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Cardiovascular Research 2003 57(1):71-81; doi:10.1016/S0008-6363(02)00609-0
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Copyright © 2003, European Society of Cardiology

Combined phospholamban ablation and SERCA1a overexpression result in a new hyperdynamic cardiac state

Wen Zhaoa, Konrad F Franka, Guoxiang Chua, Michael J Gersta, Albrecht G Schmidta, Yong Jib, Muthu Periasamyc and Evangelia G Kraniasa,*

aDepartment of Pharmacology and Cell Biophysics, University of Cincinnati, College of Medicine, 231 Albert Sabin Way, Cincinnati, OH 45267-0575, USA
bDepartment of Molecular and Cellular Physiology, University of Cincinnati, College of Medicine, Cincinnati, OH 45267-0575, USA
cDepartment of Physiology and Cell Biology, Ohio State University, Columbus, OH 43210, USA

litsa.kranias{at}uc.edu

* Corresponding author. Tel.: +1-513-558-2377; fax: +1-513-558-2269

Received 3 May 2002; accepted 12 July 2002


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Acknowledgments
 References
 
Objective: Phospholamban ablation or ectopic expression of SERCA1a in the heart results in significant increases in cardiac contractile parameters. The aim of the present study was to determine whether a combination of these two genetic manipulations may lead to further augmentation of cardiac function. Methods: Transgenic mice with cardiac specific overexpression of SERCA1a were mated with phospholamban deficient mice to generate a model with SERCA1a overexpression in the phospholamban null background (SERCA1OE/PLBKO). The cardiac phenotype was characterized using quantitative immunoblotting, sarcoplasmic reticulum calcium uptake and single myocyte mechanics and calcium kinetics. Results: Quantitative immunoblotting revealed an increase of 1.8-fold in total SERCA level, while SERCA2 was decreased to 50% of wild types. Isolated myocytes indicated increases in the maximal rates of contraction by 195 and 125%, the maximal rates of relaxation by 200 and 124%, while the time for 80% decay of the Ca2+-transient was decreased to 43 and 75%, in SERCA1OE/PLBKO hearts, compared to SERCA1a overexpressors and phospholamban knockouts, respectively. These mechanical alterations reflected parallel alterations in Vmax and EC50 for Ca2+ of the sarcoplasmic reticulum Ca2+ transport system. Furthermore, there were no significant cardiac histological or pathological alterations, and the myocyte contractile parameters remained enhanced, up to 12 months of age. Conclusions: These findings suggest that a combination of SERCA1a overexpression and phospholamban ablation results in further enhancement of myocyte contractility over each individual alteration.

KEYWORDS Ca-pump; Contractile function; Myocytes; SR (function)


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 Acknowledgments
 References
 
The sarcoplasmic reticulum (SR) Ca2+-ATPase (SERCA) is the major regulator of Ca2+ homeostasis and contractility in cardiac and skeletal muscle. SERCA1a is the predominant isoform in adult fast twitch skeletal muscle [1], while SERCA2a is primarily expressed in cardiac and slow-twitch skeletal muscles [2]. SERCA1a and SERCA2a are ~84% identical at the protein level, and they have essentially identical topologies and tertiary structure [3]. In vitro overexpression of the SERCA1 or SERCA2a isoforms in chicken embryonic cardiac myocytes resulted in 5.0- or 2.6-fold increases in SR calcium uptake rates, respectively, indicating a higher kinetic turnover by SERCA1 compared with SERCA2a [4,5]. To further determine whether SERCA1a can functionally substitute SERCA2a in the heart in vivo, Loukianov et al. [6] created a transgenic mouse model with cardiac-specific expression of SERCA1a. Ectopic expression of SERCA1a in the heart resulted in a 2.5-fold increase in the total SERCA protein, compared to the 1.2–1.5-fold increases obtained by SERCA2a overexpression [7,8]. Furthermore, the maximal rates of contraction and relaxation were significantly higher in SERCA1a overexpressing hearts [6,9] compared to SERCA2a overexpressing hearts [7,8]. Interestingly, the phospholamban expression levels were not altered and addition of the phospholamban antibody to SR Ca2+-uptake assays increased the apparent Ca2+-affinity to the same extent in SERCA1a overexpressing and wild type hearts [9], suggesting that at least part of the ectopically expressed SERCA1a molecules may be regulated by phospholamban in the transgenic hearts.

Phospholamban is mainly expressed in the SR of cardiac muscle, and in low levels in slow-twitch skeletal and smooth muscles. In cardiac muscle, dephosphorylated phospholamban is closely associated with SERCA2a and acts as an inhibitor of the pump's Ca2+-affinity [10,11]. Phosphorylation of phospholamban by cAMP-dependent protein kinase or Ca2+/calmodulin-dependent protein kinase II can relieve this inhibition [12,13]. The role of phospholamban in the regulation of cardiac function has been elucidated through the development and characterization of genetically altered mouse models. A phospholamban-deficient mouse, generated by gene targeting in embryonic stem cells, displayed enhanced Ca2+-kinetics in isolated myocytes and hyperdynamic function in perfused hearts [14,15], as well as enhanced cardiac contractility in intact mice, using either invasive or noninvasive techniques [16,17]. Interestingly, the hyperdynamic function persisted over the long term, indicating that compensatory changes did not develop through the aging process to diminish the enhanced function [18]. On the other hand, cardiac-specific overexpression of phospholamban resulted in depressed Ca2+-cycling associated with depressed contractile parameters [19]. Furthermore, ectopic expression of phospholamban in mouse fast-twitch skeletal muscle was associated with a decrease in the affinity of SERCA1a for Ca2+ and decreased maximal rates of relaxation of the muscle [20], suggesting that phospholamban is capable of interacting with SERCA1 in vivo. Thus, phospholamban is a major regulator of both SERCA1a and SERCA2a in vivo and as such, it is a key determinant of basal contractile parameters. Interestingly, phospholamban-knockout hearts exhibit such an enhanced basal state, that β-agonist stimulation can only minimally stimulate their hyperdynamic contractility [14].

