Activation of protease-activated receptor-2 (PAR-2) induces cardioprotection in anaesthetised mice

S.Y. Lim1, C.L. Wainwright2, S. Kennedy1, K.A. Kane1. 1Department of Physiology & Pharmacology, University of Strathclyde, Glasgow G4 0NR, U.K. 2School of Pharmacy, The Robert Gordon University, Aberdeen AB10 1FR, U.K.

PAR2 activation has been shown to mediate cardioprotection in isolated rat hearts (Napoli et al., 2000). The present study aimed to determine whether the PAR2 agonist, trypsin and the activating peptide (SLIGRL-NH2, SL-NH2), could protect the heart from acute myocardial ischaemia and reperfusion injury (MIRI) in vivo. To confirm an action through PAR2, studies were performed in wild type (PAR2+/+) and PAR2 knockout (PAR2-/-) mice. The adenosine A1 receptor agonist, 2-chloro-N6-cyclopentyl-adenosine (CCPA), was studied as a cardioprotective agent acting via a PAR2 independent mechanism.

PAR2-/- mice were produced by back-crossing 5 times with C57BL/ 6 mice and the knockout confirmed by genotyping. 14-16 week old PAR2+/+ and PAR2-/- mice (18-34g) were anaesthetised with a combination of ketamine, xylazine and atropine (intraperitoneally) and artificially ventilated with O 2. The left anterior descending coronary artery was ligated to induce 30 min of occlusion followed by 120 min of reperfusion. Infarct size was assessed by Evan’s blue-triphenyltetrazolium chloride dual staining. Mice were pre-treated with a bolus dose (0.05 ml/ 25 g) of saline (vehicle control), trypsin (1 U/g), SL-NH2 (0.3 nmol/g) or CCPA (100 ng/g) intravenously via the tail vein 24-hour prior to surgery. Statistical significance was assessed using one way ANOVA with Dunnett’s post-hoc test.

A significant reduction in infarct size (expressed as percentage of the area at risk, AAR) was observed in PAR2+/+ mice pre-treated with trypsin (49 ± 3%; n=11), SL-NH2 (41 ± 4%; n=10) and CCPA (39 ± 4%; n=10) when compared to the vehicle control (59 ± 2%; n=14). These effects of trypsin and SLIGRL2 were not seen in PAR2-/- mice (58 ± 5% (trypsin, n=7), 59 ± 2% (SL-NH2, n=6) vs. 56 ± 4% (saline, n=9); p>0.05), although CCPA still reduced infarct size (41 ± 4%, n=8; p<0.05). The AAR was similar in hearts from all groups. In untreated mice, PAR2 deletion by itself did not modify infarct size (56 ± 3% vs. 54 ± 5% in PAR2+/+ (n=11) and PAR2-/- (n=12) mice respectively; p>0.05). The respective control arterial blood pressures (mmHg) and heart rates (beat per minute) in PAR2+/+ (123 ± 5 and 388 ± 14) and PAR2-/- (132 ± 5 and 407 ± 10) mice, prior to coronary artery occlusion, were not significantly modified by pre-treatment with trypsin, SL-NH2 or CCPA.

We conclude that exogenously applied trypsin and SL-NH 2 have a cardioprotective action on MIRI in the mouse and this effect is PAR2 mediated as demonstrated by the lack of a response in the PAR2-/- mice. However, infarct size was not modified by deletion of the PAR-2 itself.

Napoli C. et al. (2000). Proc. Natl.
Acad. Sci. USA. 97 : 3678.

S.Y. Lim holds an ORS & Strathclyde University studentship. PAR2 mice and PAR-2 activating peptide are courtesy of Prof. Robin Pelvin .