Inhibiting protease-activated receptor 4 limits myocardial ischemia/reperfusion injury in rat hearts by unmasking adenosine signaling

Abstract

Harnessing endogenous cardioprotectants is a novel therapeutic strategy to combat ischemia/reperfusion (I/R) injury. Thrombin causes I/R injury, whereas exogenous adenosine prevents I/R injury. We hypothesized that blocking thrombin receptor activation with a protease-activated receptor (PAR) 4 antagonist would unmask the cardioprotective effects of endogenous adenosine. The protective role of two structurally unrelated PAR4 antagonists, trans-cinnamoyl-YPGKF-amide (tc-Y-NH(2)) and palmitoyl-SGRRYGHALR-amide (P4pal10), were evaluated in two rat models of myocardial I/R injury. P4pal10 (10 microg/kg) treatment before ischemia significantly decreased infarct size (IS) by 31, 21, and 19% when given before, during, and after ischemia in the in vivo model. tc-Y-NH(2) (5 microM) treatment before ischemia decreased IS by 51% in the in vitro model and increased recovery of ventricular function by 26%. To assess whether the cardioprotective effects of PAR4 blockade were due to endogenous adenosine, isolated hearts were treated with a nonselective adenosine receptor blocker, 8-sulfaphenyltheophylline (8-SPT), and tc-Y-NH(2) before ischemia. 8-SPT abolished the protective effects of tc-Y-NH(2) but did not affect IS when given alone. Adenosine-mediated survival pathways were then explored. The cardioprotective effects of tc-Y-NH(2) were abolished by inhibition of Akt (wortmannin), extracellular signal-regulated kinase 1/2 [PD98059 (2'-amino-3'-methoxyflavone)], nitric-oxide synthase [N(G)-monomethyl-l-arginine (l-NMA)], and K(ATP) channels (glibenclamide). PD98059, l-NMA, and glibenclamide alone had no effect on cardioprotection in vitro. Furthermore, inhibition of mitochondrial K(ATP) channels [5-hydroxydecanoic acid (5-HD)] and sarcolemmal K(ATP) channels (sodium (5-(2-(5-chloro-2-methoxybenzamido)ethyl)-2-methoxyphenylsulfonyl)(methylcarbamothioyl)amide; HMR 1098) abolished P4pal10-induced cardioprotection in vivo. Thrombin receptor blockade by PAR4 inhibition provides protection against injury from myocardial I/R by unmasking adenosine receptor signaling and supports the hypothesis of a coupling between thrombin receptors and adenosine receptors.

Fig. 1

Experimental protocols. All hearts were subjected to 30 min of regional ischemia after a 50-min stabilization period and followed by 180 min of reperfusion. The control group (A) received no treatment. The tc-Y-NH₂ group (B) was continuously perfused with different concentrations of tc-Y-NH₂ 15 min before the onset of ischemia. The inhibitor groups were continuously perfused with an inhibitor (PAR4 AP, wortmannin, PD98059, L-NMA, or glibenclamide) for 30 min before ischemia with (C) or without (D) the addition of tc-Y-NH₂ 15 min before ischemia. E, 8-SPT was perfused with tc-Y-NH₂ for the 15 min before ischemia.

Fig. 2

Analysis of PAR4 expression by reverse transcription-PCR and immunoblotting rat heart tissue. A, PCR of rat heart cDNA with PAR4-specific primers. Lane 1, 100-bp ladder. PAR4 (559 bp, lane 2) was detected in pooled cDNA from three rat hearts. Lane 3, negative control that contains the PAR4 primers but no cDNA. β-Actin (838 bp, lane 4) is an internal control. B, determination of PAR4 protein by immunoblot analysis of rat heart ventricles (lanes 2 and 3) and isolated cardiomyocytes (lane 5) and cardiac fibroblasts (lane 1). Glyceraldehyde-3-phosphate dehydrogenase is the loading control.

Fig. 3

Analysis of the cardioprotective effects of PAR4 inhibition in vivo. A, dose-response curve of P4pal10, a PAR4 antagonist. Rats were treated with either saline or increasing doses of P4pal10 (0.1, 1, 3, 10, 30, and 100 μg/kg) administered as an i.v. bolus 15 min before ischemia. B, phase action of P4pal10 on cardioprotection in the rat. Rats were treated with either saline or increasing doses of P4pal10 (10 μg/kg) 15 min before ischemia, 15 min after onset of ischemia, or 10 s after onset of reperfusion. Data are mean ± S.D., n = 6/group. *, P < 0.05, treated versus control.

Fig. 4

Analysis of the cardioprotective effects of PAR4 inhibition in vitro. Concentration-response curve of tc-Y-NH₂, a PAR4 antagonist. Rat hearts were perfused with buffer with increasing concentrations of tc-Y-NH₂ (0, 1, 5, and 10 μM) for 15 min before 30-min regional ischemia and 180-min reperfusion. A, infarct size expressed as a percentage of AAR. B, recovery of LVDP. The protective effects of tc-Y-NH₂ were abolished by a PAR4-activating peptide. Rat hearts were perfused with PAR4 AP (20 μM) for 15 min before the addition of tc-Y-NH₂ (5 μM) and perfused an additional 15 min before 30 min of regional ischemia and 180-min reperfusion. C, infarct size expressed as a percentage of AAR. D, recovery of LVDP. Data are mean ± S.D., n = 6/group.*, P < 0.05, treated versus control.

