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Cardiovascular Research 2003 57(4):1044-1051; doi:10.1016/S0008-6363(02)00810-6
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Copyright © 2003, European Society of Cardiology

Cardiac ischemia causes inhibition of the Na/K ATPase by a labile cytosolic compound whose production is linked to oxidant stress

William Fuller, Vina Parmar, Philip Eaton, James R Bell and Michael J Shattock*

Cardiac Physiology, Centre for Cardiovascular Biology and Medicine, King's College London, The Rayne Institute, St. Thomas’ Hospital, London SE1 7EH, UK

* Corresponding author. Tel.: +44-20-7928-9292x3376; fax: +44-20-7928-0658. michael.shattock{at}kcl.ac.uk

Received 12 August 2002; accepted 13 November 2002


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
Objective: Intracellular Na rises rapidly during cardiac ischemia and this has been attributed to the combination of increased influx of Na via sodium–proton exchange and decreased activity of the Na/K ATPase. The aim of these studies was to investigate the effects of ischemia on Na/K ATPase function in Langendorff-perfused rat hearts. Methods: Na/K ATPase activity was determined by measuring ouabain-sensitive phosphate generation from ATP by cardiac homogenates. Results: Global ischemia (15 and 30 min) caused a substantial reduction in Na/K ATPase function despite high substrate availability in the assay. When sarcolemmal membranes were purified away from the cytosol a profound activation of the Na/K ATPase was revealed following ischemia, indicating that the inhibition was due to the cytosolic accumulation of an inhibitor of Na/K ATPase. The half-life of the inhibitor in cardiac homogenates was 10±3 min at room temperature. Perfusion with the antioxidant MPG (1 mmol/l) reduced the accumulation of this inhibitor, however MPG was without effect on Na/K ATPase function when added directly to the Na/K ATPase activity assay. While the inhibitor reduced the activity of cardiac and brain forms of the Na/K ATPase in bioassay experiments, no effect was observed on the renal and skeletal muscle forms of the enzyme. Conclusions: An unstable cardiac and brain-specific inhibitor of the Na/K ATPase whose production is linked to oxidant stress, accumulates intracellularly during ischemia. Intracellular Na is a primary determinant of electro-mechanical recovery on reperfusion, so inhibition of the Na/K ATPase by this compound may be crucial in determining recovery from ischemia.

KEYWORDS HRP, horseradish peroxidase; MPG, mercaptopropionylglycine; PAGE, polyacrylamide gel electrophoresis; PMSF, phenylmethylsulfonyl fluoride; SDS, sodium dodecylsulfate; SLP, sarcolemma/particulate fraction


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 References
 
In excitable tissues, the activity of the plasmalemmal Na/K ATPase is crucial for the maintenance of normal electrical activity and ion gradients. In cardiac muscle, the transarcolemmal sodium (Na) gradient established by Na/K ATPase activity is essential not only for generating the rapid upstroke of the action potential but also for driving a number of ion-exchange and transport processes crucial for normal cellular function, ion homeostasis and the control of cell volume. These Na-dependent membrane transporters include those responsible for the regulation of other ions (such as the Na/Ca exchanger, Na/H exchanger and Na–HCO3 cotransporter) [1], as well as those involved in the movement of substrates and amino acids [2]. Interventions that influence either the activity of the Na/K ATPase, or indirectly the transmembrane Na gradient, can therefore profoundly affect normal cellular function.

Many studies have investigated the effects of ischemia on intracellular Na [3–6]. Studies, using NMR techniques have reported that Na begins to rise immediately after the onset of ischemia and then progressively increases reaching levels as high as 30 mmol/l after 30 min [3,5,6]. This rise in intracellular Na during ischemia has been attributed to a combination of an increased cellular influx of Na via the Na/H exchanger and a decrease in Na extrusion by the Na/K ATPase [3,6]. The ischemia-induced inhibition of the Na/K ATPase and the failure of intracellular Na to recover completely on reperfusion have been shown to be important determinants of electrical and contractile dysfunction in the ischemic/reperfused myocardium [5,7].

