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(Circulation Research. 1998;83:1205-1214.)
© 1998 American Heart Association, Inc.

Original Contributions

Targeted Overexpression of the Sarcoplasmic Reticulum Ca²⁺-ATPase Increases Cardiac Contractility in Transgenic Mouse Hearts

Debra L. Baker, Katsuji Hashimoto, Ingrid L. Grupp, Yong Ji, Thomas Reed, Evgenij Loukianov, Gunter Grupp, Ajit Bhagwhat, Brian Hoit, Richard Walsh, Eduardo Marban, Muthu Periasamy

From the Division of Cardiology (D.L.B., Y.J., T.R., E.L., A.B., B.H., R.W., M.P.) and Department of Pharmacology and Cell Biophysics (I.L.G., G.G.), University of Cincinnati College of Medicine, Cincinnati, Ohio, and Section of Molecular and Cellular Cardiology (K.H., E.M.), The John Hopkins University, Baltimore, Md.

Correspondence to Muthu Periasamy, Division of Cardiology, University of Cincinnati College of Medicine, 231 Bethesda Avenue, ML542, Cincinnati, OH 45267. E-mail muthu.periasamy{at}uc.edu

	Abstract

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Abstract—Cardiac hypertrophy and heart failure are known to be associated with a reduction in Ca²⁺-ATPase pump levels of the sarcoplasmic reticulum (SR). To determine whether, and to what extent, alterations in Ca²⁺ pump numbers can affect contraction and relaxation parameters of the heart, we have overexpressed the cardiac SR Ca²⁺-ATPase specifically in the mouse heart using the -myosin heavy chain promoter. Analysis of 2 independent transgenic lines demonstrated that sarco(endo)plasmic reticulum Ca²⁺-ATPase isoform (SERCA2a) mRNA levels were increased 3.88±0.4-fold and 7.90±0.2-fold over those of the control mice. SERCA2a protein levels were increased by 1.31±0.05-fold and 1.54±0.05-fold in these lines despite high levels of mRNA, suggesting that complex regulatory mechanisms may determine the SERCA2a pump levels. The maximum velocity of Ca²⁺ uptake (V_max) was increased by 37%, demonstrating that increased pump levels result in increased SR Ca²⁺ uptake function. However, the apparent affinity of the SR Ca²⁺-ATPase for Ca²⁺ remains unchanged in transgenic hearts. To evaluate the effects of overexpression of the SR Ca²⁺ pump on cardiac contractility, we used the isolated perfused work-performing heart model. The transgenic hearts showed significantly higher myocardial contractile function, as indicated by increased maximal rates of pressure development for contraction (+dP/dt) and relaxation (–dP/dt), together with shortening of the normalized time to peak pressure and time to half relaxation. Measurements of intracellular free calcium concentration and contractile force in trabeculae revealed a doubling of Ca²⁺ transient amplitude, with a concomitant boost in contractility. The present study demonstrates that increases in SERCA2a pump levels can directly enhance contractile function of the heart by increasing SR Ca²⁺ transport.

Key Words: sarcoplasmic reticulum Ca²⁺-ATPase • transgenic mice • Ca²⁺ uptake • working heart model

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
The sarcoplasmic reticulum (SR) plays a central role in the contraction-and-relaxation cycle of the heart by regulating intracellular calcium (Ca²⁺) concentrations (reviewed in Reference 1¹ ). Ca²⁺ release from the SR via the ryanodine receptor initiates muscle contraction, whereas Ca²⁺ reuptake into the lumen of the SR leads to muscle relaxation. The Ca²⁺ uptake function of the SR is driven by an ATP-dependent Ca²⁺ transport pump, the sarco(endo)plasmic reticulum Ca²⁺-ATPase (SERCA). Molecular cloning analyses have identified a family of SERCA pumps encoded by 3 highly homologous genes (SERCA1, SERCA2, and SERCA3).^{2 3 4 5 6 7 8}

The SERCA2 gene encodes 2 isoforms, SERCA2a and SERCA2b, which differ at the COOH terminus as a result of alternative splicing (SERCA2a comprises 4 amino acids, and SERCA2b comprises 49 amino acids).^{5 6 7} SERCA2a is the primary SERCA isoform expressed in the heart and is also present in slow-twitch skeletal muscle, smooth muscle, and fetal fast-twitch muscle.^{9 10} In the rat heart, SERCA2a expression can be detected as early as 10 days postcoitum, and its expression levels continue to increase postnatally.^{10 11} SERCA2b is expressed in most cell types but is present at high levels in cardiac and smooth muscle of the rat and rabbit.^{7 9} SERCA2a and SERCA2b isoforms not only possess different patterns of expression but also differ in in vitro functional characteristics. The SERCA2a isoform exhibits a lower affinity for Ca²⁺ and a higher rate of turnover for Ca²⁺ transport compared with the SERCA2b isoform. These differences in pump function have been attributed to the terminal 12 amino acids of SERCA2b.¹²

Previous studies from our laboratory and others have shown that SERCA2a pump expression levels are regulated during muscle development and in response to a variety of pathophysiological conditions (reviewed in Reference 13¹³ ). Several studies have reported that thyroid hormone markedly increases SR Ca²⁺-ATPase levels and produces enhanced myocardial function.^{14 15 16 17 18 19} In contrast, decreased SR calcium transport is observed in chronic pressure overload hypertrophy and was shown to be due to decreased expression of the SERCA pump.^{14 20 21 22} Studies on human hearts also suggest that SR Ca²⁺ transport function is altered in end-stage human heart failure.^{23 24} Intracellular Ca²⁺ measurements using aequorin showed that Ca²⁺ transients in muscle samples from failing human hearts are markedly prolonged in both Ca²⁺ release and uptake phases.²⁴ Using tissue samples from failing human hearts, we and others have found that the expression levels of SERCA was decreased both at the mRNA^{25 26 27 28} and the protein levels²⁹ in end-stage heart failure. The decrease in the levels of SERCA can be closely correlated with a decreased myocardial function.^{29 30} These studies provide correlative evidence to suggest that a decrease in SERCA levels may contribute to contractile dysfunction, but they fail to establish a cause-effect relationship.

