omit detailed description of already published data, instead we will focus on what we have really learned from these models so far. Increased SERCA pump expression Transgenic animals with increased SERCA pump levels in the heart were generated by several investigators. Two independent mouse lines overexpressing SERCA2a protein (cardiac isoform) 1.2fold44 and 1.5-fold45 were described. One intriguing finding of these experiments was that despite much higher mRNA levels for SERCA2a (2.6-fold and 1055 eight-fold, respectively) the increase in SERCA2a protein level was only modest above the endogenous expression. The basis for this observation is still not known. There might be competition between the exogenous and the endogenous protein and/or there are powerful post-transcriptional mechanisms working to maintain a physiological SERCA level. Still, the moderate increase in SERCA protein led to significant functional changes in both models. He et al.44 described faster rates of Ca2+ decline as well as increased maximal rates of shortening and relengthening in isolated myocytes. The SR Ca2+ content of caffeine-sensitive stores was increased by 29%. The relaxation time of isolated papillary muscles was shorter and maximal rates of contraction and relaxation, evaluated by in vivo cardiac catheterization, were higher. Baker et al.45 described an increase of 37% in the maximum velocity of Ca2+ uptake function and SERCA level dependent increased maximal rates of contraction and relaxation in the isolated work-performing heart preparation. Recently, SERCA2a overexpression in transgenic rats was shown to increase parameters of contraction and relaxation.46 Taken together these studies demonstrate that (i) it is possible to increase the SERCA2a pump level in vivo, (ii) increases in SERCA2a pump levels alter Ca2+ homeostasis and enhance contractile function of the heart and (iii) SERCA2a overexpression is not pathological. Furthermore, these findings are not limited to increases in the expression of SERCA2a isoform. Very similar results were obtained in transgenic mice ectopically expressing the fast-twitch skeletal muscle Ca2+ ATPase SERCA1a.47,48 The SERCA1a pump is a pump with faster kinetics in vitro.49,50 Overexpression of this pump (under the MHCpromoter) increased SERCA levels to 2.5-fold and Ca2+ uptake to 1.9-fold, but the apparent affinity for Ca2+ was unchanged. The measured functional parameters of these hearts were clearly enhanced, but only slightly pronounced in comparison to the mouse-line with 1.5-fold SERCA2a protein expression. It seems that the SERCA1a expressing hearts reach a maximal contractile limit and other factors do not allow further enhancement of cardiac function, but this hypothesis needs to be tested under varying experimental conditions. Interestingly, the expression of SERCA1a led to a 50% downregulation of the endogenous SERCA2a pump, as detected by isoform specific antibodies (see Fig. 1). This suggests a competition of SERCA2a and 1a for functional sites in the SR, but might also be due to a compensatory downregulation of the endogenous SERCA2a isoform. The reduced SERCA2a 1056 M. Periasamy and S. Huke Figure 1 Western blot analysis of SERCA1a TG mouse hearts. Increasing amounts of protein from TG and control hearts were separated in 10% SDS-PAGE, transferred onto nitrocellulose membrane and probed with SERCA1a-, SERCA2a-, calsequestrin-, -sarcomeric actin-, and tropomyosin-specific antibodies.47 levels and the enhanced cardiac function in these mice clearly demonstrate that SERCA1a can functionally substitute for SERCA2a in the heart. Recently, we generated transgenic mice with increased cardiac levels of SERCA2b pump.51 SERCA2b has a higher Ca2+ affinity than SERCA2a and it is endogenously expressed in the heart, but in a much smaller amount than SERCA2a. In these mice the 8–10-fold overexpression of SERCA2b did not cause a reduction of SERCA2a pump (unlike SERCA1a overexpressing mice), most likely due to a distinct distribution of SERCA2b within the SR. This was further validated by