A more recent version of this article appeared on July 20, 2001

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(Circulation Research. 2001;0:hh1401.093584.)
© 2001 American Heart Association, Inc.

Article

Sarcoplasmic Reticulum Ca²⁺ ATPase (SERCA) 1a Structurally Substitutes for SERCA2a in the Cardiac Sarcoplasmic Reticulum and Increases Cardiac Ca²⁺ Handling Capacity

M. Jane Lalli, Ji Yong, Vikram Prasad, Katsuji Hashimoto, Dave Plank, Gopal J. Babu, Darryl Kirkpatrick, Richard A. Walsh, Mark Sussman, Atsuko Yatani, Eduardo Marbán Muthu Periasamy

From the Division of Cardiology (M.J.L., J.Y., V.P., G.J.B., M.P.), University of Cincinnati College of Medicine, Cincinnati, Ohio; Division of Cardiology, Department of Internal Medicine (D.K., R.A.W.), Case Western College of Medicine, Cleveland, Ohio; Division of Molecular Cardiovascular Biology (D.P., M.S.), Children’s Hospital Research Center, Cincinnati, Ohio; Cardiovascular Research Institute (A.Y.), University of Medicine and Dentistry of New Jersey, Hackensack, NJ; and Institute of Molecular Cardiobiology (K.H., E.M.), The Johns Hopkins University, Baltimore, Md. Present affiliation of M.P. is Department of Physiology and Cell Biology, College of Medicine and Public Health, Ohio State University, Columbus, Ohio.

Correspondence to Muthu Periasamy, Department of Physiology and Cell Biology, College of Medicine and Public Health, Ohio State University, 302 Hamilton Hall, 1645 Neil Ave, Columbus, OH 43210-1218. E-mail periasamy.1{at}osu.edu

Abstract

Abstract—Ectopic expression of the sarcoplasmic reticulum (SR) Ca²⁺ ATPase (SERCA) 1a pump in the mouse heart results in a 2.5-fold increase in total SERCA pump level. SERCA1a hearts show increased rates of contraction/relaxation and enhanced Ca²⁺ transients; however, the cellular mechanisms underlying altered Ca²⁺ handling in SERCA1a transgenic (TG) hearts are unknown. In this study, using confocal microscopy, we demonstrate that SERCA1a protein traffics to the cardiac SR and structurally substitutes for the endogenous SERCA2a isoform. SR Ca²⁺ load measurements revealed that TG myocytes have significantly enhanced SR Ca²⁺ load. Confocal line-scan images of field-stimulated SR Ca²⁺ release showed an increased rate of Ca²⁺ removal in TG myocytes. On the other hand, ryanodine receptor binding activity was decreased by 30%. However, TG myocytes had a greater rate of spontaneous ryanodine receptor opening as measured by spark frequency. Whole-cell L-type Ca²⁺ current density was reduced by 50%, whereas the time course of inactivation was unchanged in TG myocytes. These studies provide important evidence that SERCA1a can substitute both structurally and functionally for SERCA2a in the heart and that SERCA1a overexpression can be used to enhance SR Ca²⁺ transport and cardiac contractility.

Key Words: transgenic • contractility • gene therapy • Ca²⁺ load • Ca²⁺ uptake • sparks

The sarcoplasmic reticulum (SR) Ca²⁺-ATPase (SERCA) is a major component of beat-to-beat Ca²⁺ cycling in the heart. Reduction of SERCA pump expression and activity has been linked to diastolic dysfunction in hypertrophied^{1 2} and failing hearts.^{3 4 5 6 7 8 9} Previous studies from our laboratory and others have shown that overexpression of both SERCA1a and SERCA2a in the mouse heart leads to enhanced Ca²⁺ transport with a concomitant boost in contractility.^{10 11 12 13 14 15} Similarly, adenovirus-mediated gene transfer of SERCA gene into adult myocytes results in increased contractility, with an increased rate of Ca²⁺ uptake and release.^{16 17 18 19} A recent study by Miyamoto et al²⁰ shows that adenoviral gene transfer of SERCA2a improves cardiac function in an aortic-banded rat heart failure model. Taken together, these in vivo and in vitro studies suggest that increased expression is feasible and results in enhanced Ca²⁺ transport and contractility.

