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(Circulation Research. 2003;92:881.)
© 2003 American Heart Association, Inc.

Cellular Biology

Ca²⁺ Uptake by the Sarcoplasmic Reticulum in Ventricular Myocytes of the SERCA2^b/b Mouse Is Impaired at Higher Ca²⁺ Loads Only

Gudrun Antoons, Mark Ver Heyen, Luc Raeymaekers, Peter Vangheluwe, Frank Wuytack, Karin R. Sipido

From the Laboratories of Experimental Cardiology (G.A., K.R.S.) and Physiology (M.V.H., L.R., P.V., F.W.), University of Leuven, Belgium.

Correspondence to Karin R. Sipido, MD, PhD, Laboratory of Experimental Cardiology, KUL, Campus Gasthuisberg O/N 7th floor, Herestraat 49, B-3000 Leuven, Belgium. E-mail Karin.Sipido{at}med.kuleuven.ac.be

	Abstract

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SERCA2a is the cardiac-specific isoform of Ca²⁺-ATPase of the sarcoplasmic reticulum (SR). A reduction of SERCA2a has been implicated in the contractile dysfunction of heart failure, and partial knockout of the SERCA2 gene (Atp2a2^+/- mice) reiterated many of the features of heart failure. Yet, mice with a mutation of Atp2a2, resulting in full suppression of the SERCA2a isoform and expression of the SERCA2b isoform only (SERCA2^b/b), showed only moderate functional impairment, despite a reduction by 40% of the SERCA2 protein levels. We examined in more detail the Ca²⁺ handling in isolated cardiac myocytes from SERCA2^b/b. At 0.25 Hz stimulation, the amplitude of the [Ca²⁺]_i transients, SR Ca²⁺ content, diastolic [Ca²⁺]_i, and density of I_CaL were comparable between WT and SERCA2^b/b. However, the decline of [Ca²⁺]_i was slower (t_1/2 154±7 versus 131±5 ms; P<0.05). Reducing the amplitude of the [Ca²⁺]_i transient (eg, SR depletion), removed the differences in [Ca²⁺]_i decline. In contrast, increasing the Ca²⁺ load revealed pronounced reduction of SR Ca²⁺ uptake at high [Ca²⁺]_i. There was no increase in Na⁺-Ca²⁺ exchange protein or function. Theoretical modeling indicated that in the SERCA2^b/b mouse, the higher Ca²⁺ affinity of SERCA2b partially compensates for the 40% reduction of SERCA expression. The lack of SR depletion in the SERCA2^b/b may also be related to the absence of upregulation of Na⁺-Ca²⁺ exchange. We conclude that for SERCA isoforms with increased affinity for Ca²⁺, a reduced expression level is better tolerated as Ca²⁺ uptake and storage are impaired only at higher Ca²⁺ loads.

Key Words: ventricular myocytes • transgenic mice • sarcoplasmic reticulum Ca²⁺-ATPase • excitation-contraction coupling • Na⁺-Ca²⁺ exchange

	Introduction

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The Ca²⁺-transport ATPase of the sarco- and endoplasmic reticulum (SERCA) is essential for reuptake of Ca²⁺ into the intracellular stores. In cardiac cells, the SERCA2a splice variant maintains and regulates the Ca²⁺ content of the sarcoplasmic reticulum (SR). As Ca²⁺ release is the major source for the transient increase in [Ca²⁺]_i during excitation-contraction coupling, the amount of Ca²⁺ available for release, and thus SERCA activity, are important in regulation of cardiac contractility (see review^1,2). SERCA activity is regulated by phospholamban (PLB), which decreases the affinity for Ca²⁺and thus acts as an inhibitor (see review³). Phosphorylation of PLB by protein kinase A (PKA) during adrenergic stimulation removes this inhibition, enhances SR Ca²⁺ uptake, and contributes to the positive inotropic and lusitropic effect of adrenergic stimulation (see review⁴). This is accompanied by an increase in SR Ca²⁺ content.⁵ Phosphorylation of PLB, or SERCA itself, by Ca²⁺/calmodulin kinase has been implicated in the enhanced Ca²⁺ uptake at higher stimulation frequencies.^6,7

