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Am J Physiol Heart Circ Physiol 286: H1146-H1153, 2004. First published November 20, 2003; doi:10.1152/ajpheart.00720.2003
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Accelerated onset of heart failure in mice during pressure overload with chronically decreased SERCA2 calcium pump activity

Jo El J. Schultz,1,2 Betty J. Glascock,3 Sandra A. Witt,3 Michelle L. Nieman,4,5 Kalpana J. Nattamai,6 Lynne H. Liu,6 John N. Lorenz,4,5 Gary E. Shull,1 Thomas R. Kimball,3 and Muthu Periasamy6

1Department of Molecular Genetics, Biochemistry, and Microbiology, 2Department of Pharmacology and Cell Biophysics, 4Department of Molecular and Cellular Physiology; 5Noninvasive Cardiac Imaging and Hemodynamic Research Laboratory, Division of Cardiology, and 3Department of Pediatrics, Children's Hospital Medical Center, Cincinnati 45229; and 6Department of Physiology and Cell Biology, The Ohio State University, Columbus, Ohio 43210

Submitted 28 July 2003 ; accepted in final form 10 November 2003


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
We recently developed a mouse model with a single functional allele of Serca2 (Serca2+/–) that shows impaired cardiac contractility and relaxation without overt heart disease. The goal of this study was to test the hypothesis that chronic reduction in sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA)2 levels in combination with an increased hemodynamic load will result in an accelerated pathway to heart failure. Age-matched wild-type and Serca2+/– mice were subjected to 10 wk of pressure overload via transverse aortic coarctation surgery. Cardiac hypertrophy and heart failure were assessed by echocardiography, gravimetry/histology, hemodynamics, and Western blotting analyses. Our results showed that ~64% of coarcted Serca2+/– mice were in heart failure compared with 0% of coarcted wild-type mice (P < 0.05). Overall, morbidity and mortality were greatly increased in Serca2+/– mice under pressure overload. Echocardiography assessment revealed a significant increase in left ventricular (LV) mass, and LV hypertrophy in coarcted Serca2+/– mice converted from a concentric to an eccentric pattern, similar to that seen in human heart failure. Coarcted Serca2+/– mice had decreased contractile/systolic and relaxation/diastolic performance and/or function compared with coarcted wild-type mice (P < 0.05), despite a similar duration and degree of pressure overload. SERCA2a protein levels were significantly reduced (>50%) in coarcted Serca2+/– mice compared with noncoarcted and coarcted wild-type mice. Our findings suggest that reduction in SERCA2 levels in combination with an increased hemodynamic load results in an accelerated pathway to heart failure.

sarco(endo)plasmic reticulum calcium-adenosine 5'-triphosphatase pump; knockout mice; echocardiography; cardiac dysfunction

SARCO(ENDO)PLASMIC RETICULUM Ca2+-ATPase (SERCA) in the heart plays a pivotal role in the beat-to-beat function of the heart. The SERCA pump promotes muscle relaxation by lowering the cytosolic Ca2+ concentration and through active Ca2+ transport restores the intracellular Ca2+ stores, thus providing Ca2+ needed for the next contraction. Decreases in SERCA pump expression and activity have been observed in a variety of pathological conditions. In particular, defects in the sarcoplasmic reticulum (SR) Ca2+ uptake function have been identified in animal models of heart disease and have been shown to correlate with altered contractile function. It has been shown that the expression level of SERCA and Ca2+ transport is significantly decreased in pressure overload-induced hypertrophy and during heart failure (1, 9, 18, 23, 28, 31). Studies on human hearts, primarily explant hearts, suggest that SR Ca2+ transport function is altered in end-stage human heart failure (2, 11, 12). A decrease in the level of SERCA (mRNA, protein, or activity) was closely correlated with decreased myocardial function and impaired force-frequency response (1, 3, 12, 24, 30). These studies suggest that the SERCA pump level is critical for the maintenance of cardiac function, but the true relationship between SERCA levels and cardiac function is difficult to establish. Interpretation of studies using tissues from end-stage failing hearts is difficult because multiple changes occur within myocytes during the progression into heart failure. Therefore, whether decreased SERCA expression and/or activity is a cause or a consequence of heart failure is under debate.

To understand how decreased SERCA levels affect cardiac physiology, we generated Serca2 knockout mice with genetargeting techniques (29, 16). As expected, the disruption of both copies of Serca2 gene resulted in early embryonic lethality, whereas heterozygous mice (Serca2+/–) with a single functional allele are alive and reproduce well. These mice have provided us with an in vivo model system to study how a decrease in SERCA pump levels affects the contraction-relaxation cycle of the heart. Serca2+/– mice showed decreased SERCA2a protein levels as well as a lower maximal velocity of SR Ca2+ uptake, both reduced by ~35% (16, 29). Analyses of in vivo cardiac function with a Millar catheter revealed decreased maximal rates of contraction and relaxation and lower mean arterial pressure in Serca2+/– mice. Interestingly, the heart weight-to-body weight ratio was normal in Serca2+/– mice. The hearts of Serca2+/– mice did not show any signs of cardiac hypertrophy or fibrosis as analyzed by light microscopy. Despite a reduction in SERCA pump levels, there was no evidence of cardiac pathology in Serca2+/– mice. We therefore reasoned that Serca2+/– hearts were able to meet the functional requirements under normal conditions. However, it was not known whether Serca2+/– hearts would be able to adapt well to a pathological stress such as increased hemodynamic load. In the present study, we tested the hypothesis that chronic reduction in SERCA2 levels in combination with an increased hemodynamic load will result in an accelerated pathway to heart failure. Our results show that heart failure occurs more rapidly with reduced SERCA2 levels in conjunction with pressure overload.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Mice were housed in a specific pathogen-free facility and handled in accordance with standard use protocols and animal welfare regulations. The generation of Serca2+/– mice was described previously (29). All protocols were approved by the University of Cincinnati Institutional Animal Care and Use Committee.

