Novel approach for quantification of endoplasmic reticulum Ca²⁺ transport

Abstract

The type 2a sarco-/endoplasmic reticulum Ca²⁺-ATPase (SERCA2a) plays a key role in Ca²⁺ regulation in the heart. However, available techniques to study SERCA function are either cell destructive or lack sensitivity. The goal of this study was to develop an approach to selectively measure SERCA2a function in the cellular environment. The genetically encoded Ca²⁺ sensor R-CEPIA1er was used to measure the concentration of Ca²⁺ in the lumen of the endoplasmic reticulum (ER) ([Ca²⁺]_ER) in HEK293 cells expressing human SERCA2a. Coexpression of the ER Ca²⁺ release channel ryanodine receptor (RyR2) created a Ca²⁺ release/reuptake system that mimicked aspects of cardiac myocyte Ca²⁺ handling. SERCA2a function was quantified from the rate of [Ca²⁺]_ER refilling after ER Ca²⁺ depletion; then, ER Ca²⁺ leak was measured after SERCA inhibition. ER Ca²⁺ uptake and leak were analyzed as a function of [Ca²⁺]_ER to determine maximum ER Ca²⁺ uptake rate and maximum ER Ca²⁺ load. The sensitivity of this assay was validated by analyzing effects of SERCA inhibitors, [ATP]/[ADP], oxidative stress, phospholamban, and a loss-of-function SERCA2a mutation. In addition, the feasibility of using R-CEPIA1er to study SERCA2a in a native system was evaluated by using in vivo gene delivery to express R-CEPIA1er in mouse hearts. After ventricular myocyte isolation, the same methodology used in HEK293 cells was applied to study endogenous SERCA2a. In conclusion, this new approach can be used as a sensitive screening tool to study the effect of different drugs, posttranslational modifications, and mutations on SERCA function.

NEW & NOTEWORTHY The aim of this study was to develop a sensitive approach to selectively measure sarco-/endoplasmic reticulum Ca²⁺-ATPase (SERCA) function in the cellular environment. The newly developed Ca²⁺ sensor R-CEPIA1er was used to successfully analyze Ca²⁺ uptake mediated by recombinant and native cardiac SERCA. These results demonstrate that this new approach can be used as a powerful tool to study new mechanisms of Ca²⁺ pump regulation.

INTRODUCTION

The sarco-/endoplasmic reticulum Ca²⁺-ATPase (SERCA) is an essential component of Ca²⁺ signaling (7). During the SERCA transport cycle, hydrolysis of one ATP molecule provides energy for translocation of two Ca²⁺ ions from the cytosol into the endoplasmic reticulum (ER) lumen. As a result of SERCA activity, the ER luminal Ca²⁺ concentration ([Ca²⁺]_ER) can reach millimolar concentration, while cytosolic [Ca²⁺] ([Ca²⁺]_i) is maintained at the submicromolar level. This large Ca²⁺ gradient is the foundation for Ca²⁺ signaling, as activation of ER Ca²⁺ release can produce a robust intracellular Ca²⁺ transient that is essential for activation of many cellular processes, including growth, migration, secretion, metabolism, neurotransmission, and contraction. Three SERCA genes with 10 isoforms have been identified (31). Despite a significant homology between SERCA types, they have specific differences in kinetics and Ca²⁺ sensitivity uniquely fitted to their function in different cell types. Here, we focus on the cardiac isoform SERCA2a.

As the muscle contraction-relaxation cycle is controlled by intracellular Ca²⁺ dynamics, Ca²⁺ transport is a particularly important point of cardiac regulation. The rate at which SERCA2a removes Ca²⁺ from the cytosol into the sarcoplasmic reticulum (SR) is a major determinant of the rate of cardiac muscle relaxation. SERCA2a activity also plays a key role in setting diastolic [Ca²⁺]_i, which affects the compliance of the heart muscle as the ventricles fill with blood. SERCA2a also sets the total amount of Ca²⁺ in the SR (i.e., Ca²⁺ load), which determines the magnitude of SR Ca²⁺ release with each heartbeat. This latter function of SERCA2a is the primary determinant of the strength of the cardiac contraction. Thus SERCA2a activity must be closely regulated to match the varying demands of exercise and rest. This is accomplished through the action of the transmembrane peptide phospholamban (PLB). This peptide is the major regulator of the SR Ca²⁺ pump (24), inhibiting SERCA2a activity by lowering the apparent affinity of the pump for Ca²⁺ (20). Phosphorylation of PLB by PKA relieves this inhibition, increasing the Ca²⁺ pumping rate by several-fold (36). Stimulation of SR Ca²⁺ uptake by PLB phosphorylation is the major mechanism of acceleration of relaxation (lusitropy) and increased contraction strength (inotropy) during adrenergic stimulation (15). Because of the central role of SERCA2a in the cardiac contractile cycle, it is not surprising that impaired SERCA2a function has been reported in a number of pathological conditions, including heart failure (44). The impaired SERCA2a function in heart failure can be a consequence of SERCA2a downregulation at the mRNA and protein levels (13, 19, 30) or a result of posttranslational modifications of SERCA2a and PLB (1, 13, 22, 33). Moreover, it has been shown that several mutations of PLB contribute to cardiomyopathies (18). Consequently, the SERCA2a/PLB complex has attracted attention as a potential target for therapeutic approaches to improve heart function during diseases (17). Despite this interest in understanding SERCA-regulatory mechanisms, it remains problematic to selectively measure the SERCA-mediated Ca²⁺ uptake in living cells due to a contribution of different ion channels, exchangers, and pumps to Ca²⁺ homeostasis.

