AJP - Heart Calcium Transients and Cell-Sarcomere
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Heart Circ Physiol 294: H1426-H1434, 2008. First published January 18, 2008; doi:10.1152/ajpheart.01016.2007
0363-6135/08 $8.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
294/3/H1426    most recent
01016.2007v1
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 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.

Is reduced SERCA2a expression detrimental or beneficial to postischemic cardiac function and injury? Evidence from heterozygous SERCA2a knockout mice

M. A. Hassan Talukder,1 Anuradha Kalyanasundaram,2 Li Zuo,1 Murugesan Velayutham,1 Yoshinori Nishijima,1 Muthu Periasamy,2 and Jay L. Zweier1,2

1Davis Heart and Lung Research Institute and The 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

Submitted 3 September 2007 ; accepted in final form 17 January 2008


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Recent studies have demonstrated that increased expression of sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) 2a improves myocardial contractility and Ca2+ handling at baseline and in disease conditions, including myocardial ischemia-reperfusion (I/R). Conversely, it has also been reported that pharmacological inhibition of SERCA might improve postischemic function in stunned hearts or in isolated myocardium following I/R. The goal of this study was to test how decreases in SERCA pump level/activity affect cardiac function following I/R. To address this question, we used a heterozygous SERCA2a knockout (SERCA2a+/–) mouse model with decreased SERCA pump levels and studied the effect of myocardial stunning (20-min ischemia followed by reperfusion) and infarction (30-min ischemia followed by reperfusion) following 60-min reperfusion. Our results demonstrate that postischemic myocardial relaxation was significantly impaired in SERCA2a+/– hearts with both stunning and infarction protocols. Interestingly, postischemic recovery of contractile function was comparable in SERCA2a+/– and wild-type hearts subjected to stunning. In contrast, following 30-min ischemia, postischemic contractile function was reduced in SERCA2a+/– hearts with significantly larger infarction. Rhod-2 spectrofluorometry revealed significantly higher diastolic intracellular Ca2+ in SERCA2a+/– hearts compared with wild-type hearts. Both at 30-min ischemia and 2-min reperfusion, intracellular Ca2+ levels were significantly higher in SERCA2a+/– hearts. Electron paramagnetic resonance spin trapping showed a similar extent of postischemic free-radical generation in both strains. These data provide direct evidence that functional SERCA2a level, independent of oxidative stress, is crucial for postischemic myocardial function and salvage during I/R.

myocardial ischemia-reperfusion; sarco(endo)plasmic reticulum Ca2+-ATPase; calcium handling; free radicals; postischemic function

THE SARCO(ENDO)PLASMIC RETICULUM Ca2+-ATPase (SERCA) is primarily responsible for sarcoplasmic reticulum (SR) Ca2+ uptake during the contraction-relaxation cycle of the heart (15). SR Ca2+ uptake activity not only determines the speed of muscle relaxation but also the SR Ca2+ content and therefore the amount of Ca2+ released for subsequent contraction (27). Acute myocardial ischemia-reperfusion (I/R) injury is associated with contractile dysfunction, arrhythmias, myocardial infarction, and sudden death (5). Several studies suggest that intracellular Ca2+ overload with reduced expression and/or activity of SERCA2a plays a prominent role in myocardial I/R injury (29, 35, 44). 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 (19, 22, 48, 49). Thus cytosolic free Ca2+ overload and oxidative stress, either independently or cooperatively, are major contributors to I/R-induced injury.

It is now well established that increased expression of SERCA2a improves myocardial contractility and Ca2+ handling at baseline and in disease conditions including myocardial I/R (2, 8, 10, 18, 28). Conversely, it has also been reported that pharmacological inhibition of SERCA might improve postischemic function in the stunned heart or in isolated myocardium following I/R (1, 13, 47). However, the results with the SERCA inhibitors thapsigargin and cyclopiazonic acid (CPA) were inconsistent in terms of effectiveness, narrow dose range, and timing of administration (1, 13). The advent of gene-modified models with either targeted deletion or transgenic overexpression of SERCA has improved the characterization of cardiac effects more directly in different experimental settings (20, 32, 37, 43). Therefore, to further explore this issue, we utilized heterozygous SERCA2a knockout (SERCA2a+/–) mice with decreased SERCA levels (21, 31) to study the effect of reduced SERCA2a expression and activity on postischemic myocardial function and injury.

It was observed that reduction in SERCA2a expression in the heart worsened the postischemic myocardial recovery with impaired postischemic diastolic function, increased intracellular Ca2+ overload, and enlarged myocardial infarction.


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

Mice. The generation and characterization of heterozygous SERCA2a+/– mice have been described previously (31). Deletion of both alleles was lethal, so experiments were performed on SERCA2a+/– mice. SERCA2a+/– mice have a normal life span and reproduce well. SERCA2a+/– mice were bred with Black Swiss mice to establish germ-line transmission. Each mouse was genotyped when it was weaned, and the respective littermates served as controls for SERCA2a+/– mice. The experiments were performed in young (16–20 wk) male mice, and tail clips were kept to reconfirm the genotypes.