In human heart failure, a decrease in SERCA function is likely to be important in the slowed relaxation and diastolic dysfunction,^8–10 although it may not be the only mechanism.¹¹ Reduced SERCA function can also contribute to the decreased systolic function, as it would lower SR Ca²⁺ content. Such a decrease in SR Ca²⁺ content was observed in human heart failure,^12,13 and in some^14,15 but not all animal models of heart failure.¹⁶ Enhanced expression of the Na⁺-Ca²⁺ exchanger may help to maintain diastolic function,^14,17,18 but may also enhance loss of Ca²⁺ from the cell as observed during overexpression in cultured rabbit cardiomyocytes.¹⁹ The importance of SERCA in heart failure is supported by experiments where overexpression of the cardiac isoform of SERCA, ie, SERCA2a, could restore systolic and diastolic function.²⁰

Transgenic mouse models have further explored the pivotal role of SERCA2a in excitation-contraction coupling. Overexpression of SERCA2a,^21,22 or removing the inhibition of PLB on SERCA2a,^23,24 enhanced cardiac function and increased SR Ca²⁺ content. A knockout of the Atp2a2 gene, encoding SERCA2, was lethal in utero for homozygous mice.²⁵ Heterozygous mice, Atp2a2^+/-, had a 50% downregulation of SERCA2 protein and reduced in vivo contractility. Isolated myocytes had smaller [Ca²⁺]_i transients and reduced SR Ca²⁺ content, but the rate of [Ca²⁺]_i decline was unaffected.²⁶ It was postulated that in these mice, the observed upregulation of Na⁺-Ca²⁺ exchange compensated for the loss of Ca²⁺ removal by SERCA.

Ver Heyen et al²⁷ recently described a gene-targeted mouse with full substitution of the cardiac isoform SERCA2a by SERCA2b (further indicated as SERCA2^b/b). SERCA2b, the isoform that is found in most cell types and therefore considered to be the housekeeping form, has a nearly 2-fold higher affinity for Ca²⁺ but a lower maximal catalytic turnover rate than SERCA2a. Unexpectedly, total cardiac SERCA2 protein levels in the SERCA2^b/b were found to be decreased by 40% as compared with the WT, whereas phospholamban levels (PLB) were doubled. Ca²⁺-uptake studies showed a decrease of V_max of 40%, but despite the increase in PLB, the affinity was significantly higher for the SERCA2b (K_d of 0.19±0.01 µmol/L in SERCA2b versus 0.28±0.02 µmol/L in WT). Adult SERCA2^b/b mice showed a mild cardiac hypertrophy and a slowed relaxation and contraction in vivo, but no signs of heart failure. Myocytes isolated from SERCA2^b/b mice presented a slower decline of [Ca²⁺]_i compared with WT, although the difference was small. In the present study, we further investigated Ca²⁺ handling in SERCA2^b/b in order to understand the mild phenotype.

	Materials and Methods

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Cell Isolation
Wild-type mice (WT) were compared with SERCA2^b/b mice, expressing SERCA2b but no SERCA2a.²⁷ The exclusive expression of SERCA2b, and not SERCA2a, was obtained by mutation of the Atp2a2 gene. The 5'D1 donor splicing site was inactivated, and a strong polyadenylation site was added such that class 1 mRNA encoding SERCA2a, could not be produced. Single ventricular myocytes from hearts of 3- to 4-month-old mice were enzymatically isolated as described in.²⁸ All animal handling was conformed with the Guide for the Care and Use of Laboratory Animals (National Institute of Health, USA), and experimental protocols were approved by the in-house ethical committee. Cells were stored at room temperature and used for experiments within 6 hours after isolation.