Several established experimental approaches are currently used for the quantification of SERCA2a function. In intact myocytes, SERCA-mediated Ca²⁺ uptake can be inferred from the decay of the cytosolic Ca²⁺ transient induced by an action potential (6) or from the total amount of Ca²⁺ stored in the SR (14, 40). Because the cytosolic measurements can be contaminated by plasmalemmal and mitochondrial Ca²⁺ transport, these indirect approaches are not selective for SERCA2a activity. Moreover, these measurements depend on high-affinity cytosolic Ca²⁺ indicators (e.g., Fluo-3) that can be easily saturated by SR Ca²⁺ release. In vitro assays of SERCA function based on ATP consumption (29) or Ca²⁺ uptake into ER/SR vesicles (34) are more direct and quantitative measures of SERCA2a function, but they require cell homogenization and relatively large amounts of protein. Recently, another approach has been developed which allows measuring the SR luminal [Ca²⁺] ([Ca²⁺]_SR) directly in intact cells (11, 21, 35). This approach is based on the ability of low-affinity Ca²⁺ dyes, such as Fluo-5N, to measure SR luminal Ca²⁺. Thus SERCA2a-mediated Ca²⁺ uptake can be measured directly from the rate of [Ca²⁺]_SR recovery during the Ca²⁺ transient (10, 46). However, this method also suffers from several limitations, including dye extrusion, nonspecific dye localization, and species limitation. Thus an alternative approach is needed to quantify SERCA2a transport kinetics in living cells.

The aim of this study was to develop an approach to selectively measure SERCA2a function. We used the genetically encoded ER Ca²⁺ sensor R-CEPIA1er to directly measure SR Ca²⁺ uptake mediated by recombinant and native SERCA2a. The approach was validated by testing effects of well-known SERCA inhibitors and regulators. The obtained results demonstrate that this new method can be used as a sensitive assay to study SERCA function in the cellular environment.

MATERIAL AND METHODS

Vector Production

The vector encoding the human RyR2 cDNA fused to green fluorescent protein (GFP) at the NH₂-terminus domain was kindly provided by Dr. Christopher George (University of Cardiff). pCMV R-CEPIA1er was a gift from Dr. Masamitsu Iino (Addgene plasmid 58216; http://n2t.net/addgene:58216; RRID: Addgene_58216). The vector encoding human SERCA2a cDNA was kindly provided by Dr. David Thomas (University of Minnesota). The SERCA2a cDNA was cloned into the mCerulean-M1 modified plasmid (Addgene) using KpnI and NotI restriction enzymes, yielding a recombinant SERCA2a protein fused to a modified cerulean fluorescent protein (mCer) at the NH₂-terminus. SERCA2a cDNA was also cloned into the inducible expression vector pcDNA5/FRT/TO using KpnI and EcoRV for the generation of a SERCA2a stable cell line. D426A/E429A/E435A (AAA) and E876Q/D877N/E882Q (QNQ) SERCA2a mutants were generated by site-directed mutagenesis (Q5 site-directed mutagenesis kit, NEB). The sequences were all verified by single-pass primer extension analysis (ACGT).

Generation of SERCA2a Stable Inducible Flp-In T-Rex-293 Cell Line

The stable inducible Flp-In T-Rex-293 cell line expressing SERCA2a was generated using the Flp-In T-REx Core Kit (Invitrogen). Briefly, Flp-In T-REx-293 cells were cotransfected with the pOG44 vector encoding the Flp recombinase and the expression vector pcDNA5/FRT/TO containing the SERCA2a cDNA (without a fluorescent tag) in a ratio of 9:1 using polyethylenimine (PEI; 1 μg/ml). Forty-eight hours after transfection, the growth medium was replaced with a selection medium containing 100 μg/ml hygromycin (Invitrogen). The selection medium was changed every 2 days until the stable line cell foci could be isolated. The hygromycin-resistant cell foci were selected and expanded. Stable line cells were cultured in high-glucose DMEM supplemented with 100 U/ml penicillin, 100 mg/ml streptomycin, and 10% fetal bovine serum at 5% CO₂ and 37°C.

Characterization of hSERCA2a Stable Cell Line

Expression of SERCA2a in the stable cell line was verified by Western blot analysis. Briefly, samples were collected 48 h after induction of recombinant SERCA2a expression with 1 μg/ml tetracycline. Samples were lysed in a buffer containing 1% Triton and protease inhibitors. Equal amounts of protein were run on a SDS-PAGE and blotted on nitrocellulose using the turbo transfer system (Bio-Rad). Membranes were incubated with primary antibody anti-SERCA2 (IID8, Santa Cruz Biotechnology) and developed using with the horseradish peroxidase (HRP)-conjugated secondary antibody. Western blots were imaged in a ChemiDoc (Bio-Rad) apparatus after incubation with the HRP chemiluminescent substrate (Millipore).

