EXPERIMENTAL STUDY
Atorvastatin, administered at the onset of reperfusion, and independent oflipid lowering, protects the myocardiumby up-regulating a pro-survival pathway
Robert M. Bell, MBBS, BSc, PhD* and
Derek M. Yellon, PhD, DSc, Hon FRCP, FACC*,*
* The Hatter Institute for Cardiovascular Studies, Academic and Clinical Cardiology, Division of Medicine, University College Hospitals and Medical School, University College, London, London, United Kingdom
Manuscript received August 9, 2002;
revised manuscript received September 19, 2002,
accepted October 31, 2002.
* Reprint requests and correspondence: Dr. Derek M. Yellon, The Hatter Institute for Cardiovascular Studies, Academic and Clinical Cardiology, Division of Medicine, University College Hospitals and Medical School, University College, London, Grafton Way, London, UK WC1E 6DB.
hatter-institute{at}ucl.ac.uk
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Abstract
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OBJECTIVES: The purpose of this study was to determine whether atorvastatin, a 3-hydroxy-3-methylglutaryl (HMG)-co-enzyme A (CoA) reductase inhibitor, limits myocardial necrosis when administered as an adjunct to reperfusion.
BACKGROUND: Statins inhibit HMG-CoA reductase to reduce the synthesis of cholesterol. However, it is proposed that statins have cardiovascular effects beyond their ability to lower cholesterol, possibly via recruitment of phosphatidyl inositol 3-kinase (PI3K) and the serine/threonine kinase, Akt. This signaling pathway has recently been linked to growth factor–mediated reperfusion salvage.
METHODS: Isolated perfused mouse hearts were subjected to 35 min of global ischemia and reperfused for 30 min in the presence of incremental concentrations of atorvastatin. Infarct size was determined by triphenyltetrazolium chloride staining, and the activity of the PI3K signaling cascade was determined by Western blot analysis.
RESULTS: We found that there was a profound dose-dependent reduction of infarct size with atorvastatin in the range of 25 to 100 µmol/l (optimal protection was seen at 50 µmol/l with infarct size of 16 ± 2% vs. control, 33 ± 2%, p < 0.01). Moreover, this protection was sensitive to inhibition with the PI3 kinase inhibitor, wortmannin, and was absent in endothelial nitric oxide synthase (eNOS) knockout mice. Western blot analysis revealed that atorvastatin resulted in rapid activation of the PI3K/Akt signaling cascade (within 5 min) and that both Akt and eNOS phosphorylation were significantly increased by 4.1-fold and 2.9-fold, respectively (p < 0.01). Moreover, phosphorylation of the PI3K substrates was abrogated by the administration of wortmannin.
CONCLUSIONS: Atorvastatin attenuates lethal reperfusion-induced injury in a manner that is reliant on PI3K and Akt activity and the presence and activity of eNOS.
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Abbreviations and Acronyms
| | AU | | arbitrary units | | DMSO | | dimethyl sulfoxide | | eNOS | | endothelial nitric oxide synthase | | HMG-CoA | | 3-hydroxy-3-methylglutaryl-co-enzyme A | | KO | | knockout | | PI3K | | phosphatidyl inositol 3-kinase | | TTC | | triphenyl tetrazolium chloride |
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Reperfusion is a prerequisite to myocardial salvage after coronary occlusion. Although reperfusion is undeniably beneficial, it is perhaps not without risk. While there is some controversy regarding reperfusion injury as an event independent of the antecedent ischemia (1,2), we and others propose that this form of injury may be amenable to pharmacologic manipulation (3,4). Recently, we have identified a potential role for the phosphatidyl inositol 3-kinase (PI3K) and the downstream target, the serine/threonine kinase, Akt pathway in ameliorating reperfusion injury (5,6). Indeed, using the growth factors, insulin and insulin-like growth factor-1, it has been shown that recruitment of the PI3K/Akt signaling cascade upon reperfusion may be beneficial in limiting myocardial/myocyte cell death (5–8). Moreover, over-expression of Akt in rats attenuates cell death and myocardial dysfunction after injurious ischemia (9).
3-Hydroxy-3-methylglutaryl (HMG)-co-enzyme A (CoA) reductase inhibitors have been increasingly prescribed for patients with cardiovascular disease after robust outcome results from both primary (10) and secondary prevention studies (11). It is, however, becoming clear that statins appear to have pleiotropic effects beyond their ability to lower cholesterol (12). Indeed, statins have been shown to be beneficial in such diverse conditions as Alzheimer’s disease (13) and osteoporosis (14).
The signaling sequelae of HMG-CoA reductase remain unclear. In attenuating HMG-CoA reductase activity, statins reduce cellular content of the products of the mevalonate metabolic pathway that leads to the synthesis of cholesterol. Many of these products, the isoprenoids, are thought to be essential for the growth and proliferation of eukaryotic cells. One important isoprenoid is farnesiol, which is capable of binding covalently to a number of guanosine triphosphate–binding proteins such as p21ras; a potentially important component of growth factor–stimulated signaling (15). Another is geranylgeranyl pyrophosphate, which through geranylgeranylation of the guanosine triphosphatase Rho appears to down-regulate endothelial nitric oxide synthase (eNOS) messenger ribonucleic acid (mRNA) (16). This effect may help to explain, in part, why statins lead to increased eNOS mRNA in thrombocytes (17) and are able to reverse the down-regulation of eNOS by low-density lipoprotein cholesterol (18). Up-regulation of eNOS activity is of potential interest in reperfusion injury; recent work with nitric oxide (NO) donors suggests that NO attenuates myocardial dysfunction after an injurious ischemia/reperfusion insult (19,20).