The goal of this study was to demonstrate whether alterations in SERCA levels can affect cardiac contractility. In vitro studies have shown that adenovirus-mediated gene transfer of SERCA into fetal/neonatal myocytes can increase Ca²⁺ transport function and enhance contractility.^{31 32} In this study, we chose a transgenic (TG) approach to address this question. The TG approach allows us to specifically alter SERCA pump levels within the intact heart. The TG mouse model also provides us with an in vivo system to test the functional consequences of altered SERCA expression on cardiac muscle physiology.

In a recent study, He et al³³ used the chicken ß-actin promoter linked to the viral (cytomegalovirus [CMV]) enhancer to overexpress SERCA2a in a TG mouse model. In this model the amounts of SERCA2a mRNA and protein were increased 2.6-fold and 1.2-fold, respectively. Functional analysis of calcium handling and contractile parameters in isolated cardiac myocytes indicated that the intracellular calcium decline (t_1/2) and myocyte relengthening (t_1/2) were accelerated by 23% and 22%, respectively. In addition, the rate of myocyte shortening was also significantly faster, and cardiac function measured in vivo demonstrated significantly accelerated contraction and relaxation in SERCA2 TG mice.

However, in the above study, SERCA2a was overexpressed using the chicken ß-actin promoter (driven by the viral CMV enhancer), which is not tissue specific. This transgene could drive SERCA2a expression in multiple tissues (including muscle and nonmuscle tissues). In particular, overexpression of SERCA2a in other tissues, including vascular smooth muscle, may complicate the interpretation of the results by altering Ca²⁺ homeostasis in multiple tissues. To avoid these complications, we chose to use the cardiac tissue–specific mouse -myosin heavy chain (-MHC) promoter^{34 35} to target pump overexpression specifically to the heart. This promoter has been shown by many investigators to consistently overexpress high levels of the desired protein to the cardiac compartment.

In this study, we report the generation and characterization of 2 independent SERCA2a TG lines that vary in transgene copy number and levels of SERCA2a mRNA and protein. Overexpression of the SERCA2a mRNA resulted in an increase in the total amount of SERCA pumps in a copy number-dependent manner. In response to increased protein expression, SERCA2a TG hearts showed increased SR Ca²⁺ uptake (V_max). However, the apparent affinity of the SERCA for Ca²⁺ remained unchanged in TG hearts. Functional analysis revealed that TG hearts were hyperdynamic, exhibiting increased rates of contraction and relaxation. These findings suggest that the increased expression of SERCA pumps directly enhances cardiac muscle contractility.

	Materials and Methods

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SERCA2a Transgene
The SERCA2a transgene was constructed by cloning the rat SERCA2a cDNA⁶ downstream of the 5.5-kb -MHC gene promoter³⁵ at the SalI and HindIII sites. The SERCA2a cDNA contained the entire coding sequence (starting at the AUG codon, NcoI site) and the complete 3' untranslated region. Linkers were added to the 3' and 5' ends of the cDNA to facilitate cloning into 5.5-kb -MHC promoter vector. An additional human growth hormone polyadenylation site (Hgh poly A) was included to ensure that the transcript would be polyadenylated. The transgene was removed from the vector through restriction digestion (with BamHI), isolated from an agarose gel, and purified in a cesium chloride gradient. The resulting DNA was resuspended in 5 mmol/L Tris-HCl, pH 7.4, and 0.1 mmol/L EDTA at a concentration of 5 µg/mL for microinjection into mouse oocytes.

Production of SERCA2a TG Mice
TG mice were generated by the Transgenic Core Facility, University of Cincinnati, following established procedures.³⁶ Transgene DNA was microinjected into the pronuclei of fertilized mouse oocytes derived from superovulated FVB/N females. Injected embryos were implanted into the oviducts of pseudopregnant foster females. TG mice were identified using polymerase chain reaction (PCR)³⁷ and Southern blot analysis³⁸ of genomic DNA from tail biopsies. PCR primers derived from the second intron of the -MHC promoter (5'-GCCCACACCAGAAATGACAGA-3') and SERCA2a cDNA exon 4 (5'-TCACCTTCTTCGAACCAAGC-3') produced a 390-bp fragment in TG samples. Six positive founder mice were obtained and bred with non-TG (NTG) cohorts to establish stable TG lines. The transgene copy number was determined using Southern blot analysis in comparison with endogenous -MHC gene. In brief, 10 µg of genomic DNA prepared from tail biopsies was digested with EcoRI and electrophoresed. The blots were hybridized with the NdeI-to-SalI fragment of the mouse -MHC promoter (NTG samples gave a 3.0-kb endogenous band, whereas TG samples gave 3.0-kb and 2.7-kb bands). Intensities of the TG band were compared with the endogenous band to determine copy number.

Morphological Studies
Histological evaluation was performed on the hearts from 10- to 12-month-old SERCA2a TG and wild-type mice. The tissues were fixed in 10% formalin, dehydrated, and embedded in paraffin.³⁹ Longitudinal sections (5 µm) of the heart (cut at 50-µm intervals) were stained with hematoxylin and eosin. Longitudinal sections were made to demonstrate all 4 heart chambers and valves. Hearts were examined for hypertrophy, hyperplasia, inflammation, septal defects, necrosis, mineralization, fibrosis, cytoplasmic vacuolization, and altered cell orientation.

