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(Circulation. 2004;110:3553-3559.)
© 2004 American Heart Association, Inc.

Molecular Cardiology

Excessive Sarcoplasmic/Endoplasmic Reticulum Ca²⁺-ATPase Expression Causes Increased Sarcoplasmic Reticulum Ca²⁺ Uptake but Decreases Myocyte Shortening

Nils Teucher; Juergen Prestle, PhD; Tim Seidler, MD; Susan Currie, PhD; Elspeth B. Elliott; Deborah F. Reynolds, PhD; Peter Schott, MD; Stefan Wagner; Harald Kogler, MD; Giuseppe Inesi, PhD; Donald M. Bers, PhD; Gerd Hasenfuss, MD; Godfrey L. Smith, PhD

From the Department of Cardiology and Pneumology, University of Göttingen, Göttingen, Germany (N.T., J.P., T.S., P.S., S.W., H.K., G.H.); Biochemistry and Molecular Biology, University of Maryland, Baltimore (G.I.); the Department of Physiology, Loyola University Chicago, Stritch School of Medicine, Maywood, Ill (D.M.B.); and the Institute of Biomedical and Life Sciences, University of Glasgow, Glasgow, UK (S.C., E.B.E., D.F.R., G.L.S.).

Correspondence to Gerd Hasenfuss, MD, Department of Cardiology and Pneumology, University of Goettingen, Robert-Koch-Strasse 40, 37075 Goettingen, Germany. E-mail hasenfus{at}med.uni-goettingen.de

Received June 20, 2003; de novo received December 22, 2003; revision received March 30, 2004; accepted April 4, 2004.

	Abstract

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Background— Increasing sarcoplasmic/endoplasmic reticulum (SR) Ca²⁺-ATPase (SERCA) uptake activity is a promising therapeutic approach for heart failure. We investigated the effects of different levels of SERCA1a expression on contractility and Ca²⁺ cycling. We tested whether increased SERCA1a expression levels enhance myocyte contractility in a gene-dose–dependent manner.

Methods and Results— Rabbit isolated cardiomyocytes were transfected at different multiplicities of infection (MOIs) with adenoviruses encoding SERCA1a (or ß-galactosidase as control). Myocyte relaxation half-time was decreased by 10% (P=0.052) at SERCA1a MOI 10 and by 28% at MOI 50 (P<0.05). Myocyte fractional shortening was increased by 12% at MOI 10 (P<0.05) but surprisingly decreased at MOI 50 (–22%, P<0.05) versus control. SR Ca²⁺ uptake (in permeabilized myocytes) demonstrated a gene-dose–dependent decrease in K_m by 29% and 46% and an increase in V_max by 37% and 72% at MOI 10 and MOI 50, respectively (all P<0.05 versus control). Ca²⁺ transient amplitude was increased in Ad-SERCA1a–infected myocytes at MOI 10 (by 121%, P<0.05), but at MOI 50, the Ca²⁺ transient amplitude was not significantly changed. Caffeine-induced Ca²⁺ transients indicated significantly increased SR Ca²⁺ content in Ad-SERCA1a–infected cells, by 72% at MOI 10 and by 87% at MOI 50. Mathematical simulations demonstrate that the functional increase in SR Ca²⁺-ATPase uptake activity at MOI 50 (and increased cytosolic Ca²⁺ buffering) is sufficient to curtail the Ca²⁺ transient amplitude and explain the reduced contraction.

Conclusions— Moderate SERCA1a gene transfer and expression improve contractility and Ca²⁺ cycling. However, higher SERCA1a expression levels can impair myocyte shortening because of higher SERCA activity and Ca²⁺ buffering.

