AJP - Heart BIOPAC complete lab solutions
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Heart Circ Physiol 293: H2418-H2428, 2007. First published July 13, 2007; doi:10.1152/ajpheart.00663.2007
0363-6135/07 $8.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
293/4/H2418    most recent
00663.2007v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Web of Science (5)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Talukder, M. A. H.
Right arrow Articles by Zweier, J. L.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Talukder, M. A. H.
Right arrow Articles by Zweier, J. L.

Expression of SERCA isoform with faster Ca2+ transport properties improves postischemic cardiac function and Ca2+ handling and decreases myocardial infarction

M. A. Hassan Talukder,1 Anuradha Kalyanasundaram,2 Xue Zhao,1,3 Li Zuo,1 Poornima Bhupathy,2 Gopal J. Babu,2 Arturo J. Cardounel,1 Muthu Periasamy,2 and Jay L. Zweier1,2

1Davis Heart and Lung Research Institute and Division of Cardiovascular Medicine, Department of Internal Medicine, and 2Department of Cell Biology and Physiology, The Ohio State University College of Medicine and Public Health, Columbus, Ohio; and 3Department of Cardiology, Shanghai Changzheng Hospital, Shanghai, China

Submitted 7 June 2007 ; accepted in final form 10 July 2007


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Myocardial ischemia-reperfusion (I/R) injury is associated with contractile dysfunction, arrhythmias, and myocyte death. Intracellular Ca2+ overload with reduced activity of sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) is a critical mechanism of this injury. Although upregulation of SERCA function is well documented to improve postischemic cardiac function, there are conflicting reports where pharmacological inhibition of SERCA improved postischemic function. SERCA2a is the primary cardiac isoform regulating intracellular Ca2+ homeostasis; however, SERCA1a has been shown to substitute SERCA2a with faster Ca2+ transport kinetics. Therefore, to further address this issue and to evaluate whether SERCA1a expression could improve postischemic cardiac function and myocardial salvage, in vitro and in vivo myocardial I/R studies were performed on SERCA1a transgenic (SERCA1a+/+) and nontransgenic (NTG) mice. Langendorff-perfused hearts were subjected to 30 min of global ischemia followed by reperfusion. Baseline preischemic coronary flow and left ventricular developed pressure were significantly greater in SERCA1a+/+ mice compared with NTG mice. Independent of reperfusion-induced oxidative stress, SERCA1a+/+ hearts demonstrated greatly improved postischemic (45 min) contractile recovery with less persistent arrhythmias compared with NTG hearts. Morphometry showed better-preserved myocardial structure with less infarction, and electron microscopy demonstrated better-preserved myofibrillar and mitochondrial ultrastructure in SERCA1a+/+ hearts. Importantly, intraischemic Ca2+ levels were significantly lower in SERCA1a+/+ hearts. The cardioprotective effect of SERCA1a was also observed during in vivo regional I/R with reduced myocardial infarct size after 24 h of reperfusion. Thus SERCA1a+/+ hearts were markedly protected against I/R injury, suggesting that expression of SERCA 1a isoform reduces postischemic Ca2+ overload and thus provides potent myocardial protection.

myocardial ischemia-reperfusion; sarco(endo)plasmic reticulum calcium-adenosine triphosphatase; free radicals; postischemic function

THE SARCO(ENDO)PLASMIC RETICULUM Ca2+-ATPase (SERCA) plays a central role in muscle contraction and relaxation by maintaining intracellular Ca2+ concentration (13). The sarcoplasmic reticulum (SR) Ca2+-uptake activity determines not only the speed of Ca2+ removal for relaxation but also the SR Ca2+ content and, therefore, the amount of Ca2+ released for subsequent contraction (30). The rate of active muscle relaxation is determined largely by the Ca2+ uptake function of SERCA2a (35). Acute myocardial ischemia-reperfusion (I/R) injury is associated with contractile dysfunction, arrhythmias, myocardial infarction, and sudden death (5). Although the causes of myocardial I/R injury are multifactorial, it is well known that intracellular Ca2+ overload with reduced expression and/or activity of SERCA2a plays a prominent role (32, 37, 42). High levels of oxygen-derived free radicals are generated during myocardial I/R and have been shown to damage SERCA2a, potentially contributing to cellular Ca2+ overload and myocardial injury (16, 22, 49). Thus cytosolic free Ca2+ overload and oxidative stress, either independently or cooperatively, are major contributors to I/R-induced injury.

SERCA2a is the cardiac-specific isoform and is the major component of beat-to-beat Ca2+ cycling during excitation-contraction coupling (39). Recently, it has been reported that SERCA1a, the isoform normally expressed in fast skeletal muscle but not the heart (35), is more resistant to oxidative stress (43) and acidosis (46). SERCA1a and SERCA2a possess ~84% sequence homology, and transgenic expression of SERCA1a in the mouse heart has been shown to substitute for SERCA2a both structurally and functionally with concomitant increases in SR Ca2+ uptake and cardiac contractility (19, 23, 26). Confocal microscopy demonstrated specific trafficking of SERCA1a to cardiac SR and an increased rate of Ca2+ removal from cytosol with increased intracellular Ca2+ transients in cardiomyocytes of SERCA1a-overexpressed mice (23). The striking observation is that with its unique antigenicity and equivalent upregulation, SERCA1a has twofold greater velocity of Ca2+ transport than SERCA2a (6, 40). Chronic expression of SERCA1a in vivo did not result in significant morphological differences in the hearts, with similar mortality curves seen (23).

Several studies have consistently demonstrated that increased expression of SERCA2a improves myocardial contractility and Ca2+ handling at baseline and in disease conditions including myocardial I/R (3, 7, 9, 15, 31, 33). Conversely, several investigators have controversially reported that pharmacological inhibition of SERCA may improve postischemic function in stunned heart or in isolated myocardium following I/R (2, 12). The results with SERCA inhibitors, thapsigargin or cyclopiazonic acid, were inconsistent in terms of effectiveness, narrow dose range, and timing of administration (48). Interestingly, enhanced SR function in mice overexpressing SERCA1a recently has been shown to partially rescue the heart from hydroxyl radical-induced injury (17).

Despite its unique antigenicity and increased Ca2+ pumping kinetics (6, 40), it is not known whether SERCA1a overexpression can provide myocardial protection against I/R as has been shown with SERCA2a overexpression (7, 9). Therefore, to further address this issue, the aim of the present study was to test the hypothesis that expression of SERCA1a in the heart will provide cardioprotection characterized by improved postischemic contractile function, reduced intracellular Ca2+ overload, fewer ventricular arrhythmias, and smaller myocardial infarction.


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
This study was reviewed and approved by the Institutional Laboratory Animal Care and Use Committee at The Ohio State University, carried out according to the approved guidelines, and conforms with the Guidelines for the Care and Use of Laboratory Animals published by the National Institutes of Health [DHHS Publication (NIH) No. 85-23, Revised 1996].

Mice. Details regarding the generation and characterization of SERCA1a transgenic (SERCA1a+/+) mice have been described previously (26). Briefly, rat SERCA1a cDNA was linked to the mouse cardiac {alpha}-MHC promoter, and SERCA1a+/+ mice were generated. They were bred with FVB/N wild-type mice to establish germ line transmission. Each mouse was genotyped when it was weaned, and only respective littermates served as controls. The experiments were performed in young (16–20 wk) male mice, and tail clips were kept to reconfirm the genotypes.

