Role of O‐GlcNAcylation in Regulating Mitophagy in Cardiomyocytes

Abstract

Acute increases in O‐GlcNAcylation in the heart have been shown to be cardioprotective, which was associated with O‐GlcNAcylation of mitochondrial proteins and increased tolerance of mitochondria to oxidative stress. Mitophagy, a specific type of cargo‐mediated autophagy, is critical in removing damaged mitochondria and has also been shown to contribute to protection against ischemia/reperfusion injury. Therefore, the goal of this study was to determine whether O‐GlcNAcylation contributed to the regulation of mitophagy. Mitophagy was induced in AC16 cardiomyocytes, using 2,3‐dimethoxy‐1,4‐napthoquinone (DMNQ, 20 µM), a redox cycling agent which increases mitochondrial reactive oxygen species (ROS). Protein O‐GlcNAc levels were increased by treatment with the O‐GlcNAcase inhibitor, Thiamet‐G (3 µM). DMNQ treatment had no significant effect on either O‐GlcNAc levels or protein levels of PINK1, which recruits Parkin to damaged mitochondria to initiate mitophagy; however, Thiamet‐G significantly increased total PINK1 levels 2‐fold. Preliminary studies showed that PINK1 is O‐GlcNAcylated under basal conditions, which is markedly decreased by DMNQ; whereas Thiamet‐G prevents the loss of PINK O‐GlcNAcylation.Treatment of AC16 cells with either DMNQ or 4‐Hydroxynonenal (HNE, 30 µM) resulted in significantly decreased mitochondrial maximal oxygen consumption and reserve capacity, which was partially improved if thiamet‐G was added following treatment. Surprisingly pre‐treatment with Thiamet‐G had no protective effect. These results support the concept that protein O‐GlcNAcylation may play a role in regulating mitophagy and protect mitochondria against oxidative stress.