"Introduction Table of ContentsIntroduction............................................................................................................................................. 3Review of Literature ................................................................................................................................ 8CONCEPT OF PRECONDITIONING ................................................................................................. 12MOLECULAR MECHANISM OF ISCHEMIC PRECONDITIONING ............................................. 14CLINICAL ASPECTS OF ISCHEMIC PRECONDITIONING .......................................................... 18DIABETES MELLITUS AND CARDIOPROTECTIVE EFFECT OF ............................................... 20ISCHEMIC PRECONDITIONING ...................................................................................................... 20BIOLOGY OF CAVEOLAE ................................................................................................................ 24ROLE OF CAVEOLIN IN ISCHEMIC PRECONDITIONING .......................................................... 26HEME OXYGENASE .......................................................................................................................... 28REGULATION OF ACTIVITY ........................................................................................................... 28HEME OXYGENASE-1 IN MYOCARDIAL ISCHEMIA-REPERFUSION INJURY ...................... 30HEME OXYGENASE-1 IN DIABETES ............................................................................................. 31EXPERIMENTAL DIABETES MELLIUS ......................................................................................... 33ESTIMATION OF SERUM GLUCOSE .............................................................................................. 33ISOLATED PERFUSED RAT HEART ............................................................................................... 34ISCHEMIC PRECONDITIONING ...................................................................................................... 34ASSESSMENT OF MYOCARDIAL INJURY .................................................................................... 351. ASSESSMENT OF MYOCARDIAL INFARCT SIZE ............................................................... 352. ESTIMATION OF LACTATE DEHYDROGENASE (LDH) RELEASE LDH ..................... 363. ESTIMATION OF CREATINE KINASE-MB (CK-MB) RELEASE ......................................... 36NITRITE ESTIMATION ...................................................................................................................... 37EXPERIMENTAL PROTOCOL .......................................................................................................... 38REAGENTS AND CHEMICALS ........................................................................................................ 42STATISTICAL ANALYSIS ................................................................................................................ 42Results ................................................................................................................................................... 45Discussion ............................................................................................................................................. 49Summary and Conclusion ..................................................................................................................... 55Bibliography ......................................................................................................................................... 58 IntroductionCoronary artery disease is a leading cause of morbidity and mortality worldwide(Murray and Lopez, 1997; Yusuf et al., 2001). Inadequate blood flow to themyocardium leads to ischemia (Michael, 2006) and therefore, early reperfusion isnecessary for the viability of myocardium (Napoli et al., 2002). However, reperfusionafter a period of prolonged ischemia is not without risk, it produces damage ofmyocardium and is known as Ischemia-Reperfusion (I/R)-injury (Collard andGelman, 2001).The cardioprotective strategy against Ischemia-Reperfusion injury was given byMurry in 1986 and is known as ischemic preconditioning (IPC) i.e. a powerfulendogenous protective phenomenon in which short intermittent cycles of sublethalischemia followed by reperfusion before the subsequent prolonged ischemic insult,improves