Functions Of Demethylase In The Differentiation Of Embryonic Stem Cells To Cardiomyocytes And The Underlying Molecular Mechanisms

Aim: To investigate the function of histone demethylase in the differentiation of embryonic stem cells into cardiomyocytes and illustrate the underlying molecular mechanismContents: We identifed the histone demethylase that is potentially involved in the differentiation of embryonic stem cells to cardiomyocytes. Then we demonstrated its function during the differentiation process and illustrated the underlying molecular mechanism.Methods: We first used Q-PCR to identify the histone demethylases that demonstrate specific expression pattern during cardiomyocytes differentiation and found PHF8 as a candidate gene. Then we knockdown PHF8 in mouse embryonic stem cell by RNAi and knockout PHF8 by homologous recombination. The self-renewal ability of knockout cell line was examined by analyzing the expression of multi-potential markers and alkaline phosphatase staining. The survivability was evaluated by MTT assay. The proliferation of knockout cells was examined by Brd U labeling and cell cycle analysis. Apoptosis of knockout cell was evaluated by annexin V stainging and TUNEL assay. The differentiation ability of knockout cell to the three germ layers was examined by analyzing the expression of markers of early differentiated cells and terminal differentiated cells.Results: By screening the expression pattern of 18 histone dymethylases during the differentiation of embryonic stem cell to cardiomyocytes, we found that the expression level of PHF8 increased dramatically in the first day of differentiation and then decreased progressively during latter stages of differentiation. The unique expression pattern indicates that PHF8 may play a critical role in cardiomyocyte differentiation. Through analyzing the PHF8 knockout embryonic stem cells, we found that PHF8 was not critical for self-renew of ES cells. PHF8 deficiency promoted the differentiation of ES cells to mesoderm and endoderm but inhibited the differentiation of ES cells to ectoderm. Therefore, PHF8 knockoutES cells demonstrate greater ability to differentiate into cardiomyocytes. Further investigation illustrated that PHF8 deficiency inhibited the apoptosis of ES cells during the differentiation process. Especially, it inhibited the apoptosis of mesoderm progenitor cells at early stage. The mechanism study demonstrated that although PHF8 deficiency promoted the expression of caspase 3, it did not induce caspase 3 mediated apoptosis. Finally, we found that PHF8 deficiency inhibited the expression of apoptosis induce factor(AIF) which in turn inhibited the apoptosis of ES cells during differentiation.Conclusion: PHF8 deficiency did not affect the self-renew of ES cells. However, it promoted the differentiation of ES cells into mesoderm via inhibiting the apoptosis of ES cells during the differentiation process, which in turn promote the differentiation of ES cells to cardiomyocytes.