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Cardiovascular Research 2001 51(2):294-303; doi:10.1016/S0008-6363(01)00303-0
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Copyright © 2000, European Society of Cardiology

Lipid hydroperoxide modification of proteins during myocardial ischaemia

Philip Eaton*, David J Hearse and Michael J Shattock

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

* Corresponding author. Tel.: +44-171-928-9292 (ext. 2749); fax: +44-171-922-8139 philip.eaton{at}kcl.ac.uk

Received 25 October 2000; accepted 29 March 2001


   Abstract
Top
 Abstract
1 Introduction
 2 Methods
 3 Results
 4 Discussion
 5 Conclusions
 References
 
Objective: Lipid hydroperoxides (LOOH) are lipid peroxidation products formed during oxidative stress. A component of their cytotoxicity is mediated by the direct modification of proteins. Objectives: (i) To assess whether ischaemia and reperfusion in the isolated rat heart generates LOOH–protein (ii) to characterise the extent, time-course and subcellular localization of any protein adducts formed. Methods: Using a well-characterised antibody which binds to LOOH-modified proteins and densitometry of Western blots, we quantified the amounts of LOOH–protein in control aerobically perfused rat hearts and those subjected to ischaemia with and without reperfusion. Results: Hearts (n=3/4 group), analysed after various periods (0, 5, 10, 20 and 30 min) of zero-flow global ischaemia, exhibited a time-dependent increase in the LOOH-mediated modification of a number of proteins. In hearts subjected to 30 min of ischaemia and then reperfused for various times (0, 5, 10, 20, 30 or 60 min) no changes in LOOH–protein content achieved during the proceeding ischaemic episode were detected. Reperfusion after short periods of ischaemia (5 or 10 min) also did not result in reperfusion-induced LOOH–protein formation. Administration of mercaptopropionylglycine (1 mM) to hearts for 5 min before the onset of 30 min ischaemia efficiently attenuated the formation of LOOH–protein, maintaining the modified proteins at control levels. These Western immunoblot results were confirmed by additional in situ immunofluorescent studies which showed marked LOOH–protein immunostaining in ischaemic tissue around the sarcolemmal membrane. Conclusions: We conclude that the modification of proteins (particularly those associated with sarcolemmal membranes) by LOOH during ischaemia may contribute to the pathophysiology of ischaemic injury. In addition, these modifications may be initiators of oxidant-induced signal transduction pathways. These findings are consistent with an oxidant stress occurring during ischaemia which is not exacerbated or reduced during the first 60 min of reperfusion.

KEYWORDS LOOH, lipid hydroperoxide; HNE, hydroxynonenal; MPG, mercaptopropionylglycine


   1 Introduction
Top
 Abstract
 1 Introduction
2 Methods
 3 Results
 4 Discussion
 5 Conclusions
 References
 
Ischaemia [9,10,17,22,26,28] and reperfusion [19,30,33,36] exert an oxidant stress on the heart which contributes to both electrical and contractile dysfunction [16,20]. Proteins, particularly those associated with membranes (e.g. ion translocators) have been proposed as direct molecular targets of damage of this oxidative insult [4,5,11].

Lipid hydroperoxides (LOOH) are primary lipid peroxidation products formed when omega-6 polyunsaturated fatty acids, such as linoleic acid, react with free radical species [29,48]. Consequently, lipid hydroperoxides are produced as a result of ischaemia and reperfusion in tissues, including the heart [6,7,45,48]. The reactivity of lipid hydroperoxides is such that they are damaging to proteins within tissues and the ability of these species when applied exogenously to precipitate cardiac dysfunction is established [14,24]. Since LOOH is produced in the heart during oxidative stress and it is able to elicit cardiac dysfunction, it is possible that a component of the injury which accompanies ischaemia and reperfusion might be as a result of LOOH modifying cardiac proteins. A growing number of studies have shown a role for reactive lipid oxidation products, such as LOOH and its breakdown product hydroxynonenal, in the initiation of redox-sensitive signal transduction pathways [8,23,25].