Recently, HMG-CoA reductase inhibitors have been shown to recruit the PI3K and Akt signaling cascade in endothelial cell lines, although not in neonatal rat cardiac myocytes (21). However, simvastatin has been shown to possess infarct sparing properties and is capable of attenuating reperfusion myocardial dysfunction in adult rats (22,23). Evidence to support a potential role of statins in recruiting the cell-survival PI3K/Akt pathway in the cardiovascular system is twofold. First, atorvastatin administration to aortic endothelium results in a decrease in caveolin abundance and, thus, reduced inhibitory binding of eNOS. This is correlated with recruitment of eNOS into an eNOS/heat stress protein 90/Akt complex, leading to the phosphorylation of eNOS at serine 1177 (24). Second, statins have been shown to have both a proliferative and anti-proliferative effect on the vasculature. At low concentrations, statins have angiogenic properties, whereas at higher concentrations they are anti-proliferative (25); the angiogenic process has been linked to the recruitment of the PI3K/Akt pathway and phosphorylation of eNOS (21).
Thus, we hypothesized that exposure of the normo-cholesterolemic adult heart to the HMG-CoA reductase inhibitor, atorvastatin, after a period of injurious ischemia, would recruit the PI3K pathway and lead to cardioprotection. Because the optimal concentration of atorvastatin that would prove to be protective was unknown, the initial aim was to construct a dose–response curve using concentrations of atorvastatin that are clinically representative of those used to reduce cholesterol in hypercholesterolemic patients. Secondary aims were to determine whether there was a role for the PI3K/Akt signaling cascade, whether recruitment of eNOS occurs, and whether the activity of this pathway is essential to HMG-CoA reductase inhibitor–induced protection.
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Methods
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All work was conducted in accordance with the Guidelines on the Operation of the Animals (Scientific Procedures) Act 1986, published by The Stationery Office (London, UK). Wortmannin was purchased from Sigma (Poole, UK). Dimethyl sulfoxide (DMSO; BDH Laboratory Supplies, Poole, UK) was used as the solvent for wortmannin and used at a final concentration in the perfusion buffer of 0.001%. Atorvastatin was kindly supplied by Pfizer and was dissolved in methanol (BDH Laboratory Supplies, Poole, UK) at 5 mg/100 µl. Final concentration of methanol in the perfusion buffer did not exceed 0.001%.
Mice were obtained from the breeding colony kept at University College, London, bred under license from Jackson Laboratories. Both eNOS wild-type (parental wild-type, based on C57BL/6J background) and eNOS knockout (KO) mice were used in the present study; a total of 75 mice were used. Three mice were excluded before starting the experimental protocol. No hearts were excluded after the onset of the ischemia/reperfusion protocol.
Langendorff perfusion.
Male and female eNOS KO mice (3 to 4 months of age, 20 to 30 g weight) were randomly attributed to either control or treatment groups. Heart isolation and Langendorff perfusion (with modified Krebs-Henseleit buffer consisting of NaCl 118 mmol/l, NaCO3 24 mmol/l, glucose 10 mmol/l, KCl 4 mmol/l, NaH2PO4 1.0 mmol/l, Na2 ethylenediaminetetraacetic acid 0.5 mmol/l, MgCl2 1.2 mmol/l, CaCl2 2.5 mmol/l) were performed as previously described (26). In brief, mice were anesthetized with an intra-peritoneal injection of 60 mg/kg pentobarbitone. Hearts were then harvested via a para-medial thoracotomy and rapidly transferred to a dissection dish filled with ice cold Krebs-Henseleit buffer. The aorta was cannulated with a 21-gauge murine cannula and transferred to the Langendorff rig. Hearts were paced throughout the stabilization period and during the final 20 min of reperfusion (600 beats/min).
Western blotting
Western blot analysis was performed as previously described (26). Lanes were loaded at 60 µg/lane. Loading was confirmed both by Ponceau staining of the membranes after transfer from the polyacrylamide gel and by quantification of the relevant non-phosphorylated protein (Akt or eNOS) by Western blot analysis. The membranes were probed with rabbit polyclonal antibodies for Akt, phospho- (serine 473) Akt, eNOS, and phospho- (serine 1177) eNOS antibodies purchased from Cell Signalling (Hitchin, UK) and used in accordance with the manufacturer’s instructions. Quantification was achieved using the enhanced chemiluminescence technique, with exposure to photographic film. The developed films were scanned using a flatbed document scanner, and the relative densitometry was assessed using the National Institutes of Health (NIH) Shareware program, NIH Image 1.62.
Experimental protocols
Langendorff perfusions were split into two phases: phase A for infarct size analysis and phase B for Western blot analysis. All hearts were subjected to 35 min of global, normothermic ischemia. Hearts were then either reperfused for 30 min and stained with triphenyl tetrazolium before planimetry in phase A, or they were reperfused for 5, 10, or 15 min and snap-frozen in phase B.
Phase A
The eNOS wild-type and eNOS KO hearts were randomized to receive one of the following treatments from the onset of reperfusion: 1) vehicle (n = 6 each for Krebs-Henseleit buffer, 0.001% methanol, or 0.001% DMSO); 2) atorvastatin (n = 6 each for 5, 25, 50, and 100 µmol/l); and 3) wortmannin 100 nmol/l in the presence or absence of 25 µmol/l atorvastatin.
Phase B
These experiments were divided into two groups as follows: 1) The eNOS wild-type hearts were randomized into 5-, 10-, or 15-min reperfusion groups in the presence or absence of 25 µmol/l atorvastatin; 2) The eNOS wild-type hearts were randomized to receive either vehicle or 25 µmol/l atorvastatin in the presence or absence of wortmannin, 100 nmol/l, whereas eNOS KO hearts were randomized to receive either vehicle or 25 µmol/l atorvastatin.