Wild-type and Serca2+/– mice (10–12 wk of age) were randomly assigned to the present study, and 11 wild-type and 11 Serca2+/– mice subjected to aortic coarctation (AC) completed the study, which included echocardiography, blood pressure and left ventricular (LV) measurements, gravimetry/histology, and SERCA2a and calsequestrin (Csq) protein determination. After AC, four Serca2+/– mice died because of heart failure. No mortality was observed in wild-type mice committed to the 10-wk study.

AC surgery. Age-matched wild-type and Serca2+/– mice of either sex weighing 22–26 g were anesthetized intraperitoneally with 2.5% Avertin (0.017 ml/g). A tracheotomy was performed, and the mouse was artificially ventilated with room air (120 breaths/min with a tidal volume of 0.3 ml) with a Harvard Apparatus mouse ventilator (model 687; South Natick, MA). AC surgery was performed as described previously (35), and the surgeon was blinded to the identity of the mice. In brief, a blunt dissection was performed at the second intercostal space to expose the aortic arch. A 7-0 silk suture was placed around the aorta between the left common carotid artery and brachiocephalic trunk. A 27-gauge needle, bent to an L shape, was placed on top of the exposed transverse aorta. Tying the suture around this needle and subsequent removal of the needle produced banding of the aorta. The lungs were reexpanded with positive pressure, and the chest and skin incisions were closed with 5-0 silk and 5-0 Ti-cron, respectively. The trachea incision was closed with 7-0 silk. The mice were monitored daily for 10 wk.

Echocardiography. Echocardiography was performed preoperatively and once a week postoperatively for 10 wk on wild-type and Serca2+/– mice as previously described (35). Intra- and interobserver variability was similar to that previously noted in our laboratory (36), and the sonographers were blinded to the identity of the mice. Two-dimensional and M-mode measurements of LV end-diastolic and end-systolic chamber size, septal wall thickness and LV posterior wall thickness (PWT) in diastole and systole, and R-R interval were made from original tracings, as recommended by the American Society of Echocardiography (33). LV mass was estimated with the cube formula, and cardiac function (fractional shortening, FS) was calculated (35). Changes in diastolic function were assessed by calculating the peak E-to-A ratio and isovolumic relaxation time (IVRT). Peak E (rapid ventricular filling) velocity and peak A (filling during atrial contraction) velocity through the mitral valve were measured from the apical four-chamber view at the tips of the mitral leaflets. Peak E/A is an indicator of LV filling. IVRT was obtained from continuous-wave Doppler of the LV outflow and inflow tracts. IVRT is the time interval from aortic valve closure to mitral valve opening and is a measure of LV relaxation as related to left atrial pressure.

In vivo hemodynamic measurements. Blood pressure measurements and cardiac function were obtained with a Millar catheter as described previously (34, 35). After echocardiographic assessment, age- and sex-matched noncoarcted (NC) and 10-wk AC wild-type and Serca2+/– mice were surgically instrumented as described previously (21). Average values for heart rate, mean arterial blood pressure, systolic blood pressure, and left intraventricular pressure (LVP) were measured directly from the pressure waveforms. Maximum and minimum change in pressure over time (dP/dt) were derived from a differential tracing of LVP. At the end of the experimental protocol, mice were killed, organs including the heart, lungs, kidney, and colon were harvested, and the tissue was weighed and normalized to mouse body weight and tibia length. The tissue was either placed in liquid nitrogen and stored at –80°C for Western blot analysis or placed in 4% paraformaldehyde for histological determination.

Western blot analysis. To determine the protein levels, hearts from AC and NC animals were homogenized in 10 mM imidazole (pH 7.0), 0.3 M sucrose, 0.3 mM phenylmethylsulfonyl fluoride, 1 mM dithiothreitol, and 10 mM sodium fluoride. Protein concentration was determined by the Bradford method with bovine serum albumin for the standard curve. The homogenates were electrophoretically separated on a 10% SDS polyacrylamide gel, blotted onto a 0.22-µm nitrocellulose membrane, and probed with polyclonal antibody raised against SERCA2a (16). In addition, antibodies specific for phospholamban (PLB) and Csq were used to probe the Western blots (polyclonal PLB and anti-Csq antibodies were from Affinity BioReagents, Golden, CO). Csq was used to correct loading variations between samples because it did not vary significantly between control and banded samples. The signal obtained for each sample was quantified by densitometry with a UMAX Astra 1200s scanner and NIH Image software (version 1.62) (16).