The most frequent explanation for the inhibition of the Na/K ATPase in early ischemia is the decline in cytosolic ATP limiting energy supply to the pump. However, in early ischemia, intracellular Na rises at a time when the total ATP concentration greatly exceeds the Km for the pump (~0.1–0.8 mmol/l) [8,9] and the free energy of ATP exceeds that required for pump activity (~44 kJ/mol) [8]. As ATP is depleted during ischemia, ATP delivery to cellular compartments may be limited, and this could lead to local depletion of ATP, for example in the subsarcolemmal space. Studies suggest that it is glycolytically-derived ATP, rather than the absolute cytosolic concentration, that is essential for maintaining pump function both during and immediately following ischemia [10,11] and that the failure of the pump occurs co-incident with glycogen depletion [10].

Ischemia not only depletes bulk ATP but also induces a progressive series of cellular changes, all of which may modulate Na/K ATPase activity. These include: acidosis [12], Ca overload [13], activation of proteases [14], accumulation of lipid metabolites [15–17], and, in some but not all tissues, the acute translocation of pump protein away from surface membranes [18,19]. In addition, there is a substantial literature describing the regulation of Na/K ATPase activity in a variety of cell types following activation of protein kinases A and C [20–23], although not all such studies suggest regulation is through direct phosphorylation of the enzyme (for example, see Ref. [21]).

In the present study, we have observed a novel unstable inhibitor of the Na/K ATPase that accumulates intracellularly during cardiac ischemia. This inhibitor appears to be specific for isoforms of the Na/K ATPase derived from the heart and the brain, and may be an important determinant of cell injury during cardiac ischemia.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 References
 
2.1 Preparations and protocols
2.1.1 Isolated-perfused rat heart
Male Wistar rats (200–250 g) were used in all studies. Animals received humane care in compliance with the ‘Principles of Laboratory Animal Care’ formulated by the National Society for Medical Research and the Guide for the Care and Use of Laboratory Animals prepared by the National Academy of Sciences and published by the National Institutes of Health (NIH Publication no. 85-23). Hearts were perfused in the Langendorff mode as described previously [24].

2.1.2 Effects of ischemia on Na/K ATPase
Hearts were maintained at 37 °C in a water-jacketed chamber and were aerobically perfused for a 30-min stabilization period. Hearts were then rendered globally ischemic for either 15 or 30 min by completely ceasing the flow of the perfusion buffer. Hearts were removed from the cannula and immediately snap-frozen. In some studies, mercaptopropionylglycine (MPG) was perfused at 1 mmol/l for the final 10 min of aerobic perfusion.

2.2 Protein extraction and subcellular fractionation
Fractionation was carried out as described previously [24]. Briefly, hearts were diced in a high salt solution (2 mol/l NaCl, 20 mmol/l HEPES, pH 7.4) and agitated for 30 min at 4 °C to depolymerize the myofilaments. Tissue was rinsed, then homogenized in buffer A containing (in mmol/l): HEPES 20, sucrose 250, EDTA 2, MgCl2 1, 10 ml/g wet weight of heart, pH 7.4 using a ground glass homogenizer. To isolate a purified sarcolemmal/particulate fraction (SLP), homogenates were centrifuged at 20,000xg for 30 min. The purified sarcolemmal/particulate (SLP) fraction prepared in this way is experimentally useful as it excludes the other membrane compartment that harbors functional Na/K ATPase (the endosomes) [24]. This purified SLP fraction contains all of the sarcolemmal membranes, in addition to other subcellular compartments which do not contain Na/K ATPase, including SR, nuclei, residual myofilaments and mitochondria. An important point to note is that soluble cytosolic molecules are excluded from this fraction. We have designated this fraction SLP to reflect its origin.

2.3 Quantitative immunoblotting
SDS–PAGE and quantitative immunoblotting were carried out as described previously [24].

2.4 Na/K ATPase activity
Two 50-µl aliquots of either the unfractionated homogenate or purified SLP (resuspended in buffer A) were taken and one mixed with 50 µl of reaction buffer 1 containing (in mmol/l): Tris–HCl 200, MgCl2 30, NaCl 200, KCl 60, EGTA 10 pH 7.5 and the other with reaction buffer 2 (as reaction buffer 1+2 mmol/l ouabain). Each reaction also contained 100 µg/ml PMSF, 2 µg/ml apronitin and 2 µg/ml pepstatin A. Tubes were prewarmed to 37 °C, and the reaction started by the addition of 10 mmol/l ATP. Reactions proceeded for 5 min at 37 °C, and 10 µl of 100% (w/v) trichloroacetic acid were added to stop the reaction. Samples were left on ice for 1 h and centrifuged at 20,000xg for 30 min to pellet precipitated protein. Supernatants were assayed for Pi using an ammonium molybdate spectrophotometric assay kit (Sigma). The difference between the two samples represents the ouabain inhibitable phosphatase (i.e. Na/K ATPase) activity.