SERCA1a has been shown to have faster Ca²⁺ transport kinetics, and it is associated with faster rates of contraction and relaxation.²¹ To investigate whether SERCA1a expression in the heart leads to faster Ca²⁺ cycling and increased contractility, we generated SERCA1a transgenic (TG) mice.¹³ SERCA1a overexpression results in a 2.5-fold increase in total SERCA pump levels in TG heart and a 2-fold increase in SR Ca²⁺ uptake function.¹⁴ SERCA1a overexpression levels are consistently maintained at 2.5-fold above controls in successive generations, indicating that expression levels do not diminish with germline transmission/aging.

A major goal of this study was to investigate how high levels of SERCA pump overexpression in the heart alter intracellular Ca²⁺ homeostasis. Specifically, we investigate the following: (1) whether SR Ca²⁺ stores, Ca²⁺ release, and Ca²⁺ uptake functions are altered; (2) whether SERCA1a is structurally and functionally an integral component of cardiac SR; (3) whether long-term expression of SERCA1a can be detrimental to myocyte structure and function; and (4) whether overexpression of SERCA pump leads to alterations in the protein levels or functional performance of other SR and sarcolemmal Ca²⁺ handling proteins.

Materials and Methods

Isolation of Ventricular Myocytes
Ventricular myocytes were isolated from wild-type (WT) and SERCA1a hearts from adult mice (10 to 16 weeks old) using a Langendorff perfusion system with 0.8 mg/mL collagenase type 2 for 12 to 20 minutes until the heart became soft.¹²

Fluorescent Antibody Staining/ Confocal Microscopy
Adult cardiac myocytes were fixed in 4% paraformaldehyde/PBS, permeabilized with 0.2% Triton X-100, exposed overnight to appropriate antibody, and viewed by confocal microscopy.²² Slides were quickly scanned to determine an appropriate intensity setting, and this setting was used for all samples.

Intracellular Ca²⁺ Measurements
Intracellular free Ca²⁺ transients were measured as ratio of 340 to 380 nm excitation fluorescence of fura-2 acetoxymethyl ester (AM) (emission wavelength, 510 nm) using a photo scan dual spectrophotometer in individual myocytes loaded with 7.5 µmol/L fura-2 AM at 37°C for 10 to 15 minutes in the dark.¹² Cells were field stimulated at 0.5 Hz (Grass SD9 stimulator) until twitch characteristics were repeatable. Caffeine was applied for 10 seconds.

Line-Scan Imaging and Ca²⁺ Spark Analyses
Line-scan imaging was performed using fluo-3 Ca²⁺ fluorescent indicator and confocal microscopy (Molecular Dynamics). A single cell was scanned repetitively at 500 Hz for 5 seconds along a horizontal line. Ca²⁺ sparks were recorded from a central region of the cell using the x-y scan mode with pixels set to 512x512.

L-Type Ca²⁺ Channel Current Measurements
Whole-cell Ca²⁺ channel currents were recorded as reported.^{23 24 25 26} External solution contained the following (in mmol/L): CaCl₂ 2 or BaCl₂ 2, MgCl₂ 1, TEA-Cl 135, 4-aminopyridine 5, glucose 10, and HEPES (pH 7.3) 10. Pipette solution contained (in mmol/L) cesium aspartate 100, CsCl 20, MgCl₂ 1, MgATP 2, GTP 0.5, EGTA 5, and HEPES 5 (pH 7.3). Membrane capacitance was measured using voltage ramps of 0.8 V/second from a holding potential of -50 mV. Rapid solution changes were made using a modified Y-tube.²³

[³H]Ryanodine Receptor (RyR) Binding Assay
Total ryanodine binding to cardiac homogenates was measured after incubation with [³H]ryanodine (56.9 Ci/mmol, DuPont New Research Products) for 90 minutes at 37°C.²⁷ Binding data were analyzed by a radioligand analysis program (G.A. McPherson, Elsevier-BIOSOFT).