Cell Line Transfection and Experimental Protocol

Two different cell lines (HEK293 and Flp-In T-Rex-293) were used to study the SERCA2a function. HEK293 cells at 60–80% confluence were transiently cotransfected with plasmids containing the cDNA of RyR2, SERCA2a, and R-CEPIA1er. Experiments were conducted 48 h after transfection to obtain the optimal level of recombinant proteins expression. Flp-In T-Rex-293 stably expressing SERCA2a were cotransfected with plasmids containing the cDNA of RyR2 and R-CEPIA1er, using the same conditions for the HEK293 cells. Before each experiment, cells cultured on a laminin-coated glass coverslip were washed with a solution containing (in mM) 150 K-aspartate, 0.25 MgCl₂, 0.1 EGTA, 10 HEPES, pH 7.2. Afterward, cells were permeabilized with saponin (0.005%). Experiments were conducted after washout of saponin with a saponin-free experimental solution (in mM): 100 K-aspartate, 15 KCl, 5 KH₂PO₄, 5 MgATP, 0.35 EGTA, 0.22 CaCl₂, 0.75 MgCl₂, and 10 HEPES; 2% dextran (MW: 40,000), pH 7.2. Free [Ca²⁺] and [Mg²⁺] of this solution were 200 nM and 1 mM, respectively.

Adenovirus Production

The R-CEPIA1er was subcloned into pShuttle-CMV by standard methods and verified by sequencing. Adenoviral vectors of all the cDNAs were made using the Stratagene AdEasy XL System. Amplified viruses were purified by CsCl gradient ultracentrifugation for 2 h at 22,000 rpm. The resultant adenovirus band was dialyzed and spectrophotometrically read at 260 nm to determine the titer value before addition of 10% glycerol. The final adenovirus density was 5 × 10¹⁰ particles/ml.

In Vivo Gene Delivery of R-CEPIA1er

All animal husbandry and surgical procedures were performed under animal protocols approved by the Institutional Animal Care and Use Committee and conformed to the Guide for the Care and Use of Laboratory Animals (NIH Publication 85-23, Revised 1985). C57BL/6J mice (Jackson) were maintained on a 12-h:12-h light-dark cycle and were given ad libitum food and water throughout the experiments. Eight- to ten-week-old mice, weighing 20–25 g, were used in the experiments. Mice were anesthetized with isoflurane at a dose of 1–3% with oxygen (0.5%). Artificial respiration was maintained with a rodent ventilator. The heart was exposed upon opening of the left pleural cavity by cutting the intercostal muscles and by positioning a retractor between the left third and fourth ribs. The R-CEPIA1er adenovirus (5 × 10¹⁰ particles/ml) was administered by direct injection in the left ventricular wall of the myocardium (6 spots, 8 μl/site) using a syringe fitted with a 29-gauge needle. After the injection, the chest was closed in layers. Mice were carefully monitored for postoperative recovery and given free access to the analgesic carprofen. Expression of the R-CEPIA1er transgene was optimal at 6 days postinjection.

Myocyte Isolation

Six days after the injection with the R-CEPIA1er adenovirus, the mice underwent terminal surgery for cardiomyocytes isolation. Mice were anesthetized using isoflurane (1%). Following thoracotomy, hearts were quickly excised, immersed in Ca²⁺ free buffer, mounted on a Langendorff apparatus, and retrogradely perfused with liberase H (Roche)-containing solution at 37°C according to the procedure previously described (25). The left ventricle was excised from the digested heart, placed in stop buffer containing BSA (1 mg/ml), cut into several pieces (average size 1 mm), and gently triturated into single cells. Myocytes were pelleted by gravity (∼0.1 ml) and resuspended in low-Ca Tyrode’s buffer (in mM): 140 NaCl, 4 KCl, 0.1 CaCl₂, 1 MgCl₂, 10 glucose, and 10 HEPES; pH 7.4. [Ca²⁺] was gradually adjusted to 1 mM. Isolated cardiomyocytes were stored at room temperature (20°C). All chemicals and reagents were purchased from Sigma-Aldrich (St. Louis, MO).

Confocal Microscopy

Expression of recombinant proteins and changes in the luminal ER [Ca²⁺] ([Ca]_ER) were measured with laser scanning confocal microscopy (Radiance 2000 MP, Bio-Rad) equipped with a ×40 oil-immersion objective lens (N.A. = 1.3).

Expression of recombinant proteins.

To verify and quantify expression of SERCA2a, mCer was excited with the 457-nm line of the argon laser and the signal was collected at >485 nm. To verify and quantify expression of RyR2, the GFP was excited with the 488-nm line of the argon laser, and the signal was collected at >515 nm. Two-dimensional images were collected at a speed of 6 ms/line.

[Ca²⁺]_ER measurements.