Western blot analysis. Tail genotyping (PCR) and SERCA1a and SERCA2a expressions in cardiac homogenates were performed as described previously (26). Hearts from age-matched mice were homogenized in the lysis buffer, and protein concentrations were determined by Bio-Rad protein assay. The relative expression of SERCA2a and SERCA1a proteins in SERCA1a transgenic (SERCA1a+/+) vs. nontransgenic (NTG) mice were determined by immunoblotting. Equal amounts (10 µg) of protein extract were separated by SDS-PAGE on polyacrylamide gel and then transferred to nitrocellulose membrane using a Bio-Rad transblot apparatus. Membranes were incubated with a polyclonal antibody for SERCA2a or SERCA1a (1:5,000 dilution) at room temperature for 1 h. The membranes were washed with TBS-T (20 mM Tris·HCl, 137 mM NaCl, and 0.05% Tween 20) six times (10 min each). Secondary antibodies were peroxidase-labeled anti-rabbit IgG (Kirkegaard & Perry Laboratories) at room temperature at a dilution of 1:5,000 for 45 min. After extensive washing with TBS-T, antibody signals were detected using an enhanced chemiluminescence kit (Pierce).

Langendorff-perfused heart preparation. Hearts were isolated from age-matched mice of both strains as described previously (41, 44). Briefly, mice were anesthetized with pentobarbital (50 mg/kg ip), and hearts were excised, aortas were cannulated, and hearts were perfused in a Langendorff mode at a constant pressure of 80 mmHg with a modified Krebs-Henseleit buffer (KHB) equilibrated with 95% O2-5% CO2 at 37°C. The constituents of KHB were (in mM) 120 NaCl, 5.9 KCl, 25 NaHCO3, 1.2 MgCl2, 2.5 CaCl2, 0.5 EDTA, and 16.7 glucose. A fluid-filled balloon was inserted into the left ventricle (LV) across the mitral valve and connected to a pressure transducer permitting continuous measurement of LV pressure (LVP). Hearts were immersed in a water-jacketed bath maintained at 37°C, and the LV balloon was filled with water to yield a LV diastolic pressure of 3–6 mmHg. Coronary flow was continuously monitored via a Doppler flow probe (T206; Transonic Systems, Ithaca, NY) placed in the aortic perfusion line. Aortic pressure and LV developed pressure (LVDP) were recorded on a PowerLab/400 multichannel data acquisition system (ADInstruments; Castle Hill, Australia). The LVP signal was digitally processed (using PowerLab Chart software version 4.2; ADInstruments) to yield diastolic and systolic pressures as well as heart rate. Hearts having unexpected arrhythmias during equilibration were excluded from the study. There was no preischemic arrhythmia with SERCA1a+/+ hearts; however, 2 of 26 NTG hearts developed preischemic arrhythmias and were excluded from the study.

Following 30 min of equilibration, hearts underwent 30 min of global ischemia, followed by 45 min of reperfusion. At the end of reperfusion, hearts were processed for myocardial infarct size measurement, histopathological examination, Western blot analysis, and electron microscopy. One subset of hearts underwent only 5 min of reperfusion to measure free radical generation.

Criteria used to determine arrhythmias. Ventricular tachyarrhythmias, such as ventricular fibrillation (VF) and ventricular tachycardia (VT), with episodes of mechanical alternans are common occurrences with reperfusion after 30 min of global ischemia. Because these hearts were unpaced, a change in heart rate reflects an important pathophysiological impact of reperfusion on these hearts. Identification of rhythm abnormalities was derived from synchronized recordings of LVP tracings and heart rate; therefore, differentiation of supraventricular and ventricular arrhythmias was not possible in relation to the P wave. According to Merillat et al. (28) and Kawahara et al. (21), VF was defined as 1) the development of chaotic, irregular, rapid LVP recordings, 2) the loss of pulsatile LVP, and 3) the loss of grossly observable regular ventricular contraction. VT was defined as a rapid and regular cyclic change in LVP with smaller amplitude than control. Mechanical alternans (34) characterized by alternate large and small contractions were defined according to the previously described hallmarks such as 1) impaired and incomplete relaxation of the strong contraction, 2) higher end-diastolic pressure of the strong contraction, and 3) smaller peak systolic pressure of the weak contraction. When we recorded abnormal contractile rhythms for >30 min of reperfusion, we considered them as persistent rhythm abnormalities.

Light microscopy. Standard hematoxylin and eosin (HE) staining was used for morphological evaluation. Hearts were fixed in 10% neutral buffered formalin and embedded in paraffin, and serial cross sections (6 µm) were made for staining. Digital images of each slide were randomly taken for morphometric evaluation using Spot Basic Software 4.0.4 (Diagnostics International).

Electron microscopy. Small tissue blocks (~1 mm3) were cut from the LV free wall and fixed in 2.5% glutaraldehyde plus 2% paraformaldehyde in 0.1 M sodium cacodylate buffer (pH 7.4) overnight at 4°C. The samples were rinsed three times in 0.1 M sodium cacodylate buffer, postfixed in 1% osmium tetroxide in the same buffer for 1 h at room temperature, and stained in 1% uranyl acetate for 1 h. The samples were then dehydrated in ascending concentrations of alcohol, treated with propylene oxide, embedded in Spur resin, and heat polymerized. After polymerization, ultrathin sections (~70 nm) were cut using a Leica electron microscope UC6 ultramicrotome, mounted on uncoated copper grids, and stained in 2% uranyl acetate followed by Reynold's lead citrate. Samples were examined in a Philips CM12 transmission electron microscope at 80 kV.

Electron paramagnetic resonance spectroscopy and spin trapping. Spin-trapping measurements of oxygen radical generation from SERCA1a+/+ and NTG hearts were performed as described previously (44, 45). Hearts were infused with 50 mM 5,5-dimethyl-1-pyrroline N-oxide (DMPO), and effluent was sampled before ischemia and during the first 3 min of reperfusion. Relative quantitation of radical signals was performed by double integration.

Immunohistochemistry for nitrotyrosine. Immunohistochemistry for nitrotyrosine were performed as described previously (44, 45, 47). Briefly, the formalin-fixed paraffin sections (6 µm) from preischemic and postischemic hearts were incubated with rabbit polyclonal anti-nitrotyrosine antibody (1:400 dilution; Upstate), then with the biotinylated secondary, and again with the tertiary, ExtrAvidin alkaline phosphatase (1:800 dilution). Slices from the mouse heart injected with peroxynitrite (1 mM, 0.05 ml; Cayman Chemical) into the LV were taken as the positive control, and slices with rabbit IgG instead of anti-nitrotyrosine antibody served as the negative control. Color was developed with fast red.

Ca2+ uptake assay. Ca2+ uptake assays were performed as described previously (20). Heart tissue from SERCA1a+/+ and NTG mice was homogenized in the following mixture (in mM): 50 potassium phosphate, pH 7.0, 10 NaF, 1 EDTA, 300 sucrose, 0.3 phenylmethylsulfonyl fluoride, and 0.5 DTT. The SR Ca2+ uptake activity was measured using a modified Millipore filtration method. Cardiac homogenates (100 µg/ml) were incubated at 37°C in 1.5 ml of reaction mixture containing 40 mM imidazole, pH 7.0, 100 mM KCl, 5 mM MgCl2, 5 mM NaN3, 5 mM potassium oxalate, 1 µM ruthenium red, and 0.5 mM EGTA to yield a free Ca2+ concentration in the range of 0.03 to 3 µM (containing 1 µCi/µmol 45Ca) as determined using Calcium Titration Program computer software. The uptake reaction was started by adding 5 mM ATP to the reaction mixture. After 1 min, 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 Ca2+ concentration required for one-half of the maximum velocity for Ca2+ uptake was determined by nonlinear curve fitting using GraphPad Prism 4.0 software.

Rhod-2 spectrofluorometry. Intracellular free Ca2+ (Cai2+) changes were estimated by loading the isolated beating hearts with a Ca2+-sensitive fluorescence probe, rhod-2 AM (Molecular Probes, Eugene, OR), as previously described with slight modification (10, 11, 27). Rhod-2 AM is membrane permeable and becomes Ca2+ sensitive and trapped in the cytosol when deesterified to rhod-2 intracellularly. Because the amplitude of rhod-2 fluorescence transients depends on Cai2+ (11), the estimation of its value allows monitoring the Cai2+ changes over time.