Infarct size analysis
Infarct size was determined by the triphenyl tetrazolium chloride (TTC) staining technique as previously described (26). In brief, the hearts at the end of the experimental protocol were infused with TTC in phosphate-buffered solution (pH 7.4). This turns viable tissue a dark red color, whereas non-viable, necrotic myocardium appears pale. The hearts were incubated for 10 min in TTC before destaining in 10% formaldehyde solution to increase contrast between necrotic and viable myocardium. Infarct size is expressed as an infarct-to-risk-zone ratio (when the risk zone in the global ischemic heart is the whole ventricular volume). Data are presented as a percentage ± SEM.
Statistical analysis
All values are expressed as mean ± SEM. Differences in continuously distributed variables between predetermined experimental groups were analyzed using one-way analysis of variance followed by Fisher’s protected test of least significant difference. A p value of 0.05 was considered to be at the limit of statistical significance.
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Results
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Does atorvastatin limit infarct size when administered as an adjunct to reperfusion?.
To determine whether atorvastatin is capable of attenuating myocardial infarction (MI) in this isolated mouse model of ischemia and reperfusion, we compared wild-type hearts, in the presence or absence of the solute vehicle, methanol (0.001%) added at reperfusion, with incremental concentrations of atorvastatin administered upon reperfusion. Over a concentration range of 25 to 100 µmol/l, atorvastatin significantly attenuated infarction (p < 0.01), with maximal protection observed with the 50 µmol/l dose. Infarct-to-risk-zone ratios were 26 ± 3%, 19 ± 2%, 16 ± 2%, and 21 ± 3% for 5, 25, 50, and 100 µmol/l, versus Krebs-Henseleit and methanol vehicle control 32 ± 2% and 33 ± 2%, respectively (Fig. 1).
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Figure 1 Atorvastatin dose–response curve. Incremental concentrations of atorvastatin resulted in a dose-dependent reduction of infarct size, peaking with 50 µmol/l (**p < 0.001 vs. control). Protection with 100 µmol/l was, however, less than that seen at 50 µmol/l (*p < 0.05 vs. control).
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Does atorvastatin administration at reperfusion result in increased activity of PI3K and Akt?
To determine whether atorvastatin-mediated reperfusion salvage is mediated by the recruitment of the PI3K/Akt signaling cascade, we measured the phosphorylation of the substrates of each of these protein kinases, Akt and eNOS, respectively. In control hearts, modest phosphorylation of Akt and eNOS was observed 10 min after the onset of reperfusion (Figs. 2 and 3), although over the time period examined (15 min of reperfusion ) there was no statistically significant increase in the activity of either PI3K or Akt. In contrast, treatment of the hearts at reperfusion with 25 µmol/l atorvastatin resulted in a significant increase of phosphorylation at all time points measured, suggesting that atorvastatin results in a rapid and more robust activation of the PI3K signaling cascade. Densitometry data indicate that the total Akt and eNOS phosphorylation after reperfusion in the atorvastatin group were 4.1-fold and 2.9-fold greater, respectively (p < 0.001), than over the same 15-min period in control hearts. Performing a simple regression plot between the quantification of phospho-Akt and phospho-eNOS resulted in a robust correlation (r2= 0.7, p < 0.001 with a gradient of 0.8) indicating a direct relationship between the phosphorylation of Akt and the phosphorylation of eNOS.
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Figure 2 The Akt phosphorylation time-course. Administration of 25 µmol/l atorvastatin to wild-type hearts at the start of reperfusion after a period of ischemia resulted in a robust and rapid increase in Akt phosphorylation (4.1-fold greater than that seen in control hearts, p < 0.001).
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Figure 3 The eNOS phosphorylation time-course. Administration of 25 µmol/l atorvastatin at the start of reperfusion after a period of ischemia resulted in a similar pattern of phosphorylation of eNOS to that seen with Akt, with a significant 2.8-fold increase over control (p < 0.001).
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Is increased PI3K activity essential for reperfusion salvage, or an epiphenomenon?
To determine whether the increase in PI3K activity is functionally related to atorvastatin reperfusion salvage or merely an epiphenomenon, we used the PI3K inhibitor, wortmannin (100 nmol/l) upon reperfusion in the presence or absence of atorvastatin (25 µmol/l). Wortmannin alone had no impact on infarct size (32 ± 2% vs. control, 34 ± 2%). However, wortmannin completely abrogated the protection observed with atorvastatin (33 ± 2% vs. atorvastatin alone, 19 ± 2% and control 34 ± 2%, p < 0.05, Fig. 4).
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Figure 4 The PI3K inhibitor, wortmannin, inhibits the reperfusion salvage seen with 25 µmol/l atorvastatin. Wortmannin administered alone had no impact on infarction (*p < 0.05, atorvastatin group vs. control and wortmannin plus atorvastatin).
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Is eNOS required for atorvastatin-mediated protection?
To determine whether eNOS is essential in the mediation of atorvastatin reperfusion salvage, we administered the cardioprotective dose of atorvastatin (25 µmol/l) to eNOS KO hearts. In contrast to the eNOS wild-type hearts previously studied, we found no protection (32 ± 1% vs. control, 31 ± 1%, Fig. 5).
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Figure 5 Reperfusion salvage seen with 25 µmol/l atorvastatin is absent in eNOS KO mice. Although atorvastatin results in significant protection in the wild-type hearts (WT), this protection was completely absent in the eNOS KO hearts (*p < 0.001 atorvastatin vs. WT control).
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Is PI3K activity modified in eNOS KO hearts and inhibited by wortmannin?
To determine whether the loss of atorvastatin-mediated protection that was observed in eNOS KO hearts and eNOS wild-type hearts treated with wortmannin was meditated by inhibition of PI3K activity, we assayed Akt phosphorylation in KO and wortmannin-treated hearts. Wortmannin administration significantly attenuated atorvastatin-triggered Akt phosphorylation to below the level seen in control hearts (143 ± 6 AU vs. atorvastatin alone, 772 ± 31 AU and control, 422 ± 52 AU, p < 0.001 and p < 0.01, respectively, Fig. 6).
