Primary and Secondary Signaling Pathways in Early Preconditioning That Converge on the Mitochondria to Produce Cardioprotection

http://www.100md.com 《循环研究杂志》2004年第1期

     From the Laboratory of Signal Transduction, National Institute of Environmental Health Sciences, National Institutes of Health, US Department of Health and Human Services, Research Triangle Park, NC.+, http://www.100md.com

    This Review is part of a thematic series on Mitochondrial Dysfunction in Ischemia, which includes the following articles:+, http://www.100md.com

    Role of the Mitochondrial Permeability Transition in Myocardial Disease+, http://www.100md.com

    Primary and Secondary Signaling Pathways in Early Preconditioning That Converge on the Mitochondria to Produce Cardioprotection+, http://www.100md.com

    Evidence for Mitochondrial K⁺ Channels and Their Role in Cardioprotection+, http://www.100md.com

    Mitochondrial Death Pathways+, http://www.100md.com

    Roberto Bolli Editor+, http://www.100md.com

    Abstract+, http://www.100md.com

    Cardioprotective mechanisms such as acute or early preconditioning activate several primary signaling pathways that seem to converge on mitochondrial targets, leading to altered cell metabolism and inhibition of apoptosis. Acute preconditioning leads to generation of agonists, which bind to G protein–coupled receptors, and initiates a signaling cascade that involves activation of phosphoinositide-3-kinase, endothelial NO synthase, protein kinase C, glycogen synthase kinase 3ß, mitogen-activated protein kinases, and other signaling pathways. Activation of these signaling pathways along with generation of reactive oxygen species leads to alterations in the activity of key mitochondrial proteins such as mitochondrial ATP-sensitive K⁺ channels, the mitochondrial permeability transition pore, and bcl-2 family members. Alterations in these mitochondrial proteins results in altered metabolism and inhibition of cell death, thus resulting in cardioprotection.

    Key Words: apoptosis cardioprotection signaling pathways preconditioning mitochondria5i}}&r, 百拇医药

    Introduction5i}}&r, 百拇医药

    Over the years, several cardioprotective drugs and interventions have been described and studied. A major goal of this research effort has been to understand the mechanisms involved in cell death so that one can intervene to block cell death. The recent recognition of the role of apoptosis, a regulated form of cell death, in cardiac cells has enhanced the expectation that one can pharmacologically intervene to block or modulate cell death. Cell death is a primary factor in the pathogenesis of ischemia/reperfusion, as well as in the myocyte loss associated with heart failure. Most studies in cardioprotection have focused on reducing myocyte death during an acute episode of ischemia and reperfusion. Many drugs have been shown to be cardioprotective when given before ischemia^1–6; however, because patients typically present after the onset of ischemia, many of these drugs are of limited clinical benefit. There is an obvious clinical need for drugs that reduce cell death and dysfunction when given at the time of reperfusion. There are a few reports that addition of cardioprotective agents at the immediate onset of reperfusion can reduce infarct size.^6–10 These drugs may offer promise when given with angioplasty-associated reperfusion. It has also been suggested that a gene therapy approach to upregulate cardioprotective genes is a plausible strategy for protecting at-risk populations.^11,12 A better understanding of the signaling mechanisms in cardioprotection and how they ultimately lead to reduced cell death will enhance our ability to make cardioprotection a useful clinical strategy.

    This review focuses on the signaling pathways involved in cardioprotection. Cardioprotection can be divided conceptually into cardioprotection that requires new transcription or translation and cardioprotection that occurs as the result of addition of a drug or intervention that modulates cell metabolism or signaling pathways directly or via posttranslational modification of proteins. This review focuses on acute cardioprotection, which does not require new protein synthesis. As mentioned, there are many drugs or agents that have been reported to be cardioprotective. These drugs initiate cardioprotection by modulating diverse signaling pathways. However, there is emerging evidence that these diverse signals converge on a few final common effectors or signaling pathways that ultimately ameliorate cell death With analogy to the apoptosis pathway, activation of effector caspases are the mediators of apoptosis; however, many different signaling pathways can lead to caspase activation. Thus, this review is divided into two parts. The first part reviews some of the common primary signaling pathways or mediators of cardioprotection, and the second part focuses on how these primary signals may converge on secondary cardioprotective signals or the end effectors of cardioprotection.

