Loss of O-GlcNAc Transferase activity impairs the dynamic formation of protective stress granules in ischemic cardiomyocytes

Stress granules are transient biomolecular condensates of proteins and RNA formed during stress. Stress granules support cell survival by sequestering components of the translational machinery, suppressing pro-apoptotic signaling, and conserving cellular resources. O-GlcNAcylation, a dynamic modification of cytoplasmic proteins, is an important regulator of stress responses, including stress-granule assembly. O-GlcNAcylation is protective in acute myocardial stress; however, whether O-GlcNAcylation regulates stress granules to protect cardiomyocytes remains unclear. Here, we investigated whether O-GlcNAcylation regulates stress granule formation in cardiomyocytes exposed to sodium arsenite stress or ischemia/reperfusion injury. G3BP1 is an RNA-binding protein that is essential in stress granule formation. Using a G3BP1-EGFP reporter, we found that OGT, the enzyme responsible for installing O-GlcNAc on target proteins, is necessary for efficient stress-granule formation in cardiomyocytes. Importantly, we identified T268 of G3BP1 as an O-GlcNAcylation site in these cells and demonstrated by mutagenesis its functional importance for stress granule assembly. In primary cardiomyocytes subjected to simulated ischemia/reperfusion, stress granules formed transiently during ischemia, and this response was decreased by OGT-knockdown. Similarly, stress granules were transiently detected at the ischemic phase of ischemia/reperfusion injury, and their numbers were reduced in OGT-deficient hearts. Furthermore, pretreatment with sodium arsenite before ischemia/reperfusion was cardioprotective. Likewise, pretreatment with sodium arsenite protected cells from staurosporine-induced death and this effect was decreased by OGT knockdown. Together, these findings identify OGT and O-GlcNAcylation as important regulators of stress-granule assembly in cardiomyocytes, define G3BP1 as an O-GlcNAc-modified effector, and demonstrate that stress granules can act as cytoprotective mediators during acute cardiac injury.