It is clear that multiple signalling pathways regulate the critical balance between cell death and survival in myocardial ischaemia-reperfusion. Recent attention has focused on the activation of survival or salvage kinases, particularly during reperfusion, as a common mechanism of many cardioprotective interventions. The phosphatidyl inositol 3 0 -hydroxy kinase/Akt complex (PI3K/Akt) and p42/p44 mitogen-activated protein kinase cascades have been widely promoted in this respect but the cyclic guanosine 3 0 ,5 0 -monophosphate/cGMP-dependent protein kinase (cGMP/PKG) signal transduction cassette has been less systematically investigated as a survival cascade. We propose that activation of the cGMP/PKG signalling pathway, following activation of soluble or particulate guanylate cyclases, may play a pivotal role in survival signalling in ischaemiareperfusion, especially in the classical preconditioning, delayed preconditioning and postconditioning paradigms. The resurgence of interest in reperfusion injury, largely as a result of postconditioning-related research, has confirmed that the cGMP/PKG pathway is a pivotal salvage mechanism in reperfusion. Numerous studies suggest that the infarct-limiting effects of preconditioning and postconditioning, exogenously donated nitric oxide (NO), natriuretic peptides, phosphodiesterase inhibitors, and other diverse drugs and mediators such as HMG co-A reductase inhibitors (statins), Rho-kinase inhibitors and adrenomedullin, whether given before and during ischaemia, or specifically at the onset of reperfusion, may be mediated by activation or enhancement of the cGMP pathway, either directly or indirectly via endogenous NO generation downstream of PI3K/Akt. Putative mechanisms of protection include PKG regulation of Ca 2 þ homeostasis through the modification of sarcoplasmic reticulum Ca 2 þ uptake mechanisms, and PKG-induced opening of ATP-sensitive K þ channels during ischaemia and/or reperfusion. At present, significant technical obstacles in defining the precise roles played by cGMP/PKG signalling include the heavy reliance on pharmacological PKG inhibitors of uncertain selectivity, difficulties in determining PKG activity in intact tissue, and the growing recognition that intracellular compartmentalisation of the cGMP pool may contribute markedly to the nucleotide's biological actions and biochemical determination. Overall, the body of experimental evidence suggests that cGMP/PKG survival signalling ameliorates irreversible injury associated with ischaemia-reperfusion and may be a tractable therapeutic target.

Natriuretic peptides are regulatory autacoids in the mammalian myocardium whose functions, mediated via particulate guanylyl cyclase/cGMP, may include cytoprotection against ischaemia-reperfusion injury. Previous work has identified that B-type natriuretic peptide (BNP) limits infarct size when administered prior to and during coronary occlusion through a K(ATP) channel-dependent mechanism. The present study examined the hypothesis that the protection afforded by BNP is mediated specifically at reperfusion in a postconditioning-like manner. Langendorff-perfused rat hearts were subjected to 35 min coronary artery occlusion and 120 min reperfusion, and infarct size was determined by tetrazolium staining. Postconditioning was effected by applying six 10-second periods of global ischaemia at the onset of reperfusion.Treatment with either BNP 10 nM or the NO donor S-nitroso-N-acetylpenicillamine (SNAP) 1-10 microM was commenced 5 min prior to reperfusion and continued until 10 min after reperfusion. Control infarct size (% of ischaemic risk zone) was 40.8 +/- 3.7%.BNP at reperfusion induced a significant limitation of infarct size (BNP 22.9 +/- 4.1% P<0.05 vs. control). Co-treatment at reperfusion with BNP and the K(ATP) channel blockers 5-hydroxydecanote (5HD, 100 microM), glibenclamide (Glib; 10 microM) or HMR1098 (10 microM) abolished the infarct-limiting effect of BNP (BNP + 5HD 41.0 +/- 3.9%, BNP + Glib 39.8 +/- 5.6%, BNP + HMR 1098 46.0 +/- 7.1%,P < 0.05 vs. BNP). BNP given together with L-NAME (100 microM) at reperfusion resulted in a marked loss of protection (BNP + L-NAME 53.1 +/- 3.8% P < 0.001 vs. BNP). In a second series of experiments, SNAP (1-10 microM) given at reperfusion was found not to be protective (SNAP 1 microM 30.2 +/- 4.9%, SNAP 2 microM 27.5 +/- 9.5%, SNAP 5 microM 39.2 +/- 5.7%, SNAP 10 microM 33.7 +/- 6.4%, not significant vs. control). In a third series of experiments, postconditioning significantly limited infarct size (14.9 +/- 3.6 % vs. control 34.5 +/- 4.9%, P < 0.01) and this effect of postconditioning was abolished in the presence of isatin (100 microM), a non-specific blocker of particulate guanylyl cyclases (35.1 +/- 6%, P < 0.05 vs. postconditioning). In conclusion, pharmacological activation of pGC by BNP can effectively induce protection against reperfusion injury, by mechanisms involving K(ATP) channel opening and endogenous NO synthase activation. Furthermore, endogenous activation of pGC could play a role in the mechanism of postconditioning.