A fiber optic probe was gently positioned against the LV wall to obtain emission signal from the heart. To suppress motion-induced artifacts and minimize the effects from the curvature of the epicardium, the distance between heart and cable surface was adjusted by monitoring tissue autofluorescence before rhod-2 loading. Rhod-2 was loaded after spontaneous cardiac contractility became regular (20 min) and no clear signs of damage were evident. Rhod-2 (50 µg) was added to 25 µl of DMSO, thoroughly mixed, and diluted up to 2 ml with perfusion buffer constituting 25 µg/ml. Rhod-2 dye (2 ml) was added without recirculation through a parallel infusion line just above the aortic cannula. During bolusing, the bolus line was opened and the other line was closed. Dye loading was followed by a 10-min washout period with normal KHB to remove any extracellular dye. LVDP and heart rate were monitored during loading and the washout period. Typically, there was a 30–50% decline in LVDP during rhod-2 loading, followed by complete recovery within 5 min of dye-free perfusion. Hearts having arrhythmias during the washout period were excluded from the study.

Fluorescence was excited with a 150-W xenon arc lamp through excitation/emission filters in a modified tissue fluorometer (C&L Instruments, Hummelstown, PA), and the light was directly focused on the photomultiplier tube (PMT). To reduce light interference, the tissue chamber was housed in a solid dark metal box. The area of the light guide facing the heart was 28 mm2, providing a complete observation window of the whole heart. The PMT shutters were kept closed except during data acquisition to minimize photobleaching and photooxidation of rhod-2. The PMT output was collected via personal computer using an analog-to-digital converter. The emitted signal was detected, digitized, and recorded at a rate of 125 Hz for analysis (FluorMeasure version 2.7 acquisition software). The excitation/emission parameter for rhod-2 is 531 ± 20 nm/593 ± 20 nm. The sampling time was set at 8 ms per data point per filter. After 10 min of washout (37°C), Cai2+ fluorescence data was acquired. The heart was then subjected to 30 min of global ischemia, and Cai2+ fluorescence data was collected at end ischemia just before reperfusion. Upon reperfusion, gradual loss and washout of rhod-2 occurred, precluding subsequent measurement of Cai2+ fluorescence.

Cai2+ was calculated according to previous literature (10) with rhod-2 calibration in the isolated heart, that is: Cai2+ = Kd(F – Fmin)/(Fmax – F), where Kd (710 nM) is the dissociation coefficient for rhod-2 and F is the fluorescence signal detected by PMT at a specific time point, Fmax is the maximal fluorescence after digitonin treatment, and Fmin is the blank fluorescence. The Fmin was acquired before rhod-2 loading, and Fmax was obtained after 15 µM digitonin (Sigma) infusion at the end of each experiment. The calculated myocardial CaFormula in our experimental conditions with control FVB/N mice was 370 ± 6 and 753 ± 22 nM in diastole and systole, respectively. These results agree well with those previously reported with rhod-2 (11) and aequorin (14) in the mouse heart. The diastolic or mean ischemic levels of rhod-2 fluorescence were used to evaluate CaFormula changes and were expressed as relative units (RU).

In vivo myocardial I/R. In vivo myocardial I/R was performed as described previously (47). Briefly, mice were anesthetized with a mixture of intraperitoneal ketamine (55 mg/kg) and xylazine (15 mg/kg). After adequate anesthesia and aseptic preparations, mice were intubated and ventilated with room air by a MiniVent (type 845; Harvard Apparatus). The respiratory rate was maintained at 100 breaths/min with a tidal volume of 0.25 ml for a 25-g mouse. After the chest was opened and the heart visualized, the left anterior descending (LAD) coronary artery was ligated 2 mm below the tip of the left auricle with a 7-0 silk ligature. Occlusion was confirmed by the dramatic change in color (red to pallor) and restricted ventricular motion. After 30 min of LAD artery occlusion, the knot was released to start reperfusion, and reperfusion was confirmed by return of the pink-red color and motion of the anterior wall of the LV. The chest was closed in layers with subcutaneous administration of Penicillin G Procaine. Buprenorphine (0.1 mg/kg, <0.5 ml in volume) was given subcutaneously to reduce postoperative acute pain. The rectal temperature of the mouse was maintained at 37°C by a thermo heating pad. When mice resumed a normal breathing pattern and started walking, the ventilator was taken off and mice were transferred to a clean cage with free access to food and water.

Myocardial infarct size measurement. In vitro myocardial infarction was measured by 2,3,5-triphenyltetrazolium chloride (TTC; Sigma) staining of heart sections as reported previously (44). Briefly, the heart was immediately removed after I/R, wrapped in polyethylene, and frozen for 10 min for hardening. The heart was then serially sectioned into transverse slices (1 mm) with a heart slicer (Zivic laboratories) and incubated in 1% TTC in phosphate-buffered saline for 15 min at room temperature to demarcate the viable (brick red) and infarcted (pale) myocardium. Heart slices are then fixed overnight in 10% neutral buffered formaldehyde for better color contrast and digitally imaged. Computerized planimetry (with image analysis software Meta Vue, version 6.0) of each section was used to determine the percent infarction from the total cross-sectional area of the LV.

In vivo myocardial infarction was measured at 24 h postreperfusion as previously described with slight modification (47). Mice were anesthetized, intubated, and ventilated, and the chest was opened along the previous incision line and the left main coronary artery was religated at the same location as before. Evans blue dye (0.2 ml of a 4.0% solution) was injected directly into the inferior vena cava for visualization of the nonischemic zone. The area of the myocardium not stained with Evans blue was defined as the area at risk (AAR). The heart was then rapidly excised, wrapped in polyethylene, frozen for 10 min for hardening, serially sectioned along the short axis (1 mm thick) with a heart slicer, and incubated with 1% TTC for 15 min at room temperature for demarcation of the viable and nonviable myocardium within the AAR. Both sides of each myocardial slice were photographed, and the area of infarction, AAR, and nonrisk area were determined by computerized planimetry as described above.

Data analysis. All results are means ± SE. Data were analyzed using either two-tailed Student's t-test for paired data from the same experiment and unpaired data from different experiments or ANOVA followed by Fisher's post hoc test. Values of P < 0.05 were considered to be statistically significant.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Characterization of SERCA1a and SERCA2a expression. Transgenic expression of SERCA1a was confirmed by tail biopsy genotyping and Western blot analysis of cardiac homogenates. Consistent with previous data (26), PCR results demonstrated the distinct band of SERCA1a gene in the transgenic mice, whereas no band was seen in the NTG mice (Fig. 1A). Similarly, SERCA1a+/+ hearts demonstrated prominent SERCA1a protein expression (Fig. 1B), and this was associated with a concomitant decrease in SERCA2a (Fig. 1C). In SERCA1a+/+ hearts, the total amount of SERCA was markedly increased compared with NTG hearts. As demonstrated previously with quantitative immunoblotting (26), the level of SERCA1a appeared to be twice the level of SERCA2a in NTG, whereas the level of SERCA2a was decreased by ~50%.


Figure 1
View larger version (12K):
[in this window]
[in a new window]

  		Fig. 1. Characterization of sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) gene and protein expression in nontransgenic (NTG) and SERCA1a transgenic (SERCA1a+/+) mice. A: PCR analysis with tail sample from offspring of transgenic breeders. Genomic DNA with SERCA1a primer shows a distinct band of SERCA1a gene in the transgenic mice but not in the NTG mice. B and C: immunoblots for SERCA1a (B) and SERCA2a protein levels (C) in cardiac homogenates.

 
Pre- and postischemic cardiac function in perfused hearts. The functional characteristics of the isolated-perfused hearts are summarized in Table 1. There were no significant differences in body weight (32 ± 1 vs. 30 ± 1 g, n = 8/group) or heart weight-to-body weight ratio (0.49 ± 0.01 vs. 0.48 ± 0.01%, n = 8/group) between SERCA1a+/+ and NTG mice (Table 1). The intrinsic heart rates were identical; however, baseline coronary flow, LVDP, and maximal rates of contraction (+dP/dt) and relaxation (–dP/dt) were significantly higher in SERCA1a+/+ than in NTG hearts.