[Ca²⁺]_ER was recorded as changes in fluorescence intensity of the genetically encoded ER-targeted Ca²⁺ sensor R-CEPIA1er. R-CEPIA1er was excited with a 514-nm line of the argon laser, and the signal was collected at >560 nm. Line scan images were collected at a speed of 10 ms/line. The R-CEPIA1er signal (F) was converted to [Ca²⁺]_ER by the following formula: [Ca²⁺]_SE = K_d × [(F − F_min)/(F_max − F)]. F_max was recorded in 5 mM Ca²⁺ and 5 μM ionomycin, and F_min was recorded after ER Ca²⁺ depletion with 5 mM caffeine. For HEK293 cell experiments, the K_d (Ca²⁺ dissociation constant) was 564 μM (38). For myocyte experiments, the K_d was 609 μM (9). SERCA-mediated Ca²⁺ uptake was calculated as the first derivative of [Ca²⁺]_ER refilling (d[Ca²⁺]_ER/dt) after RyR2 inhibition with ruthenium red (15 μM) and tetracaine (1 mM). It has been shown the inhibitors used in these concentrations completely inhibit RyR2 activity in bilayer experiments (27, 45). RyR2-independent Ca²⁺ leak was analyzed as the first derivative of [Ca²⁺]_ER decline (d[Ca²⁺]_ER/dt) after inhibition of RyR2 and SERCA. The rates of SERCA2a uptake and Ca²⁺ leak were plotted as a function of [Ca²⁺]_ER to analyze maximum ER Ca²⁺ uptake rate and maximum ER Ca²⁺ load. In intact ventricular myocytes, [Ca²⁺]_SR dynamics were measured in Tyrode’s solution (in mM): 140 NaCl, 4 KCl, 1.5 CaCl₂, 1 MgCl₂, 10 glucose, and 10 HEPES, pH 7.4. Action potentials were induced by electrical field stimulation using a pair of platinum electrodes, which were connected to a Grass stimulator (Astro-Med). R-CEPIA1er expression did not affect the SR Ca²⁺ buffer capacity (9). All two-dimensional images and line scan measurements for [Ca²⁺]_SR were analyzed with ImageJ software (National Institutes of Health).

Statistics

Data are presented as means ± SE of n measurements. Statistical comparisons between groups were performed with Student's t-test for paired or unpaired data sets. Differences were considered statistically significant at P < 0.05. Significance between multiple groups was determined by two-way ANOVA followed by a Newman-Keuls post hoc test. Statistical analysis and graphical representation of averaged data were carried out with OriginPro7.5 software (OriginLab).

RESULTS

Model System to Study SERCA Function

The Ca²⁺ sensor R-CEPIA1er was used to directly measure [Ca]_ER in HEK293 cells expressing human SERCA2a tagged with mCer. SERCA2a expression in HEK293 cells was verified by Western blot analysis, revealing an approximately fivefold higher expression over endogenous SERCA (Fig. 1). Endogenous RyR and PLB were undetectable in HEK293 cells (Fig. 1). To study ER Ca²⁺ uptake over the entire physiological range of [Ca²⁺]_ER, we cotransfected these cells with cardiac RyR2 tagged with GFP (Fig. 2A). The plasma membrane of these cells was permeabilized with saponin to control cytosolic [Ca²⁺] at 200 nM and [ATP] at 5 mM. Coexpression of SERCA2a and RyR2 resulted in periodic Ca²⁺ waves due to spontaneous activation of RyR2 (Fig. 2B, cell #1). Ca²⁺ waves did not occur in HEK cells with low expression of SERCA2a (Fig. 2B, cell #2), suggesting a threshold level of SERCA activity is required to bring [Ca²⁺]_ER to a critical level that can trigger “cardiac-like” Ca²⁺ waves. After recording spontaneous Ca²⁺ release events, full depletion of ER Ca²⁺ was accomplished by applying 5 mM caffeine (Fig. 2B, Caf). The caffeine action was rapidly reversed by exchanging the caffeine-containing solution with a solution containing RyR2 inhibitors (15 μM ruthenium red and 1 mM tetracaine) (Fig. 2B, RR+Tetr). With this approach, we were able to reliably switch RyR2-mediated Ca²⁺ release from full activation to full inhibition within a few seconds. With RyR2 blocked, SERCA-mediated Ca²⁺ uptake was measured as the rate of [Ca²⁺]_ER refilling. ER Ca²⁺ uptake was analyzed as the first derivative of [Ca²⁺]_ER refilling during RyR2 inhibition. Finally, the RyR2-independent Ca²⁺ leak was measured for each cell after inhibition of SERCA by thapsigargin (Fig. 2B) while maintaining RyR2 blockade. The RyR2-independent Ca²⁺ leak was analyzed as the first derivative of [Ca²⁺]_ER decline after inhibition of RyR2 and SERCA. Figure 2C shows the dependence of ER Ca²⁺ uptake (black) and Ca²⁺ leak (gray) on [Ca²⁺]_ER. RyR2-independent Ca²⁺ leak was subtracted from Ca²⁺ uptake to determine maximum ER Ca²⁺ uptake rate (Ca²⁺ uptake against a minimal Ca²⁺ gradient) and maximum ER Ca²⁺ load that SERCA could generate (Fig. 2D). For each cell, SERCA2a expression was estimated from the mCer fluorescence for normalization of the effects of different drugs and SERCA mutations on ER Ca²⁺ uptake.

