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. 2013 Dec;9(4):633-42.
doi: 10.1007/s11302-013-9374-3. Epub 2013 Jul 5.

P2Y2 receptor agonist with enhanced stability protects the heart from ischemic damage in vitro and in vivo

Edith Hochhauser  1 , Ronit Cohen, Maayan Waldman, Anna Maksin, Ahuva Isak, Dan Aravot, P Suresh Jayasekara, Christa E Müller, Kenneth A Jacobson, Asher Shainberg
Affiliations

P2Y2 receptor agonist with enhanced stability protects the heart from ischemic damage in vitro and in vivo

Edith Hochhauser et al. Purinergic Signal. 2013 Dec.

Abstract

Extracellular nucleotides acting via P2 receptors play important roles in cardiovascular physiology/pathophysiology. Pyrimidine nucleotides activate four G protein-coupled P2Y receptors (P2YRs): P2Y2 and P2Y4 (UTP-activated), P2Y6, and P2Y14. Previously, we showed that uridine 5'-triphosphate (UTP) activating P2Y2R reduced infarct size and improved mouse heart function after myocardial infarct (MI). Here, we examined the cardioprotective role of P2Y2R in vitro and in vivo following MI using uridine-5'-tetraphosphate δ-phenyl ester tetrasodium salt (MRS2768), a selective and more stable P2Y2R agonist. Cultured rat cardiomyocytes pretreated with MRS2768 displayed protection from hypoxia [as revealed by lactate dehydrogenase (LDH) release and propidium iodide (PI) binding], which was reduced by P2Y2R antagonist, AR-C118925 (5-((5-(2,8-dimethyl-5H-dibenzo[a,d][7]annulen-5-yl)-2-oxo-4-thioxo-3,4-dihydropyrimidin-1(2H)-yl)methyl)-N-(1H-tetrazol-5-yl)furan-2-carboxamide). In vivo, echocardiography and infarct size staining of triphenyltetrazolium chloride (TTC) in 3 groups of mice 24 h post-MI: sham, MI, and MI+MRS2768 indicated protection. Fractional shortening (FS) was higher in MRS2768-treated mice than in MI alone (40.0 ± 3.1 % vs. 33.4 ± 2.7 %, p < 0.001). Troponin T and tumor necrosis factor-α (TNF-α) measurements demonstrated that MRS2768 pretreatment reduced myocardial damage (p < 0.05) and c-Jun phosphorylation increased. Thus, P2Y2R activation protects cardiomyocytes from hypoxia in vitro and reduces post-ischemic myocardial damage in vivo.

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Figures

Fig. 1
Fig. 1
Effect of MRS2768 on cardiomyocytes subjected to hypoxia. a Cultured cardiomyocytes, 5 days in vitro, were washed twice with glucose-free PBS and incubated with two concentrations of MRS2768 (10 and 50 μM) for 1 h prior to hypoxic stress at 37 °C. (*p < 0.001 vs. hypoxia). b Viability of MRS2768-treated cardiomyocytes subjected to hypoxia as tested with PI staining. (*p < 0.001 vs hypoxia). Values are means ± SE. n = 5 in each group
Fig. 2
Fig. 2
Effect of AR-C118925 and MRS2768 on cardiomyocytes subjected to hypoxia. Cultured cardiomyocytes, 5 days in vitro, were washed twice with glucose-free PBS and incubated with two concentrations of AR-C118925 (3 and 10 μM). After 1 h, MRS2768 (50 μM) was included to the same dishes for 1 h prior to hypoxic stress at 37 °C. (*p < 0.001 vs. hypoxia, # p < 0.01 vs hypoxia+MRS2768+AR-C118925). Values are means ± SE. n = 5 in each group
Fig. 3
Fig. 3
Echocardiographic studies. Echocardiographic studies 24 h after MI demonstrated left ventricular (LV) end systolic diameter (LVSD) and LV end diastolic diameter (LVDD) of sham and of infarcted hearts compared with the MRS2768-pretreated mice (a, b). In addition, FS of sham and of infarcted hearts were compared with MRS2768-pretreated mice (c). Heart rate at sham and post-MI of control (saline treated) and MRS2768-treated (d). Representative M mode images are presented in Fig. 2e.Values represent means ± SE, n = 7 in each group. *p < 0.05 vs MI
Fig. 4
Fig. 4
Measurements of Troponin T, infarct size, and TNF-α. a The release of Troponin T to the serum 24 h post-LAD ligation. Values are means ± SE. n = 12 hearts in each group. *p < 0.05 vs. MI. b The percent of irreversible injury was determined by scanning the images of mice hearts ventricular sections with triphenyl tetrazolium chloride (TTC). Representative images of the two different groups, revealing different degrees of myocardial ischemia (white to red zones after TTC staining). Hearts were subjected to 24 h of LAD ligation. A significant size of damaged tissue was noted in the myocardium of MI group. Mice pretreated with MRS2768 had significantly smaller infarct size. This figure represents the percent of irreversible injury from the total area of the section at 24 h post-LAD ligation. Values represent means ± SE. n = 7 hearts in each group. c The levels of TNF-α in the serum 24 h post-LAD ligation. Values are means ± SE. n = 4 in each group. *p < 0.05 vs MI
Fig. 5
Fig. 5
Histological study of hearts 24 h post-MI: Hematoxylin and eosin (H&E) staining showed neutrophil infiltration to the hearts 24 h post-MI. There was a significant infiltration of neutrophils to the heart tissue in both MI and MI pretreated with MRS2768 mice, n = 4/group (a–c)
Fig. 6
Fig. 6
Histological study of hearts 24 h post-MI using neutrophil marker: Immunostaining showed the infiltration of neutrophils to the infarcted area 24 h post-MI. There was a significant infiltration of neutrophils to the heart tissue compared to sham control (a) in both MI (b) and MI pretreated with MRS2768 (c) mice (n = 4/group). Quantification of immunostaining for neutrophils was done by counting 10 fields per slide. Data are means ± SE, n = 4/group. A significant difference was observed between MI and MI+MRS2768 in neutrophil infiltration (d), p < 0.05
Fig. 7
Fig. 7
Western blot analyses of phosphorylated c-Jun (p-c-Jun) and IκB. Cardiac ischemic extracts were subjected to immunoblotting with antibodies to c-Jun phosphorylated at Ser63. a MI caused elevation in the expression of p-c-Jun. MRS2768-pretreated mice before MI also showed elevation in the expression of p-c-Jun. These figures are representative of western blot analyses of three similar separate experiments in each group. b The activation of IκB in the ischemic cardiac tissue was similar in MI and MRS2768-treated groups
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