Previous studies have indicated that overexpression of SERCA2a, or downregulation of phospholamban, may enhance SR Ca2+-uptake and cardiac contractile function in animal models or end-stage failing hearts [21–24]. Moreover, overexpression of SERCA2a by adenovirus, in vivo, improved not only systolic and diastolic performance in failing hearts but also survival and cardiac energetics [25]. Thus, it was of special interest to determine whether: (a) a combination of SERCA1a overexpression, which is associated with greater enhancement of cardiac function than SERCA2a overexpression, and phospholamban ablation, may lead to a greater hyperdynamic cardiac state, than each intervention alone; and (b) the long term effects of such hyperdynamic function may be detrimental to myocyte structure and function. To achieve this, the SERCA1a overexpressing mouse was mated with the phospholamban knockout mouse and the effects of the two genetic manipulations on cardiac function were examined at 3 months and 12 months of age. Our findings indicate that a combination of SERCA1a overexpression with phospholamban ablation results in further enhancement of contractile parameters, than each intervention alone, and the hyperdynamic cardiac function is maintained over the long term.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 Acknowledgments
 References
 
This study was approved by the ethics committee of the University of Cincinnati. The investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the US National Institute of Health (NIH Publication No.85-23, revised, 1996), and the Responsible Care and Use of Animal in Research and Education manual published by the University of Cincinnati. Inbred FVB/N and Svj129/CF1 mice were used.

2.1 Mouse models
Phospholamban knockout (PLBKO) mice in the Svj129/CF1 background were backcrossed with FVB/N wild types for six generations in order to transfer phospholamban deficiency from the Svj129/CF1 background to the FVB/N background. Phospholamban knockout heterozygotes were then crossed to obtain phospholamban-homozygotes in the FVB/N background. SERCA1a overexpression (SERCA1OE) mice (FVB/N background) were mated with PLBKO mice (FVB/N background) to acquire heterozygous phospholamban offspring carrying the SERCA1a transgene, which were then backcrossed with PLBKO to obtain SERCA1a overexpression transgenic mice deficient in phospholamban (SERCA1OE/PLBKO). Genomic DNA was isolated from mouse-tail clips and mice with SERCA1a overexpression and phospholamban deficiency were identified using PCR [6,14].

2.2 Sarcoplasmic reticulum enriched-microsomal protein analysis
SR-enriched microsomes were prepared from mouse hearts [19]. Equal amounts of SERCA1OE, SERCA1OE/PLBKO and wild type microsomal proteins were separated in SDS–PAGE and stained with Coomassie blue. The identity of the SERCA band was confirmed by Western blot analysis with SERCA2a-specific polyclonal antibody or a monoclonal SERCA1a-specific antibody, A-52. The stained gels were scanned and analyzed [6]. Three different preparations of SERCA1OE, SERCA1OE/PLBKO and wild type microsomes were used, and at least three separate gels were obtained per preparation. The total amount of SERCA protein in SERCA1OE/PLBKO hearts was determined by standard protein curves.

2.3 Quantitative immunoblotting
Hearts from wild-type, SERCA1a overexpression, phospholamban-knockout and SERCA1OE/PLBKO mice were homogenized and subjected to quantitative immunoblotting with specific primary monoclonal antibodies to phospholamban, SERCA1a and calsequestrin (Affinity Bioreagents, Golden, CO, USA) and polyclonal antibody to SERCA2a [26]. An alkaline phosphatase-conjugated anti-mouse (phospholamban and SERCA1a) or anti-rabbit (SERCA2a and calsequestrin) secondary antibodies (Cappel Division of Organon Teknika) were then used according to the standard procedures [26].