To date no studies have demonstrated the production of LOOH-modified proteins in hearts subjected to ischaemia and reperfusion. The aim of these studies was therefore to assess whether protein-LOOH formation can be detected in the isolated perfused rat heart both during ischaemia and following reperfusion and whether any such adduct formation can be ameliorated by an antioxidant. This study extends our previous investigations into cardiac protein modification by reactive lipid degradation products where we have demonstrated ischaemia-induced modification of proteins by the major LOOH breakdown product 4-hydroxynonenal (HNE) [2]. In the present study we exploit the availability of a characterised antibody which has been used for Western immunochemical analysis, immunoassay and immunohistochemical detection of LOOH-modified proteins [31].


   2 Methods
Top
 Abstract
 1 Introduction
 2 Methods
3 Results
 4 Discussion
 5 Conclusions
 References
 
2.1 Animals
Male Wistar rats (200–250 g) were used throughout this study and were obtained from BK Universal. The animals were maintained humanely in compliance with the Principles of Laboratory Animal Care formulated by the National Society for Medical Research and Guide for the Care and Use of Laboratory Animals prepared by the National Academy of Sciences and published by the National Institute of Health (NIH publication No. 85-23, revised 1985).

2.2 Isolated heart preparations
Rats were anesthetised with diethyl ether and injected with 200 I.U. of sodium heparin via the femoral vein. Hearts were rapidly excised, placed in cold (4°C) bicarbonate buffer and the aorta cannulated. The hearts were then perfused with bicarbonate buffer gassed with 95% O2–5% CO2 at 37.0°C (pH 7.4). Perfusion was in the non-recirculating Langendorff mode at a constant pressure equivalent to 100 cm of H2O. The bicarbonate buffer contained (in mM) NaCl 118.5, KCl 3.1, KH2PO4 1.18, NaHCO3 25.0, MgCl2 1.2, CaCl2 1.4 and glucose 10.0.

2.3 Perfusion protocols
The perfusion protocols used in this study are summarised in Fig. 1. During ischaemia, hearts were maintained in air in a thermostatically-controlled chamber at 37.0°C. Mercaptopropionylglycine (MPG) solutions were prepared immediately before use by dissolving MPG to a final concentration of 1 mM in bicarbonate buffer. Hearts were perfused with this solution during the last 5 min of the initial aerobic perfusion period. In all protocols, three or four hearts were analysed in each treatment group.


Figure 1
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  		Fig. 1 Perfusion protocols used in this study. Cardiac tissue was prepared following ischaemia or reperfusion at the times shown by the arrows. Mercaptopropionylglycine (MPG, 1 mM) was administered 5 min prior to the onset of 30 min ischaemia.

 
2.4 Preparation of LOOH-modified protein
A 0.1-ml volume of bovine serum albumin (fraction V, 1 mg/ml) was incubated with 2 mg of lipid hydroperoxide (13(S)-hydroperoxyoctadeca-9Z,11E-dienoic acid (Biomol Research Laboratories, USA) for 60 min at 37°C. The reaction was terminated by the addition of sodium dodecylsulphate (SDS) sample buffer.

2.5 Protein analysis
Cardiac protein was prepared by grinding tissue under liquid nitrogen and then solublising 100 mg of this in 2 ml of SDS sample buffer without boiling. SDS–polyacrylamide gel electrophoresis (PAGE) was carried out using the Bio-Rad mini protean II system on 10% polyacrylamide gel with 30 µg of protein loaded per lane.

After electrophoresis, samples were transferred to PVDF membrane (Amersham, UK) using a Pharmacia system semi-dry blotter. A characterised antibody which recognises LOOH–protein (kindly donated by Dr. S. Parthasarathy) was used to detect the modified proteins. An anti-rabbit secondary antibody coupled to horseradish peroxidase (Amersham) was used with the enhanced chemiluminescent (ECL) reagent (Amersham) to visualise primary antibody binding. Western blots were digitised using a flat-bed scanner (HP Scanjet 11C). The digitised image was then quantitatively analysed for total LOOH modified proteins in each sample lane using the NIH IMAGE software (Freeware, NIH, Baltimore, USA).