Key Words: heart failure • gene therapy • calcium • sarcoplasmic reticulum

	Introduction

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Changes in myocardial excitation-contraction coupling are important in the pathophysiology of heart failure. Increased expression of Na⁺-Ca²⁺ exchanger (NCX), altered ryanodine receptor (RyR) function, and decreased expression of SR Ca²⁺-ATPase (SERCA) contribute to reduced SR Ca²⁺ accumulation. Furthermore, an increased phospholamban (PLB)/SERCA2a ratio reduces SERCA activity.¹

Decreased SR Ca²⁺ uptake activity slows relaxation and depresses contractility (because of reduced SR Ca²⁺ loading). In mice, disruption of 1 copy of the SERCA2 gene reduced the maximum velocity of SR Ca²⁺ uptake in homogenates, SR Ca²⁺ content and contractility in isolated cardiomyocytes, and in vivo cardiac performance.^2,3

Thus, restoring SR Ca²⁺ uptake may be valuable therapeutically in heart failure. Accordingly, transgenic mice overexpressing SERCA2a exhibited improved cardiac function and Ca²⁺ handling.^4,5 In neonatal rat cardiomyocytes with normal and depressed SERCA2a expression, adenovirus-mediated overexpression of SERCA2a resulted in enhanced SR Ca²⁺ uptake and accelerated decay of Ca²⁺ transients.^6,7 Furthermore, catheter-based transfection with an adenovirus encoding SERCA2a restored cardiac function in rats in transition to heart failure⁸ and improved survival.⁹ In human cardiomyocytes isolated from end-stage failing hearts, adenovirus-mediated augmented expression of SERCA2a resulted in enhanced contractility and Ca²⁺ handling.¹⁰

Here, we tested the hypothesis that increasing levels of SERCA expression progressively enhance contractility and relaxation rate. We used adenovirus-mediated gene transfer of SERCA1a into isolated rabbit ventricular cardiomyocytes. SERCA1a is a splice transcript of the SERCA1 gene (expressed in adult fast-twitch skeletal muscle) that has faster Ca²⁺ transport kinetics¹¹ and might achieve higher levels of SERCA activity than equivalent upregulation of SERCA2a.¹² Rabbit ventricular myocytes were used because excitation-contraction coupling in rabbit myocardium is similar to that in humans.^13,14

	Methods

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Primary Culture of Rabbit Ventricular Myocytes and Adenovirus Transfection
Ventricular cardiomyocytes were isolated from adult female Chinchilla Bastard rabbits (2.0 to 2.5 kg) as previously described.¹⁵ Myocytes were counted, and adenoviral transfection was performed at the indicated multiplicity of infection (MOI) while the cells were plated on laminin-coated 35-mm dishes at a density of 5x10⁵ rod-shaped myocytes/dish. This study was designed and performed in accordance with institutional guidelines for the care and use of animals.

Recombinant Adenovirus
Adenovirus containing the SERCA1a gene (Ad-SERCA1a) was generated as described previously.¹⁶ Briefly, cDNA of the chicken SERCA1a gene was expressed as a fusion protein with a c-terminal c-myc tag under control of the constitutively active cytomegalovirus promoter.

Verification of Transgene Expression and Expression Pattern
Reverse transcriptase–polymerase chain reaction (RT-PCR) was performed after 48 hours of culture. Specific primers for chicken SERCA1a and rabbit calsequestrin were used for amplification. For Western immunoblot, a polyclonal anti–c-myc antibody (PA1-981, ABR) was used at a dilution of 1:5000 to detect the expressed protein.

Single Myocyte Shortening and Intracellular Ca²⁺ Measurements
The stimulation frequency was 1 Hz, and myocytes were continuously perfused with Krebs buffer (2 mmol/L CaCl₂ at 36°C to 37°C). In separate experiments, intracellular Ca²⁺ concentration ([Ca²⁺]_i) was measured with Fura-2 (-AM loaded) was used as previously outlined.¹⁷ After 60 to 90 seconds of continuous 1-Hz stimulation, SR Ca²⁺ content was estimated by the transient rise in [Ca²⁺]_i induced by rapid application of 10 mmol/L caffeine for 3 to 5 seconds.¹⁸

Measurements of SR Ca²⁺ Uptake Characteristics
Myocytes (5x10⁵/mL, 48 hours after transfection) were permeabilized using 0.1 mg/mL ß-escin. Myocyte suspensions (4x10⁵ cells/mL) were stirred, and Fura-2 (10 µmol/L, Molecular Probes) was used to monitor [Ca²⁺] (20°C to 22°C). Oxalate (10 mmol/L) was included to maintain low and constant intra-SR [Ca²⁺] and ruthenium red (2.7 µmol/L) to block SR Ca²⁺ efflux.