immunostaining of isolated myocytes. The distribution pattern shows a preferential localization for SERCA2b around the T-tubules, whereas SERCA2a is distributed transversely and longitudinally in the SR membrane. The maximal velocity of Ca2+ uptake was not changed in hearts from SERCA2b mice, but the apparent Ca2+ affinity was increased. The heart function determined by working heart preparations was enhanced, indicating that SERCA2b contributes to SR Ca2+ transport on a beat-tobeat basis. Decreased SERCA pump expression To understand how decreased SERCA levels affect cardiac function, we chose to disrupt the SERCA2 gene by homologous recombination.52 As expected, the disruption of both copies of SERCA2 gene is lethal, whereas heterozygous mice with a single functional allele are alive and reproduce well. SERCA2a protein levels and the maximal velocity of SR Ca2+ uptake were reduced by >35%. In spite of that heterozygous mice do not develop cardiac hypertrophy or other signs of heart failure, so far examined for up to 12 months of age. In the absence of cardiac pathology, the peak amplitude of the Ca2+ transients in isolated single myocytes was decreased by more than 30%, resulting in decreased rates of cell shortening and relengthening in heterozygous mice.53 Measurements of in vivo cardiovascular function via transducers in the left ventricle and right femoral artery of the anesthetized mouse revealed reductions in heart rate, mean arterial pressure, systolic ventricular pressure, and the absolute values of both maximal rates of contraction and relaxation. These results SERCA Pump and Cardiac Contractility 1057 Figure 2 Representative recordings of cardiac performance in isolated work-performing hearts from SERCA1a TG, FVB/N controls (WT) and SERCA2 heterozygous (SERCA2-HET) mice. Mean aortic pressure was set to 50 mmHg and venous return to 5 ml/min. Aortic-, left ventricular pressure and the rate of contraction (dP/dt) is shown. demonstrate that two functional copies of the SERCA2 gene are required to maintain “normal” levels of SERCA2a protein, Ca2+ sequestering activity, and Ca2+ homeostasis, and that the loss of one functional SERCA2 gene depresses cardiac function. These studies using transgenic models with altered SERCA levels/activity suggest that there is a direct correlation between SERCA level and contractile state of the heart. Alterations in the SERCA level, an increase or a decrease, modulate cardiac contractility (see Fig. 2). Therefore we conclude that the level of functional SERCA protein in the SR is one of the fundamental determinants of cardiac contractility. Is there a cross-talk between SERCA and other Ca2+ transport proteins? SERCA pump is a major determinant of SR Ca2+ transport and changes in its level alter intracellular Ca2+ homeostasis. Thus, this could have a profound effect on the expression or activity of other Ca2+ handling proteins that participate in the maintenance of the intracellular Ca2+ homeostasis. These include proteins in the SR membrane, such as phospholamban (PLB), an inhibitory regulator of SERCA pump and the SR Ca2+ release channel (ryanodine receptor, RyR). In addition, sarcolemmal Ca2+ handling proteins, the Na+–Ca2+ exchanger (NCX) and the L-type Ca2+ channel could be altered due to changes in SERCA expression. Some of the following interpretations are supported by a limited amount of data and need further confirmation. Does the SERCA level affect the PLB protein levels and its interaction with SERCA pump? A change in SERCA level could affect the dynamics of SERCA/PLB interaction. An increase in SERCA pump may shift the equilibrium of SERCA/PLB ratio in favor of more free pump. On the other hand, a decrease in SERCA may result in higher PLB ratio to SERCA resulting in maximal inhibition of SERCA pump. However, it is not yet clear if the expression of one protein determines the expression and/or activity of the other. Recently, we reported that chronic alterations in SERCA2a pump levels modify the PLB protein level.53 We found that in SERCA2 heterozygous hearts, where