Statistical analysis of data. All values are expressed as means ± SE. Tissue weight-to-body weight ratios, in vivo hemodynamic measurements, and SERCA2a and Csq protein levels were compared by one-way ANOVA with Fisher's least-significant difference post hoc test. Differences between echocardiographic data were compared with two-way ANOVA for time and treatment with repeated measures with Fisher's least-significant difference test. Statistical differences were considered significant at P < 0.05.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Gravimetric analysis reveals a heart failure phenotype in Serca2+/– mice after 10 wk of pressure overload. Wild-type and Serca2+/– mice were subjected to 10 wk of pressure overload via transverse AC surgery. Echocardiography was performed at 1-week intervals to follow the development and progression of cardiac hypertrophy and heart failure. Gravimetric, histological, hemodynamic, and molecular measurements were also obtained for age- and sex-matched NC and 10-wk AC wild-type and Serca2+/– mice.

Gravimetric analysis showed that both 10-wk AC wild-type and Serca2+/– mice had a significant increase in whole heart weight- and LV weight-to-body weight ratio compared with NC cohorts (P < 0.05; Fig. 1). Interestingly, there was a further significant increase in whole heart weight- and LV weight-to-body weight ratio in AC Serca2+/– mice compared with AC wild-type mice (P < 0.05; Fig. 1). Heart weights were not significantly different between NC wild-type and Serca2+/– mice, whereas AC animals had a significant increase in heart weight (NC wild type = 128 ± 11 mg; NC Serca2+/– = 127 ± 13 mg; AC wild type = 152 ± 10 mg; AC nonfailing Serca2+/– = 160 ± 14 mg; AC failing Serca2+/– = 159 ± 9 mg). Body weights were not different between NC and AC wild-type and Serca2+/– mice (NC wild type = 31 ± 3 g; NC Serca2+/– = 30 ± 4 g; AC wild type = 30 ± 2 g; AC nonfailing Serca2+/– = 27 ± 2 g; AC failing Serca2+/– = 24 ± 4 g), indicating that the increase in whole heart weight- and LV weight-to-body weight ratio in AC Serca2+/– mice was not the result of diminished murine body weight. Wet lung weight-to-body weight ratio, an experimental measure of pulmonary edema, revealed a heart failure phenotype in AC Serca2+/– mice (P < 0.05; Fig. 1). The heart failure phenotype was determined retrospectively and was based on the criterion of wet lung weight-to-body weight ratio (NC wild type range 5.4–7.1; NC Serca2+/– range 5.6–6.9; 10-wk AC wild type range 4.0–6.3; 10-wk AC Serca2+/– range 5.1–17.7). In fact, the incidence of heart failure was genotype dependent, as 64% of AC Serca2+/– mice had pulmonary edema (i.e., significant increase in wet lung weight-to-body weight ratio) compared with 0% of AC wild-type mice (P < 0.05; Fig. 2). Dry lung weight-to-body weight ratio was not significantly different in NC and AC wild-type and Serca2+/– mice, confirming that the excess wet lung weight was due to edema (data not shown). No mortality occurred in 10-wk AC wild-type mice; however, four AC Serca2+/– mice died between 4 and 8 weeks of pressure overload (i.e., 1 mortality at 4 wk of AC, 2 mortalities at 6 wk of AC, and 1 mortality at 8 wk of AC). Histological assessment of 10-week NC and AC wild-type and Serca2+/– hearts indicated that AC wild-type hearts had mild myocyte hypertrophy with mild perivascular fibrosis whereas AC Serca2+/– hearts had diffuse, moderate myocyte hypertrophy and moderate multifocal fibrosis (data not shown).



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  		Fig. 1. Tissue weight-to-body-weight (BW) ratios for age- and sex-matched noncoarcted (NC) and 10-wk aortic coarctation (AC) wild-type and Serca2+/– mice. Mice were subjected to sham surgery or AC for 10 wk to induce a chronic pressure overload. Tissues were harvested to identify mice that underwent cardiac hypertrophy and/or had signs of pulmonary edema, an indication of heart failure. Values are expressed as means ± SE; n = 4 for NC wild-type mice, 4 for NC Serca2+/– mice, 11 for AC wild-type mice, and 7 for AC Serca2+/– mice. Four AC Serca2+/– mice died during the course of the study and were not included in the tissue weight analysis. LV, left ventricle.

 


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  		Fig. 2. Percentage of Serca2+/– mice in failure (based on observed pulmonary edema from lung weight measurements) during chronic pressure overload. No wild-type mice went into failure during the course of the study.

 

Echocardiographic assessment demonstrates LV hypertrophy and systolic and diastolic dysfunction in Serca2+/– mice after pressure overload. Echocardiography showed that AC wild-type and nonfailing Serca2+/– mice had similar increases in LV mass; however, the increase in LV mass was significantly greater for failing Serca2+/– mice compared with AC wild-type mice at 5 and 10 wk of AC (P < 0.05; Fig. 3). There were no differences in LV mass between wild-type and Serca2+/– mice before AC. No significant LV chamber dilation occurred in AC wild-type and nonfailing Serca2+/– mice (Table 1). However, there was a significant increase in LV chamber dilation and PWT in AC failing Serca2+/– mice (P < 0.05; Table 1), demonstrating an eccentric, dilated form of hypertrophy. A significant increase in PWT in AC wild-type and nonfailing Serca2+/– mice was also observed (P < 0.05; Table 1), indicating a concentric form of hypertrophy in these mice.



