Published online before print March 20, 2008, doi: 10.1161/CIRCRESAHA.107.167072
© 2008 American Heart Association, Inc.
Cellular Biology |
Inhibition of Mitochondrial Permeability Transition Pore Opening by Ischemic Preconditioning Is Probably Mediated by Reduction of Oxidative Stress Rather Than Mitochondrial Protein Phosphorylation
From the Department of Biochemistry and the Bristol Heart Institute, University of Bristol, Bristol, United Kingdom. Present address for M.D.: Cardiovascular Research Center, University of Connecticut, School of Medicine, Farmington.
Correspondence to Professor Andrew P. Halestrap, Department of Biochemistry and the Bristol Heart Institute, University of Bristol, Bristol BS8 1TD, United Kingdom. E-mail a.halestrap{at}bristol.ac.uk
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Abstract |
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Inhibition of mitochondrial permeability transition pore (MPTP) opening at reperfusion is critical for cardioprotection by ischemic preconditioning (IP). Some studies have implicated mitochondrial protein phosphorylation in this effect. Here we confirm that mitochondria rapidly isolated from preischemic control and IP hearts show no significant difference in calcium-mediated MPTP opening, whereas IP inhibits MPTP opening in mitochondria isolated from IP hearts following 30 minutes of global normothermic ischemia or 3 minutes of reperfusion. Analysis of protein phosphorylation in density-gradient purified mitochondria was performed using both 2D and 1D electrophoresis, with detection of phosphoproteins using Pro-Q Diamond or phospho-amino-specific antibodies. Several phosphoproteins were detected, including voltage-dependent anion channels isoforms 1 and 2, but none showed significant IP-mediated changes either before ischemia or during ischemia and reperfusion, and neither Western blotting nor 2D fluorescence difference gel electrophoresis detected translocation of protein kinase C (
, 
, or 
isoforms), glycogen synthase kinase 3β, or Akt to the mitochondria following IP. In freeze-clamped hearts, changes in phosphorylation of GSK3β, Akt, and AMP-activated protein kinase were detected following ischemia and reperfusion but no IP-mediated changes correlated with MPTP inhibition or cardioprotection. However, measurement of mitochondrial protein carbonylation, a surrogate marker for oxidative stress, suggested that a reduction in mitochondrial oxidative stress at the end of ischemia and during reperfusion may account for IP-mediated inhibition of MPTP. The signaling pathways mediating this effect and maintaining it during reperfusion are discussed.
Key Words: mitochondrial permeability transition • preconditioning • reperfusion injury • protein phosphorylation • oxidative stress
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Introduction |
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A critical factor mediating reperfusion injury of the heart is the mitochondrial permeability transition pore (MPTP), the opening of which causes mitochondrial swelling with release of proapoptotic proteins and uncoupling of mitochondrial oxidative phosphorylation. The resulting ATP deprivation causes disruption of ionic homeostasis and contractile function and ultimately sarcolemma rupture and necrosis.1 Inhibition of MPTP opening during reperfusion protects hearts from reperfusion injury.1 Effective cardioprotection is also mediated by ischemic preconditioning (IP) before prolonged ischemia is initiated,2 and this also involves inhibition of MPTP opening.3–6
Extensive evidence points to protein kinase (PK)C playing a central role in IP, although controversy remains over which PKC isoform(s) are involved and their translocation to mitochondria.7,8 The strongest evidence implicates PKC because PKC-knockout mice do not exhibit IP and transgenic mice with cardiac-specific overexpression of PKC or expression of a PKC activator are protected from reperfusion injury.8 Several studies have reported PKC translocation to the particulate fraction, including mitochondria,9,10 where it may phosphorylate putative components of the MPTP such as the voltage-dependent anion channel (VDAC).9–11 Others have proposed that activation of cyclic-GMP–dependent PK (PKG) by nitric oxide activates a mitochondrial intermembrane pool of PKC, leading to opening of mitochondrial ATP-sensitive potassium channels, followed by reactive oxygen species (ROS) formation, activation of a distinct mitochondrial PKC pool, and, finally, inhibition of the MPTP.12,13 Activation of prosurvival kinases such as Akt, especially during reperfusion, has been implicated by others,14 whereas Sollott and colleagues15 have proposed that protection by all these kinase may converge to phosphorylate and inhibit glycogen synthase kinase (GSK)3β. However, no mitochondrial phosphoprotein has been identified that may mediate protection by these kinases.