Ribonuclease Protection Assays
The riboprobes for mouse cardiac RyR and rat SERCA1a were generated from respective cDNA clones. Ribonuclease protection assay was performed using the RPAIII kit (Ambion, Inc), and protected fragments were separated by electrophoresis in a 5% denaturing polyacrylamide gel.²⁸

Quantitative Immunoblotting
Quantitative immunoblotting of cardiac homogenates was used to determine the protein levels of SERCA1a, RyR, sodium-calcium exchanger, triadin, L-type channel, and actin.^{12 13} Homogenates were electrophoretically separated, blotted to membrane, and probed with appropriate antibodies. Quantification of the signals was performed by densitometry (UMAX Astra 1200) and analyzed (NIH Image, version 6.1).^{12 13}

Simultaneous Intracellular Ca²⁺ and Twitch Force Measurements
Geometrically regular trabeculae (dimensions in mm, 1.03±0.20 length, 0.26±0.09 width, and 0.13±0.04 thickness) were mounted to force transducers and superfused with buffer.^{29 30 31 32} After equilibration, trabeculae were stimulated at 1.0 Hz using a Grass SD9 stimulator. Fura-2 potassium salt was microinjected³³ and [Ca²⁺]_i was determined by measuring the epifluorescence of fura-2 signal. [Ca²⁺]_i was calculated using the Grynkiewicz equation.³⁴

Statistical Analyses
WT and SERCA1a parameters were compared using the Student t test and/or ANOVA. Results are expressed as mean±SEM.

Results

SERCA1a Structurally Substitutes for SERCA2a in the Heart
To link enhanced Ca²⁺ transport with SERCA1a overexpression, it was necessary to show at the subcellular level that SERCA1a is an integral component of the cardiac SR. Immunostaining of adult mouse myocytes with tropomodulin, an actin binding protein, gave a uniform striated pattern in rod-shaped cells (Figure 1, top and middle, green); this striated pattern was used as a selection criterion. Cells were costained with SERCA1a antibody (red) to determine the pattern of SERCA1a distribution. WT cells showed no specific staining with SERCA1a antibody (Figure 1, upper left), whereas SERCA1a TG myocytes showed a distinctive horizontal and vertical pattern (Figure 1, middle and bottom), which was indistinguishable from that seen with SERCA2a antibody staining (Figure 1, bottom, green). Thus, these data demonstrate that SERCA1a and SERCA2a exhibit subcellular colocalization within the limits of resolution of confocal microscopy (200 nm).

View larger version (64K): [in this window] [in a new window]   Figure 1. Immunofluorescent staining of isolated myocyte from WT (top) and SERCA1a (middle and bottom). A-band was stained with tropomodulin (green, top and middle), which gave a uniform striated pattern in healthy cells and costained with SERCA1a (red, all panels). To confirm that SERCA1a and SERCA2a give a similar pattern of staining, myocytes from SERCA1a hearts (bottom) were stained with SERCA1a-antibody (red) and SERCA2a-antibody (green).

To determine possible long-term detrimental effects of SERCA1a overexpression, blinded comparative histological studies were performed from 20-month-old animals. Both WT hearts (Figure 2A, bottom panels) and TG hearts (Figure 2A, top panels) showed mild myocyte hypertrophy, fibrosis, and nuclear rowing. No significant morphological differences were evident at the gross anatomic level (Figure 2A, left panels, x10; right panels, x40). In addition, both control and TG mice had the same mortality curves. Consistent with histological analysis, there was no change in size of left ventricular myocytes as estimated by the cell capacitance (127.2±3.4 pF [n=60, WT] versus 118.9±3.3 pF [n=78, TG]). Thus, SERCA1a overexpression does not result in pathology or hypertrophy. To confirm that these aged hearts still expressed SERCA1a at high levels, Western blot analysis was performed. Figure 2B clearly shows robust expression of SERCA1a in both male (M) and female (F) mice at 20 months.

View larger version (91K): [in this window] [in a new window]   Figure 2. A, Histological staining of SERCA1a heart (top) and WT (bottom) at x10 (left) and x40 (right) magnification. B, Western blot analysis of SERCA1a protein in 20-month-old mice, both male (M) and female (F).

Ca²⁺ Transient Amplitude and Contractility Are Altered in SERCA1a Hearts
A major target of Ca²⁺ released from the SR is troponin-C, which on binding Ca²⁺ undergoes a conformational change and initiates actin-myosin interaction. By mass action, increased [Ca²⁺]_i leads to increased Ca²⁺–troponin-C complex activation and results in greater force production. Thus, to determine how Ca²⁺ cycling and force were altered in SERCA1a overexpressing heart muscle, contractile force and intracellular Ca²⁺ were measured simultaneously in fura-2–loaded isometrically contracting trabeculae. Figure 3A shows representative tracings of Ca²⁺ transients (top) and twitch force (bottom) in muscles from WT (left) and SERCA1a TG (right) hearts. Trabeculae from TG versus WT hearts exhibited an increase in Ca²⁺ transient amplitude (1.25±0.21 versus 0.9±0.11 µmol/L), whereas the time course of Ca²⁺ removal was significantly shorter (74.9±3.5 versus 142.3±7.1 ms). Force generation was also increased in TG trabeculae (28.1±1.4 versus 14.8±3.0 mN/mm² [n=4, TG and WT], P<0.01), whereas relaxation times were unchanged. Diastolic [Ca²⁺]_i was not different between the two groups. In summary, trabeculae data support isolated myocyte data and show that SERCA1a TG hearts have greater rates of Ca²⁺ cycling than do WT hearts.