Echocardiographic evaluation of left ventricle function. In vivo cardiac dimension and contractile function were evaluated by transthoracic M-mode echocardiography under light isoflurane anesthesia as described previously (41). In a related study, we checked the concentration-dependent effects of isoflurane on the heart rate and contractility in C57BL mice. Isoflurane markedly decreased both heart rate and fractional shortening in a concentration-dependent manner. Heart rate with 5, 3, and 1% isoflurane was 263, 361, and 503 bpm, respectively. Similarly, fractional shortening with 5, 3, and 1% isoflurane was 25, 36, and 54%, respectively. Therefore, in the present study, we took care to use isoflurane levels of 1% or less. Briefly, with a GE Vivid7 echocardiography system and intraoperative epicardial probe (Model i13L; frequency 14 MHz), the two-dimensional short-axis view was used as a guide, and left ventricle (LV) M-mode tracings were obtained close to the papillary muscle. LV end-diastolic and end-systolic internal diameter (LVEDD and LVESD, respectively) were measured with the American Society of Echocardiography leading-edge method (33). LV fractional shortening (%) was calculated as (LVEDD – LVESD)/LVEDD x 100.

Langendorff perfused heart preparation. Hearts were isolated from age-matched mice of both strains as described previously (43, 45). Briefly, mice were anesthetized with pentobarbital (50 mg/kg ip), hearts were excised, and aorta were cannulated and 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 mmol/l): 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 LV across the mitral valve and was 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 and LV developed pressures (LVDP) were recorded on a PowerLab/400 multichannel data-acquisition system (ADInstruments, Castle Hill, Australia). The LVP signal was digitally processed (PowerLab Chart v. 4.2; ADInstruments) to yield diastolic and systolic pressures as well as heart rate. LVDP was calculated as the difference between systolic and diastolic pressures. Changes in the LVDP and LV systolic pressure were expressed as percent change from preischemic (PI) baseline value.

Following 30 min of equilibration, hearts underwent either 20 or 30 min of global ischemia, followed by 60 min of reperfusion. At the end of the 60-min reperfusion, hearts were processed for myocardial infarct size measurement and histopathological examination. One subset of hearts underwent only 5 min of reperfusion to measure free-radical generation.

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 (43, 45). Briefly, the heart was immediately removed after I/R, wrapped in polyethylene wrap, and frozen for 10 min for hardening. Then the heart was serially sectioned into transverse slices (1 mm) by a heart slicer (Zivic Laboratories) and was 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 were then fixed overnight in 10% neutral-buffered formaldehyde for better color contrast and were digitally imaged. Computerized planimetry (with image-analysis software Meta Vue, v. 6.0) of each section was used to determine percent infarction from the total cross-sectional area of the LV.

Light microscopy. Standard hematoxylin and eosin (H&E) 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 by using MetaMorph 7.0r4 (Universal Imaging).

Rhod-2 spectrofluorometry. Intracellular free Ca2+ (Ca2+i) 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 (11, 12, 25, 43). 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 Ca2+i (12), the estimation of its value allows monitoring the Ca2+i changes over time.

A fiberoptic 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. These two milliliters of rhod-2 containing perfusate were infused without recirculation through a parallel infusion line just above the aortic cannula. During bolus infusion, 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 washout. 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 washout 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), 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 computer by using an analog-to-digital converter. The emitted signal was detected, digitized, and recorded at a rate of 125 Hz for analysis (FluorMeasure v. 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 washout (37°C), Ca2+i fluorescence data was acquired. Then the heart was subjected to 30-min global ischemia, and Ca2+i fluorescence data was collected at end ischemia just before reperfusion. With this model, gradual loss and washout of the fluorescence dye occurs following several minutes of reperfusion and thus precludes subsequent measurement of Ca2+i fluorescence. Therefore, Ca2+i-fluorescence data was collected just until 2 min of reperfusion.

Ca2+i was calculated according to previous literature (11) with rhod-2 calibration in the isolated heart; that is: Ca2+i = Kd (F – Fmin)/(Fmax – F), where Kd (710 nM) is the dissociation coefficient for rhod-2, 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 blank fluorescence was acquired before rhod-2 loading, and the maximal fluorescence was obtained after 15 µM digitonin (Sigma) infusion at the end of each experiment. The calculated myocardial Ca2+i fluorescence in our experimental conditions with control Black Swiss mice were 388 ± 27 nM and 652 ± 18 nM in diastole and systole, respectively. These results agree well with those previously reported with rhod-2 (12, 43) and aequorin (17) in the mouse heart. Although sharp rise and rapid fall of amplitude for rhod-2 Ca2+ 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. Therefore, we took the mean of rhod-2 Ca2+ fluorescence at 30 min of ischemia and termed this mean ischemic (IS) Ca2+ fluorescence. The diastolic or mean IS levels of rhod-2 fluorescence were used to evaluate Ca2+i changes and were expressed as relative units (RU).