Northern Blot Analysis
Total RNA from cardiac tissue was isolated, using Ultra Spec II (RNA isolation kit; Biotecx Laboratories, Inc), from 11- to 14-week old mice. SERCA2a mRNA levels were estimated using Northern blot analysis of 10 µg of total cardiac RNA following established procedures.⁴⁰ For dot blots, serial dilutions (1.25, 2.5, and 5 µg) of total RNA were blotted onto nitrocellulose membranes. Both Northern blots and dot blots were hybridized with an NcoI fragment from the rat SERCA2 cDNA. Dot blots were also hybridized with a mouse GAPDH.

Western Blot Analysis
Hearts from adult TG mice and littermates (11 to 14 weeks old) were homogenized in the following (in mmol/L): imidazole 10, pH 7.0, sucrose 300, DTT 1, and sodium bisulfate 1.⁴¹ Protein concentrations were determined by Bio-Rad protein assay. The relative SERCA2, phospholamban, and cardiac actin protein levels in TG versus NTG samples were determined by quantitative immunoblotting as described below. Equal amounts of protein extract from both TG and NTG littermates (1, 2, and 4 µg) were separated by SDS-PAGE on a 13% polyacrylamide gel and then transferred to nitrocellulose using a Bio-Rad transblot apparatus. Filters were incubated with a polyclonal antibody for SERCA⁴² (1:500 dilution), a cardiac actin monoclonal antibody (1:2500; Sigma), or phospholamban monoclonal antibody (1:1000; Affinity Bioreagents) overnight. Secondary antibodies were peroxidase-labeled anti-rabbit or anti-mouse IgG (Kirkegaard & Perry Laboratories). Antibody signals were detected through an enhanced chemiluminescence kit (Kirkegaard & Perry Laboratories).

Ca²⁺ Uptake Assays
Heart tissue from TG and NTG mice was homogenized in the following (in mmol/L): potassium phosphate 50, pH 7.0, NaF 10, EDTA 1, sucrose 300, phenylmethylsulfonyl fluoride 0.3, and DTT 0.5. The SR Ca²⁺ uptake activity was measured using a modified Millipore filtration method.^{43 44} Cardiac homogenates (100 µg/mL) were incubated at 37°C in 1.5 mL of reaction mixture containing 40 mmol/L imidazole, pH 7.0, 100 mmol/L KCl, 5 mmol/L MgCl₂, 5 mmol/L NaN₃, 5 mmol/L potassium oxalate, 1 µmol/L Ruthenium red, and 0.5 mmol/L EGTA to yield free Ca²⁺ concentration in the range of 0.03 to 3 µmol/L (containing 1 µCi/µmol ⁴⁵Ca) as determined by the computer program (Calcium Titration Program).⁴⁵ The uptake reaction was started by adding 5 mmol/L ATP to the reaction mixture. At serial time intervals, 300-µL samples were vacuum filtered through Millipore (0.45 µm HAWP) nitrocellulose membrane, and the vesicles remaining on the filters were washed, dissolved, and then processed for liquid scintillation counting. The rates of Ca²⁺ uptake were calculated by the least-squares linear regression analysis of uptake values at 30, 60, and 90 s. The calcium concentration required for half of the maximum velocity for Ca²⁺ uptake (K_0.5) was determined by nonlinear curve fitting using Origin 3.5 (MicroCal Software Inc).

Functional Analysis of TG Hearts Using Isolated Work-Performing Heart Preparations
The anterograde, work-performing heart preparation has been previously described.⁴⁶ In brief, 12- to 16-week-old, age-matched mice of either sex were anesthetized with 30 mg/kg pentobarbital sodium IP and injected with 500 U/kg heparin sodium (Elkins Sinn) to prevent intracardiac blood coagulation. A 12-lead ECG and heart rate were recorded for each mouse before opening the chest and removing the heart. Hearts were removed via midsternal incision, immediately suspended on a 20-gauge stainless steel cannula, and then connected to the perfusion apparatus. Retrograde perfusion with heated (37.4°C) and oxygenated Krebs-Henseleit (KH) buffer (containing [in mmol/L] NaCl 118, KCl 4.7, CaCl₂ 2.5, MgSO₄ 1.2, KH₂PO₄ 1.2, sodium EDTA 0.5, NaHCO₃ 25, and glucose 11) was started to clear the coronary vasculature of the remaining blood and to insert the intraventricular catheter for pressure measurements. After a short period of stabilization on retrograde perfusion, the pulmonary vein was cannulated, and perfusion of the heart was then switched from retrograde to anterograde. Venous return (preload) was adjusted and regulated with a micrometer and continuously measured using a dual-channel Transonic flowmeter. Aortic pressure (afterload) was adjusted with a second micrometer. To set basal loading conditions, flow was adjusted to deliver an initial "venous return" (preload) of 5 mL/min cardiac output and an afterload of 50 mm Hg aortic pressure, providing a basal workload of 250 mL/minxmm Hg/s. Cardiac function curves were performed after stabilization of the heart at basal loading conditions by increasing and decreasing the aortic pressure (pressure loading) stepwise, while keeping the venous return constant. Following pressure loading, volume loading was performed by stepwise increasing and decreasing of the venous return at a constant aortic resistance. Because ventricular end-diastolic and left atrial pressures were monitored continuously, any developing cardiac failure was recognized and corrected immediately. All hearts were compared under identical loading conditions ranging from 250 mm HgxmL/min (50 mm Hg mean aortic pressure) to 400 mm HgxmL/min (80 mm Hg mean aortic pressure). The perfusion fluid, oxygenation, and temperature were constant throughout the experiment. Heart rate, aortic pressure, left intraventricular (systolic, diastolic, and end-diastolic) pressure, and atrial pressure were continuously monitored, and the first derivative of left intraventricular pressure, +dP/dt and –dP/dt, time to peak pressure (TPP)/mm Hg, and time to half relaxation (RT_1/2)/mm Hg were calculated with a custom-designed computer program. Cardiac output and coronary flow were computer calculated.