    fig.ommitted|95w}qx, 百拇医药

    Primary and secondary signaling pathways in preconditioning. Primary signaling pathways: preconditioning leads to release of adenosine, opioids, or bradykinin, which bind to GPCR and initiate a signaling cascade that involves activation of PI3K, perhaps via transactivation of the EGF receptor.²² PI3K results in signaling via downstream targets such as PKB, eNOS, GSK3ß, PKC, mTOR, and p70S6. GPCR activation also leads to release of Gß, which, with other receptor signaling components, leads to activation of the ERK pathways, likely via an endosomal signaling pathway. PC also leads to generation of ROS, which may also play a role in activation of PKC and perhaps other signaling events (such as activation of mitoK_ATP). Secondary signaling pathways: these primary signaling pathways seem to converge on a few related mitochondrial proteins. ERK has been reported to phosphorylate mitochondrial-associated BAD, which would cause it to dissociate from bcl-2, leaving bcl-2 free to bind and inhibit VDAC and the MPT. PKC, NO, 12-LO metabolites, and ROS have all been shown to activate the mitoK_ATP channel, which has been shown to reduce apoptosis and cell death. MitoK_ATP has been reported to maintain mitochondrial structure such that VDAC is in a low conductance state. This would synergize with the bcl-2 associated with VDAC to maintain VDAC in a low conductance state. VDAC in a closed state would reduce breakdown of glycolytic ATP and would reduce apoptosis (either via VDAC association with BAX or as part of the MPT). PC signaling involves several redundant signaling pathways that appear to converge on a few mitochondrial targets, such as mitoK_ATP, VDAC, and MPT. As discussed in the text, there may be additional primary targets. For example, delayed PC has additional targets, such as nuclear factor-{kappa} B, inducible NOS, COX-2, and p70s6K, that lead to increased expression of cardioprotective genes.

    Primary Signaling Pathways in Cardioprotection2rt%:), http://www.100md.com

    This review focuses on cardioprotection related to signaling pathways resulting in posttranslational modification. Acute cardioprotective agents can protect either by decreasing the development of injury or by attenuating cell death pathways. Some approaches, such as reducing temperature, mediate cardioprotection by decreasing metabolism and the consumption of ATP.¹³ On the other end of the spectrum, caspase inhibitors seem to protect by attenuating mechanisms of cell death.¹⁴ Most cardioprotective agents work by both decreasing the development of injury (eg, preserving ATP) and by inhibition of cell death mechanisms. For example, limiting Ca²⁺ overload may serve both to conserve ATP and reduce mitochondrial-induced apoptosis. Drugs such as Na⁺-H⁺ exchange inhibitors reduce the development of injury if given before ischemia.⁶ Na⁺-H⁺ exchange inhibitors reduce the ischemia-reperfusion–induced rise in intracellular Na⁺ and intracellular Ca²⁺.¹⁵ The reduction in the rise in Ca²⁺ would be expected to reduce Ca²⁺ activation of proteases and to reduce ATP degradation related to stimulation of Ca²⁺ ATPases. The reduction in Ca²⁺ would also reduce Ca²⁺ accumulation by intracellular organelles such as sarcoplasmic reticulum and mitochondria, and decreased mitochondrial Ca²⁺ would mediate cardioprotection via a reduction in mitochondrial-dependent apoptosis. Activation of the phosphoinositide-3-kinase (PI3K) or protein kinase C (PKC) pathways seems to protect primarily by inhibiting cell death mechanisms,^16,17 although alterations in cell metabolism and energy utilization may also play a role.

    Many of the cardioprotective signaling pathways have been elucidated through studies of preconditioning. Brief intermittent periods of ischemia and reperfusion, referred to as preconditioning (PC), have been shown to reduce injury during a subsequent sustained period of ischemia. shows signaling pathways involved in PC.m(#q1dd, http://www.100md.com

    Overview of PC Signalingm(#q1dd, http://www.100md.com

    Considerable data suggest that PC leads to release of hormones or agonists that bind to receptors and activate signaling pathways.^{1,2,4,18–21} Released factors include adenosine,¹ opioids,^2,19 and bradykinin.⁴ These agonists bind to G protein–coupled receptors and activate signaling pathways.^22,23 PC and most of these agonists activate PI3K, which generates phosphoinositides that localize kinases, such as phosphoinositide-dependent kinase (PDK), with substrates, leading to activation of downstream kinases such as protein kinase B (PKB, also known as Akt) and p70S6-kinase (p70S6K). PI3K is also important in receptor internalization and endosomal signaling,²³ which, as discussed below, may play a role in PC. Activation of PI3K has been reported to be upstream of PKC,²⁴ GSK3ß,²⁵ p70S6K,²⁶ generation of NO,²⁴ and activation of mitochondrial ATP-sensitive K channels (mitoK_ATP).²¹ However, Qin et al²⁷ have shown that different signaling pathways can vary somewhat depending on the agonist. Details of these signaling elements are discussed.

    G Protein–Coupled Receptor:]z\/7c, http://www.100md.com

    Administration of many G protein–coupled receptor (GPCR) agonists, especially those that signal via G{alpha} _i, such as adenosine, opioids, bradykinin, and acetylcholine, has been shown to reduce infarct size.^{1,2,4,19–22} Furthermore, inhibition of G{alpha} _i signaling by pertussis toxin (PTX) treatment blocks the protection afforded by PC in rabbits¹⁸ and in some²⁸ but not all studies in rats.²⁹ Activation of GPCR leads to signaling via G{alpha} and Gß{gamma} . As discussed below, signaling through Gß{gamma} can lead to activation of PI3K²³ and also recruits G protein receptor kinase, which leads to receptor internalization. Activation of GPCR can also lead to activation of the mitogen-activated protein kinase (MAPK) pathway via G{alpha} -dependent signaling, by transactivation of the epidermal growth factor (EGF) receptor, or via ß-arrestin–dependent endosomal signaling.³⁰ Pertussis toxin inhibition of PC suggests a possible role for G{alpha} _i-mediated signaling in PC. G{alpha} _i has been shown to inhibit adenyl cyclase or to lead to activation of membrane channels.