Fig. 1.

Expression levels of the Ca²⁺ release channel ryanodine receptor (RyR2), sarco-/endoplasmic reticulum Ca²⁺-ATPase (SERCA), and phospholamban (PLB) in mouse ventricular myocytes (VM) and HEK293 cells (HEK). A: RyR2, SERCA, and PLB expression levels in VM and HEK. HEK lane 1 is untransfected HEK293 cells; lane 2 is HEK293 cells transfected with PLB-modified cerulean fluorescent protein (mCer); lane 3 is HEK293 cells transfected with SERCA2a-mCer and RyR2-green fluorescent protein (GFP). B: SERCA expression in HEK293 cells before (endogenous; End) and after transfection with the SERCA2a-mCer plasmid (End+SERCA2a). These data were obtained by averaging results from 6 experiments. *P < 0.05 vs. End.

Fig. 2.

Model system to study SERCA function. A: images of HEK293 cells cotransfected with mCer-SERCA2a and GFP-RyR2. B: experimental protocol to measure SERCA-mediated endoplasmic reticulum (ER) Ca²⁺ uptake. Line-scan image of R-CEPIA1er during an initial 60-s recording is shown (marked by a gray box) and corresponding profile of changes in ER Ca²⁺ concentration ([Ca²⁺]_ER; during entire experiment: 250 s). C: for each individual cell, ER Ca²⁺ uptake and RyR2-independent Ca²⁺ leak were analyzed as the first derivative (d[Ca²⁺]_ER/dt) and plotted as a function of ER Ca²⁺ load ([Ca²⁺]_ER). D: RyR2-independent Ca²⁺ leak was subtracted from ER Ca²⁺ uptake to determine maximum ER Ca²⁺ uptake rate and maximum ER Ca²⁺ load.

Effects of SERCA Inhibitors, [ATP]/[ADP] Ratio, and Oxidative Stress on ER Ca²⁺ Uptake

To quantify the contribution of the endogenous SERCA pump to ER Ca²⁺ uptake, we studied HEK293 cells that were not transfected with SERCA2a. Compared with SERCA2a, the maximum ER Ca²⁺ uptake rate by endogenous SERCA was about six times slower (Fig. 3, A and D), consistent with the lower expression level of endogenous SERCA (Fig. 1). Spontaneous Ca²⁺ waves were not detected in those cells, presumably because endogenous SERCA did not increase [Ca²⁺]_ER to a level at which spontaneous Ca²⁺-induced Ca²⁺ release can occur. As expected, ER Ca²⁺ uptake by endogenous and SERCA2a was sensitive to thapsigargin and cyclopiazonic acid (CPA) (Fig. 3A and B). ER Ca²⁺ uptake in cells with endogenous and SERCA2a was completely blocked by 100 nM thapsigargin (n = 6) and 25 μM cyclopiazonic acid (n = 5). ER Ca²⁺ uptake was also sensitive to the cytosolic [ATP]/[ADP] ratio (Fig. 3B). Decreasing [ATP] from 5 mM to 2.5 mM and increasing [ADP] to 2.5 mM (ADP) slowed ER Ca²⁺ uptake by 69% (n = 8) (Fig. 3D). The changes in the [ATP]/[ADP] ratio also decreased the thermodynamic limit of SERCA (or maximum ER Ca²⁺ load) by 58% (n = 8; Fig. 3E). It is known that SERCA is sensitive to redox modifications (39, 42). We tested whether this approach can be used to study the effect of oxidative stress on SERCA function. SERCA2a oxidation with diamide (100 μM) decreased ER Ca²⁺ uptake rate by 38% (n = 12), without a significant effect on ER Ca²⁺ load (n = 12; Fig. 3, C and E). Low doses of diamide (≤50 μM), however, did not have a significant effect on SERCA function.

Fig. 3.

Effects of SERCA inhibitors, [ATP]/[ADP] ratio, and oxidative stress on ER Ca²⁺ uptake. A: representative traces of ER Ca²⁺ uptake mediated by endogenous and recombinant type 2a SERCA (SERCA2a) expressed in HEK293 cells. B: effect of the low [ATP]/[ADP] ratio (ADP) on ER Ca²⁺ uptake. Thapsigargin (TG; 100 nM) and cyclopiazonic acid (CPA; 20 μM) completely inhibited ER Ca²⁺ uptake by SERCA. C: effect of diamide (Diam; 100 μM) on ER Ca²⁺ uptake. Maximum [Ca²⁺]_ER uptake rate (D) and maximum [Ca²⁺]_ER load (E) mediated by endogenous SERCA (n = 6 cells), recombinant SERCA2a in control conditions (Ctrl; n = 20 cells), in the presence of the low [ATP]/[ADP] ratio (ADP; n = 8 cells), and Diam (n = 12 cells). *P < 0.05 vs. control. #P < 0.05 vs. control.