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  		Fig. 3. Serial echocardiographic results for wild-type and Serca2+/– mice before and after AC. Calculated, estimated LV mass for prebanded and AC wild-type and Serca2+/– mice is shown. LV mass was calculated from the echocardiographic measurements of septal wall thickness, posterior wall thickness, and LV end-diastolic chamber size. Values are expressed as means ± SE. n = 11 for wild-type and 7 for Serca2+/– mice.

 

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  		Table 1. Echocardiographic measures before and during pressure overload

 

Cardiac function assessed by echocardiography demonstrated that AC Serca2+/– mice had significant systolic (measured as FS) and diastolic (measured as IVRT and E/A) dysfunction compared with AC wild-type mice (P < 0.05; Figs. 4 and 5). There was a 51% reduction in FS in 10-wk AC failing Serca2+/– mice compared with AC wild-type and nonfailing Serca2+/– mice (Fig. 4A). Serca2+/– mice had a slower IVRT before AC compared with wild-type mice (P < 0.05; Fig. 4B), which supports our earlier findings (29) of a slower rate of relaxation in Serca2+/– mice. After AC, IVRT was further elevated in Serca2+/– mice, indicating severe diastolic dysfunction (Fig. 4B). Another measure of diastolic function is E/A, which is a measure of ventricular filling, both passive (E) and via atrial contraction (A), and is influenced by the speed of LV relaxation and LV compliance. Before AC, Serca2+/– mice had an elevated E/A compared with wild-type mice (P < 0.05; Fig. 5B). After 10 wk of AC, failing Serca2+/– mice had a significant increase in E/A compared with 10-wk AC wild-type mice and prebanded Serca2+/– mice (P < 0.05; Fig. 5B). Although not significant, there was also an elevation of E/A of nonfailing Serca2+/– mice after AC compared with wild-type mice and prebanded Serca2+/– mice. Different E/A patterns of diastolic filling were observed in coarcted Serca2+/– mice (Fig. 5A). The images depicted in Fig. 5A are representative of E/A patterns observed in coarcted Serca2+/– at different time points of AC. The E/A reversal pattern was observed at early time points (between 2 and 4 wk) in AC Serca2+/– mice, but this pattern was not observed in AC wild-type mice. At later stages (8–10 wk) of pressure overload, the E/A pattern demonstrated restrictive LV relaxation in Serca2+/– mice but not in AC wild-type mice.



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  		Fig. 4. Echocardiographic determination of fractional shortening (FS) and isovolumic relaxation time (IVRT) for prebanded and AC wild-type and Serca2+/– mice. A: FS. AC wild-type mice showed a preservation of function suggesting a compensated stage of hypertrophy. However, after 10 wk of AC, a subpopulation of Serca2+/– mice had a significant reduction in FS, indicating systolic dysfunction and a decompensatory stage of hypertrophy. FS was determined from the echocardiographic measurements of LV end-diastolic and end-systolic chamber size. B: IVRT was significantly slower in NC and 10-wk AC Serca2+/– mice. Values are expressed as means ± SE; n = 11 for wild-type and 7 for Serca2+/– mice.

 


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  		Fig. 5. Echocardiographic assessment of LV filling in prebanded and AC wild-type and Serca2+/– mice. A: LV filling patterns of coarcted Serca2+/– mice. B: E-to-A ratio representing early filling (E) and filling by atrial contraction (A). After 10-wk AC, Serca2+/– mice had a significant impairment in LV filling, indicating diastolic dysfunction. Values are expressed as means ± SE; n = 11 for wild-type and 7 for Serca2+/– mice.

 

There was no difference in the heart rate or pressure gradient produced in wild-type and Serca2+/– mice to account for the cardiac hypertrophy and/or remodeling and functional differences observed (see Tables 1 and 2). The echocardiographic measurements of pressure gradient after AC are comparable to the gradients obtained from catheterizations of the LV and femoral artery as shown previously (35).


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  		Table 2. Hemodynamic measurements in noncoarcted and 10-week AC wild-type and Serca2+/– mice

 

LV catheterization study reveals systolic and diastolic dysfunction in Serca2+/– mice after pressure overload. As previously observed (29), LV catheterization demonstrated that age-and sex-matched NC Serca2+/– mice had a significant decrease in +dP/dt and –dP/dt compared with NC wild-type mice (P < 0.05; Fig. 6). Consistent with the functional assessment obtained by echocardiography, a subpopulation of 10-wk AC Serca2+/– mice had significant systolic and diastolic cardiac dysfunction, as measured by the rates of contraction and relaxation (+dP/dt and –dP/dt, respectively), compared with AC wild-type mice (P < 0.05; Fig. 6), despite a similar duration and degree of pressure overload (Table 2). In addition, peak LV systolic pressure, as measured by the Millar catheter placed proximal to the aortic coarctation, was not different between Serca2+/– mice and wild-type mice after 10 wk of AC (Table 2), confirming matched hemodynamic loads. Both AC wild-type and nonfailing Serca2+/– mice had enhanced rates of contraction and relaxation, suggesting that these mice were in a compensatory stage during pressure overload. LV end-diastolic pressure was significantly elevated in AC failing Serca2+/– mice compared with AC wild-type mice (6.2 ± 1.7 mmHg in AC failing Serca2+/– vs. 2.1 ± 0.9 mmHg in AC wild type; P < 0.05).



