Protection of the ischaemic heart: investigations into the phenomenon of ischaemic preconditioning

Abstract

Exposure of the heart to one or more short episodes of ischaemia/reperfusion protects the heart against a subsequent prolonged period of ischaemia, as evidenced by a reduction in infarct size and an improvement in functional recovery during reperfusion. Elucidation of the mechanism of this endogenous protection could lead to the development of pharmacological mimetics to be used in the clinical setting. The aim of our studies was therefore to gain more information regarding the mechanism of ischaemic preconditioning, using the isolated perfused working rat heart as model. A preconditioning protocol of 1 x 5 or 3 x 5 min of ischaemia, interspersed with 5 min of reperfusion was found to protect hearts exposed to 25 min of global ischaemia or 35-45 min of regional ischaemia. These models were used throughout our studies. In view of the release of catecholamines by ischaemic tissue, our first aim was to evaluate the role of the alphaadrenergic receptor in ischaemic preconditioning. However, using a multi-cycle ischaemic preconditioning protocol, we could not find any evidence for alpha-1 adrenergic or PKC activation in the mechanism of preconditioning. Cyclic increases in the tissue cyclic nucleotides, cAMP and cGMP were found, however, to occur during a multi-cycle preconditioning protocol, suggesting roles for the beta-adrenergic signalling pathway and nitric oxide (NO) as triggers of cardioprotection. This was substantiated by the findings that (1) administration of the beta-adrenergic agonist, isoproterenol, or the NO donors SNAP or SNP before sustained ischaemia also elicited cardioprotection similar to ischaemic preconditioning; (2) beta-adrenergic blockade or nitric oxide synthase inhibition during an ischaemic preconditioning protocol abolished protection. Effectors downstream of cAMP, such as p38MAPK and CREB, were also demonstrated to be involved in the triggering process. Our next step was to evaluate intracellular signalling during sustained ischaemia and reperfusion. Our results showed that ischaemic preconditioned-induced cardioprotection was associated with a significant reduction in tissue cAMP, attenuation of p38MAPK activation and increased tissue cGMP levels and HSP27 activation, compared to non-preconditioned hearts. The role of the stress kinase p38MAPK was further investigated by using the inhibitor SB203580. Our results suggested that injury by necrosis and apoptosis share activation of p38MAPK as a common signal transduction pathway and that pharmacological targeting of this kinase offers a tenable option to manipulate both these processes during ischaemia/reperfusion injury.

Fig. 1.

Tissue cAMP and cGMP levels during a multi-cycle preconditioning protocol. PC1-, PC2-, PC3-: first, second and third episodes of 5 min of global ischaemia. PC1+, PC2+, PC3+: first, second and third episodes of reperfusion after ischaemia.

Fig. 2.

Functional performance during reperfusion after 25 min of global ischaemia. Non-PC: non-preconditioned hearts; PC: ischaemic preconditioned (3 × 5 min); PC + Alp: ischaemic preconditioned (3 × 5 min) + alprenolol (7.5 × 10^-5 M) added during the preconditioning protocol; Iso: beta-adrenergic preconditioning with isoproterenol (1 × 5 min; 10^-6 M).

Fig. 3.

Effect of ischaemic (IPC) and beta-adrenergic preconditioning (BPC) on infarct size after 35 min of regional ischaemia. nPC: non-preconditioned hearts. Infarct size is expressed as a percentage of the area at risk.

Fig. 4.

Pharmacological preconditioning with NO donors. SNAP: S-nitroso-N-penicillamine, 50 μM; SNP: Sodium nitroprusside, 100 μM; L-Arg: L-arginine (10 mM).

Fig. 5.

Functional performance during reperfusion after 25 min of global ischaemia: effect of NOS inhibition with LNAME [N(omega)-nitro-L-arginine methyl ester], (50 μM).

Fig. 6.

Phosphorylation pattern of p38 MAPK during an ischaemic preconditioning protocol. PC1-, PC3-: first and third episodes of 5 min of ischaemia; PC1+, PC3+: first and third episodes of 5 min of reperfusion after ischaemia.

Fig. 7.

Phosphorylation pattern of HS P27 in cytosolic fraction during an ischaemic preconditioning protocol. PC1-, PC3-: first and third episodes of 5 min of ischaemia; PC1+, PC3+: first and third episodes of 5 min of reperfusion after ischaemia.

Fig. 8.

Phosphorylation of CREB by ischaemia/reperfusion and forskolin (1 μM). Inhibition of ischaemia/reperfusion-induced phosphorylation of CREB by propranolol (0.1 μM), prazosin (0.3 μM), DPCPX (adenosine A1 receptor blocker, 1 μM) and MRS-1191 (adenosine A3 receptor blocker, 5 μM).

Fig. 9.

Signalling pathways involved in CREB phosphorylation by endogenous catecholamines, adenosine and phospholipase A2 activation.

Fig. 10.

Tissue cAMP and cGMP changes during sustained global ischaemia: effect of prior ischaemic preconditioning (3 × 5 min).

Fig. 11.

p38 MAPK phosphorylation during sustained global ischaemia: effect of prior ischaemic preconditioning (3 × 5 min).

Fig. 12.

p38 MAPK phosphorylation during reperfusion after 25 min global ischaemia: effect of prior ischaemic preconditioning (3 × 5 min).

Fig. 13.

HS P27 phosphorylation during sustained global ischaemia: effect of prior ischaemic preconditioning (3 × 5 min).