View larger version (14K): [in this window] [in a new window]   Figure 3. A, Simultaneous Ca2+ amplitude and force measurements in isolated trabeculae from SERCA1a and WT hearts. B, Ca2+ transients during 10-second caffeine pulse in isolated myocytes from SERCA1a (TG) and WT hearts; all Ca2+ removal results from either the NCX, the sarcolemmal Ca2+ pump, or other slow mechanisms.

SERCA1a Overexpression Results in a Significant Increase in SR Ca²⁺ Load
Both trabeculae (Figure 3) and isolated myocytes¹³ from SERCA1a hearts showed increased amplitude of calcium signal on field stimulation. Thus, we sought to test whether there was also an increased SR Ca²⁺ load. Caffeine binds to the RyR, keeping it open, thereby emptying the SR of Ca²⁺, giving a measure of total SR Ca²⁺ load (Figure 3B). We used 10-second pulses of caffeine to empty Ca²⁺ from the SR. Peak Ca²⁺ signal from SERCA1a myocytes was 2-fold greater than that seen in WT cells as measured by fura-2; thus, SERCA1a overexpression results in a significant increase in SR Ca²⁺ stores (Table 1).

View this table: [in this window] [in a new window]   Table 1. Peak Ca2+ Amplitude and Rate of Decay After Caffeine Stimulation

The rate of Ca²⁺ removal after caffeine exposure is a measure of the ability of non-SR Ca²⁺ extrusion mechanisms to operate. We hypothesized that an increase in SR Ca²⁺ uptake function might diminish the role played by other Ca²⁺ removal mechanisms, such as the Na⁺-Ca²⁺ exchanger (NCX).^{35 36} The exponential rate of decay of the Ca²⁺ signal in the presence of caffeine, which is a measure of NCX activity, showed no significant difference in the first or second time constants (Figure 3B, Table 1). In addition, Western blot analysis (Figure 4A) revealed that there was no significant difference in NCX protein levels (2.59±0.08 versus 2.81±0.18, NS). This finding, coupled with the lack of change in the rates of Ca²⁺ removal after caffeine exposure, implies that NCX is not altered by SERCA1a overexpression.

View larger version (28K): [in this window] [in a new window]   Figure 4. A, Western blot analysis of NCX protein levels. B, Ribonuclease protection analysis of RyR mRNA levels. C, Western blot analysis of RyR levels. D, RyR binding studies using [3H]ryanodine. Left, Scatchard plot of specific ryanodine binding in cardiac homogenates. Data are pooled from 6 hearts from SERCA1a (•) and WT () mice and are fitted with a single class of binding sites. Right, Ca2+ dependence of RyR binding. NTG indicates nontransgenic.

RyR Levels Are Decreased in SERCA1a TG Hearts
We next sought to determine whether increased Ca²⁺ release was also partly due to alterations in RyR expression. Quantification of mRNA by ribonuclease protection assays revealed that there was no change in message levels of RyR (Figure 4B). However, quantitative immunoblot analysis using specific anti-RyR2 antibody revealed that RyR protein levels were decreased by 20% (1.08±0.03 nontransgenic versus 0.89±0.03 TG, P<0.01) (Figure 4C).

To confirm these findings and to determine whether functional RyR levels were downregulated in SERCA1a TG mice, radioligand receptor binding studies were performed (Figure 4D). RyR binding was significantly decreased in SERCA1a hearts versus WT over various ranges of [³H]ryanodine from 0.1 to 30 nmol/L. Scatchard plot analysis showed that the maximal binding (B_max) of the receptor is decreased by 32.5% in SERCA1a, whereas the K_d value remained unchanged (Table 2). RyR binding is Ca²⁺-dependent; thus, to investigate whether the decrease of the B_max was due to a change in receptor sensitivity to Ca²⁺, we determined RyR binding in the presence of increasing free Ca²⁺ concentrations. The Ca²⁺ sensitivity of ryanodine binding (K_0.5) was unchanged. Thus, the difference in B_max is due to decreased RyR level in SERCA1a hearts (Figure 4D; Table 2).

