Abstract 3386: Differentiation of Adult Cardiac Stem Cells is Induced by Inhibition of Redox Effector Factor-1
Redox effector factor-1 (Ref-1) plays an essential role in DNA repair and redox regulation of several transcription factors including activator protein-1, p53 etc. In the present study we assessed the role of Ref-1 in maintaining the redox status and the survivability of adult cardiac stem cells challenged with sub-toxic level of hydrogen peroxide (H2O2) under inhibition of Ref-1 by RNA interference. Treatment of cardiac stem cells with low concentration (10 μM) of H2O2 for 24 hrs induced the expression of Ref-1 followed by survival signaling via phosphorylation of Akt at Ser 473. However, inhibition of Ref-1 expression via Ref-1 siRNA treatment followed by addition of H2O2 extensively induced the level of intracellular reactive oxygen species (ROS) identified using redox sensitive dye 5-(and-6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate, acetyl ester. Further, the components NADPH oxidase like p22 phox, p47 phox and Nox4 were significantly activated in cells treated with Ref-1 siRNA and H2O2. The mRNA expression of cardiac differentiation markers (Nkx2.5, MEF2C, GATA4 and α-actinin) were significantly elevated in cells treated with Ref-1 siRNA followed by H2O2 treatment. Moreover, our confocal microscopic images showed that the expression of Nox4 and Nkx2.5 were colocalized; and the expression of Nkx2.5 was found to be colocalized with α-actinin in differentiated cardiac stem cells previously treated with Ref-1 siRNA and H2O2. The release of cell death marker enzyme lactate dehydrogenase into the culture medium, and the percentage of cells undergoing apoptosis analyzed by terminal deoxynucleotidyl transferase dUTP nick end labeling were significantly elevated in Ref-1 siRNA followed by H2O2 treated stem cells. Ref-1 inhibition elevated the level of p53 (both mRNA and protein) and at the same time the phosphorylation of Akt was diminshed. Treatment with ROS scavenger N-acetyl-L-cysteine attenuated Ref-1 siRNA-mediated induction of NADPH oxidase and cardiac differentiation. In conclusion, our results indicate that Ref-1 plays an important role in maintaining the redox status of cardiac stem cells and protects them from oxidative injury-mediated cell death and differentiation.