Electron paramagnetic resonance spectroscopy and spin trapping. Spin-trapping measurements of oxygen radical generation from SERCA2a+/– and WT hearts were performed as described previously (45, 46). 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.

Data analysis. All results are expressed as means ± SE. Data were analyzed either by two-tailed Student's t-test for paired data from the same experiment and unpaired data from different experiments or by 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
 REFERENCES
 
Baseline cardiac function in wild-type and SERCA2a+/– mice. Table 1 summarizes in vivo echocardiographic parameters in anesthetized mice and baseline functional characteristics in isolated hearts perfused at constant pressure. Consistent with a previous report (31), there were no significant differences in echocardiographic parameters between wild-type (WT) and SERCA2a+/– mice, and in vitro baseline coronary flow, LVDP, and intrinsic heart rates were comparable between WT and SERCA2a+/– hearts.


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

  		Table 1. In vivo echocardiographic data, body weight, and baseline hemodynnamic parameters in wild-type and SERCA2a+/– mice

 
Functional effects of I/R in WT and SERCA2a+/– hearts. Cardiac function was assessed from respective PI or IS levels in WT and SERCA2a+/– hearts subjected to 20- or 30-min global ischemia and 60-min reperfusion, stunning or infarction protocol, respectively. In SERCA2a+/– hearts, postischemic ventricular relaxation was significantly impaired in both protocols (Fig. 1, A and B). LV end-diastolic pressure (LVEDP, an index of ventricular relaxation) tended to be higher in SERCA2a+/– hearts during ischemia with both protocols (Fig. 1, C and D). Importantly, on reperfusion, postischemic ventricular relaxation was significantly impaired in SERCA2a+/– hearts with both protocols. Gradual attenuation of LVEDP without postischemic contracture was seen in both WT and SERCA2a+/– hearts after 20-min ischemia (Fig. 1A). In contrast, when the hearts were subjected to 30-min global ischemia, LVEDP sharply rose on reperfusion and remained persistently elevated throughout reperfusion in SERCA2a+/– hearts, whereas it gradually decreased to a significantly lower level in WT hearts (Fig. 1B). Figure 2 shows development of postischemic systolic pressure from PI levels with stunning and infarction protocols. SERCA2a+/– hearts demonstrated increased recovery of systolic pressure in both protocols. The recovery of systolic function in SERCA2a+/– hearts with stunning was slightly but nonsignificantly higher through reperfusion than WT hearts; however, with the infarction protocol WT hearts continued to improve systolic function through reperfusion, and the final recovery was comparable in the two strains. Recovery of postischemic LVDP (an index of global contractile function) from PI level was strikingly different between the two protocols. With the stunning protocol, the magnitude of postischemic LVDP recovery was comparable in both strains (Fig. 3A), whereas with the infarction protocol the magnitude of postischemic LVDP recovery was significantly reduced in SERCA2a+/– hearts (Fig. 3B).


Figure 1
View larger version (19K):
[in this window]
[in a new window]

  		Fig. 1. Ventricular relaxation function in isolated hearts subjected to 20- and 30-min global ischemia (IS) followed by 60-min reperfusion (RP). A and B: time course for recovery of postischemic left ventricle (LV) end-diastolic pressure (LVEDP) after 20- and 30-min global ischemia, respectively. C and D: average LVEDP during preischemia (PI), IS, and RP. Values are means ± SE. Differences in recovery of LVEDP in sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) 2a+/– and wild-type (WT) hearts were significant, with *P < 0.05 (ANOVA); n = 6/group.

 

Figure 2
View larger version (11K):
[in this window]
[in a new window]

  		Fig. 2. Ventricular systolic function in isolated hearts subjected to 20- and 30-min global IS followed by 60-min RP. Time course for recovery of postischemic LV systolic pressure (LVSP) as percentage of preischemic value after 20- (A) and 30-min (B) global IS. Values are means ± SE; n = 6/group.

 

Figure 3
View larger version (7K):
[in this window]
[in a new window]

  		Fig. 3. Myocardial contractile function in isolated hearts subjected to 20- and 30-min global IS followed by 60-min RP. Postischemic contractile recovery as final LVDP after 60-min RP either following 20-min ischemia (stunning protocol; A) or 30-min ischemia (infarction protocol; B). Values are means ± SE; **P < 0.01 vs. WT; n = 6/group.

 
Infarct size in SERCA2a+/– hearts. To correlate postischemic LV function with irreversible myocardial injury, myocardial infarct size was determined after 30-min ischemia and 60-min reperfusion (Fig. 4). TTC staining revealed that isolated SERCA2a+/– hearts had markedly larger LV infarction compared with WT hearts, with infarct size of 26 ± 3% in SERCA2a+/– hearts compared with 13 ± 1% in WT hearts (P < 0.01).