SERCA2a pump levels are decreased 1058 M. Periasamy and S. Huke by >35%, PLB protein levels are decreased by >40%. It seems possible that a cross-talk exists between these two proteins to maintain a constant PLB:SERCA2a ratio. Similarly, in mice ectopically expressing SERCA1a in the heart (total SERCA level 2.5-fold) the PLB protein levels were decreased by >50%, in line with a 50% reduction in the endogenous SERCA2a pump levels (Prasad et al., unpublished data). This rather intriguing finding suggests that SERCA2a pump level, but not SERCA1a level, may modulate PLB protein level. Recently, Ver Heyen et al. generated a mouse model in which the SERCA2a isoform is completely replaced by SERCA2b by exon-specific gene targeting methods.54 In this model PLB protein levels are increased over two-fold despite a decrease in total SERCA protein (SERCA2b) to around 50% of nontransgenic controls. This was most intriguing considering that SERCA levels are drastically reduced in these hearts. This leaves the impression that in the absence of its endogenous partner SERCA2a the regulation of PLB protein level is lost. Based on these studies one could speculate that SERCA2a is the virtual partner for PLB. However, studies have shown that PLB can interact with SERCA1 and other SERCA2 isoforms when co-expressed.55–57 These data could provide support to the idea that chronic alterations in SERCA2a pump levels can influence PLB levels thereby maintaining an optimal PLB:SERCA2a ratio. It is unclear whether simple protein ratio can be equated to a functional ratio of PLB to SERCA, since PLB exists both as a monomer and pentamer and the PLB monomer is shown to be the more active inhibitor.58,59 Changes in SERCA pump level could influence the pentamer to monomer ratio, although this has not been carefully studied. Additionally, it has been well established that phosphorylation of PLB relieves the inhibition and therefore accelerates SERCA pump activity.60,61 An important finding in SERCA2 heterozygous hearts is the enhanced basal phosphorylation status of PLB (in vivo) at both Ser-16 and Thr-17 in spite of decreased PLB protein level.53 These data suggest that a decrease in PLB protein level and an increase in PLB phosphorylation are adaptive changes to partially compensate for the decrease in SERCA pump level and Ca2+ uptake function. The phosphorylation status of PLB in models where SERCA pump is increased is not known. shown by an increase in the amplitude of the Ca2+ transients.48 This is likely due to an increase in SR Ca2+ load.62 Interestingly, in SERCA1a expressing hearts the amount of Ca2+ release channel (RyR) protein is decreased (>30%). This rather indicates a compensatory downregulation in the RyR protein expression to regulate the amount of Ca2+ released during each contraction.62 A similar finding was described for PLB-null mice, where RyR protein levels are decreased (25%) while mRNA levels are unchanged.63 Taken together these findings suggest that alterations in the level of ryanodine receptors (likely due to post-transcriptional regulation) may be a general protective mechanism to regulate Ca2+ release from the SR in models with increased SR Ca2+ load. Alterations in sarcolemmal proteins, Na+–Ca2+ exchanger and L-type Ca2+ channel activity Previous studies have shown that there is a reciprocal/inverse relationship between SERCA and Na+–Ca2+ exchanger protein expression. This is particularly observed during heart development, in hyper- and hypothyroid hearts and in heart failure.19,64–66 It is generally believed that the Na+–Ca2+ exchanger might play a compensatory role for loss of SERCA pump activity. Indeed, in SERCA2 heterozygous hearts, the Na+–Ca2+ exchanger protein level and the exchanger current activity were increased53 (see Fig. 3). The exact physiological significance of this finding is not completely understood. On the other hand, in mice overexpressing SERCA pump, the Na+–Ca2+ exchanger levels are not altered, but functional measurements are needed.62 Our preliminary studies on L-type Ca2+ channel