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Ischaemic conditioning comprises mechanical administration of sublethal ischaemia either directly to the organ of interest or to a distant organ, in the case of remote ischaemic conditioning. Significant cardioprotection is elicited when ischaemic conditioning is applied either prior (pre-), during (per-) or after (post-) the main ischaemic insult. Extensive research has suggested that the molecular pathways elicited by these conditioning protocols are similar (reviewed by Hausenloy and Yellon, 2007); as such, pharmacological modulation of these pathways may provide a clinically applicable therapeutic intervention for cardioprotection. The discussion of ischaemic conditioning here will focus on IPC since this is the most widely studied and easily reproducible conditioning protocol to date.

i

Classical ischaemic preconditioning

Ischaemic preconditioning consists of a short sublethal period of myocardial ischaemia (usually 3 to 5 minutes) followed by reperfusion (usually 5 minutes) immediately prior to the main ischaemic insult. Cardioprotection by IPC was first demonstrated by Murry et al. in the in vivo dog heart (Murry et al., 1986) and has since been shown to be efficacious in every species tested (Jennings, 2011). Indeed, IPC is recognised as the most potent cardioprotective intervention after early myocardial reperfusion (Yellon and Downey, 2003). The molecular pathways initiated by IPC are described below.

ii

Molecular signalling of ischaemic conditioning

The signalling pathways elicited by ischaemic pre- and postconditioning have been largely elucidated and demonstrate striking convergence, although the precise details of the specific interactions between these components remains the subject of extensive research (reviewed by Hausenloy and Yellon, 2007). Understanding the molecular basis of ischaemic conditioning may allow cardioprotection by pharmacological modulation.

Direct effects: Ischaemic conditioning stimulates release of extracellular triggers, such

as adenosine and bradykinin, which ultimately elicit cardioprotection. The primary action of these signalling autocoids is mediated via binding to G-protein coupled receptors (GPCRs) which initiate intracellular signal transduction. The multiple signalling autocoids and GPCR binding interactions provide functional redundancy to the conditioning stimulus. GPCR binding activates the intracellular reperfusion injury salvage kinase (RISK) signalling cascades: phosphoinositide 3-kinase (PI3K), Akt and Erk1/2. RISK pathway activation mediates intracellular signal transduction to the end-effector of ischaemic conditioning, believed to be the mPTP (Hausenloy et al., 2004b) (

Figure 1.8).

Figure 1.8: Ischaemic conditioning

Ischaemic conditioning produces autocoids which bind to GPCRs which activate intracellular signalling cascades. Activation of the RISK pathway relays the conditioning signal to mitochondrial targets which converge to prevent mPTP opening and thus reduce cell death. Constructed from information provided by Hausenloy and Yellon (2007).

EXTRACELLULAR TRIGGERS

E.g. Adenosine, Bradykinin, Noradrenaline, Opioids

GPCR binding

Ras Tyrosine kinase

Mek1/2 Erk1/2 PI3K Akt eNOS PKCε MitoKATP Signalling ROS mPTP EXTRACELLULAR INTRACELLULAR MITOCHONDRIAL GSK3β PKG NO

The precise molecular pathways triggered by activation of the RISK pathway may vary between conditioning protocols and species, however, a number of common downstream effectors of ischaemic conditioning have been identified.

PI3K-Akt-eNOS-PKG: Phosphorylation of Akt activates endothelial nitric oxide synthase (eNOS) resulting in increased nitric oxide, which activates cytosolic protein kinase G (PKG). This nitric oxide may also directly inhibit mPTP opening (Hausenloy et al., 2009). MitoKATP: PKG in turn phosphorylates mitochondrial PKC-epsilon (PKCε) which has been linked to opening of the mitochondrial membrane ATP sensitive potassium channel (mitoKATP) (Costa et al., 2005). MitoKATP channel opening upon reperfusion is controversial, it has however, been linked with production of signalling ROS and prevention of cell death by inhibition of mPTP opening (Hausenloy et al., 2007).

GSK3β: Akt and Erk1/2 activation cause inactivation of glycogen synthase kinase-3β (GSK3β) by phosphorylation. Juhaszova et al. (2004) implicated GSK3β as a point of convergence of cardioprotection on the mPTP. Recent evidence shows active GSK3β phosphorylates cyclophilin-D (Cyp-D) to enhance mPTP opening; thus inactivation of GSK3β reduces Cyp-D activity and mPTP opening (Rasola et al., 2010); section 1.4.1.

Indirect effects: Ischaemic conditioning may also indirectly inhibit mPTP opening by

modulating mitochondrial conditions to favour mPTP closure:

Reduced calcium overloading: The precise mechanisms of reduced calcium overloading upon ischaemic conditioning have not been confirmed although there is evidence that it is linked to reduced acidosis which in turn reduces calcium influx through sodium- calcium exchange (see Figure 1.2) (Steenbergen et al., 1993). Ischaemic conditioning may also mediate increased calcium uptake by the sarcoplasmic reticulum to reduce intracellular and mitochondrial calcium overloading (Murphy and Steenbergen, 2011). Reduced mitochondrial calcium levels reduce mPTP opening.

Reduced detrimental ROS: Ischaemic conditioning reduces ROS production upon reperfusion, although the mechanism of this is not clear. Since oxidative stress is an important activator of mPTP opening, the effect of ischaemic conditioning on reducing ROS may in turn reduce mPTP opening (Murphy and Steenbergen, 2011). The roles of ROS in the setting of mPTP opening are complicated by the divergence of ‘signalling’ and ‘detrimental’ ROS, since it is well established that IPC-mediated ROS release is an essential signal transduction mechanism of the RISK pathway. These divergent effects of ROS may result from differences in the localisation, quantity, identity or timing of ROS release (Hausenloy and Yellon, 2007).

