Abstract 18056: Activation of Nox4 Promotes Cardiomyocyte Autophagy and Survival During Nutrient Starvation
Autophagy is an adaptive mechanism for cardiomyocytes (CMs) during energy deprivation (ED), such as myocardial ischemia. The signaling mechanism activating autophagy during ED is not fully understood. ED is accompanied by increases in reactive oxygen species (ROS). Nox4, an enzyme purposefully generating O2-/H2O2, is a major source of ROS in the heart. We investigated whether Nox4 is involved in the regulation of autophagy and survival in CMs during ED. After 4 hours of glucose deprivation (GD) in cultured CMs, ROS were increased, as indicated by dihydroethidium (DHE) and 2',7'-dichlorofluorescin diacetate (DCF) staining (1.5 ± 0.2 and 2.3 ± 0.2 fold, p<0.05), which was accompanied by upregulation of Nox4 (2.4 fold, p<0.05). Downregulation of Nox4 by shRNA significantly reduced DHE and DCF staining (0.5 and 0.4 fold, p<0.05). Nox4-dependent production of O2- in response to GD was not increased in mitochondria. However, it was significantly increased in the microsomal fraction (3.4 ± 0.8 fold, p<0.05), indicating that Nox4 activation in the endoplasmic reticulum (ER) contributes to the increase in ROS during GD. Consistently, immunostaining analysis showed that Nox4 was co-localized with ER during GD. Survival of CMs after 24 hours of GD was significantly lower in the presence of Nox4 knock-down than in its absence (32 ± 4% vs. 56 ± 3%, p<0.05), suggesting that Nox4 activation during GD is protective. GD-induced activation of autophagy was significantly attenuated in the presence of Nox4 knock-down, as indicated by suppression of LC3II expression (0.4 fold vs. control GD, p<0.05) and accumulation of p62 (2.5 fold vs. control GD, p<0.05). Reactivation of autophagy by Atg7 overexpression significantly rescued the survival of CMs during GD in the absence of Nox4 (2.2 ± 0.1 fold, p<0.05), suggesting that downregulation of Nox4 promotes cell death through suppression of autophagy. Mice with cardiac-specific Nox4 deletion exhibited a significant reduction in cardiac function (LVFS, control 43 ± 2 vs. KO 29 ± 3 %, p<0.05) and autophagy (LC3II, 0.5 fold vs. control, p<0.05) during starvation, suggesting that Nox4 controls autophagy and plays an adaptive role during ED in vivo. In conclusion, Nox4-dependent ROS production promotes autophagy and survival in CMs during ED.
- © 2012 by American Heart Association, Inc.