Limiting infarct size by timely reperfusion is critical to improve outcomes in patients with myocardial infarction. In contrast, reperfusion may increase infarct size, a phenomenon known as ischemia/reperfusion (I/R) injury. Exogenously delivered pharmacological agents, such as adenosine, have been shown to limit infarct size resulting from I/R in experimental studies. However translating these studies into clinical practice has been disappointing. Therefore, harnessing endogenous cardioprotectants may be a novel therapeutic strategy to limit I/R injury.
Adenosine is an endogenous nucleoside present in normal conditions; however, during ischemia, the production of adenosine increases severalfold (Shryock and Belardinelli, 1997
). Adenosine activates four well characterized G protein-coupled receptors (GPCRs) to produce various physiological effects that attenuate many of the proposed mechanisms of I/R injury (Headrick et al., 2003a
). However, the specific adenosine receptor (AR) subtype (A1AR, A2aAR, A2bAR, or A3AR) responsible for adenosine-mediated cardioprotective effects remains unclear. The signaling pathway for adenosine-mediated cardioprotection involves the activation of PI3
K/Akt, ERK1/2, nitric-oxide synthase (NOS), and the sarcolemmal (sarc) or mitochondrial (mito) ATP-sensitive potassium channels (KATP
channels) (Headrick et al., 2003b
). In experimental models as well as in small clinical studies, administration of adenosine with reperfusion results in a marked reduction in infarct size and improvement in ventricular function. In the larger AMISTAD and AMISTAD II trials, a 3-h infusion of adenosine as an adjunct to reperfusion resulted in a striking reduction in infarct size (55–65%) but did not significantly affect clinical outcome (Mahaffey et al., 1999
; Ross et al., 2005
). Despite these potential cardioprotective actions, adenosine treatment has not been incorporated into standard clinical practice. Adenosine has a very short half-life, necessitating a continuous infusion. Not only is this inconvenient, but it increases the chance of side effects, such as bradycardia and hypotension. Harnessing endogenous adenosine provides a novel treatment strategy to reduce I/R, which would eliminate the side effects and expense of adenosine infusions.
Thrombin contributes to I/R injury independently of its effects on platelets and fibrinogen (Chong et al., 2003
; Strande et al., 2007
). Thrombin activates the seven trans-membrane G protein-coupled protease-activated receptors (PARs) 1 and 4 by proteolytic cleavage of the N terminus to expose a tethered ligand, which then transactivates the receptor (Ossovskaya and Bunnett, 2004
). Thrombin is a non-selective activator of PARs; therefore, in experimental systems, synthetic activating peptides (APs) containing 5- to 14-amino acid residues that correspond to the tethered ligand are used to selectively activate each receptor. The peptide AYPGKF-amide is a PAR4 AP. PAR1 and PAR4 were originally detected in platelets where their activation causes platelet activation and aggregation. As a result, they have been identified in a variety of cell types and tissues and have been shown to modulate a variety of physiological processes. We have recently shown that PAR1 inhibition protects against myocardial I/R injury by recruiting cardioprotective pathways including PI3
K/Akt, NOS, and KATP
channels (Strande et al., 2007
). However, the role of PAR4 in determining the extent of myocardial I/R injury is not known. Therefore, we focused on PAR4 in this study to determine whether PAR4 inhibition is cardioprotective.
Adenosine has been shown to modulate thrombin signaling in various models. For instance, in vein endothelial cells, adenosine is able to down-regulate tissue factor induction by thrombin (Deguchi et al., 1998
). Additionally in corneal endothelium, adenosine opposes thrombin-mediated inhibition of calcium wave propagation (D’Hondt et al., 2007
). However, the mechanism of this adenosine and thrombin receptor “cross-talk” is poorly understood. Furthermore, it is not known whether this receptor cross-talk occurs in the myocardium. Therefore, we proposed that by blocking thrombin activation pathways with a PAR4 antagonist, endogenous adenosine would be allowed to exert its cardioprotective effects.
We used both an in vivo rat model and an in vitro isolated rat heart model of I/R injury to assess the effects of PAR4 blockade on infarct size. The absence of blood in the isolated heart model eliminated the possible interference of coagulation as well as platelet activation in mediating injury. We studied the ability of two structurally different PAR4 inhibitors, trans-cinnamoyl-YPGKF-amide (tc-Y-NH2) and palmitoyl-SGRRY-GHALR-amide (P4pal10), to protect the heart against injury from I/R. We then assessed the role of adenosine by blocking its effects with a nonselective adenosine receptor antagonist. Furthermore, based on the assumption that antagonizing PAR4 signaling would unmask AR signaling, we confirmed the signaling pathway leading to protection was similar to that previously reported for AR activation. Therefore, we explored the adenosine-mediated cardioprotective pathways including Akt, ERK1/2, NOS, sarc, and mitoKATP channels.