activity show that SERCA overexpression results in a significant reduction in L-type Ca2+ current density, suggesting altered Ca2+ channel activity or expression.62 Experiments are in progress to determine the Ca2+ channel protein expression in transgenic models with increased or decreased SERCA protein expression. It is noteworthy to mention that in myocytes from PLB ablated mice the decay of Ltype Ca2+ current was faster than in control cells.67 These and other studies suggest that alterations in SERCA expression and activity can affect sarcolemmal Ca2+ ion transporters, but the exact mechanism remains to be understood. How do SERCA levels affect SR Ca2+ release function? SERCA as a Therapeutic Agent In SERCA1a transgenic myocytes more Ca2+ is released from the SR during each contraction, as In recent years gene therapy for heart diseases has become a promising area of research. Heart failure SERCA Pump and Cardiac Contractility Figure 3 Western blot analysis of SERCA2 heterozygous hearts. Increasing amounts of protein were separated by SDS-PAGE and probed with a Na+–Ca2+ exchanger (NCX) specific antibody. A representative immunoblot is shown. The bar graph shows the mean±... of six individual hearts.53 provides an attractive candidate for gene therapy, because a number of protein targets have been identified as defective or functionally impaired.68 In particular, abnormalities in excitation–contraction coupling have received much attention. A decrease in SR Ca2+ ATPase expression and/or activity seems to be a major defect responsible for impaired function of the failing heart. Therefore, a number of recent studies were focused on restoring SERCA pump activity by adenoviral mediated gene transfer. Hajjar et al.69 and Giordano et al.70 have elegantly demonstrated that the overexpression of SERCA2a in isolated myocytes through adenoviral gene transfer results in increased contractility and a faster decay of the cytosolic Ca2+ transient. Inesi and colleagues71,72 have further documented that both SERCA2a and SERCA1a can be expressed at high levels in embryonic chicken and neonatal rat cardiomyocytes by adenoviral vectors. These authors showed that despite identical protein expression 1059 levels SERCA1a activity was two-fold greater than SERCA2a activity due to intrinsic differences in turnover rates. The decay of cytosolic Ca2+ transients in cells expressing SERCA1a was faster without a significant change in resting Ca2+ or peak amplitudes. Furthermore, two independent studies tested the benefit of higher SERCA levels during pressureoverload induced cardiac remodeling in genetically altered animal models.46,73 Transgenic mice and rats overexpressing SERCA2a were subjected to aortic stenosis for 7 weeks or abdominal aortic banding for 10 weeks, respectively. Ito et al.73 found decreased mortality in banded transgenic mice v banded controls and both studies demonstrate a beneficial effect of increased SERCA levels in terms of enhanced contractile reserve or higher cardiac contractility after the treatment. Recently, the feasibility of restoring function both in failing cardiac myocytes and in intact animal hearts subjected to experimental heart failure was tested. Del Monte et al.74 showed that overexpression of SERCA2a in human ventricular myocytes from patients with end-stage heart failure can increase SERCA pump activity and enhance contraction and relaxation velocity. The negative frequencyresponse was normalized in cardiomyocytes overexpressing SERCA2a. In addition, Hajjar and colleagues have used a catheter-based technique of adenoviral gene transfer to achieve global myocardial transduction of SERCA2a in vivo.75,76 The authors chose to restore SERCA2a activity in an animal model of pressure-overload hypertrophy in transition to failure. SERCA2a levels and activity were decreased and severe contractile dysfunction was evident. Overexpression of SERCA2a by gene transfer in vivo restored both systolic and diastolic dysfunction to normal levels. SERCA overexpression decreased left ventricular size and restored the