Measurements of [Ca²⁺]_i and Membrane Currents
The setup for combined fluorescence and membrane current measurements was as described before.²⁹ Myocytes were studied under voltage clamp, using the ruptured whole-cell recording technique.³⁰ Membrane currents were filtered at 2 kHz and sampled and digitized at 4 kHz. [Ca²⁺]_i was monitored with fluo-3, included in the pipette solution. Fluorescence values were calibrated using the approach of Cheng et al³¹ with a K_d of 600 nmol/L, and after establishing that resting [Ca²⁺]_i was similar for WT and SERCA2^b/b.²⁷ In a number of cells, F_max was measured at the end of the experiment by repeated depolarizing steps to +120 mV and/or gently pushing down the cell on the bottom of the chamber with the patch pipette. This resulted in a rapid increase of fluorescence and cell contracture.

Solutions and Experimental Protocols
To study the general properties of the [Ca²⁺]_i transient and the frequency response, the external solution was a normal Tyrode solution (in mmol/L: NaCl 137, KCl 5.4, MgCl₂ 0.5, CaCl₂ 1.8, Na-HEPES 11.8, and glucose 10; pH 7.4, temperature 37°C), and the pipette solution was K⁺-based (in mmol/L: 120 K-aspartate, 20 KCl, 10 K-HEPES, 5 MgATP, 10 NaCl, and 0.05 K₅-fluo-3; pH 7.2). Cells were stimulated with 25-ms depolarizing pulses from -70 to +20 mV, mimicking the short action potentials of mouse ventricular myocytes.

For all other experiments, K⁺ currents were blocked by replacing K⁺ with Cs⁺. The bath solution contained (in mmol/L) NaCl 130, CsCl 10, MgCl₂ 0.5, CaCl₂ 1.8, Na-HEPES 11, and glucose 10; pH 7.4, temperature 24°C; the pipette solution contained (in mmol/L) Cs-aspartate 120, TEACl 10, Cs-HEPES 10, MgCl₂ 0.5, MgATP 5, and K₅-fluo-3 0.05; pH 7.2.

Cells were stimulated at 0.25 Hz with a depolarizing step of 50 ms from -70 to -40 mV to inactivate Na⁺ current, followed by a 100-ms step from -40 to +20 mV to activate I_CaL. I_NCX was measured as the inward current 15 ms after repolarization to -70 mV. Currents were normalized to cell membrane capacitance. Half-relaxation time of [Ca²⁺]_i transients (t_1/2) was measured from peak to half maximal relaxation.

To assess releasable SR Ca²⁺ content, cells were clamped at -70 mV and abruptly superfused for 8 seconds with 10 mmol/L of caffeine after a train of conditioning pulses at 0.25 Hz to induce stable loading. From the resulting inward I_NCX, the amount of Ca²⁺ extruded was calculated and normalized to cell volume using a surface/volume relationship of 8.44 pF/pL (for rat³²), an accessible cell volume fraction of 65%, and a correction factor for Ca²⁺ removal by other pathways.^28,33

High cellular Ca²⁺ loads were obtained by blocking Ca²⁺ extrusion via the Na⁺-Ca²⁺ exchanger. After depleting the SR with a fast caffeine (10 mmol/L) application, cells were superfused with Na⁺-free solution (in mmol/L: NMDGCl 120, TEACl 20, HEPES 11, MgCl₂ 0.5, CaCl₂ 5.4, and glucose 10; pH 7.4) and repeatedly depolarized (100-ms steps from -70 to 0 mV, at 0.25 Hz) activating Ca²⁺ entry via I_CaL.

Immunoblot
Protein levels of the Na⁺-Ca²⁺ exchanger were determined in total cardiac homogenates by Western blot analysis, using a polyclonal rabbit antibody (8–10, detailed in the expanded Materials and Methods section in the online data supplement available at http://www.circresaha.org).

Statistics
For group comparisons, unpaired t test was used. For frequency-dependent and time-dependent changes, ANOVA for repeated measurements was used with Bonferroni post hoc testing; P<0.05 was considered as significant. Results are shown as mean±SEM.