2.4 Isolation of mouse left ventricular myocytes and measurements of cell shortening and Ca2+ transients
Isolation of mouse left ventricular myocytes was carried out as described previously [27]. Cell shortening and Ca2+ transients were measured separately from cardiomyocytes at room temperature. Briefly, mouse hearts were excised from anesthetized (pentobarbital sodium, 70 mg/kg, i.p.) adult mice, mounted in a Langendorff perfusion apparatus, and perfused with Ca2+-free Tyrode solution at 37 °C for 3 min. The normal Tyrode solution contained 140 mM NaCl, 4 mM KCl, 1 mM MgCl2, 10 mM glucose, and 5 mM HEPES, pH 7.4. Perfusion was then switched to the same solution containing 75 units/ml type 1 collagenase (Worthington), and perfusion continued until the heart became flaccid (~10–15 min). The left ventricular tissue was excised, minced, pipette-dissociated, and filtered through a 240-µm screen. The cell suspension was then sequentially washed in 25, 100, 200 µm and 1 mM Ca2+-Tyrode and resuspended in 1.8 mM Ca2+-Tyrode for further analysis. To obtain intracellular Ca2+ signals, cells were incubated with the acetoxymethyl ester form of fura-2 (Fura-2/AM; 2 µM) for 30 min at room temperature and resuspended in 1.8 mM Ca2+-Tyrode solution. The myocyte suspension was placed in a Plexiglas chamber, which was positioned on the stage of an inverted epifluorescence microscope (Nikon Diaphot 200), and perfused with 1.8 mM Ca2+-Tyrode solution at room temperature (22–23 °C). Myocyte contraction was field-stimulated by a Grass S5 stimulator (0.5 Hz, square waves), and contractions were videotaped and digitized on a computer. A video edge motion detector (Crescent Electronics) was used to measure myocyte length and cell shortening, from which the percent fractional shortening {[(resting cell length-maximal cell shortening length)/resting cell length]x100} and maximal rates of contraction and relaxation (±dL/dt) were calculated [28]. For Ca2+ signal measurements, the cells were alternately excited at 340 and 380 nm by a Delta Scan dual-beam spectrophotofluorometer (Photon Technology). Ca2+ transients were recorded as the 340:380 nm ratio of the resulting 510-nm emissions. Baseline and amplitude, estimated by the 340:380 nm ratio, and the times for 50 and 80% decay of the Ca2+ signal were acquired. All data were analyzed using software from FELIX and IONWIZARD. In some experiments, 2.5 mM Ca2+-Tyrode was used, as indicated in the text.

2.5 Calcium uptake analysis
Oxalate-supported Ca2+ uptake measurements in cardiac homogenates were performed as described [14] by a modified Millipore filtration technique. Briefly, frozen cardiac powder was homogenized in 50 mM potassium phosphate (pH 7.0), 10 mM NaF, 1 mM EDTA, 0.3 M sucrose, 0.3 M phenylmethylsulfonyl fluoride, and 0.5 mM dithiothreitol. Ca2+ uptake in tissue homogenates (0.1 mg/ml) was measured by the Millipore filtration technique. The reaction mixture contained (in mM): imidazole 40 (pH 7.0), KCl 100, MgCl2 5, NaN3 5, potassium oxalate 5, EGTA 0.5 and various concentrations of CaCl2 to yield 0.03–3 µM free Ca2+ (containing 1 µCi/µM 45Ca2+) as determined by a computer program. The reaction was initiated by the addition of 5 mM ATP. The rates of Ca2+ uptake were calculated by least squares linear regression analysis of uptake values at 30, 60 and 90 s. The results were analyzed using PRIZM software.

2.6 Morphological studies
Histological evaluation was performed on the hearts of 12-month-old mice [28]. The tissue was fixed in 10% formalin, dehydrated through graded alcohols, and embedded in paraffin. Longitudinal sections (5 µm) of the heart (cut at 50-µm intervals) were stained with hematoxylin and eosin.

2.7 Statistics
Results are expressed as mean±S.E. The number (n) of mice used is indicated. Comparisons were made between wild type, SERCA1a overexpression, phospholamban knockout and SERCA1OE/PLBKO mice, using the Student's t test. Values of P<0.05 are considered statistically significant.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 Acknowledgments
 References
 
3.1 Phospholamban ablation in the FVB/N background
The phospholamban-knockout mouse was originally generated in the Svj129/CF1 genetic background and previous characterization studies have been done using that model. However, it was important to transfer the phospholamban-deficient alleles to another genetic background, and determine whether the hyperdynamic cardiac function is still present. Thus, we chose the FVB/N mouse strain, since it is commonly used for transgenesis to facilitate studies on the role of phospholamban ablation in various myopathies, based on crosses of the phospholamban deficient mouse with the various transgenic models, exhibiting depressed cardiac function and remodeling. In our studies, the phospholamban knockout mice were backcrossed with FVB/N wild type mice for six generations and phospholamban heterozygotes were then bred to homozygosity. Characterization studies at 10 weeks of age, indicated that the EC50 of SR Ca2+-uptake for Ca2+ was decreased to 0.11±0.01 µM compared to 0.23±0.02 µM in wild types. Analysis of myocyte mechanics revealed increases of 1.9-fold in percent fractional shortening, 2- and 2.5-fold in the maximal rates of contraction (+dL/dt) and relaxation (–dL/dt), respectively, and 48% decreases in the time for 50% decay (t50) of the Ca2+-signal. Echocardiography also revealed an increase of 30% in Vcfc and a shortening of 20% in ejection time. These findings indicate that the increases in cardiac contractile parameters upon phospholamban ablation in the FVB/N background are similar to those previously reported in the Svj129/CF1 background [14,15,29].

3.2 Model with SERCA1a overexpression and phospholamban ablation
Phospholamban deficient mice (FVB/N) were mated with mice expressing SERCA1a in the heart (FVB/N) to generate the SERCA1OE/PLBKO model. To determine the expression levels of SERCA, SR-enriched cardiac microsomes were isolated and subjected to SDS–PAGE followed by Coomassie blue staining. The ~110-kDa protein band, which was confirmed to be SERCA by Western blots, was increased by 1.8-fold in cardiac microsomes from SERCA1OE/PLBKO mice (n=3), when compared to wild type microsomes (n=3) (Fig. 1). Thus, ectopic expression of SERCA1a in the phospholamban-null background resulted in a lower (~1.8-fold) increase in the total SERCA protein compared to its overexpression (~2.5-fold) in the wild type background [6].