    PI3K:*p3, http://www.100md.com

    Activation of the PI3K pathway has been shown in many models to be cardioprotective.^{7,16,22,24,31–35} PC has been shown to lead to activation of PKB and p70S6K, kinases downstream of PI3K, consistent with a role for PI3K in PC.^24,31 Furthermore, inhibitors of PI3K, such as wortmannin and LY294002, attenuate the protection afforded by PC.^24,31,33:*p3, http://www.100md.com

    How is PI3K activated? GPCR can activate PI3K via release of Gß, which activates PI3K{gamma} .³⁶ To test whether this mechanism was responsible for PC-induced activation of PI3K, studies were performed using mice with a cardiac-specific expression of ßARKct, a peptide that sequesters Gß.³⁷ Expression of ßARKct inhibits Gß{gamma} -dependent signaling and has been shown to inhibit GPCR activation of PI3K.²³ Using hearts from ßARKct mice, Tong et al³⁸ reported that sequestration of Gß{gamma} blocks PC; however, sequestering Gß did not block PC-induced activation of kinases downstream of PI3K, such as PKB and p70S6K. These data suggest that activation of PKB and p70S6 kinase per se are not sufficient for protection; however, the data do not rule out the possibility that activation of these signals may be required.

    Krieg et al²² have recently reported that acetylcholine-induced protection leads to activation of the PI3K pathway via GPCR transactivation of the EGF receptor. Krieg et al²² reported that acetylcholine-induced phosphorylation of PKB was blocked by the src inhibitor PP2 and the EGF receptor inhibitor AG-1478. Sequestration of Gß{gamma} has been reported to inhibit transactivation of the EGF receptor.³⁰ If this is the case in PC, one would expect that expression of ßARKct would block transactivation of the EGF receptor and subsequent activation of PI3K. Additional studies will be necessary to resolve the precise mechanism of activation of PI3K in PC. It is worth noting that hydrogen peroxide has been reported to activate PI3K,³⁹ suggesting that ROS may play a role in PI3K activation in PC.8, 百拇医药

    What is the role of Gß{gamma} in PC? Gß{gamma} is known to be required for receptor internalization and associated endosomal signaling, and there are emerging data suggesting that inhibition of endosomal trafficking blocks activation of cytosolic extracellular-regulated kinase (ERK).^30,40,41 Recent studies have shown also that inhibition of endosomal trafficking blocks PC.³⁸

    What are the signals downstream of PI3K and how does activation of PI3K mediate cardioprotection? Because there are no inhibitors of PKB, it is unclear whether activation of PKB, which has been shown consistently to accompany PC, is required for PC. The data showing that sequestration of Gß{gamma} blocked PC-induced protection, but not activation of PKB or p70S6K, suggest that activation of PKB and p70S6K is not sufficient for protection.³⁸ However, PKB is reported to phosphorylate and modulate several pathways involved in cardioprotection, suggesting that it may have a role in PC, even though its activation is not sufficient to induce PC. PKB phosphorylates and activates endothelial NO synthase (eNOS) and p70S6K and phosphorylates and inactivates GSK3ß and the proapoptotic BAD (phosphorylation of BAD causes it to be sequestered by 14-3-3).⁴² NO and eNOS have been shown to be important in PC.^43,44 NO has been shown to activate the mitoK_ATP channel.⁴⁵ There are also data showing a role for GSK3ß, a kinase downstream of PKB, in preconditioning and cardioprotection.²⁵ GSK3ß is phosphorylated and inactivated by PKB. Expression of a mutant GSK that cannot be phosphorylated blocked the antiapoptotic action of PI3K.⁴⁶ Phosphorylation of GSK3ß has been shown to occur during PC; thus, PC would lead to inactivation of GSK3ß. Consistent with a role for inhibition of GSK3ß in cardioprotection, Tong et al²⁵ showed that pharmacological inhibition of GSK3ß with SB216763 reduced infarct size and improved recovery of postischemic function. Opioid protection has also been shown to involve GSK3ß.⁴⁷ The targets downstream of GSK-3ß in PC remain to be elucidated, but GSK-3ß has been reported to mediate the antiapoptotic actions of PI3K.⁴⁶

    Activation of PI3K was also shown to be upstream of PKC.²⁵ Wortmannin was shown to block the PC-induced translocation of PKC{epsilon} . Furthermore, the protective effect of pharmacological activation of PKC with DOG was not blocked by inhibitors of PI3K. Also consistent with activation of PKC downstream of PI3K, DOG did not enhance phosphorylation of PKB.*^e, 百拇医药