Figure 4
View larger version (18K):
[in this window]
[in a new window]

  		Fig. 4. Myocardial infarction in hearts subjected to 30-min global IS and 60 min RP. Hearts were stained with 2,3,5-triphenyltetrazolium chloride, and representative sections are shown in A. B: percentage of infarct size over total LV area. Values are means ± SE; **P < 0.01 vs. WT; n = 5/group.

 
Structural changes following I/R injury. To determine if alterations in myocardial structure mirrored the differences in functional and infarction data, light-microscopic histological examination was performed. No differences in histological structure were found in H&E-stained sections of PI SERCA2a+/– and WT hearts, and both appeared structurally intact with no evidence of fibrosis (Fig. 5, A and B). After I/R, frequent interstitial spacing, thinning and waving of myofibrils, and contraction band-type changes were evident in SERCA2a+/– hearts (Fig. 5D, blue arrow), but only modest changes were seen in WT hearts (Fig. 5C).


Figure 5
View larger version (125K):
[in this window]
[in a new window]

  		Fig. 5. Representative histological photographs of PI and postischemic hearts from duplicate experiments. PI myocardium in WT (A) and SERCA2a+/– (B) mice are shown. With 60-min RP after 30 min IS, grossly distorted structure with frequent interstitial spacing, thinning and waving of myofibrils, and frequent appearance of contraction bands (blue arrows) are noted in SERCA2a+/– hearts (D), whereas these changes are much less in WT hearts (C). Bar = 50 µM. Magnification, x200.

 
Electron paramagnetic resonance measurements of free-radical generation. To correlate postischemic contractile dysfunction and myocardial infarction with oxidative stress, the magnitude of free-radical generation was determined in postischemic hearts of both strains. Electron paramagnetic resonance (EPR) and spin-trapping studies were performed with coronary effluent using the spin trap DMPO. Before ischemia (PI), no significant radical generation was seen in either strain (Fig. 6, A and B, top traces). However, over the first minute of reperfusion, both SERCA2a+/– and WT hearts exhibited increased free-radical signals consisting of a 1:2:2:1 quartet of DMPO-OH. The reperfusion-associated increase in myocardial free-radical generation was not significantly different between the WT and SERCA2a+/– hearts (Fig. 6, A and B, bottom traces, and Fig. 6C).


Figure 6
View larger version (13K):
[in this window]
[in a new window]

  		Fig. 6. Electron paramagnetic resonance (EPR) spin-trapping measurement of free-radical generation in PI and postischemic hearts. Hearts were infused with 50 mM 5,5-dimethyl-1-pyrroline N-oxide (DMPO) before IS and first 45 s of RP following 30 min of global IS, and measurements were performed with coronary effluents. Representative EPR signals during PI and first minute of RP in WT (A) and SERCA2a+/– hearts (B) are shown. C: average normalized data in arbitrary units (AU) on free-radical generation during RP in both strains. Values are means ± SE; n = 3/group.

 
Myocardial Ca2+ fluorescence levels during PI and I/R. To determine Ca2+i in isolated beating mouse hearts, rhod-2 Ca2+i fluorescence was measured before (PI) and at 30-min of ischemia (IS) and during early reperfusion. Fig. 7A shows averaged rhod-2 Ca2+i fluorescence tracings in both WT and SERCA2a+/– hearts during PI (n = 6). PI diastolic Ca2+i fluorescence (CaF) was markedly higher in the SERCA2a+/– hearts compared with WT hearts (718 ± 72 vs. 388 ± 27 RU, P < 0.01, n = 6), and the peak systolic values were also higher in the SERCA2a+/– hearts compared with WT (981 ± 86 vs. 652 ± 18 RU, P < 0.05, n = 6). Furthermore, mean IS CaF was significantly higher in the SERCA2a+/– hearts compared with WT hearts (1,766 ± 23 vs. 1,463 ± 39 RU, P < 0.001, n = 6; Fig. 7B). At 2-min reperfusion, diastolic CaF remained significantly elevated in the SERCA2a+/– hearts compared with WT hearts (964 ± 120 vs. 624 ± 58 RU, P < 0.05, n = 6; Fig. 7B).


Figure 7
View larger version (10K):
[in this window]
[in a new window]

  		Fig. 7. Rhod-2 spectrofluorometry of Ca2+i in relative units (RU) during PI and after IS. A: averaged peak and diastolic rhod-2 intracellular Ca2+ (Ca2+i) fluorescence signals before IS. B: average diastolic Ca2+i levels during PI, mean Ca2+i levels at 30-min IS, and average diastolic Ca2+i levels at 2-min RP. Values are means ± SE. *P < 0.05 vs. WT at 2-min RP; **P < 0.01 vs. WT at PI; ***P < 0.001 vs. WT at 30-min IS; n = 6/group.

