Excessive production of nicotinamide adenine dinucleotide phosphate oxidase 4-dependent reactive oxygen species suppresses cardiomyocyte differentiation from embryonic stem cells

BACKGROUND: Reactive oxygen species (ROS), including superoxide anion (O2) and hydrogen peroxide (H2O2), have been recognized as specific second messengers in signaling cascades involved in the growth and differentiation of cells.The generation of excessive ROS initiates cardiomyocyte apoptosis. This paper is aimed to corroborate the hypothesis that excessive amounts of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX) 4-dependent ROS can suppress cardiomyocyte differentiation from embryonic stem (ES) cells by induction of apoptosis.OBJECTIVE: To investigate the role of NOX4 ROS on cardiomyocyte differentiation.DESIGN: Randomized and controlled trial observation.SETTING: Beijing Institute of Geriatrics, Beijing Hospital, Key Laboratory of Geriatrics, Ministry of Health.MATERIALS: ES cells were preserved by the laboratory of Beijing Institute of Geriatrics, Beijing Hospital, Key Laboratory of Geriatrics, Ministry of Health, while other reagents were purchased from Sigma Company without specific notification.METHODS: The experiment was carried out in the Beijing Institute of Geriatrics, Beijing Hospital, Key Laboratory of Geriatrics, Ministry of Health from October 2003 to August 2005. ①Mouse ES cells were differentiated into embryoid bodies (Ebs). At day 4, Ebs were managed for one hour with different concentrations (1, 10, 100, 1 000 nmol/L) of H2O2, and generation of cardiomyocytes was observed at day 8 to analyze the effect of ROS on cardiomyocyte differentiation.② CGB8 cells transfected with pcDNA3.1 and pcDNA3.1-NOX4 respectively were adopted, while those untransfected CGB8 cells were taken as controls. A quantitative NBT (nitro blue tetrazolium) test was used to measure NOX4 ROS generation. The levels of NOX4 mRNA and MLC2v protein were assayed by RT-PCR and western blotting, respectively.③ROS scavenger N-acetylcysteine (5 mmol/L) and catalase (200 U/mL) managed for 2 hours before transcription were taken as controls to observe the apoptosis of CGB8 cell after NOX4 overexpressed and the expressions of p21, p53 and Bcl-2 of NOX4-transfected CGB8 cells simultaneously. And the apoptosis of ES cells was detected by Hoechst staining, TUNEL assay and DNA fragmentation.MAIN OUTCOME MEASURES: ①the role of ROS on cardiomyocyte differentiation.②the expression of NOX in ES cells.③ the production of ROS, differentiation of cardiomyocyte and apoptosis of ES cells after NOX4 overexpressed.④the feasibility that p53, p21 and Bcl-2 are involved in the apoptosis induced by the overexpression of NOX4.RESULTS: ①Different concentrations of ROS play different roles on cardiomyocyte differentiation. The exposure of Ebs to 1-100 nmol/L H2O2 for 2 hours at day 4 of culture leaded to an enhanced beating activity (P ＜ 0.01 or 0.001), whereas 1 μmol/L H2O2 depressed cardiomyocyte differentiation as compared with control conditions (P ＜ 0.001). ②NOX4 was highly expressed in ES cells, while NOX1 and NOX2 were absent and only a weak band of NOX3 was detected. The results from RT-PCR revealed that NOX4 overexpressed in CGR8 transfected with pcDNA3.1-NOX4 as compared to the control.③NBT result proved that highly-expressed NOX4 produced the excessive amounts of ROS (P ＜ 0.05). The beating activity was remarkably reduced in NOX4-overexpressing Ebs as compared to wild-type or mock-transfected Ebs (P ＜0.01). Moreover, a severe reduction of MLC2v protein in NOX4-overexpressing Ebs was also observed by western blot analysis.④p21 and p53 may be responsible for the NOX4-induced apoptosis. P53-/- cells R72D27 transfected by NOX4 did not induce apoptosis. NOX4 overexpression induced ES cell apoptosis could be prevented by overexpression of Bcl-2.CONCLUSION: The findings of this paper highlight the role of NADPH oxidase NOX4 in cardiomyocyte differentiation from ES cells. P53, p21 and Bcl-2 could be involved in the apoptotic pathway.