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Figure 6 Akt phosphorylation with atorvastatin after 10 min of reperfusion: the effect of wortmannin and the absence of eNOS. Administration of atorvastatin to wild-type and KO hearts resulted in a robust increase in phosphorylation of Akt, although no cardioprotection is seen in eNOS KO hearts (**p < 0.001 vs. control hearts). The administration of the PI3K inhibitor, wortmannin, significantly reduced Akt phosphorylation compared with both control and atorvastatin-treated hearts ( p < 0.01 vs. both control and atorvastatin groups).
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Although no protection was observed as a result of atorvastatin administration in eNOS KO hearts, a similar level of Akt phosphorylation was nonetheless observed in KO hearts as it was in wild-type hearts (866 ± 32 AU vs. wild-type atorvastatin-treated hearts, 772 ± 31 AU, p > 0.05). Thus PI3K activity is indeed increased by atorvastatin-treated hearts despite the absence of cardioprotection.
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Discussion
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This study identifies the HMG-CoA reductase inhibitor, atorvastatin, as a drug capable of attenuating MI when administered as an adjunct to reperfusion, in a setting that is independent of cholesterol lowering. The data demonstrate, for the first time, that atorvastatin administered as an adjunct to reperfusion results in significant, dose-dependent cardioprotection, with an optimal concentration range between 25 and 100 µmol/l and maximal protection at 50 µmol/l. This study also identifies a key signaling pathway by which this protection at reperfusion is wrought: the PI3K signaling cascade—the first time that statins have been shown to regulate this kinase pathway in myocardium. Previous work has shown that statins may have quite opposing effects on tissue Akt activation and cellular survival, with up-regulation demonstrated in endothelial cells (21), no effect on neonatal myocytes (21), and a down-regulation in smooth muscle cells (27,28). Interestingly, our preliminary data in unstressed, Langendorff perfused, adult mouse myocardium indicated that brief (10-min) administration of 25 µmol/l atorvastatin did not enhance PI3K activity, as determined by the quantification of Akt phosphorylation by Western blotting (data not shown). However, we show here that 25 µmol/l atorvastatin does significantly increase phosphorylation of both Akt and eNOS in the post-ischemic myocardium (although the specific cell types have not been delineated). These data provide evidence that the PI3K may be playing an important role during the early reperfusion period, as has been suggested by recent work by ourselves and others with insulin both in vitro and in vivo (6,29). The PI3K hypothesis of reperfusion salvage is further supported by the data obtained after the administration of the PI3K inhibitor wortmannin. Not only does wortmannin lead to a significant attenuation of Akt phosphorylation associated with atorvastatin administration, wortmannin also abrogates atorvastatin-mediated protection. Thus, it appears that PI3K activity is essential in attenuating reperfusion injury.
A significant number of downstream targets for the PI3K signaling cascade have been identified (30), although those that are essential for early reperfusion salvage are not clear. One candidate that we have recently identified is the ribosomal p70S6 kinase (6). In this study, using insulin to trigger reperfusion salvage via insulin tyrosine kinase linked receptor, we were able to maintain the pro-apoptotic Bcl-2 family member, Bad, in a phosphorylated and, therefore, inactivated state (6,31). In the present study, we have investigated the role of an alternative target of Akt, namely eNOS (32). Concurrent with the rapid increase in Akt phosphorylation observed in hearts treated with atorvastatin administered at reperfusion, we see a significant and parallel increase of the phosphorylation of eNOS. Comparing the phosphorylation densitometry of both Akt and eNOS blotted from the same membrane, we find a significant and high correlation between the two events, implying that phosphorylation of eNOS is closely related to phosphorylation and activity of Akt. Significantly, the protection wrought by the administration of atorvastatin is completely absent in the eNOS KO heart. This suggests that eNOS is pivotal to the PI3K signaling cascade; Western blot analysis demonstrates that eNOS KO hearts are not deficient in the activity of PI3K, as measured by Akt phosphorylation. Therefore, it would seem that in this model of ischemia and reperfusion, PI3K activation alone is not sufficient to provide protection against infarction in the reperfused heart.
The concentrations of atorvastatin used in the present investigation are representative of top-end oral administration of the drug used in the management of hypercholesterolemic patients. Lower concentrations of atorvastatin have been found to be beneficial in promoting angiogenesis; it is interesting to note, however, that the concentrations used in the present study are anti-angiogenic according to recent work by Weis et al. (25). The duration of administration is the determining factor in producing this effect; in the present model we are investigating the immediate direct myocardial protective properties of statins, whereas the angiogenic effects are found with long-term administration, suggesting a dichotomy of beneficial concentration depending on the period of the treatment regime. That a "high dose" of statin is appropriate in the short-term setting of reperfusion injury management is supported by the recent work of Di Napoli et al. (22) with 25 µmol/l simvastatin in a working heart model of ischemia/reperfusion. In that study, simvastatin was administered with the onset of 15 min of global ischemia and at 180 min of reperfusion; high-dose simvastatin was found to be cardioprotective and, moreover, dependent on NO synthase activity.
Isolated, crystalloid perfused hearts were used in the present study to remove the potentially confounding effects of serum cholesterol, of actions on thrombocytes, and of the neutrophil/endothelial interaction previously hypothesized to be the mechanism of statin-mediated protection (17,33). In this study, we demonstrate that atorvastatin is capable of conferring protection on the reperfused myocardium independently of these factors, suggesting a direct effect on the myocardium of increasing the resistance to ischemia/reperfusion injury, mediated by PI3K, Akt, and eNOS. However, the isolated perfused model of ischemia and reperfusion has limitations specifically related to the limited time during which an isolated heart may be reperfused. Although this limitation should be recognized in the context of evolving MI and remodeling, we believe that the data presented here provide an insight into the mechanisms by which statins appear to confer additional protection on the ischemic heart beyond the drugs’ ability to reduce serum cholesterol (34,35).