In this report, we investigate which PKs associate with carefully purified mitochondria from control and IP hearts and analyze whether consistent changes in mitochondrial protein phosphorylation can be detected. Neither approach provided evidence for IP mediating its effects by mitochondrial protein phosphorylation, but we confirm that mitochondrial protein oxidation is decreased by IP at the end of ischemia and early reperfusion. We propose that this reduction in oxidative stress experienced by mitochondria, rather than protein phosphorylation, may be responsible for IP-mediated inhibition of MPTP at the start of reperfusion.
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Materials and Methods |
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Materials
The sources of all materials are described in online data supplement, available at http://circres.ahajournals.org.
Heart Perfusion
All procedures conformed to the UK Animals (Scientific Procedures) Act 1986 and the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health (NIH Publication No. 85-23, revised 1996). Langendorff perfusions of hearts from male Wistar rats (250 to 260 g) were performed as described previously3,16 and are detailed in the expanded Methods section in the online data supplement. All hearts experienced 35 minutes of preischemia, which included the required treatment shown schematically in Figure 1. Perfusate was sampled for determining lactate dehydrogenase (LDH) activity. Table I in the online data supplement presents data on hemodynamic function and LDH release, confirming cardioprotection by IP similar to that observed previously.3–5,17,18 At the required time (Figure 1), hearts were either rapidly homogenized for the preparation of mitochondria or freeze-clamped using liquid nitrogen-cooled tongues, ground under liquid nitrogen, and stored at –80°C for later analysis.
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Isolation and Analysis of Particulate and Mitochondrial Fractions
All procedures were carried out at 0°C to 4°C in buffers containing protease and phosphatase inhibitors. Two protocols were used to prepare mitochondrial and particulate fractions from homogenized hearts or frozen heart powder as detailed in the expanded Methods section.
PKC Translocation and Protein Phosphorylation
Fractions (10 to 25 µg protein) were analyzed by SDS-PAGE and Western blotting with antibodies against both specific phosphoproteins and the corresponding total protein and then quantification by scanning (see the expanded Methods section). The ratio of the band intensity for phosphoprotein to total protein was used as a measure of phosphorylation state. Purity of mitochondrial fractions was assessed by Western blotting with antibodies against the adenine nucleotide translocase and monocarboxylate transporter 1, a specific plasma membrane marker.19
Two-Dimensional Gel Electrophoresis and Two-Dimensional Difference Gel Electrophoresis
These were performed in the University of Bristol Proteomics Facility as described in the expanded Methods section, and gels were visualized fluorescently (difference gel electrophoresis) or stained for phosphoproteins and total protein (Pro-Q Diamond and Sypro–Ruby, Invitrogen).
Measurement of MPTP Opening In Vitro and Protein Carbonylation Assays
MPTP opening was determined at 25°C under deenergized conditions by monitoring A520,3 whereas protein carbonyls were analyzed by derivatization with dinitrophenylhydrazine, followed by Western blotting,5,18 as detailed in the expanded Methods section.
Statistical Analysis
Data are presented as means±SE. Statistical significance was evaluated using 1-way ANOVA, followed by 2-tailed Student’s t test (for simple comparisons between control and preconditioned hearts) or Tukey’s multiple comparison post hoc test (for comparisons among multiple groups of hearts) using GraphPad Prism version 4.0 software. Differences were considered significant at P<0.05.
