When mammalian cells are exposed to induced electric fields (IEFs), the outer plasma membrane (PM) potential increases, reaching a critical value of 0.2–1 V [1]. This enhances PM permeability to molecules and electrical current [2], a process termed electroporation (EP) or electropermeabilization. EP research has expanded into medical applications, including electrochemotherapy (ECT), gene electrotransfer (GET), and tissue ablation. Reversible EP (RE) is used in ECT and GET, while irreversible EP (IRE) is applied in ablation. Improving the safety and efficacy of these methods requires a deeper understanding of cellular responses to EP, including membrane integrity restoration and homeostasis recovery. Electropermeabilization activates repair mechanisms like exocytosis, pore sealing, and membrane shedding [3]. However, the role of Annexin A4 in pore sealing remains understudied. Annexins, a multifunctional protein superfamily, regulate apoptosis (via PS externalization and Ca²⁺ signaling [4]) and necrosis (via membrane repair and inflammatory responses [5]). Despite their importance, studies on Annexin A4’s role in post-EP membrane repair are limited. Thus, we investigated Annexin A4’s significance in electropermeabilization efficacy, PM integrity recovery, and cell viability after EP.
In this study, we used wild-type MCF7-WT (WT) and MCF7-ANXA4- (KO) cells, in which Annexin A4 protein expression was knocked out using the CRISPR/Cas9 system. The efficiency of electrotransfer and the dynamics of pore resealing were assessed using flow cytometry by evaluating propidium iodide uptake, while cell viability was determined using the MTS assay.
Our findings demonstrate that WT cells exhibit higher viability than KO cells under equivalent pulse intensities. Furthermore, KO cell line demonstrated greater permeabilization levels. However, compared to the WT cell line, KO cell pore closure efficiency had significantly decreased. Additionally, it was observed that the presence of CaCl₂ in the EP medium did not affect electropermeabilization efficiency but negatively impacted the ability of both WT and KO cells to repair plasma membrane and reduced cell viability within the tested parameter range. Notably, KO cells were more sensitive to EP exposure, despite WT cells being morphologically larger. Based on the Shwan equation, larger cells are more susceptible to electric pulses and suggest that the WT cell line should have been more sensitive than the KO line. The increased sensitivity of KO cells to EP indicates that the role of the ANX A4 protein in membrane recovery and maintaining cell viability process after EP is far more significant than previously thought. In summary, this study demonstrated the importance of the Annexin A4 protein in plasma membrane recovery and cell viability maintenance after electroporation.