Oxygen Consumption in Work-Performing Hearts
Coronary arterial perfusion buffer and venous effluent samples were collected anaerobically, and the PO₂ and PCO₂ values of these samples were measured using an automated blood gas analyzer (model 248, CIBA Corning Diagnostics Corp). Oxygen consumption (MVO₂) by the perfused hearts was computed by multiplying the coronary flow by the arteriovenous difference in oxygen content and normalized per gram of tissue mass as follows:

(1)

where PaO₂ and PvO₂ represent the perfusate and venous partial pressures of O₂ (mm Hg), respectively, and C=0.0239 (Bunsen solubility coefficient of oxygen dissolved in perfusate at 37°C, in mL O₂ · atm^–1 · mL^–1).

Unanesthetized Blood Pressure and Heart Rate Measurements
The blood pressures were measured in the unanesthetized mice via tail cuff (Natsume model KN-210 Photosensor unit, modified for mice; cuff size 7 mm) and recorded on a Grass P7 polygraph. Heart rate was computed from the amplitude of the pulse signal. The mice were acclimated to the chamber before the actual measurements of the pressures were taken. A set of 30 measurements at 3 different time periods was averaged for each mouse.

Intracellular Ca²⁺ and Twitch Force Measurements in Intact Trabeculae
The intact mouse cardiac muscle preparation has been previously described.⁴⁷ In brief, 12- to 19-week-old mice of either sex were anesthetized with 10 to 20 mg of pentobarbital sodium (IP) and injected with 20 to 30 mg of heparin sodium. The hearts were retrogradely perfused with modified KH buffer with high K⁺ (20 mmol/L) and gassed with a 95% O₂-5% CO₂ gas mixture in a dissection dish at room temperature. Trabeculae suitable for force measurements and fura-2 microinjection were quickly dissected from the right ventricle and mounted between a force transducer and a micromanipulator in a perfusion bath. Geometrically suitable (long, thin, nonbranched) trabeculae were found in a minority of the hearts, so that the overall success rate of these experiments was 20%. The trabeculae were superfused with KH buffer (containing the following [in mmol/L]: NaCl 112, KCl 5, CaCl₂ 0.5, MgSO₄ 1.2, NaH₂PO₄ 2, NaHCO₃ 28, and glucose 10) equilibrated with 95% O₂-5% CO₂. The perfusion rate was 5 to 10 mL/min, and the preparations were stimulated at 0.5 Hz. All experiments were performed at room temperature (22°C). The preparations were field stimulated with 5-ms pulses, with a Grass SD9 stimulator. After stabilization of the preparations, fura-2 potassium salt was microinjected iontophoretically into one cell and allowed to spread throughout the muscle via gap junctions. [Ca²⁺]_i was measured as described previously.^{48 49} [Ca²⁺]_i was determined by measuring the epifluorescence of fura-2 excited at 380 and 340 nm. The fluorescent light was collected at 510 nm by a photomultiplier tube (model R2693; Hamamatsu). The output of the photomultiplier tube was filtered at 100 Hz, collected by an analog-to-digital converter, and stored in the computer for later analysis. Intracellular [Ca²⁺] was given by the following equation (after subtraction of the autofluorescence of the muscle):

(2)

where R is the observed ratio of fluorescence (340 nm/380 nm), K_d is the apparent dissociation constant, R_max is the ratio of 340 nm/380 nm at saturating [Ca²⁺], and R_min is the ratio of 340 nm/380 nm at 0 [Ca²⁺]. The values for K_d, R_max, and R_min were 3.5, 7.20, and 0.47 µmol/L, respectively. After equilibration of the loaded fura-2, intracellular free Ca²⁺ concentration and contractile force were measured.

Statistical Analyses
TG and NTG parameters were compared using either one-way ANOVA followed by a Dunnett's/Student-Newman-Keuls post hoc test or a 2-way ANOVA as appropriate using SigmaStat software. Results are expressed as the mean±SEM. The level of statistical significance was P0.05.

	Results

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Characterization of SERCA2a TG Mouse Lines
In this study the cardiac -MHC gene promoter was used to direct the rat SERCA2a cDNA expression specifically to the heart (Figure 1A). PCR and Southern blot analysis of founder mice identified 6 TG lines containing 2 to 13 copies of the SERCA2a transgene. Of these 6 founder lines, 4 mice (founder mice 100, 19, 16, and 3) showed germ-line transmission and expression of the transgene. The TG lines 3 (copy 13) and 100 (copy 8), expressing high levels of SERCA2a mRNA, were chosen for detailed characterization (Figure 1B). Limited characterization was also performed on TG line 16 (copy 2). Founder 21 was sterile, and founder 90 showed mosaic expression of the SERCA2a transgene; these two mice were discarded.