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Advances in molecular biology with the advent of knockout and transgenic animal models have enabled specific hypotheses regarding the mechanisms of disease to be tested at the gene level, and this approach can provide important information about modulation of critical intracellular signaling pathways. Therefore, to understand how decreased SERCA2a levels affect cardiac function both under physiological and pathological conditions, we used gene targeting to generate a mouse carrying a single allele of the SERCA2a gene. The major goal of this study was to test whether a reduction in SERCA2a expression and activity worsens or improves postischemic myocardial recovery. We observed that SERCA2a+/– hearts that have decreased SERCA2a levels exhibit similar recovery of postischemic myocardial function following shorter periods of ischemia that induce myocardial stunning. However, importantly with longer periods of ischemia, they exhibit markedly increased I/R injury with postischemic contractile dysfunction and larger infarction. This is the first report that demonstrates the deleterious effect of reduced SERCA2a levels on myocardial stunning and infarction. The effect of decreased SERCA2a levels in the myocardial-infarction model did not alter postischemic free-radical generation; thus the observed effects were independent of difference in oxidative stress. However, decreased SERCA2a levels were associated with increased postischemic Ca2+i overload. Thus these results verify the hypothesis that optimal levels of SERCA2a are critical for postischemic myocardial function and that decreased SERCA2a activity worsens postischemic myocardial recovery and infarction.

Functional SERCA2a levels and postischemic myocardial recovery. Myocardial I/R injury is associated with severe contractile dysfunction and myocardial death (5, 29, 44). An elevated level of Ca2+i has been related to postischemic mechanical dysfunction in both reversibly and irreversibly injured myocardium (7, 30, 39). The postischemic elevation of Ca2+i could be due to impaired Ca2+ sequestration and/or extrusion. In mammalian ventricular myocytes, SERCA2a and Na+/Ca2+ exchanger are mainly involved in Ca2+ removal from the cytosol (3, 14). Thus reduced SERCA2a will result in decreased sequestration of cytosolic Ca2+, leading to cytosolic Ca2+ overload and diastolic dysfunction.

Interestingly, even with impaired diastolic function, postischemic global contractile recovery with stunning was comparable in SERCA2a+/– and WT hearts. We observed that SERCA2a+/– hearts had mildly increased postischemic systolic function compared with WT hearts (112 ± 8% vs. 96 ± 6% PI). Thus it is apparent that the increased systolic function helped to maintain a comparable level of postischemic global function with stunning. The effective postischemic contractile recovery with stunning in SERCA2a+/– hearts and the improved aortic-output recovery reported in rat hearts treated with SERCA inhibitor CPA are probably mediated by different mechanisms. Whereas CPA markedly decreased IS contracture in rat hearts (13), we observed that diastolic pressure in SERCA2a+/– hearts remained significantly elevated through reperfusion in (Fig. 1). The cellular mechanisms of contractile failure in stunned myocardium are thought to involve altered Ca2+ sensitivity of the myofilaments and/or maximal Ca2+-activated force rather than inadequate delivery of activator Ca2+ (16, 23). Although we did not directly determine the pressure-Ca2+ relation curves with stunning, the improved systolic contraction in SERCA2a+/– hearts could be due to direct myofilament activation from cytosolic free Ca2+.

In contrast, the scenario in 30-min ischemia was different where postischemic global contractile recovery was significantly reduced in SERCA2a+/– hearts, with markedly impaired diastolic function and increased myocardial infarction. Although the postischemic systolic function recovered well in early reperfusion, it was not sustained and adequate enough in the face of persistent diastolic dysfunction through reperfusion. The decreased contractile recovery and increased infarct size in SERCA2a+/– hearts were associated with greater myocardial structural damage seen by light microscopy. These striking differences between SERCA2a+/– and WT hearts during reperfusion clearly showed that reduced SERCA2a renders the heart more susceptible to lethal I/R injury. Our observation is quite different from that of Avellanal et al. (1), where pharmacological inhibition of SERCA2a protected the isolated rabbit myocardium against I/R injury only if CPA was administered before ischemia. They reported that PI treatment with CPA significantly lessened IS contracture and increased the postischemic contractile function. This discrepancy could be related to differential or nonspecific myocardial effects of this SERCA inhibitor vs. the selective disruption of SERCA in SERCA2a+/– hearts, to species differences (40), to the different experimental conditions (isometric muscle strip vs. isovolumic whole heart contraction with different perfusate calcium concentrations), or to all of the above. Of note, using a mouse model of SERCA1a overexpression, we have recently reported (43) that increased functional levels of SERCA can greatly enhance postischemic myocardial recovery.

SERCA2a is under direct control of an intrinsic SR protein, phospholamban (PLB). The nonphosphorylated form of PLB inhibits the activity of SERCA2a and SR Ca2+ transport, whereas phosphorylation of PLB at either Ser16 by protein kinase A or Thr17 by Ca2+-calmodulin protein kinase removes its inhibitory effects on SERCA2a, thereby accelerating Ca2+ uptake by the SR (6, 42). In heterozygous SERCA2a hearts, both SERCA2a and PLB protein levels were decreased by ~35% to maintain an optimal PLB:SERCA2a ratio (20, 21). Interestingly, basal phosphorylation status of PLB was significantly enhanced at both Ser16 and Thr17 despite decreased PLB protein level. These data thus suggested that a decrease in PLB protein level and an increase in PLB phosphorylation status work concurrently to partially compensate for the decrease in SERCA pump level and Ca2+ uptake under normal physiological conditions.

