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Ischaemic preconditioning: can the protection be bottled?
C, Friedman H. Differing macrophage and lymphocyte roles in resistance to Legionella pneumophila infection. J Immunol 1992; 148: 584-89. Moffat JF, Tompkins LS. A quantitative model of intracellular growth of Legionella pneumophila in Acanthamoeba castellanii. Infect Immun
10 Yamamoto Y, Klein TW, Newton
11
1992; 60: 296-301. 12
Bhopal RS. Wagstaffe R. Prospects for the elimination of legionnaires’ disease. J Infect 1993; 26: 239-43.
Ischaemic
preconditioning: can the
protection
be bottled?
Several years ago it would have been incomprehensible for a rational scientist or physician to conclude that myocardial ischaemia might somehow act to protect the heart from necrosis. Then Murry et all reported their improbable observations that an isolated 40-min occlusion in dogs resulted in an infarct whose mass could be decreased by 75% if the animals were pretreated with four cycles of 5-min coronary occlusion plus 5 min of reflow. Preconditioning with brief periods of ischaemia has now been shown to increase the heart’s resistance to infarction in numerous species, including rabbit, rat, and pig. Recognition of the benefits of ischaemic preconditioning has unleashed efforts to define its characteristics, study its mechanism, and apply the principles clinically. In rabbit heart, full protection by ischaemic preconditioning is afforded by a single 5-min ischaemic period, but there is no protection with two 2-5-min ischaemic periods; this observation suggests a very sharp response threshold.2 The reflow period following this brief ischaemia may be as short as 1 min, but protection wanes if the reflow is extended beyond 1 h.2A second preconditioning stimulus in rabbits can fully reinstate protection after the initial protection has waned.3 Recent studies have given us insight into the mechanism of the anti-infarct effect of ischaemic preconditioning. Adenosine is a ubiquitous product of ischaemia, and occupation of Al adenosine receptors seems to be the first step in preconditioning.4 Agents that block adenosine receptors prevent the protective effect of preconditioning whereas intracoronary infusion of adenosine or AI-selective agonists mimics the effect. Although adenosine receptors couple to several signalling systems in myocytes we now believe that protein kinase C (PKC) is the important one for preconditioning.5 Inhibitors of this pathway such as staurosporine or polymyxin B block protection, and pretreatment with activators including phorbol myristate acetate or oleyl acetyl glycerol mimics preconditioning.S One of the novel findings of this research is the mechanism of the memory. We believe that the heart "remembers" that it has been preconditioned through a translocation of PKC from the cytosol into the cell membranes. Population of A1 receptors activates phospholipase C, which releases diacylglycerol in the membrane and this, in turn, triggers translocation and activates any PKC residing in the membrane. Cytosolic PKC is inactive and can phosphorylate protein only after it has been translocated to the membrane. Thus, the first ischaemic episode primarily translocates PKC into the membranes, where it will stay for about an hour. Repopulation of adenosine receptors during the second occlusion can then result in phosphorylation of proteins early in the ischaemic period and these mediate the protection. Colchicine, which disrupts the microtubules responsible for pulling PKC into the membrane, blocks protection completely. Conversely, 6
initiating translocation with phorbol myristate acetate protects, but that protection can still be blocked if an adenosine receptor antagonist is then administered; repopulation of adenosine receptors during the second coronary occlusion is required to reactivate the translocated PKC. Preliminary data suggest that ischaemic preconditioning also occurs in human beings. For example, the second coronary occlusion during the course of coronary angioplasty results in less evidence of myocardial ischaemia than the first.6 Similarly, a bout of angina may be protective; but the protection may well be lost with recurrent angina. Biological systems become tolerant to most Acadenosinemediated responses if receptors are continuously populated, and PKC is also known to downregulate quickly. Thus, we were not surprised when we found that the effect of preconditioning was lost in rabbits that had forty or more 5-min occlusions over 3-4 days (unpublished observation). Moreover, we could not maintain a continuously preconditioned state by infusing an AI-selective adenosine agonist into rabbits for 3 days.7 How might one design a pharmacological approach to confer preconditioning’s anti-infarct effect to patients? Occupation of any receptor coupled to phospholipase C (and hence to PKC) in the heart should theoretically precondition