2.6 Immunofluorescence
In this study control tissue was prepared by snap-freezing (in liquid nitrogen and isopentane) ventricular tissue after 20 min aerobic perfusion. Ischaemic tissue was obtained by terminating flow after 20 min aerobic perfusion during which the heart was thermostatically maintained at 37°C. An ischaemic duration of 20 min was used for these histochemical studies as this is sufficient to allow near maximal amounts of LOOH–protein to be formed but at a time when there is little necrosis and tissue disruption. Extending the ischaemic time to 30 min caused ultrastructural degeneration and made the immunofluorescent localisation of the modified proteins more difficult. The effect of the antioxidant MPG was investigated by administering it at 1 mM for 5 min during aerobic perfusion immediately prior to the onset of ischaemia. Frozen sections of 12 µm were cut and immunofluorescent analysis carried out as previously described [2]. An immunofluorescent positive control was generated by incubating a control tissue section with 13(S)-hydroperoxyoctadeca-9Z,11E-dienoic acid (5 µg/ml in phosphate buffered saline) for 10 min at room temperature, after which sections were washed three times with phosphate buffered saline. Specific antibody binding was detected using FITC-conjugated goat anti-rabbit IgG (Sigma, UK). Laser scanning confocal microscopy was carried out using a Bio-Rad confocal microscope. The distribution of FITC-conjugated antibodies was visualised by excitation with a laser line at 488 nm and stored digitally. Immunostaining experiments were repeated on three hearts per group and at least ten sections examined per heart. Representative results are shown in Fig. 4 and a similar patter was seen in all sections from all hearts within an experimental group.


Figure 4
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  		Fig. 4 Immunofluorescent images of tissue stained with the anti protein–LOOH antibody. The top four panels show transmitted light images, with the corresponding fluorescent images directly below. Tissue from control preparations was not stained by the antibody, however, control tissue which has been incubated with the lipid hydroperoxide 13(S)-hydroperoxyoctadeca-9Z,11E-dienoic acid (positive control) became immunoreactive, showing staining over the entire tissue section — with the exception of the nuclei. Ischaemic tissue sections showed very clear immunoreactivity around the sarcolemmal membrane, when either transverse or longitudinal sections are viewed. Thus, the ischaemia-induced modifications of proteins by LOOH observed using Western blotting (Fig. 2b) are confirmed with immunohistochemical analysis, which localised the modified proteins to the sarcolemma. At higher magnification of ischaemic sections, in addition to sarcolemmal staining, a regular filamentous staining pattern is present. Immunostaining experiments were replicated three times with representative results shown. A 20-µm scale bar is shown in each image.

 
2.7 Statistics
Results are presented as mean±S.E.M. (n=3 or 4 per group). Differences between groups were assessed using ANOVA, followed by a Dunnett Test. Differences were considered significant at the 95% confidence level.


   3 Results
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
4 Discussion
 5 Conclusions
 References
 
3.1 Confirmation of anti-sera specificity
The antibody used in this study has been characterised and its utility and specificity in immunoblotting, immuohistochemistry and ELISA have been already demonstrated [31]. To confirm the antibodies ability to detect specifically LOOH modified protein in this laboratory, we derivatised BSA with LOOH. It is evident from Fig. 2A that only LOOH-modified BSA is recognised by the antisera.


Figure 2
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  		Fig. 2 (A) Western immunoblot showing the ability of the LOOH–protein antibody to specifically recognise bovine serum albumin after it is derivatised with the lipid hydroperoxide 13(S)-hydroperoxyoctadeca-9Z, 11E-dienoic acid. Thus, the modified BSA shows the specificity of the antisera and acts as a positive control. (B) Western immunoblot showing the formation of LOOH-modified cardiac proteins after 30 min of ischaemia. Clearly, the antioxidant MPG efficiently attenuates the formation of LOOH-modified proteins during ischemia. (C) Coomassie blue stained PVDF membrane used to produce the Western immunoblots shown in Fig. 2B. The amount of total protein loaded is the same in all lanes.

 
3.2 LOOH–protein formation during ischaemia and ischaemia with reperfusion
The Western blot shown in Fig. 2B, where each lane represents a sample from different hearts, demonstrates that there is an accumulation of proteins modified by LOOH during ischaemia. It is clear that there is a high-molecular-weight protein which shows significant immunostaining in control samples. This may represent non-specific staining; but as this band increases in ischaemic samples, it is probably a protein which shows high basal modification. Quantitative analysis (of the total signal in an entire lane) of digitised Western immunoblots (Fig. 3A) shows that the ischaemia-induced accumulation of these modified proteins is time-dependent and that the LOOH–protein signal becomes maximal after 20 min and is not increased if ischaemia is extended to 30 min. This quantitative analysis also shows that reperfusion (5, 10, 20, 30 and 60 min) following 30 min of ischaemia does not alter the amount of LOOH–protein generated during ischaemia. Fig. 3B shows reperfusion after short periods of ischaemia (5 or 10 min), where the amount of LOOH–protein was submaximal, did not result in reperfusion-induced cardiac protein oxidation by LOOH.