	Results

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Slowed Relaxation, but no Decrease in SR Ca²⁺ Content
Figure 1A shows a typical example of [Ca²⁺]_i transients in a SERCA2^b/b versus a WT cell, using experimental conditions approaching physiological conditions. The amplitude of the [Ca²⁺]_i transient showed a negative frequency dependence for both SERCA2^b/b and WT and was not significantly different between the groups (Figure 1B). However, the rate of decline of the [Ca²⁺]_i transient was slower in SERCA2^b/b at the lower frequencies (Figure 1C), but not at the higher frequencies where the amplitude of the [Ca²⁺]_i transient was smaller.

View larger version (23K): [in this window] [in a new window]   Figure 1. Frequency dependence of amplitude and decline of [Ca2+]i transients in SERCA2b/b vs WT. A, Examples of [Ca2+]i transients recorded in a SERCA2b/b cell and WT cell during steady-state stimulation with 25-ms depolarizing steps from -70 to +20 mV at 1 Hz in near physiological conditions (K+-based solutions, 37°C). B, Pooled data of peak [Ca2+]i, showing a decrease at higher stimulation frequencies and no difference between SERCA2b/b, n=12 () vs WT, n=7 (). C, Half-relaxation time (t1/2) of [Ca2+]i transients (*P<0.05 for SERCA2b/b vs WT).

At lower temperature and with K⁺-free solutions (see Materials and Methods), the difference in rate of [Ca²⁺]_i decline between SERCA2^b/b and WT remained (Figure 2A). A slower rate of Ca²⁺ uptake into the SR could lead to a decreased SR Ca²⁺ content. However, as shown in Figure 2B, there was no difference of SR Ca²⁺ content. Differences in Ca²⁺ influx via I_CaL could affect SR Ca²⁺ loading, but we could not detect a significant difference in peak current densities at +20 mV (4.17±0.73 and 3.73±0.3 pA/pF for respectively SERCA2^b/b and WT). Consistent with this absence of differences in I_CaL and SR content, the amplitude of the [Ca²⁺]_i transient was not significantly different (Figure 2C).

View larger version (26K): [in this window] [in a new window]   Figure 2. Slowed relaxation, but no decrease in SR Ca2+ content in SERCA2b/b. A, Average data of half-relaxation time (t1/2), and (C) amplitude of [Ca2+]i transients, [Ca2+]i, during steady-state stimulation at 0.25 Hz in K+-free solutions at 24°C (n=17 for SERCA2b/b vs n=15 for WT). B, SR Ca2+ content, calculated as described in Materials and Methods (n=12 for SERCA2b/b and for WT).

Function and Expression of the Na⁺-Ca²⁺ Exchanger
In the Atp2a2^+/- mouse studied by Periasamy and coworkers,²⁵ Na⁺-Ca²⁺ exchange was upregulated. We evaluated Na⁺-Ca²⁺ exchange function in the SERCA2^b/b mice from the rate of Ca²⁺ removal from the cytosol in the presence of 10 mmol/L caffeine, which prevents sequestration of Ca²⁺ in the SR. Figure 3A shows a typical example of a [Ca²⁺]_i transient in this condition. The decline of the [Ca²⁺]_i transient was fitted with a single exponential. The values for are shown in Figure 3B and do not differ between the groups. Western blot analysis showed that the protein levels of the Na⁺-Ca²⁺ exchanger were also not different between the groups (Figure 4A). We also measured peak current densities of I_NCX, as the inward current on repolarization to -70 mV, normalized to cell capacitance (Figure 4B) and to [Ca²⁺]_i at the time of peak current (Figure 4C), and found no significant differences. These data indicate that in contrast to the findings in the Atp2a2^+/- mice, the Na⁺-Ca²⁺ exchanger is not upregulated in the SERCA2^b/b.

View larger version (16K): [in this window] [in a new window]   Figure 3. Function of the Na+-Ca2+ exchanger in the absence of SR Ca2+ uptake. A, Typical example of a [Ca2+]i transient induced by 8-second application of 10 mmol/L of caffeine after a train of conditioning pulses, fitted with a single exponential. B, Pooled data of the time constant, , of the decline of the caffeine-induced transients in SERCA2b/b (n=7) and WT (n=9, P=NS).