The role of PLB phosphorylation in I/R injury is not clearly understood. Recently, it has been reported that phosphorylation of PLB sites are crucial for the mechanical and Ca2+i recovery in the stunned mouse and rat hearts (34). In our stunning model, identical recovery in the postischemic contractile function in both WT and SERCA2a+/– hearts thus supports the notion that despite having less endogenous SERCA2a, increased endogenous PLB phosphorylation status might have contributed significantly in SERCA2a+/– hearts. In contrast, PLB phosphorylation is reported to decrease after ≥30 min of ischemia (36), and postischemic myocardial recovery and Ca2+i overload were significantly worsened in SERCA2a+/– hearts compared with WT hearts in the infarction model. Together, these findings suggest that PLB phosphorylation might play a differential role in myocardial stunning and infarction.

SERCA2a expression and myocardial Ca2+ handling. It has been known for quite a long time that Ca2+i overload with reduced expression and/or activity of SERCA2a plays a pivotal role in myocardial I/R injury (27, 30, 35, 44), and thus it is of crucial importance in the transition from reversible to irreversible injury during I/R. Longer duration of ischemia is associated not only with prolonged exposure to raised Ca2+i during ischemia but with very large Ca2+i increases and/or large oscillations of Ca2+i on reperfusion (24). We observed that SERCA2a+/– hearts had markedly higher diastolic or mean Ca2+i at baseline, after ischemia (30 min), and during early reperfusion (Fig. 7). This cytosolic Ca2+ overload is likely due to decreased cytosolic Ca2+ removal by SR in SERCA2a+/– hearts because SERCA2a+/– hearts are reported to have lower Ca2+-uptake activity than WT hearts (31), and this could explain the observed acute impairment in ventricular relaxation in SERCA2a+/– hearts. In this regard, we have recently demonstrated that upregulation of SERCA in a mouse model strongly protects the heart against I/R injury with efficient Ca2+ removal and improved diastolic function (43). Thus the present study directly shows for the first time that impaired (reduced) SERCA activity with resultant cytosolic Ca2+ overload is detrimental during acute lethal myocardial I/R.

Reactive oxygen species and postischemic myocardial recovery. Reactive oxygen species have long been recognized to cause oxidative protein modifications and to act as a major mediator of I/R injury (4, 9, 48, 49). Potent oxidants and free radicals such as superoxide anion, hydroxyl radical, and peroxynitrite are formed during postischemic reperfusion and reach highest concentrations during the first minutes of reperfusion (4, 45, 48, 49). Oxygen-derived free radicals have been reported to damage the SERCA, potentially contributing to Ca2+ overload with concurrent myocardial damage and ventricular arrhythmias on reperfusion (19, 22). With EPR spin-trapping studies, similar DMPO-OH adduct signals, indicative of superoxide-derived hydroxyl radicals (48, 49), were seen in both WT and SERCA2a+/– hearts over the first minute of reperfusion (Fig. 6, A and B), and the observed levels of free radicals were indistinguishable (Fig. 6C) between the two strains. Despite the presence of comparable free radicals, SERCA2a+/– hearts exhibited decreased recovery of postischemic contractile function with larger myocardial infarction. Thus the increased postischemic functional deterioration in SERCA2a+/– hearts compared with WT hearts is not related to the levels of oxygen radicals but to the levels of functional SERCA2a.

Implications of SERCA modulation in postischemic myocardial recovery. If the rise in postischemic Ca2+i or Ca2+i oscillation is attributable to SR Ca2+ content, then according to Avellanal et al. (1) and du Toit et al. (13) reduced SERCA2a function in SERCA2a+/– hearts will decrease the rise in Ca2+i and thus improve postischemic mechanical function. However, in striking contrast, we observed the reverse findings. Figure 8 shows the possible cascade of events under normal physiological conditions and during myocardial I/R in relation to SERCA2a function, as well as the hypothetical deleterious effects of reduced SR Ca2+ uptake activity on myocardial Ca2+i handling and contractile function after lethal I/R. Our overall findings provide clear, direct evidence that reduced SERCA2a levels, independent of the effect of oxidant stress that accompanies reperfusion, worsen postischemic myocardial contractile function and infarction following lethal I/R by significantly increasing intracellular Ca2+ overload. In the future, it will be important to verify these observations in an in vivo model of myocardial I/R.


Figure 8
View larger version (14K):
[in this window]
[in a new window]

  		Fig. 8. Cascade of events by which ischemia-reperfusion (I/R)-induced impairment in SERCA function can influence myocardial function, Ca2+i levels, and infarction following lethal I/R. SR, sarcoplasmic reticulum.