2.5 Bioassay technique
Brain, kidney and soleus muscle were homogenized 10 ml/g wet weight in buffer A. Basal Na/K ATPase activity in these tissues was measured exactly as described for cardiac homogenates. The effect of ischemic homogenate on Na/K ATPase activity in brain, kidney and soleus muscle was assessed by measuring ouabain inhibitable phosphatase activity in an assay containing equal volumes of the test preparation and ischemic homogenate.

2.6 Materials and antibodies
All Na/K ATPase subunit antibodies were supplied by Upstate Biotechnology (Buckinghamshire, UK). Anti-rabbit and anti-mouse HRP-linked secondary antibodies were supplied by Amersham (Amersham, UK). Unless specified, all chemicals used were of AnalaR grade and were obtained from Sigma (Poole, UK).

2.7 Statistical analysis
Quantitative data are shown as means±standard errors of the means. Differences in mean measurements between experimental groups were tested by ANOVA followed by a t-test with a Bonferroni correction and differences were considered significant at the 95% level.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 References
 
3.1 Effect of ischemia on Na/K ATPase activity
Na/K ATPase activity was assayed in crude homogenates from control and ischemic hearts in the presence of 10 mmol/l ATP, such that the ATP concentration was not limiting throughout the course of the reaction. Fig. 1 shows the ouabain-sensitive phosphatase activity in crude homogenates is significantly depressed as ischemia progresses, despite the availability of substrate. Crucially, when the purified ischemic SLP is isolated from the crude homogenate and resuspended in buffer A, the Na/K ATPase activity is no longer depressed, indicating it is most likely inhibited by a factor which is eliminated when SLP is separated by centrifugation. Indeed, not only is the effect of this inhibitor lost, but a significant activation of sarcolemmal Na/K ATPase is revealed by this separation, from 2.4±0.4 µmol phosphate/min/g wet weight in aerobic SLP, to 3.9±0.5 and 7.2±0.5 following 15 and 30 min ischemia, respectively. This activation is sensitive to kinase inhibitors, and appears to be mediated by phosphorylation of an accessory subunit of the Na/K ATPase (Fuller et al., in preparation).


Figure 1
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  		Fig. 1 Effect of ischemia on Na/K ATPase activity: activities are shown in crude cardiac homogenates (filled bars), and purified SLP (open bars). Ischemia (30 min) causes an approximate threefold depression of Na/K ATPase function in crude homogenates, however purification of the SLP reveals that Na/K ATPase activity more than doubles in the sarcolemma following 30 min ischemia (n=5).

 
3.2 Effect of an anti-oxidant on Na/K ATPase activity during ischemia
It is well-established that oxidative stress during cardiac ischemia may cause the accumulation of substances which inhibit membrane transporters such as the Na/K ATPase [15,16,25,26]. Indeed, oxidative stress and specifically the superoxide anion has been reported to inhibit the Na/K ATPase in various tissues and species, including the rat [26]. Hearts were treated with an anti-oxidant during ischemia to determine the contribution of oxidant stress to the inhibition of the Na/K ATPase observed in crude ischemic homogenates. The anti-oxidant MPG (1 mmol/l) was introduced for the final 10 min of aerobic perfusion. Hearts were then made ischemic for 30 min, homogenized, and Na/K ATPase activity assayed. Accumulation of the Na/K ATPase inhibitor was attenuated by perfusion with this anti-oxidant prior to ischemia (Fig. 2).


Figure 2
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  		Fig. 2 Effect of MPG during ischemia on Na/K ATPase activity in crude homogenates: the anti-oxidant MPG (1 mmol/l) was perfused for the final 10 min of aerobic perfusion, and hearts were made ischemic for 30 min. Data for untreated hearts are shown for comparison. The accumulation of the soluble inhibitor of the Na/K ATPase is attenuated by MPG (n=3).