Mathematical Modeling and Analysis
Simulations were based on mean measured Ca²⁺ transient parameters (see Table 2). Equations were derived to describe the intrinsic [Ca²⁺]_i dependence of Ca²⁺ transport by the SR Ca²⁺ pump (J_SR-Pump) and NCX (J_NCX), reported in µmol/L cytosol per second (or simply µmol/L/s).¹⁹ Ca²⁺ transients were simulated using these fluxes plus fluxes for SR Ca²⁺ release (J_Rel) and Ca²⁺ current (I_Ca) as required to produce the measured Ca²⁺ transient characteristics.

Statistics
Data are presented as mean±SEM based on the number of animals (ie, number of transfections) to allow the comparison of the Ca²⁺ uptake characteristics from cell aggregates. The number of cells was used to compare contractility and [Ca²⁺]_i measurements; the number of animals (ie, transfections) is also reported. Statistical analysis used paired Student t test or Wilcoxon signed-rank test as appropriate. A value of P<0.05 was considered significant.

Details of all aspects of the techniques used in this study are given in the online-only Data Supplement.

	Results

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Efficiency of Transgene Expression
Transgene expression of SERCA1a was confirmed by use of RT-PCR and Western blot immunodetection. Both methods demonstrated a dose-dependent increase in transgene expression (Figure 1) without changes in calsequestrin levels, which served as an internal standard. Immunohistochemical studies demonstrated a similar striated pattern of expression in both the MOI 10 and MOI 50 Ad-SERCA1a groups (see online-only Data Supplement), suggesting that there were no differences in the trafficking of SERCA1a at these 2 MOI values.

View larger version (53K): [in this window] [in a new window]   Figure 1. Dose-dependent SERCA1a expression after 48 hours. A, RT-PCR revealed a single band at 382 bp that increased with virus dose. Amplification with calsequestrin-specific primers using same probes demonstrates equal cDNA loading. B, Western immunoblot analysis showing virus dose–dependent increase in SERCA1a protein levels.

Contractile Parameters
Single-cell shortening was measured 48 hours after transfection (Figure 2, Table 1). At MOI 10, time to peak cell shortening (TTP) was reduced by 10%, relaxation time (RT₅₀) was reduced by 11%, and fractional shortening was 12% higher in Ad-SERCA1a–infected myocytes versus control (Figure 2, Table 1). Increasing Ad-SERCA1a to MOI 50 caused further abbreviation of time to peak shortening (by 23%) and RT₅₀ (by 28%), but fractional shortening was decreased by 22% at the higher level of SERCA1a expression compared with control (Figure 2, Table 1). The difference in shortening observed in the Ad-LacZ groups (MOI 10 versus MOI 50) is not a result of the different MOIs (not shown) but is likely to be a result of variation in isolated myocytes batches, in which MOI 10 studies (±SERCA1a) were performed in 1 set of cells and the MOI 50 studies were performed with separate batches of cells on different days. To exclude the influence of isolation and culture, mean values for each experimental day (for LacZ and SERCA) were compared using a paired t test (Table 1). Ca²⁺ transient measurements were made separately, and both MOI values were studied at the same time.

View larger version (37K): [in this window] [in a new window]   Figure 2. SERCA1a dose–dependent changes in myocyte contraction. Contraction parameters were normalized against Ad-LacZ–transfected controls (at same MOI). A representative recording from a control cell is shown. Fractional shortening is expressed relative to diastolic cell length; TTP, time to peak shortening; RT50, time from peak shortening to half relaxation. *P<0.05 vs Ad-LacZ at corresponding MOI; P=0.052 vs Ad-LacZ at corresponding MOI.