View larger version (37K): [in this window] [in a new window]   Figure 4. Protein levels of Na+-Ca2+ exchanger in cardiac homogenates and current density. A, Left, Western blot analysis on total cardiac homogenates probed with a polyclonal antibody for the Na+-Ca2+ exchanger (NCX). Right, Pooled data expressed as mean±SEM relative to the average WT levels (n=14 for SERCA2b/b vs n=15 for WT); P=NS. B, Density of the NCX current measured as the inward current 15 ms after repolarization to -70 mV, and (C) density of the NCX current normalized to [Ca2+]i at the time of measurement (n=17 for SERCA2b/b, n=15 for WT).

Ca²⁺ Removal in Cells Heavily Loaded With Ca²⁺
Although there are significant differences in the rate of Ca²⁺ removal between SERCA2^b/b and WT, at least at the low frequencies of stimulation, these differences are not very pronounced despite the reduction of SERCA protein by 40%.²⁷ We hypothesized that this relatively mild effect was related to the documented higher Ca²⁺ affinity of SERCA2b compared with SERCA2a.²⁷ Indeed, a leftward shift in the Ca²⁺-activation curve of SERCA2b compared with SERCA2a would compensate for decreased levels of SERCA2 at lower [Ca²⁺]_i. However, one can expect a more severe impairment of Ca²⁺ removal at higher Ca²⁺ loads (see Ca²⁺ uptake curve of Figure 3C in Ver Heyen et al²⁷). We therefore examined the decline of [Ca²⁺]_i transients during Ca²⁺ loading in Na⁺-free solutions with high [Ca²⁺]_o (5.4 mmol/L). A typical example is shown in Figure 5A with superimposed [Ca²⁺]_i transients for a WT and a SERCA2^b/b cell. The amplitude of the [Ca²⁺]_i transients increased with successive pulses (Figure 5B), due to cumulative influx via I_CaL, which was comparable for both cell types (Figure 5C). [Ca²⁺]_i transients at high Ca²⁺ loads had a very slow initial decline of [Ca²⁺]_i, followed by a more rapid phase. We ruled out that dye saturation distorted the time course of the [Ca²⁺]_i transient as F_max measured immediately after the Na⁺-free loading protocol was clearly higher than the fluorescence values obtained during the experiment. As a first approach to compare the differences in Ca²⁺ removal, we measured the half-time of [Ca²⁺]_i decline from the onset of repolarization for successive pulses. The half-time for the SERCA2^b/b increased well above the half-time of the controls, and the curves diverged as the amplitude of [Ca²⁺]_i increased (Figure 6A, compare to Figure 5C for [Ca²⁺]_i). A plot of the half-time values versus the peak [Ca²⁺]_i for all individual traces confirms that the half-time of SERCA2^b/b is longer in particular for the larger values of [Ca²⁺]_i (Figure 6B). We next measured the actual SR Ca²⁺ flux during a single large [Ca²⁺]_i transient³⁴ (see online data supplement). Figure 6C shows the time course for total [Ca²⁺] after 12 seconds in Na⁺-free solution. The derivative of [Ca²⁺]_tot, after subtraction of the sarcolemmal flux, is a direct indicator of the SR Ca²⁺ flux³⁴ (Figure 6D). Shortly after repolarization, when [Ca²⁺]_i is still high, the rate of Ca²⁺ uptake is clearly lower in the SERCA2^b/b (a, corresponding to a free [Ca²⁺]_i of 450 nmol/L), but as [Ca²⁺]_tot declines, the rate becomes similar (b, corresponding to a free [Ca²⁺]_i of 200 nmol/L, consistent with the Ca²⁺-uptake curve in Ver Heyen et al²⁷). The somewhat lower SR Ca²⁺ release in SERCA2^b/b in Figure 6D suggests that at this time SR Ca²⁺ loading is also compromised. These data are consistent with the hypothesis that Ca²⁺ reuptake in SERCA2^b/b is more impaired at higher Ca²⁺ loads.

