Generation and Characterization of an Inducible Cx43 Overexpression System in Mouse Embryonic Stem Cells

Abstract

Connexins (Cx) are a large family of membrane proteins that can form intercellular connections, so-called gap junctions between adjacent cells. Cx43 is widely expressed in mammals and has a variety of different functions, such as the propagation of electrical conduction in the cardiac ventricle. Despite Cx43 knockout models, many questions regarding the biology of Cx43 in health and disease remain unanswered. Herein we report the establishment of a Cre-inducible Cx43 overexpression system in murine embryonic stem (ES) cells. This enables the investigation of the impact of Cx43 overexpression in somatic cells. We utilized a double reporter system to label Cx43-overexpressing cells via mCherry fluorescence and exogenous Cx43 via fusion with P2A peptide to visualize its distribution pattern. We proved the functionality of our systems in ES cells, HeLa cells, and 3T3-fibroblasts and demonstrated the formation of functional gap junctions based on dye diffusion and FRAP experiments. In addition, Cx43-overexpressing ES cells could be differentiated into viable cardiomyocytes, as shown by the formation of cross striation and spontaneous beating. Analysis revealed faster and more rhythmic beating of Cx43-overexpressing cell clusters. Thus, our Cx43 overexpression systems enable the investigation of Cx43 biology and function in cardiomyocytes and other somatic cells.

Understanding the Role of Connexins in Hepatocellular Carcinoma: Molecular and Prognostic Implications

“…This discrepancy is explained later herein. Cx43 is mainly located in the cytoplasm and barely expressed in areas connecting non-adjacent cells on the cell membrane [148]. In HCC, studies have shown that Cx43 expression is associated with the presence of histological differentiation, multiple foci, vascular tumor thrombosis, and early recurrence [149].…”

18-α-glycyrrhetinic acid alleviates oxidative damage in periodontal tissue by modulating the interaction of Cx43 and JNK/NF-κB pathways

“…Among the 21 connexin isomers identified in the human genome, Connexin43 (Cx43) is the most widely expressed and critical connexin. It plays an essential role in regulating GJs function, controlling cell migration and proliferation, wound repair, and immune regulation (Turovsky et al, 2021;Niemann et al, 2022). In 2013, Kato et al first demonstrated the existence of functional GJs in hPDLCs (Kato et al, 2013).…”