View larger version (28K): [in this window] [in a new window]   Figure 1. Structure of the SERCA2a transgene construct and determination of copy number. A, The SERCA2a transgene consists of the rat SERCA2a cDNA entire coding sequence and the complete 3' untranslated region cloned downstream of the 5.5 -MHC gene promoter at the SalI and HindIII sites. An additional polyadenylation site (Hgh poly A) was included to ensure that the transcript would be polyadenylated. B, Southern blot analysis of genomic DNA isolated from mouse tail biopsies. Genomic DNA (10 µg) was digested with EcoRI and electrophoresed. The blots were hybridized with the NdeI-to-SalI fragment of the mouse -MHC promoter (NTG samples gave a 2.6-kb endogenous band, whereas TG samples gave 3.0-kb and 2.6-kb bands).

The SERCA2a TG mice showed no evidence of pathology. The heart weight/body weight ratio was not significantly different between TG and NTG mice. Heart sections from SERCA2a TG mice and wild-type littermates were evaluated using light microscopy for differences in gross morphology as described in Materials and Methods. Under low magnification, TG hearts were morphologically indistinguishable from NTG hearts. No significant differences in heart size, chamber size, wall thickness, or architecture could be identified in these hearts. Histological analysis of the TG hearts revealed no evidence of hypertrophy. The overall myocyte organization and structure were well maintained in both groups (data not shown).

To determine whether SERCA2a overexpression has an effect on in vivo blood pressures and heart rates, measurements were performed on unanesthetized TG and NTG mice. The blood pressures and heart rates were slightly higher in TG mice (blood pressure, 116.96±3.09 mm Hg [control], 130.00±7.25 mm Hg [TG 3], and 124.58±0.98 mm Hg [TG 100]; heart rate, 591.58±24.34 bpm [control], 661.33±16.34 bpm [TG 3], and 631.83±29.28 bpm [TG 100]), but the differences were not statistically significant.

SERCA2a mRNA Levels Are Increased Severalfold in TG Hearts
To detect SERCA2a transgene expression, Northern blot analysis was performed using a rat cDNA probe on total RNA from TG (lines 16, 3, and 100) and NTG hearts. Northern blot analysis indicated that SERCA2a mRNA levels were significantly increased in TG hearts (Figure 2A). The rat SERCA2a message migrated at the same level as the endogenous mouse (4-kb) transcript; however, overexpression of the transgene can be recognized by the abundance of SERCA2a mRNA levels. Furthermore, the SERCA2a overexpression was limited to the heart (data not shown). Precise quantification by dot-blot analysis revealed that the SERCA2a mRNA level was increased 3.88±0.4-fold in line 100 and 7.9±0.2-fold in line 3 (Figure 2B). The SERCA2a mRNA levels were normalized for loading variations by a GAPDH cDNA probe. Our data show that line 3, with the highest transgene copy number (13 copies), expressed the highest SERCA2a mRNA level (8-fold).

View larger version (48K): [in this window] [in a new window]   Figure 2. The SERCA mRNA levels are increased severalfold in SERCA2a TG mouse hearts. A, Northern blot analysis. Total RNA (10 µg) from wild-type and SERCA2a TG mouse hearts were fractionated on formaldehyde gels and hybridized with a 32P-labeled SERCA2a-specific probe. B, Representative dot blot of SERCA mRNA levels in wild-type and SERCA2a TG mice (line 100). Total RNA (5 µg) from wild-type and TG hearts were blotted onto nitrocellulose membranes and probed with SERCA2- and GAPDH-specific sequences.

SERCA Protein Levels Are Increased in TG Hearts
To determine whether SERCA2a protein levels were increased in TG hearts, Western blot analysis was performed using cardiac homogenates from age-matched TG and NTG littermates.⁴¹ SERCA2a protein was quantitated using a polyclonal antibody raised against SERCA2a protein.⁴² A representative Western blot is shown in Figure 3. Our protein analysis revealed that total SERCA2a protein was increased 1.31±0.05-fold in TG 100 and 1.54±0.05-fold in TG 3. These data suggest that there is no strict correlation between SERCA2a mRNA and protein levels in the TG mice. However, the line that produced the highest SERCA2a mRNA levels (TG 3) also shows the greatest increase in SERCA2a protein levels. To determine whether increased expression of SERCA2a has affected the expression of other proteins, we performed quantitative immunoblotting on phospholamban (a regulator of the SERCA pump) and actin (a myofilament protein) levels using specific antibodies. The levels of phospholamban and actin were not altered in TG hearts (Figure 3).

View larger version (36K): [in this window] [in a new window]   Figure 3. SERCA protein levels are increased in SERCA2a TG hearts. A representative Western blot analysis comparing protein levels from NTG (control) and TG (lines 3 and 100) is shown. Protein from whole heart homogenate (1 and 2 µg) was analyzed. Relative SERCA2, phospholamban, and actin levels were determined by immunoblotting.

Maximal Velocity (V_max) of SR Ca²⁺ Uptake Is Increased in SERCA2a TG Hearts
To study the effects of SERCA2a overexpression on SR Ca²⁺ transport function, the initial rates of ATP-dependent, oxalate-facilitated SR Ca²⁺ uptake were assessed using cardiac homogenates under conditions that restrict Ca²⁺ uptake to the SR.^{43 44} Ca²⁺ uptake rates for TG mice were higher than for their NTG littermates (Figure 4A). The maximum velocity of Ca²⁺ uptake (V_max) was significantly higher (37%; n=5) in TG mice (line 100) than in NTG (age- and sex-matched) littermates (n=5). This increase in SR Ca²⁺ uptake is consistent with increases in SERCA2a protein levels in TG hearts. To determine whether the apparent Ca²⁺ affinity is altered in TG hearts, the Ca²⁺ dependence of Ca²⁺ uptake rates (K_0.5) were determined. The data are expressed as the percentage of the maximum uptake rates obtained at pCa 6.0 for each preparation. Analysis of the data did not reveal a significant difference in the K_0.5 of TG samples (Figure 4B).

