Abstract Body: Background: Oxidative stress is a hallmark of cardiovascular (CV) diseases. The potential involvement of Galectin-3 (Gal-3) has been proposed in CV pathological conditions, but its role in reactive oxygen species (ROS)-induced cardiomyocyte damage remains elusive.

Hypothesis: Oxidative stress leads to ruptures of membrane-bound organelles including lysosome. Intracellular Gal-3 of cardiomyocytes may interact with glycans exposed by ROS-damaged organelles to modulate downstream cellular signaling.

Aims: To investigate whether intracellular Gal-3-glycan interactions may occur in ROS-treated cardiomyocytes and how such interactions may regulate cell survival or death.

Methods and Results: Using cardiomyoblast H9c2 cells exposed to hydrogen peroxide (H₂O₂) to be the cell model, we found that oxidative stress induced significant lysosomal damage followed by cell death. The immunofluorescence imaging showed that cytosolic Gal-3 aggregated into small puncta, which were colocalized with LAMP2. To examine whether the aggregation of Gal-3 was contributed by ROS-induced damage, catalase was ectopically expressed in H₂O₂-treated cells and the formation of Gal-3 puncta was attenuated. These results suggest that intracellular Gal-3 interacts with glycans presented by the ruptured lysosomes in H₂O₂-treated H9c2 cells, leading to puncta formation. To further delineate whether these Gal-3 puncta show a functional role in cell survival under oxidative stress, we knocked endogenous Gal-3 down in H9c2 cells by RNAi and found that Gal-3 ablation protected the cells from ROS-induced injuries. Moreover, the ectopic expression of N-terminally truncated Gal-3, which loses its protein-interaction function but preserved the carbohydrate-recognition ability, also prevented H9c2 cells from oxidative stress-caused damage.

Conclusions: ROS-induced lysosomal rupture leads to intracellular Gal-3-glycan interactions, further recruiting cytosolic proteins and activating downstream cell death signaling cascades. Such intracellular Gal-3-glycan interactions may contribute to the cardiomyocyte injuries under oxidative stress.