View this table:
[in this window]
[in a new window]

  		Table 1. Hemodynnamic parameters before and after in vitro myocardial I/R

 
Figure 2, A and B, shows the time course of the recovery of LVDP and LVEDP in SERCA1a+/+ and NTG hearts subjected to 30 min of global ischemia and 45 min of reperfusion. Upon reperfusion, improved recovery of postischemic LV function was seen in SERCA1a+/+ hearts with significantly higher LVDP (Fig. 2A) and greater recovery in both +dP/dt and –dP/dt than in the NTG hearts (Table 1). Although a similar rise in LVEDP was seen in both groups during ischemia, upon reperfusion LVEDP decreased in SERCA1a+/+ hearts, whereas it increased above ischemic levels in NTG hearts. Throughout the time course of reperfusion, LVEDP remained elevated in NTG compared with SERCA1a+/+ hearts (Fig. 2B). SERCA1a+/+ hearts started to beat much earlier than NTG hearts and showed diminished incidence of reperfusion arrhythmias (Table 1). The diminished propensity of abnormal cardiac rhythms in SERCA1a+/+ hearts resulted in a markedly shorter mean duration of arrhythmias (Fig. 2C; 22 ± 4 vs. 42 ± 2 min, P < 0.001).


Figure 2
View larger version (19K):
[in this window]
[in a new window]

  		Fig. 2. Contractile function and myocardial infarction in isolated hearts subjected to 30 min of global ischemia followed by reperfusion. A and B: time courses of recovery of left ventricular developed pressure (LVDP; A) and LV end-diastolic pressure (LVEDP; B). The differences in recovery of LVDP and LVEDP in SERCA1a+/+ and NTG hearts are highly significant. ***P < 0.001; n = 8/group. C: mean duration of abnormal cardiac rhythms in NTG and SERCA1a+/+ hearts after 45 min of reperfusion. ***P < 0.001 vs. NTG; n = 8/group. D and E: myocardial infarction in hearts subjected to 30 min of global ischemia and 60 min of reperfusion. Hearts were stained with 2,3,5-triphenyltetrazolium chloride (TTC), and representative sections (D) and the percentage of infarct size over total LV area (E) are shown. **P < 0.01 vs. NTG; n = 6/group. All values are means ± SE. PI, preischemia.

 
Myocardial infarction after in vitro I/R. To correlate postischemic LV function with irreversible myocardial injury, we determined myocardial infarct size after I/R (Fig. 2, D and E). TTC staining revealed that isolated SERCA1a+/+ hearts had markedly smaller LV infarct size compared with NTG hearts, with an infarct size of 20 ± 2% in SERCA1a+/+ hearts compared with 39 ± 5% in NTG hearts (P < 0.01).

Myocardial histology before and after in vitro I/R. To determine whether alterations in myocardial structure paralleled the differences in functional recovery, we performed light microscopic histological examination. No differences in histological structure were found in HE-stained sections of nonischemic SERCA1a+/+ and NTG hearts, and both appeared structurally normal (Fig. 3, A and B). After I/R, frequent interstitial edema, nuclear vacuolation, and contraction band-type changes (arrow) were evident in NTG hearts as early as the first minute of reperfusion (Fig. 3C) and persisted for 45 min (Fig. 3E), but only modest changes were seen in SERCA1a+/+ hearts (Fig. 3, D and F).


Figure 3
View larger version (115K):
[in this window]
[in a new window]

  		Fig. 3. Representative histological photographs of nonischemic and postischemic hearts. Nonischemic myocardium is shown in NTG (A) and SERCA1a+/+ mice (B). With reperfusion after 30 min of ischemia, a grossly distorted structure, interstitial spacing, and frequent appearance of contraction bands (arrows) are noted in NTG hearts (C and E), whereas structure was largely preserved in SERCA1a+/+ hearts (D and F). Bar, 50 µm; magnification, x400; n = 3/group. NI, nonischemic; RP, reperfusion.

 
Myocardial ultrastructure before and after in vitro I/R. To further correlate myocardial infarction and histology findings with myocardial ultrastructure, we performed electron microscopic examination. Concordant with light microscopy, no qualitative differences in myocardial ultrastructure were seen between nonischemic SERCA1a+/+ and NTG hearts (Fig. 4, A and B). Orderly distributed myofibrils with normal sarcomere and dark mitochondria containing tightly arranged cristae were seen in both strains. However, after I/R, NTG hearts showed markedly severe ultrastructural damage (Fig. 4C) with thinning, discontinuation, or lysis of myofibrils associated with intracellular edema and misregistration of Z bands, whereas little change was seen in SERCA1a+/+ hearts (Fig. 4D). Most of the mitochondria in NTG hearts were swollen and larger in size with abnormal cristae and loss of matrix, whereas no abnormalities were seen in SERCA1a+/+ hearts.


Figure 4
View larger version (82K):
[in this window]
[in a new window]

  		Fig. 4. Representative electron micrographs of nonischemic and postischemic hearts of two separate experiments. In nonischemic hearts, normal myocardial ultrastrucure is shown in both NTG (A) and SERCA1a+/+ mice (B). After 30 min of ischemia and 45 min of reperfusion, grossly distorted structures of myofibrils and mitochondria are noted in NTG (C) but not SERCA1a+/+ hearts (D). Bar, 1 µm; magnification, x12,500.

 
Electron paramagnetic resonance measurements of free radical generation. To determine the magnitude of free radical generation in postischemic hearts, we performed electron paramagnetic resonance (EPR) and spin-trapping studies using the spin trap DMPO. Before ischemia, no radical generation was seen in either strain (Fig. 5, A and B, top tracings). However, over the first minute of reperfusion, both SERCA1a+/+ and NTG hearts exhibited radical signals consisting of a 1:2:2:1 quartet of DMPO-OH. The reperfusion-associated increase in radical generation was not different between the NTG and SERCA1a+/+ hearts (Fig. 5, A and B, bottom tracings).


Figure 5
View larger version (9K):
[in this window]
[in a new window]

  		Fig. 5. Electron paramagnetic resonance (EPR) spin trapping measurement of free radical generation in nonischemic and postischemic hearts. Hearts were infused with 50 mM DMPO before ischemia and upon the first 45 s of reperfusion following 30 min of global ischemia, and measurements were performed on the coronary effluents. Representative EPR signals were recorded during nonischemia and reperfusion in NTG (A) and SERCA1a+/+ hearts (B). Data are averages normalized on free radical generation during reperfusion in both strains (n = 3/group).

 
Immunohistochemistry for nitrotyrosine. Immunohistochemical staining of nitrotyrosine, a marker of peroxynitrite formation (32, 33) was performed in nonischemic and postischemic hearts. In positive controls with peroxynitrite infusion, strong dense red nitrotyrosine staining of the vascular bed and weaker staining within the myocytes was seen throughout the heart (Fig. 6A). There was no staining in the negative control (Fig. 6B). In nonischemic myocardium, there was no positive staining for nitrotyrosine (Fig. 6, C and D). In postischemic myocardium, focal positive nitrotyrosine staining was detectable upon reperfusion within the myocytes of both SERCA1a+/+ and NTG hearts at 1 min of reperfusion (Fig. 6, E and F). The intensity of nitrotyrosine staining in reperfused myocardium was similar in both strains.


Figure 6
View larger version (88K):
[in this window]
[in a new window]

  		Fig. 6. Representative immunostaining for nitrotyrosine product in isolated nonischemic and postischemic heart slices. Hearts were obtained before ischemia (nonischemia) and at the first minute of reperfusion following 30 min of global ischemia. A: a positive control heart infused with 1 mM peroxynitrite. B: a negative control of the same slice without anti-nitrotyrosine primary antibody. There is an absence of positive red staining in the nonischemic NTG (C) and SERCA1a+/+ hearts (D); however, focal positive red staining (arrow) is shown after 1 min of reperfusion in both NTG (E) and SERCA1a+/+ hearts (F). Magnification, x400; n = 3/group.