View larger version (13K): [in this window] [in a new window]   Figure 4. Maximal velocity (Vmax) of SR Ca2+ uptake is increased in SERCA2a TG hearts. SR Ca2+ uptake function was assayed using cardiac homogenates from SERCA2a TG () and NTG () hearts from littermates. A, Initial rates of Ca2+ uptake were assayed over a range of Ca2+ concentrations. Vmax (nmol · mg–1 · min–1) was 92.96±5.29 for NTG hearts and 125.24±7.00 (P<0.01) for SERCA2a TG hearts. B, Data are represented as the percentage of the maximal Ca2+ uptake rates at pCa6.0. K0.5 (µmol/L) was 0.23±0.01 for NTG hearts and 0.22±0.01 for SERCA2a TG hearts. Values are mean±SEM (TG, n=5; NTG, n=5).

SERCA2a TG Hearts Show a Significant Increase in the Rates of Contraction and Relaxation
To determine whether an increase in SERCA2a pump levels affects the overall cardiac function, we used the isolated work-performing heart preparation to measure contractile and relaxation function. This model allows us to compare myocardial contractile parameters in individual mouse hearts under identical afterload conditions (50 mm Hg mean aortic pressure) and preload conditions (5 mL/min venous return, an approximation of preload), producing a cardiac minute work (mean aortic pressurexcardiac output) of 250 mm Hgx mL/min. Although isolated TG hearts showed similar heart rates (360 to 390 bpm) and end diastolic pressures compared with age-matched NTG hearts, both systolic and diastolic intraventricular pressures were increased under identical load conditions (Table). Furthermore, the maximal rate of pressure development for contraction (+dP/dt) was significantly increased by 35% (TG 3) and 12% (TG 100), and the maximal rate of pressure decline (–dP/dt) was increased by 41% (TG 3) and 14% (TG 100) compared with NTG hearts (Figure 5). Moreover, TG hearts showed 26% (TG 3) and 9% (TG 100) shorter TPP, and the RT_1/2 was shortened by 26% in TG 3 and by 12% in TG 100. The decreased TPP indicates that the SERCA2a hearts have significantly higher contractility (shorter time to develop peak ventricular pressure), while increased RT_1/2 shows that relaxation is also shortened. Additionally, there was a trend toward increased MVO₂; for TG line 3, MVO₂ was 173±8.9; for TG line 100, 180.5±19; and for NTG, 159.48±9.4. However, this increase was not significant.

View this table: [in this window] [in a new window]   Table 1. Cardiac Parameters of SERCA2a Overexpression Versus Controls (FVB/N)

View larger version (28K): [in this window] [in a new window]   Figure 5. SERCA2a TG hearts show a significant increase in the rates of contraction and relaxation. Isolated work-performing heart preparations were used to measure contractile and relaxation function of SERCA2a-overexpressing mouse hearts. Contractile parameters of TG and NTG hearts were measured under identical afterload (50 mm Hg mean aortic pressure) and preload conditions (5 mL/min venous return, approximation of preload). The maximal rates of pressure development for contraction (+dP/dt) (A) and relaxation (–dP/dt) (B), TPP (C), and RT1/2 (D) are indicated for wild type (NTG), TG line 3, and TG line 100. Data are mean±SEM.

Effect of Pressure and Volume Loading on Contractile Parameters of TG Hearts
To evaluate the capacity of SERCA2a TG mouse hearts to respond to increased work, we performed pressure loading on 7 TG 100, 4 TG 3, and 6 NTG hearts. We determined left ventricular functional response to increased pressure-loading conditions by plotting cardiac minute work (from 250 to 400 mm HgxmL/min) versus the rate of pressure development and relaxation (+dP/dt and –dP/dt in mm Hg/s; Figure 6). The TG hearts showed significantly higher rates of contraction and relaxation compared with control values for all pressure-loading conditions examined. Our results also demonstrate that both control and TG hearts respond to increased cardiac minute work with increased contractility, as evidenced by increased +dP/dt. However, the TG and NTG hearts did not respond to increased pressure loading with increased rates of relaxation. The hearts from TG 3 rather showed a decline in the rate of relaxation under high-pressure loading conditions.

View larger version (46K): [in this window] [in a new window]   Figure 6. Effect of pressure loading on contractile parameters in TG and NTG hearts. The effects of pressure loading on ventricular function were examined in TG 100 (n=7), TG 3 (n=4), and NTG (n=6) hearts. The left ventricular function was plotted as cardiac minute work (mean aortic pressure x cardiac output) vs the rates of contraction and relaxation (+dP/dt and –dP/dt in mm Hg/s). Comparisons were made between and among control and TG groups under different loading conditions. Data are mean±SEM.