Effect of SERCA2a Mutations on ER Ca²⁺ Uptake

We also used this approach to quantify the function of two SERCA2a mutants (Fig. 4A). We have previously shown the functional significance of the SERCA cytosolic Nβ5–β6 loop, as mutation of three negatively charged amino acids to alanine (D426A/E429A/E435A “AAA-SERCA”) decreased ATPase and ER Ca²⁺ uptake (32) as a result of impaired headpiece closure kinetics (37). ER Ca²⁺ uptake by WT-SERCA2a and by the loss-of-function AAA-SERCA2a was only analyzed in cells that had a similar level of mCer fluorescence and, therefore, comparable SERCA2a expression levels. The mean mCer signal was 78 ± 4 arbitrary units (a.u.; n = 11) in cells expressing wild-type (WT) and 76 ± 7 a.u. (n = 10) in cells expressing the AAA mutant. We found that these cytosolic mutations decreased maximum ER Ca²⁺ uptake rate by 55% (n = 11) and maximum ER Ca²⁺ load by 15% (n = 10; Fig. 4, B and C), which is consistent with our previous observations (32). We also tested whether three negatively charged amino acids facing the ER lumen can regulate SERCA2a activity by a Ca²⁺-dependent mechanism. These amino acids were replaced with polar amino acid (E876Q/D877N/E882Q) to generate the QNQ-SERCA2a mutant. Analysis of ER Ca²⁺ uptake by WT and the QNQ mutant revealed that these mutations did not significantly affect maximal ER Ca²⁺ uptake and load (Fig. 4, B and C). The mCer signal was 86 ±  7 a.u. (n = 9) in WT cells and 79 ± 6 a.u. (n = 7) in cells expressing the QNQ mutant.

Fig. 4.

Effect of SERCA2a mutations on ER Ca²⁺ uptake. A: representative traces of ER Ca²⁺ uptake mediated by wild-type recombinant SERCA2a (WT) and 2 SERCA2a mutants (QNQ and AAA). Maximum [Ca]_ER uptake rate (B) and maximum [Ca²⁺]_ER load (C) mediated by WT (n = 20 cells), AAA (n = 13 cells), and QNQ (n = 10 cells) SERCA2a mutants. *P < 0.05 vs. WT.

Regulation of SERCA2a by PLB

We tested whether this approach could detect changes in SERCA function in response to physiological regulation. PLB is the most important regulator of SERCA2a function in ventricular myocytes (24). In these experiments, the effect of PLB on ER Ca²⁺ uptake was studied in the inducible human SERCA2a stable line (T-Rex-293 cells). Two days after SERCA2a gene induction with tetracycline, SERCA2a expression increased >10 times (Fig. 5A). PLB (tagged with yellow fluorescent protein) expression in these cells showed a similar pattern as CEPIA-1er (Fig. 5B), indicating that PLB is localized predominantly in the ER membrane. We found that PLB coexpression with SERCA2a decreased ER Ca²⁺ uptake and ER Ca²⁺ load (Fig. 5, D and E; Ctrl vs. PLB). Moreover, PLB decreased Ca²⁺ wave frequency by 43% (n = 7) and [Ca²⁺]_ER recovery during Ca²⁺ waves by 35% (n = 15). The PKA catalytic subunit (10 U/ml) reversed the effect of PLB on SERCA2a function (Fig. 5, D and E; PLB vs. PLB+PKA). The PKA catalytic subunit, however, did not significantly affect ER Ca²⁺ uptake in HEK cells that didn’t express PLB (Fig. 5, D and E; Ctrl vs. +PKA).

Fig. 5.

Regulation of SERCA by phospholamban (PLB). A: SERCA2a expression in T-Rex-293 stable line cells after 24 and 48 h of SERCA2a gene induction with tetracycline. B: images of T-Rex-293 cell cotransfected with PLB-yellow fluorescent protein (YFP; left) and R-CEPIA1er (right). C: representative traces of ER Ca²⁺ uptake mediated by SERCA2a cotransfected with PLB in control conditions (PLB) and in the presence of the PKA catalytic subunit (PLB+PKA). Maximum [Ca]_ER uptake rate (D) and maximum [Ca]_ER load (E) mediated by SERCA2a in control conditions (Ctrl; n = 9 cells) and in the presence of PKA (+PKA; n = 6 cells), and by SERCA2a cotransfected with PLB (PLB; n = 11 cells) alone and in the presence of PKA (PLB+PKA; n = 5 cells). *P < 0.05 vs. Ctrl. ^#P < 0.05 vs. PLB.