 
Rhod-2 spectrofluorometry for myocardial Ca2+ and SR Ca2+ uptake activity. To study Ca Formula in the isolated beating mouse heart, we measured rhod-2 CaFormula fluorescence before (preischemia) and after 30 min of ischemia. Figure 7 shows representative rhod-2 CaFormula fluorescence tracings in both NTG (A) and SERCA1a+/+ hearts (B) during preischemia and after ischemia. Figure 7C shows average systolic and diastolic rhod-2 CaFormula fluorescence tracings after 10 min of rhod-2 loading and washout. Preischemic peak systolic rhod-2 CaFormula fluorescence was significantly higher (954 ± 28 vs. 753 ± 40 RU, P < 0.05, n = 5), whereas diastolic CaFormula fluorescence was significantly lower (295 ± 11 vs. 376 ± 4 RU, P < 0.05, n = 5) in the SERCA1a+/+ hearts compared with NTG hearts (Fig. 7C). Although a sharp rise and rapid fall of amplitude for rhod-2 CaFormula fluorescence transients were seen in both strains before ischemia, with the onset of ischemia the amplitude of these transients decreased as diastolic values increased, with only minimal transients seen after 30 min of global ischemia. After 30 min of ischemia, the CaFormula fluorescence values were markedly elevated in both strains with decreased or no rhythmic amplitude, and mean CaFormula fluorescence (Fig. 7D) was significantly less in the SERCA1a+/+ hearts compared with NTG hearts (1,147 ± 45 vs. 1,528 ± 93 RU, P < 0.05, n = 5).


Figure 7
View larger version (19K):
[in this window]
[in a new window]

  		Fig. 7. Rhod-2 spectrofluorometry of intracellular free Ca2+ (Cai2+) in relative units (RU), and sarcoplasmic reticulum (SR) Ca2+ uptake activity during preischemia and ischemia. A and B: representative rhod-2 Ca2+ fluorescence signals for NTG and SERCA1a+/+ hearts, respectively. C: averaged peak and diastolic rhod-2 Cai2+ signals in NTG and SERCA1a+/+ hearts before ischemia. D: bar graphs show average diastolic rhod-2 Cai2+ levels during preischemia and mean rhod-2 Cai2+ levels at 30 min of ischemia. *P < 0.05 vs. NTG; n = 5/group. E and F: ATP-dependent SR Ca2+ uptake activity in NTG and SERCA1a+/+ hearts, respectively. The rate of SR Ca2+ uptake increased significantly over a wide range of free Ca2+ concentration (pCa 7.5 to 5.5) at both preischemia and ischemia. The average of 3 separate experiments, each performed in duplicate, is shown. IS, ischemia; PI, preischemia.

 
The ATP-dependent Ca2+ uptake was determined in NTG (Fig. 7E) and SERCA1a+/+ (Fig. 7F) cardiac homogenates from nonischemic (30-min equilibration) hearts and ischemic (30-min global ischemia) hearts. The rate of SR Ca2+ uptake in SERCA1a+/+ hearts was increased significantly over a wide range of free Ca2+ concentration (pCa 7.5 to 5.5) at both preischemia and ischemia. The maximal velocity of SR Ca2+ uptake was less affected by ischemia in SERCA1a+/+ hearts (181 to 157 nmol·mg–1·min–1, or ~13%) than that observed in NTG hearts (40 to 28 nmol·mg–1·min–1, or ~29%).

Myocardial infarction after in vivo I/R. Myocardial infarction (Fig. 8, A–C) was assessed 24 h after 30 min of LAD ligation. Under anesthesia, both the NTG and SERCA1a+/+ mice had blood pressure between 70 and 75 mmHg and heart rate between 335 and 400 beats/min, as reported previously (18). Although NTG and SERCA1a+/+ hearts exhibited similar values (~65% in both strains) for AAR/LV (Fig. 8B), much smaller infarct size was observed in the SERCA1a+/+ hearts with little discernable infarction (Fig. 8, A and C). The calculated infarct area per AAR was 12 ± 4.5% in NTG hearts and 0.6 ± 0.4% in SERCA1a+/+ hearts (P < 0.05). Of note, the FVB/N strain has been shown to be relatively resistant to in vivo postischemic injury with smaller infarction seen than in other mouse strains, such as C57BL/6 (29).


Figure 8
View larger version (38K):
[in this window]
[in a new window]

  		Fig. 8. Measurement of infarct size in hearts subjected to in vivo regional myocardial ischemia and reperfusion. Evans blue infusion was performed to visualize the nonrisk region and TTC staining to visualize infarction (A). Percentages of LV area at risk (AAR/LV) and myocardial infarct size over the area at risk (IA/AAR) are shown in B and C, respectively. Values are means ± SE. *P < 0.05 vs. NTG; n = 5/group.

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The major goal of this study was to determine whether cardiac expression of SERCA1a can protect the heart against I/R injury and the mechanism of this protection. We observed that SERCA1a+/+ expression resulted in 1) accelerated and markedly improved postischemic myocardial contractile function, 2) diminished incidence of abnormal ventricular rhythms upon reperfusion, 3) significantly smaller myocardial infarct size, and 4) significantly reduced accumulation of intracellular Ca2+ in the postischemic hearts compared with NTG mice. Concordant with these in vitro findings, we also observed much less myocardial infarction in SERCA1a+/+ mice compared with NTG mice subjected to in vivo regional I/R. Thus these results verify the hypothesis that upregulation of SERCA function is beneficial for postischemic myocardial recovery and salvage, and they suggest that expression of SERCA1a could substitute for SERCA2a under conditions where SERCA2a expression and/or activity are reduced. The mechanism of protection was shown to be primarily due to enhanced Ca2+ handling with prevention of postischemic cytosolic Ca2+ overload.

Beneficial role of SERCA upregulation in cardiac protection. Whereas increased SERCA2a expression improves Ca2+ cycling and cardiac function in different experimental conditions (3, 15, 31, 33), the use of SERCA1a to improve cardiac contractility has been only recently attempted (8). This is the first study to investigate the efficacy of cardiac SERCA1a overexpression in protecting the heart against I/R injury both in vitro and in vivo. We chose to study the effect of SERCA1a, since it has been shown to have higher Ca2+ transport velocity than SERCA2a and is associated with faster rates of contraction and relaxation in adult cardiomyocytes compared with equivalent SERCA2a overexpression (3, 6, 26, 40). Importantly, diastolic intracellular Ca2+ is decreased with SERCA1a expression (8) compared with SERCA2a overexpression (31).

Consistent with previous reports on various myocardial preparations (17–19, 23, 26), we also observed markedly enhanced baseline contractile function (LVDP) with significantly faster rates of contraction and relaxation in SERCA1a+/+ hearts compared with NTG hearts (Table 1). Interestingly, baseline coronary flow was significantly higher in SERCA1a+/+ compared with NTG hearts. Recently, SERCA2a gene transfer also has been shown to increase the coronary blood flow in rat heart (38). The enhanced contractility in SERCA1a+/+ hearts was associated with markedly higher rates of SR Ca2+ uptake (Fig. 6, E and F), although heart rates were identical in the two strains. SERCA1a+/+ hearts have reduced levels of SERCA2a (Fig. 1) and its reversible inhibitor, phospholamban (PLB) (18). Thus, with SERCA1a expression, there is an increased SERCA/PLB ratio in favor of more free SERCA1a pump, and this in addition to the faster kinetics of SERCA1 likely explains the enhanced baseline contractile function in SERCA1a+/+ hearts.