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  		Fig. 6. LV catheterization assessment of contractile (+dP/dt; A) and relaxation (–dP/dt; B) performance in NC and 10-wk AC wild-type and Serca2+/– mice. Hemodynamic measurements were obtained in closed-chest mice. A fluid-filled catheter was placed in the femoral artery for systemic blood pressure measurements, and a Millar catheter was placed in the LV for intraventricular pressure measurements. Values are expressed as means ± SE; n = 4 for NC wild-type mice, 4 for NC Serca2+/– mice, 11 for AC wild-type mice, and 7 for AC Serca2+/– mice. WT, wild type; +/–, Serca2+/–; NF, nonfailing; F, failing.

 

Pressure overload results in a further decrease in SERCA2 protein levels in Serca2+/– mice. Western blot analysis revealed that 10-wk AC wild-type mice had a 14% reduction in SERCA2a protein levels compared with their NC cohorts (P < 0.05; Fig. 7B). AC Serca2+/– mice had a further 36% reduction in SERCA2a protein levels (for a total reduction of 56% compared with NC wild-type mice) compared with their NC cohorts, which started with an initial 20% reduction in SERCA2a protein levels (P < 0.05; Fig. 7B). Compared with AC wild-type mice, SERCA levels were significantly decreased in Serca2+/– mice after 10 wk of pressure overload. On the other hand, Csq protein levels were not significantly different between NC and AC cohorts. Our analysis of PLB expression showed that PLB was not significantly different between AC and NC samples in wild-type and Serca2+/– mice (data not shown). These data suggest that the severity of heart failure may relate to the greater decrease in SERCA levels observed in the AC Serca2+/– mice.



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  		Fig. 7. Western blot analysis depicting % sarco(endo) plasmic reticulum Ca2+-ATPase (SERCA)2a levels in NC and 10-wk AC wild-type and Serca2+/– mice. A: representative image of Western blot for SERCA2a and calsequestrin (Csq) levels in NC and 10-wk AC wild-type and Serca2+/– mice. B: quantitation of the SERCA2a protein levels in NC and 10-wk AC wild-type and Serca2+/– mice. Values are expressed as means ± SE; n = 3 for NC wild-type mice, 3 for NC Serca2+/– mice, 3 for AC wild-type mice, and 3 for AC Serca2+/– mice. The 4 AC Serca2+/– mice that died during the course of the study were not included in this analysis.

 


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
A number of animal models of cardiac hypertrophy and human heart failure have decreased levels of SERCA2 mRNA and/or protein (2, 5, 6, 9, 20, 24). However, the question remained as to whether there was a causal relationship between decreased levels of SERCA2 and heart failure. We reported previously (16, 29) that disruption of a single copy of the SERCA2 gene (Serca2+/– mice) results in decreased SERCA2 protein and activity and causes an impairment of basal cardiac performance and altered Ca2+ homeostasis, with no overt signs of cardiac disease.

Our present study has demonstrated that heart failure occurs more rapidly and more frequently in mice with chronically reduced SERCA2 levels in conjunction with a pathological stress (i.e., increased hemodynamic load). Systolic and diastolic performance is significantly decreased in 10-wk AC Serca2+/– mice. Also, these mice (Serca2+/–) had a significant degree of cardiac hypertrophy and dilatation compared with wild-type mice after pressure overload, which suggests that Ca2+ handling via the SERCA2 pump may not only regulate cardiac function but also influence cardiac remodeling during disease events. The functional and remodeling parameters were significantly different between AC wild-type and Serca2+/– mice despite a similar duration and degree of pressure overload. Western blot analysis revealed that Serca2+/– mice subjected to pressure overload had a further reduction in SERCA2, which may account for the systolic and diastolic dysfunction observed in AC Serca2+/– mice (see Fig. 7). These data suggest that depressed levels of SERCA2 protein and its activity below a certain threshold (e.g., >35% reduction in SERCA2 protein and activity) contribute to the onset and progression of heart failure. Our findings correlate well with recent studies showing that a defect in SR Ca2+ uptake function can contribute to cardiac dysfunction and pathology. Dash et al. (4) showed that fourfold overexpression of PLB (inhibitor of SERCA) in transgenic mice resulted in depressed Ca2+ transport (as evidenced by prolonged Ca2+ transients) and mechanical function. Young transgenic animals showed no signs of heart failure and maintained a full response to isoproterenol in vivo. However, on aging (15–18 mo), transgenic mice exhibited a progression to cardiac hypertrophy, LV chamber dilatation, and heart failure and sudden death (4). In addition, treatment of mice with ryanodine, an inhibitor of the SR Ca2+ release channel, resulted in cardiac hypertrophy and contractile dysfunction, suggesting that defects in SR Ca2+ handling can cause cardiac pathology (25). Although we have suggested that the level of SERCA2 protein and/or activity may impact the heart's ability to handle chronically increased pressure load, ultimately leading to heart failure, modifying other signaling mechanisms involved in cardiac remodeling and/or contractility could exhibit profound effects on the maintenance of cardiac function in response to hemodynamic overload. For example, mitogen-activated protein kinase and ERK kinase kinase 1 knockout mice were shown to be susceptible to pressure overload and to developing cardiac dysfunction compared with control wild-type mice, although it is not known whether SERCA2 levels were decreased in this model as they were not studied (32). However, a study by Kiriazis and colleagues (17) demonstrated that SERCA2 protein levels were markedly decreased (50%) in coarcted failing wild-type and PLB knockout mice, whereas coarcted nonfailing wild-type mice had a 24% reduction in SERCA2 protein, and these levels of SERCA2 protein correlated with FS. Our data are consistent with those of Kiriazis et al. (17) in that we detected a 56% reduction in SERCA2 protein levels in AC failing Serca2+/– mice and AC nonfailing wild-type mice had only a 14% reduction in SERCA2 (see Fig. 7).