SERCA2a Ca²⁺ Transport in Ventricular Myocytes

We assessed whether this approach can be used to study endogenous SERCA2a function in ventricular myocytes. We used in vivo gene delivery of the R-CEPIA1er cDNA with an adenovirus in mice hearts. Six days after adenovirus injection, myocytes were isolated from the left ventricle. Myocytes expressing R-CEPIA1er showed periodic peaks of fluorescence approximately every 1.8 μm, which corresponds to the sarcomere spacing of the junctional SR (Fig. 6A). First, we analyzed the effect of adrenergic receptor stimulation on SERCA2a function in intact ventricular myocytes (Fig. 6B). [Ca²⁺]_SR was measured under control conditions and in the presence of isoproterenol (ISO; 0.1 μM). Myocytes were electrically stimulated at a constant frequency 0.5 Hz. Action potentials caused regular [Ca²⁺]_SR depletion followed by a fast recovery of Ca²⁺ due to SERCA2a activity. ISO increased SR Ca²⁺ release amplitude (Fig. 6B), the rate of [Ca²⁺]_SR recovery, and SR Ca²⁺ load (Fig. 6C). Adrenergic stimulation also increased the propensity of spontaneous Ca²⁺ waves. We next used a similar approach to study the effect of PKA activation on SR Ca²⁺ uptake in permeabilized ventricular myocytes. After SR Ca²⁺ depletion with caffeine, RyR2 inhibitors (RR+Tetr) were applied to measure SR Ca²⁺ uptake (Fig. 6D). Similar to experiments presented in Fig. 1, the same analysis was performed to calculate maximal SR Ca²⁺ uptake rate and maximal SR Ca²⁺ load generated by endogenous SERCA2a (Fig. 6E). We have shown previously that endogenous PKA can be activated by cAMP after membrane permeabilization (8). cAMP (10 μM) significantly increased maximal SR Ca²⁺ uptake and SR Ca²⁺ load (Fig. 6E). The effects of PKA activation on endogenous SERCA2a function were similar to those observed for recombinant SERCA2a (Fig. 5). Taken together, these results demonstrate that this new approach can be used as a sensitive tool to quantify SERCA function in expressing cell systems as well in cardiomyocytes.

Fig. 6.

SERCA2a properties in ventricular myocytes. A: transmitted light (left) and confocal (middle) images of left ventricular myocytes isolated from a mouse heart infected with the R-CEPIA1er adenovirus. Zoomed-out image of the R-CEPIA1er signal (right) is defined by the white dashed box in the middle image. Myocyte expressing R-CEPIA1er showed a periodic pattern of the SR network together with the nucleus envelope. B: changes in [Ca²⁺]_SR during electrical filed stimulations (marked by triangles) in control conditions (Ctrl) and during isoproterenol (ISO) application. Ca²⁺ waves are marked by red asterisks. Caffeine (Caf; 10 mM) was applied to completely deplete [Ca²⁺]_SR. C: effect of ISO on tau of [Ca²⁺]_SR recovery and sarcoplasmic reticulum (SR) Ca²⁺ load (n = 13 myocytes, 4 animals). D: representative traces of SR Ca²⁺ uptake in left ventricular myocytes mediated by SERCA2a in control conditions (Ctrl) and in the presence of cAMP (cAMP). E: maximum [Ca²⁺]_SR uptake rate and maximum [Ca]_SR load mediated by SERCA2a in control conditions (Ctrl; n = 7 myocytes) and in the presence of cAMP (cAMP; n = 6 myocytes). *P < 0.05 vs. Ctrl.

DISCUSSION

Cardiac cytosolic Ca²⁺ dynamics are of great interest as they represent the central signals that orchestrate the cycle of contraction and relaxation. They have been intensively scrutinized using fluorescence microscopy and Ca²⁺-sensitive indicators that report the concentration of Ca²⁺ in the cytoplasm, yielding insight into the biophysical mechanisms that govern Ca²⁺ release from the SR during systole. These methods have also provided indirect assessment of the subsequent reuptake of Ca²⁺ into the SR during diastole. For example, SERCA transport activity can be inferred from the rate of decay of the cytosolic Ca²⁺ transient (4, 5, 41). SERCA activity could also be estimated from ATP consumption (29) or Ca²⁺ uptake by ER/SR vesicles (34). However, these semiquantitative measures suffer from several significant limitations, as addressed in the following paragraphs.

First, they do not control for changes in SR Ca²⁺ content, and how these changes might affect the transporter function. To address this issue, low-affinity Ca²⁺ indicators such as Fluo-5N have been used to directly measure [Ca²⁺]_SR in myocytes (11, 46), but unfortunately this technique does not work in myocytes isolated from small rodents that are commonly used for transgenic studies. In the present study, we circumvented some of the drawbacks of previous approaches by using the genetically encoded ER Ca²⁺ sensor R-CEPIA1er to directly measure Ca²⁺ dynamics within the ER lumen in expressing cell systems as well as in freshly isolated cardiomyocytes. It has been previously established that R-CEPIA1er has an appropriate affinity and sufficient dynamic range to reliably track [Ca²⁺]_ER in both non-muscle (38) and muscle cells (9). As an example of the versatility of R-CEPIA, we created a cardiomimetic Ca²⁺-handling system in non-muscle cells by coexpressing SERCA2a together with the cardiac Ca²⁺ release channel RyR2. This enabled fast, complete, and rapidly reversible depletion of ER Ca²⁺ load (with caffeine), so that the subsequent ER Ca²⁺ uptake could be measured over the entire physiological range of [Ca²⁺]_ER, starting from fully empty to maximally loaded ER.