the tolerance against ischemia-reperfusion-induced injury (Murry et al.,1986; Tomai et al., 1999a). Ischemic preconditioning mediated cardioprotection hasbeen documented in various species including human beings (Cohen et al., 1991;Tomai et al., 1999a). Ischemic preconditioning is a biphasic phenomenon, associatedwith two forms of myocardial protection i.e., early phase is short-lived and wanes offgradually within 2-3 hours (Downey and Cohen, 1997). The other one is delayedphase, it‘s effect is more prolonged, becomes apparent after 12-24 hours of IPCstimulus and lasts for 3-4 days (Gross, 1997). Further, administration of variouspharmacological agents such as bradykinin (Li et al., 1990), adenosine (Liu et al.,1991), potassium channel openers (Garlid et al., 1997), sildenafil (Kukreja et al.,2005) mimic preconditioning like cardioprotection which is termed aspharmacological preconditioning.Ischemic Preconditioning stimulates the generation of endogenous ligandswhich bind to their respective G-protein coupled receptors (Baines et al., 1999;1 IntroductionMurphy, 2004) and initiate a signalling cascade i.e., activation of PI3K (Downey etal., 2007) and phospholipase C (Tyagi and Tayal, 2002; Ardehali, 2006).Administration of pharmacological agents such as insulin, erythropoietin also activatePI3K (Lawlor and Alessi, 2001) and subsequently activate the protein kinase B (Akt)(Stokoe et al., 1997; Hausenloy et al., 2004a; Garg et al., 2010). Activation of Aktphosphorylate inhibits a number of substrates including proapoptotic members of theBcl-2 family, caspases-9, and enhance the generation of nitric oxide (NO)(Ferdinandy et al., 2007; Murphy and Steenbergen, 2008). The nitric oxide (NO) leadsto activation of protein kinase G (PKG) via elevation of intracellular cGMP level(Gross et al., 2004; Costa et al., 2005). Moreover, PKG phosphorylates andinactivates glycogen synthase kinase-3ß (GSK-3ß) and consequently inhibit theopening of mPTP during reperfusion phase and mediate the cardioprotection againstischemia reperfusion-induced injury (Tong et al., 2002; Gross et al., 2004; Juhaszovaet al., 2004; Hausenloy et al., 2004b; Yadav et al.,2010). Akt is also known tophosphorylate and activate ubiquitin ligase murine double minute 2 (mdm2) whichbinds with proapoptotic p53 (Mocanu and Yellon, 2003) and mediate thecardioprotective effect of ischemic preconditioning (Fayard et al., 2005).Diabetes mellitus is a heterogenous pathological condition that is characterisedby the abnormalities in carbohydrate, lipid and protein metabolism which ultimatelyleads to several acute and chronic complications (Mahgoub and Abd-Elfattah, 1998).Ischemic heart disease is occurring at an alarming rate in diabetic patients(Lopaschuk, 2001). The cardioprotective effect of ischemic preconditioning isattenuated in diabetic myocardium and it may be due to hyperglycaemia (Kersten etal., 2000), impairment of K channel (del Valle et al., 2002), impairment of PI- ATP 3K/AKT pathway (Tsang et al., 2005; Wynne et al., 2007) and altered activation of2 IntroductionJAK/STAT and MAPK, GSK-3ß (Gross et al., 2007; Yadav et al., 2010). Hence, thecardioprotective effect of IPC in diabetic myocardium remains controversial.Caveolae are the specialised membrane domains, triton insoluble, cholesteroland sphingolipids enriched proteins (Williams and Lisanti, 2004) which serve asorganizing centres for cellular signal transduction (Shaul and Anderson, 1998; Patel etal., 2007). The caveolin gene family consists of three members which differs in theirpattern of expression in different cell types. Caveolin-1and caveolin-2 is co-expressedin many cell types including adipocytes, endothelial cells, epithelial cells andfibroblasts (Scherer et al., 1994, Scherer et al., 1997) whereas Caveolin-3 is restrictedto skeletal, smooth muscles and cardiac myocytes (Scherer et al., 1994; Minetti et al.,1998; Galbiati et al., 1999). Various signalling molecules have been shown to localizewithin caveolae. These include src family, tyrosine kinase, GPCR, members of Ras- MAPK cascade and nitric oxide synthase (Ostrom and Insel, 2004; Insel et al., 2005).These signalling molecules may be involved in the cardioprotective effect of IPCthrough interaction with scaffolding domain of caveolin (Krajewska and Maslowska,2004). Further, Caveolin is a well known negative regulator of endothelial nitric oxidesynthase and hence decreased availability of NO (Feron et al., 1996; Feron andBalligand, 2006). In