    P70S6K, another kinase downstream of PI3K, has also been reported to be involved in PC.²⁶ PDK1 phosphorylation of p70S6K on thr 229 is required for activation of P70S6K (reviewed by Murphy et al⁴⁸). Prior phosphorylation of thr 389 by mammalian target of rapamycin (mTOR) is necessary before PDK1 can phosphorylate thr 229. PKB is also reported to phosphorylate p70S6K. As discussed below, p70S6K may integrate signals from several diverse pathways.*^e, 百拇医药

    PKC*^e, 百拇医药

    Numerous studies have demonstrated a role for PKC{epsilon} in cardioprotection and PC.^5,49–54 Ping et al⁵⁰ demonstrated that PC leads to translocation of PKC{epsilon} . Inhibition of PKC has been shown by several investigators to block the protection afforded by PC, and pharmacological activators of PKC have been shown to be cardioprotective.^5,49,52,53 Transgenic mice with cardiac-specific overexpression of PKC{epsilon} or expression of an activator of PKC{epsilon} have been shown to exhibit endogenous protection.^51,54,55 Taken together, there is strong evidence suggesting a role for PKC in PC.

    Several mechanisms have been suggested to be involved in PC-induced activation of PKC. Reactive oxygen species (ROS), which are generated by PC, have been reported to be involved in activation of PKC.⁵⁶ As discussed above, inhibition of PI3K blocks translocation of PKC,²⁴ suggesting a role for PI3K-dependent signaling. The precise role of PI3K in activation of PKC has not been determined. Phosphoinositide products have been reported to activate several isoforms of PKC.⁵⁷ Also, Ping et al⁴⁴ have reported that NO is involved in PC-induced translocation of PKC{epsilon} ; they also report that NO donors lead to translocation of PKC{epsilon} and that inhibitors of NOS, such as L-NAME, block the PC-induced translocation of PKC{epsilon} . Because inhibition of PI3K was shown to reduce generation of NO,²⁴ these data would be consistent with PI3K activation of PKC{epsilon} via an eNOS-mediated mechanism.1+iun(^, http://www.100md.com

    Considerable effort has been directed toward elucidating the downstream targets of PKC in cardioprotection. PKC activation has been shown to be important in activation of the mitoK_ATP channel.⁵⁸ PKC{epsilon} also forms a complex with components of the mitochondrial permeability transition (MPT) pore.¹⁷ However, the association of PKC{epsilon} with components of the MPT does not demonstrate that this association is important in modulating the MPT or in cardioprotection. Additional studies are need to fill in the details. Baines et al³³ have additionally reported that ERK and PKC{epsilon} are contained in a multimeric mitochondrial signaling complex and that PKC{epsilon} may lead to activation of ERK in this complex. It will be of interest to determine whether ERK has a role in activation of the mitoK_ATP channel. PKC activation has also been shown to be upstream of 12-LO signaling, and both are upstream of mitoK_ATP.⁵⁹

    ERK:, 百拇医药

    Most^38,60–62 but not all⁶³ investigators report that PC leads to activation of ERK. However, there are conflicting data regarding the role of ERK in preconditioning. Strohm et al⁶² reported that inhibition of the ERK pathway with PD98059 blocked PC, and Fryer et al⁶⁰ similarly reported that PD98059 blocked protection by PC or opioid-induced protection.¹⁹ In contrast, Mocanu et al³¹ reported that PD98059 did not block PC-induced protection. Johassen et al⁷ have suggested that inhibition of ERK on reperfusion blocks cardioprotection.:, 百拇医药

    How are the ERKs activated? Ping and colleagues^61,64 reported that PC-induced phosphorylation of ERK is mediated by PKC. Gross et al⁶⁵ report that delay preconditioning initiated by K_ATP channel openers is abolished by inhibition of the ERK pathway using the MEK1/2 inhibitor PD 98059, suggesting that K_ATP channel opening can lead to activation of ERK, likely secondary to ROS generated by opening of the mitoK_ATP channel. Tong et al³⁸ showed that ERK phosphorylation is blocked by sequestering Gß{gamma} , inhibition of PI3K, or inhibition of endosomal trafficking, consistent with a role for receptor-mediated endosomal activation of ERK. Endosomal activation of ERK is not inconsistent with ERK activation by PKC, and it is also possible that endosomal-dependent signals lead to mitochondrial targeting of ERK. Regarding the mechanism by which the ERKs mediated protection, Baines et al⁶⁴ suggested that ERK is part of a mitochondrial complex involved in phosphorylation of BAD.

    p70S6K@2|, 百拇医药

    Kinases from different pathways such as PI3K, PDK1, PKC, ERK, mTOR, and PKB are involved in the phosphorylation and activation of p70S6K. p70S6K therefore can integrate signals from diverse pathways. Kis et al²⁶ recently reported that inhibition of PI3K or inhibition of mTOR with rapamycin blocked the second window or delayed PC. Johassen et al⁷ have reported that insulin administered at reperfusion is cardioprotective and that rapamycin abolished this protection. The role of p70S6K in acute PC has not been addressed.@2|, 百拇医药

    p38 MAPK@2|, 百拇医药

    The role of p38 MAPK in PC has been controversial and has been recently reviewed elsewhere.⁶⁶ Briefly, there are data suggesting that inhibition of p38 MAPK with SB202190 or SB203580 blocks the protection afforded by PC; however, there are also data showing that p38 MAPK inhibitors do not block PC. Furthermore, some groups find that in the absence of PC, inhibition of p38 MAPK before and during ischemia is cardioprotective, whereas others find inhibition of p38 MAPK to have no effect. The reasons for these differences are not clear and are discussed elsewhere.⁶⁶