View this table: [in this window] [in a new window]   Table 2. RyR Binding Studies

SERCA1a Myocytes Showed Greatly Increased Frequency of Ca²⁺ Spark Activity and Enhanced Rate of Ca²⁺ Removal
RyR activity is a critical determinant of SR Ca²⁺ release. In SERCA1a TG myocytes, RyR protein levels were decreased, yet global SR Ca²⁺ release was not decreased but rather increased (Figure 3). To determine whether the decrease in RyR protein level was offset by an increase in RyR channel opening, Ca²⁺ spark analysis was performed. Ca²⁺ sparks were recorded from healthy, quiescent WT and TG cells (Figure 5). SERCA1a myocytes showed greatly increased frequency of spark activity under basal conditions (2- to 4-fold increase over WT); spark amplitude was also approximately double in SERCA1a myocytes versus WT (1.83±0.48 versus 0.92±0.37 units; F/Fo). Often TG myocytes showed an "intense burst" of Ca²⁺ sparks after field stimulation, which lasted for 10 to 15 seconds. Cells then returned to a quiescent state and could be again field-stimulated.

View larger version (62K): [in this window] [in a new window]   Figure 5. Top, Line-scan image of calcium transient in SERCA1a (TG) and WT myocytes. Trace 1 shows typical Ca2+ release and removal on field stimulation in WT myocytes. Trace 2 shows narrow transient in the SERCA1a TG myocyte, indicating an increased rate of Ca2+ removal from cytosol. Bottom, Ca2+ sparks in WT and SERCA1a TG myocytes at rest. Note the increased frequency of spark activity in TG myocytes.

In addition, we recorded line-scan images of Ca²⁺ transients from WT and transgenic myocytes loaded with fluo-3 and field-stimulated cells (Figure 5 top). Line-scan images were thinner in SERCA1a myocytes compared with WT; thus, the time course of Ca²⁺ removal in SERCA1a TG myocytes was significantly faster than in WT myocytes (109.8±13.7 ms [n=22, TG] versus 204.5±33.8 ms [n=36, WT]; P<0.001). This is consistent with trabecula data in Figure 3 and shows that overexpression of SERCA1a isoform leads to increased Ca²⁺ removal.

L-Type Ca²⁺ Channel Current Amplitude Is Significantly Decreased in SERCA1a Myocytes, but Inactivation Time Is Unchanged
The cellular Ca²⁺ transients and contraction elicited by electric excitation are strongly influenced by the amount of Ca²⁺ influx through the L-type Ca²⁺ channel. Thus, we next determined whether L-type Ca²⁺ channel properties were altered. Interestingly, peak Ca²⁺ current amplitude, normalized relative to cell capacitance (pA/pF), was significantly decreased in SERCA1a myocytes compared with WT (3.8±0.2 pA/pF [n=65] versus 8.5±0.4 pA/pF [n=48]; P<0.001; Figure 6A). There was no change in the voltage range for current activation (Figure 6B). In both groups, the current began to activate around -30 mV and reached its maximum value near +10 mV. At this potential, Ca²⁺ current inactivated rapidly during maintained depolarization in both groups. Consistent with electrophysiological analysis, Western blotting analysis showed that channel protein expression was decreased 30 to 35% in TG hearts (70.5±3.2% normalized to WT; Figure 6D). This finding was corroborated by the amplitude of Ba²⁺ current through the L-type Ca²⁺ channels. Because Ba²⁺ can permeate the L-type Ca²⁺ channels, but Ba²⁺ itself cannot inactivate the channel or trigger the release of Ca²⁺ from the SR, it can be used as an effective charge carrier to record maximal channel amplitude. In the presence of Ba²⁺, maximal current in SERCA1a myocytes was significantly smaller than in WT (6.3±0.6 versus 10.9±0.9, P<0.001, data not shown).