View this table: [in this window] [in a new window]   TABLE 1. Mechanical Parameters of Isotonically Contracting Myocytes

Stimulated and Caffeine-Induced Ca²⁺ Transients
As shown in Figures 3 and 4 and Table 2, the Ca²⁺ transient amplitude in cells infected with Ad-SERCA1a at MOI 10 was increased significantly, by 121%, versus control (Ad-LacZ), whereas the 18% increase in Ca²⁺ transient amplitude at MOI 50 was not significant (versus control). At MOI 10, diastolic [Ca²⁺]_i was not significantly different, but peak [Ca²⁺]_i was increased by 75%. At MOI 50, diastolic [Ca²⁺]_i was decreased significantly, by 32%, but peak [Ca²⁺]_i was not significantly different from that with Ad-LacZ-50. At both MOIs, the rate constant of the decline of [Ca²⁺]_i was increased in the Ad-SERCA1a group compared with the control cells (Table 2). As illustrated in Figures 3 and 4, SR Ca²⁺ content (assessed by rapid caffeine application) after 1-Hz stimulation was 72% higher in Ad-SERCA1a (MOI 10) than in control Ad-LacZ-10 myocytes. At MOI 50, caffeine-induced Ca²⁺ transient was 87% higher compared with the Ad-LacZ control group (Figure 4, Table 2). Thus, there is a SERCA1a gene-dose–dependent increase in the rate of diastolic [Ca²⁺]_i decline, consistent with increased SR Ca²⁺-ATPase function, but Ca²⁺ transient amplitude did not increase at the higher gene dose, and contraction decreased.

View larger version (28K): [in this window] [in a new window]   Figure 3. Ca2+ transients. A, [Ca2+]i recorded 48 hours after transfection with (i) Ad-LacZ (MOI 10), (ii) Ad-SERCA1a (MOI 10), and (iii) Ad-SERCA1a (MOI 50). Last 3 steady-state twitches at 1 Hz and caffeine-induced Ca2+ transients are shown. B, Average and normalized twitch Ca2+ transients from these groups.

View larger version (26K): [in this window] [in a new window]   Figure 4. Mean Ca2+ transient amplitudes (±SEM). *P<0.05 vs Ad-LacZ.

View this table: [in this window] [in a new window]   TABLE 2. Parameters of SR-Ca2+ Uptake Activity and Intracellular Ca2+ Measurements

SR Ca²⁺ Uptake Rates
As shown in Figure 5, oxalate-supported Ca²⁺ uptake was measured in permeabilized cardiomyocytes after transfection with Ad-LacZ and Ad-SERCA1a. The decay of the [Ca²⁺] in the cuvette after addition of an aliquot of CaCl₂ (50 nmol) was faster after Ad-SERCA1a transfection compared with the Ad-LacZ control, indicating increased Ca²⁺ uptake rate (Figure 5C). A quantitative description of SERCA activity was obtained by calculating the changes of total [Ca²⁺] on the basis of the known Ca²⁺ buffering capacity of the extracellular solution (Figure 5A). Differentiation of the total [Ca²⁺] signal reveals the rate of Ca²⁺ uptake that can be plotted against the associated free [Ca²⁺] to obtain a sigmoidal relationship (Figure 5B). This relationship was fitted with a logistic curve to estimate the free [Ca²⁺] that generated half the maximal Ca²⁺ uptake rate (K_m) and the value of the maximal rate of Ca²⁺ uptake (V_max). Table 2 shows mean values of V_max and K_m for Ad-LacZ and Ad-SERCA1a transfection at the 2 MOIs used. Cells infected with Ad-LacZ (MOI 10 versus MOI 50) exhibited comparable Ca²⁺ affinity (K_m) and V_max. Transfection with Ad-SERCA1a decreased the K_m of SERCA-mediated SR Ca²⁺ uptake by 29% and 46% (at MOI 10 and MOI 50, respectively, versus controls). The V_max increased by 37% and 71% at MOI 10 and MOI 50, respectively (versus controls). This is consistent with a gene-dose–dependent increase in SR Ca²⁺-ATPase function.