 
3.3 Effect of anti-oxidants on Na/K ATPase activity within the Na/K ATPase assay
To further characterize the inhibitor we supplemented the Na/K ATPase activity assay with the anti-oxidant MPG (1 mmol/l) and the reducing agent dithiothreitol (DTT, 1 mmol/l). Fig. 3 indicates that these substances were without effect on the activity of the Na/K ATPase in ischemic homogenate, indicating that inhibition is neither due to the maintained presence of an oxidizing species, nor a result of reversible protein oxidation during ischemia.


Figure 3
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  		Fig. 3 Effect of anti-oxidants within the assay on Na/K ATPase activity: the anti-oxidant MPG (1 mmol/l) and the protein reducing agent DTT (1 mmol/l) were added to homogenates from ischemic (30 min) hearts, and Na/K ATPase activity measured. Data for homogenates from aerobic hearts are shown for comparison. Neither agent has any effect on Na/K ATPase activity following ischemia (n=3).

 
3.4 Stability of the Na/K ATPase inhibitor
The stability of the inhibitor of the Na/K ATPase was measured at room temperature. Homogenates from hearts made ischemic for 30 min were incubated at 25 °C for up to 60 min, and Na/K ATPase activity was measured. Under these conditions relief from inhibition was rapid (Fig. 4). The data were fitted to a single exponential as described in the legend to Fig. 4, with a correlation coefficient of 0.95. The time constant for decay was calculated to be 0.067±0.02 min–1, giving a half-life of 10±3 min. Hence the inhibitor is relatively unstable.


Figure 4
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  		Fig. 4 The inhibitor of the Na/K ATPase is unstable at room temperature: ischemic homogenate was incubated for the indicated times at room temperature and Na/K ATPase activity assayed. The data are fitted to the curve activity=A0(1–e–{lambda}t)+B where A0 is the underlying Na/K ATPase activity, B is the residual activity at time zero, and {lambda} is the time constant for degradation of the inhibitor. The half-life of the inhibitor is 10 min (n=5).

 
3.5 Effect of ischemic homogenate on Na/K ATPase from different tissues
Inhibition of the Na/K ATPase during ischemia was further investigated using a bioassay technique. Crude homogenate from a heart made ischemic for 30 min was applied to different preparations: SLP purified from aerobic and ischemic hearts, and crude rat kidney, brain and soleus muscle homogenates. Fig. 5 indicates that while cardiac and brain Na/K ATPase activity was inhibited by the presence of ischemic homogenate during the assay, Na/K ATPase derived from the kidney and soleus muscle was unaffected.


Figure 5
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  		Fig. 5 Bioassay of ischemic cardiac homogenate on Na/K ATPase activity from different sources: the activity in homogenates from hearts made ischemic for 30 min is shown for reference. The effect of applying buffer A (filled bars) and this ischemic homogenate (open bars) to aerobic SLP, ischemic SLP and rat kidney, brain and soleus muscle homogenates (10% w/v in buffer A) is shown. The inhibitor present in ischemic homogenate is specific for cardiac and brain Na/K ATPase (n=5).

 
In order to purify and identify this inhibitor we separated total cardiac membranes from the cytosol by centrifugation of ischemic homogenate at 100,000xg for 60 min, as previously described [24]. No inhibitory activity was detected in the supernatant from these spins, yet activation of the Na/K ATPase in the total membrane fraction was of a similar order of magnitude to that seen in purified SLP (not shown). This suggests that even at 4 °C this inhibitor is relatively labile, such that following centrifugation for 60 min little activity remains.

Additional bioassays were performed using cardiac coronary effluent collected during reperfusion of the heart immediately following 30 min ischemia. Aliquots of reperfusate were collected up to 5 min of reperfusion, and applied to SLP purified from aerobic hearts. Given the measured instability of the inhibitor, hearts were reperfused hypoxically as well as aerobically, to ensure the inhibitor was not rapidly oxidized by the reperfusing buffer. Both aerobic and hypoxic reperfusion failed to wash out of the heart any substance with an inhibitory effect on cardiac Na/K ATPase activity (Fig. 6), suggesting that the inhibitor described accumulates intra- rather than inter-cellularly.


