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

Dynein‐mediated autophagosome ( AP ) trafficking was recently demonstrated to contribute to the formation of autophagolysosomes ( APL s) and autophagic flux process in coronary arterial myocytes ( CAM s). However, 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 whether the dynein‐mediated autophagy maturation is regulated by a redox signalling associated with lysosomal Ca 2+ release machinery. In primary cultures of CAM s, reactive oxygen species ( ROS ) including H 2 O 2 and O 2 −. (generated by xanthine/xanthine oxidase) significantly increased dynein ATP ase activity and AP movement, which were accompanied by increased lysosomal fusion with AP and APL formation. Inhibition of dynein activity by (erythro‐9‐(2‐hydroxy‐3‐nonyl)adenine) ( EHNA ) or disruption of the dynein complex by dynamitin ( DCTN 2) overexpression blocked ROS ‐induced dynein activation, AP movement and APL formation, and resulted in an accumulation of AP along with a failed breakdown of AP . Antagonism of nicotinic acid adenine dinucleotide phosphate ( NAADP )‐mediated Ca 2+ signalling with NED ‐19 and PPADS abolished ROS ‐enhanced lysosomal Ca 2+ release and dynein activation in CAM s. In parallel, all these changes were also enhanced by overexpression of NADPH oxidase‐1 (Nox1) gene in CAM s. Incubation with high glucose led to a marked O 2 −. production compared with normoglycaemic CAM s, while Nox1 inhibitor ML 117 abrogated this effect. Moreover, ML 117 and NED ‐19 and PPADS significantly suppressed dynein activity and APL formation caused by high glucose. Taken together, these data suggest that ROS function as important players to regulate dynein‐dependent AP trafficking leading to efficient autophagic maturation in CAM s.