In summary, we present evidence that the HMG-CoA reductase inhibitor, atorvastatin, is capable of attenuating ischemia/reperfusion injury when administered at the onset of reperfusion. Moreover, we show that the mechanism of action by which this protection is achieved is dependent on the recruitment and activation of a signaling cascade involving PI3K, Akt, and eNOS. Moreover, this protection appears to be mediated directly via the myocardium, independently of circulating thrombocytes, neutrophils, and other inflammatory moieties, and independently of a cholesterol-lowering effect.
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Footnotes
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Supported by a grant from the British Heart Foundation. Robert M. Bell was supported by a clinical fellowship from Pfizer Pharmaceuticals.
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References
|
|---|
1. Hearse DJ. Reperfusion-induced injury: a possible role for oxidant stress and its manipulation. Cardiovasc Drugs Ther. 1991;5:225–235
2. Kloner RA. Does reperfusion injury exist in humans? J Am Coll Cardiol. 1993;21:537–545[Abstract]
3. Yellon DM, Baxter GF. Reperfusion injury revisited. Is there a role for growth factor signaling in limiting lethal reperfusion injury? Trends Cardiovasc Med. 2000;9:245–249
4. Yellon DM, Baxter GF. Protecting the ischaemic and reperfused myocardium in acute myocardial infarction: distant dream or near reality? Heart. 2000;83:381–387[Free Full Text]
5. Jonassen AK, Brar BK, Mjos OD, Sack MN, Latchman DS, Yellon DM. Insulin administered at reoxygenation exerts a cardioprotective effect in myocytes by a possible anti-apoptotic mechanism. J Mol Cell Cardiol. 2000;32:757–764[CrossRef][Medline]
6. Jonassen AK, Sack MN, Mjos OD, Yellon DM. Myocardial protection by insulin at reperfusion requires early administration and is mediated via Akt and p70s6 kinase cell-survival signaling. Circ Res. 2001;89:1191–1198[Abstract/Free Full Text]
7. Fujio Y, Nguyen T, Wencker D, Kitsis RN, Walsh K. Akt promotes survival of cardiomyocytes in vitro and protects against ischemia-reperfusion injury in mouse heart. Circulation. 2000;101:660–667[Abstract/Free Full Text]
8. Yamashita K, Kajstura J, Discher DJ, et al. Reperfusion-activated Akt kinase prevents apoptosis in transgenic mouse hearts overexpressing insulin-like growth factor-1. Circ Res. 2001;88:609–614[Abstract/Free Full Text]
9. Matsui T, Tao J, del Monte F, et al. Akt activation preserves cardiac function and prevents injury after transient cardiac ischemia in vivo. Circulation. 2001;104:330–335[Abstract/Free Full Text]
10. A coronary primary prevention study of Scottish men aged 45–64 years: trial design. The West of Scotland Coronary Prevention Study Group. J Clin Epidemiol. 1992;45:849–860[CrossRef][Medline]
11. Randomised trial of cholesterol lowering in 4444 patients with coronary heart disease: the Scandinavian Simvastatin Survival Study (4S). Lancet. 1994;344:1383–1389[CrossRef][Medline]
12. Takemoto M, Liao JK. Pleiotropic effects of 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors. Arterioscler Thromb Vasc Biol. 2001;21:1712–1719[Abstract/Free Full Text]
13. Cucchiara B, Kasner SE. Use of statins in CNS disorders. J Neurol Sci. 2001;187:81–89[CrossRef][Medline]
14. Herrington DM, Potvin Klein K. Statins, hormones, and women: benefits and drawbacks for atherosclerosis and osteoporosis. Curr Atheroscler Rep. 2001;3:35–42[Medline]
15. O’Donnell MP, Kasiske BL, Kim Y, Atluru D, Keane WF. The mevalonate pathway: importance in mesangial cell biology and glomerular disease. Miner Electrolyte Metab. 1993;19:173–179[Medline]
16. Laufs U, Endres M, Liao JK. [Regulation of endothelial NO production by Rho GTPase]. Med Klin. 1999;94:211–218[Medline]
17. Laufs U, Gertz K, Huang P, et al. Atorvastatin upregulates type III nitric oxide synthase in thrombocytes, decreases platelet activation, and protects from cerebral ischemia in normocholesterolemic mice. Stroke. 2000;31:2442–2449[Abstract/Free Full Text]
18. Martinez-Gonzalez J, Raposo B, Rodriguez C, Badimon L. 3-Hydroxy-3-methylglutaryl coenzyme A reductase inhibition prevents endothelial NO synthase downregulation by atherogenic levels of native LDLs: balance between transcriptional and posttranscriptional regulation. Arterioscler Thromb Vasc Biol. 2001;21:804–809[Abstract/Free Full Text]
19. Cao Y, Hotta Y, Shioi K, Nagata Y, Kawai N, Ishikawa N. Protective effects of FK409, a novel nitric oxide donor, against postischemic myocardial dysfunction in guinea-pig hearts. J Cardiovasc Pharmacol. 2001;38:593–605[CrossRef][Medline]
20. Mohara J, Oshima K, Tsutsumi H, Takeyoshi I, Matsumoto K, Morishita Y. FK409 ameliorates ischemia-reperfusion injury in heart transplantation following 12-hour cold preservation. J Heart Lung Transplant. 2000;19:694–700[CrossRef][Medline]
