Study On Mitochondrial Mechanism Of P19 Cells Differentiating Into Cardiomyocytes

Vertebrate heart is the first formed and functional organ during embryonic development, which happens to go through a series of important morphological events, including cell decision, cell migration and cell differentiation to ensure normal fetal heart coordinated development. Slight abnormalities in this process may affect heart development, resulting in congenital heart disease. Although the study of embryonic heart formation from genetic and molecular level provides some useful clues for the mechanisms of congenital heart disease’ development, it is far from being able to meet human beings’ need for interpreting the complex mechanisms of regulating normal heart development. Among all types of cells in mammal, myocardial cells have the greatest amounts of mitochondrial density, and slight change of mitochondrial function can arouse great changes in myocardial cells. Therefore, in recent years, people have paid more and more attention to the importance of mitochondrion in heart development.This study speculates the role of mitochondria during the differentiation of P19cells (mesoderm-derived cell lines, which can be directed to differentiate into myocardial cells with the ability of beating) into cardiomyocytes by monitoring mitochondrial change of morphology and function. In addition, it is well known that carbonyl-cyanide-on-trifluoromethoxy basic hydrazone (carbonyl cyanide p-trifluoromethoxyphenylhydrazone, FCCP) is a mitochondrial uncoupler of oxidative phosphorylation, which can damage the inner mitochondrial membrane, and lead to mitochondrial membrane depolarization and a proton leakage, resulting in increased oxygen consumption and reduction in ATP production without affecting intracellular phosphorylation, thus mitochondrial uncoupler FCCP is used to investigate the effect of FCCP on the differentiation of cell-derived cardiomyocytes in P19cells. Part ⅠMorphological and functional changes of mitochondria during the differentiation of P19cells into cardiomyocytesObjective:To observe dynamic mitochondrial changes in regard to morphology and function during the differentiation of p19cells into cardiomyocytes.Methods:P19cells were induced to differentiate with1%DMSO. The ultrastructure of the mitochondria in P19cells was visualized using transmission electron microscopy after differentiation. The levels of mitochondrial DNA copy numbers were detected by real-time quantitative PCR. The levels of ATP production was detected using a photon-counting luminometer. The reactive oxygen species (ROS) were detected by DCFDA (2’,7’-Dichlorofluorescein diacetate, DCFDA) with a FACScan flow cytometer using Cell Quest software. Cellular ATP production was determined using luciferase-based luminescence assay.Results:1) During the differentiation of P19cells into myocardial cells in the suspension phase, the morphology of mitochondria changed from the granular patterns to round shape; mitochondrial DNA copy number, cellular ATP and ROS production were reduced.2) In the remaining course of P19cells into cardiac cells, the number of mitochondria increased; mitochondrial cristae became longer; mitochondrial DNA copy number, cellular ATP and ROS production gradually increased.Conclusion:In order to adapt to the functional needs of myocardial cells, a series of changes in morphology and function of mitochondria occurred in the process of P19cells differentiation into cardiomyocytes. Part ⅡEffect of FCCP on the differentiation of P19cells into cardiomyocytesObjective:To investigate the effect of FCCP on the differentiation of P19cells into cardiomyocytes.Methods:In the beginning of differentiation, P19cells were treated with5μM FCCP and untreated cells were used as control group in two days, the morphologic changes of P19cells into cardiomyocytes at indicated time during the differentiation were observed and photographed under an inverted microscope. The expressions of GATA4, NKX2.5and cTnT were monitored using Real-time PCR on the differentiated process.Results:1) Compared to the control group, the embryoid bodies formed in the FCCP treated group appeared smaller and were loosely structured.2) The expressions of GATA4, NKX2.5and cTnT could be observed in the two groups, while FCCP treated cells present with slower changes during the differentiation.Conclusion:FCCP inhibited the differentiation of P19cells to cardiac myocytes.