 
Thus the present study indicates that hearts with decreased SERCA2a are at greater risk to develop postischemic cardiac injury that in the clinical setting would be expected to lead to heart failure with greater morbidity and mortality. Therefore interventions primarily targeted at restoration of depressed contractility via enhancement of SERCA2a function could provide potential beneficial effects both in heart failure and in the setting of myocardial I/R. Currently novel pharmacological and gene therapy approaches are being explored in an effort to upregulate SERCA2a function or expression (26, 38). These are now under clinical evaluation for the treatment of heart failure but could also be of value in salvage of postischemic myocardium.

In conclusion, decreased SERCA2a expression worsens postischemic myocardial recovery with increased infarction. Therefore strategies to improve or augment SERCA expression or activity should be considered for the treatment of IS and postischemic myocardium.


   ACKNOWLEDGMENTS
 
This work was supported by National Heart, Lung, and Blood Institute Grants HL-63744, HL-65608, HL-38324, and HL-64140.


   FOOTNOTES
 

Address for reprint requests and other correspondence: J. L. Zweier, Davis Heart and Lung Research Institute, The Ohio State Univ., 473 West 12thAve., 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
 REFERENCES
 

  1. Avellanal M, Rodrigues P, Barrigon S. Protective effects of cyclopiazonic acid on ischemia-reperfusion injury in rabbit hearts. J Cardiovasc Res 32: 845–851, 1998.
  2. 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]
  3. Bers DM. Cardiac excitation-contraction coupling. Nature 415: 198–205, 2002.[CrossRef][Medline]
  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. Brittsan A, Kranias EG. Phospholamban and cardiac contractile function. J Mol Cell Cardiol 32: 2131–2139, 2000.[CrossRef][Web of Science][Medline]
  7. Carrozza JP Jr, Bentivegna LA, Williams CP, Kuntz RE, Grossman W, Morgan JP. Decreased myofilament responsiveness in myocardial stunning follows transient calcium overload during ischemia and reperfusion. Circ Res 71: 1334–1340, 1992.[Abstract/Free Full Text]
  8. 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]
  9. Dart RC, Sanders AB. Oxygen free radicals and myocardial reperfusion injury. Ann Emerg Med 17: 53–58, 1988.[CrossRef][Web of Science][Medline]
  10. 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]
  11. 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]
  12. 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]
  13. Du Toit EF, Opie LH. Inhibitors of Ca2+ ATPase pump of sarcoplasmic reticulum attenuate reperfusion stunning in isolated rat heart. J Cardiovasc Res 24: 678–684, 1994.
  14. Fabiato A. Calcium-induced release of calcium from the cardiac sarcoplasmic reticulum. Am J Physiol Cell Physiol 245: C1–C14, 1983.[Abstract/Free Full Text]
  15. 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]
  16. Gross GJ, Kersten JR, Warltier DC. Mechanisms of postischemic contractile dysfunction. Ann Thorac Surg 68: 1898–1904, 1999.[Abstract/Free Full Text]
  17. 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]
  18. 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]
  19. 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]
  20. 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]
  21. Ji Y, Lalli MJ, Babu GJ, Xu Y, Kirkpatrick DL, Liu LH, Chiamvimonvat N, Walsh RA, Shull GE, Periasamy M. Disruption of single copy of the SERCA2a gene results in altered Ca2+ homeostasis and cardiomyocytes function. J Biol Chem 275: 38073–38080, 2000.[Abstract/Free Full Text]
  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. Kusuoka H, Koretsune Y, Chacko VP, Weisfeldt ML, Marban E. Excitation-contraction coupling in postischemic myocardium. Does failure of activator Ca2+ transients underlie stunning? Circ Res 66: 1268–1276, 1990.[Abstract/Free Full Text]
  24. Lee JA, Allen DG. Mechanisms of acute ischemic contractile failure of the heart. J Clin Invest 88: 361–367, 1991.[Web of Science][Medline]
  25. 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]
  26. Micheletti R, Palazzo F, Barassi P, Giacalone G, Ferrandi M, Schiavone A, Moro B, Parodi O, Ferrari P, Bianchi G. Istaroxime, a stimulator of sarcoplasmic reticulum calcium adenosine triphosphatase isoform 2a activity, as a novel therapeutic approach to heart failure. Am J Cardiol 99, Suppl: 24A–32A, 2007.[CrossRef][Web of Science][Medline]
  27. 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]
  28. 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]
  29. 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]
  30. Mubagwa K. Sarcoplasmic reticulum function during myocardial ischemia and reperfusion. Cardiovasc Res 30: 166–175, 1995.[Free Full Text]
  31. Periasamy M, Reed TD, Liu LH, Ji y Loukianov E, Paul RJ, Nieman ML, Riddle T, Duffy JJ, Doetschman T, Lorenz JN, 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]
  32. Periasamy M, Huke S. SERCA pump level is a critical determinant of Ca(2+)homeostasis and cardiac contractility. J Mol Cell Cardiol 33: 1053–1063, 2001.[CrossRef][Web of Science][Medline]
  33. Sahn DJ, DeMaria A, Kisslo J, 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. Said M, Vittone L, Mundina-Weilenmann C, Ferrero P, Kranias EG, Mattiazzi A. Role of dual-site phospholamban phosphorylation in the stunned heart: insights from phospholamban site-specific mutants. Am J Physiol Heart Circ Physiol 285: H1198–H1205, 2003.[Abstract/Free Full Text]
  35. 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]
  36. Schoutsen B, Blom JJ, Verdouw PD, Lamers JM. Calcium transport and phospholamban in sarcoplasmic reticulum of ischemic myocardium. J Mol Cell Cardiol 21: 719–727, 1989.[CrossRef][Web of Science][Medline]
  37. Schultz JEJ, Glascock BJ, Witt SA, Nieman ML, Nattamai KJ, Liu LH, Lorenz JN, Shull GE, Kimball TR, Periasamy M. Accelerated onset of heart failure in mice during pressure overload with chronically decreased SERCA2 calcium pump activity. Am J Physiol Heart Circ Physiol 286: H1146–H1153, 2004.[Abstract/Free Full Text]
  38. Smith G. Matters of the heart: the physiology of cardiac function and failure. Exp Physiol 92: 973–986, 2007.[Abstract/Free Full Text]
  39. Steenbergen C, Fralix TA, Murphy E. Role of increased cytosolic free calcium concentration in myocardial ischemic injury. Basic Res Cardiol 88: 456–470, 1993.[CrossRef][Web of Science][Medline]
  40. Su Z, Li F, Spitzer KW, Yao A, Ritter M, Barry WH. Comparison of sarcoplasmic reticulum Ca2+-ATPase function in human, dog, rabbit, and mouse ventricular myocytes. J Mol Cell Cardiol 35: 761–767, 2003.[CrossRef][Web of Science][Medline]
  41. Suarez J, Belke DD, Gloss B, Dieterle T, McDonough PM, Kim YK, Brunton LL, Dillmann WH. In vivo adenoviral transfer of sorcin reverses cardiac contractile abnormalities of diabetic cardiomyopathy. Am J Physiol Heart Circ Physiol 286: H68–H75, 2004.[Abstract/Free Full Text]
  42. Tada M, Toyofuku T. Molecular regulation of phospholamban function and expression. Trends Cardiovasc Med 8: 330–340, 1998.[CrossRef][Web of Science][Medline]
  43. Talukder MAH, Kalyanasundaram A, Zhao X, Li Zuo L, Bhupathy P, Babu GJ, Cardounel AJ, Periasamy M, Zweier JL. Expression of SERCA isoform with faster Ca2+ transport properties improves postischemic cardiac function, Ca2+ handling, and decreases myocardial infarction. Am J Physiol Heart Circ Physiol 293: H2418–H2428, 2007.[Abstract/Free Full Text]
  44. 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]
  45. 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]
  46. 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]
  47. 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]
  48. Zweier JL, Flaherty JT, Weisfeldt ML. Direct measurement of free radical generation following reperfusion of ischemic myocardium. Proc Natl Acad Sci USA 84: 1404–1407, 1987.[Abstract/Free Full Text]
  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]