Figure 3
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  		Fig. 3 (A) Quantitative assessment of protein–LOOH adduct formation in cardiac tissue during ischaemia and reperfusion. Mercaptopropionylglycine (MPG, 1 mM) attenuated the formation of protein–LOOH following 30 min of ischaemia. (B) Reperfusion of tissue after short ischaemic durations (5 or 10 min) did not elevate protein–LOOH levels above that reached during the ischaemic period. Thus, reperfusion-induced protein–LOOH formation was undetectable.

 
3.3 Effect of MPG pretreatment on LOOH–protein adduct content of the isolated heart
Fig. 3 also shows that the antioxidant MPG (1 mM administered 5 min prior to the onset of 30 min ischaemia) reduced the formation of ischaemia-induced LOOH–protein modification with striking efficiency, maintaining the level of modified protein at near the aerobic control value.

3.4 Immunolocalisation of the LOOH–protein adducts
Fig. 4 shows immunohistochemical localization of LOOH-modified proteins in heart cryosections. Control tissue which had been subjected to aerobic perfusion was not immunoreactive for LOOH-modified protein. However, pretreatment of control tissue sections with LOOH resulted in widespread, gross immunoreactivity of the tissue section, although the lack of staining of the nuclei was notable. Tissue subjected to aerobic perfusion followed by 20 min ischaemia contained high levels of LOOH–protein localised primarily to the sarcolemmal membrane. When viewed at higher magnification (120 or 180x) ischaemic sections showed immunoreactivity in a regular filamentous pattern, which may represent modification of myofilament or cytoskeletal elements. However, overall the majority of LOOH-modified proteins was found within the sarcolemmal membrane. Immunostaining experiments were replicated three times with similar results.


   4 Discussion
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
5 Conclusions
 References
 
4.1 LOOH–protein formation in the ischaemic heart
Our studies demonstrate that as ischaemia proceeds in the isolated rat heart, proteins become increasingly subjected to modification by LOOH. As LOOH is formed by free radical-induced lipid peroxidation [29,48], we conclude that during ischaemia the hearts suffers an oxidative insult, either as a result of free radical production or antioxidant depletion, which leads to the generation of LOOH which covalently attaches to proteins. This is consistent with a recent study which showed ischaemia-induced formation of free lipid hydroperoxides [41]. LOOH modification of proteins can lead to enzymatic and cellular dysfunction, and as such, alteration of cardiac proteins observed in this study is likely to represent a contributor to ischaemic injury. The concept that LOOH formation is likely to impact negatively on the heart is supported by studies demonstrating that the exogenous application of LOOH can induce cardiac dysfunction [14,24].

The time-dependent increase in LOOH–protein as ischaemia proceeds may parallel a combination of ischaemia-induced antioxidant depletion and free radical production. Both antioxidant depletion and free radical production in ischaemic cardiac tissue proceed with a similar time-course to that observed for LOOH–protein formation [10,21,22,26]. Reduced glutathione (GSH) falls substantially within minutes of the onset of ischaemia and as a consequence renders the heart vulnerable to lipid peroxidation and subsequent formation of LOOH–protein. GSH also directly scavenges reactive products and thus its depletion may directly lead to elevated levels of LOOH which prove detrimental. This loss of GSH during ischaemia occurs because the biochemical pathway required to reduce oxidised glutathione (GSSG) and restore GSH is dependent on ATP which becomes limiting as ischaemia progresses [13]. Myocardial GSH loss compromises recovery from ischaemia [40], even after short ischaemic durations [18]. Furthermore, recovery on reperfusion is GSH-dependent [12,39]. Thus the loss of GSH during ischaemia may contribute to cellular damage resulting from LOOH accumulation.