Transient mPTP opening: The precise mechanism of reduced calcium overloading and detrimental ROS levels have not been fully elucidated, however, there is evidence that these beneficial effects of IPC may be mediated by transient opening of the mPTP to a low-conductance state (Hausenloy et al., 2004a). Transient low-conductance mPTP opening is hypothesised to create a small diameter pore allowing passage of ions and solutes up to 300 Da. This low-conductance pore would therefore permit calcium and ROS efflux from the mitochondrial matrix whilst maintaining the electrochemical gradient for oxidative phosphorylation (Zoratti and Szabo, 1995; Ichas et al., 1997) (Figure 1.9). It has been suggested that transient low-conductance mPTP opening may provide a mechanism for rapid regulation of mitochondrial matrix conditions which may in turn reduce activation of high-conductance mPTP opening and associated cell death (Hausenloy et al., 2004a) (Figure 1.9). It should also be noted that a similar effect may be mediated by transitory opening of the mPTP to a high-conductance state. The precise mechanism by which IPC triggers transient low-conductance or transient mPTP opening remains to be fully identified and has not yet been investigated as a mode of ischaemic postconditioning or pharmacological conditioning. The occurrence of transient low-conductance mPTP opening may be related to the physiological function of the mPTP, discussed subsequently (see 1.4.1).

Figure 1.9: Model of transient mPTP opening in response to ischaemic preconditioning IPC triggers transient low conductance mPTP opening which mediates Ca2+ and ROS release from the mitochondrial matrix which would otherwise trigger high conductance mPTP opening. Constructed from information provided by Zoratti and Szabo (1995) and Hausenloy et al. (2004a).

The cumulative effects of RISK pathway activation and decreased mitochondrial matrix calcium and ROS accumulation, reduce the probability of high conductance mPTP opening and cell death (Steenbergen et al., 1993; Baines, 2009a). Reduced calcium load upon ischaemic conditioning may protect against other mechanisms of cell death

MITOCHONDRIAL MATRIX OMM IMM Ca2+ ROS INTERMEM. SPACE IPC ? Ca2+ ROS Pi LOW-CONDUCTANCE HIGH-CONDUCTANCE

IPC RISK PATHWAY ACTIVATION

H+ ATP

ADP ATP synthase

iii

Delayed ischaemic conditioning

The classical early IPC phenomenon described above elicits significant cardioprotection only if the delay between conditioning stimulus and ischaemic insult is less than approximately 2 hours. Subsequent studies have shown that a second phase of protection exists approximately 12 and 72 hours after the initial IPC stimulus, which has been termed the ‘second window of protection’ or ‘delayed conditioning’ (Marber et al., 1993; Baxter et al., 1997). The phenomenon of delayed conditioning suggests that it may be possible to target both the early and delayed components of protection to maximise cardioprotective potency, although this has not yet been confirmed.

The precise molecular mechanism for delayed IPC remains unclear; however, the temporal separation of the protective phases suggests that the conditioning stimulus evokes a memory response likely to involve de novo protein synthesis. The conditioning stimulus releases protective triggers, autocoids and cytokines, which in turn activate ‘early’ mediators to activate transcription factors causing synthesis of ‘distal mediators’ 12-24 hours later. The end-effectors of delayed conditioning include inhibition of mPTP opening (summarised in Figure 1.10) (reviewed by Hausenloy and Yellon, 2010).

Figure 1.10: Delayed ischaemic conditioning

Ischaemic conditioning produces extracellular autocoids and cytokines which activate ‘early mediators’, such as the RISK pathway, which elicit early cardioprotection and induce transcription factor expression which triggers synthesis of ‘distal mediators’ 12-24 hours later. Distal mediators confer a delayed cardioprotective effect which is thought to converge on inhibition of mPTP opening. Constructed from information provided by Hausenloy and Yellon (2010).

AUTOCOIDS

E.g. Adenosine, Bradykinin

mPTP

EXTRACELLULAR

INTRACELLULAR

MITOCHONDRIAL

GPCR binding

RISK pathway activation

Transcription factors NFKβ, STAT1,3, HIF-1α MnSOD HSP COX-2 ? Less detrimental ROS Less calcium overloading CYTOKINES

E.g. TNF-α, IL-β, IL-6

CARDIOPROTECTION ? ? Prostaglandin production ? (E ar ly p ro te ct io n ) (D e la y ed p ro te ct io n )

Although the precise actions of these distal mediator proteins in cardioprotection have not been fully elucidated, the main roles identified to date are detailed below.

Manganese superoxide dismutase: MnSOD is an anti-oxidant enzyme elevated 24 hours after the initial conditioning stimulus, possibly in response to increased signalling ROS (Zhai et al., 1996). MnSOD expression is expected to decrease the levels of detrimental ROS and thus reduce mPTP opening (Hausenloy and Yellon, 2010).

Heat shock proteins: The role of heat shock proteins as mediators of cardioprotection is controversial, however, there is evidence that HSP70 may reduce calcium overloading and thus reduce mPTP opening (Marber et al., 1993; Hausenloy and Yellon, 2010). Cyclo-oxygenase 2: Upon myocardial ischaemia, COX-2 produces prostaglandins (PE), PGE2 and PGF1α, which have been shown to elicit significant cardioprotection by a yet

unknown mechanism (Shinmura et al., 2000; Hausenloy and Yellon, 2010).

Although the clinical applicability of delayed IPC is limited in acute myocardial infarction, it presents the intriguing possibility that it may be possible to pharmacologically elicit delayed cardioprotection. Furthermore, it may be possible to achieve additive cardioprotection by targeting both early and late cardioprotective pathways, for example by inhibition of mPTP opening and prostaglandin expression; pharmacological conditioning for cardioprotection is discussed below.