The second major limitation of the previous methods used to study SR Ca²⁺ uptake is that it is difficult to specifically separate Ca²⁺ transport by SERCA from Ca²⁺ fluxes mediated by other ion channels and pumps. One particularly challenging process to account for is the RyR2-independent Ca²⁺ leak. Due to the complex nature of the ER Ca²⁺ leak (12, 43), this pathway cannot be blocked completely or selectively. Instead of blocking it, we measured the RyR2-independent Ca²⁺ leak after RyR2 and SERCA inhibition (Fig. 2). We determined that the Ca²⁺ leak component was relatively small compared with ER Ca²⁺ uptake in HEK cells, suggesting that inositol 3-phosphate receptors and other Ca²⁺ leak channels are mainly inactive in the absence of appropriate agonists. Moreover, direct measurement of [Ca²⁺]_ER separates the SERCA-dependent Ca²⁺ uptake from Ca²⁺ fluxes mediated by Ca²⁺ transporters on the surface membrane and other organelles, including the mitochondria.

In addition to overcoming the two limitations associated with previous methodologies outlined above, the advantage of the present approach enables access to two key biophysical parameters of SERCA function: maximum Ca²⁺ uptake rate and maximum Ca²⁺ load (Fig. 2D). These parameters reflect the kinetic and the thermodynamic efficacy of SERCA, respectively, and various interventions differentially modulated these properties. Lowering the cytosolic ATP/ADP ratio decreased both the maximum Ca²⁺ uptake rate and the maximum Ca²⁺ load (Fig. 3B). This can be explained if high [ADP] inhibits the SERCA kinetic efficacy (28), whereas low [ATP]/[ADP] decreases the thermodynamic limit, of the pump (23). SERCA2a oxidation (39, 42) had a stronger effect on ER Ca²⁺ uptake than on reduction of the ER Ca²⁺ load (Fig. 3C). Loss-of-function mutations of the SERCA2a cytoplasmic headpiece (32) also decreased the maximal Ca²⁺ uptake rate (Fig. 4), while several mutations of luminal residues were functionally benign.

We have previously designed a method to selectively measure [Ca²⁺]_SR in intact ventricular myocytes using R-CEPIA1er (9). However, expression of the sensor requires >48 h of culturing, which significantly alters myocyte structure and function (for a review, see Ref. 26). To overcome this limitation, we used in vivo gene delivery of the R-CEPIA1er cDNA with an adenovirus in mice hearts. The same methodology to analyze SERCA2a function was used in freshly isolated myocytes (Fig. 6). The effects of PKA activation on endogenous SERCA2a were similar to those observed for recombinant SERCA2a (Fig. 5), suggesting that our heterologous expression system is a suitable model of Ca²⁺ pump regulation in cardiac myocytes. Moreover, using in vivo gene delivery of the R-CEPIA1er can open new perspectives to more directly characterize changes in SERCA2a function in different transgenic models of heart diseases.

However, the critical limitation of the in vivo gene delivery approach is that its success can be technically challenging, as it depends on the quality of animal survival surgery and myocyte isolation. In contrast to myocytes, HEK293 cells provided an ideal minimal cardiomimetic Ca²⁺-handling system for investigating SERCA2a-regulatory mechanisms. In HEK293 cells, the Ca²⁺ transients occur without eliciting contractions, which can cause motion artifacts in the myocyte experiments. Although myocyte contractility can be eliminated pharmacologically, blebbistatin is sensitive to the wavelengths used to excite fluorescence (16). and 2,3-butanedione 2-monoxime may alter Ca²⁺ handling (2). In contrast, the HEK cell model is readily and stably transfectable, it does not require generation of viral delivery vectors or transgenic animals, and it is without the confounding effects of endogenous PLB or other regulators of SERCA2a. This enabled us to observe PLB-dependent, PKA-reversible decreases in both the kinetic and thermodynamic efficacy of the pump (Fig. 5). We consider this well-defined model system to be ideal for testing the effects of disease-causing PLB mutations, or the effects of other regulatory micropeptides, some of which may specifically target SERCA catalytic efficiency (3).

In conclusion, the enhanced SERCA specificity of this new approach, together with differentiation of kinetic and thermodynamic parameters, may provide a more sensitive and discriminating assay for drugs, posttranslational modifications, and mutations that may alter human SERCA2a function.

GRANTS

This work was supported by the National Institutes of Health Grants HL-130231 (to A. V. Zima), HL-092321 (to S. L. Robia) and DK-101585 (I. Y. Kuo).

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

E.B., R.N., S.B., D.K., Q.C., J.L.M., I.Y.K., S.R., and A.V.Z. conceived and designed research; E.B., R.N., S.B., D.K., Q.C., J.L.M., and A.V.Z. performed experiments; E.B., R.N., S.B., D.K., Q.C., J.L.M., I.Y.K., S.R., and A.V.Z. analyzed data; E.B., R.N., S.B., D.K., Q.C., J.L.M., I.Y.K., S.R., and A.V.Z. interpreted results of experiments; E.B., R.N., and A.V.Z. prepared figures; E.B., R.N., I.Y.K., S.R., and A.V.Z. drafted manuscript; E.B., R.N., S.B., D.K., Q.C., J.L.M., I.Y.K., S.R., and A.V.Z. edited and revised manuscript; E.B., R.N., S.B., D.K., Q.C., J.L.M., I.Y.K., S.R., and A.V.Z. approved final version of manuscript.