21. Kureishi Y, Luo Z, Shiojima I, et al. The HMG-CoA reductase inhibitor simvastatin activates the protein kinase Akt and promotes angiogenesis in normocholesterolemic animals. Nat Med. 2000;6:1004–1010[CrossRef][Medline]
22. Di Napoli P, Antonio Taccardi A, Grilli A, et al. Simvastatin reduces reperfusion injury by modulating nitric oxide synthase expression: an ex vivo study in isolated working rat hearts. Cardiovasc Res. 2001;51:283–293[Abstract/Free Full Text]
23. Lefer AM, Siegfried MR, Ma XL. Protection of ischemia-reperfusion injury by sydnonimine NO donors via inhibition of neutrophil–endothelium interaction. J Cardiovasc Pharmacol. 1993;22(Suppl 7):S27–33
24. Brouet A, Sonveaux P, Dessy C, Moniotte S, Balligand JL, Feron O. Hsp90 and caveolin are key targets for the proangiogenic nitric oxide- mediated effects of statins. Circ Res. 2001;89:866–873[Abstract/Free Full Text]
25. Weis M, Heeschen C, Glassford AJ, Cooke JP. Statins have biphasic effects on angiogenesis. Circulation. 2002;105:739–745[Abstract/Free Full Text]
26. Bell RM, Smith CC, Yellon DM. Nitric oxide as a mediator of delayed pharmacological (A(1) receptor triggered) preconditioning; is eNOS masquerading as iNOS? Cardiovasc Res. 2002;53:405–413[Abstract/Free Full Text]
27. Weiss RH, Ramirez A, Joo A. Short-term pravastatin mediates growth inhibition and apoptosis, independently of Ras, via the signaling proteins p27Kip1 and P13 kinase. J Am Soc Nephrol. 1999;10:1880–1890[Abstract/Free Full Text]
28. Chen Z, Fukutomi T, Zago AC, et al. Simvastatin reduces neointimal thickening in low-density lipoprotein receptor-deficient mice after experimental angioplasty without changing plasma lipids. Circulation. 2002;106:20–23[Abstract/Free Full Text]
29. Gao F, Gao E, Yue TL, et al. Nitric oxide mediates the antiapoptotic effect of insulin in myocardial ischemia-reperfusion: the roles of PI3-kinase, Akt, and endothelial nitric oxide synthase phosphorylation. Circulation. 2002;105:1497–1502[Abstract/Free Full Text]
30. Datta SR, Brunet A, Greenberg ME. Cellular survival: a play in three Akts. Genes Dev. 1999;13:2905–2927[Free Full Text]
31. Datta SR, Katsov A, Hu L, et al. 14-3-3 proteins and survival kinases cooperate to inactivate BAD by BH3 domain phosphorylation. Mol Cell. 2000;6:41–51[CrossRef][Medline]
32. Dimmeler S, Fleming I, Fisslthaler B, Hermann C, Busse R, Zeiher AM. Activation of nitric oxide synthase in endothelial cells by Akt-dependent phosphorylation. Nature. 1999;399:601–605[CrossRef][Medline]
33. Lefer AM, Campbell B, Shin YK, Scalia R, Hayward R, Lefer DJ. Simvastatin preserves the ischemic-reperfused myocardium in normocholesterolemic rat hearts. Circulation. 1999;100:178–184[Abstract/Free Full Text]
34. Sacks FM, Pfeffer MA, Moye LA, et al. The effect of pravastatin on coronary events after myocardial infarction in patients with average cholesterol levels. Cholesterol and Recurrent Events Trial investigators. N Engl J Med. 1996;335:1001–1009[Abstract/Free Full Text]
35. Tonkin AM, Colquhoun D, Emberson J, et al. Effects of pravastatin in 3260 patients with unstable angina: results from the LIPID study. Lancet. 2000;356:1871–1875[CrossRef][Medline]
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G. Di Sciascio, G. Patti, V. Pasceri, A. Gaspardone, G. Colonna, and A. Montinaro
Efficacy of Atorvastatin Reload in Patients on Chronic Statin Therapy Undergoing Percutaneous Coronary Intervention: Results of the ARMYDA-RECAPTURE (Atorvastatin for Reduction of Myocardial Damage During Angioplasty) Randomized Trial
J. Am. Coll. Cardiol.,
July 1, 2009;
(2009)
j.jacc.2009.05.028v1.
[Abstract]
[Full Text]
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H. Ishii, T. Amano, T. Matsubara, and T. Murohara
Pharmacological Intervention for Prevention of Left Ventricular Remodeling and Improving Prognosis in Myocardial Infarction
Circulation,
December 16, 2008;
118(25):
2710 - 2718.
[Full Text]
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Y. Ye, Y. Lin, S. Manickavasagam, J. R. Perez-Polo, B. C. Tieu, and Y. Birnbaum
Pioglitazone protects the myocardium against ischemia-reperfusion injury in eNOS and iNOS knockout mice
Am J Physiol Heart Circ Physiol,
December 1, 2008;
295(6):
H2436 - H2446.
[Abstract]
[Full Text]
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Y. Ye, J. D. Martinez, R. J. Perez-Polo, Y. Lin, B. F. Uretsky, and Y. Birnbaum
The role of eNOS, iNOS, and NF-{kappa}B in upregulation and activation of cyclooxygenase-2 and infarct size reduction by atorvastatin
Am J Physiol Heart Circ Physiol,
July 1, 2008;
295(1):
H343 - H351.
[Abstract]
[Full Text]
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Y.-J. Yang, H.-Y. Qian, J. Huang, Y.-J. Geng, R.-L. Gao, K.-F. Dou, G.-S. Yang, J.-J. Li, R. Shen, Z.-X. He, et al.
Atorvastatin treatment improves survival and effects of implanted mesenchymal stem cells in post-infarct swine hearts
Eur. Heart J.,
June 2, 2008;
29(12):
1578 - 1590.