Nitric oxide (NO) and B-type natriuretic peptide (BNP) are protective against ischemia-reperfusion injury as they increase intracellular cGMP level via activation of soluble (sGC) or particulate guanylate cyclases (pGC), respectively. The aim of the present study was to examine if the cGMP-elevating mediators, NO and BNP, share a common downstream signaling pathway via cGMP-dependent protein kinase (PKG) in cardiac cytoprotection. Neonatal rat cardiac myocytes in vitro were subjected to 2.5 h simulated ischemia (SI) followed by 2 h reoxygenation. Cell viability was tested by trypan blue exclusion assay. PKG activity of cardiac myocytes was assessed by phospholamban (PLB) phosphorylation determined by western blot. Cell death was 34 +/- 2% after SI/reoxygenation injury in the control group. cGMP-inducing agents significantly decreased irreversible cell injury: the cGMP analog 8-bromo-cGMP (8-Br-cGMP, 10 nM) decreased it to 13 +/- 1% (p < 0.001), the direct NO-donor S-nitroso-N-acetylpenicillamine (SNAP, 1 microM) to 18 +/- 6% (p < 0.05) and BNP (10 nM) to 12 +/- 2% (p < 0.001), respectively. This protective effect was abolished by the selective PKG inhibitor KT-5823 (600 nM) in each case. As PLB is not a unique reporter for PKG activity since it is also phosphorylated by protein kinase A (PKA), we examined PLB phosphorylation in the presence of the PKA inhibitor KT-5720 (1 microM). The ratio of pPLB/PLB significantly increased after administration of both BNP and 8-Br-cGMP under ischemic conditions, which was abolished by the PKG inhibitor. This is the first demonstration that elevated cGMP produced either by the sGC activator SNAP or the pGC activator BNP exerts cytoprotective effects via a common downstream signaling pathway involving PKG activation.

The myocardium represents a major source of several families of peptide hormones under normal physiological conditions and the plasma concentrations of many of these "cardiac peptides" (or related pro-peptide fragments) are substantially augmented in many cardiac disease states. In addition to well-characterised endocrine functions of several of the cardiac peptides, pleiotropic functions within the myocardium and the coronary vasculature represent a significant aspect of their actions in health and disease. Here, we focus specifically on the cardioprotective roles of four major peptide families in myocardial ischemia and reperfusion: adrenomedullin, kinins, natriuretic peptides and the urocortins. The patterns of early release of all these peptides are consistent with roles as autacoid cardioprotective mediators. Clinical and experimental research indicates the early release and upregulation of many of these peptides by acute ischemia and there is a convincing body of evidence showing that exogenously administered adrenomedullin, bradykinin, ANP, BNP, CNP and urocortins are all markedly protective against experimental myocardial ischemia-reperfusion injury through a conserved series of cytoprotective signal transduction pathways. Intriguingly, all the peptides examined so far have the potential to salvage against infarction when administered specifically during early reperfusion. Thus, the myocardial secretion of peptide hormones likely represents an early protective response to ischemia. Further work is required to explore the potential therapeutic manipulation of these peptides in acute coronary syndromes and their promise as biomarkers of acute myocardial ischemia.