Targeted expression of the SERCA pump in transgenic mouse hearts has been shown to improve cardiac function, increase systolic Ca2+ transients, and accelerate relaxation rate (13, 22, 19). In addition, studies have examined the benefit of increased SERCA pump levels during pressure overload-induced cardiac hypertrophy in genetically altered animal models. Ito et al. (15) found decreased mortality in 7-wk banded transgenic SERCA2a mice vs. banded controls showed and that transgenic animals maintained increased SERCA levels and enhanced contractile function even after banding. However, the magnitude of hypertrophy was similar in both controls and transgenic mice (15). These studies conclude that enhanced SERCA levels maintain increased SR Ca2+ load and contractile function, which therefore modify the outcome of pressure overload-induced remodeling.

In general, genetic manipulations that improve Ca2+ transport either by SERCA gene therapy or PLB ablation have been shown to be beneficial and to prevent cardiac dysfunction and ventricular remodeling in both transgenic and surgically induced heart failure models (7, 8, 10, 14, 26, 27). Recently, it was shown that ablation of PLB could prevent heart failure in a mouse model of dilated cardiomyopathy caused by deficiency in a cytoskeletal protein the muscle-specific Lim protein (26). Further PLB ablation prevented systolic dysfunction and exercise intolerance, but not hypertrophy (10), in a hypertrophic cardiomyopathy mouse model resulting from Arg403Gln mutation in myosin heavy chain. Hoshijima et al. (14) showed that sustained expression of pseudophosphorylated PLB into Bio 14.6 cardiomyopathic hamsters improves Ca2+ transport and contractile function. Adenoviral gene transfer studies by Hajjar and colleagues (7, 8, 27) showed that SERCA2a gene transfer can improve survival, ventricular function, and energy metabolism in an experimentally induced heart failure model. These studies clearly point out that restoration of normal Ca2+ transport can be beneficial; it can prevent ventricular dysfunction as well as cardiac remodeling triggered by a defect in Ca2+ transport.

In conclusion, our findings suggest that a decrease in SERCA pump level can lead to cardiac dysfunction and heart failure when the heart is subjected to an increase in physiological demands. This study presents evidence that hearts with decreased SERCA levels are at greater risk and develop heart failure, thus suggesting that a decrease in SERCA levels may contribute to worsening function, thus leading to failure.


   ACKNOWLEDGMENTS
 
The investigators acknowledge D. Puthoff for assistance with echocardiographic data, M. Luehrmann and A. Whitaker for excellent animal husbandry, and G. P. Boivin for pathological reports.

GRANTS

This work was supported by National Heart, Lung, and Blood Institute Grants R01-HL-61974 (to G. E. Shull), TG-HL-07382 (to Arnold Schwartz, University of Cincinnati, Cincinnati, OH), and R01-HL-64140-03 (to M. Periasamy) and American Heart Association Grant 9950570 (to M. Periasamy).


