Influence of Exogenous PlGF on Apoptosis in the H9c2 Cardiomyoblast Cell Line

Abstract

Placenta growth factor (PlGF) is known to inhibit apoptosis in cells such as trophoblast or endothelial cells. Although PlGF is expressed in heart tissue in vivo and in vitro, little is known about its antiapoptotic effects in cardiomyocytes. H9c2 cells (a rat cardiomyoblast cell line) were used to determine if PlGF can protect these cells from oxidative-induced apoptosis. Cells were cultured and then treated with hydrogen peroxide (H2O2) to induce apoptosis. An H2O2 dose response curve was performed and showed increased concentrations of H2O2 were associated with increased percent apoptosis: 25uM H2O2 = 36.77±8.9%; 50uM H2O2 = 47.53±4.6%; and 100uM H2O2 = 61.36±5.1% apoptosis. Some cultures were then pre-treated with either 10ng/mL or 50ng/mL of exogenous recombinant mouse P1GF for 24 hours and then incubated with 50uM H2O2 for 2 hours. Cells were stained with 4',6-diamidino-2-phenylindole (DAPI), a fluorescent stain that binds strongly to DNA and then fixed with ice cold acetone. Apoptotic cells were recognized by the condensed, fragmented, and degraded nuclei with fluorescence microscopy. Random fields of cells from each treatment group were chosen to count the apoptotic cells and percent of apoptosis was calculated for each field. Control cultures showed 12.41±0.5% apoptosis, H2O2+0ng/mL PlGF = 46.49±1.5% apoptosis, H2O2+10ng/mL PlGF = 30.48±5.9% apoptosis, and H2O2+50ng/mL PlGF = 33.70±5.8% apoptosis. In conclusion, we have developed a consistent method to induce apoptosis in H9c2 cells and used this to show that although exogenous PlGF tended to protect H9c2 cells from oxidative-induced apoptosis, this difference did not attain statistical significance.