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Results |
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MPTP Opening Was Inhibited in Mitochondria Isolated From IP Hearts Only After Ischemia
Figure 2A confirms that mitochondria isolated from IP hearts at the end of ischemia or after 3 minutes of reperfusion exhibited less calcium-induced MPTP opening than those isolated from control hearts, whereas no differences were observed in mitochondria isolated immediately after the IP protocol.3–5 This lack of effect of IP was independent of the [Ca2+] used (supplemental Figure II). Because our protocol uses deenergized mitochondria with buffered [Ca2+] in the presence of calcium-ionophore (A23187), the IP-mediated inhibition of MPTP opening cannot be caused by either differences in mitochondrial calcium loading or membrane potential. Rather, it must reflect a change in calcium sensitivity of the MPTP. Two possible mechanisms may account for this: either phosphorylation of some MPTP component or oxidative modification of critical thiol groups on the MPTP that sensitize it to [Ca2+].1
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Mitochondrial Protein Oxidation Was Decreased by Preconditioning
The data of Figure 2B and 2C confirm that mitochondria isolated from IP hearts during reperfusion exhibit less protein carbonylation, a measure of protein oxidation and a surrogate marker of mitochondrial oxidative stress,5 whereas no effect of IP was observed in preischemic hearts. Previous work has shown a similar reduction in protein carbonylation by IP and other preconditioning stimuli at the end of ischemia5 and with temperature preconditioning, urocortin, and apomorphine at reperfusion.18,20,21
Translocation of Protein Kinases to Purified Mitochondria Was Not Detected Following IP
Activation of PKC isoforms causes their translocation to intracellular membranes, including the plasma membrane.22 Because conventionally prepared mitochondria are contaminated with such membranes,23 their complete removal is essential when studying PKC translocation to mitochondria. Two protocols were used to achieve this, as illustrated in supplemental Figure I. Either a cytosolic fraction and crude high-speed particulate fraction were prepared, followed by preparation of mitochondria from the latter (protocol 1) or a more conventional mitochondrial preparation was used (protocol 2). In both cases, contaminating plasma membranes were removed by Percoll gradient centrifugation. Figure 3 shows that protocol 1 produced a crude particulate fraction containing both mitochondria (adenine nucleotide translocase) and plasma membranes (monocarboxylate transporter 1), as did the crude mitochondrial preparation of protocol 2 (Figure 4). In both cases, Percoll purification removed plasma membranes (monocarboxylate transporter 1), with the loss of almost all the PKC and PKC. Although PKC remained, no increase was detected following IP treatment, whether mitochondria were isolated before ischemia (Figures 3A and 4
A) or during reperfusion (Figure 3C). Treatment with 50 µmol/L diazoxide, an IP mimic, did not cause detectable PKC or PKC translocation, whereas treatment with phorbol ester (200 nmol/L phorbol ester {phorbol-12-myristate-13-acetate [TPA]}) produced the anticipated loss of both isoforms from the cytosol and a slight increase in the crude particulate fraction (Figure 3A). A larger PKC increase was detected in the plasma membrane fraction (Figure 4A), ensuring a robust response in the particulate:cytosolic ratio (Figure 3B). A small increase in PKC in the mitochondrial fraction was also detected following TPA treatment, although this may reflect contamination with residual plasma membranes that are highly enriched in PKC following the substantial TPA stimulus (Figures 3A and 4A).
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The data of Figure 4B illustrate that we were also unable to detect significant amounts of either the nonphosphorylated or phosphorylated forms of AMP-activated PK (AMPK), GSK3β, or Akt in the purified mitochondrial fraction isolated from either control or IP hearts.
Determination of AMPK, GSK3β, and Akt phosphorylation in Freeze-Clamped Hearts
We investigated the phosphorylation state of AMPK, GSK3β, and Akt in freeze-clamped control and IP hearts. Extracts were rapidly prepared in the presence of phosphatase and protease inhibitors, and proteins were separated by SDS-PAGE before Western blotting with antibodies against the phosphorylated and total kinases. Representative blots and mean data for the ratio of phosphorylated to total protein are shown in Figure 5. Before index ischemia, samples from IP hearts were taken either at the end of the third brief ischemic phase of preconditioning (IP#) or after a further 5-minute normoxic recovery (IP). Significant increases in AMPK and GSK3β phosphorylation relative to control hearts were detected in both IP and IP# hearts, with the latter showing the greater effect. We confirmed the activation of AMPK by measuring an increase in phosphorylation state of acetyl-coenzyme A carboxylase (ACC). However, no significant effects of IP on Akt phosphorylation were detected before ischemia. Samples taken after 5 minutes of index ischemia showed a substantial increases in AMPK phosphorylation relative to preischemic values and small increases in the phosphorylation of ACC, GSK3β, and Akt, but in no case could we detect a significant difference between control and IP hearts. After 30 minutes of ischemia, AMPK phosphorylation remained slightly elevated compared with the preischemia, whereas ACC phosphorylation at both time points was less than preischemic values. Akt phosphorylation was slightly reduced after 30 minutes ischemia and elevated after 3 minutes of reperfusion, whereas GSK3β phosphorylation was greater than preischemic values at both the end of ischemia and 3 minutes reperfusion. Importantly, however, none of the phosphoproteins showed a significant difference between control and IP hearts, whether measured at 5 minutes of ischemia, 30 minutes of ischemia, or 3 minutes of reperfusion. Nor did we observe changes in phosphorylation of AMPK, AkT, ACC, or GSK3β in response to preconditioning by 50 µmol/L diazoxide (supplemental Figure III).
