Phospholipase A2 inhibitor attenuates NOx production and myocardial damage in the cardiac allograft

Abstract

Ischemia and reperfusion injury occurring during donor heart preservation and transplantation results in cardiac allograft damage and dysfunction. Myocyte death by apoptosis together with decreased myocyte function is caused by high concentrations of NOx (nitrite/nitrate) generated by Nitric Oxide Synthase-2 (NOS-2), which is upregulated during cardiac ischemia. Activation of Type A2 phopholipase (PLA2) and its products induces multiple inflammatory mediators which then induce the expression of NOS-2, cell death and cardiac dysfunction. We tested the efficacy of a novel PLA2 inhibitor (KH064) in reducing myocardial damage following donor heart preservation. The hearts of two groups of Lewis rats (n=6/group) were harvested and subjected to 10 hours of cold ischemia after flushing with University of Wisconsin (UW) preservative solution via the aortic root. In the treated group, 3mg/kg of PLA2 inhibitor was added to the UW solution. DNA fragmentation was evaluated using the terminal transferase UTP nick end labeling (TUNEL) assay, with double labeling for desmin to highlight apoptotic cardiac myocytes. Immunohistochemistry was used to study DNA repair (proliferative cell nuclear antigen, PCNA). The expression of NOS-2 (mRNA, protein) and production of NOx was measured. Cardiomyocyte damage was significantly reduced with myocardial apoptosis detected in 5.2±0.6/100 myocytes in untreated and 0.5±0.2/100 myocytes in treated rats (p<0.001). Endothelial apoptosis also decreased from 23.6±1.9 in untreated to 6.5±0.8 cells/high power field in treated hearts (p<0.001). PCNA immunohistochemistry showed a reduction from 18.3±1.9 in control to 12.6±1.5 positive nuclei per high power field in treated animals (p=0.02). The production of NOx from the myocardium was 1.8±0.23 in the control and 0.84±0.07 mmol/mcg in the treated group (p<0.001). These findings suggest that production of NOx, myocardial damage, and cardiomyocyte death in cardiac allografts may be reduced by the inhibition of PLA2 during donor heart preservation.