View larger version (25K): [in this window] [in a new window]   Figure 5. Individual experiments of SERCA-mediated Ca2+ uptake in permeabilized myocytes. A, Time course of cuvette [Ca2+] (4x105 cells/mL) measured using Fura-2 (10 µmol/L). Addition of 50 µmol/L total [Ca2+] causes a rapid [Ca2+] rise and subsequent decline because of SR transport. Superimposed dashed line is change in total [Ca2+] calculated from known Ca2+ buffers in solution. B, Rate of Ca2+ uptake (d[Ca2+]total/dt) vs [Ca2+]. Gray line is best fit to d[Ca2+]total/dt=Vmax/[1+([Ca2+]/Km)2]. Values of Vmax and Km are shown. C, Ca2+ uptake rates for different conditions (mean values in Table 2)

Intact Myocyte Ca²⁺ Flux Analysis
Simulations were used to clarify the lack of Ca²⁺ transient enhancement at high SERCA1a MOI. Figure 6A shows the [Ca²⁺]_i dependence of Ca²⁺ transport by the SERCA and NCX inferred from stimulated and caffeine-induced Ca²⁺ transients in intact myocytes. All 4 J_NCX curves were almost identical, so only the mean curve is shown. The V_max for J_SR-Pump was increased from 100 to 167 µmol/L/s in Ad-SERCA1a versus Ad-LacZ at MOI 10 and from 125 to 275 µmol/L/s for Ad-SERCA1a versus Ad-LacZ at MOI 50, respectively (ie, 60% and 85% increases in V_max). The apparent Ca²⁺ affinity was only slightly enhanced in the Ad-SERCA1a MOI 10 (K_m=197 versus 210 nmol/L), but the shift was greater in Ad-SERCA1a MOI 50 (164 versus 183 nmol/L). These results are consistent with the increases measured in permeabilized cells (although absolute values differ).

View larger version (24K): [in this window] [in a new window]   Figure 6. Ca2+ transport analysis and simulation. A, [Ca2+]-dependence of rate of Ca2+ transport by SERCA (JSR-Pump) (top 4 curves) and NCX (JNCX) inferred from intact cell Ca2+ transients. B, Simulated Ca2+ transients for MOI 10 (LacZ-10, SERCA-10). Broken curve demonstrates expected intracellular Ca2+ transients as a consequence of increased SR-Ca2+ content and therefore increased SR Ca2+ release (JRel) but without increased SERCA activity. C, Similar curves at MOI 50. Solid curves represent measured results. Broken curves are with higher SERCA-50 JRel but without increasing JSR-Pump activity (long dash) and without increasing JSR-Pump and without increased cytosolic Ca2+ buffering associated with additional SR Ca2+ pumps (short dash).

Figure 6B shows simulated Ca²⁺ transients for Ad-LacZ (MOI 10) and Ad-SERCA1a (MOI 10). For the SERCA-10 transient, the appropriate J_SR-Pump (and J_NCX) characteristics were used, and the SR Ca²⁺ release flux (J_Rel peak=3 mmol/L/s, integral=49 µmol/L) had to be scaled up by 97%. This exceeds the 72% increase in SR Ca²⁺ content assessed by caffeine, consistent with an increased fractional SR Ca²⁺ release at higher SR Ca²⁺ load.¹⁹ The broken curve in Figure 6B shows what would happen if we had increased only J_Rel without increasing SR Ca²⁺-ATPase activity. This suggests that the enhanced J_SR-Pump limits the rise in Ca²⁺-transient amplitude by only 13%.

Figure 6C shows similar curves for the SERCA-50 case. The lower 2 curves reflect measured results. The broken curves simulate transients with the same increase in J_Rel but without increasing SR Ca²⁺ pump function (shown), without increasing buffering (not shown), or both (top curve). Under those conditions, the Ca²⁺ transient would be larger. That is, Ca²⁺ transient amplitude for a given J_Rel is curtailed by 47% by the increased Ca²⁺-pump rate, 33% by the higher buffering, and 75% when combined. This analysis indicates that increasing SR Ca²⁺ pumps above an optimal value may buffer and curtail the Ca²⁺ transient without further raising SR Ca²⁺ load.