it. Thus, we find that the muscarinic agonist carbachol (through the M2-receptor) and the adrenergic agonist noradrenaline (through the al-receptor) are equipotent with adenosine as preconditioning agents Patients developing tolerance to adenosine-mediated protection because of recurrent anginal episodes may be amenable to preconditioning with either a muscarinic or an adrenergic agonist depending on the level in the signalling pathway at which tolerance occurs. Unfortunately, all of these agonists have serious side-effects when given parenterally, and direct activators of PKC can be tumour promoters. Perhaps additional opportunities for drug design will become apparent when we identify which phosphorylated protein is responsible for the anti-infarct effect. Putting preconditioning in a bottle remains a pharmacological challenge. Michael V Cohen, James M
Downey
Department of Physiology, University of South Alabama, Mobile, Alabama, USA 1
2 3
4
5
6
7
8
9
Murry CE, Jennings RB, Reimer KA. Preconditioning with ischemia: a delay of lethal cell injury in ischemic myocardium. Circulation 1986; 74: 1124-36. Lawson CS, Downey JM. Preconditioning: state of the art myocardial protection. Cardiovasc Res 1993; 27: 542-50. Yang X-M, Arnoult S, Tsuchida A, et al. The protection of ischaemic preconditioning can be reinstated in the rabbit heart after the initial protection has waned. Cardiovasc Res 1993; 27: 556-58. Liu GS, Thornton J, Van Winkle DM, Stanley AWH, Olsson RA, Downey JM. Protection against infarction afforded by preconditioning is mediated by A1 adenosine receptors in rabbit heart. Circulation 1991; 84: 350-56. Ytrehus K, Liu Y, Downey JM. Preconditioning protects the ischemic rabbit heart by protein kinase C activation. FASEB J 1993; 7: A418. Deutsch E, Berger M, Kussmaul WG, Hirshfeld JW Jr, Herrmann HC, Laskey WK. Adaptation to ischemia during percutaneous transluminal coronary angioplasty: clinical, hemodynamic, and metabolic features. Circulation 1990; 82: 2044-51. Tsuchida A, Thompson R, Olsson RA, Downey JM. The anti-infarct effect of an adenosine A1-selective agonist is diminished after prolonged infusion as is the cardioprotective effect of ischemic preconditioning in rabbit heart. J Mol Cell Cardiol (in press). Thornton JD, Liu GS, Downey JM. Pretreatment with pertussis toxin blocks the protective effects of preconditioning: evidence for a Gprotein mechanism. J Mol Cell Cardiol 1993; 25: 311-20. Thornton JD, Daly JF, Cohen MV, Yang X-M, Downey JM. Catecholamines can induce adenosine receptor-mediated protection of the myocardium but do not participate in ischemic preconditioning in the rabbit. Circ Res (in press).
10 Yamamoto Y, Klein TW, Newton
11
1992; 60: 296-301. 12
Bhopal RS. Wagstaffe R. Prospects for the elimination of legionnaires’ disease. J Infect 1993; 26: 239-43.
Ischaemic
preconditioning: can the
protection
be bottled?
Several years ago it would have been incomprehensible for a rational scientist or physician to conclude that myocardial ischaemia might somehow act to protect the heart from necrosis. Then Murry et all reported their improbable observations that an isolated 40-min occlusion in dogs resulted in an infarct whose mass could be decreased by 75% if the animals were pretreated with four cycles of 5-min coronary occlusion plus 5 min of reflow. Preconditioning with brief periods of ischaemia has now been shown to increase the heart’s resistance to infarction in numerous species, including rabbit, rat, and pig. Recognition of the benefits of ischaemic preconditioning has unleashed efforts to define its characteristics, study its mechanism, and apply the principles clinically. In rabbit heart, full protection by ischaemic preconditioning is afforded by a single 5-min ischaemic period, but there is no protection with two 2-5-min ischaemic periods; this observation suggests a very sharp response threshold.2 The reflow period following this brief ischaemia may be as short as 1 min, but protection wanes if the reflow is extended beyond 1 h.2A second preconditioning stimulus in rabbits can fully reinstate protection after the initial protection has waned.3 Recent studies have given us insight into the mechanism of the anti-infarct effect of ischaemic preconditioning. Adenosine is a ubiquitous product of ischaemia, and occupation of Al adenosine receptors seems to be the first step in preconditioning.4 Agents that block adenosine receptors prevent the protective effect of preconditioning whereas intracoronary infusion of adenosine or AI-selective agonists mimics the effect. Although adenosine receptors couple to several signalling systems in myocytes we now believe that protein kinase C (PKC) is the important one for preconditioning.5 Inhibitors of this pathway such as staurosporine or polymyxin B block protection, and pretreatment with activators including phorbol myristate acetate or oleyl acetyl glycerol mimics preconditioning.S One of