Research of the Annexin-A4 protein function in cell plasma membrane recovery and cell viability postelectroporation

Abstract

When short, high-voltage electric pulses are applied, the cell plasma membrane becomes permeable. This process is known as electroporation (EP). It is still unclear how exactly electropermeabilization works.[1] The duration, quantity, and intensity of electric pulses are critical characteristics that impact the effectiveness of EP; high levels of any of these parameters may have a detrimental effect on cell viability.[2] Cell survival post-EP depends on the plasma membrane recovery, underscoring the function of the annexin family proteins, especially annexin A4, which is known to be involved in the processes of cell plasma membrane repair following activation by Ca2+ ions. Although there have been many publications on the influence of calcium on cell plasma membrane recovery after EP, but there is a lack of knowledge regarding the role of proteins in the restoration of cell plasma membrane post-EP. The purpose of this research was to investigate how Ca2+ ions affect the cell plasma membrane’s ability to recover after EP. The response of MCF7 wild-type cells, which have an intact annexin A4 gene, was compared to MCF7-AnxA4-KO cells, which have defective annexin A4 gene expression. The MTS assay was utilized to assess the vitality of the cells, and flow cytometry was employed to determine the dynamics of electropermeabilization and plasma membrane repair by quantifying the amount of propidium iodide permeable cells. Fluorescence microscopy was used to monitor ANX-A4 activity. A single 100 μs electric pulse was used to operate the electroporation at different intensities, using a 2 mM concentration of CaCl2. As a result, MCF7-WT cells are less susceptible to the negative effects of electroporation in comparison to MCF7-AnxA4- (KO) cells. Furthermore, although calcium had no observable effect on cell electropermeabilization, the data indicated that calcium had a negative effect on the survivability of both cell lines. On the other hand, 35 minutes after EP, the amount of permeable cells in both cell lines decreased, which signified cell plasma membrane restoring process that was more pronounced in MCF-WT cell line. Microscopy images indicate that cells electroporated in calcium demonstrate higher AnxA4 activity and faster translocation from cytosol to plasma membrane and nucleus proving that AnxA4 protein is involved in cell plasma membrane recovery after EP.