Myocardial I/R injury is associated with severe arrhythmias, contractile dysfunction, and myocardial death (5, 32, 42); therefore, reduction of arrhythmias and limitation of myocardial infarction are of paramount importance. With reperfusion following 30 min of global ischemia, we observed that SERCA1a+/+ hearts started to beat much earlier with diminished incidence of arrhythmias and postischemic contracture compared with NTG hearts (Table 1 and Fig. 2B). This improved postischemic contractile recovery in SERCA1a+/+ hearts was associated with a 2.5-fold increase in the recovery of LVDP (Fig. 2A) and an ~2-fold decrease in myocardial infarct size compared with NTG hearts (Fig. 2E). Concordantly, LVEDP after 45 min of reperfusion was ~2-fold lower in SERCA1a+/+ hearts (Fig. 2B), and both maximal +dP/dt and –dP/dt were 8- and 6-fold higher respectively in SERCA1a+/+ hearts (Table 1). The decreased infarct size and improved contractile recovery in SERCA1a+/+ hearts are due to greater preservation of myocardial integrity with less myocardial structural damage seen by light and electron microscopy (Figs. 3 and 4). These striking differences between SERCA1a+/+ and NTG hearts during reperfusion clearly show that SERCA1a expression imparts prominent myocardial protection despite the 50% reduction in constitutive SERCA2a.

Consistent with the isolated heart data, our studies in an in vivo regional model of I/R also showed markedly smaller infarct size in SERCA1a+/+ hearts than in NTG hearts, demonstrating that expression of SERCA1a protects against myocardial injury in the complex in vivo situation. Thus these findings further extend our understanding that postischemic myocardial recovery is closely related to the functional levels of SERCA and that preservation or augmentation of SR Ca2+ uptake activity can profoundly protect the heart and prevent contractile dysfunction and myocyte death.

Together, our findings with SERCA1a expression are consistent with earlier studies showing that overexpression of SERCA2a confers myocardial protection with enhanced contractile function (9). The observed cardioprotective effect of SERCA1a is not model specific, because prominent reduction of infarct size occurred in both in vitro and in vivo models.

SERCA overexpression and myocardial Ca2+ handling. Intracellular Ca2+ overload is one of the major mechanisms of myocardial I/R injury (32, 37, 42). Cardiac contracture observed with ischemia and reperfusion is mainly due to a rise in intracellular Ca2+ levels (1, 32). It has been reported that SERCA1a+/+ hearts can tolerate supraphysiological levels of calcium without any signs of Ca2+ overload or failure (18). Despite larger baseline LVDP and larger peak systolic Ca2+ fluorescence (Fig. 7C), LVEDP in SERCA1a+/+ hearts after 30 min of ischemia was not greater than in NTG hearts. Importantly, LVEDP rapidly decreased upon reperfusion in SERCA1a+/+ hearts, whereas a further rise was seen in NTG hearts (Fig. 2B). This salutary effect is likely due to increased Ca2+ uptake activity in SERCA1a+/+ hearts, because we noted significantly lower both preischemic diastolic and mean ischemic (30 min) rhod-2 Ca2+ fluorescence in SERCA1a+/+ hearts compared with NTG hearts (Fig. 7D). Thus our results directly show for the first time that the overexpression of SERCA improves intracellular Ca2+ handling during acute myocardial ischemia.

In this context, we observed that the rate of SR Ca2+ uptake in SERCA1a+/+ hearts was much higher than in NTG hearts both at baseline and following 30 min of ischemia (Fig. 7, E and F). Consistent with prior reports that Ca2+-ATPase activity is decreased following ischemia (22), we also observed that the maximal velocity of SR Ca2+ uptake was decreased in postischemic NTG hearts with >25% loss of activity compared with <15% loss of activity in SERCA1a+/+ hearts. Together, these data suggest that expression of SERCA1a is effective in preserving SR Ca2+ uptake during I/R, and thus in decreasing intracellular Ca2+ overload.

SERCA upregulation and postischemic reactive oxygen and nitrogen species. The burst of reactive oxygen species during reperfusion is another key central mechanism of reperfusion injury. Potent oxidants and free radicals such as superoxide anion, hydroxyl radical, and peroxynitrite are formed during postischemic reperfusion and reach their highest concentrations during the first minute of reperfusion (4, 49). Oxygen-derived free radicals have been reported to damage SERCA, potentially contributing to Ca2+ overload with concurrent myocardial damage and ventricular arrhythmias upon reperfusion (16, 22). With EPR spin-trapping studies, similar DMPO-OH adduct signals, indicative of superoxide-derived hydroxyl radicals (49), were seen in both NTG and SERCA1a+/+ hearts over the first minute of reperfusion (Fig. 5), and the observed levels of radical generation were indistinguishable between the two strains. Importantly, despite comparable radical generation, SERCA1a+/+ hearts exhibited enhanced recovery of postischemic contractile function and smaller infarct size. Consistent with our findings, a recent study has shown that SERCA1a expression can protect the heart from hydroxyl radical-induced injury (17). Thus it is evident that the postischemic functional recovery in SERCA1a+/+ hearts would be less affected by oxygen radical generation during reperfusion.

It has been shown that the burst of superoxide during the early period of reperfusion reacts with nitric oxide to form the reactive nitrogen species peroxynitrite in the myocardium, subsequently aggravating reperfusion injury (4, 24, 45, 49, 50). Interestingly, it has been reported that exogenous peroxynitrite-induced nitration of SERCA2a is associated with a parallel loss of SERCA2a activity in skeletal muscle, whereas SERCA1a did not become nitrated (43). Recently, it has been reported that increased nitration of SERCA2a protein causes impaired cardiac relaxation in heart failure patients (25). Although we observed a similar degree of nitrotyrosine staining in the postischemic hearts of both strains (Fig. 6, E and F), SERCA1a+/+ hearts displayed improved postischemic contractile recovery with significantly lower LVEDP. Thus the enhanced ability to pump back Ca2+ into the SR and the resultant lower cytosolic Ca2+ levels do not affect the magnitude of free radical generation or peroxynitrite-mediated nitration.

Implications of SERCA modulation in cardioprotection. Since defective SR Ca2+ handling plays a major role in myocardial I/R-induced contractile dysfunction and arrhythmias (32, 37, 42, 48), the SR has recently been proposed as a critical primary target for reperfusion protection (36). Although currently available drugs do not improve SR Ca2+ uptake, in both transgenic mice and adenovirus-mediated cardiac myocytes, overexpression of SERCA2a or SERCA1a resulted in increased Ca2+ uptake activity, Ca2+ transient amplitude and SR Ca2+ content with increased contractility (3, 8, 13, 26, 32, 40). In this study, we have demonstrated that expression of the higher-velocity SERCA1a isoform not only can substitute for SERCA2a with enhanced SR Ca2+ uptake activity (19, 23, 25) but also strongly protects the heart against I/R injury with efficient Ca2+ removal capacity under oxidative stress. On the basis of our overall findings, we can understand the mechanisms by which SERCA overexpression enhances myocardial contractility under normal physiological conditions and how it is effective in preserving contractile function following I/R (Fig. 9). In conclusion, the present study provides clear direct evidence that cardiac expression of SERCA1a, independent of the effect of oxidant stress that accompanies reperfusion, confers potent myocardial protection against I/R injury by reducing intracellular Ca2+ overload.


Figure 9
View larger version (15K):
[in this window]
[in a new window]

  		Fig. 9. Mechanisms by which SERCA overexpression enhances myocardial contractility under normal physiological conditions and preserves contractile function following ischemia-reperfusion.

 

   GRANTS
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by National Heart, Lung, and Blood Institute Grants HL-63744, HL-65608, HL-38324, and HL-64140. P. Bhupathy is supported by an American Heart Association Predoctoral Fellowship (AHA-0415170B).