    12-Lipoxygenase.{y, 百拇医药

    12-Lipoxygenase (12-LO) metabolites have been reported in neuronal cells to activate K_ATP channels.⁶⁷ This observation led to studies to examine the role of 12-LO in PC. 12-LO metabolites are generated by PC, and inhibitors of 12-LO block the protection afforded by PC.^59,68 Furthermore, transgenic mice that are null for the leukocyte 12-LO are not protected by PC.⁶⁹ 12-LO has also been shown to be upregulated in an opioid model of PC.⁷⁰ The 12-LO pathway has been shown to be downstream of PKC and upstream of mitoK_ATP channel.⁵⁹.{y, 百拇医药

    Linear Versus Converging Pathways.{y, 百拇医药

    Several signaling molecules seem to be necessary for the protection afforded by PC . For example, there are data suggesting that adenosine, opioids, G{alpha} _i, PI3K, PKC, 12-LO, ERK, NOS, GSK3ß, and mitoK_ATP channels are all required for acute PC. Although the signaling pathway in acute PC is frequently presented as linear, the data are consistent with branching and converging pathways, as illustrated There seem to be redundant mechanisms to activate some key molecules, such as PKC. For example, PKC may be activated by ROS as well as by PI3K/eNOS, and different pathways may predominate under different conditions. The diverse pathways seem to converge on mitochondrial signals that integrate signals from different pathways. However, the diverse pathways could also act on additional targets, and the protection afforded by PC may require the modulation of multiple targets, such as inhibition of cell death, alterations in the cytoskeleton to prevent plasma membrane rupture, and reduction in the rate of ATP utilization. Also, PC activates signaling pathways that lead to the second window or delayed PC, and additional signaling pathways, such as inducible NOS, COX2, nuclear factor-{kappa} B, and JAK/STAT have been identified as components of the second window or delayed PC.^11,71,72 In most cases, the role of these pathways in acute PC has not been established.

    Compartmentation0@, 百拇医药

    Emerging data indicate that localization of signaling molecules is a key component in the phenotype of the signal.⁷³ It has been recognized that PKC translocates to different compartments and that the signaling phenotype is dependent on the location of receptors for C kinase.⁷⁴ Similarly, A-kinase anchoring proteins regulate and localize signaling for protein kinase A (PKA). Furthermore, it has been shown that global measurements of cAMP are not a reliable indicator of localized activation of PKA. Agonists can activate localized PKA and phosphorylate localized targets without a global increase in cAMP. Conversely, a global increase in cAMP does not always lead to PKA-dependent phosphorylation of all PKA targets in the cell.⁷⁵ Also, recent data show that angiotensin 1a receptor signaling leads to activation of functionally distinct pools of ERK.⁴⁰ Angiotensin II leads to activation of nuclear ERK via Gq/11-dependent signaling and activation of cytosolic ERK via ß-arrestin signaling. Consistent with the importance of localized signaling, Ping and coworkers^17,64 and Arrell et al⁷⁶ have shown a role for large signaling complexes in PC. For example, Baines et al¹⁷ have reported that PKC{epsilon} forms a complex with MAPK as well as a complex with voltage-dependent anion channel (VDAC), hexokinase II (HKII), and ANT1. Arrell et al⁷⁶ have used a proteomic approach to show altered posttranslational modifications associated with addition of adenosine. Taken together, these data suggest that measurements of total cell changes in a signaling pathway will not necessarily reflect localized changes in signaling pathways and that the signaling in localized compartments is likely to be important in cardioprotection.

    In summary, there are several signaling pathways that are involved in initiating cardioprotection. There seem to be redundant and multiple signaling branches that converge on common signaling pathways in the mitochondria. There may also be additional targets. Certainly the second window or delayed PC involves transcription factors that act on the nucleus to upregulated cardioprotective genes. Localized signaling is likely to be important, and these localized signals may not be reflected in a global measurement. It is also possible that there may be species variations in some of the signaling pathways or in their relative importance, and this may account for some of the discrepancies in the literature.:jqz9p, 百拇医药

