Excessive Sarcoplasmic/Endoplasmic Reticulum Ca2+-ATPase Expression Causes Increased Sarcoplasmic Reticulum Ca2+ Uptake but Decreases Myocyte Shortening
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 β-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 Km by 29% and 46% and an increase in Vmax 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.
Received June 20, 2003; de novo received December 22, 2003; revision received March 30, 2004; accepted April 4, 2004.
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
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 5×105 rod-shaped myocytes/dish. This study was designed and performed in accordance with institutional guidelines for the care and use of animals.
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 (≈5×105/mL, 48 hours after transfection) were permeabilized using 0.1 mg/mL β-escin. Myocyte suspensions (≈4×105 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 (JSR-Pump) and NCX (JNCX), 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 (JRel) and Ca2+ current (ICa) as required to produce the measured Ca2+ transient characteristics.
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.
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.
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.
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.
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 (Km) and the value of the maximal rate of Ca2+ uptake (Vmax). Table 2 shows mean values of Vmax and Km 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 (Km) and Vmax. Transfection with Ad-SERCA1a decreased the Km of SERCA-mediated SR Ca2+ uptake by 29% and 46% (at MOI 10 and MOI 50, respectively, versus controls). The Vmax 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.
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 JNCX curves were almost identical, so only the mean curve is shown. The Vmax for JSR-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 Vmax). The apparent Ca2+ affinity was only slightly enhanced in the Ad-SERCA1a MOI 10 (Km=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).
Figure 6B shows simulated Ca2+ transients for Ad-LacZ (MOI 10) and Ad-SERCA1a (MOI 10). For the SERCA-10 transient, the appropriate JSR-Pump (and JNCX) characteristics were used, and the SR Ca2+ release flux (JRel 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 JRel without increasing SR Ca2+-ATPase activity. This suggests that the enhanced JSR-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 JRel 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 JRel 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.
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 β-escin–permeabilized and intact cells, SR Ca2+ uptake demonstrated a gene-dose–dependent increase in Vmax and Ca2+ affinity (reduced Km) in SERCA1a-expressing cells (Figures 5C and 6⇑A and Table 2). Although SERCA1a and SERCA2a have identical Ca2+ concentration dependence11 and are both regulated by PLB,22,23 the shift in Km 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 ΔGATP (eg, ΔGSR-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.
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.
The online-only Data Supplement, which contains an additional figure, is available with this article at http://www.circulationaha.org.
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