   FOOTNOTES
 

Address for reprint requests and other correspondence: J. L. Zweier, Davis Heart and Lung Research Institute, The Ohio State Univ., 473 West 12th Ave., Columbus, OH 43210 (e-mail: jay.zweier{at}osumc.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
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Abdallah Y, Gkatzoflia A, Pieper H, Zoga E, Walther S, Kasseckert S, Schäfer M, Schlüter KD, Piper HM, Schäfer C. Mechanism of cGMP-mediated protection in a cellular model of myocardial ischemia reperfusion injury. Cardiovasc Res 66: 123–131, 2005.[Abstract/Free Full Text]
  2. Avellanal M, Rodriguez P, Barrigon S. Protective effects of cyclopiazonic acid on ischemia-reperfusion injury in rabbit hearts. J Cardiovasc Pharmacol 32: 845–851, 1998.[CrossRef][Web of Science][Medline]
  3. Baker DL, Hashimoto K, Grupp IL, Ji Y, Reed T, Loukianov E, Grupp G, Bhagwhat A, Hoit B, Walsh R, Marban E, Periasamy M. Targeted overexpression of the sarcoplasmic reticulum Ca2+-ATPase increases cardiac contractility in transgenic mouse hearts. Circ Res 83: 1205–1214, 1998.[Abstract/Free Full Text]
  4. Bolli R, Jeroudi MO, Patel BS, DuBose CM, Lai EK, Roberts R, McCay PB. Direct evidence that oxygen-derived free radicals contribute to postischemic myocardial dysfunction in the intact dog. Proc Natl Acad Sci USA 86: 4695–4699, 1989.[Abstract/Free Full Text]
  5. Bolli R, Becker L, Gross G, Mentzer R Jr, Balshaw D, Lathrop DA. Myocardial protection at a crossroads: the need for translation into clinical therapy. Circ Res 95: 125–134, 2004.[Abstract/Free Full Text]
  6. Cavagna M, O'Donnell JM, Sumbilla C, Inesi G, Klein MG. Exogenous Ca2+-ATPase isoform effects on Ca2+ transients of embryonic chicken and neonatal rat cardiac myocytes. J Physiol 528: 53–63, 2000.[Abstract/Free Full Text]
  7. Chen Y, Escoubet B, Prunier F, Amour J, Simonides WS, Vivien B, Lenoir C, Heimburger M, Choqueux C, Gellen B, Riou B, Michel JB, Franz WM, Mercadier JJ. Constitutive cardiac overexpression of sarcoplasmic/endoplasmic reticulum Ca2+-ATPase delays myocardial failure after myocardial infarction in rats at a cost of increased acute arrhythmias. Circulation 109: 1898–1903, 2004.[Abstract/Free Full Text]
  8. Chossat N, Griscelli F, Jourdon P, Logeart D, Ragot T, Heimburger M, Perricaudet M, Lompré Hatem S, Mercadier JJ. Adenoviral SERCA1a gene transfer to adult rat ventricular myocytes induces physiological changes in calcium handling. Cardiovasc Res 49: 288–297, 2001.[Abstract/Free Full Text]
  9. Del Monte F, Lebeche D, Guerrero JL, Tsuji T, Doye AA, Gwathmey JK, Hajjar RJ. Abrogation of ventricular arrhythmias in a model of ischemia and reperfusion by targeting myocardial calcium cycling. Proc Natl Acad Sci USA 101: 5622–5627, 2004.[Abstract/Free Full Text]
  10. Del Nido PJ, Glynn P, Buenaventura P, Salama G, Koretsky AP. Fluorescence measurement of calcium transients in perfused rabbit heart using rhod 2. Am J Physiol Heart Circ Physiol 274: H728–H741, 1998.[Abstract/Free Full Text]
  11. Du C, MacGowan GA, Farkas DL, Koretsky AP. Calcium measurements in perfused mouse heart: quantitating fluorescence and absorbance of Rhod-2 by application of photon migration theory. Biophys J 80: 549–561, 2001.[Web of Science][Medline]
  12. Du Toit EF, Opie LH. Inhibitors of Ca2+ ATPase pump of sarcoplasmic reticulum attenuate reperfusion stunning in isolated rat heart. J Cardiovasc Pharmacol 24: 678–684, 1994.[CrossRef][Web of Science][Medline]
  13. Frank KF, Bölck B, Erdmann E, Schwinger RHG. Sarcoplasmic reticulum Ca2+-ATPase modulates cardiac contraction and relaxation. Cardiovasc Res 57: 20–27, 2003.[Abstract/Free Full Text]
  14. Hampton TG, Amende I, Travers KE, Morgan JP. Intracellular calcium dynamics in mouse model of myocardial stunning. Am J Physiol Heart Circ Physiol 274: H1821–H1827, 1998.[Abstract/Free Full Text]
  15. He H, Giordano FJ, Hilal-Dandan R, Choi DJ, Rockman HA, McDonough PM, Bluhm WF, Meyer M, Sayen MR, Swanson E, Dillmann 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.[Web of Science][Medline]
  16. Hess ML, Manson NH. Molecular oxygen: friend and foe. The role of the oxygen free radical system in the calcium paradox, the oxygen paradox and ischemia/reperfusion injury. J Mol Cell Cardiol 16: 969–985, 1984.[CrossRef][Web of Science][Medline]
  17. Hiranandani N, Bupha-Intr T, Janssen PML. SERCA overexpression reduces hydroxyl radical injury in murine myocardium. Am J Physiol Heart Circ Physiol 291: H3130–H3135, 2006.[Abstract/Free Full Text]
  18. Huke S, Prasad V, Nieman ML, Nattamai KJ, Grupp IL, Lorenz JN, Periasamy M. Altered dose response to beta-agonists in SERCA1a-expressing hearts ex vivo and in vivo. Am J Physiol Heart Circ Physiol 283: H958–H965, 2002.[Abstract/Free Full Text]
  19. Ji Y, Loukianov E, Loukianova T, Jones LR, Periasamy M. SERCA1a can functionally substitute for SERCA2a in the heart. Am J Physiol Heart Circ Physiol 276: H89–H97, 1999.[Abstract/Free Full Text]
  20. Ji Y, Loukianov E, Periasamy M. Analysis of sarcoplasmic reticulum Ca2+ transport and Ca2+ ATPase enzyme properties using mouse cardiac tissue homogenates. Anal Biochem 269: 236–244, 1999.[CrossRef][Web of Science][Medline]
  21. Kawahara K, Takase M, Yamauchi Y. Increased vulnerability to ischemia/reperfusion-induced ventricular tachyarrhythmias by pre-ischemic inhibition of nitric oxide synthase in isolated rat hearts. Cardiovasc Pathol 12: 49–56, 2003.[CrossRef][Web of Science][Medline]
  22. Krause S, Hess ML. Characterization of cardiac sarcoplasmic reticulum dysfunction during short-term, normothermic, global ischemia. Circ Res 55: 176–184, 1984.[Abstract/Free Full Text]
  23. Lalli MJ, Yong J, Prasad V, Hashimoto K, Plank D, Babu GJ, Kirkpatrick D, Walsh RA, Sussman M, Yatani A, Marban E, Periasamy M. Sarcoplasmic reticulum Ca2+ ATPase (SERCA) 1a 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]
  24. Liu P, Hock CE, Nagele R, Wong PY. Formation of nitric oxide, superoxide, and peroxynitrite in myocardial ischemia-reperfusion injury in rats. Am J Physiol Heart Circ Physiol 272: H2327–H2336, 1997.[Abstract/Free Full Text]
  25. Lokuta AJ, Maertz NA, Meethal SV, Potter KT, Kamp TJ, Valdivia HH, Haworth RA. Increased nitration of sarcoplasmic reticulum Ca2+-ATPase in human heart failure. Circulation 111: 988–995, 2005.[Abstract/Free Full Text]
  26. Loukianov E, Ji Y, Grupp IL, Kirkpatrick DL, Baker DL, Loukianova T, Grupp G, Lytton J, Walsh RA, 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]
  27. MacGowan GA, Kirk JA, Evans C, Shroff SG. Pressure-calcium relationships in perfused mouse heart. Am J Physiol Heart Circ Physiol 290: H2614–H2624, 2006.[Abstract/Free Full Text]
  28. Merillat JC, Lakatta EG, Hano O, Guarnieri T. Role of calcium and the calcium channel in the initiation and maintenance of ventricular function. Circ Res 67: 1115–1123, 1990.[Abstract/Free Full Text]
  29. Miller DL, Van Winkle DM. Ischemic preconditioning limits infarct size following regional ischemia-reperfusion in in situ mouse hearts. Cardiovasc Res 42:680–684, 1999.[Abstract/Free Full Text]
  30. Misquitta CM, Mack DP, Grover AK. Sarco/endoplasmic reticulum Ca2+ (SERCA)-pumps: link to heart beats and calcium waves. Cell Calcium 25: 277–290, 1999.[CrossRef][Web of Science][Medline]
  31. Miyamoto MI, del Monte F, Schmidt U, DiSalvo TS, Kang ZB, Matsui T, Guerrero JL, Gwatghmey JK, Rosenzweig A, 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]
  32. Moens AL, Claeys MJ, Timmermans JP, Vrints CJ. Myocardial ischemia/reperfusion-injury, a clinical view on a complex pathophysiological process. Int J Cardiol 100: 179–190, 2005.[CrossRef][Web of Science][Medline]
  33. Muller OJ, Lange M, Rattunde H, Lorenzen HP, Müller M, Frey N, Bittner C, Simonides W, Katus HA, Franz WM. Transgenic rat hearts overexpressing SERCA2a show improved contractility under baseline conditions and pressure overload. Cardiovasc Res 59: 380–389, 2003.[CrossRef][Web of Science][Medline]
  34. Nwasokwa ON. Mechanism of mechanical alternans in ischemia-reperfusion: role of deficient relaxation of the strong twitch. Am J Physiol Heart Circ Physiol 269: H169–H175, 1995.[Abstract/Free Full Text]
  35. Periasamy M, Huke S. SERCA pump level is a critical determinant of Ca2+ homeostasis and cardiac contractility. J Mol Cell Cardiol 33: 1053–1063, 2001.[CrossRef][Web of Science][Medline]
  36. Piper HM, Kasseckert Abdallah Y. The sarcoplasmic reticulum as the primary target of reperfusion injury. Cardiovasc Res 70: 170–173, 2006.[Free Full Text]
  37. Saini HK, Dhalla NS. Defective calcium handling in cardiomyocytes isolated from hearts subjected to ischemia-reperfusion. Am J Physiol Heart Circ Physiol 288: H2260–H2270, 2005.[Abstract/Free Full Text]
  38. Sakata S, Lebeche D, Sakata Y, Sakata N, Chemaly ER, Liang L, Nakajima-Takenaka C, Tsuji T, Konishi N, del Monte F, Hajjar RJ, Takaki M. Transcoronary gene transfer of SERCA2a increases coronary blood flow and decreases cardiomyocyte size in a Type 2 diabetic rat model. Am J Physiol Heart Circ Physiol 292: H1204–H1207, 2007.[Abstract/Free Full Text]
  39. Stokes DL, Wagenknecht T. Calcium transport across the sarcoplasmic reticulum: structure and function of Ca2+-ATPase and ryanodine receptor. Eur J Biochem 267: 5274–5279, 2000.[Web of Science][Medline]
  40. Sumbilla C, Cavagna M, Zhong L, Ma H, Lewis D, Farrance I, Inesi G. Comparison of SERCA1 and SERCA2a expressed in COS-1 cells and cardiac myocytes. Am J Physiol Heart Circ Physiol 277: H2381–H2391, 1999.[Abstract/Free Full Text]
  41. Talukder MAH, Morrison RR, Mustafa SJ. Comparison of the vascular effects of adenosine in isolated mouse heart and aorta. Am J Physiol Heart Circ Physiol 282: H49–H57, 2002.[Abstract/Free Full Text]
  42. Verma S, Fedak PW, Weisel RD, Butany J, Rao V, Maitland A, Li RK, Dhillon B, Yau TM. Fundamentals of reperfusion injury for the clinical cardiologist. Circulation 105: 2332–2336, 2002.[Free Full Text]
  43. Viner RI, Ferrington DA, Williams TD, Bigelow DJ, Schöneich C. Protein modification during biological aging: selective tyrosine nitration of the SERCA2a isoform of the sarcoplasmic reticulum Ca2+-ATPase in skeletal muscle. Biochem J 340: 657–669, 1999.[CrossRef][Web of Science][Medline]
  44. Wang P, Chen H, Qin H, Sankarapandi S, Becher MW, Wong PC, Zweier JL. Overexpression of human copper,zinc-superoxide dismutase (SOD1) prevents postischemic injury. Proc Natl Acad Sci USA 95: 4556–4560, 1998.[Abstract/Free Full Text]
  45. Wang P, Zweier JL. Measurement of nitric oxide and peroxynitrite generation in the postischemic heart. Evidence for peroxynitrite-mediated reperfusion injury. J Biol Chem 271: 29223–29230, 1996.[Abstract/Free Full Text]
  46. Wolosker H, Rocha JBT, Engelender S, Panizzutti R, Miranda JD, de Meis L. Sarco/endoplasmic reticulum Ca2+-ATPase isoforms: diverse responses to acidosis. Biochem J 321: 545–550, 1997.[Web of Science][Medline]
  47. Zhao X, Chen YR, He G, Zhang A, Druhan LJ, Strauch AR, Zweier JL. Endothelial nitric oxide synthase (NOS3) knockout decreases NOS2 induction, limiting hyperoxygenation and conferring protection in the postischemic heart. Am J Physiol Heart Circ Physiol 292: H1541–H1550, 2007.[Abstract/Free Full Text]
  48. Zucchi R, Ronca F, Ronca-Testoni S. Modulation of sarcoplasmic reticulum function: a new strategy in cardioprotection? Pharmacol Ther 89: 47–65, 2001.[CrossRef][Web of Science][Medline]
  49. Zweier JL. Measurement of superoxide-derived free radicals in reperfused heart: evidence for a free radical mechanism of reperfusion injury. J Biol Chem 263: 1353–1357, 1988.[Abstract/Free Full Text]
  50. Zweier JL, Fertmann J, Wei G. Nitric oxide and peroxynitrite in postischemic myocardium. Antioxid Redox Signal 3: 11–22, 2001.[CrossRef][Web of Science][Medline]