[Abstract]
[Full Text]
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G. F. Kocsis, J. Pipis, V. Fekete, A. Kovacs-Simon, L. Odendaal, E. Molnar, Z. Giricz, T. Janaky, J. van Rooyen, T. Csont, et al.
Lovastatin interferes with the infarct size-limiting effect of ischemic preconditioning and postconditioning in rat hearts
Am J Physiol Heart Circ Physiol,
May 1, 2008;
294(5):
H2406 - H2409.
[Abstract]
[Full Text]
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Y. Birnbaum, Y. Lin, Y. Ye, R. Merla, J. R. Perez-Polo, and B. F. Uretsky
Pretreatment With High-Dose Statin, But Not Low-Dose Statin, Ezetimibe, or the Combination of Low-Dose Statin and Ezetimibe, Limits Infarct Size in the Rat
Journal of Cardiovascular Pharmacology and Therapeutics,
March 1, 2008;
13(1):
72 - 79.
[Abstract]
[PDF]
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M. J. Davidson and D. S. Baim
Percutaneous Aortic Valve Interventions
Card. Surg. Adult,
January 1, 2008;
3(2008):
963 - 971.
[Full Text]
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P. Ferdinandy, R. Schulz, and G. F. Baxter
Interaction of Cardiovascular Risk Factors with Myocardial Ischemia/Reperfusion Injury, Preconditioning, and Postconditioning
Pharmacol. Rev.,
December 1, 2007;
59(4):
418 - 458.
[Abstract]
[Full Text]
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L. Zhang, Z. G. Zhang, X. S. Liu, A. Hozeska-Solgot, and M. Chopp
The PI3K/Akt Pathway Mediates the Neuroprotective Effect of Atorvastatin in Extending Thrombolytic Therapy After Embolic Stroke in the Rat
Arterioscler. Thromb. Vasc. Biol.,
November 1, 2007;
27(11):
2470 - 2475.
[Abstract]
[Full Text]
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D. M. Yellon and D. J. Hausenloy
Myocardial Reperfusion Injury
N. Engl. J. Med.,
September 13, 2007;
357(11):
1121 - 1135.
[Full Text]
[PDF]
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R. Yin, J. Zhu, Z. Wang, H. Huang, J. Qian, Z. Li, and H. Jing
Simvastatin attenuates cardiac isograft ischemia-reperfusion injury by down-regulating CC chemokine receptor-2 expression
J. Thorac. Cardiovasc. Surg.,
September 1, 2007;
134(3):
780 - 788.
[Abstract]
[Full Text]
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R. Merla, Y. Ye, Y. Lin, S. Manickavasagam, M.-H. Huang, R. J. Perez-Polo, B. F. Uretsky, and Y. Birnbaum
The central role of adenosine in statin-induced ERK1/2, Akt, and eNOS phosphorylation
Am J Physiol Heart Circ Physiol,
September 1, 2007;
293(3):
H1918 - H1928.
[Abstract]
[Full Text]
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J. Afilalo, A. A Majdan, and M. J Eisenberg
Intensive statin therapy in acute coronary syndromes and stable coronary heart disease: a comparative meta-analysis of randomised controlled trials
Heart,
August 1, 2007;
93(8):
914 - 921.
[Abstract]
[Full Text]
[PDF]
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Y. Ye, Y. Lin, R. Perez-Polo, M.-H. Huang, M. G. Hughes, D. J. McAdoo, S. Manickavasagam, B. F. Uretsky, and Y. Birnbaum
Enhanced cardioprotection against ischemia-reperfusion injury with a dipyridamole and low-dose atorvastatin combination
Am J Physiol Heart Circ Physiol,
July 1, 2007;
293(1):
H813 - H818.
[Abstract]
[Full Text]
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A. Bulhak, J. Roy, U. Hedin, P.-O. Sjoquist, and J. Pernow
Cardioprotective effect of rosuvastatin in vivo is dependent on inhibition of geranylgeranyl pyrophosphate and altered RhoA membrane translocation
Am J Physiol Heart Circ Physiol,
June 1, 2007;
292(6):
H3158 - H3163.
[Abstract]
[Full Text]
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S. M. Davidson and M. R. Duchen
Endothelial Mitochondria: Contributing to Vascular Function and Disease
Circ. Res.,
April 27, 2007;
100(8):
1128 - 1141.
[Abstract]
[Full Text]
[PDF]
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G. Patti, V. Pasceri, G. Colonna, M. Miglionico, D. Fischetti, G. Sardella, A. Montinaro, and G. Di Sciascio
Atorvastatin Pretreatment Improves Outcomes in Patients With Acute Coronary Syndromes Undergoing Early Percutaneous Coronary Intervention: Results of the ARMYDA-ACS Randomized Trial
J. Am. Coll. Cardiol.,
March 27, 2007;
49(12):
1272 - 1278.
[Abstract]
[Full Text]
[PDF]
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Y. Ye, Y. Lin, S. Atar, M.-H. Huang, J. R. Perez-Polo, B. F. Uretsky, and Y. Birnbaum
Myocardial protection by pioglitazone, atorvastatin, and their combination: mechanisms and possible interactions
Am J Physiol Heart Circ Physiol,
September 1, 2006;
291(3):
H1158 - H1169.
[Abstract]
[Full Text]
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C. Wang, D. A. Neff, J. G. Krolikowski, D. Weihrauch, M. Bienengraeber, D. C. Warltier, J. R. Kersten, and P. S. Pagel
The influence of B-cell lymphoma 2 protein, an antiapoptotic regulator of mitochondrial permeability transition, on isoflurane-induced and ischemic postconditioning in rabbits.
Anesth. Analg.,
May 1, 2006;
102(5):
1355 - 1360.
