There are several important new findings in the present study. First, brief exposure to parstatin preconditioned the rat heart and decreased infarct size, improved the recovery of LVDP, and increased coronary perfusion. More importantly, parstatin administration after the onset of reperfusion was still cardioprotective. This underscores the translational potential of this compound. Secondly, the co-administration of an ERK1/2, p38 MAPK, NOS, sGC, and KATP inhibitors diminished the beneficial effects of parstatin-induced preconditioning. Thirdly, in normal coronary arterioles, parstatin induces a potent vasodilation through an endothelium-dependent NO and KATP mechanisms.
Preconditioning puts the heart into a state of self-preservation. Preconditioning is triggered by either brief cycles of ischaemia or by exogenous compounds (i.e. adenosine and opioids),18
which typically activate Gi
-protein-coupled surface receptors, AMP-activated kinase,19
and 3′-phosphoinositide-dependent kinase-120
to set off a complex pathway which ultimately results in cell survival. Gi
-protein activation leads to the activation of members of the MAPK family including p38 MAPK and ERK1/2; important mediators of cardioprotection.21–24
ERK1/2 is known to activate NOS25–28
and the production of NO subsequently targets sGC which results in the conversion of guanosine-5′-triphophate to the intracellular second messenger cyclic guanosine-3′,5′-monophosphate (cGMP). KATP
channels are opened in a cGMP-dependent manner.27,29,30
These steps are thought to occur prior to the index ischaemia and are able to put the heart into its preconditioned state. Opening of the KATP
channels generates reactive oxygen species which are thought to be secondary messengers for kinase activation.31
Therefore, by the time of reperfusion prosurvival kinases including ERK1/2 are quickly activated32
and protect the heart against reperfusion injury.33,34
In addition, activation of sarcolemmal KATP
channels shortens action membrane potential duration and decreases intracellular Ca2+
loading, which also leads to cardioprotection.20,35
Our data demonstrates that parstatin acts through a Gi-protein-mediated pathway to induce cardioprotection by modulating both the trigger and mediator phases of preconditioning. Blockade of p38 MAPK, ERK1/2, NOS, and KATP channels prior to ischaemia abolishes the infarct sparing and recovery of LVDP effects of parstatin. This implicates parstatin's role in mediating preconditioning through previously delineated mediators.
However, we did not detect equal contribution to parstatin-mediated cardioprotection by all factors examined. Only ERK1/2 and NOS inhibition completely reversed parstatin-mediated cardioprotection. p38 MAPK inhibition resulted in the partial reversal of parstatin cardioprotection. Furthermore, parstatin treatment before ischaemia is able to increase the phosphorylation of ERK1/2 but not p38 MAPK at reperfusion. These findings indicate that either p38 MAPK is upstream of ERK1/2 or in a divergent pathway. p38 MAPK may be an early trigger whereas ERK1/2 is most likely a trigger and mediator of parstatin-induced cardioprotection.
Furthermore, sGC and KATP
channel inhibition also resulted in the partial reversal of cardioprotection by parstatin. Thus, parstatin-mediated cardioprotection appears to be mediated in part by a pathway independent of sGC activity. Previously, it had been shown that the KATP
channel is regulated by a cGMP-dependent protein kinase signalling pathway.30
However, it has also been shown that KATP
channels can be opened in an sGC-independent manner relying instead on ERK1/2 phosphorylation.36,37
In this study, although the parstatin-mediated cardioprotection is stimulated by a cGMP-KATP
channel-dependent process, the complete action of parstatin does not require it.
On the other hand, the complete action of parstatin does require NOS and presumably NO signalling. NO can modify proteins involved in the signalling mechanism by chemical reaction such as S
-nitrosylation other than by means of activation of sGC.38 S
-Nitrosylation has also been shown to be involved in myocardial protection from ischaemia–reperfusion injury.39
Indeed, our data suggests that parstatin-mediated cardioprotection is mediated by both NO activation of sGC and NO activation of other mediating proteins. The resultant S
-nitrosylation effects of parstatin are yet to be determined.
Nitric oxide, ERK1/2, and KATP channels appear to serve some common physiological functions, such as vasodilation and cardioprotection. Indeed, the signalling pathways responsible for coronary artery vasodilation are somewhat parallel to the signalling pathways responsible for preconditioning and cardioprotection. During ischaemia and reperfusion in the isolated heart, parstatin increases coronary flow under constant coronary pressure conditions. Typically, coronary flow is tightly coupled to oxygen demand. In non-diseased coronary vessels, whenever cardiac activity and oxygen consumption increases, there is an increase in coronary blood flow that is nearly proportionate to the increase in oxygen consumption. In innervated hearts, increased metabolic activity from increased heart rate and/or contractility triggers sympathetic activation which results in coronary vasodilation and increased coronary flow. The advantage of the isolated heart model is the examination of contractility, heart rate, and vascular effects without the neuronal and hormonal complications of an intact animal model. We confirmed the vasodilatory effects of parstatin in the isolated heart under constant-flow conditions. In this model, higher perfusion pressure correlates with vasoconstriction and lower perfusion pressure correlates with vasodilation. Parstatin reduced coronary perfusion pressure during regional ischaemia and reperfusion. Furthermore, parstatin salvaged the ischaemic myocardium and restores contractile function under both constant perfusion and constant-flow conditions, suggesting that parstatin exerts its cardioprotective functions by acting on both myocardial myocytes and vasculature.
Since parstatin increased coronary flow during ischaemia and reperfusion in rat hearts, we reasoned that parstatin was mediating vasodilation of the coronary microvasculature. Our data also demonstrates that parstatin mediates vasodilation in an endothelium and NO-dependent mechanism. Paralleling our cardioprotection results, sGC and KATP
channel inhibition do not completely abolish parstatin-mediated vasodilation. Moreover, sGC inhibition does not significantly reverse parstatin's vasodilatory effects. sGC and KATP
channel inhibition are additive in abolishing parstatin-mediated vasodilation. In this case, NO may be vasodilating in a cGMP-KATP
channel-dependent and -independent manner. NO is also known to mediate vasodilation by activating other potassium channels.40,41
In conclusion, parstatin mediates endothelium-dependent vasodilation in coronary arterioles through a mechanism that involves NO and KATP
In conclusion, we have demonstrated that parstatin, the N-terminal cleavage product of PAR1, is an effective agent for cardioprotection during ischaemia and reperfusion of the rat myocardium. We have also shown that parstatin causes vasodilation in isolated rat coronary arterioles. Both cardioprotection and vasodilatory properties of parstatin is largely dependent on NOS to a lesser extent on sGC and KATP channels. Parstatin is a novel and intriguing activator of cardioprotection and vasodilation. Parstatin manifests its effects in part through known mediators of cardioprotection and vasodilation, suggesting that it is acting through multiple pathways to exert its effects. These other pathways may include S-nitrosylation or other potassium channels.
Collectively, the results of these studies in rat hearts and coronary vessels strongly suggest that parstatin serves a protective role during ischaemia–reperfusion and induces coronary vasodilation. Since the cardioprotective effects of the parstatin occurred in the absence of haemodynamic changes, there is an exciting opportunity to develop parstatin to protect against myocardial and microvascular injury in the clinical setting.
Conflict of interest: none declared.