addition, NO is responsible for cardioprotective effect ofischemic preconditioning (Prendes et al., 2007; Sun and Murphy, 2010).It has been reported that expression of caveolin is upregulated in diabeticmyocardium (Bucci et al., 2004, Penumathsa et al., 2008a) and this results in increasebinding of caveolin with eNOS. Further, inhibition of expression of caveolin bypretreatment with daidzein significantly restored the cardioprotective effect ofischemic preconditioning in diabetic rat heart (Ajmani, 2009).3 IntroductionHeme-oxygenase is the rate-limiting enzyme in the biochemical pathway++ responsible for catabolism of heme into ferrous (Fe ) iron, carbon monoxide, andbiliverdin, the latter being subsequently converted into bilirubin by biliverdinreductase (Ryter et al., 2006). HO-1 is localized in the membrane caveolae and theinner leaflet of the plasma membrane where it is interacts with caveolin (Kim et al.2004). It has been reported that, in the transgenic mice, the overexpression of Heme- oxygenase-1 shows decreased expression of caveolin (Penumathsa et al., 2008b). Ithas been reported that a decrease in the cardiospecific expression of HO-1 exacerbatesthe ischemia reperfusion-induced injury (Liu et al., 2005), while upregulation of HO-1produces cardioprotection against ischemia-reperfusion induced injury(Thirunavukkarasu et al., 2007; Penumathsa et al., 2008b). Transgenic miceexpressing cardiac-specific HO-1 are resistant (Yet et al., 2001), while the heart ofHO-1 knock-out mice is more susceptible to ischemia-reperfusion-induced injury(Yoshida et al., 2001). The increase in HO-1 in diabetic rats is associated withactivated eNOS (Li et al., 2007a, b; Peterson et al., 2007). Moreover, it has beendocumented that HO-1 facilitates release of NO by disrupting association of caveolinwith eNOS (Penumathsa et al., 2008b,c). However the expression and activity of HO- 1 is reduced in diabetes mellitus (Csonka et al., 1999).The present study was designed to investigate the role of Heme- Oxygenase-1 inattenuation of cardioprotection induced by IPC in Diabetic rat hearts.4 ReviewofLiterature Review of LiteratureCoronary artery disease or ischemic heart disease is associated with the stenosisof the coronary artery along with the arteriosclerosis. The coronary lumen becomesnarrower which lead to reduced or ceasing of blood supply to the heart that is the maincause of myocardial infarction. Coronary artery disease is the leading cause of deathmostly in industrialized countries. The likelihood of the incidence of coronary arterydisease is greater in men than in women and it increases with age. The risk factorsinclude family history, lack of exercise, obesity, diabetes, smoking, high bloodpressure and mental stress. Treatment can be carried out through drugs, percutaneoustransluminal coronary angioplasty and coronary artery bypass-graft and each of themcould be treated according to its severity and health conditions of the patients(Kownaklai, 2009).ISCHEMIA REPERFUSION INJURYIschemic heart disease is a major cause of death worldwide (Murray and Lopez,1997; Yusuf et al., 2001). Myocardial ischemia occurs due to inadequate blood flowto the heart (Michael, 2006). Myocardial reperfusion is restoration of the blood flowto the ischemic heart (Collard and Gelman, 2001). Early reperfusion minimizes theextent of damage and preserves the pumping function of the heart (Napoli et al.,2002). However, reperfusion after a prolonged period of ischemia damages themyocardium rather than restoration of normal cardiac function and it is known asischemia reperfusion injury (Buckberg, 1981; Kloner, 1993). Ischemia reperfusioninjury has been implicated in the pathogenesis of various disorders i.e. angina pectoris(Verma et al., 2002), myocardial infarction (Mc Donough et al., 1999), stroke (Oliveret al., 1990) and peripheral vascular insufficiency (Muller et al., 2002). Myocardialpreconditioning has been shown to protect the heart against ischemia reperfusioninjury (Murry et al., 1986).5 Review of LiteratureIschemia reperfusion injury (Fig. 1) has been recognized as a highly complexcascade of events (Linfert et al., 2009). During ischemia, process of oxidativephosphorylation of myocytes is impaired that decreases the tissue levels of ATP andsubsequently increase the concentration of ADP, AMP and phosphate (Solaini and Harris,2005, Powers et al., 2007). These changes activate the process of anaerobic respirationi.e. glycolysis and produces