View larger version (25K): [in this window] [in a new window]   Figure 5. Induction of high Ca2+ loads into the cell. A, Typical examples of ICaL and [Ca2+]i transients recorded during the first 5 pulses in a WT and SERCA2b/b cell (top inset, pulse protocol). Arrows on ICaL indicate the increasing rate of inactivation of ICaL. B, Average amplitude of [Ca2+]i during successive pulses in WT (n=8) and SERCA2b/b (n=9). D, Ca2+ entry via ICaL calculated as the cumulative integral of successive pulses normalized to accessible cell volume.

View larger version (26K): [in this window] [in a new window]   Figure 6. Ca2+ removal at high Ca2+ loads. A, Pooled data of half-time of [Ca2+]i transients during successive depolarizations in Na+-free solutions in WT (n=8, ) and SERCA2b/b (n=9, ). *P<0.05 for SERCA2b/b vs WT. B, Half-time values vs peak [Ca2+]i for all individual traces (, WT; , SERCA2b/b). C, Time course of total [Ca2+] for the pulse at 12 seconds in Na+-free solution (black trace is average of 8 WT, and red trace of 9 SERCA2b/b cells). C, Net SR Ca2+ flux (color code as in B). See text for a and b.

Model for Ca²⁺ Removal by SERCA2b Versus SERCA2a
We further explored the effects of substituting the WT SERCA2a by a high-affinity SERCA2b isoform in a theoretical model (detailed in the online data supplement available at http://www.circresaha.org). The model consists of Ca²⁺ influx into the cytosol via I_CaL for a fixed duration and an amplitude that decreases linearly with time. Ca²⁺ release is of fixed duration, and the rate is a linear function of the SR Ca²⁺ content. Ca²⁺ uptake by SERCA and Ca²⁺ extrusion by the Na⁺-Ca²⁺ exchanger and PMCA pumps depends on the respective K_m and V_max values. The Hill coefficient is 2 for SERCA and 1 for Na⁺-Ca²⁺ exchanger and PMCA. [Na⁺]_i is assumed to be constant.

Figure 7A shows superimposed simulated [Ca²⁺]_i transients during steady-state stimulation at 1 Hz for various properties of SERCA. Without further compensatory mechanisms, a slight increase of the affinity of the SERCA pump for Ca²⁺ (decrease of K_m from 0.28 to 0.25 µmol/L, dotted line) results in a dramatic increase of the Ca²⁺ signal. The peak amplitude can be restored to that of the WT signal by decreasing the V_max of the Ca²⁺ pump from 1000 to 640 (µmol/L) · s^-1 (dashed line). This simultaneously leads to a small but significant slowing of the decline of [Ca²⁺]_i. The steady-state SR Ca²⁺ content was practically unchanged (-0.7%). A similar effect on the amplitude could be obtained by reducing the Ca²⁺ influx from 9 to 5.7 µmol/L, but this accelerated the decline of [Ca²⁺]_i (data not shown). Thus, the first approach fits better with the experimentally observed Ca²⁺ transients in SERCA2^b/b mice and is also in agreement with the observed decrease of the number of pump sites.

View larger version (26K): [in this window] [in a new window]   Figure 7. Simulations of [Ca2+]i transients in WT and SERCA2b/b ventricular cells. A, [Ca2+]i transients during steady-state stimulation at 1 Hz. Solid line is for a control myocyte, the dotted line is for SERCA with a higher Ca2+ affinity, and the dashed line is for a reduction of SERCA protein with simultaneous increase in affinity, as in SERCA2b/b. B, Simulation of 3 consecutive [Ca2+]i transients at 0.25 Hz in conditions of increased Ca2+ influx (high [Ca2+]out and low [Na+]out) and decreased Ca2+ efflux (Na+-Ca2+ exchange activity set at zero) as in Figure 5. Solid line, WT; dashed line, SERCA2b/b (SERCA Vmax=(640 µmol/L) · s-1, Km=0.19 µmol/L). Initial amount of SR-bound Ca2+ was lowered to 30 µmol/L, mimicking preceding application of caffeine. SR-bound Ca2+ at the end of the third transient was, respectively, 177 and 178 µmol/L in WT and SERCA2b/b.