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Further evidence that preischemic phosphorylation of Akt and GSK3β may be unimportant for the triggering events of IP is presented in Figure 6. Pretreatment of hearts for 19 minutes with 0.7 nmol/L insulin and a 1 minute of washout before ischemia increased the phosphorylation of both Akt and GSK3β several-fold, yet no protection from reperfusion following 30 minutes ischemia was observed. These data are consistent with previous observations that insulin must be present during reperfusion to be protective.15 Additional evidence against a role for AMPK in IP was provided by using compound C, an established inhibitor of AMPK, which exerted no effect on the ability of IP to improve hemodynamic function or lower LDH release (supplemental Table I) but does inhibit AMPK-mediated changes in ACC phosphorylation.18 By contrast, exposure to 10 µmol/L chelerythrine blocked the IP-mediated decrease in LDH release during reperfusion (supplemental Table I and Figure IV).
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Preconditioning Gave No Detectable Changes in Mitochondrial Protein Phosphorylation
The phosphorylation state of proteins in mitochondria rapidly isolated in the presence of phosphatase and protease inhibitors was determined following their separation by 2D gel electrophoresis. Pro-Q Diamond and Sypro–Ruby were used to detect phosphoproteins and total proteins, respectively.24 Figure 7 presents phosphoprotein data for mitochondria from control and IP hearts isolated both before ischemia and after 30 minutes of ischemia and 3 minutes of reperfusion. In supplemental Figure VA, these data are overlaid (red) on the Sypro–Ruby protein data (green) to allow discrimination between truly phosphorylated proteins and proteins stained nonspecifically with the Pro-Q Diamond. Supplemental Figure VB shows similar data from another set of preischemic, end-ischemic, and reperfused hearts. A significant number of proteins were preferentially stained with the Pro-Q Diamond stain, implying that they were phosphorylated. By far the strongest signal was in the 40-kDa region (box 1) in the correct location for multiple phosphorylation states of the E1 subunit of pyruvate dehydrogenase (PDHE1) (accession no. P26284, Swiss Prot; molecular mass, 40.2 kDa) that we have previously shown to be the dominant matrix phosphoprotein whose phosphorylation turns over rapidly.25 PDHE1 is known to exhibit multiple phosphorylation states,26 and the theoretical pI values of the 0, 1, 2, and 3 phosphorylated states of 6.82, 6.52, 6.31, and 6.15 are consistent with the spots observed. In supplemental Figure IX, we provide data to confirm the identity of these spots as PDH1E by using mass spectrometry (see supplemental Table II) and by Western blotting and dephosphorylation studies. Supplemental Figure IXs and Table II also provide data on the identity of the 2 phosphoprotein spots at 
31 kDa in box 2 of Figure 7. These were shown to be singly phosphorylated forms of the VDAC isoforms 1 and 2 (VDAC1, theoretical pI 7.83; VDAC2, theoretical pI 6.68). Evidence has been presented that VDAC1 can be phosphorylated27 and that this may lead, directly11 or indirectly,28 to inhibition of MPTP opening. However, our data revealed no changes in phosphorylation of either VDAC1 or VDAC2 in response to IP.
