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IS066 Keynote Lecture

Ischemic Preconditioning and Cardioprotection: Mechanisms by which Early (First Window) and Late (Second Window) Phases of Protection are Induced
James M. Downey, M.D.
Department of Medical Physiology
University of South Alabama
Mobile, AL, USA
 
  • Proposed mechanism of preconditioning
  • Mitochondrial KATP
  • New hypothesis

  • Loss of contractile myocardium is a serious consequence of acute myocardial infarction. Loss of more than a third of the left ventricle results in congestive heart failure and is associated with high mortality. Therefore, an intervention that would protect the myocardium and reduce the amount of infarction with ischemia reperfusion has been sought for several decades.

    The theoretical possibility of modifying the amount of cell death in the setting of an infarction was first proven with ischemic preconditioning in 1986 in the dog model. Charles Murray demonstrated that a 40-minute coronary artery occlusion resulted in about 28% infarction of the ischemic region. If the animals were pretreated with four 5-minute coronary occlusions, each followed by 5-minute reperfusion, they were highly protected with only about10% infarction. The preconditioning the myocardium with intermittent periods of ischemia had caused it to adapt itself to become very resistant to infarction. This very powerful anti-infarct effect lasts about 2 hours after preconditioning. It then disappears for about one day, after which the protective effect returns (the so-called second window of protection). This lecture will primarily focus on the classic or early preconditioning period and will propose a new hypothesis for the mechanism of preconditioning.

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    Proposed mechanism of preconditioning


    Figure 1. The traditional view of preconditioning's signal transduction pathway with receptors triggering kinases which terminate on the mitochondrial KATP channel.
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    Fig 1 shows the traditional hypothesis of preconditioning's mechanism. During ischemia adenosine is released from the heart as ATP is broken down to AMP, and 5'nucleotidasereleases free adenosine which then occupies adenosine receptors on the surface of the heart. Bradykinin is also released and occupies bradykinin B2 receptors. Opioids and endorphins produced by the heart occupy the delta receptors. These G protein-coupled receptors all activate phospholipases in the heart that eventually activate protein kinase C (PKC); a very important aspect of this mechanism. In a positive feedback manner PKC increases 5'nucleotidase activity, which increases adenosine release and further activates the kinases. Recently, there has been great interest in the mitochondrial KATP (mKATP) channel as the end-effector of this protection.

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    Mitochondrial KATP


     

    Opening the mKATP has been suggested to be protective and to be the mechanism of preconditioning. The channel on the mitochondria inner membrane is a different isoform from that found on the sarcolemma, thus it can be selectively opened with diazoxide and selectively closed with 5 hydroxydecanoate (5-HD). Diazoxide is 1000-fold more selective for the mKATP channel than for the sarcolemmal channel. Furthermore studies from Marban's laboratory reveal that both diazoxide and 5-HD are reversible substances that wash out very quickly.


    Figure 2. The two selective tools for the mitochondrial KATP channel are diazoxide which opens the channel and 5 hydroxydecanoate (5HD) which closes it. Here intravenous diazoxide (DIAZ) mimics preconditioning when given as a pretreatment (PRE) in an open-chest rabbit and 5HD can block that protection. Note that diazoxide was not protective when given 10 min after ischemia had started (POST). (American Journal of Physiology 1999; 276:H1361-H1368.)
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    Diazoxide was shown in the open chest rabbit model to be nearly as protective as preconditioning in studies by Baines et al. (Fig 2). In the control rabbits infarction was about 40% and in the rabbits pretreated with diazoxide, only about 20%. Giving 5-HD with diazoxide blocks the channel and the protection. 5-HD given alone has no effect on infarction. A somewhat surprising finding was that diazoxide does not seem to be protective after ischemia has begun. Because it was thought that the mKATP was the end-effector of protection it follows that it should be possible to treat very late with diazoxide and still obtain protection. That was not the case, however.