This article has been cited by other articles:


Home page
 Hum Mol GenetHome page
G. Coppola, D. Marmolino, D. Lu, Q. Wang, M. Cnop, M. Rai, F. Acquaviva, S. Cocozza, M. Pandolfo, and D. H. Geschwind
Functional genomic analysis of frataxin deficiency reveals tissue-specific alterations and identifies the PPAR{gamma} pathway as a therapeutic target in Friedreich's ataxia
Hum. Mol. Genet., July 1, 2009; 18(13): 2452 - 2461.
[Abstract] [Full Text] [PDF]

Home page
 Circ. Res.Home page
P. Nicolaou, P. Rodriguez, X. Ren, X. Zhou, J. Qian, S. Sadayappan, B. Mitton, A. Pathak, J. Robbins, R. J. Hajjar, et al.
Inducible Expression of Active Protein Phosphatase-1 Inhibitor-1 Enhances Basal Cardiac Function and Protects Against Ischemia/Reperfusion Injury
Circ. Res., April 24, 2009; 104(8): 1012 - 1020.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
M. A. H. Talukder, A. Kalyanasundaram, L. Zuo, M. Velayutham, Y. Nishijima, M. Periasamy, and J. L. Zweier
Is reduced SERCA2a expression detrimental or beneficial to postischemic cardiac function and injury? Evidence from heterozygous SERCA2a knockout mice
Am J Physiol Heart Circ Physiol, March 1, 2008; 294(3): H1426 - H1434.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
293/4/H2418    most recent
00663.2007v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Web of Science (5)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Talukder, M. A. H.
Right arrow Articles by Zweier, J. L.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Talukder, M. A. H.
Right arrow Articles by Zweier, J. L.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2007 by the American Physiological Society.