View larger version (23K): [in this window] [in a new window]   Figure 6. A, Representative whole-cell Ca2+ currents recorded in WT and SERCA1a (TG) myocytes. Currents were elicited from -50 mV to the indicated test potentials. B, I-V relationships in WT and TG myocytes. Peak inward Ca2+ current was normalized to cell capacitance (pA/pF). C, Whole-cell Ca2+ currents shown before and after application of ryanodine (10 µmol/L). D, Western blot analysis of L-type channel -subunit.

It has previously been shown that L-type Ca²⁺ current inactivation in mouse myocytes involves voltage-dependent and Ca²⁺-dependent mechanisms and that the local increase in Ca²⁺ released from the SR promotes Ca²⁺-dependent inactivation.^{24 25} Thus, we next analyzed the time to half-decay of the current (T_1/2). Although the L-type channel current amplitude was significantly reduced in SERCA1a myocytes, there was no significant difference in inactivation time between the two groups (22.8±1.2 ms [n=60, TG] versus 20.9±1.2 ms [n=45 WT]).

Decreased current amplitude is often attended by prolonged inactivation time, as less Ca²⁺ entering via the L-type channel means there is less Ca²⁺ available for Ca²⁺-induced channel inactivation. Thus, to further explore why channel inactivation is not prolonged in SERCA1a myocytes, L-type currents were recorded in the presence of 10 µmol/L ryanodine. Inactivation time is prolonged in the presence of ryanodine, because ryanodine holds RyR in an open state, effectively depleting the SR of Ca²⁺. Thus, the only Ca²⁺ available for channel inactivation in the presence of ryanodine is that which enters via the L-type channel itself. In the presence of ryanodine, inactivation time was longer in SERCA1a myocytes than in WT (39.6±2.3 versus 34.6±1.7 ms; Figure 6C). This implies that SR Ca²⁺ load and release play a role in shortening the L-type channel inactivation time in SERCA1a hearts.

Discussion

SERCA1a Traffics to the Cardiac SR and Structurally Substitutes for the Endogenous SERCA2a Isoform
The purpose of this study was to investigate the molecular basis for enhanced Ca²⁺ uptake and increased contractility in SERCA1a TG hearts. In this study we convincingly demonstrate that SERCA overexpression results not only in an increased rate of SR Ca²⁺ transport but increased SR Ca²⁺ load and release. We show for the first time that SERCA1a traffics to the cardiac SR and structurally substitutes for the endogenous SERCA2a isoform. This is consistent with our previous observation that SERCA1a overexpression results in a 50% reduction in endogenous SERCA2a pump levels,¹² which tends to argue that SERCA1a and SERCA2a compete for the same "sites" within the SR. This finding is corroborated by the confocal immunostaining of isolated myocytes; SERCA1a and SERCA2a proteins show distribution patterns that are indistinguishable from each other. Recent studies using adenovirus-mediated gene transfer into embryonic cardiac myocytes showed that SERCA1a was targeted to intracellular membranes; cytosolic Ca²⁺ transients were greatly increased and rates of shortening and relengthening were faster.¹⁸ These data suggest that SERCA1a, a protein not endogenously found in cardiac tissue, can be ectopically expressed yet properly trafficked into the cardiac SR and can functionally substitute for SERCA2a.

Increase in SERCA Pump Level Contributes to Increased SR Ca²⁺ Load
Using caffeine to empty the SR free of Ca²⁺, we demonstrate that SR Ca²⁺ load is increased 2.0-fold in SERCA1a TG myocytes. This further confirms that ectopically expressed SERCA pump is functional in the cardiac environment. Our study suggests that an important effect of SERCA pump overexpression is to increase the SR Ca²⁺ load, which in turn is responsible for the increased intracellular Ca²⁺ transients.^{37 38} Previous studies have also shown a positive correlation between SERCA level and SR Ca²⁺ load.^{39 40 41 42 43} Thyroid hormone treatment of rats and guinea pigs leads to upregulated SERCA level and increased Ca²⁺ transients.^{44 45} Studies by Santana et al⁴⁶ and Houser et al⁴⁷ have shown that accelerated Ca²⁺ uptake causes increased SR Ca²⁺ load in phospholamban knockout hearts. In SERCA1a TG hearts, this increased SR Ca²⁺ load could be due to (1) increased Ca²⁺-ATPase levels, (2) a change in the apparent affinity of SERCA1a for Ca²⁺, or (3) faster pump kinetics.¹⁸ Our previous study shows that pump affinity for Ca²⁺ is unchanged in SERCA1a TG hearts.¹³ Therefore, the increase in SR Ca²⁺ load is primarily due either to an increase in pump density and/or to the faster kinetics of SERCA1a pump.¹⁸