    The heart appears to somehow remember it is in a preconditioned state as it is possible to reperfuse for up to an hour after a 5-minute occlusion and still have protection. The memory is thought to reside in the signal transduction pathway above the kinases. If opening the KATP channel is the end-effector of protection, then the non-protected state should return as soon as the channel closed. In an experiment using isolated rabbit hearts, we found that a five minute infusion of diazoxide fully protected the heart even after up to 30-minutes of wash out of the drug. When the experiment was repeated with pinacidil, which is a non-selective opener for both the mitochondrial and sarcolemmal channels, protection was also obtained. Thus transient opening of the mKATP channel unexpectedly protected long after the drug was washed out and the channel had presumably closed again. Also the memory effect is not specific to diazoxide.


    Figure 3. The protocols used to determine whether an inhibitable step is a trigger or a mediator of preconditioning.
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    Figure 4. Staurosporine, a blocker of PKC's kinase activity only blocks preconditioning's protection if given in the late protocol indicating that PKC is a mediator of protection. (Journal of Molecular and Cellular Cardiology 1997; 29:991-999.)
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    Figure 5. The MAP kinases. Note that all are activated by MAP kinase kinases (MKKs). MKKs are dual kinases simultaneously phosphorylating a threonine and a tyrosine two amino acids apart.
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    Triggers and mediators of preconditioning

    Fig 3 reveals that the trigger and mediator functions of preconditioning can be separated by giving inhibitors either early to bracket the preconditioning ischemia or late to load the heart with inhibitor just before the long ischemic period. Giving an adenosine receptor antagonist using the early protocol completely blocks protection, showing that adenosine works as a trigger. On the other hand staurosporine, a PKC blocker will only abort preconditioning if given in the late protocol indicating that PKC is a mediator (see Fig 4). The question is; does opening the mKATP channels act as a trigger or a mediator of protection? Before we address that question we need some more background information on preconditioning's pathway.

    Location of tyrosine kinase

    Studies by Baines in my laboratory showed that preconditioning involves a tyrosine kinase. Genistein a tyrosine kinase inhibitor only blocked protection from preconditioning if the late protocol was used indicating that the tyrosine kinase acts as a mediator like PKC. We had reason to believe that the location of the tyrosine kinase in question was at p38 MAPK. Activation is by an upstream kinase called a MEK (see Fig 5) which phosphorylates p38 MAPK on its tyrosine 182 and threonine 180. The MAPKs are thought to be turned on only in a preconditioned heart and will have both immediate protective effects as well as to cause gene expression; presumably important in creating the second window by causing a protective protein to be produced that protects the heart long-term.

    In an isolated rabbit heart model we showed there was no activation of p38 MAPK during ischemia in the non-preconditioned heart. When the heart is preconditioned there was no significant activation of p38 MAPK just prior to ischemia. But, during ischemia, the so-called mediator phase, a 3-fold increase in p38 MAPK activity was seen at 10 minutes and 20 minutes. If protection was blocked with an adenosine receptor blocker so that the preconditioned hearts were not actually protected, the increase in phosphorylation was lost. Thus, an increase in p38 MAPK activation during ischemia is associated with protection.

    We wanted to measure an index of p38 MAPK's kinase activity. To accomplish this we developed an assay for the downstream kinase, MAPKAPK2 (mitogen activated protein kinase activated protein kinase 2). Tissue homogenate was separated by liquid chromatography into serial fraction using a Mono-S column. The ability of each fraction to phosphorylate a substrate peptide specific for MAPKAPK2 was then measured. At either baseline or after 20 minutes of ischemia there is no appreciable activity in any fraction from non-preconditioned hearts. However, there were two very prominent peaks of MAPKAPK2 activity in the preconditioned hearts after 20 minutes of ischemia. Western blot analyses of the fractions against the two known isoforms of MAPKAPK2 show they are both present in the first of the two activity peaks. Good evidence again that p32 MAPK is being turned on during ischemia in the preconditioned heart and that it is acting as a mediator. Work by Yellon looking at SB203580, a p38 MAPK blocker, shows that giving it with the early protocol has no effect against preconditioning's protection in the rat heart, but giving it late completely blocks the preconditioning protection. Again confirming this mediator role.

