The Mitochondrial Mechanism Underlying NECA’s Cardioprotection Against Ischemia/reperfusion Injury

ObjectiveTo study the mitochondrial mechanism underlying the protective effect of5’-(N-ethylcarboxamido) adenosine(NECA) on ischemia/reperfusion injury in cardiac H9c2 cells.MethodsA simulated ischemia/reperfusion injury model was developed in cardiac H9c2 cells.CCK-8 assay was used to evaluate cell viability. Mitochondrial membrane potential(△Ψm) was measured using Mitochondrial membrane potential assay kit with JC-1(JC-1). Amplex red hydrogen peroxide/peroxidase assay Kit was used to detect the level of intracellular H2O2. Intracellular reactive oxygen species(ROS) levels were determined with DCFH-DA. Mitochondrial ROS were detected with Mitosox red mitochondrial superoxide indicator. Complex I enzyme activity assay kit was used to evaluate the activity of complex I.Results1. Compared to the I/R group, NECA at different concentrations(0.1, 1, 10 μmol/L)prevented the reduction of cell viability(P < 0.05, respectively) with the maxium effect at 1 μmol/L and thus we used this concentration in all the subsequent studies.2. The protective effect of NECA on cell viability in cells experienced I/R was blocked by the adenosine A2 A receptor antagonist SCH58261 and the adenosine A2 B receptor antagonist MRS1706 as well as the combination of the antagonists(P < 0.05, respectively), indicating that the protective action of NECA is attributable to both adenosine A2 A and A2 B receptors.3. I/R reduced △Ψm compared to the control group, and this was reversed by NECA(P < 0.05), suggesting that NECA can modulate the mitochondrial permeablity transition pore(m PTP) opening triggered by I/R. However, SCH58261 and MRS1706, and the combination of the antagonists inhibited the effect of NECA(P< 0.05, respectively), suggesting that NECA may prevent the m PTP opening via both A2 A and A2 B receptors.4. The fluorescence intensities of DCF, Amplex Red and Mito Sox Red were much higher in the I/R group compared to the normal control group, and this was inhibited by NECA(P < 0.05, respectively), pointing to that NECA can prevent mitochondrial ROS generation caused by I/R. This effect of NECA was inihibited by the adenosine A2 A antagonist SCH58261 or/and the A2 B receptor antagonist MRS1706(P < 0.05, respectively), suggesting that NECA suppresses mitochond-rial ROS generation in the setting of I/R via both the adenosine A2 A and A2 B receptors.5. NECA reduced mitochondrial complex I activity at reperfusion compared to the I/R group(P < 0.05), and this effect was blocked by SCH58261 and MRS1706, or the combination of the two antagonists(P < 0.05, respectively), implying that NECA may prevent mitochondrial ROS generation by modulating complex I activity via both the A2 A and A2 B receptors.6. The selective Src tyrosine kinase inhibitor PP2 blocked the effect of NECA on cell viability of cells exposed to I/R(P < 0.05), pointing to that Src tyrosine kinase may mediate the protection of NECA.7. PP2 inhibited the effect of NECA on △Ψm(P < 0.05), indicating that the protective action of NECA on mitochondria is mediated by Src tyrosine kinase.8. PP2 prevented the effects of NECA on the fluorescence intenstities of DCF,Amplex Red and Mito Sox Red(P < 0.05, respectively), suggesting that Src tyrosine kinase may mediate the inhibitory effect of NECA on mitochondrial ROS generation.Conclusions1. Adenosine A2 A and A2 B receptors work in concert to mediate the cardioprotective effect of NECA.2. NECA may induce cardioproteciton by modulating the m PTP opening through inhibiton of mitochondrial ROS generation.3. The inhibitory effect of NECA on complex I activity may accout for the reducedmitochondrial ROS generation.4. Src tyrosine kinase may play a role in the protective action of NECA.