This article has been cited by other articles:


Home page
 Cardiovasc ResHome page
J. Inserte, J. A. Barrabes, V. Hernando, and D. Garcia-Dorado
Orphan targets for reperfusion injury
Cardiovasc Res, July 15, 2009; 83(2): 169 - 178.
[Abstract] [Full Text] [PDF]

Home page
 J. Pharmacol. Exp. Ther.Home page
L. Zuo, Y.-R. Chen, L. A. Reyes, H.-L. Lee, C.-L. Chen, F. A. Villamena, and J. L. Zweier
The Radical Trap 5,5-Dimethyl-1-Pyrroline N-Oxide Exerts Dose-Dependent Protection against Myocardial Ischemia-Reperfusion Injury through Preservation of Mitochondrial Electron Transport
J. Pharmacol. Exp. Ther., May 1, 2009; 329(2): 515 - 523.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Respir. Crit. Care Med.Home page
S. Ahmad, A. Ahmad, E. S. Dremina, V. S. Sharov, X. Guo, T. N. Jones, J. E. Loader, J. R. Tatreau, A.-L. Perraud, C. Schoneich, et al.
Bcl-2 Suppresses Sarcoplasmic/Endoplasmic Reticulum Ca2+-ATPase Expression in Cystic Fibrosis Airways: Role in Oxidant-mediated Cell Death
Am. J. Respir. Crit. Care Med., May 1, 2009; 179(9): 816 - 826.
[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
 EuropaceHome page
G. Antoons and K. R. Sipido
Targeting calcium handling in arrhythmias
Europace, December 1, 2008; 10(12): 1364 - 1369.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
294/3/H1426    most recent
01016.2007v1
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 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 © 2008 by the American Physiological Society.