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In supplemental Figure VI, we confirm that we were able to detect IP-mediated changes in cytosolic protein phosphorylation. As additional confirmation that IP exerted no effect on mitochondrial protein phosphorylation, we used 2D fluorescence difference gel electrophoresis in which mitochondrial proteins of control and IP hearts were labeled with red and green fluorescent probes before mixing and separating on the same 2D gel. Proteins unchanged by IP treatment run in the same place and show as yellow spots, whereas any differences are revealed as red or green spots. Data are shown in supplemental Figure VII and Table II for mitochondria from preischemic, end-ischemic, and reperfused hearts, but, once again, no consistent IP-mediated changes were revealed that may account for MPTP inhibition.
We considered that IP may cause changes in the phosphorylation state of proteins, such as the adenine nucleotide translocase, that do not readily enter the isoelectric focusing gel and so would not be detected using the 2D gels. Thus, we also performed 1D SDS-PAGE with Pro-Q Diamond staining but, again, found no evidence for changes in any protein phosphorylation following IP (supplemental Figure VIII, A). We have also used antibodies against phosphotyrosine, phosphoserine, and phosphothreonine as an alternative strategy to detect IP-mediated changes in protein phosphorylation. Here too, no effects of IP were observed (supplemental Figure VIII, B through D).
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Discussion |
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Mitochondrial Protein Phosphorylation May Not Be Required for IP-Mediated Inhibition of MPTP Opening
Mitochondria isolated immediately after IP showed no decrease in sensitivity to calcium-induced MPTP opening (Figure 2A and elsewhere3,5). These data argue against phosphorylation of a component of the MPTP during the IP protocol mediating inhibition of pore opening, and our inability to detect a change in mitochondrial protein phosphorylation at this time (Figure 7 and supplemental Figures V, VII, and IX) support this conclusion. However, we were also unable to detect an IP-induced change in the phosphorylation state of any mitochondrial protein in IP hearts at the end of ischemia or during reperfusion (Figure 7 and supplemental Figures V and VII through IX), at which time MPTP opening was inhibited by IP (Figure 2A and elsewhere4,5). Thus, it seems unlikely that inhibition of MPTP opening at reperfusion is mediated by protein phosphorylation. Our inability to detect translocation of AMPK, Akt, GSK3β, or PKC isoforms to the mitochondria (Figures 3 and 4
) or changes in their phosphorylation state (Figure 4 and supplemental Figures VII and VIII) is consistent with this conclusion. Although several groups have reported that IP causes translocation of PKC isoforms, particularly PKC, to the particulate fraction,9,11,29–33 not all reported PKC translocation to mitochondria.18,31,32 This may be because PKC translocation is transient in the rat heart, being lost after 3 brief ischemic periods, as used in the present study.34 Some studies have reported IP-mediated PKC translocation to mitochondria.31,32 Although we confirmed the presence of PKC in the mitochondria we did not observe IP-mediated translocation (Figure 3).
We have not attempted to identify all the phosphoproteins detected in the mitochondria, but phosphorylated forms of PDHE1 were the dominant spots (Figure 7 and supplemental Figure IX). PDHE1 is the major phosphoprotein in the mitochondrial matrix known to be rapidly phosphorylated and dephosphorylated, together with a small amount of the E1 subunit of branch-chain keto-acid dehydrogenase (BCKDH E1).25 The additional phosphoproteins detected by us and others24 may turn over more slowly and so are not detected using rapid labeling with 32P. Alternatively, they may represent proteins integral to the outer mitochondrial membrane such as VDAC1 and VDAC2 (supplemental Figure IX and elsewhere27) or bound to it using scaffolding proteins.35,36 Their phosphorylation could be regulated by cytosolic kinases associating weakly with mitochondria but not remaining bound during isolation. Indeed, we have shown previously that when mitochondria are isolated from hepatocytes incubated with 32Pi, additional phosphoproteins are observed with 2 proteins of 30 to 35 kDa, demonstrating increased phosphorylation following glucagon treatment.37,38 Whatever their identity, it seems unlikely that the phosphoproteins we detect are involved in IP-mediated inhibition of MPTP opening because their phosphorylation is not changed by IP. This includes VDAC1, the phosphorylation of which has been proposed by others to regulate the MPTP.11,28
We cannot totally rule out a role for protein phosphorylation regulating the MPTP because there may be phosphoproteins present at levels below the detection limit of Pro-Q Diamond, difference gel electrophoresis, or phospho–amino acid–specific antibodies. Additionally, dephosphorylation may have occurred during the mitochondrial preparation despite the presence of phosphatase inhibitors, although this is unlikely because we detected many phosphoproteins in both mitochondrial and cytosolic fractions with IP-mediated changes in the latter (supplemental Figure VI).