   FOOTNOTES
 

Address for reprint requests and other correspondence: M. Periasamy, Dept. of Physiology and Cell Biology, The Ohio State Univ. College of Medicine, 304 Hamilton Hall, 1645 Neil Ave., Columbus, OH 43210 (E-mail: periasamy.1{at}osu.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Arai M, Alpert NR, MacLennan DH, Barton P, and Periasamy M. Alterations in sarcoplasmic reticulum gene expression in human heart failure: a possible mechanism for alterations in systolic and diastolic properties of the failing myocardium. Circ Res 72: 463–469, 1993.[Abstract/Free Full Text]
  2. Arai M, Matsui J, and Periasamy M. Sarcoplasmic reticulum gene expression in cardiac hypertrophy and heart failure. Circ Res 74: 555–564, 1994.[Free Full Text]
  3. Bavendiek U, Brixius K, Munch G, Zobel C, Muller-Ehmsen J, and Schwinger RH. Effect of inotropic interventions on the force-frequency relation in the human heart. Basic Res Cardiol 93, Suppl 1: 76–85, 1998.
  4. Dash R, Kadambi V, Schmidt AG, Tepe NM, Biniakiewicz D, Gerst MJ, Canning AM, Abraham WT, Hoit BD, Liggett SB, Lorenz JN, Dorn GW 2nd, and Kranias EG. Interactions between phospholamban and {beta}-adrenergic drive may lead to cardiomyopathy and early mortality. Circulation 103: 889–896, 2001.[Abstract/Free Full Text]
  5. De Boer RA, Henning RH, Suurmeijer AJ, Pinto YM, Olthof E, Kirkels JH, van Gilst WH, Crijns HJ, and van Veldhuisen DJ. Early expression of natriuretic peptides and SERCA in mild heart failure: association with severity of the disease. Int J Cardiol 78: 5–12, 2001.[CrossRef][ISI][Medline]
  6. De la Bastie D, Levitsky D, Rappaport L, Mercadier JJ, Marotte F, Wisnewsky C, Brovkovich V, Schwartz K, and Lompre AM. Function of the sarcoplasmic reticulum and expression of its Ca2+-ATPase gene in pressure overload-induced cardiac hypertrophy in the rat. Circ Res 66: 554–564, 1990.[Abstract/Free Full Text]
  7. Del Monte F, Hajjar RJ, and Harding SE. Overwhelming evidence of the beneficial effects of SERCA gene transfer in heart failure. Circ Res 88: E66–E67, 2001.[ISI][Medline]
  8. Del Monte F, Williams E, Lebeche D, Schmidt U, Rosenzweig A, Gwathmey JK, Londowski DE, and Hajjar RJ. Improvement of survival and cardiac metabolism after gene transfer of sarcoplasmic reticulum Ca2+ ATPase in a rat model of heart. Circulation 104: 1424–1429, 2001.[Abstract/Free Full Text]
  9. Feldman AM, Weinberg EO, Ray PE, and Lorell BH. Selective changes in cardiac gene expression during compensated hypertrophy and the transition to cardiac decompensation in rats with chronic aortic banding. Circ Res 73: 184–192, 1993.[Abstract]
  10. Freeman K, Lerman I, Kranias EG, Bohlmeyer T, Bristow MR, Lefkowitz RJ, Iaccarino G, Koch WJ, and Leinwand LA. Alterations in cardiac adrenergic signaling and calcium cycling differentially affect the progression of cardiomyopathy. J Clin Invest 107: 967–974, 2001.[ISI][Medline]
  11. Hasenfuss G. Alterations of calcium-regulatory proteins in heart failure. Cardiovasc Res 37: 279–289, 1998.[Free Full Text]
  12. Hasenfuss G, Reinecke H, Studer R, Meyer M, Pieske B, Holtz J, Holubarsch C, Posival H, Just H, and Drexler H. Relation between myocardial function and expression of sarcoplasmic reticulum Ca2+-ATPase in failing and nonfailing human myocardium. Circ Res 75: 434–442, 1994.[Abstract/Free Full Text]
  13. He H, Giordano FJ, Hilal-Dandan R, Choi DJ, Rockman HA, McDonough PM, Bluhm WF, Meyer M, Sayen MR, Swanson E, and Dillman WH. Overexpression of the rat sarcoplasmic reticulum Ca2+ ATPase gene in the heart of transgenic mice accelerates calcium transients and cardiac relaxation. J Clin Invest 100: 380–389, 1997.[ISI][Medline]
  14. Hoshijima M, Ikeda Y, Iwanaga Y, Minamisawa S, Date MO, Gu Y, Iwatate M, Li M, Wang L, Wilson JM, Wang Y, Ross J Jr, and Chien KR. Chronic suppression of heart-failure progression by a pseudophosphorylated mutant of phospholamban via in vivo cardiac rAAV gene delivery. Nat Med 8: 864–871, 2002.[ISI][Medline]
  15. Ito K, Yan X, Feng X, Manning WJ, Dillmann WH, and Lorell BH. Transgenic expression of sarcoplasmic reticulum Ca2+ ATPase modifies the transition from hypertrophy to early heart failure. Circ Res 89: 422–429, 2001.[Abstract/Free Full Text]
  16. Ji Y, Lalli MJ, Babu GJ, Xu Y, Kirkpatrick DL, Liu LH, Chiamvimonvat N, Walsh RA, Shull GE, and Periasamy M. Disruption of a single copy of the SERCA2 gene results in altered Ca2+ homeostasis and cardiomyocyte function. J Biol Chem 275: 38073–38080, 2000.[Abstract/Free Full Text]
  17. Kiriazis H, Sato Y, Kadambi VJ, Schmidt AG, Gerst MJ, Hoit BD, and Kranias EG. Hypertrophy and functional alterations in hyperdynamic phospholamban-knockout mouse hearts under chronic aortic stenosis. Cardiovasc Res 53: 372–381, 2002.[Abstract/Free Full Text]
  18. Kiss E, Ball NA, Kranias EG, and Walsh RA. Differential changes in cardiac phospholamban and sarcoplasmic reticular Ca2+-ATPase protein levels. Effects on Ca2+ transport and mechanics in compensated pressure-overload hypertrophy and congestive heart failure. Circ Res 77: 759–764, 1995.[Abstract/Free Full Text]