    End Effectors of Cardioprotection:jqz9p, 百拇医药

    Cardioprotection is ultimately mediated by mechanisms that reduce cell death, both apoptotic and necrotic. Thus, the end effectors or secondary signaling pathways must somehow reduce cell death. Necrotic cell death is characterized by loss of plasma membrane integrity, whereas apoptotic cell death typically occurs initially with an intact plasma membrane via mitochondrial or FAS receptor–mediated activation of the caspase pathway. We will consider pathways that inhibit apoptosis as well as necrosis, because cardioprotective mechanisms seem to reduce death by both pathways. Thus, the cardioprotective signaling pathways must ultimately lead to secondary signals that maintain plasma membrane integrity and inhibit cell death. Agents that inhibit apoptosis, such as bcl-2 overexpression, also inhibit necrosis.^78,79This is not surprising, because mitochondria are the primary organelles involved in ATP production, which is important in maintaining cell integrity, and mitochondria are also key players in apoptosis. There are data suggesting a role for many mitochondrial proteins as end effectors; the mitoK_ATP channel,^3,80–82 apoptotic proteins such as BAD,⁶⁴ and the mitochondrial permeability transition pore^17,83–85 are all possible candidates as end effectors. Because of their common mitochondrial location, it is possible that the mechanisms involved in the regulation of these different mitochondrial proteins are related. As discussed, it is also possible that there are nonmitochondrial targets. For example, cytoskeletal or membrane proteins would be plausible targets, because they might help maintain cell integrity. In fact, cardioprotection may be mediated by the integration of multiple pathways rather than a final end-effector. For example, cardioprotection or preconditioning could modulate several targets, both mitochondrial and nonmitochondrial, to achieve protection. This review focuses on the mitochondrial targets.

    MitoK_ATP\%8ri], 百拇医药

    There are considerable data, which will be reviewed as part of this thematic series (O’Rourke, Evidence for Mitochondrial K⁺ Channels and Their Role in Cardioprotection), suggesting that activation of the mitoK_ATP channel is important in preconditioning and other mechanisms of cardioprotection.^3,80,81,86 Selective activators of the mitoK_ATP channel that do not open the plasma membrane K_ATP channel have been shown to be protective, and selective mitoK_ATP channel inhibitors block the protection afforded by PC.^3,80,87 Several recent publications have questioned whether the cardioprotective effects of diazoxide are mediated via the mitoK_ATP channel.^88–91Diazoxide also inhibits succinate dehydrogenase (SDH), and it has been suggested that this may initiate cardioprotection, perhaps via generation of ROS. As discussed in detail elsewhere,⁸² the observation that other K⁺ channel activators that do not inhibit SDH are cardioprotective argues against diazoxide protection being mediated by SDH inhibition.

    The mechanism by which activation of the mitoK_ATP results in cardioprotection is not well understood. Three mechanisms have been suggested by which activation of mitoK_ATP could reduce cell death. These include inhibition of mitochondrial calcium uptake, regulation of mitochondrial volume that could alter mitochondrial permeability of the VDAC or the MPT, and modulation of ROS.#, 百拇医药

    Inhibition of Mitochondrial Ca²⁺ Uptake#, 百拇医药

    Holmuhamedov et al⁹² showed that addition of K⁺ channel openers to isolated cardiac mitochondria depolarized the mitochondrial inner membrane and reduced mitochondrial Ca²⁺ influx. They additionally showed that pinacidil activated release of Ca²⁺ from the mitochondria. Murata et al⁹³ reported that diazoxide attenuated the accumulation of mitochondrial Ca²⁺ during simulated ischemia and reperfusion in rabbit ventricular myocytes, using rhod-2 to measure mitochondrial Ca²⁺. Ishida et al⁹⁴ reported that diazoxide depolarized the mitochondrial membrane and attenuated ouabain-induced calcium overload in mitochondria in rat ventricular myocytes. These studies support a role for altered mitochondrial Ca²⁺ regulation associated with activation of mitoK_ATP channels. However, in these studies, one cannot distinguish whether diazoxide reduced injury, which in turn reduced mitochondrial Ca²⁺ overload, or whether a primary action of diazoxide was to reduce Ca²⁺ overload, which in turn reduced injury.

    Generation of ROS!:ll;1h, 百拇医药

    There are data showing that addition of diazoxide results in generation of ROS.^95,96 Furthermore, inhibition of diazoxide-induced ROS by addition of antioxidants blocks the protection afforded by diazoxide, suggesting that the ROS generated by the K_ATP channel are required for protection.^95,96 An alternative interpretation to consider is that an oxidized redox state may be important for the binding of diazoxide to the mitoK_ATP channel, because the channel can be regulated by redox.^97,98 Although most investigators find that diazoxide results in an initial increase in ROS generation, there are data suggesting that diazoxide addition to mitochondria reduced generation of ROS at reperfusion and improved ADP-dependent oxygen consumption.⁹⁹ Interestingly, Ozcan et al⁹⁹ report that the effects of K⁺ channel openers on ROS production were maintained in nominally K⁺-free medium, suggesting that diazoxide is modulating reperfusion ROS by a mechanism that is independent of K⁺ channel activity. These studies were performed on isolated mitochondria, and it is possible that these isolated mitochondria are in a different state than in situ mitochondria (see the study by Dos Santos et al¹⁰⁰).