4.2 Lack of reperfusion-induced LOOH–protein formation
Reperfusion neither reduced nor exacerbated the content of LOOH–protein formed during the preceding period of ischaemia. This was the case even when short period of ischaemia followed by reperfusion were assessed. Free LOOH in the myocardium has been shown to increase during reperfusion, above that attained during ischaemia [41]. It is unclear exactly why we did not detect increased amounts of protein–LOOH during reperfusion. Increased protein–LOOH might have been anticipated as a consequence of free radical formation during the early minutes of reperfusion [19,30]. Oxidative tissue damage is, however, not determined by radical production alone, but also by the levels of cellular antioxidants which eliminate these harmful species. Protein–LOOH modifications during ischaemia may reflect the rapid depletion of antioxidant defences and the resulting pro-oxidative conditions. It is clear that ischaemia in itself shifts the redox state of the heart to oxidising [9,10,15,17,22,26,28,41]. These facts are somewhat at odds with the conventional dogma which reserves reperfusion as the time of oxidative stress within tissues. A lack of reperfusion-induced modification of cardiac proteins by LOOH is in agreement with our previous observation that HNE-modified proteins only form during ischaemia and not during reperfusion. This was despite the use of detailed and numerous perfusion protocols to try and detect reperfusion-induced HNE–protein formation [2].

The failure of reperfusion to modify tissue protein–LOOH content might be explained by the restoration of coronary flow, as cytotoxic species are detoxified via GSH conjugation and then exported from the cell [35,39]. This detoxification system is dependent on the maintenance of an outward concentration gradient of the GSH-conjugated product to be transported, which requires coronary flow to wash away the exported GSH conjugated cytotoxins. Consequently, during ischaemia when the flow stops, export the GSH-conjugates is halted. This may explain the lack of LOOH–protein formation in the reperfused heart.

4.3 Antioxidant attenuation of LOOH–protein formation
MPG was efficient at inhibiting ischaemia-induced LOOH–protein modifications. MPG can terminate free radical chain reactions as well as directly sequester free LOOH before it modifies proteins. Antioxidants, such as MPG, are established cardioprotective agents [3,16,20] and the protection afforded by them may conceivably involve limiting LOOH–protein formation during ischaemia.

4.4 Localisation of the LOOH-modified proteins
Immunohistochemical analysis of cryosections showed that 20 min of ischaemia resulted in increased amounts of protein–LOOH. The immunofluorescence observed was primarily localised to the sarcolemma. Since it is the membrane that contains the lipid substrate for LOOH production, it is perhaps, unsurprising that LOOH–protein appears concentrated in this sarcolemmal region. However, the sarcolemmal staining observed in ischaemia is clearly not discrete but appears as a diffuse halo around the cell periphery. LOOH once formed will not only modify proteins in its immediate environment but may also diffuse away from its site of production and modify proteins in a wider area. This may account for the diffuse staining around the sarcolemma. Since LOOH may be generated on either face of the membrane (or may diffuse through it) it is likely proteins both in the interstitial space as well as intracellularly in the sub-sarcolemmal region may be modified in this way.

4.5 LOOH–protein formation correlates with hydroxynonenal-protein formation
Previously [2] we have reported the modification of proteins by HNE during cardiac ischaemia and these previous findings corroborate the observations reported here. Thus, HNE–protein formation occurred during ischaemia, but not during reperfusion; and formation was inhibited by the pre-ischaemic administration of MPG. Likewise, HNE-modified proteins also were localised to the sarcolemma. As HNE is a breakdown product of LOOH, it is perhaps not surprising that a similar pattern of events has been revealed in this study.

4.6 Potential signalling roles for reactive lipid oxidation products
Whilst the past decade has established oxidants as mediators of cellular injury, in recent years there has been growing recognition that these agents have a dual role as signal transduction molecules [8,23,25]. The extent of the redox imbalance may determine whether oxidants simply kill cells or play a more subtle role in signal transduction. Certainly, there is substantial evidence for oxidants as signalling molecules under both normal and pathophysiological situations [47,50]. Reactive lipid oxidation products play a key role in oxidant signalling and are important initiators of early upstream signalling events involving receptors [34,37,43,49]. Reactive lipid oxidation products can covalently attach membrane receptors, such as the epidermal growth factor receptor (EGFR) — where it mimics the action of EGF itself [27,38,42]. In response to reactive lipid oxidation products, EGFR becomes phosphorylated, triggers phosphorylation of the adapter protein Shc and eventually stimulates the ERK pathway to regulate cell growth and differentiation [1,37,38,43,49]. The effects of reactive lipid oxidation products on growth seem to be under transcriptional control, and these reactive lipids can modulate the expression of cell cycle proteins and transcription factors [32,37]. Reactive lipid oxidation products also control processes such as apoptosis and the phosphorylation of cytoskeletal elements [44,46]. A common observation from the developing literature is a role for the MAP kinase family of proteins in the downstream transduction reactive lipid oxidation product initiated signal.