Figure 1
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  		Fig. 1 SERCA levels in wild type (WT), SERCA1a overexpressing (SERCA1OE) and SERCA1OE/PLBKO hearts. Increasing amounts of SR enriched microsomal proteins from wild type (WT, n=3), SERCA1OE (n=3) and SERCA1OE/PLBKO (n=3) hearts were separated by 6% SDS–PAGE. (A) Coomassie blue staining of representative gels. The order of loading the samples was reversed for ascending concentrations in the SERCA1OE/PLBKO to facilitate comparison between the two groups. (B) Quantitation of total SERCA protein levels. Values are mean±S.E.; M, protein molecular weight standard. *, P<0.05, versus WT, #, P<0.05, versus SERCA1OE.

 
Quantitative immunoblotting was then employed to determine the protein expression levels of SERCA1a and SERCA2a as well as phospholamban and calsequestrin. The SERCA1a level in the SERCA1OE/PLBKO (n=4) hearts was decreased to 65% of the levels present in the SERCA1a overexpression model (n=3). However, the levels of SERCA2a, which were reduced upon SERCA1a overexpression in the wild type background, remained similarly reduced in the phospholamban-deficient background (Fig. 2B). No phospholamban was found in the phospholamban knockout (n=4) and SERCA1OE/PLBKO hearts. In addition, the levels of calsequestrin were not altered in these hearts (Fig. 2A).


Figure 2
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  		Fig. 2 Representative quantitative immunoblots. Cardiac homogenates from wild type (WT, n=3), SERCA1a overexpressing (SERCA1OE, n=3), phospholamban knockout (PLBKO, n=4) and SERCA1OE/PLBKO (n=4) mice were subjected to 13% SDS–PAGE and electroblotted on nitrocellulose membranes. The membranes were probed with phospholamban (PLB), SERCA1a, SERCA2a and calsequestrin (CSQ)-specific antibodies. Pooled wild-type mouse cardiac homogenates were used as standard (5, 10, 15 and 20 µg) to generate a regression line for quantification of the protein expression levels. (A) Representative immunoblots. (B) Quantitation of SERCA1a and SERCA2a relative protein levels. Values are mean±S.E.; *, P<0.05, versus OE (B, left) or WT (B, right).

 
3.3 Sarcoplasmic reticulum Ca2+ uptake
The initial rates of SR calcium uptake were assessed at various calcium concentrations, similar to those present in the heart during relaxation and contraction. The curve representing the rates in SERCA1OE/PLBKO hearts (n=5) was shifted to the left, when compared with wild types (n=5) and SERCA1a overexpressors (n=4), indicating that the apparent Ca2+ affinity of SERCA was increased (Fig. 3). The EC50 value for Ca2+ dependence of Ca2+ uptake in SERCA1OE/PLBKO mice was decreased to 0.12±0.013 µM, similar to previous findings in the PLBKO (n=4), while this value was 0.23±0.02 µM (P<0.01) in wild types and 0.26±0.02 µM (P<0.01) in SERCA1a overexpressors.


Figure 3
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  		Fig. 3 SR Ca2+ transport. Hearts from wild type (WT, n=5), phospholamban knockout (PLBKO, n=4), SERCA1a overexpressing (SERCA1OE, n=4), and SERCA1OE/PLBKO (, n=5) mice were homogenized, and the initial rates of oxalate-supported SR Ca2+ uptake were measured. The Vmax values (nmol/mg) were: 86.5±8.6 in SERCA1OE/PLBKO mice; 92.4±7.9 in SERCA1a overexpressors; 61.6±2.7 in phospholamban knockouts; and 55.7±2.9 in WTs. Values are mean±S.E.; n, number of experiments. Each experiments was performed in triplicate using pooled hearts.