Figure 7B simulates the experiments of Figure 5 by reducing the capacity of the Na⁺-Ca²⁺ exchanger by 90% and setting the initial values for SR content low. It can be seen that at the low Ca²⁺ load, the decline of [Ca²⁺]_i for SERCA2^b/b is not markedly different, but that Ca²⁺ removal is most impaired at the higher [Ca²⁺]_i transient amplitudes. For the high values of SR content, the actual onset of decline of [Ca²⁺]_i in the SERCA2^b/b is also delayed, as in Figure 5.

We also examined the effects of a SERCA2-isoform switch in the more elaborated guinea-pig myocyte model, developed by Rudy et al.^35,36 Similar results were obtained (data not shown). Reduction of the K_d increased the amplitude of the [Ca²⁺]_i transient, and simultaneous reduction of the V_max by 40% brought the amplitude back to control levels, although with slowed relaxation, as experimentally observed. In neither of the models, Na⁺-Ca²⁺ exchange function had to be altered to reproduce the experimentally observed results.

	Discussion

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We have investigated Ca²⁺ handling in SERCA2^b/b mice, which express the SERCA2b isoform in the heart instead of the SERCA2a isoform. These mice have a reduction of total cardiac SERCA protein of 40%.²⁷ In SR vesicles, maximal Ca²⁺-uptake rate is reduced, but Ca²⁺ affinity is increased.²⁷ In single ventricular myocytes of SERCA2^b/b mice, [Ca²⁺]_i decline is slower, but SR Ca²⁺ content and amplitude of [Ca²⁺]_i transients are not significantly different. There is no increase in Na⁺-Ca²⁺ exchange protein or function. However, at high Ca²⁺ load, Ca²⁺ removal by the SR in myocytes of SERCA2^b/b is severely compromised. Simulations of [Ca²⁺]_i transients can reproduce these observations by incorporating into the model (1) the specific properties of SERCA2b, (2) the downregulation of the total number of pumps, and (3) the absence of upregulation of Na⁺-Ca²⁺ exchange.

Phenotype of the SERCA2^b/b Heart: Why Cellular Function Is Not More Impaired
In the present concept of cellular mechanisms of heart failure, a decrease in SERCA protein is a pivotal event, responsible for slowing of relaxation, decreased SR content, and smaller [Ca²⁺]_i transients. Although the total amount of SERCA2 protein in the SERCA2^b/b is decreased by 40%, the slowing of decline of [Ca²⁺]_i in baseline conditions is not very pronounced. In the SERCA2^b/b, we propose that the nearly 2-fold increase in affinity of the SERCA2b isoform, which makes up all of the SERCA2 protein in the mouse ventricle, compensates for the reduction of total pump capacity at lower Ca²⁺ loads. Consistent with this postulate, Ca²⁺ removal is not impaired for small [Ca²⁺]_i transients, as seen at the higher stimulation frequency (Figure 1), or after depletion of the SR following a caffeine application (Figure 6A, onset of stimulation in Na⁺-free). Conversely, Ca²⁺ removal at the higher Ca²⁺ loads is more significantly reduced (Figures 6B through 6D). This effect of the increased affinity could be reproduced during model simulations, supporting the hypothesis.

A comparison with the heterozygous SERCA KO mouse, Atp2a2^+/-, yields some interesting perspectives. In this latter model, the total SERCA protein is reduced to similar levels as in our SERCA2^b/b.²⁵ However, cardiac myocytes from Atp2a2^+/- did not show slowing of the decline of [Ca²⁺]_i. This could be explained by a compensatory upregulation of Na⁺-Ca²⁺ exchange.²⁶ In this same Atp2a2^+/- model, SR Ca²⁺ content was reduced, and the amplitude of [Ca²⁺]_i transients was smaller. It is tempting to speculate that this is due to the concomitant upregulation of Na⁺-Ca²⁺ exchange, which will favor Ca²⁺ extrusion. Such upregulation is absent in the SERCA2^b/b, and we do not see a decrease in SR content, suggesting that the lack of upregulation of Na⁺-Ca²⁺ exchange may protect the cells from Ca²⁺ loss. It is also important to note that in the transgenic mouse with overexpression of Na⁺-Ca²⁺ exchanger, there was no reduction of SR Ca²⁺ content.^37,38 This suggests that in the Atp2a2^+/-, it is the combination with the reduced SERCA, and not Na⁺-Ca²⁺ exchange alone, which leads to increased loss of Ca²⁺. In contrast, in cultured rabbit ventricular myocytes, overexpression of Na⁺-Ca²⁺ exchanger could by itself reduce SR Ca²⁺ content and amplitude of [Ca²⁺]_i transients.^19,39 Differences in [Na⁺]_i between mouse and rabbit, and/or alterations in SERCA with culture, could explain these differences.