   5 Conclusions
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
 5 Conclusions
References
 
The present study demonstrates the structural modification of cardiac proteins by lipid hydroperoxides during ischaemia and the fact that these modifications are not reversed or exacerbated by reperfusion. The formation of LOOH-modified proteins may represent potentially deleterious modifications which contribute to cardiac injury. However, there is growing evidence that modification of proteins by reactive lipid oxidation products have integral roles in initiating signal transduction pathways, a mechanism which may allow for cardiac adaptation to ischaemia and reperfusion. This may be consistent with studies which have shown that oxidative stress is integral in the triggering of the cardioprotection offered by ischaemic preconditioning. Future identification of the proteins modified and the amino acids oxidised will allow us to determine the extent to which protein–LOOH formation contributes to ischaemic heart damage and the initiation of signal transduction in response to oxidative stress.

Time for primary review 27 days.


   Acknowledgements
 
This work was supported by grants from the Wellcome Trust and the British Heart Foundation. We thank Dr. S. Parthasarathy for the gift of the LOOH–protein antibody.


   References
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
 5 Conclusions
 References
 

  1. Dianzani M.U., Barrera G., Parola M. 4-Hydroxy-2,3-nonenal as a signal for cell function and differentiation. Acta Biochim Pol (1999) 46(1):61.[Web of Science][Medline]
  2. Eaton P., Li J.M., Hearse D.J., et al. Formation of 4-hydroxy-2-nonenal-modified proteins in ischemic rat heart. Am J Physiol (1999) 45:H935.
  3. Jeroudi M.O., Hartley C.J., Bolli R. Myocardial reperfusion injury — role of oxygen radicals and potential therapy with antioxidants. Am J Cardiol (1994) 73:B2.[CrossRef][Medline]
  4. Kim M.S., Akera T. O–2 free radicals — cause of ischemia reperfusion injury to cardiac Na+–K+-ATPase. Am J Physiol (1987) 252:H252.[Web of Science][Medline]
  5. Haddock P.S., Shattock M.J., Hearse D.J. Modulation of cardiac Na+–K+ pump current — role of protein and nonprotein sulfhydryl redox status. Am J Physiol (1995) 38:H297.
  6. Takayama F., Egashira T., Kudo Y., et al. Chemiluminescence HPLC assay of phosphatidylcholine hydroperoxide generated by ischemia reperfusion in the liver of rats. Biochem Pharmacol (1992) 44:2412.[CrossRef][Web of Science][Medline]
  7. Takayama F., Egashira T., Kudo Y., et al. Effects of anti free radical interventions on phosphatidylcholine hydroperoxide in plasma after ischemia reperfusion in the liver of rats. Biochem Pharmacol (1993) 46:1749.[CrossRef][Web of Science][Medline]
  8. Herrlich P., Bohmer F.D. Redox regulation of signal transduction in mammalian cells. Biochem Pharmacol (2000) 51(1):35.
  9. Timoshin A.A., Lakomkin V.L., Ruuge E.K. Free radical centers in isolated rat heart tissue in norma, during ischemia and reperfusion. Biofizika (1993) 38:179.[Medline]
  10. Maupoil V., Rochette L., Tabard A., et al. Evolution of free radical formation during low-flow ischemia and reperfusion in isolated rat heart. Cardiovasc Drugs Ther (1990) 4:791.[CrossRef][Web of Science][Medline]
  11. Hearse D.J. Stunning — a radical review. Cardiovasc Drugs Ther (1991) 5:853.[CrossRef][Web of Science][Medline]
  12. Chatham J.C., Seymour A.M.L., Harmsen E., et al. Depletion of myocardial glutathione — its effects on heart function and metabolism during ischemia and reperfusion. Cardiovasc Res (1988) 22:833.[Abstract/Free Full Text]