the novel findings of this research is the mechanism of the memory. We believe that the heart "remembers" that it has been preconditioned through a translocation of PKC from the cytosol into the cell membranes. Population of A1 receptors activates phospholipase C, which releases diacylglycerol in the membrane and this, in turn, triggers translocation and activates any PKC residing in the membrane. Cytosolic PKC is inactive and can phosphorylate protein only after it has been translocated to the membrane. Thus, the first ischaemic episode primarily translocates PKC into the membranes, where it will stay for about an hour. Repopulation of adenosine receptors during the second occlusion can then result in phosphorylation of proteins early in the ischaemic period and these mediate the protection. Colchicine, which disrupts the microtubules responsible for pulling PKC into the membrane, blocks protection completely. Conversely, 6
initiating translocation with phorbol myristate acetate protects, but that protection can still be blocked if an adenosine receptor antagonist is then administered; repopulation of adenosine receptors during the second coronary occlusion is required to reactivate the translocated PKC. Preliminary data suggest that ischaemic preconditioning also occurs in human beings. For example, the second coronary occlusion during the course of coronary angioplasty results in less evidence of myocardial ischaemia than the first.6 Similarly, a bout of angina may be protective; but the protection may well be lost with recurrent angina. Biological systems become tolerant to most Acadenosinemediated responses if receptors are continuously populated, and PKC is also known to downregulate quickly. Thus, we were not surprised when we found that the effect of preconditioning was lost in rabbits that had forty or more 5-min occlusions over 3-4 days (unpublished observation). Moreover, we could not maintain a continuously preconditioned state by infusing an AI-selective adenosine agonist into rabbits for 3 days.7 How might one design a pharmacological approach to confer preconditioning’s anti-infarct effect to patients? Occupation of any receptor coupled to phospholipase C (and hence to PKC) in the heart should theoretically precondition it. Thus, we find that the muscarinic agonist carbachol (through the M2-receptor) and the adrenergic agonist noradrenaline (through the al-receptor) are equipotent with adenosine as preconditioning agents Patients developing tolerance to adenosine-mediated protection because of recurrent anginal episodes may be amenable to preconditioning with either a muscarinic or an adrenergic agonist depending on the level in the signalling pathway at which tolerance occurs. Unfortunately, all of these agonists have serious side-effects when given parenterally, and direct activators of PKC can be tumour promoters. Perhaps additional opportunities for drug design will become apparent when we identify which phosphorylated protein is responsible for the anti-infarct effect. Putting preconditioning in a bottle remains a pharmacological challenge. Michael V Cohen, James M
Downey
Department of Physiology, University of South Alabama, Mobile, Alabama, USA 1
2 3
4
5
6
7
8
9
Murry CE, Jennings RB, Reimer KA. Preconditioning with ischemia: a delay of lethal cell injury in ischemic myocardium. Circulation 1986; 74: 1124-36. Lawson CS, Downey JM. Preconditioning: state of the art myocardial protection. Cardiovasc Res 1993; 27: 542-50. Yang X-M, Arnoult S, Tsuchida A, et al. The protection of ischaemic preconditioning can be reinstated in the rabbit heart after the initial protection has waned. Cardiovasc Res 1993; 27: 556-58. Liu GS, Thornton J, Van Winkle DM, Stanley AWH, Olsson RA, Downey JM. Protection against infarction afforded by preconditioning is mediated by A1 adenosine receptors in rabbit heart. Circulation 1991; 84: 350-56. Ytrehus K, Liu Y, Downey JM. Preconditioning protects the ischemic rabbit heart by protein kinase C activation. FASEB J 1993; 7: A418. Deutsch E, Berger M, Kussmaul WG, Hirshfeld JW Jr, Herrmann HC, Laskey WK. Adaptation to ischemia during percutaneous transluminal coronary angioplasty: clinical, hemodynamic, and metabolic features. Circulation 1990; 82: 2044-51. Tsuchida A, Thompson R, Olsson RA, Downey JM. The anti-infarct effect of an adenosine A1-selective agonist is diminished after prolonged infusion as is the cardioprotective effect of ischemic preconditioning in rabbit heart. J Mol Cell Cardiol (in press). Thornton JD, Liu GS, Downey JM. Pretreatment with pertussis toxin blocks the protective effects of preconditioning: evidence for a Gprotein mechanism. J Mol Cell Cardiol 1993; 25: 311-20. Thornton JD, Daly JF, Cohen MV, Yang X-M, Downey JM. Catecholamines can induce adenosine receptor-mediated protection of the myocardium but do not participate in ischemic preconditioning in the rabbit. Circ Res (in press).