The contractile function in vivo of the SERCA2^b/b mice is more impaired than might be expected from the properties of the isolated myocytes reported here.²⁷ Several factors could explain this apparent discrepancy. The presence of adrenergic stimulation in vivo will increase the Ca²⁺ load and amplitude of the [Ca²⁺]_i transients, with consequently more impaired relaxation and potentially reduced SR Ca²⁺ content (Figure 6D). In addition, the presence of hypertrophy²⁷ will further impair ventricular relaxation and filling. This hypertrophy is currently unexplained, but may be a consequence of the slower Ca²⁺ removal.²⁷

Dissociation Between Downregulation of SERCA and Upregulation of Na⁺-Ca²⁺ Exchange
It has been proposed that in heart failure there is a reexpression of the fetal gene pattern, which includes a lower level of SERCA and upregulation of Na⁺-Ca²⁺ exchange.⁴⁰ A review of available literature data however indicates that upregulation of Na⁺-Ca²⁺ exchange is not a consistent finding,¹⁸ although the data on SERCA downregulation are more consistent.⁴¹ The present observations again show that low expression levels of SERCA are not necessarily associated with increased expression of Na⁺-Ca²⁺ exchange, even in the presence of hypertrophy.²⁷ This indicates that regulation of expression of these Ca²⁺-handling proteins can be controlled by independent signaling pathways, at least in hypertrophy, and is consistent with the recently demonstrated complexity of the signaling in hypertrophy.⁴² This does not exclude that certain stimuli may affect the Na⁺-Ca²⁺ exchanger and SERCA in a concerted way, as was demonstrated for the thyroid hormone.⁴³

We can as yet not explain why the total SERCA protein is decreased in the SERCA2^b/b mouse. This may, at least partially, result from a lower stability of the SERCA2b mRNA versus the SERCA2a mRNA.⁴⁴ A provocative hypothesis is that this downregulation could actually be a protective mechanism. As the modeling results show, an increase in SERCA Ca²⁺ affinity without concomitant downregulation of protein expression would lead to an increase in the [Ca²⁺]_i transients and the SR Ca²⁺ content.

Our findings underline the importance of the SERCA Ca²⁺ affinity in regulating the SR Ca²⁺ uptake. With a higher affinity, a lower number of pumps can maintain Ca²⁺ homeostasis, at least at low to moderate Ca²⁺ loads. This predicts that for eg, SERCA1, modest expression levels may be sufficient. The feasibility of introducing SERCA1, as well as the importance of controlling expression levels were recently reported.^45–47

Conclusions
Our findings in the SERCA2^b/b mouse illustrate that a reduced expression level of SERCA isoforms with increased affinity for Ca²⁺ is better tolerated than for SERCA2a as Ca²⁺ uptake and storage are impaired only at higher Ca²⁺ loads. The lack of upregulation of Na⁺-Ca²⁺ exchange may help to minimize Ca²⁺ loss, and indicates that expression levels of SERCA and of Na⁺-Ca²⁺ exchange are independently regulated. Expression of higher-affinity SERCA isoforms may have strategic advantages as efficient Ca²⁺ removal can be obtained at lower expression levels.

	Acknowledgments

 
This study was supported by the FWO, the Flanders Fund for Scientific Research.

Received October 8, 2002; revision received March 14, 2003; accepted March 18, 2003.

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