lactic acid resulting in decrease of intracellular pH and+ + + activation of Na /H antiporter (Buja, 2005). The Na that enters via this route would+ + normally be pumped out again by the Na /K ATPase but the greatly reduced [ATP]+ inhibits this efflux leading to a progressive rise in intracellular [Na ] and subsequentincrease in intracellular concentration of calcium ion (Abdallah and Schaffer, 2004). Thisin turn causes impaired ionic homeostasis, the decrease in [ATP] leads to a large increasein [AMP] through the action of adenylate kinase, and some of this AMP is converted intoadenosine and then inosine and xanthine through a purine degradation pathway (Szocs,2004). Xanthine may be further oxidised by xanthine oxidase which produces superoxidethat is further metabolised to other damaging reactive oxygen species (ROS), particularlyhydrogen peroxide and hydroxyl radicals (Vasquez-Vivar et al., 2000; Zweier andTalukder, 2006). There are additional potential sources of ROS which include therespiratory chain, nitric oxide synthase and NADPH oxidase (Becker, 2004). Since2+ increased [Ca ] triggers opening of the mPTP (mitochondrial permeability transitionpore) under conditions of oxidative stress. mPTP are multiprotein complexes that formnon-selective pores in the inner mitochondrial membrane due to certain factors like2+ acidosis, Ca overload and production of ROS which increase the risk of mPTP opening(Powers et al., 2007, Baines, 2009). Opening of mPTP cause a depolarisation of innermitochondrial membrane (? ? ) and transform mitochondria from ATP producers to ATPm consumers6 Review of Literaturevia reversal of F F -ATPase, which is used to generate mitochondrial membrane1 0 potential (? ?) (Honda et al., 2005). To maintain this ? during ischemia it willm 2+ uptake Ca . Furthermore, on reperfusion, oxygen is returned to the cell resulting ingeneration of ROS, regeneration of ? with uptake of potentially large amounts of2+ Ca into the mitochondria and restoration of normal pH results in activation of theMPTP, which in turn totally depletes the ATP of the cell (Murphy and Steenbergen,2+ 2008). Nevertheless, the depletion of ATP and elevated [Ca ] that occurs duringischemia and reperfusion will lead to a gradual decline in cellular integrity asdegradative enzymes such phospholipases (PLA2) (Ford, 2002) and calcium-activatedproteases (calpains) (Chen et al., 2002) are activated at the same time as ATP- dependent repair processes are inhibited by the lack of ATP. There may also be someBAX translocation to the mitochondria leading to permeabilisation of the outermembrane and cytochrome C release (Capano and Crompton, 2006) which couldinitiate apoptosis (Halestrap et al., 2004). In contrast, activation of these proteolyticpathways coupled with the total loss of ATP and ROS mediated damage synergise,leading to rapid loss of cellular integrity (Murphy and Steenbergen, 2008).Opening of mPTP leads to the release of cytochrome C into the cytoplasmwhich initiates the process of apoptosis by activating caspase 9 (Cardone et al., 1998)and subsequently caspase 3 (Zou et al., 1997; Weiland et al., 2000). Apoptosis is anenergy-dependent process that results in chromatin condensation, DNA fragmentationand apoptotic body formation, preserved cell membrane integrity, without anassociated inflammatory response (Fiers et al., 1999). Mitochondria integrate diversepro- apoptotic signals like Bax, Bid, PUMA, and NOXA and anti-apoptotic signalslike BclXl, Mcl-1, A1, Bcl-W, and CED-9 signals (Desagher and Martinou, 2000).The BH3-only proteins respond to a variety of cellular stress signals including8 Review of Literatureacidosis, elevated calcium levels, hypoxia and DNA damage (Zong et al., 2001;Gustaffson et al., 2004). The effect of BH3 is to disrupt the sequestration of Bax byBcl-2 or Bcl-XL which liberates Bax and triggering the further release of cytochromeC (Gustaffson et al., 2004). There are several kinases such as JNK (Aoki et al., 2002)and p38MAPK (Irving and Bamford, 2002), STAT1 (Stephanou et al, 2000; Barry etal., 2006) and Rho kinase (Hu et al., 2004) which are involved in the process ofapoptosis during ischemia and reperfusion injury (Hausenloy and Yellon, 2004).The mediators such as calcium and reactive oxygen species (ROS) which areinvolved in apoptosis have been further reported to contribute to necrosis (Zong andThompson, 2006). Necrosis is characterised by membrane disruption, massive cellswelling, cell lysis and