 
3.4 Myocyte contractions and Ca2+ transients
To determine whether the hearts with SERCA1a overexpression and phospholamban deficiency may exhibit greater enhancement of myocyte contractility and intracellular calcium kinetics, compared to either phospholamban ablation or SERCA1a overexpression, left ventricular myocytes were isolated from wild type (n=6), SERCA1a overexpression (n=5), phospholamban knockout (n=5) and SERCA1OE/PLBKO (n=8) mice, and were studied in parallel (Fig. 4, Table 1). The data indicated that in SERCA1OE/PLBKO cells: (a) +dL/dt increased to ~2.6-fold of wild types, ~2.0-fold of SERCA1a overexpressors and ~1.3-fold of phospholamban knockouts; (b) –dL/dt increased to ~3.2-fold of wild types, ~2.0-fold of SERCA1a overexpressors and ~1.2-fold of phospholamban knockouts; (c) percent fractional shortening increased to ~1.9-fold of wild types and ~1.3-fold of SERCA1a overexpressors. Furthermore, the amplitude of the Ca2+ transient was increased by ~1.8-fold compared to wild-types, ~1.4-fold compared to SERCA1a overexpressors, and ~1.13-fold compared to phospholamban knockouts. The time for 80% decay of the Ca2+-transient (t80) was also decreased by ~3.1-fold compared to wild types, ~2.3-fold compared to SERCA1a overexpressors, and ~1.3-fold compared to phospholamban knockouts. There was no significant difference in the cell percent fractional shortening between phospholamban knockout and the SERCA1OE/PLBKO mice observed, when the extracellular Ca2+ concentration was 1.8 mM (Table 1). Interestingly, when the extracellular Ca2+ concentration was increased from 1.8 to 2.5 mM, which is considered sufficient to induce maximal myofilament activation [30], the percent fractional shortening showed ~1.3-fold increase in the SERCA1OE/PLBKO myocytes (n=4) compared to phospholamban knockouts (n=3), indicating that the increased SERCA levels and activity in the SERCA1OE/PLBKO myocytes were able to handle higher calcium than the phospholamban knockouts. However, the increases in +dL/dt (~1.3-fold of phospholamban knockout) and –dL/dt (~1.3-fold of phospholamban knockout mice) were similar to those observed in 1.8 mM of extracellular Ca2+ concentration (Fig. 5A and B). These data suggest that the cardiac myocytes from SERCA1OE/PLBKO mice exhibit a hyperdynamic state, which exceeds that of either the phospholamban knockouts or the SERCA1a overexpressors.


Figure 4
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  		Fig. 4 Representative traces of cardiac myocyte mechanics and calcium transients. (A) Myocyte percent fractional shortening (FS%); (B) Maximal rates of contraction and relaxation (±dL/dt); (C) Calcium transients indicated by the fura-2 ratio (340:380 nm). The perfusate calcium was 1.8 mM. WT, wild-type; SERCA1OE, SERCA1a overexpression; PLBKO, phospholamban-knockout; SERCA1OE/PLBKO, SERCA1a overexpression in phospholamban deficient background.

 

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  		Table 1 Mechanical parameters and calcium transients of isolated cardiomyocytes from wild type, SERCA1a overexpressing, phospholamban knockout and SERCA1a overexpressing/PLB knockout mice at 1.8 mM calcium in the perfusate

 

Figure 5
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  		Fig. 5 Mechanical parameters of isolated cardiac myocytes. Isolated cardiomyocytes from phospholamban knockout (PLBKO, n=3) and SERCA1OE/PLBKO (n=4) hearts were paced at 0.5 Hz, with extracellular calcium at 2.5 mM. (A) Representative traces of myocyte percent fractional shortening (FS,%) and maximal rates of contraction and relaxation (±dL/dt); (B) Myocyte percent fractional shortening (FS, %) and±dL/dt. Values are mean±S.E. At least five cells per heart were used for triplicate measurements, and each animal was considered as a single n. *, P<0.05, versus KO.

 
3.5 Long term effects of cardiac hyperdynamic function
To further examine whether the cardiac hyperdynamic state of the SERCA1OE/PLBKO mice can be maintained in the long term, or whether it becomes detrimental to the mouse, we examined cardiomyocyte contractile parameters, cardiac morphology and heart to body weight ratios in 10–12 month old mice. There were no significant differences in percent fractional shortening (15.6±0.7 and 15.9±1.2), +dL/dt (329.5±12.3 and 325.1±15.2) and –dL/dt (278.3±7.1 and 278.2±9.6) between 3-month-old (n=8) and 10–12-month-old (n=7) SERCA1OE/PLBKO cardiac myocytes (P0.05). Furthermore, microscopic examination of ventricles, atria, and valves revealed no significant differences in gross morphology among the hearts from 12-month-old SERCA1OE/PLBKO (n=4), wild type (n=5), SERCA1a overexpressing (n=5) and phospholamban knockout (n=5) mice. At low (x10) magnification (Fig. 6A), characteristics which were similar in wild type, SERCA1a overexpressing, phospholamban knockout and SERCA1OE/PLBKO mice, included atrial wall thickness, atrial diameter, ventricular free wall and septal thicknesses, valve position, morphology and heart size. At higher (x40) magnification (Fig. 6B), the myocytes exhibited similar characteristics of cytoplasmic density, nuclear size and chromatin pattern, myocyte size, myocyte density, cell orientation, and striations. These results indicate that the hyperdynamic hearts of the SERCA1OE/PLBKO mice do not show significant morphological alterations up to 12 months of age, compared to wild type mice. Furthermore, the heart weight/body weight ratios between 3 and 12 months were not significantly different in the four groups (P0.05) (Fig. 6C).


Figure 6
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  		Fig. 6 Cardiac morphology and heart to body weight ratios. 12-month-old mouse hearts from wild-type (WT, n=5), SERCA1a overexpressing (SERCA1OE, n=5), phospholamban knockout (PLBKO, n=5), and SERCA1OE/PLBKO (n=4) mice were fixed overnight in 10% formalin and processed for morphological evaluation, as described in Methods. (A) Low magnification (x10); (B) high magnification (x40); (C) Heart weight:body weight (mg/g) at 3 months ({square}) and 12 months ({blacksquare}) in the four groups. Values are mean±S.E.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
Acknowledgments
 References
 