slope of the end-diastolic pressure-dimension relationship to control levels. Similarly, infection of senescent rat hearts with adenovirus carrying SERCA2a increased Ca2+ uptake activity and improved ratedependent contractility and diastolic function.75 These studies provide strong evidence that increased SERCA expression can be used to restore Ca2+ transport and contractility. Furthermore, these data suggest the feasibility of cardiac gene transfer into failing hearts as a therapeutic intervention. Summary and Conclusions The SERCA manipulation studies described above in mice and using adenoviral gene transfer clearly 1060 M. Periasamy and S. Huke demonstrate that it is possible to increase SERCA protein level in cardiac myocytes. The SR membrane is not fully saturated and can incorporate additional Ca2+ pumps. A pump expression of up to 2.5-fold is not pathological in the heart. The exogenously expressed pumps are functional and can alter Ca2+ transport activity. Increased pump levels lead to increases in SR Ca2+ uptake, SR Ca2+ load and enhanced contractile function. A decrease in SERCA level elicits the opposite effects. These data convincingly demonstrate that SERCA protein level is a critical determinant of intracellular calcium homeostasis and contractility in the mammalian heart. However, alterations in SERCA expression are not without consequences. Several compensatory adaptations do occur in transgenic mice at various levels both in sarcolemmal and SR membrane. Alterations in the expression and/or activity of other Ca2+ regulatory proteins, including RyR, PLB, Na+–Ca2+ exchanger and L-type Ca2+ channels, were found, suggesting that there is a cross-talk and/or functional dependence between different Ca2+ handling proteins. In particular, such compensatory changes might be needed to maintain sufficient cardiac function and prevent the development of cardiac hypertrophy in SERCA2 heterozygous mice. The SERCA manipulation studies also raise many additional questions: What is the “normal” physiological SERCA level and how is it maintained? What are the effects of long-term changes in SERCA pump level over the physiological range? What is the mechanism for sensing perturbations in SERCA pump level/activity? How do alterations in SERCA affect the dynamics of SERCA/PLB interaction? What is the relationship, if any, between the SERCA and the Na+–Ca2+ exchanger? Can data from transgenic mice be extrapolated to higher mammals, including humans? Nevertheless, the SERCA manipulation studies provide us with promising results and move us a step forward towards employing SERCA protein as a therapeutic agent to rescue function in human failing hearts. Acknowledgements The authors wish to thank Lynne H. Liu, Yong Ji, M. Jane Lalli, Vikram Prasad, Tom Reed, Kalpana Nattamai, Gopal J. Babu, Atai Watanabe and Pei Hong Dong for their contributions and for helpful discussions. We are grateful to Roger Hajjar and Guoxiang Chu for critical reading of this manuscript. M. Periasamy is supported by grant RO1 HL 64140-02 and S. Huke is supported by the DFG. References 1. F A. Calcium-induced release of calcium from the cardiac sarcoplasmic reticulum. Am J Physiol 1983; 245: C1–C14. 2. B DM, P-R E. Ca channels in cardiac myocytes: structure and function in Ca influx and intracellular Ca release. Cardiovasc Res 1999; 42: 339–360. 3. ML DH. Purification and properties of an adenosine triphosphatase from sarcoplasmic reticulum. J Biol Chem 1970; 245: 4508–4518. 4. S HK, J LR. Phospholamban: protein structure, mechanism of action, and role in cardiac function. Physiol Rev 1998; 78: 921–947. 5. H LV, P KD. Sodium-calcium exchange: recent advances. Basic Res Cardiol 1997; 92 (Suppl. 1): 45–51. 6. H SR, P V, W J. Abnormalities of calcium cycling in the hypertrophied and failing heart. J Mol Cell Cardiol 2000; 32: 1595–1607. 7. A M, M H, P M. Sarcoendoplasmic reticulum gene expression in cardiac hypertrophy and heart failure. Circ Res 1994; 74: 555–564. 