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

Xu M, Li XX, Chen Y, Pitzer AL, Zhang Y, Li PL
Source: J Cell Mol Med
Publication Date: (2014)
Issue: 18(11): 2165-75
Research Area:
Gene Expression
Basic Research
Culture Media:
4D-Nucleofector™ X-Unit
CAMs coronary arterial myocytes) were trypsinized and centrifuged at 90 9 g for 10 min. The cell pellet was resuspended in 100 ll P1 Nucleofection solutions (Lonza) for Nucleofection (with the program code CM137). The program was chosen based on the fact that Nucleofection efficiency was over 80% as analysed by flow cytometry by using control GFP plasmids. For each Nucleofection sample, 2 µg plasmid DNA was added in 100 ll P1 Nucleofection solution. After Nucleofection, the cells were cultured in DMEM medium for 24 hrs and then were ready for treatment.
Dynein-mediated autophagosome (AP) trafficking was recently demonstrated to contribute to the formation of autophagolysosomes (APLs) and autophagic flux process in coronary arterial myocytes (CAMs). 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 CAMs, reactive oxygen species (ROS) including H2 O2 and O2 (-.) (generated by xanthine/xanthine oxidase) significantly increased dynein ATPase 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 (DCTN2) 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 CAMs. In parallel, all these changes were also enhanced by overexpression of NADPH oxidase-1 (Nox1) gene in CAMs. Incubation with high glucose led to a marked O2 (-.) production compared with normoglycaemic CAMs, while Nox1 inhibitor ML117 abrogated this effect. Moreover, ML117 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 CAMs.