Increase in SR Ca²⁺ Load Contributes to a Higher Frequency of RyR Channel Opening in SERCA1a Myocytes
An important finding of the SERCA1a hearts is that the amplitude of intracellular Ca²⁺ transients is increased in isolated myocytes¹² and trabeculae. Several factors contribute to Ca²⁺ release from the SR, including (1) the Ca²⁺ gradient from the SR lumen to the cytosol,^{37 38 39 40 41 42 43} (2) the number of RyR channels and/or their frequency of opening and inactivation, and (3) Ca²⁺ entry via L-type Ca²⁺ channel.⁴⁸ In SERCA1a TG myocytes, the Ca²⁺ gradient from the SR to the lumen is increased, and thus there is an increased driving force for Ca²⁺ to exit the SR. However, RyR levels are decreased 30% (Figure 4D), yet global SR Ca²⁺ release is increased (Figure 3). Ca²⁺ spark analysis in WT and TG myocytes showed that RyR channels have a greater frequency of spontaneous opening in SERCA1a myocytes under basal conditions (Figure 5). This argues that an increase in SR Ca²⁺ load contributes to a higher frequency of RyR channel opening and results in greater Ca²⁺ release observed in SERCA1a TG myocytes, which is in agreement with previous studies.³⁷ It is interesting to note that phospholamban ablation is also associated with significant increase in SR Ca²⁺ transport and release,⁴⁹ an increase in spark frequency,⁴⁶ a decrease in RyR protein levels, and unchanged RyR mRNA levels.²⁷ Thus, it may be that RyR downregulation compensates for the increased SR Ca²⁺ load and thereby finely regulates Ca²⁺ release during excitation-contraction coupling.

Altered L-Type Channel Expression and Properties Help to Regulate Excitation-Contraction Coupling in SERCA1a Hearts
Ca²⁺ influx through the L-type Ca²⁺ channel is a critical trigger for SR Ca²⁺ release. In SERCA1a hearts, peak Ca²⁺ current amplitude (pA/pF) was significantly decreased (50%; Figure 6A). Western blotting analysis showed that L-channel -subunit expression levels were decreased (30% to 35%). This finding was corroborated by measuring Ba²⁺ currents through the L-type Ca²⁺ channel. In the phospholamban knockout model, L-type current amplitudes are unchanged, whereas Ca²⁺ transients and increased SR Ca²⁺ load are similar to those seen in the SERCA1a TG model. It is unclear whether these differences are due to different genetic background (FVBN versus SVJ/BL6) and/or are model dependent.

Although the L-type Ca²⁺ current amplitude in SERCA1a TG myocytes was decreased 50% in comparison with WT myocytes, surprisingly, there was no change in time course of Ca²⁺ current inactivation. Because Ca²⁺ entry via the channel itself plays a role in channel inactivation, it is often the case that decreased channel amplitude is accompanied by prolonged channel inactivation. However, another important source of Ca²⁺ for channel inactivation is that released from the SR.^{24 50 51} To address this question, Ca²⁺ currents were recorded in the presence of ryanodine to essentially remove the SR Ca²⁺-release component. L-type Ca²⁺-channel inactivation is prolonged in the presence of ryanodine because the only Ca²⁺ available for channel inactivation is Ca²⁺ that enters through the channel itself. In the presence of ryanodine, T_1/2 was prolonged in SERCA1a myocytes. Thus, these data allow us to conclude that SR Ca²⁺ load and release plays an important role in channel inactivation in SERCA1a myocytes and that privileged communication may exist in this case between the SR and the L-type channel.⁵²

In conclusion, we show that SERCA1a can substitute both structurally and functionally for SERCA2a in the heart and that SERCA1a overexpression can be used to enhance SR Ca²⁺ transport and cardiac contractility. Thus, SERCA1a represents an attractive candidate for gene therapy in patients with impaired cardiac contractility.

Acknowledgments

This work was funded by NIH Grants R01 6414001, HL 61476, F32 HL1001803, and GM54169.

Footnotes

Original received March 28, 2000; resubmission received March 23, 2001; revised resubmission received May 21, 2001; accepted May 22, 2001.

This manuscript was sent to Francois M. Abboud, Consulting Editor, for review by expert referees, editorial decision, and final disposition.

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