This study indicates that the hyperdynamic cardiac function of phospholamban knockout or SERCA1a overexpressing hearts can be further enhanced by combination of both genetic manipulations. In a previous study [6], it was shown that the fast-twitch skeletal muscle SR Ca2+-ATPase (SERCA1a) gene can be expressed in the mouse heart and the total SERCA level can increase to ~2.5-fold of that in wild types, resulting in enhanced cardiac cell contractility and Ca2+ transients. In that model, SERCA2a was downregulated to 50% of wild types, and the relative levels of SERCA2a and SERCA1a were 20 and 80%, respectively. In the present study, when the SERCA1a overexpressing mice were mated with the phospholamban deficient mice, the SERCA2a was downregulated to 50% of wild type levels, similar to previous observations [6], but the total SERCA level was only 1.8-fold higher than wild types. Thus, the relative levels of SERCA2a to SERCA1a were about 30 to 70%, respectively. The reason for the relative decrease in SERCA1a levels in the SERCA1OE/PLBKO model is not clear at present, and the mechanisms regulating SERCA pump synthesis, integration and turnover need further investigation. However, it is interesting to speculate that the new hyperdynamic state of the heart, associated with SERCA1a overexpression and phospholamban ablation, provided a signal for the reduced levels of SERCA1a molecules in the SERCA1OE/PLBKO model.

The role of phospholamban in the regulation of the SERCA2a activity and myocardial contractility has been clearly established through the generation and characterization of genetically altered mouse models [14,15,26]. Specifically, the relative stoichiometry of phospholamban-to-SERCA ratio in the range of 0.4–2 relative to wild type (1.0), was shown to linearly correlate with alterations in SERCA's Ca2+-affinity [15]. However, either increases (2.5-fold) or decreases (35%) in SERCA levels, corresponding to phospholamban-to-SERCA ratios of 0.4 or 1.0, did not alter SERCA's Ca2+-affinity [6,31]. Thus, changing the relative stoichiometry of phospholamban-to-SERCA by phospholamban differs from changing it by SERCA. These apparently distinct effects may be due to alterations in the fraction of ‘active phospholamban species’ or monomers. It is possible that alterations in phospholamban levels are not associated with any changes in the equilibrium between phospholamban monomers and pentamers, while increases in SERCA levels favor the phospholamban monomeric state, which is a more effective inhibitor of SERCA [32]. Interestingly, ablation of phospholamban resulted in similar decreases in the EC50 values regardless of the levels of SERCA expressed in the heart (1.0 in wild types and 1.8 in SERCA1OE/PLBKO). These findings point to the complex nature of the phospholamban inhibitory effects on SERCA.

In the SERCA1OE/PLBKO mouse model, the maximal rates of contraction and relaxation in isolated myocytes were significantly increased compared to either SERCA1a overexpressors or phospholamban knockouts. Phospholamban ablation has been previously shown to result in enhanced basal myocyte mechanics and Ca2+ kinetics [27,29]. Phospholamban deficient myocytes also exhibited a higher apparent Ca2+ affinity of SR Ca2+ uptake (54%) [14] and SR Ca2+ load (37%) [27,33]. The hyperdynamic cardiac contractile function of the phospholamban knockout mouse was also observed in the intact organ [14] and animal levels [16,17]. On the other hand, ectopic expression of SERCA1a in the mouse heart resulted in increased SR Ca2+ transport and enhanced maximal rates of contraction and relaxation [6]. Addition of a phospholamban antibody to the SR Ca2+-uptake assays increased the apparent Ca2+ affinity by 2.4-fold in the SERCA1a overexpressing hearts [9]. Since 80% of the total SERCA pumps are represented by SERCA1a in this model, the observed shift in the Ca2+ affinity is largely determined by SERCA1a, suggesting that phospholamban can interact with SERCA1a and inhibit it, when expressed in the heart. Furthermore, ectopic phospholamban expression in the mouse fast twitch skeletal muscle also presented evidence that SERCA1a can be regulated by phospholamban in vivo, resulting in depressed maximal rates of muscle relaxation [20]. Indeed, the major binding site for phospholamban, identified in SERCA2a (Asp370 through Lys400) is highly conserved between the SERCA1 and SERCA2 isoforms [10]. Taken together, these studies suggest that both SERCA1a and SERCA2a are subject to regulation by phospholamban. Therefore, ablation of phospholamban in the SERCA1a overexpression model may result in further increases in basal cardiac contractile function, as observed in the present study.

It is of special interest that in the presence of 1.8 mM extracellular Ca2+, the myocyte percent fractional shortening was similar between phospholamban knockout and SERCA1OE/PLBKO mice, but when the extracellular Ca2+ was increased to 2.5 mM, which is considered sufficient to induce maximal myofilament activation [30], the percent fractional shortening in SERCA1OE/PLBKO myocytes was significantly enhanced, compared to phospholamban knockouts. This indicated that the increased SERCA levels in the SERCA1OE/PLBKO myocytes were able to handle higher calcium than the phospholamban knockouts. Thus, the myocyte contractility can be further enhanced by a combination of increases in the Ca2+ affinity and Vmax of the SR Ca2+-transport system, elicited by phospholamban ablation [14] and SERCA1a overexpression [6], respectively.