    MitoK_ATP and VDACg!5!/&, http://www.100md.com

    Dos Santos et al¹⁰⁰ have suggested that opening the mitoK_ATP channel results in slight mitochondrial swelling, which maintains mitochondrial structure and thus maintains VDAC in a low permeability state. Reduced VDAC permeability would reduce ATP entry into the mitochondria during ischemia and therefore reduce consumption of glycolytic ATP by the F1-F0 ATPase. However, there are recent data that report that the changes in mitochondrial volume do not correlate with protection.⁹¹ This is an important area in which future studies are needed. VDAC, also known as porin, is an abundant protein in the outer mitochondrial membrane that forms a channel, allowing passage of small metabolites such as ATP and ADP. In vitro, VDAC has been shown to decrease conductance when either a positive or negative potential is applied.¹⁰¹ VDAC in concert with the adenine nucleotide transporter (ANT) controls transport of adenine nucleotides into and out of the mitochondria. Mitochondrial generation of ATP occurs via the F₁F₀-ATPase, which uses the proton gradient generated by electron transport to provide the driving force for converting ADP to ATP.¹⁰² Cell energetics depend on the appropriate transport of ADP into the mitochondria and the transport of ATP to the cytosol.

    The hypothesis that activation of the mitoK_ATP channel induces protection via modulation of VDAC is interesting in light of a recent study suggesting that overexpression of bcl-2 modulates cardioprotection via inhibition of VDAC.¹⁰³ Cardiac-specific overexpression of bcl-2 has been shown to reduce myocyte death after ischemia and reperfusion.^79,104 Bcl-2 overexpression, in addition to reducing ischemia-reperfusion injury, also reduced the rate of decline in ATP during ischemia and reduced ischemic acidification; these data are consistent with bcl-2–mediated inhibition of consumption of glycolytically generated ATP.¹⁰³ During ischemia, when the lack of oxygen inhibits mitochondrial electron transport and mitochondrial generation of ATP, the F₁F₀-ATPase can run in reverse and consume glycolytically generated ATP.^105,106 The reduction in acidification and the rate of decline in ATP during ischemia was dependent on the activity of the mitochondrial F₁F₀-ATPase in wild-type hearts but not in bcl-2–overexpressing hearts,¹⁰³ suggesting that the bcl-2–induced reduction in the ischemic decline in ATP and pH was mediated by consumption of ATP via the F₁F₀-ATPase. Bcl-2–mediated inhibition of consumption of glycolytically generated ATP could be accomplished by blocking ATP entry into the mitochondria by inhibition of VDAC or ANT or by direct inhibition of the F₁F₀-ATPase. Consistent with a role for VDAC, there was an increased association of VDAC and bcl-2 in hearts overexpressing bcl-2.¹⁰³ It is interesting that previous studies have ruled out a role for altered F₁F₀-ATPase activity in the protection afforded by PC.¹⁰⁷

    Emerging data suggest that proapoptotic family members such as BAX enter the outer mitochondrial membrane and form a large conductance channel that allows release of cytochrome c.^108–110 The precise mechanism by which BAX mediates release of cytochrome c is not well understood but has been suggested to involve channels formed by BAX or by BAX in association with other mitochondrial proteins, such as VDAC, or MPT, which is suggested to be composed of the ANT, VDAC, and cyclophilin. It has been suggested that antiapoptotic family members such as bcl-2 oppose release of cytochrome c either by binding and sequestering proapoptotic members¹⁰⁸ or by binding to proteins such as VDAC and blocking the formation or opening of a cytochrome c release pathway.^109,110 Bcl-2 is localized to the outer mitochondrial membrane, and bcl-2 family proteins have been reported to interact with VDAC.¹⁰⁹${5, http://www.100md.com

    Similar to the effects of bcl-2 overexpression, inhibition or reduction of VDAC activity might explain many of the changes associated with preconditioning, such as the reduced rate of decline in ATP, reduced acidification, and reduced cell death. Diazoxide addition has also been reported to reduce acidification during ischemia,⁹⁵ and perhaps this effect is mediated via K_ATP-dependent closure of VDAC, consistent with the suggestion of Dos Santos et al.¹⁰⁰ Dos Santos et al attributed the diazoxide-induced decrease in permeability of the mitochondria to ADP to the confirmation of VDAC conferred by diazoxide-induced mitochondrial swelling. However, in conflict with this hypothesis, Lim et al⁹¹ did not find diazoxide-induced mitochondrial swelling.

    HKII has also been reported to bind to and reduce permeability of VDAC.^17,111 Elevated glucose-6-phosphate (G-6-P) occurs during ischemia, which would reverse HKII closure of VDAC, and MPT. Interestingly, both PC and diazoxide have been reported to decrease G-6-P levels during ischemia,⁹⁵ providing another mechanism by which PC and diazoxide might reduce permeability of VDAC. Future studies will be needed to prove or disprove this speculation. Diazoxide has also been reported to reduce apoptosis,⁸⁶ and this could be mediated by closing of VDAC as part of the MPT. Alteration of VDAC by mitoK_ATP or bcl-2 family members (eg, ERK phosphorylation of BAD) could conserve ATP levels during ischemia, preserve mitochondrial function, and inhibit cell death on reperfusion, adding up to cardioprotection. VDAC has also recently been reported to modulate mitochondrial Ca²⁺ uptake. Thus, decreased VDAC activity might explain decreased mitochondrial Ca²⁺ uptake observed with diazoxide.^92,93 It is also interesting that VDAC is related to a bacterial channel that closes under conditions of cell stress. Thus, the closing of VDAC and other mitochondrial modifications may be evolutionarily conserved from bacteria.