8. ML DH, B CJ, K B, G NM. Amino-acid sequence of a Ca2+/Mg2+-dependent ATPase from rabbit muscle sarcoplasmic reticulum, deduced from its complementary cDNA sequence. Nature 1985; 316: 696–700. 9. B CJ, L S, M DR, ML DH. Adult forms of the Ca2+ ATPase of the sarcoplasmic reticulum. Expression in developing skeletal muscle. J Biol Chem 1987; 262: 4768–4774. 10. Z-H A, ML DH, P M. Characterization of rabbit cardiac sarco(endo)plasmic reticulum Ca2+ ATPase gene. J Biol Chem 1990; 265: 4670–4677. 11. G-H AM, G J, S GE. A novel Ca2+ pump expressing in brain, kidney, and stomach is encoded by an alternative transcript of the slowtwitch muscle sarcoplasmic reticulum Ca2+-ATPase gene. J Biol Chem 1988; 263: 15032–15040. 12. L J, Z-H A, P M, ML DH. Molecular cloning of the mammalian smooth muscle sarco(endo)plasmic reticulum Ca2+ ATPase gene. J Biol Chem 1988; 263: 15032–15040. 13. J LM, L JD, C P. Differential modulation of SERCA2 isoforms by calreticulin. J Cell Biol 1998; 142: 963–969. 14. A M, S JL, M F, W F, R L, L AM. In situ mRNA distribution of sarco(endo)plasmic reticulum Ca2+-ATPase mRNA isoform during ontogeny in the rat. J Mol Cell Cardiol 1994; 26: 101–102. 15. W KD, L WS, W J, B D, L J. Localization and quantification of endoplasmic reticulum Ca2+-ATPase isoform transcripts. Am J Physiol 1995; 269: C775–C784. 16. L I, B P, J LR, K U, K J, SERCA Pump and Cardiac Contractility 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. L B, L H, M A, M FR, S W, V U, N J. Expression of cardiac calcium regulatory proteins in atrium v ventricle in different species. J Mol Cell Cardiol 1999; 31: 1299–1314. B P, U C, K U, K U, K-W O, K J, L B, L H, M FU, S W, V U, Z N, J LR, N J. Regional expression of phospholamban in the human heart. Cardiovasc Res 1999; 43: 67–76. M A, K A, P K, S E, L AM, V V, V-C R. Sarcoplasmic reticulum function in determining atrioventricular contractile differences in rat heart. Am J Physiol 1997; 273: H2498–H2507. R TD, B GJ, J Y, Z A, V H M, W F, P M. The expression of SR calcium transport ATPase and the Na/Ca exchanger are antithetically regulated during mouse cardiac development and in hypo/hyperthyroidism. J Mol Cell Cardiol 2000; 32: 453–464. C F, D S, L BS, W GT. Sarcoplasmic reticulum Ca(2+)ATPase and cell contraction in developing rabbit heart. J Mol Cell Cardiol 2000; 32: 745–755. F DJ, T CA, P S. Developmental regulation of the sarcoplasmic reticulum pump in the rabbit heart. Pediatr Res 1992; 31: 474–479. G I, B P, K U, K J, L H, M FU, M T, V U, S W, B GS, N J. Postnatal changes in contractile time parameters, calcium regulatory proteins, and phosphatases. Am J Physiol 1998; 274: H2123–H2132. T GE, P TT, B DL, E ML, P HJ, B RJ. The calcium uptake of the rat heart sarcoplasmic reticulum is altered by dietary lipid. J Membr Biol 1993; 131: 963–998. C BS, M DR, J KS, W JF, M X, C JC J, B A, H AH. Human SERCA2a levels correlate inversely with age in senescent human myocardium. J Am Coll Cardiol 1998; 32: 458–467. G IL, S A, H TE, R J, G G. Comparison of normal, hypodynamic, and hyperdynamic mouse hearts using isolated workperforming heart preparations. Am J Physiol 1993; 265: H1401–H1410. K E, B AG, E I, G IL, G G, K EG. Thyroid hormone-induced alterations in phospholamban-deficient mouse hearts. Circ Res 1998; 83: 608–613. H JM, H K, K HW, F DG, K EG. Coordinate regulation of SR Ca(2+)ATPase and phospholamban expression in developing murine heart. Am J Physiol 1997; 272: H57–H66. N-G B, M V. Molecular basis of cardiac performance. J Clin Invest 1989; 84: 1693– 1700. S B. Developmental and functional adaptation of contractile proteins in cardiac and skeletal muscles. Physiol Rev 1986; 66: 710–771. N R, H AZ, B CJ, F J, T M, 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 1061 ML DH, A NR, P M. Regulation of myocardial Ca2+ ATPase and phospholamban mRNA expression in response to pressure overload and thyroid hormone. Proc Natl Acad Sci USA 1989; 86: 2966–2970. F AM, W EO, R PE, L BH. Selective changes in cardiac gene expression during compensated hypertrophy and the transition to cardiac decompensation in rats with chronic aortic banding. Circ Res 1993; 73: 