Reduction in Oxidative Stress May Explain the Inhibition of MPTP Opening by IP
Preconditioning by a variety of means reduces oxidative stress following ischemia and reperfusion,39–43 and we have previously shown that this is associated with less oxidative damage to mitochondria as monitored by protein carbonylation.5,18,20,21 Here we confirm that this is the case for IP (Figure 2B and 2C). Because thiol oxidation greatly sensitizes the MPTP to [Ca2+],1 an IP-mediated decrease in oxidative damage provides sufficient explanation for the observed inhibition of MPTP opening at reperfusion, without the need to invoke mitochondrial protein phosphorylation. This would explain why there is no decrease in calcium sensitivity of MPTP opening immediately following IP, when there is no significant oxidative damage (Figure 2, supplemental Figure II, and elsewhere4,5). Such a mechanism is also consistent with the strong cardioprotection afforded by antioxidants specifically targeted to mitochondria.44 Thus, we propose that decreasing oxidative stress at the end of ischemia and during early reperfusion represents the common mechanism by which preconditioning stimuli inhibit MPTP opening.
The Signaling Pathways by Which Ischemic Preconditioning Reduces Oxidative Stress
Our data lead us to conclude that AMPK, Akt, or GSK3β are unlikely to be involved in reducing oxidative stress at the end of ischemia and early in reperfusion, because we found no appropriate changes in their phosphorylation state in response to IP (Figure 5) or diazoxide (supplemental Figure III). Nor did the AMPK inhibitor compound C prevent protection by IP as measured by either hemodynamic function or LDH release (supplemental Table I). Indeed, if anything, this reagent enhanced the hemodynamic recovery of IP hearts, which may reflect a metabolic effect of the inhibitor such as inhibition of fatty acid oxidation.18 By contrast, the inability to precondition PKC-knockout mice,45 the ability of chelerythrine (supplemental Table I) and other PKC inhibitors to prevent IP, and the ability of PKC activation to mimic preconditioning7,29,46,47 all argue for a critical role of PKC in this process. Furthermore, the decrease in oxidative stress at reperfusion caused by both urocortin21 and temperature preconditioning18 are mediated by PKC. However, the mechanism by which this is achieved and whether decreased ROS production or increased ROS removal is involved remains to be elucidated.
Additional Signaling Pathways May Inhibit MPTP Opening as Reperfusion Continues
Activation of prosurvival kinases such as members of the mitogen-activated protein kinase family and the phosphatidylinositol 3-kinase/Akt cascade during reperfusion have been proposed to play a key role in IP-mediated cardioprotection.48 Maintenance of the MPTP inhibition during reperfusion appears to be critical for cardioprotection because treatment with cyclosporin-A and sanglifehrin-A (MPTP inhibitors) within the first 15 minutes of reperfusion is sufficient to produce a profound reduction in infarct size.49,50 This may reflect an ongoing cascade of MPTP opening whereby the initial pore opening at reperfusion stimulates ROS production that goes on to cause further MPTP opening as reperfusion continues.51–52 This MPTP-mediated ROS production could involve cytochrome c release, caspase activation, and subsequent cleavage of the p75 subunit of complex I.53,54 Prevention of MPTP opening at the start of reperfusion, as occurs through IP reduction in ROS levels, will also prevent the subsequent MPTP opening and thus represents a protective mechanism with "memory," as defined by Sollott and colleagues.15 By contrast, those stimuli, such as insulin, that affect the survival kinase pathway may act on the caspase-mediated pathway and so work only during reperfusion and thus lack "memory." In Figure 8, we present a scheme that summarizes these proposals.
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Acknowledgments |
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Sources of Funding
This work was supported by British Heart Foundation programme grant RG/03/002 (to A.P.H.).
Disclosures
None.
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Footnotes |
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*Both authors contributed equally to this work.
Original received July 4, 2007; resubmission received October 31, 2007; revised resubmission received February 8, 2008; accepted March 7, 2008.
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References |
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