Figure 6
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  		Fig. 6 The inhibitor of the Na/K ATPase present in cardiac homogenate is not washed out of the heart upon reperfusion: coronary effluent was added to the Na/K ATPase assay. A sample of effluent was taken immediately prior to ischemia (A). The effect of five 1-ml samples of cardiac effluent from early reperfusion (1–5) on Na/K ATPase activity is shown. Hearts were reperfused both aerobically and hypoxically. No inhibitory substance was detected in any fraction (n=4).

 
3.6 Subunit distribution of the Na/K ATPase
In order to determine whether the specificity of the soluble inhibitor of the Na/K ATPase for enzyme derived from heart and brain has its basis in the subunit composition of the enzyme in these tissues, quantitative immunoblotting was undertaken to assess the distribution of {alpha}1, {alpha}2, {alpha}3, β1 and β2 subunits in the tissues examined in Fig. 5. Typical immunoblots are shown in panel A of Fig. 7, and mean data in panel B. All values have been normalised to the expression of each subunit in the brain.


Figure 7
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  		Fig. 7 Subunit distribution of the Na/K ATPase in brain, heart, kidney and soleus muscle. The distribution of {alpha}1, {alpha}2, {alpha}3, β1 and β2 subunits of the Na/K ATPase in brain (B), heart (H), kidney (K) and soleus muscle (SM) was assessed by quantitative immunoblotting. Typical blots are shown in panel A, and mean data in panel B (n=3). All values in panel B are normalized to expression in the brain.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
References
 
In the present study, we have observed opposing mechanisms that regulate the Na/K ATPase during global ischemia in the Langendorff perfused rat heart. A potent, reversible, soluble inhibitor accumulates during ischemia, production of which is attenuated by an anti-oxidant. Separation of sarcolemmal membranes from the cytosol by centrifugation reveals a substantial underlying activation of the Na/K ATPase in the sarcolemma during ischemia. The widely reported increases in intracellular Na that occur during ischemia can, at least in part, be explained by the accumulation of this soluble inhibitor leading to depressed Na/K ATPase function. It is perhaps this inhibitor, rather than decreased ATP availability, that is the primary determinant of Na (and therefore Ca) overload during ischemia, and is therefore a crucial determinant of cell injury during cardiac ischemia.

It is noteworthy that inhibition of the Na/K ATPase by the soluble inhibitor is very substantial. Hearts were homogenized 10% w/v in buffer A: a heart with mass 1 g was homogenized in 10 ml buffer. Homogenates were then further diluted twofold in order to assay Na/K ATPase activity, yet the effects of the inhibitor on Na/K ATPase activity within the assay are still profound. It seems likely that although we have recorded a net inhibition of ~6 µmol phosphate/min/g wet weight following 30 min ischemia (Fig. 1), enzyme activity may be depressed considerably more within myocytes during ischemia, because the inhibitor is present at at least a 10-fold higher concentration than in these in vitro assays.

We have been unable to identify this soluble inhibitor of the Na/K ATPase. Clearly, its production is related to oxidant stress during ischemia (Fig. 2), and there is ample evidence that oxidant stress can inhibit the Na/K ATPase [25–28]. In addition, anti-oxidants are protective to the heart during ischemia [29,30], and this may be due in part to maintenance of Na/K ATPase function. However, the inhibitor is not itself an oxidizing compound, as addition of anti-oxidants to ischemic homogenate when assaying Na/K ATPase activity is without effect (Fig. 3).

The fact that we are unable to detect the inhibitor in cardiac effluent following reperfusion indicates either that it accumulates intracellularly, or that it is inactivated extremely rapidly on reperfusion. Given these characteristics, we would suggest that this substance is not an endogenous ouabain-like compound [31,32]. Its absence from cardiac effluent renders identification analytically using methods such as HPLC and mass spectroscopy difficult. In addition, its relative instability (Fig. 4) also hampers identification. Metabolism (or otherwise) of this inhibitor upon reperfusion will clearly determine the recovery of Na/K ATPase function. Indeed, there is some evidence that the recovery of intracellular Na may be slow or even incomplete after even relatively short periods of ischemia [5], however we are yet to characterize this in our preparation.