fragmentation, with an associated acute inflammatoryresponse (Hausenloy and Yellon, 2004). Necrosis results in rapid loss of plasmamembrane integrity due to increased oxidative stress, cytosolic calcium level anddecrease level of ATP (Ermak and Davies, 2002; Bartosz, 2009). Moreover, increasedROS level facilitate the release of proteolytic enzymes ie. calpains (Cao et al., 2003;Liu et al., 2004b) and cathespins (Kagedal et al., 2005). In addition, increasedoxidative stress overactivates the expression of poly ADP-ribose polymerase (PARP)which further causes necrosis (Beneke, 2008). Further, diminished ATP productionleads to perturbation of intracellular ion homeostasis subsequently mitochondrialdysfunction which further compromises the cellular energetics (Halestrap et al., 2004;Halestrap, 2006; Leung and Halestrap, 2008). Necrostatins are the powerful toolsagainst necrotic cell death block tumour necrosis factor-induced necrosis through theinhibition of RIP1 (receptor interacting protein kinase1) activity (Vandenabeele et al,2008).9 Review of LiteratureIschemia reperfusion injury has been well demonstrated to cause organ damagein the brain, heart, lungs, liver, kidneys and skeletal muscle (Novgorodov and Gudz,2009). A number of therapeutic strategies such as controlled reperfusion,preconditioning, postconditioning and several pharmacological interventions forexample adenosine (Lozza et al., 1997; Moukarbel et al., 2004;), renin-angiotensinsystem antagonist (Paz et al., 1998), calcium antagonists (Segawa, 2000), antioxidants(Marczin et al., 2003), sodium-hydrogen exchange inhibitors (Hennan et al., 2006),iron chelators (Tang et al., 2008), N-methylated synthetic sphingolipid analogue(Gundewar and Lefer, 2008), flavanoids (Akhlaghi and bandy, 2009) and exenatide(Timmers et al., 2009) have been shown to reduce ischemia reperfusion-inducedmyocardial injury.CONCEPT OF PRECONDITIONINGIschemic preconditioning (IPC) is a powerful endogenous protectivephenomenon in which short intermittent cycles of sublethal ischemia followed byreperfusion before the subsequent prolonged ischemic insult, improve the toleranceagainst ischemia reperfusion induced injury. The cardioprotective phenomenon ofischemic preconditioning was first demonstrated by Murry and his colleagues in 1986in dog heart (Murry et al., 1986) and later on documented in other species includingpigs (Schott et al., 1990), rabbit (Cohen et al., 1991), rat (Steenbergen et al., 1993),sheep (Burns et al., 1995), chicken (Liang and Gross, 1999), mouse (Mocanu et al.,2000) as well as in human beings (Tomai et al., 1999a; Fernandes et al., 2001;Granefeldt et al., 2009). In addition, cardioprotective effect of IPC is also observed inisolated cardiomyocytes (Ikonomidis et al., 1997). Moreover , IPC mediatedprotection has been well reported in various organs such as heart (Murry et al., 1986;Lochner et al., 2009), lung (Ying et al., 1996; Turan et al., 2008), brain (Barone et al.,10 Review of Literature1998; Sharp et al., 2004), skeletal muscles (Hopper et al., 2000; Sayan et al., 2009),liver (Koti et al., 2003 ), kidney (Salehipour et al., 2007), intestine (Santos et al.,2008) and neurons (Lee et al., 2009).Ischemic preconditioning is a biphasic phenomenon, associated with two formsof myocardial protection i.e., early effect of preconditioning is a powerful protectivephenomenon but is short lived and wanes off gradually within 2-3 hours. It is alsocalled as classical preconditioning (Downey and Cohen, 1997; Yellon and Downey,2003). The other one is second window of protection (SWOP) although not aspowerful as early phase but its effect is more prolonged, becomes apparent after 12- 24 hours of IPC stimulus and lasts for 3-4 days. It is called as late effect ofpreconditioning (Gross, 1997; Yellon and Downey, 2003). Early preconditioningprotects the myocardium against infarction only while late preconditioning alsodemonstrates anti-stunning effect of heart (Ferdinandy et al., 2007, Sisikian, 2008).Moreover, administration of various pharmacological agents such as bradykinin (Li etal., 1990), adenosine (Liu et al., 1991), angiotensin (Liu et al., 1995), nor-epinephrine(Cohen et al., 1997), potassium channel openers (Garlid et al., 1997), isoflurane(Belhomme et al., 1999), morphine (Liang and Gross, 1999), , naloxone (Tomai et al.,1999b), estrogen (Lee et al., 2002), nitroglycerin (Du et al., 2004), sildenafil (Kukrejaet al., 2005) mimic preconditioning like cardioprotection which