    Timing of KATP channel opening

    To determine when the KATP channel must be open for protection, we performed a similar study to the experiment in figure 3 using the mKATP blocker 5HD. Isolated rabbit hearts were used. A 5-minute diazoxide infusion was followed by a 10-minute wash out and then 5-HD was given either early or late. The early protocol (bracketing the diazoxide infusion) completely blocked protection, but when 5HD was given late there was no effect on protection. It appears that the KATP channel does not need to be open during the 30-minute period of ischemia. Clearly mKATP opening from diazoxide is acting as a trigger.

    We repeated the experiment using ischemic preconditioning (5 minutes of coronary occlusion plus 10 minutes of reperfusion) in isolated rabbit hearts. 5-HD when given early completely blocked preconditioning's protection. But, when 5-HD was given late there was no effect on protection. The mKATP channel opening is clearly acting as a trigger with ischemic preconditioning as well as with diazoxide treatment.

    Is mKATP channel opening a signal transduction step?

    If mKATP channel opening acts as a signal transduction step, diazoxide treatment would be expected to activate the downstream kinases. A study was done to determine whether a kinase blocker would block protection from diazoxide. When diazoxide was given with chelerythrine, a PKC blocker, to isolated hearts, neither the early nor the late protocol had any effect on protection. But, a tyrosine kinase blocker, genistein, completely blocked protection from diazoxide when given with the late protocol. Clearly diazoxide causes kinase-dependent protection, indicating that the mKATP channel is upstream from at least one tyrosine kinase. Opening the mKATP channel triggers protection, which causes activation of the kinases during the index ischemia.

    Free radicals

    In perhaps the most important experiment, diazoxide was shown to protect in a free radical-dependent manner. N-2-mercaptopropionylglycine (MPG), an intracellular free radical scavenger, was given 5 minutes prior to diazoxide and continued until halfway through the ischemic period. MPG completely blocked protection. A recent paper by Yao probably explains this effect. Dichlorofluorescein, a dye that becomes fluorescent when it reacts with free radicals, was used as an index of free radical production. In the cardiomyocyte acetylcholine occupies a Gi-coupled receptor (M2) that is very similar to the adenosine A1 receptor, and because it activates PKC, acetylcholine can mimic preconditioning. After neonatal chicken myocytes were given acetylcholine, there was a burst of free radicals. When myxothiazol, an electron transport blocker, was given the production of free radicals from acetylcholine was blocked. This indicates that free radicals were coming from the mitochondria. When MPG was given, the free radicals are soaked up and the signal removed. Most importantly, when the mKATP channel was blocked by 5-HD the burst of free radicals was lost. This indicates that giving a receptor agonist causes opening of mKATP channels which causes a burst of free radicals.

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    New hypothesis


    Figure 6. The new hypothesis now puts the mitochondrial KATP channel between the receptors and the kinases. The end effector is unknown.
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    The major problem that has haunted the mKATP hypothesis of preconditioning is why should opening this channel be protective? Channel opening causes the mitochondria to swell and to be slightly uncoupled. Neither effect should be protective. The observation that diazoxide's protection is dependent on free radical production is significant in that both PKC as well as the p38 MAPK pathway are known to be activated by the presence of free radicals. If the hypothesis is true then PKC would have to be in parallel with the tyrosine kinase containing step (p38 MAPK) rather than in series as originally proposed (see Fig 6). Thus the data now indicate that opening of the mKATP channel acts as a signal transduction step that triggers preconditioning's protection rather than as the end-effector. In summary we proposed a new hypothesis: Occupation of the surface receptors activate the mKATP channels causes the mitochondria to produce free radicals. The free radicals in turn trigger the kinases which then cause protection by phosphorylating some as yet unknown end-effector.

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