[Abstract]
[Full Text]
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Y. Abdallah, A. Gkatzoflia, D. Gligorievski, S. Kasseckert, G. Euler, K.-D. Schluter, M. Schafer, H.-M. Piper, and C. Schafer
Insulin protects cardiomyocytes against reoxygenation-induced hypercontracture by a survival pathway targeting SR Ca2+ storage
Cardiovasc Res,
May 1, 2006;
70(2):
346 - 353.
[Abstract]
[Full Text]
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K. Iwakura, H. Ito, S. Kawano, A. Okamura, T. Kurotobi, M. Date, K. Inoue, and K. Fujii
Chronic pre-treatment of statins is associated with the reduction of the no-reflow phenomenon in the patients with reperfused acute myocardial infarction
Eur. Heart J.,
March 1, 2006;
27(5):
534 - 539.
[Abstract]
[Full Text]
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N. V. Solenkova, V. Solodushko, M. V. Cohen, and J. M. Downey
Endogenous adenosine protects preconditioned heart during early minutes of reperfusion by activating Akt
Am J Physiol Heart Circ Physiol,
January 1, 2006;
290(1):
H441 - H449.
[Abstract]
[Full Text]
[PDF]
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Y. Abdallah and C. Schafer
Insulin: An overall cardiovascular protector?
Cardiovasc Res,
January 1, 2006;
69(1):
4 - 6.
[Full Text]
[PDF]
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J. Herrmann
Peri-procedural myocardial injury: 2005 update
Eur. Heart J.,
December 1, 2005;
26(23):
2493 - 2519.
[Abstract]
[Full Text]
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K. K. Ray and C. P. Cannon
The Potential Relevance of the Multiple Lipid-Independent (Pleiotropic) Effects of Statins in the Management of Acute Coronary Syndromes
J. Am. Coll. Cardiol.,
October 18, 2005;
46(8):
1425 - 1433.
[Abstract]
[Full Text]
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D. Weihrauch, J. G. Krolikowski, M. Bienengraeber, J. R. Kersten, D. C. Warltier, and P. S. Pagel
Morphine Enhances Isoflurane-Induced Postconditioning Against Myocardial Infarction: The Role of Phosphatidylinositol-3-Kinase and Opioid Receptors in Rabbits
Anesth. Analg.,
October 1, 2005;
101(4):
942 - 949.
[Abstract]
[Full Text]
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A. Tsang, D. J. Hausenloy, and D. M. Yellon
Myocardial postconditioning: reperfusion injury revisited
Am J Physiol Heart Circ Physiol,
July 1, 2005;
289(1):
H2 - H7.
[Full Text]
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I. S. Ali and K. J. Buth
Preoperative statin use and in-hospital outcomes following heart surgery in patients with unstable angina
Eur. J. Cardiothorac. Surg.,
June 1, 2005;
27(6):
1051 - 1056.
[Abstract]
[Full Text]
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K. Mensah, M. M. Mocanu, and D. M. Yellon
Failure to protect the myocardium against ischemia/reperfusion injury after chronic atorvastatin treatment is recaptured by acute atorvastatin treatment: A potential role for phosphatase and tensin homolog deleted on chromosome ten?
J. Am. Coll. Cardiol.,
April 19, 2005;
45(8):
1287 - 1291.
[Abstract]
[Full Text]
[PDF]
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R. Schulz
Pleiotropic effects of statins: Acutely good, but chronically bad?
J. Am. Coll. Cardiol.,
April 19, 2005;
45(8):
1292 - 1294.
[Full Text]
[PDF]
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S. Sanada, H. Asanuma, T. Minamino, K. Node, S. Takashima, H. Okuda, Y. Shinozaki, A. Ogai, M. Fujita, A. Hirata, et al.
Optimal Windows of Statin Use for Immediate Infarct Limitation: 5'-Nucleotidase as Another Downstream Molecule of Phosphatidylinositol 3-Kinase
Circulation,
October 12, 2004;
110(15):
2143 - 2149.
[Abstract]
[Full Text]
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M. Campbell, W. E. Allen, C. Sawyer, B. Vanhaesebroeck, and E. R. Trimble
Glucose-Potentiated Chemotaxis in Human Vascular Smooth Muscle Is Dependent on Cross-Talk Between the PI3K and MAPK Signaling Pathways
Circ. Res.,
August 20, 2004;
95(4):
380 - 388.
[Abstract]
[Full Text]
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M. B. Harris, M. A. Blackstone, S. G. Sood, C. Li, J. M. Goolsby, V. J. Venema, B. E. Kemp, and R. C. Venema
Acute activation and phosphorylation of endothelial nitric oxide synthase by HMG-CoA reductase inhibitors
Am J Physiol Heart Circ Physiol,
August 1, 2004;
287(2):
H560 - H566.
[Abstract]
[Full Text]
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F. Eefting, B. Rensing, J. Wigman, W. J. Pannekoek, W. M. Liu, M. J. Cramer, D. J Lips, and P. A Doevendans
Role of apoptosis in reperfusion injury
Cardiovasc Res,
February 15, 2004;
61(3):
414 - 426.
[Abstract]
[Full Text]
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D. J Hausenloy and D. M Yellon
New directions for protecting the heart against ischaemia-reperfusion injury: targeting the Reperfusion Injury Salvage Kinase (RISK)-pathway
Cardiovasc Res,
February 15, 2004;
61(3):
448 - 460.
[Abstract]
[Full Text]
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E. A. Woodcock
Unc-II and Unc-III Are Cardioprotective against Ischemia Reperfusion Injury: An Essential Endogenous Cardioprotective Role for CRFR2 in the Murine Heart
Endocrinology,
January 1, 2004;
145(1):
21 - 23.
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Abstract
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