J Physiol Volume 585, Number 3, 649-650, December 15, 2007 DOI: 10.1113/jphysiol.2007.146209
Temperature preconditioning: a cold-hearted answer to ischaemic–reperfusion injury?
Iffath A. Ghouri1,
Ole J. Kemi1 and
Godfrey L. Smith1
1 Institute of Biomedical and Life Sciences, University of Glasgow, Glasgow G12 8QQ, UK Email: i.ghouri.1{at}research.gla.ac.uk
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Introduction
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Restoration of blood flow following a prolonged period of ischaemia results in irreversible tissue damage and myocardial dysfunction. Such ischaemic–reperfusion (I/R) injury occurs following myocardial infarction and is a risk in a number of surgical procedures, including coronary bypass and transplant surgery. Numerous studies have shown that ischaemic preconditioning (IP) is a powerful means of protecting the heart against this type of injury. This involves subjecting the heart to brief periods of ischaemia interspersed by periods of normal perfusion prior to prolonged ischaemia, and it has been shown to reduce myocardial cell death as well as having a number of other cardioprotective effects (Murry et al. 1986). Similar beneficial effects are produced by hypothermia, which is used in a number of surgical procedures to protect against an expected ischaemic insult (Riess et al. 2004).
In a recent issue of The Journal of Physiology, Khaliulin et al. (2007) described the novel protocol of temperature preconditioning (TP), which combines the principles of ischaemic preconditioning and hypothermia. The study compared the effectiveness of TP with IP and delivery of a single hypothermic perfusion (SHP) in protecting the heart against I/R injury.
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Experimental procedure
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The study by Khaliulin et al. (2007) used Langendorff perfused isolated rat hearts divided into groups receiving the IP, TP or SHP protocol during the preischaemic period. A separate group of hearts were perfused for 40 min at 37°C and acted as controls. IP hearts were subjected to three cycles of 2 min global ischaemia interspersed by 3 min normal perfusion. The TP protocol involved delivering three cycles of 2 min hypothermic perfusion at 26°C interspersed by 3 min normothermic perfusion and the SHP protocol involved delivering a single 6 min perfusion at 26°C. After 40 min preischaemia, all four groups were subjected to 25 min global ischaemia followed by 60 min reperfusion at 37°C. The cardioprotective effect of each protocol was assessed in terms of haemodynamic recovery, incidence of arrhythmias, lactate dehydrogenase release (indicating tissue necrosis), Ca2+ induced mitochondrial swelling and protein carbonylation (a surrogate marker for oxidative stress). Experiments then went on to investigate possible signalling pathways involved in this protection.
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Preservation of cardiac function
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Measurements of haemodynamic function showed that postischaemic haemodynamic recovery, in terms of increase in left ventricular developed pressure (LVDP) and work index, was poor in control hearts, reaching only 
35% of initial values. IP, TP and SHP all improved haemodynamic recovery, but improvement was greatest in TP hearts, with LVDP and work index being essentially restored to initial values. During reperfusion, TP hearts also displayed the fewest incidents of arrhythmia and the lowest levels of lactate dehydrogenase (LDH) release, indicating less necrotic damage. Analysis of tissue metabolite levels revealed that preischaemic ATP levels were significantly increased in TP but not IP hearts compared to controls, although creatine phosphate levels were significantly increased in both groups. After 60 min reperfusion, total adenine nucleotide levels were significantly higher in IP and TP hearts compared to controls, implying better preservation of energy metabolism.
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Role of the mitochondrial permeability transition pore
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It is thought that opening of the mitochondrial permeability transition pore (MPTP) is likely to represent the point of no return in reperfusion injury (Halestrap, 2006). Prolonged opening of the pore results in cell death by necrosis due to the inability of the cell to maintain ATP levels. A less severe ischaemic insult causes short-term opening of the pore and results in death by apoptosis. Pore opening allows release of the pro-apoptotic factor cytochrome c. A subsequent pore closure allows resumption in production of the ATP required for apoptosis. Opening of the MPTP is triggered by mitochondrial Ca2+ overload, lack of adenine nucleotides, high inorganic phosphate levels and abundance of oxygen free radicals. All these conditions are met following ischaemia, making opening of the MPTP and cell death a likely result. The study therefore looked at the effectiveness of the protocols at inhibiting opening of the MPTP.
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Inhibition of pore opening
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Mitochondria were isolated after 3 min reperfusion and opening of the MPTP was determined by measuring the amplitude and rate of mitochondrial swelling following Ca2+ addition. IP hearts displayed a 2-fold decrease in the amplitude and rate of swelling compared to controls. However, TP was shown to be even more effective at inhibiting pore opening, causing a 4-fold decrease in mitochondrial swelling compared to controls. This perhaps explains the reduced necrotic damage and better bioenergetic recovery in TP hearts. In addition, the study also looked at levels of protein carbonylation after 60 min reperfusion as a surrogate marker for oxidative stress, a contributing factor towards pore opening. Again it was shown that levels were lowest in TP hearts, comparable in fact to levels in non-ischaemic control hearts.