184–192. M H, ML DH, A N, P M. Sarcoplasmic reticulum gene expression in pressure overload-induced cardiac hypertrophy in rabbit. Am J Physiol 1995; 268: C252–C258. Q M, S TR, E DE, B DM, S AM. Downregulation of sarcoplasmic reticulum Ca2+-ATPase during progression of left ventricular hypertrophy. Am J Physiol 1997; 272: H2416– H2424. A T, Y K, E Y, M A, Y I, S S, M S, H Y, B DL, P M. The sarcoplasmic reticulum Ca2+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: 919–926. K E, B NA, K EG, W RA. Differential changes in cardiac phospholamban and sarcoplasmic reticular Ca(2+)-ATPase protein levels. Effects on Ca2+ transport and mechanics in compensated pressure-overload hypertrophy and congestive heart failure. Circ Res 1995; 77: 759–764. G JK, C L, ML R, S FJ, F MD, G W, M JP. Abnormal intracellular calcium handling in myocardium from patients with end-stage heart failure. Circ Res 1987; 61: 70–76. B DJ, N M, E E. Intracellular calcium handling in isolated ventricular myocytes from patients with terminal heart failure. Circulation 1992; 85: 1046–1055. A M, A NR, ML DH, B P, P M et al. Alterations in sarcoplasmic reticulum gene expression in human heart failure: A possible mechanism for alterations in systolic and diastolic properties of the failing myocardium. Circ Res 1993; 72: 463–469. H G, R H, S R, M M, P B, H J, H C, P H, J H, D H. Relation between myocardial function and expression of sarcoplasmic reticulum Ca2+-ATPase in failing and nonfailing human myocardium. Circ Res 1994; 75: 434–442. H G. Alteration of calcium-regulatory proteins in heart failure. Cardiovasc Res 1998; 37: 279– 289. M JJ, L AM, D P, B KR, F JB, W C, A PD, K M, S K. Altered sarcoplasmic reticulum Ca(2+)ATPase gene expression in the human ventricle during end-stage heart failure. J Clin Invest 1990; 85: 305–309. A NR, H G, L BJ, I FP, P B, M LA. A mechanistic analysis of reduced mechanical performance in human heart failure. Jpn Heart J 2000; 41: 103–115. 1062 M. Periasamy and S. Huke 43. P B, K B, M M, H C, W J, P H, M K, J H, H G. Alterations in intracellular calcium handling associated with the inverse force-frequency relation in human dilated cardiomyopathy. Circulation 1995; 92: 1169–1178. 44. H H, G FJ, H-D R, C DJ, R HA, MD PM, B WF, M M, S MR, S E, D 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. 45. B DL, G IL, J Y, R T, L E, G G, B A, H B, W R, P M. Targeted overexpression of the sarcoplasmic reticulum Ca2+ ATPase increases cardiac contractility in transgenic mouse hearts. Circ Res 1998; 83: 1205–1214. 46. M OJ, R H, L M, L HP, D M, F WM. Transgenic rats overexpressing SERCA2a compensate heart failure in aortic banding model. Circulation 2000; 102 (Suppl. II): 460. 47. L E, J Y, G IL, K DL, B DL, L T, G G, L J, W RA, P 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. 48. J Y, L E, L T, J LR, P M. SERCA1a can functionally substitute for SERCA2a in the heart. Am J Physiol 1999; 276: H89–H97. 49. C M, O’D M, S C, I G, K MG. Exogenous Ca2+-ATPase isoform effects on Ca2+ transients of embryonic chicken and neonatal rat cardiac myocytes. J Physiol 2000; 528: 53–63. 50. S C, C M, Z L, M H, L D, F I, I G. Comparison of SERCA1 and SERCA2a expressed in COS-1 cells and cardiac myocytes. Am J Physiol 1999; 277: H2381–H2391. 51. G AL, L MJ, J Y, B GJ, G IL, S M, P M. Overexpression of SERCA2b in the heart leads to an increase in sarcoplasmic reticulum calcium transport function and increased cardiac contractility. J Biol Chem 2000; 275: 24722–24727. 52. P M, R TD, L LH, J Y, L E, P RJ, N ML, R T, D JJ, D T, L JN, S GE. Impaired cardiac performance in heterozygous mice with a null mutation in the sarco(endo)plasmic reticulum Ca2+-ATPase isoform 2 (SERCA2) gene. J Biol Chem 1999; 274: 2556–2562. 53. J Y, L MJ, B GJ, X Y, K DL, L LH, C N, W RA, S GE, P M. Disruption of a single copy of the SERCA2 gene results in altered Ca2+ homeostasis and cardiomyocyte function. J Biol Chem 2000; 275: 38073–38080. 54. V-H E, H S, A G, R T, P M, A B, L J, V P, D M, C D, S K, C P, 55. 56. 57. 58. 59. 60. 61. 62. 63. 64. 65. 66. 67. 