Enhancement of dynein‐mediated autophagosome trafficking and autophagy maturation by ROS in mouse coronary arterial myocytes (1077.10)

Abstract

We have recently demonstrated that dynein‐mediated autophagosome (AP) trafficking contributes to the formation of autophagolysosomes (APLs) and autophagic flux process in coronary arterial myocytes (CAMs). It remains unknown how the function of dynein as a motor protein for AP trafficking is regulated under physiological and pathological conditions. The present study tested a hypothesis that the dynein‐mediated autophagy maturation is regulated by a redox signaling mechanism associated with NADPH oxidase. First, we showed that in primary cultures of CAMs reactive oxygen species (ROS) including H2O2 and O2‐. generated by xanthine/xanthine oxidase (X/XO) significantly increased APs as shown by largely increased level of LC3B, an AP marker. It was also found that ROS dramatically increased the number of APLs in CAMs as shown by flow cytometic analysis of APLs using acridine orange. Furthermore, fluorescent confocal microscopy demonstrated that both ROS increased the fusion of APs with lysosomes in CAMs as detected by increased colocalization of LC3B with lysosome marker Lamp1. All these changes in APs formation and autophagic flux were also enhanced by overexpression of NADPH oxidase‐1 (Nox1) gene in CAMs after cDNA transfection. Mechanistically, we found that enhanced autophagic flux by ROS was associated with activation of the motor protein‐dynein, which increased APs movement in CAMs. This ROS‐induced APs movement was abolished by either inhibition of dynein activity with EHNA or disruption of the dynein complex. Lastly, another stimulus, high glucose, was used to increase the APLs formation in CAMs, which was abolished by either Nox1 inhibitor, ML117 or dynein inhibitor, EHNA. Taken together, these data suggest that ROS increases autophagic flux through enhanced dynein‐dependent APs trafficking in CAMs.Grant Funding Source: HL057244, HL091464 and HL075316