Fig. 5

The cardioprotective effect of PAR4 and thrombin inhibition is dependent on endogenous adenosine. Inhibition of adenosine receptors (8-SPT) abolished the cardioprotective effects of the PAR4 antagonist tc-Y-NH₂. Hearts were perfused with 8-SPT (1 μM) with or without tc-Y-NH₂ (5 μM) for 15 min before 30-min regional ischemia and 180-min reperfusion. A, infarct size expressed as a percentage of AAR. B, percentage of recovery of LVDP. Data are mean ± S.D., n = 6/group. *, P < 0.05, treated versus control. The protective effect of thrombin inhibition is abolished by 8-SPT. Rat hearts were perfused with 8-SPT (1 μM) with or without lepirudin (1 U/ml) for 15 min before 30 min of regional ischemia and 180-min reperfusion. C, infarct size expressed as a percentage of AAR. D, percentage recovery of LVDP. Data are mean ± S.D., n = 6/group. *, P < 0.05, treated versus control.

Fig. 6

The cardioprotective effects of PAR4 inhibition are dependent on PI₃K/Akt activation. Inhibition of PI₃K/Akt [wortmannin (Wort)] abolished the cardioprotective effects of the PAR4 antagonist tc-Y-NH₂. Hearts were perfused with Wort (100 nM) for 15 min before the addition of tc-Y-NH₂ (5 μM) and perfused an additional 15 min before 30-min regional ischemia and 180-min reperfusion. A, infarct size expressed as a percentage of AAR. B, percentage of recovery of LVDP. Data are mean ± S.D., n = 6/group. *, P < 0.05, treated versus control. PAR4 inhibition did not increase activation of Akt after 5 min of reperfusion as measured by phosphorylation of Akt. Rat hearts were perfused with Wort (100 nM) or -tc-Y-NH₂ (5 μM) for 15 min before 30min regional ischemia. The free wall of the left ventricle was harvested for protein extraction after 5 min of reperfusion. C, immunoblot for phosphorylated Akt. β-actin is the loading control. D, quantitation of phosphorylated Akt when normalized to β-actin. Data are mean ± S.D., n = 3/group. *, P < 0.05, treated versus control.

Fig. 7

ERK1/2 is required for the cardioprotective effects of PAR4 inhibition, but it is not activated by tc-Y-NH₂. Inhibition of ERK1/2 (PD98059) abolished the cardioprotective effects of the PAR4 antagonist tc-Y-NH₂. Hearts were perfused with PD98059 (10 μ M) for 15 min before the addition of tc-Y-NH₂ (5 μM) and perfused an additional 15 min before 30-min regional ischemia and 180-min reperfusion. A, infarct size expressed as a percentage of AAR. B, percentage of recovery of LVDP. Data are mean ± S.D., n = 6/group. *, P < 0.05, treated versus control. PAR4 inhibition did not increase activation of ERK1/2 after 5 min of reperfusion as measured by phosphorylation of ERK1/2. Rat hearts were perfused with PD98059 (10 μM) or tc-Y-NH₂ (5 μM) for 15 min before 30 min of regional ischemia. The free wall of the left ventricle was harvested for protein extraction after 5 min of reperfusion. C, immunoblot for phosphorylated ERK1/2. β-actin is the loading control. D, quantitation of phosphorylated ERK2 when normalized to β-actin. E, quantitation of phosphorylated ERK1 when normalized to β-actin. Data are mean ± S.D., n = 3/group. *, P < 0.05, treated versus control.

Fig. 8

Nitric oxide is required for the cardioprotective effects of PAR4 inhibition. Inhibition of nitric oxide synthase (L-NMA) abolished the cardioprotective effect of the PAR4 antagonist tc-Y-NH₂. Hearts were perfused with L-NMA (100 μM) for 15 min before the addition of tc-Y-NH₂ (5 μM) and perfused an additional 15 min before 30-min regional ischemia and 180-min reperfusion. A, infarct size expressed as a percentage of AAR. B, percentage of recovery of LVDP. Data are mean ± S.D., n = 6/group. *, P < 0.05, treated versus control.

Fig. 9

The cardioprotective effects of PAR4 inhibition are mediated through opening of K_ATP channels. Inhibition of K_ATP channels [glibenclamide (Glib)] abolished the cardioprotective effect of the PAR4 antagonist tc-Y-NH₂. Hearts were perfused with Glib (3 μM) for 15 min before the addition of tc-Y-NH₂ (5 μM) and perfused an additional 15 min before 30-min regional ischemia and 180-min reperfusion. A, infarct size expressed as a percentage of AAR. B, percentage of recovery of LVDP. Data are mean ± S.D., n = 6/group. *, P < 0.05, treated versus control. The protective effects of P4pal10 was abolished by blocking either sarcolemmal K_ATP channels (HMR; 3 mg/kg) or mitochondrial K_ATP channels (5-HD; 10 mg/kg) before ischemia (C) and before reperfusion (D). In these studies, infarct size was determined after 30 min of regional ischemia and 120-min reperfusion. Data are mean ± S.D., n = 6/group. *, P < 0.05, treated versus control.