  13. Wolfe C.L., Donnelly T.J., Sievers R., et al. Myocardial protection with verapamil during ischemia and reperfusion — dissociation between myocardial salvage and the degree of ATP depletion during ischemia. Cardiovasc Res (1991) 25:101.[Abstract/Free Full Text]
  14. Thollon C., Iliou J.P., Cambarrat C., et al. Nature of the cardiomyocyte injury induced by lipid hydroperoxides. Cardiovasc Res (1995) 30:648.[Abstract/Free Full Text]
  15. Chen W., Gabel S., Steenbergen C., et al. A redox based mechanism for cardioprotection induced by ischemic preconditioning in perfused rat heart. Circ Res (1995) 77:424.[Abstract/Free Full Text]
  16. Myers M.L., Bolli R., Lekich R.F., et al. Enhancement of recovery of myocardial function by oxygen free radical scavengers after reversible regional ischemia. Circulation (1985) 72:915.[Abstract/Free Full Text]
  17. Grill H.P., Flaherty J.T., Weisfeldt M.L., et al. Direct measurement of free radicals in an in vivo model of regional ischemia and reperfusion. Circulation (1987) 76:196.
  18. Blaustein A., Deneke S.M., Stolz R.I., et al. Myocardial glutathione depletion impairs recovery after short periods of ischemia. Circulation (1989) 80:1449.[Abstract/Free Full Text]
  19. Garlick P.B., Davies M.J., Hearse D.J., et al. Direct detection of free radicals in the reperfused rat heart using electron spin resonance spectroscopy. Circ Res (1987) 61:757.[Abstract/Free Full Text]
  20. Hearse D.J., Tosaki A. Free radicals and reperfusion induced arrhythmias: protection by spin trapping agent PBN in rat heart. Circ Res (1987) 60:375.[Abstract/Free Full Text]
  21. Chen W., Gabel S., Steenbergen C., et al. A redox based mechanism for cardioprotection induced by ischemic preconditioning in perfused rat heart. Circ Res (1995) 77:424.[Abstract/Free Full Text]
  22. Obata T., Hosokawa H., Soeda T., et al. Myocardial microdialysis of salicylic acid to detect hydroxyl radical generation during ischemia. Comp Biochem Physiol (1995) 110:277.[CrossRef][Medline]
  23. Finkel T. Oxygen radicals and signaling. Curr Opin Cell Biol (1998) 10(2):248.[CrossRef][Web of Science][Medline]
  24. Iliou J.P., Thollon C., Robin F., et al. Protective effect of S12340 on cardiac cells exposed to oxidative stress. Eur J Pharmacol (1993) 248:263.[Web of Science][Medline]
  25. Rhee S.G. Redox signaling:hydrogen peroxide as intracellular messenger. Exp Mol Med (1999) 31(2):53.[Web of Science][Medline]
  26. Timoshin A.A., Tskitishvili O.V., Serebryakova L.I., et al. Microdialysis study of ischemia-induced hydroxyl radicals in the canine heart. Experientia (1994) 50:677.[CrossRef][Web of Science][Medline]
  27. Suc I., Meilhac O., LajoieMazenc I., et al. Activation of EGF receptor by oxidised LDL. FASEB J (1998) 12(9):665.[Abstract/Free Full Text]
  28. Arroyo C.M., Kramer J.H., Leiboff R.H., et al. Spin trapping of oxygen and carbon-centered free radicals in ischemic canine myocardium. Free Radic Biol Med (1987) 3:313.[Web of Science][Medline]
  29. Esterbauer H., Schaur R.J., Zollner H. Chemistry and biochemistry of 4-hydroxynonenal, malonaldehyde and related aldehydes. Free Radic Biol Med (1991) 11:81.[CrossRef][Web of Science][Medline]
  30. Blasig I.E., Shuter S., Garlick P., et al. Relative time profiles for free radical trapping, coronary flow, enzyme leakage, arrhythmias, and function during myocardial reperfusion. Free Radic Biol Med (1994) 16:35.[CrossRef][Web of Science][Medline]
  31. Kim J.G., Sabbagh F., Santanam N., et al. Generation of a polyclonal antibody against lipid peroxide-modified proteins. Free Radic Biol Med (1997) 23:251.[CrossRef][Web of Science][Medline]
  32. Pizzimenti S., Barrera G., Dianzani M.U., et al. Inhibition of D1, D2 and A cyclin expression in HL 60 cells by the lipid peroxidation product 4-hydroxynonenal. Free Radic Biol Med (1999) 26(11–12):1578.[CrossRef][Web of Science][Medline]
