Therapeutic effect of DJ1 plasmid vector for cardiovascular ischemic injury gene therapy

Abstract

ABSTRACT: Ischemia/reperfusion injury is a tissue injury which occurs because of reperfusion in tissues with pre-existing ischemic damage. A good blood supply to tissues aids in the survival of ischemic tissue, however because of prolonged ischemia the levels of ATP decrease and pH declines and leading to acidosis. Reduced ATP level leads to an increase in the AMP/ATP ratio, this causes the cessation of intracellular calcium transport and hence induces calcium overload and cell death consequently. In this study we demonstrate the synergistic and antagonistic effect of DJ1 and microR-214 [miR-214] in rescuing myoblast C2C12 cells after ischemia/reperfusion in an in vitro model. Both DJ1 and miRNA-214 were cloned into a hypoxic inducible expression cassette and transfected into the C2C12 myoblast cell-line. We demonstrated that DJ1 and miRNA-214 have synergistic effects in reducing intracellular lactate dehydrogenase [LDH] and intracellular transient calcium levels after reoxygenation compared to control cells [P<0.0001]. We further showed that DJ1 and miRNA-214 reduced cell death via necrosis [P<0.0001]. Western Blotting revealed a decrease in autophagosome formation in LC3II/I ratio and an increase in AKT expression [P<0.0001] in cells transfected with DJ1 and miRNA-214. Using quantitative real time PCR [qRT-PCR] we demonstrated that DJ1 and miRNA-214 significantly reduced the expression of pro-apoptotic factors and autophagy compared to the hypoxia/ reoxygenation control. Our results indicated DJ1 is an endogenous oxidative stress molecule and miRNA-214 is a potent inhibitor of the sodium calcium exchanger channel in reverse mode has different effects on myoblast cell recovery after hypoxia/reoxygenation. DJ1 has the greatest effect to inhibit mitochondrial cell death pathways by possibly acting as a modulator of autophagy. Additionally, we have concluded that miRNA-214 has an inhibitory effect on extrinsic cell death pathways such as necrosis and autophagy. However, they had overlap in some pathways.