    Phosphorylation of BAD or Modification of MPT\:/.@)?, http://www.100md.com

    BAD is a proapoptotic protein that binds and sequesters bcl-2, such that bcl-2 can no longer inhibit apoptosis. Phosphorylation of BAD causes it to be bound to 14-3-3, thereby freeing bcl-2 to inhibit apoptosis. Baines et al⁶⁴ have reported that mice overexpressing PKC{epsilon} contain a mitochondrial localized signaling complex containing PKC{epsilon} and MAPK; this signaling complex was associated with increased mitochondrial phosphorylation of BAD. Baines et al¹⁷ have also reported that PKC{epsilon} forms a complex with components of the MPT, including VDAC, ANT, and HKII. Modulation of the MPT by phosphorylation or complex formation is another potential end effector (see the study by Weiss et al⁸⁵). However, whether the association of these protein regulates pore opening requires additional study.\:/.@)?, http://www.100md.com

    Using isolated mitochondria, Korge et al¹¹² have reported that diazoxide prevents opening of the MPT induced by Ca²⁺ addition. They additionally reported that PKC activation mimicked the effect of diazoxide on the MPT and was blocked by 5HD. Furthermore, VDAC is a component of the MPT; therefore, if mitoK_ATP channel opening leads to inhibition of VDAC, this could promote inhibition of the MPT and might provide a mechanism by which diazoxide inhibits cell death.

    Nonmitochondrial Targets1/?*c, 百拇医药

    As discussed, there are strong data supporting several mitochondrial targets as the end effectors of PC. It is possible that these mitochondrial targets mediate all the effects of cardioprotection, because mitochondria have a key role in regulating cell death and ATP generation, key aspects of cardioprotection. It is also possible that the same signaling kinases that modify mitochondrial proteins also modify nonmitochondrial proteins and modulate other aspects of cell metabolism. Notably, PC has been reported to alter connexins^113,114 and Ca²⁺ handling by sarcoplasmic reticulum.^115,116 PC has been abolished by cytochalasin D,¹¹⁷ suggesting a role for actin cytoskeleton; however, neither vinculin¹¹⁸ nor {alpha} B crystallin¹¹⁹ seem to be involved in PC. Furthermore, PC is known to result in acute and delayed cardioprotection, and the delayed cardioprotection involves upregulation of gene function; this likely occurs via PC-induced signaling pathways that do not directly involve the mitochondria. By analogy with hormone agonist signaling, it maybe naive to assume that there is only one end effector. In fact, it is likely that PC has pleiotropic effects that are mediated via different end effectors. However, activation of the mitoK_ATP channel with diazoxide has been shown to be as protective as PC, suggesting that the mitoK_ATP channel may be the primary end effector. Alternatively, the mitoK_ATP channel could function as a trigger as well as an end effector, consistent with data showing that opening mitoK_ATP generates ROS, which can trigger protective signaling pathways, thereby explaining why mitoK_ATP channel openers induce full protection.

    How Do Primary Signals Lead to Activation of Secondary Signals or the End Effector?}(8, 百拇医药

    In acute cardioprotection, the mechanisms by which activation of the trigger/mediator signaling pathways lead to activation of the end effectors, such as the mitoK_ATP channel or other mitochondrial proteins, must involve posttranslational modification. Because many of the primary signaling pathways involve kinases, it is likely that phosphorylation plays a role. NOS is also activated by PC; therefore, nitrosylation or nitration of proteins could also be involved. In fact, both PKC and NO have been reported to regulate the activity of K_ATP channels. Redox modulation of thiol groups could also be involved. Alterations in macromolecular complexes are also likely to modulate the function of mitochondrial proteins. Baines et al¹⁷ have reported that PKC{epsilon} interacts with VDAC, ANT, and HKII. They additionally showed that overexpression of PKC{epsilon} increased the formation of this complex, and they presented in vitro data showing that PKC can phosphorylate VDAC. These macromolecular complexes may modulate function directly or may modulate function via phosphorylation or other posttranslational modifications. VDAC activity has been shown to be regulated by binding to ANT, HKII, and PKB.¹²⁰

    In summary, cardioprotective mechanisms such as PC activate several signaling pathways that seem to converge on a few mitochondrial targets, leading to altered cell metabolism and inhibition of apoptosis. Future studies will determine whether there are other end targets in addition to the mitochondria.f, 百拇医药

    Acknowledgmentsf, 百拇医药

    Dr Murphy is supported by a National Institute of Environmental Health Sciences intramural program.f, 百拇医药

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