  19. Lalli MJ, Yong J, Hashimoto K, Babu GJ, Kirkpatrick D, Loukianov E, Walsh RA, Sussman M, Marban E, and Periasamy M. SERCA1a structurally substitutes for SERCA2a in the cardiac sarcoplasmic reticulum and increases cardiac Ca2+ handling capacity. Circ Res 89: 160–167, 2001.[Abstract/Free Full Text]
  20. Linck B, Boknik P, Eschenhagen T, Muller FU, Neumann J, Nose M, Jones LR, Schmitz W, and Scholz H. Messenger RNA expression and immunological quantification of phospholamban and SR-Ca2+-ATPase in failing and nonfailing human hearts. Cardiovasc Res 31: 625–632, 1996.[CrossRef][ISI][Medline]
  21. Lorenz JN and Robbins J. Measurement of intraventricular pressure and cardiac performance in the intact closed-chest anesthetized mouse. Am J Physiol Heart Circ Physiol 272: H1137–H1146, 1997.[Abstract/Free Full Text]
  22. Loukianov E, Ji Y, Grupp IL, Kirkpatrick DL, Baker DL, Loukianova T, Grupp G, Lytton J, Walsh RA, and Periasamy M. Enhanced myocardial contractility and increased Ca2+ transport function in transgenic hearts expressing the fast-twitch skeletal muscle sarcoplasmic reticulum Ca2+ ATPase. Circ Res 83: 889–897, 1998.[Abstract/Free Full Text]
  23. Matsui H, MacLennan DH, Alpert NR, and Periasamy M. Sarcoplasmic reticulum gene expression in pressure overload-induced cardiac hypertrophy in rabbit. Am J Physiol Cell Physiol 268: C252–C258, 1995.[Abstract/Free Full Text]
  24. Mercadier JJ, Lompre AM, Duc P, Boheler KR, Fraysse JB, Wisnewsky C, Allen PD, Komajda M, and Schwartz K. Altered sarcoplasmic reticulum Ca2+-ATPase gene expression in the human ventricle during end-stage heart failure. J Clin Invest 85: 305–309, 1990.[ISI][Medline]
  25. Meyer M, Trost SU, Bluhm WF, Knot HJ, Swanson E, and Dillmann WH. Impaired sarcoplasmic reticulum function leads to contractile dysfunction and cardiac hypertrophy. Am J Physiol Heart Circ Physiol 280: H2046–H2052, 2001.[Abstract/Free Full Text]
  26. Minamisawa S, Hoshijima M, Chu G, Ward CA, Frank K, Gu Y, Martone ME, Wang Y, Ross J Jr, Kranias EG, Giles WR, and Chien KR. Chronic phospholamban-sarcoplasmic reticulum calcium ATPase interaction is the critical calcium cycling defect in dilated cardiomyopathy. Cell 99: 313–322, 1999.[CrossRef][ISI][Medline]
  27. Miyamoto MI, Del Monte F, Schmidt U, Matsui T, Guerrero JL, Gwathmey JK, Rosenzweig A, and Hajjar RJ. Adenoviral gene transfer of SERCA2a improves left ventricular function in aortic-banded rats in transition to heart failure. Proc Natl Acad Sci USA 97: 793–798, 2000.[Abstract/Free Full Text]
  28. Nagai R, Zarain-Herzberg A, Brandl CJ, Fujii J, Tada M, MacLennan DH, Alpert NR, and Periasamy M. Regulation of myocardial SR Ca2+-ATPase and phospholamban mRNA expression in response to pressure overload and thyroid hormone. Proc Natl Acad Sci USA 86: 2966–2970, 1989.[Abstract/Free Full Text]
  29. Periasamy M, Reed TD, Liu LH, Ji Y, Loukianov E, Paul RJ, Nieman ML, Riddle T, Duffy JJ, Doetschman T, Lorenz JN, and Shull GE. Impaired cardiac performance in heterozygous mice with a null mutation in the sarco(endo)plasmic reticulum Ca2+-ATPase isoform 2 (SERCA2) gene. J Biol Chem 274: 2556–2562, 1999.[Abstract/Free Full Text]
  30. Pieske B, Kretschmann B, Meyer M, Holubarsch C, Posival H, Just H, and Hasenfuss G. Alterations in intracellular calcium handling associated with the inverse force-frequency relation in human dilated cardiomyopathy. Circulation 92: 1169–1178, 1995.[Abstract/Free Full Text]
  31. Qi M, Shannon TR, Euler DE, Bers DM, and Samarel AM. Down-regulation of sarcoplasmic reticulum Ca2+-ATPase during progression of left ventricular hypertrophy. Am J Physiol Heart Circ Physiol 272: H2416–H2424, 1997.[Abstract/Free Full Text]
  32. Sadoshima J, Montagne O, Wang Q, Yang G, Warden J, Liu J, Takagi G, Karoor V, Hong C, Johnson GL, Vatner DE, and Vatner SF. The MEKK1-JNK pathway plays a protective role in pressure overload but does not mediate cardiac hypertrophy. J Clin Invest 110: 271–279, 2002.[CrossRef][ISI][Medline]
  33. Sahn DJ, DeMaria A, Kisslo J, and Weyman A. Recommendations regarding quantitation in M-mode echocardiography: results of a survey of echocardiographic measurements. Circulation 58: 1072–1083, 1978.[Abstract/Free Full Text]
  34. Schultz JE, Witt SA, Glascock BJ, Nieman ML, Reiser PJ, Nix SL, Kimball TR, and Doetschman T. TGF-{beta}1 mediates the hypertrophic cardiomyocyte growth induced by angiotensin II. J Clin Invest 109: 787–796, 2002.[CrossRef][ISI][Medline]
  35. Schultz JE, Witt SA, Nieman ML, Reiser PJ, Engle SJ, Zhou M, Pawlowski SA, Lorenz JN, Kimball TR, and Doetschman T. Fibroblast growth factor-2 mediates pressure-induced hypertrophic response. J Clin Invest 104: 709–719, 1999.[ISI][Medline]
  36. Williams RV, Lorenz JN, Witt SA, Hellard DT, Khoury PR, and Kimball TR. End-systolic stress-velocity and pressure-dimension relationships by transthoracic echocardiography in mice. Am J Physiol Heart Circ Physiol 274: H1828–H1835, 1998.[Abstract/Free Full Text]



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