SERCA2a TG Hearts Show a Sizable Increase in Ca²⁺ Transient Amplitude, With a Concomitant Boost in Contractility
An increase in SERCA2a expression might logically be predicted to increase the ability of the SR to store calcium, such that more calcium is available to be released during each heartbeat. If so, the increased contractility in SERCA2a TG hearts would be attributable to an increase in activator Ca²⁺ availability. To determine whether this was the case, we measured intracellular Ca²⁺ and contractile force simultaneously in fura-2-loaded isometrically contracting trabeculae. The experiments were performed in 2 mmol/L [Ca²⁺]_o at a stimulation rate of 2 Hz (22°C). Figure 7 shows typical records of Ca²⁺ transients (top) and the corresponding twitch force (bottom) in muscles from hearts of normal (left) and SERCA2a TG (right) mice. The TG hearts exhibit a sizable increase in Ca²⁺ transient amplitude relative to the normal hearts, with a concomitant increase in force generation. Figure 8 shows pooled data for [Ca²⁺]_i (A) and force (B) from 4 normal and 4 TG trabeculae. The SERCA2a TG muscles showed significantly increased systolic [Ca²⁺]_i and force, but no changes in the diastolic values. These data enable us to conclude that the increase in contractility indeed reflects an increase in activator Ca²⁺ availability. In addition, it is notable that the large increases in systolic [Ca²⁺]_i and force occur with no evidence of diastolic calcium overload or diastolic dysfunction, consistent with the enhanced capacity of the SR to sequester calcium.

View larger version (21K): [in this window] [in a new window]   Figure 7. Ca2+ transients (top) and twitch force (bottom) in representative mouse ventricular trabeculae of normal (left) and TG (right) hearts. The muscles were superfused with modified KH buffer at 2 mmol/L [Ca2+] and stimulated at 2 Hz (22°C). SERCA2a TG hearts showed 1.84 µmol/L systolic [Ca2+]i and 30.4 mN/mm2 systolic force, whereas those of NTG hearts were 1.02 µmol/L and 18.0 mN/mm2, respectively.

View larger version (17K): [in this window] [in a new window]   Figure 8. SERCA2a TG hearts showed a sizable increase in Ca2+ transients and contractility. Pooled data are shown for systolic and diastolic [Ca2+]i (A) and force (B) in trabeculae from normal control (n=4) and TG hearts (n=4). Both systolic [Ca2+]i and force were significantly higher in muscles from TG hearts with no significant changes in diastolic [Ca2+]i and force. All muscles were superfused with KH buffer containing 2 mmol/L [Ca2+]o and stimulated at 2 Hz. Data are mean±SEM.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
The goal of the present study was to determine whether and to what extent alterations in SERCA2a pump levels affect cardiac contractility, using a TG mouse model. In this study, we used the cardiac tissue–specific -MHC promoter to direct the cardiac tissue–specific overexpression of the SERCA2a pump. Our analyses of SERCA2a TG hearts revealed that SERCA2a mRNA levels were increased severalfold (3.88±0.4-fold and 7.90±0.2-fold), whereas, SERCA2a protein levels were increased by 1.31±0.05-fold and 1.54±0.05-fold over those of controls. The maximum velocity (V_max) of SR membrane Ca²⁺ uptake was increased in TG hearts in correspondence with increased protein levels. Our studies using the isolated work-performing heart model demonstrate that TG hearts have increased rates of contraction/relaxation with decreased TPP and RT_1/2 under resting conditions and respond to increased cardiac load with increased myocardial contractility. Reduction in RT_1/2 indicates enhanced SR Ca²⁺ uptake in TG hearts, whereas a reduction in the TPP is indicative of increased Ca²⁺ availability. Our results suggest that increased Ca²⁺ uptake in the TG hearts results in an increased availability of Ca²⁺ for contraction. Thus, by increasing SERCA2a levels, we have not only increased the rate of relaxation but also the rate of contraction. These data provide convincing evidence that the SERCA2a pump level is an important determinant of myocardial contractility.

Level of SERCA2a Pump Expression Can Directly Affect Cardiac Contractility
In this study, we have analyzed 2 independent SERCA2a TG lines that vary in transgene copy number, SERCA2a mRNA expression, and SERCA protein levels. Data from both lines 3 and 100 independently demonstrate an increase in contractile function in response to SERCA2a overexpression. More importantly, this study demonstrates that different levels of pump expression can affect contractile parameters to different extents. Although there is no strict correlation between SERCA2a mRNA and protein levels, TG 3 mice that produced the highest overexpression of the SERCA2a message (7.9-fold) and SERCA2a protein levels (1.54-fold) also demonstrated the highest level of cardiac performance, whereas TG 100, which produced a 3.88-fold increase in SERCA2a message and protein levels (1.31-fold), exhibited a lesser but significant increase in cardiac contractility. Thus, analysis of these 2 lines allows us to conclude that changes in Ca²⁺-ATPase levels can directly affect cardiac contractility. Our studies using the intact mouse ventricular trabeculae method revealed directly that muscles from TG hearts exhibit an increased Ca²⁺ transient amplitude, with a concomitant boost in twitch force. This experimental approach, which is the most direct one available for the characterization of calcium cycling and contractile activation in mice, verifies that SERCA2a overexpression is capable of increasing the calcium-sequestering ability of the SR in a physiologically significant manner. Recently, He et al³³ reported that SERCA2a overexpression (1.2-fold) in transgenic mouse (using the CMV enhancer and the chicken ß-actin promoter) resulted in increased Ca²⁺ transients in isolated myocytes and increased contractile function. Furthermore, using adenovirus-mediated gene transfer of SERCA into fetal/neonatal myocytes, Hajjar et al³¹ and Inesi et al³² have shown that increased SERCA protein levels produced an increase in peak Ca²⁺ levels, shorter Ca²⁺ transients, and enhanced contractility. These studies together provide strong evidence that SERCA pump level is a critical determinant of myocardial function.

