UTP reduces infarct size and improves mice heart function after myocardial infarct via P2Y2 receptor.

Pyrimidine nucleotides are signaling molecules, which activate G protein-coupled membrane receptors of the P2Y family. P2Y(2) and P2Y(4) receptors are part of the P2Y family, which is composed of 8 subtypes that have been cloned and functionally defined. We have previously found that uridine-5'-triphosphate (UTP) reduces infarct size and improves cardiac function following myocardial infarct (MI). The aim of the present study was to determine the role of P2Y(2) receptor in cardiac protection following MI using knockout (KO) mice, in vivo and wild type (WT) for controls. In both experimental groups used (WT and P2Y(2)(-/-) receptor KO mice) there were 3 subgroups: sham, MI, and MI+UTP. 24h post MI we performed echocardiography and measured infarct size using triphenyl tetrazolium chloride (TTC) staining on all mice. Fractional shortening (FS) was higher in WT UTP-treated mice than the MI group (44.7±4.08% vs. 33.5±2.7% respectively, p<0.001). However, the FS of P2Y(2)(-/-) receptor KO mice were not affected by UTP treatment (34.7±5.3% vs. 35.9±2.9%). Similar results were obtained with TTC and hematoxylin and eosin stainings. Moreover, troponin T measurements demonstrated reduced myocardial damage in WT mice pretreated with UTP vs. untreated mice (8.8±4.6 vs. 12±3.1 p<0.05). In contrast, P2Y(2)(-/-) receptor KO mice pretreated with UTP did not demonstrate reduced myocardial damage. These results indicate that the P2Y(2) receptor mediates UTP cardioprotection, in vivo.

Bax deficiency reduces infarct size and improves long-term function after myocardial infarction.

We have previously found that, following myocardial ischemia/reperfusion injury, isolated hearts from bax gene knockout mice [Bax(-/-)] exhibited higher cardioprotection than the wild-type. We here explore the effect of Bax(-/-), following myocardial infarction (MI) in vivo. Homozygotic Bax(-/-) and matched wild-type were studied. Mice underwent surgical ligation of the left anterior descending coronary artery (LAD). The progressive increase in left-ventricular end diastolic diameter, end systolic diameter, in Bax(-/-) was significantly smaller than in Bax(+/+) at 28 d following MI (p < 0.03) as seen by echocardiography. Concomitantly, fractional shortening was higher (35 +/- 4.1% and 27 +/- 2.5%, p < 0.001) and infarct size was smaller in Bax(-/-) compared to the wild-type at 28 days following MI (24 +/- 3.7 % and 37 +/- 3.3%, p < 0.001). Creatine kinase and lactate dehydrogenase release in serum were lower in Bax(-/-) than in Bax(+/+) 24 h following MI. Caspase 3 activity was elevated at 2 h after MI only in the wild-type, but reduced to baseline values at 1 and 28 d post-MI. Bax knockout mice hearts demonstrated reduced infarct size and improved myocardial function following permanent coronary artery occlusion. The Bax gene appears to play a significant role in the post-MI response that should be further investigated.

Cathepsin B inactivation attenuates the apoptotic injury induced by ischemia/reperfusion of mouse liver.

BACKGROUND: A major mechanism underlying warm ischemia/reperfusion (I/R) injury during liver transplantation is the activation of the caspase chain, which leads to apoptosis. Recently, it was demonstrated that the release of cathepsin B, a cysteine protease, from the cytosol in liver injury induces mitochondrial release of cytochrome c and the activation of caspase-3 and -9, thereby leading to apoptosis. The aim of this study was to ascertain if cathepsin B inactivation attenuates the apoptotic injury due to I/R in mouse liver. METHODS: A model of segmental (70%) hepatic ischemia was used. Eighteen mice were anesthetized and randomly divided into three groups: (1) CONTROL GROUP: sham operation (laparotomy); (2) Ischemic group: midline laparotomy followed by occlusion of all structures in the portal triad to the left and median lobes for 60 min (ischemic period); (3) STUDY GROUP: like group 2, but with intraperitoneal administration of a pharmacological inhibitor of cathepsin B (4 mg/100 g) 30 min before induction of ischemia. Serum liver enzyme levels were measured by biochemical analysis, and intrahepatic caspase-3 activity was measured by fluorometric assay; apoptotic cells were identified by morphological criteria, the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) fluorometric assay, and immunohistochemistry for caspase-3. RESULTS: Showed that at 6 h of reperfusion, there was a statistically significant reduction in liver enzyme levels in the animals pretreated with cathepsin B inhibitor (p<0.05). On fluorometric assay, caspase-3 activity was significantly decreased in group 3 compared to group 2 (p<0.0001). The reduction in postischemic apoptotic hepatic injury in the cathepsin B inhibitor -treated group was confirmed morphologically, by the significantly fewer apoptotic hepatocyte cells detected (p<0.05); immunohistochemically, by the significantly weaker activation of caspase-3 compared to the ischemic group (p<0.05); and by the TUNEL assay (p<0.05). CONCLUSION: The administration of cathepsin B inhibitor before induction of ischemia can attenuate postischemic hepatocyte apoptosis and thereby minimize liver damage. Apoptotic hepatic injury seems to be mediated through caspase-3 activity. These findings have important implications for the potential use of cathepsin B inhibitors in I/R injury during liver transplantation.

Effect of adenosine A2A receptor agonist (CGS) on ischemia/reperfusion injury in isolated rat liver.

Ischemia/reperfusion injury during liver transplantation is a major cause of primary nonfunctioning graft for which there is no effective treatment other than retransplantation. Adenosine prevents ischemia-reperfusion-induced hepatic injury via its A2A receptors. The aim of this study was to investigate the role of A2A receptor agonist on apoptotic ischemia/reperfusion-induced hepatic injury in rats. Isolated rat livers within University of Wisconsin solution were randomly divided into four groups: (1) continuous perfusion of Krebs-Henseleit solution through the portal vein for 165 minutes (control); (2) 30-minute perfusion followed by 120 minutes of ischemia and 15 minutes of reperfusion; (3) like group 2, but with the administration of CGS 21680, an A2A receptor agonist, 30 microg/100 ml, for 1 minute before ischemia; (4) like group 3, but with administration of SCH 58261, an A2A receptor antagonist. Serum liver enzyme levels were measured by biochemical analysis, and intrahepatic caspase-3 activity was measured by fluorometric assay; apoptotic cells were identified by morphological criteria, the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) fluorometric assay, and immunohistochemistry for caspase-3. Results showed that at 1 minute of reperfusion, there was a statistically significant reduction in liver enzyme levels in the animals pretreated with CGS (p < 0.05). On fluorometric assay, caspase-3 activity was significantly decreased in group 3 compared to group 2 (p < 0.0002). The reduction in postischemic apoptotic hepatic injury in the CGS-treated group was confirmed morphologically, by the significantly fewer apoptotic hepatocyte cells detected (p < 0.05); immunohistochemically, by the significantly weaker activation of caspase-3 compared to the ischemic group (p < 0.05); and by the TUNEL assay (p < 0.05). In conclusion, the administration of A2A receptor agonist before induction of ischemia can attenuate postischemic apoptotic hepatic injury and thereby minimize liver injury. Apoptotic hepatic injury seems to be mediated through caspase-3 activity.