Connexin 43 (Cx43) is the main gap junction protein found in the heart. Previous studies have shown that the gene that codes for Cx43 (GJA1) produces multiple alternatively translated proteins, the most abundant product of this alternative translation is 20kd in size and is named GJA1-20k (20k). Recent work has identified GJA1-20k as a stress response protein and its expression in the setting of ischemic injury reduces infarction size and oxygen demand in the heart. The AC16 cell line is a human cardiomyocyte derived cell line that readily divides in culture, making it an ideal cell model for myocardial cell signaling studies. The purpose of this study is to characterize GJA1-20k expression in this cell line to facilitate mechanistic understanding of the pathways regulating GJA1-20k signaling. We performed western blotting to determine that GJA1-20k is present in the AC16 cell line and this was confirmed with GJA1 siRNA knockdown. When compared to other cell types, the GJA1-20k band in AC16 cells appears as a doublet which disappears with phosphatase treatment indicating that GJA1-20k is phosphorylated in the AC16 cell line. As GJA1-20k has been established as a stress response protein, we treated AC16 cells with Ionomycin and TPA to induce oxidative stress and determine whether 1) endogenous GJA1-20k expression increased and 2) whether exogenous GJA1-20k expression reduces the degree of cell injury death in response to oxidative stress in this cell line. The ultimate goal of this project is to determine whether the AC16 cell line recapitulates the phenotype observed in vivo in response to GJA1-20k expression.