It is clearly significant that we cannot identify any inhibitory activity of this substance on the Na/K ATPase prepared from the kidney and soleus muscle (Fig. 5). The most logical explanation for this phenomenon is that a soluble factor is present in ischemic homogenate that specifically inhibits cardiac and brain Na/K ATPase. It is highly unlikely that the inhibitor is membrane associated rather than soluble: for an inhibitory effect to be observed, it would have to ‘jump’ from the membranes of the ischemic homogenate to those in the test preparation when these two preparations were mixed. Our data clearly indicate that this inhibitor is freely diffusable within the cardiac homogenate, however we cannot exclude the possibility that it is pelleted, but inactivated, during the preparation of the SLP.

The nature of the specificity of the substance for the cardiac enzyme may be related to the subunit composition of the enzyme, or the phosphorylation (or other post-translational modification) status of the protein. However we have been unable to identify a combination of subunits in the heart and brain that is not found in soleus muscle and kidney (Fig. 7). Inhibition of the Na/K ATPase in the heart is most likely through an action on the {alpha}1 subunit, as this is the most abundant subunit expressed. The {alpha}1 subunit is also substantially expressed in the kidney (Fig. 7), yet renal Na/K ATPase is insensitive to the inhibitor (Fig. 5). Hence the specificity of the inhibitor cannot be explained on the basis of expression of different catalytic subunits of the Na/K ATPase. Differential distribution of the β subunits of the Na/K ATPase is also unable to explain the basis of the specificity, as neither β subunit is unique to the heart and brain over soleus muscle and kidney. One substantial difference between brain, cardiac and renal forms of the enzyme is the expression of the {gamma} subunit in the kidney. Whether the presence of the {gamma} subunit is able to confer resistance to this substance on the {alpha}1 subunit in the kidney remains to be determined. In addition, the recent observation that phospholemman (FXYD1) associates with the cardiac Na/K ATPase [33] offers another subunit that may confer sensitivity to the inhibitor. Phospholemman is expressed in the heart and brain, but not the kidney [34].

Clearly it is desirable to identify this inhibitor of the Na/K ATPase. However, given the physical and chemical properties we have described, such identification is not possible using a simple analytical approach. While it would undoubtedly be possible to simply screen chemicals for their ability to inhibit the Na/K ATPase, such a ‘needle in a haystack’ approach is of questionable value. We have investigated the effect of a small number of compounds on Na/K ATPase activity without identifying one with the correct properties and activity profile. Increased levels of lipid metabolites in the ischemic heart may be one of the causes of ischemic injury [35], and some [36] but not other [15] authors have reported that a long chain acyl carnitine, palmitoyl carnitine, inhibits the cardiac Na/K ATPase. We have been unable to measure an inhibitory effect of palmitoyl carnitine in our system (data not shown). However, if the soluble inhibitor is a lipid-derived compound, the basis of its specificity for the cardiac and brain forms of the Na/K ATPase described in Fig. 5 may lie in its ability to modify the lipid environment of the enzyme in these tissues only, as a result of differences in membrane composition of the heart and brain compared to the kidney and soleus muscle.

The Na/K ATPase activity assay used in this study is designed to measure the Vmax of the enzyme, with no reference to the Km. Substrate concentrations (ATP, sodium, potassium and magnesium) are not rate-limiting. We have confirmed that the enzymatic reaction is linear over the 5-min assay period (data not shown). Hence the soluble inhibitor reduces the Vmax of the Na/K ATPase, either through a reduction in Vmax of every functional Na/K ATPase, or a complete inhibition of some functional units in the enzyme population. The high substrate availability in the assay implies the inhibition may be non-competitive in nature, but this warrants further investigation.

To conclude, we have observed a potent, reversible, labile inhibitor of the cardiac and brain forms of the Na/K ATPase that accumulates during cardiac ischemia. The basis of the specificity is unclear, however it is intriguing to note that this inhibitor is effective against Na/K ATPase derived from the two organs most sensitive to ischemia–reperfusion injury. Given the well-established link between intracellular sodium during ischemia and functional recovery upon reperfusion, accumulation of this inhibitor is undoubtedly an important determinant of cardiac injury during ischemia.

Time for primary review 23 days.


   Acknowledgements
 
This work was supported by grants from the British Heart Foundation and the Wellcome Trust.


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

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