SERCA2a Overexpression Results in an Increased Rate of SR Ca²⁺ Uptake Without a Change in the Apparent Ca²⁺ Affinity
Our studies show that Ca²⁺ uptake rates (V_max) were significantly higher in TG hearts (37% increase over NTG hearts). However, the Ca²⁺ dependence of SR Ca²⁺ uptake (apparent affinity for Ca²⁺) was not altered in TG hearts, despite an increase in SERCA pump level. This is particularly interesting, since the level of phospholamban is unaltered in the TG hearts. On the basis of the increased expression of the SERCA2a protein, one might expect that the phospholamban/SERCA2 ratio might be altered. Phospholamban/SERCA2a ratios have been shown to influence the Ca²⁺ dependence of Ca²⁺ uptake in studies in which the phospholamban level has been altered. A recent report by Hajjar et al⁵⁰ has indicated that alterations in phospholamban/SERCA2a ratios by adenoviral gene transfer of phospholamban result in altered Ca²⁺ affinity of the Ca²⁺ pump and Ca²⁺ transients. Furthermore, ablation of the phospholamban gene results in an increase in the SERCA pump affinity for calcium and increased rates of contraction and relaxation.⁵¹ On the other hand, overexpression of phospholamban in TG mice results in decreased SERCA pump affinity for Ca²⁺ and decreased rates of relaxation and contraction.⁵²

In this study, we show that the apparent SERCA2a affinity for Ca²⁺ remains unchanged in TG hearts despite an increase in SERCA2a protein levels. Two possible explanations are the following. (1) Although the total phospholamban level has not been altered, the monomer/pentamer ratio for phospholamban maybe adjusted for increased SERCA2a. (2) Phospholamban exists at saturating levels in the mouse heart, and thus increases in SERCA2a levels do not result in a compensatory change in the affinity of the pump for Ca²⁺. Our study suggests that increases in the SERCA2a pump level may not necessarily produce a shift in the apparent affinity of the pump for Ca²⁺. Currently, the in vivo functional ratio between phospholamban and SERCA2a is unknown. It is also not known whether phospholamban is in excess or in a limited quantity in the heart. Studies have shown that phospholamban exists both as a monomer and as a pentamer, and the equilibrium between these 2 states is highly regulated.⁵³ There are still controversies as to whether the active form of phospholamban is a monomer or a pentamer,^{53 54} although some studies have suggested that the monomer is the more active form⁵³ (David D. Thomas, oral communication, January 1998).

Similarities and Differences Between SERCA2a and SERCA1a TG Overexpression Models
In a parallel study, we have overexpressed the fast skeletal muscle SERCA1a pump in the TG mouse heart to determine whether the SERCA1a pump is functionally distinct from the SERCA2a pump.⁵⁵ Ectopic expression of the SERCA1a pump resulted in a 2.5-fold increase in total SERCA pump level but decreased the endogenous SERCA2a level to 50% as compared with NTG hearts. In contrast, SERCA2a overexpression resulted in a severalfold increase in the mRNA level, but only a modest increase (1.3- to 1.5-fold) in protein levels. The different levels of SERCA protein expression observed with these models may be due to differences in the model system or to isoform-specific differences. It is well known that the density of SERCA1a pumps is 3- to 5-fold greater in fast skeletal muscle SR as compared with SERCA2a in slow-twitch or cardiac muscle. Both models demonstrate that increases in Ca²⁺-ATPase levels can directly modify cardiac contractility, although the magnitude of the cardiac contractility is model dependent. We also found that the SERCA1a pump can be regulated by phospholamban. However, it remains to be determined whether these models differ in their response to neurohormonal regulation.

In conclusion, we have demonstrated that SERCA2a is a critical determinant of cardiac contractility. Our studies demonstrate that different levels of SERCA2a pump can affect cardiac contractility to different extents. Taken together, the results from our studies and those of He et al,³³ Inesi et al,³² and Hajjar et al³¹ demonstrate that alteration of SR Ca²⁺-ATPase levels can affect Ca²⁺ transport and cardiac function. The SERCA2a TG mice with increased Ca²⁺-ATPase levels in the heart provide us with an interesting model to study the role of SERCA in Ca²⁺ homeostasis and heart failure.

	Acknowledgments

 
This study was supported in part by National Institutes of Health (NIH) grant NIH-HL52318 (Specialized Center of Research on Heart Failure), NIH grant NIH-HL22619-17, National Research Service Award Postdoctoral Fellowship F32-HL09409 (to D.L.B.), NIH training grant HL07382-20 (to T.R.), and a Marion Merrell Dow Research Foundation gift. The work on calcium cycling and contractile activation in mouse trabeculae was supported by NIH grant R01 HL44065 (to E.M.) and by a fellowship from the Sumitomo Life Foundation of Japan (to K.H.). We thank Dr Gary Shull (Department of Molecular Genetics, University of Cincinnati) for providing the SERCA2a cDNA, Dr Jeffrey Robbins (Division of Molecular Cardiovascular Biology, Childrens Hospital Research Foundation, Cincinnati, Ohio) for providing the -MHC gene promoter, Dr Evanglia Kranias (Department of Pharmacology, University Cincinnati) for providing a polyclonal antibody raised against SERCA2, and Dr Frank Wuytack (Laboratorium voor Fysiologie, Universiteit Leuven, Leuven, Belgium) for providing a polyclonal antibody raised against SERCA2a. We would also like to thank J. Neumann (Transgenic Core Facility, University of Cincinnati) and G. Boivin, DMV (Animal Pathology Core, University of Cincinnati) for their contributions to the production and analysis of the TG mice. We are also grateful to Alla Zilberman, Darryl Kirkpatrick, Gilbert Newman, Jason Straus, and Traci Jackson for their technical support.

	Footnotes

 
This manuscript was sent to Michael R. Rosen, Consulting Editor, for review by expert referees, editorial decision, and final disposition.

Received January 28, 1998; accepted October 5, 1998.

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