is termed aspharmacological preconditioning. Moreover, brief episode of ischemia followed byreperfusion to one organ (remote organ) produces protection against ischemiareperfusion induced injury on heart and it is known as remote preconditioning(Pryzklenk et al., 1993). Remote preconditioning is also known as preconditioning indistant organ or inter-organ preconditioning (Riksen et al., 2004). Further, briefocclusion of anterior mesenteric artery provides protection to heart against infarction11 Review of Literatureis known as mesenteric preconditioning (Gho et al., 1996; Santos et al., 2008). Briefocclusion of renal artery provides protection to heart against infarction is known asrenal preconditioning (Takaoka et al., 1999; Diwan et al., 2008). Similarly, briefepisodes of aortic occlusion provides protection against infarction to heart is known asaortic remote preconditioning (Weinbrenner et al., 2002, Weinbrenner et al., 2004;Khanna et al., 2008). Brief episodes of occlusion and reperfusion of left circumflexartery salvage the myocardium region supplied by left anterior descending coronaryartery from subsequent prolonged ischemia. This phenomenon is termed asintracardiac preconditioning (Pryzklenk et al., 2003; Petrishchev et al., 2007).Paradoxically, the transfer of coronary effluent from preconditioned heart to non- preconditioned heart limits infarct size in the latter heart against ischemia reperfusioninjury. This phenomenon is termed as intercardiac preconditioning (Pryzklenk et al.,2003). Remote preconditioning has also been reported to occur in human beings(Kloner and Jennings, 2001; Walsh et al., 2007).MOLECULAR MECHANISM OF ISCHEMIC PRECONDITIONINGThe mechanisms underlying the early and late ischemic preconditioning havenot been completely understood. The signal transduction mechanism involved in theearly phase of IPC (Fig 2.) triggers the activation of various endogenous ligands(Baines et al., 1999; Murphy, 2004) such as acetylcholine (Yao and Gross, 1993;Kreig et al., 2004), nor-epinephrine (Banerjee et al., 1993; Seyfarth et al., 1996),adenosine (Liu et al., 1994; Cohen and Downey, 2007), bradykinin (Goto et al., 1995;Cohen et al., 2007), angiotensin (Liu et al., 1995; Sharma and Singh, 1999), opioids(Schultz et al., 1995; Peart et al., 2008), endothelin (Wang et al., 1996; Duda et al.,2007) which bind to their respective G-protein coupled receptors and initiate asignalling cascade which involves the activation of PI3K (Mocanu et al., 2002) and12 Review of Literaturephospholipase C (Tyagi and Tayal, 2002). Administration of pharmacological agentssuch as insulin, erythropoietin also shown to activate PI3K (Lawlor and Alessi, 2001)which generate phosphatidyl-inositol 3, 4,5 triphosphate (PIP ) from cell membrane3 lipid phosphatidyl inositol 3,4-biphosphate (PIP ) leading to activation of2 phosphoinositide-dependent kinase (PDK1) and subsequent activation of proteinkinaseB (Akt) (Stokoe et al., 1997; Hausenloy et al., 2004a). The activated Aktphosphorylate a number of substrates including proapoptotic members of the Bcl-2family, caspases-9, glycogen synthase kinase (GSK-3ß) and endothelial nitric oxidesynthase (eNOS) (Ferdinandy et al., 2007; Murphy and Steenbergen, 2008). The nitricoxide (NO) generated from eNOS (Muscari et al., 2004; Prendes et al., 2007) leads toactivation of protein kinase G (PKG) via elevation of intracellular cGMP level (Grosset al., 2004; Costa et al., 2005). This PKG phosphorylate and inactivates glycogensynthase kinase which increase the threshold of mPTP opening in cardiomyocytes,thereby, augmenting the cardioprotective effect of ischemic preconditioning (Tong etal., 2002; Gross et al., 2004; Hausenloy et al., 2004b; Davidson et al., 2006). Akt isalso known to activate p70S6K (Jonassen et al., 2004). In addition, PI3K stimulate theactivation of mTOR which further activates p70S6K and provide cardioprotectionthrough opening of mitochondrial potassium ATP channel (Murphy and Steenbergen,2008). Moreover, PI3K also activates PKC and produces cardioprotection (Tong etal., 2000).The activated phospholipase C through receptor dependent mechanism catalyzesthe hydrolysis of PIP into inositol triphosphate (IP ) and diacylglycerol (DAG) (Tyagi2 3 and Tayal, 2002). The DAG translocates PKC from cytosol to perinuclear membraneleading to its activation (Mitchell et al., 1995; Tong et al., 2004). PKC also appears to beactivated by ROS generation during preconditioning (Baines, 1997;14 "