68. W F. Targeted disruption of muscle-specific SR Ca2+-ATPase isoform (SERCA2a) splicing causes malformations and impairs contraction–relaxation of the heart. J Clin Invest in revision. S G, K HW, C J, K EG. Regulation of the skeletal sarcoplasmic reticulum Ca2+ pump by phospholamban in reconstituted phospholipid vesicles. Membr Biochem 1990; 9: 191–202. S JP, G IL, G DG, R N, K EG. Ectopic expression of phospholamban in fast-twitch skeletal muscle alters sarcoplasmic reticulum Ca2+ transport and muscle relaxation. J Biol Chem 1997; 272: 18862–18868. V H, W F, D S H, H B, C R. Functional difference between SERCA2a and SERCA2b Ca2+ pumps and their modulation by phospholamban. Biochem J 1992; 286: 591–595. K Y, K K, T M, ML DH. Phospholamban inhibitory function is activated by depolymerization. J Biol Chem 1997; 272: 15061– 15064. C RL, J LR, A JM, T DD. Mutation and phosphorylation change the oligomeric structure of phospholamban in lipid bilayers. Biochemistry 1997; 36: 2960–2967. T M, K MA, R DI, K AM. The stimulation of calcium transport in cardiac sarcoplasmic reticulum by adenosine 3′:5′-monophosphate-dependent protein kinase. J Biol Chem 1974; 249: 6174–6180. L W, G IL, H J, P S, G G, D JJ, D T, K EG. Targeted ablation of the phospholamban gene is associated with markedly enhanced myocardial contractility and loss of beta-agonist stimulation. Circ Res 1994; 75: 401–409. L MJ, J Y, P D, P V, H K, B GJ, K D, L E, W RA, S M, Y A, M E, P M. SERCA1a structurally substitutes for SERCA2a in the cardiac sarcoplasmic reticulum and increases cardiac Ca2+ handling capacity. Circ Res submitted. C G, F DG, E I, K E, S Y, K EG. Phospholamban ablation and compensatory responses in the mammalian heart. Ann N Y Acad Sci 1998; 853: 49–62. V R, S R, R H, K F, O I, D H. Reciprocal changes in the postnatal expression of the sarcolemmal Na(+)-Ca(2+)exchanger and SERCA2 in rat hearts. J Mol Cell Cardiol 1995; 27: 1689–1701. C J, K F, P V, K B, V R. Thyroid control of sarcolemmal Na+/ Ca2+ exchanger and SR Ca2+-ATPase in developing rat heart. Am J Physiol 1998; 275: H264–H273. S R, R H, B J, E T, B M, H G, J H, H J, D H. Gene expression of the cardiac Na(+)-Ca(2+) exchanger in end-stage human heart failure. Circ Res 1994; 75: 443–453. S LF, K EG, L WJ. Calcium sparks and excitation-contraction coupling in phospholamban-deficient mouse ventricular myocytes. J Physiol 1997; 503: 21–29. H RJ, M F, M T, R A. SERCA Pump and Cardiac Contractility 69. 70. 71. 72. Prospects for gene therapy for heart failure. Circ Res 2000; 86: 616–621. H RJ, K JX, G JK, R A. Physiological effects of adenoviral gene transfer of sarcoplasmic reticulum calcium ATPase in isolated rat myocytes. Circulation 1997; 95: 423–429. G FJ, H HP, MD P, M M, S MR, D WH. Adenovirus-mediated gene transfer reconstitutes depressed sarcoplasmic reticulum Ca2+-ATPase levels and shortens prolonged cardiac myocyte Ca2+ transients. Circulation 1997; 96: 400–403. I G, L D, S C, N A, S C, H KW, R TB, J DC, K PD, O CP. Cell-specific promoter in adenovirus vector for transgenic expression of SERCA1 ATPase in cardiac myocytes. Am J Physiol 1998; 274: C645–C653. C M, O’D MJ, S C, I G, K MG. Exogenous Ca2+-ATPase isoform effects on Ca2+ transients of embryonic chicken and neonatal rat cardiac myocytes. J Physiol 2000; 523: 53–63. 1063 73. I K, Y X, F X, D WH, L BH. Effects of SERCA2 overexpression on contractile function during the transition from hypertrophy to early failure. Circulation 2000; 102 (Suppl. II): 459. 74. D M F, H SE, S U, M T, K ZB, D GW, G JK, R A, H RJ. Restoration of contractile function in isolated cardiomyocytes from failing human hearts by gene transfer of SERCA2a. Circulation 1999; 100: 2308–2311. 75. H RJ, S U, M T, G JL, L KH, G JK, D GW, S MJ, R A. Modulation of ventricular function through gene transfer in vivo. Proc Natl Acad Sci USA 1998; 95: 5251–5256. 76. M MI, D M F, S U, M T, G JL, G JK, R A, H RJ. Adenoviral gene transfer of SERCA2a improves left ventricular function in aortic-banded rats in transition to heart failure. Proc Natl Acad Sci USA 2000; 97: 793–798.