To determine whether the hyperdynamic phenotype may persist over the long term or whether compensatory changes may develop in the myocardium of the SERCA1OE/PLBKO mice, heart function and morphology were assessed at 10–12 months of age. Previous studies have shown that either PLB ablation or SERCA1 overexpression does not result in any cardiac pathology or hypertrophy in the aging mouse [18,34]. Importantly, the present findings also indicate that the combination of phospholamban ablation and SERCA1a overexpression was not associated with any significant morphological alterations at the gross anatomic level and the heart to body weight ratio was maintained up to 10–12 months of age. Furthermore, the hyperdynamic maximal rates of contraction and relaxation in the SERCA1OE/PLBKO cardiac myocytes were not diminished at 10–12 months of age. Thus, overexpression of SERCA1a in the phospholamban null background was not detrimental to cardiac performance and did not result in cardiac remodeling leading to hypertrophy.

In summary, the combination of SERCA1a overexpression with phospholamban ablation creates a new cardiac hyperdynamic state, which is maintained over the long term without any apparent cardiac remodeling. The SERCA1OE/PLBKO model may provide a unique system to further: (a) investigate the mechanisms of interaction between SERCA1a and SERCA2a in the absence of their negative regulator, phospholamban; and (b) gain new insights into combination of therapeutic approaches to target both SERCA, which is downregulated, and phospholamban, which is dephosphorylated, in heart failure.

Time for primary review 31 days.


   Acknowledgments
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Acknowledgments
References
 
This work was supported by National Institutes of Health Grants HL-26057, HL-64018, RR12358, and HL-52318 (EGK). We are grateful to Dr. Donald M. Bers and Dr. Gerald W. Dorn II for excellent suggestions regarding this work.