	Discussion

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The present study demonstrates that dissociation between SR Ca²⁺ content and myocyte fractional shortening occurs at high levels of SERCA1a overexpression. At MOI 10, myocytes expressing SERCA1a (versus Ad-LacZ controls) revealed enhanced SR Ca²⁺ uptake, relaxation rates, SR Ca²⁺ content, isotonic shortening, and Ca²⁺ transient amplitude. At higher SERCA expression levels (at MOI 50), myocytes exhibited further increases in SR Ca²⁺ uptake, relaxation rate, and SR Ca²⁺ content but showed depressed contraction amplitude and no Ca²⁺ transient enhancement versus control. We infer that high SERCA activity causes a paradoxical decreased contractile activation because of greater Ca²⁺ removal from the cytosol.

Several studies have shown that increased SR Ca²⁺-ATPase expression improves Ca²⁺ cycling and myocardial function. Here, the fast-twitch skeletal muscle SERCA1a was used because it has faster Ca²⁺ transport kinetics versus SERCA2a.^11,16 Furthermore, SERCA1a transfection in cultured neonatal cardiomyocytes generated higher SERCA protein levels than parallel SERCA2a studies.¹² SERCA1a targets to intracellular membranes after adenovirus-mediated gene transfer into embryonic cardiac myocytes in a pattern identical to that of SERCA2a.²⁰ Confocal studies on transgenic mice expressing SERCA1a in the heart showed traffic of SERCA1a to cardiac SR.²¹ A similar expression pattern was found in the present study.

In both our ß-escin–permeabilized and intact cells, SR Ca²⁺ uptake demonstrated a gene-dose–dependent increase in V_max and Ca²⁺ affinity (reduced K_m) in SERCA1a-expressing cells (Figures 5C and 6A and Table 2). Although SERCA1a and SERCA2a have identical Ca²⁺ concentration dependence¹¹ and are both regulated by PLB,^22,23 the shift in K_m in SERCA1 cells may indicate an increased SERCA/PLB ratio, with more Ca²⁺ pumps unregulated by PLB.

At MOI 10, improved SR Ca²⁺ uptake was accompanied by larger Ca²⁺ transients, contractions, SR Ca²⁺ content, and rates of [Ca²⁺]_i decline and relaxation. These findings are consistent with previous studies with SERCA1a transfection in chicken or rat cardiomyocytes.^12,20,21

Interestingly, higher virus titer (MOI 50) and SERCA1a protein expression decreased twitch contraction amplitude and failed to enhance Ca²⁺ transient amplitude, despite enhanced SERCA activity, SR Ca²⁺ load, twitch [Ca²⁺]_i decline, and relaxation rates. This indicates that above an optimum SERCA1a expression level (for Ca²⁺ transient amplitude), greater SERCA1a expression may limit Ca²⁺ transient amplitude. The higher rate of [Ca²⁺]_i decline after SERCA1a overexpression is paralleled by the increase in caffeine-releasable Ca²⁺. This strongly suggests that the additional efflux of Ca²⁺ from the cytosol is directed into the SR and not the sarcolemma. The unaltered NCX function (based on rate constants of [Ca²⁺]_i in the presence of caffeine) indicates that there were no compensatory changes in NCX transport during these studies. Thus, the net effect of high levels of SERCA1a overexpression is to enhance relaxation and, by shortening of the duration of activation of the contractile proteins, to reduce the amplitude of myocyte shortening.