  33. Bolli R., Mccay P.B. Use of spin traps in intact animals undergoing myocardial ischemia reperfusion — a new approach to assessing the role of oxygen radicals in myocardial stunning. Free Radic Res Commun (1990) 9:169.[Web of Science][Medline]
  34. Dianzani M.U. 4-Hydroxnonenal and cell signaling. Free Rad Res (1998) 28(6):553.[Web of Science][Medline]
  35. Ishikawa T., Sies H. Cardiac transport of glutathione disulfide and S-conjugate — studies with isolated perfused rat heart during hydroperoxide metabolism. J Biol Chem (1984) 259:3838.[Abstract/Free Full Text]
  36. Zweier J.L., Kuppusamy P., Williams R., et al. Measurement and characterization of postischemic free radical generation in the isolated perfused heart. J Biol Chem (1989) 264:18890.[Abstract/Free Full Text]
  37. Uchida K., Shiraishi M., Naito Y., et al. Activation of stress signaling pathways by the end product of lipid peroxidation — 4-hydroxy-2-nonenal is a potential inducer of intracellular peroxide production. J Biol Chem (1999) 272(4):2234.
  38. Liu W., Akhand A.A., Kato M., et al. 4-Hydoxynonenal triggers an epidermal growth factor receptor linked signal pathway for growth inhibition. J Cell Sci (1999) 112(14):2409.[Abstract]
  39. Ceconi C., Curello S., Cargnoni A., et al. The role of glutathione status in the protection against ischemic and reperfusion damage — effects of N-acetyl cysteine. J Mol Cell Cardiol (1988) 20:5.[Web of Science][Medline]
  40. Werns S.W., Fantone J.C., Ventura A., et al. Myocardial glutathione depletion impairs recovery of isolated blood perfused hearts after ischaemia. J Mol Cell Cardiol (1992) 24:1215.[CrossRef][Web of Science][Medline]
  41. Palace V., Kumar D., Hill M.F., et al. Regional differences in non-enzymatic antioxidants in the heart under control and oxidative stress conditions. J Mol Cell Cardiol (1999) 31:193.[CrossRef][Web of Science][Medline]
  42. Blanc E.M., Kelly J.F., Mark R.J., et al. 4-Hydroxynonenal, an aldehydic product of lipid peroxidation, impairs signal transduction associated with muscarinic, acetylcholine and metabotropic glutamate receptors: possible action on G {alpha} (q/11). J Neurochem (1997) 69(2):570.[Web of Science][Medline]
  43. Ruef J., Hu Z., Bode C., et al. Involvement of 4-hydroxynonenal in vascular smooth muscle cell signaling and atherosclerosis. J Am Coll Cardiol (1998) 32(2SA):A72. Abstract.
  44. Compton C.N., Franko A.P., Murray M.T., et al. Signaling in apoptotic lung injury by lipid hydroperoxides. J Trauma Inj Infect Crit Care (1998) 44(5):783.[CrossRef]
  45. Schumacher M., Eber B., Tatzber F., et al. Reperfusion injury due to oxygen-derived free radicals in acute myocardial infarction. Medizinische Welt (1997) 48:275.[Web of Science]
  46. Mattson M.P., Fu W.M., Waeg G., et al. 4-Hydroxynonenal, a product of lipid peroxidation, inhibits dephosphorylation of the microtubule associated protein tau. Neuroreport (1997) 8(9–10):2275.[Web of Science][Medline]
  47. Adler V., Yin Z.M., Tew K.D., et al. Role of redox potential and reactive oxygen species in stress signaling. Oncogene (1999) 18(45):6104.[CrossRef][Web of Science][Medline]
  48. Esterbauer H. Estimation of peroxidative damage — a critical review. Pathol. Biol. (1996) 44:25.[Web of Science][Medline]
  49. Keller J.N., Mattson M.P. Roles of lipid peroxidation in modulation of cellular signaling pathways, cell dysfunction, and death in the nervous system. Rev Neurosci (1998) 9(2):105.[Web of Science][Medline]
  50. Abe J., Berk B.C. Reactive oxygen species as mediators of signal tranduction in cardiovascular disease. Trends Cardiovasc Med (1998) 8(2):59.[CrossRef][Web of Science]

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