Abstract 16705: Nox4-Derived ROS Production in the Endoplasmic Reticulum Promotes Autophagy During Cardiomyocyte Nutrient Deprivation Through the PHD4/ PERK Pathway
Autophagy is an essential survival mechanism during energy stress in the heart. The signaling mechanism through which energy stress leads to activation of autophagy is poorly understood. We investigated whether Nox4, an enzyme that purposefully generates reactive oxygen species (ROS) at intracellular membranes, is involved in the regulation of autophagy during energy stress in cardiomyocytes (CMs).
Four hours of glucose deprivation (GD) selectively increased production of ROS in the endoplasmic reticulum (ER) but not in mitochondria in CMs, as indicated by the increase in the fluorescence of ER-targeted (1.4 fold, p<0.05), but not of mitochondria-targeted, HyPer, a ROS biosensor. Protein levels and activity of Nox4 were also increased in the ER (3.4 and 2.1 fold, p<0.05 for both), but not in mitochondria, in response to GD. Short hairpin RNA-mediated Nox4 knockdown abrogated GD-induced ROS production (-54%, p<0.05), indicating that Nox4 is largely responsible for ROS production during GD. Selective knockdown of Nox4, but not Nox2, abrogated GD-induced autophagy (-36% of autophagosomes, -67% of autolysosomes, p<0.05). Selective reduction of ROS in the ER with ER-targeted catalase also inhibited autophagy during GD (0.47 fold of LC3II/I ratio, p<0.05), but reduction of ROS in mitochondria with peroxiredoxin3 did not, indicating that Nox4-derived ROS in the ER are required for autophagy activation. Nox4 promoted activation of the PERK pathway by suppressing prolyl hydroxylase 4 during GD. PERK reactivation in Nox4-depleted CMs restored autophagy activation during GD. Nox4 knockdown reduced CM survival (-44%, p<0.05) during GD, which was completely rescued by reactivation of autophagy through Atg7 overexpression. In vivo, Nox4 was activated during nutrient starvation in the mouse heart. Cardiac-specific Nox4 knockout mice presented a defect in cardiac autophagy and reduced systolic function during starvation (FS: LacZ CT 43 ± 3% vs. LacZ KO 27 ± 4%, p<0.05), which was partially rescued by autophagy reactivation through adenovirus-mediated Atg7 cardiac overexpression (FS: Atg7 KO 37 ± 6%, p<0.05). Thus, Nox4 critically mediates autophagy in response to energy stress in CMs by increasing ROS in the ER and by stimulating the PERK pathway.
- © 2013 by American Heart Association, Inc.