On the basis of the rate constants of [Ca²⁺]_i decline,¹⁹ the contribution of NCX to [Ca²⁺]_i decline was 25% to 26% in Ad-LacZ–infected myocytes, as in previous data in rabbit.¹³ However, with enhanced SERCA1a expression (MOI 10 and MOI 50), NCX contribution decreased to 20% and 13%, respectively. This is consistent with the stronger competition of SERCA versus NCX. The Ca²⁺ flux analysis and simulations help to explain the unaltered Ca²⁺ transient amplitude and decreased isotonic shortening despite increased SR Ca²⁺ load and pump function at high SERCA overexpression. Greater SR Ca²⁺ pump expression can limit the Ca²⁺ transient by curtailing the peak. The impact of this effect is shown in Figure 6, B and C. Analogous data in rabbit myocytes showed that acute SERCA block with thapsigargin (without unloading the SR) increased Ca²⁺ transient amplitude by 20%.¹³ A second means by which increased SERCA expression can limit [Ca²⁺]_i is that it adds to cytosolic Ca²⁺ buffering. After troponin C (70 µmol/L), the SR Ca²⁺ pump is the most prominent cytosolic Ca²⁺ buffer (50 µmol/L), with equally high affinity.¹⁹ Thus, a 50% increase in SR Ca²⁺ pump expression would compete for Ca²⁺ binding to troponin C and limit the amplitude of the Ca²⁺ transient (see Figure 6C). The lower diastolic [Ca²⁺] in SERCA-overexpressing cells will also contribute to the absence of improved inotropy, because a larger SR Ca²⁺ release would be required to achieve peak systolic [Ca²⁺] levels that were comparable to control. Third, more SR Ca²⁺ pumps can increase the rate of SR Ca²⁺ uptake but may not increase maximal SR Ca²⁺ load for a given [Ca²⁺]_i.^24–26 This is because the SR Ca²⁺-ATPase can only build a [Ca²⁺] gradient (or potential energy) related to G_ATP (eg, G_SR-Pump=2 RT ln ([Ca²⁺]_SR/[Ca²⁺]_i) or [Ca²⁺]_SR/[Ca²⁺]_i 7000).²⁵ Thus, higher SR Ca²⁺ pump expression may allow a closer approach toward this limit (and get there faster), but there is a limit. It is notable that the diastolic [Ca²⁺]_i was significantly lower and SR Ca²⁺ load significantly higher in SERCA-50 versus LacZ-50, and this may represent an approach to this limiting [Ca²⁺]_SR/[Ca²⁺]_i gradient. Thus, at high SERCA levels, one may approach a maximal SR Ca²⁺ load that more SERCA cannot further improve.

Very high levels of SERCA1a gene transfer into neonatal rat and embryonic chicken cardiomyocytes could also produce cytotoxic effects, possibly related to SR dysfunction.²⁷ This did not occur here, where we saw enhanced SR Ca²⁺ transport and load and reduced diastolic [Ca²⁺]_i at the highest SERCA1a expression levels.

In conclusion, the dose of SERCA1a overexpression is critical for improved myocardial function. Although increased SR Ca²⁺ pump expression can enhance SR Ca²⁺ content, Ca²⁺ transient amplitude, and diastolic function, there may be an optimum value above which limiting factors prevent further increase in Ca²⁺ transients and can even reduce myocyte shortening. Although we used chicken SERCA1a and rabbit myocytes, we believe that the present findings are transferable to other species and SERCA isoforms. Increasing SR Ca²⁺ pump function in failing hearts (with low functional SERCA2a expression) may be beneficial, but the same SERCA1a expression in myocardium with normal SERCA2a levels could be detrimental. We conclude that the use of SERCA1a for gene therapy in heart failure requires careful control of transfection efficiency and induced expression levels.

	Acknowledgments

 
This study was supported by the Deutsche Forschungsgemeinschaft (graduate program 521), SFB Transregio II (project C4), the German National Genome Research Network (NGFN, Cardiovascular Diseases, Goettingen), the British Heart Foundation, and the National Institutes of Health (HL-30077). The help of Ying Sun is gratefully acknowledged. We gratefully acknowledge the expert technical assistance of M.A. Craig, M. Kothe, S. Ott-Gebauer, A. Rankin, and J. Spitalieri.

	Footnotes

 
The online-only Data Supplement, which contains an additional figure, is available with this article at http://www.circulationaha.org.

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