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Protective signalling mechanisms
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These results suggest that TP is strongly protective against I/R injury, even more so than IP. It seems that it is the short cycles of cooling and rewarming that induces resistance to injury, as a single 6 min hypothermic perfusion is less effective at conferring protection. The cardioprotective effects appear to be due to inhibition of the MPTP, and the paper then goes on to describe possible signal transduction mechanisms involved in this protection. Preischaemic perfusion of TP hearts with chelerythrine (an inhibitor of protein kinase C), compound C (an inhibitor of AMP-activated protein kinase – AMPK) and the free-radical scavenger N-(2-mercaptopropionyl)glycine (MPG) all either reduced or abolished the cardioprotection afforded by TP. This implicates protein kinase C, AMPK and reactive oxygen species (ROS) in the signalling pathway. They also showed that TP increased protein kinase C 
(PKC) translocation to the particulate fraction and increased activation (phosphorylation) of AMPK after 5 min index ischaemia (but not before ischaemia). Thus they propose that the protective pathway involves a modest increase in reactive oxygen species (ROS), leading to activation of PKC
and possibly activation of AMPK. The observed protection in both IP and TP was attributed to reduced accumulation of ROS at the end of ischaemia, resulting in reduced opening of the MPTP, reduced tissue necrosis and subsequently improved contractile function.
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Additional mitochondrial involvement?
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This study considers a variety of parameters as evidence of protection and supports the idea that TP provides strong overall cardioprotection, more powerful than IP. However, it is unclear whether the metabolic conditions that exist during the brief ischaemic periods and the brief hypothermic periods are comparable. During periods of ischaemia, ATP levels are known to fall and it is unclear what happens to ATP levels during hypothermia. It may be that ATP levels are better preserved during the short hypothermic periods and that this is the reason for the significantly higher than control preischaemic ATP levels seen in TP but not IP hearts. Further investigations could assess how mitochondrial metabolic activity changes during periods of ischaemia and reperfusion, and whether TP and IP are having any direct effect on cellular metabolism. This would perhaps help explain the differences in metabolite levels seen in the different groups. In addition, future experiments could investigate cytosolic and mitochondrial Ca2+ levels in cells isolated from each group. This may give an indication as to whether reduced opening of the MPTP in TP and IP hearts was also due to reduced mitochondrial Ca2+ uptake and subsequent overload, as well as lower levels of oxidative stress.
Finally, it should be noted that as no measurements of coronary flow rate were made during the temperature preconditioning protocol, the effects of hypothermia on coronary vessel tone are not clear. Thus, if the response to hypothermia is vasoconstriction, temperature preconditioned hearts would have also been exposed to reduced flow and possibly ischaemic conditions.
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Clinical relevance
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The cardioprotective effects of IP against I/R injury are well established; however, its use in the clinical setting is limited by the risks of inducing direct ischaemia to potentially vulnerable organs. Recently, Ali et al. (2007) demonstrated that remote ischaemic preconditioning applied to the lower limb reduced myocardial injury following abdominal aortic aneurysm repair. This protection is thought to be due to the action of circulating mediators released at the remote ischaemic site and avoids the risks of applying ischaemia directly to the myocardium. It must still be established if the cardioprotection afforded by TP can be reproduced in humans and the feasibility of its use in the clinical setting would need to be assessed. One problem that may limit the clinical usefulness of TP is that it may be difficult to keep the heart hypothermic with short periods of cold perfusion in a closed chest situation, whilst in contact with warm organs such as the lungs and great vessels.
However, the study by Khaliulin et al. (2007) provides the basis for future investigation into a potential alternative mechanism for the prevention of I/R injury, and understanding the protective signalling pathways involved in TP may lead to the development of more effective pharmacological interventions.
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References
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Ali ZA, Callaghan CJ, Lim E, Ali AA, Reza Novraei SA, Akthar AM, Boyle JR, Varty K, Kharbanda RK, Dutka DP & Gaunt ME (2007). Remote Ischemic Preconditioning Reduces Myocardial and Renal Injury After Elective Abdominal Aortic Aneurysm Repair: A Randomized Controlled Trial. Circulation 116, I98–I105.[CrossRef][Medline]Halestrap AP (2006). Calcium, mitochondria and reperfusion injury: a pore way to die. Biochem Soc Trans 34, 232–237.[CrossRef][Medline]
Khaliulin I, Clarke SJ, Lin H, Parker J, Suleiman MS & Halestrap AP (2007). Temperature preconditioning of isolated rat hearts – a potent cardioprotective mechanism involving a reduction in oxidative stress and inhibition of the mitochondrial permeability transition pore. J Physiol 581, 1147–1161.[Abstract/Free Full Text]
Murry CE, Jennings RB & Reimer KA (1986). Preconditioning with ischemia: a delay of lethal cell injury in ischemic myocardium. Circulation 74, 1124–1136.[Abstract/Free Full Text]
Riess ML, Camara AKS, Kevin LG, An J & Stowe DF (2004). Reduced reactive O2 species formation and preserved mitochondrial NADH and [Ca2+] levels during short-term 17 °C ischemia in intact hearts. Cardiovasc Res 61, 580–590.[Abstract/Free Full Text]
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Introduction
Experimental procedure