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Acknowledgments
 References
 

  1. Brandl C.J, deLeon S, Martin D.R, MacLennan D.H. Adult forms of the Ca2+ATPase of sarcoplasmic reticulum. Expression in developing skeletal muscle. J. Biol. Chem. (1987) 262:3768–3774.[Abstract/Free Full Text]
  2. Zarain-Herzberg A, MacLennan D.H, Periasamy M. Characterization of rabbit cardiac sarco(endo)plasmic reticulum Ca2+- ATPase gene. J. Biol. Chem. (1990) 265:4670–4677.[Abstract/Free Full Text]
  3. Brandl C.J, Green N.M, Korczak B, MacLennan D.H. Two Ca2+ ATPase genes: homologies and mechanistic implications of deduced amino acid sequences. Cell (1986) 44:597–607.[CrossRef][ISI][Medline]
  4. Inesi G, Lewis D, Sumbilla C, et al. Cell-specific promoter in adenovirus vector for transgenic expression of SERCA1 ATPase in cardiac myocytes. Am. J. Physiol. (1998) 274:C645–653.[ISI][Medline]
  5. Sumbilla C, Cavagna M, Zhong L, et al. Comparison of SERCA1 and SERCA2a expressed in COS-1 cells and cardiac myocytes. Am. J. Physiol. (1999) 277:H2381–2391.[ISI][Medline]
  6. Loukianov E, Ji Y, Grupp I.L, et al. Enhanced myocardial contractility and increased Ca2+ transport function in transgenic hearts expressing the fast-twitch skeletal muscle sarcoplasmic reticulum Ca2+-ATPase [see comments]. Circ Res (1998) 83:889–897.[Abstract/Free Full Text]
  7. He H, Giordano F.J, Hilal-Dandan R, et al. Overexpression of the rat sarcoplasmic reticulum Ca2+ ATPase gene in the heart of transgenic mice accelerates calcium transients and cardiac relaxation. J Clin Invest (1997) 100:380–389.[ISI][Medline]
  8. Baker D.L, Hashimoto K, Grupp I.L, et al. Targeted overexpression of the sarcoplasmic reticulum Ca2+-ATPase increases cardiac contractility in transgenic mouse hearts. Circ Res (1998) 83:1205–1214.[Abstract/Free Full Text]
  9. Ji Y, Loukianov E, Loukianova T, Jones L.R, Periasamy M. SERCA1a can functionally substitute for SERCA2a in the heart. Am J Physiol (1999) 276:H89–97.[ISI][Medline]
  10. James P, Inui M, Tada M, Chiesi M, Carafoli E. Nature and site of phospholamban regulation of the Ca2+ pump of sarcoplasmic reticulum. Nature (1989) 342:90–92.[CrossRef][Medline]
  11. Kim H.W, Steenaart N.A, Ferguson D.G, Kranias E.G. Functional reconstitution of the cardiac sarcoplasmic reticulum Ca2+-ATPase with phospholamban in phospholipid vesicles. J Biol Chem (1990) 265:1702–1709.[Abstract/Free Full Text]
  12. Kranias E.G. Regulation of Ca2+ transport by cyclic 3',5'-AMP-dependent and calcium–calmodulin-dependent phosphorylation of cardiac sarcoplasmic reticulum. Biochim Biophys Acta (1985) 844:193–199.[Medline]
  13. Tada M, Inui M, Yamada M, et al. Effects of phospholamban phosphorylation catalyzed by adenosine 3':5'-monophosphate- and calmodulin-dependent protein kinases on calcium transport ATPase of cardiac sarcoplasmic reticulum. J Mol Cell Cardiol (1983) 15:335–346.[CrossRef][ISI][Medline]
  14. Luo W, Grupp I.L, Harrer J, et al. Targeted ablation of the phospholamban gene is associated with markedly enhanced myocardial contractility and loss of β-agonist stimulation. Circ Res (1994) 75:401–409.[Abstract/Free Full Text]
  15. Luo W, Wolska B.M, Grupp I.L, et al. Phospholamban gene dosage effects in the mammalian heart. Circ Res (1996) 78:839–847.[Abstract/Free Full Text]
  16. Hoit B.D, Khy S.F, Kranias E.G, Ball N, Walsh R.A. In vivo echocardiographic detection of enhanced left ventricular function in gene-targeted mice with phospholamban deficiency. Circ Res (1995) 77:632–637.[Abstract/Free Full Text]
  17. Lorenz J.N, Kranias E.G. Regulatory effects of phospholamban on cardiac function in intact mice. Am J Physiol (1997) 273:H2826–2831.[ISI][Medline]
  18. Slack J.P, Grupp I.L, Dash R, et al. The enhanced contractility of the phospholamban-deficient mouse heart persists with aging. J Mol Cell Cardiol (2001) 33:1031–1040.[CrossRef][ISI][Medline]
  19. Kadambi V.J, Ponniah S, Harrer J.M, et al. Cardiac-specific overexpression of phospholamban alters calcium kinetics and resultant cardiomyocyte mechanics in transgenic mice. J Clin Invest (1996) 97:533–539.[ISI][Medline]
  20. Slack J.P, Grupp I.L, Ferguson D.G, Rosenthal N, Kranias E.G. Ectopic expression of phospholamban in fast-twitch skeletal muscle alters sarcoplasmic reticulum Ca2+ transport and muscle relaxation. J Biol Chem (1997) 272:18862–18868.[Abstract/Free Full Text]
  21. Minamisawa S, Hoshijima M, Chu G, et al. Chronic phospholamban–sarcoplasmic reticulum calcium ATPase interaction is the critical calcium cycling defect in dilated cardiomyopathy. Cell (1999) 99:313–322.[CrossRef][ISI][Medline]
  22. Davia K, Bernobich E, Ranu H.K, et al. SERCA2A overexpression decreases the incidence of aftercontractions in adult rabbit ventricular myocytes. J Mol Cell Cardiol (2001) 33:1005–1015.[CrossRef][ISI][Medline]
  23. Eizema K, Fechner H, Bezstarosti K, et al. Adenovirus-based phospholamban antisense expression as a novel approach to improve cardiac contractile dysfunction: comparison of a constitutive viral versus an endothelin-1-responsive cardiac promoter. Circulation (2000) 101:2193–2199.[Abstract/Free Full Text]
  24. del Monte F, Harding S.E, Dec G.W, Gwathmey J.K, Hajjar R.J. Targeting phospholamban by gene transfer in human heart failure. Circulation (2002) 105:904–907.[Abstract/Free Full Text]
  25. del Monte F, Hajjar R.J, Harding S.E. Overwhelming evidence of the beneficial effects of SERCA gene transfer in heart failure. Circ Res (2001) 88:E66–67.[ISI][Medline]
  26. Chu G, Luo W, Slack J.P, et al. Compensatory mechanisms associated with the hyperdynamic function of phospholamban-deficient mouse hearts. Circ Res (1996) 79:1064–1076.[Abstract/Free Full Text]
  27. Li L, Chu G, Kranias E.G, Bers D.M. Cardiac myocyte calcium transport in phospholamban knockout mouse: relaxation and endogenous CaMKII effects. Am J Physiol (1998) 274:H1335–1347.[ISI][Medline]
  28. Chu G, Lester J.W, Young K.B, Luo W, Zhai J, Kranias E.G. A single site (Ser16) phosphorylation in phospholamban is sufficient in mediating its maximal cardiac responses to β-agonists [In Process Citation]. J Biol Chem (2000) 275:38938–38943.[Abstract/Free Full Text]
  29. Wolska B.M, Stojanovic M.O, Luo W, Kranias E.G, Solaro R.J. Effect of ablation of phospholamban on dynamics of cardiac myocyte contraction and intracellular Ca2+. Am J Physiol (1996) 271:C391–397.[ISI][Medline]
  30. Hampton T.G, Amende I, Travers K.E, Morgan J.P. Intracellular calcium dynamics in mouse model of myocardial stunning. Am J Physiol (1998) 274:H1821–1827.[ISI][Medline]
  31. Ji Y, Lalli M.J, Babu G.J, et al. Disruption of a single copy of the SERCA2 gene results in altered Ca2+ homeostasis and cardiomyocyte function. J Biol Chem (2000) 275:38073–38080.[Abstract/Free Full Text]
  32. Kimura Y, Kurzydlowski K, Tada M, MacLennan D.H. Phospholamban inhibitory function is activated by depolymerization. J Biol Chem (1997) 272:15061–15064.[Abstract/Free Full Text]
  33. Santana L.F, Kranias E.G, Lederer W.J. Calcium sparks and excitation–contraction coupling in phospholamban-deficient mouse ventricular myocytes. J Physiol (1997) 503:21–29.[Abstract/Free Full Text]
  34. Jane Lalli M, Yong J, Prasad V, et al. Sarcoplasmic reticulum Ca2+ ATPase (serca) 1a structurally substitutes for serca2a in the cardiac sarcoplasmic reticulum and increases cardiac Ca2+ handling capacity. Circ Res (2001) 89:160–167.[Abstract/Free Full Text]

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