Forkhead box O (FOXO) 3 modulates hypoxia-induced autophagy through AMPK signalling pathway in cardiomyocytes.

Yunpeng Chi,Conghong Shi,Chengjun Guo,Yang Zhao

doi:10.1042/bsr20160091

Abstract

Autophagy is promoted as a response to such environmental stress conditions as ATP depletion and excessive accumulation of reactive oxygen species (ROS). Multiple signalling pathways, including AMP-activated protein kinase (AMPK), are indicated to promote autophagy in ischaemic/hypoxic (I/R) heart. However, it's far more to clarify the orchestrated cross-talk between AMPK and other signalling pathways in the autophagy. In the present study, we investigated the autophagy induction by hypoxia in Rat H9C2 cardiomyocytes with LC3-EGFP reporter, EM and Western blot analysis. Then, we examined the promotion of forkhead box O (FOXO) 3, one member of FOXO transcriptional protein family, by hypoxia in Rat H9C2 cells and determined the mediation of FOXO 3 in the hypoxia-induced autophagy in H9C2 cells. In addition, we investigated the role of AMPK signalling in the FOXO3-mediated, hypoxia-induced autophagy in H9C2 cells. It was demonstrated that hypoxia induced significant autophagy in H9C2 cells, via promoting autophagic vesicles, inducing the conversion of LC3-I to LC3-II and up-regulating autophagy-related (ATG) markers. Moreover, FOXO3 was up-regulated by the hypoxia in H9C2 cells; and the knockdown of FOXO3 significantly reduced the hypoxia-induced autophagy. In addition, AMPK signalling was significantly promoted by hypoxia in H9C2 cells, and the chemical manipulation of AMPK exerted significant influence on the hypoxia-induced autophagy and on the FOXO3 level. In conclusion, FOXO3 regulated the hypoxia-induced autophagy in cardiomyocytes, and AMPK mediated the FOXO3 promotion during the autophagy induction by hypoxia, implying the key regulatory role of FOXO3 and AMPK signalling in the hypoxia-induced autophagy in cardiomyocytes.

Highlights

Ischaemic/hypoxic (I/R) heart disease is widely conceived as the single leading cause of death worldwide
To confirm the autophagy induction by hypoxia, we examined the autophagosome in the H9C2 cells under hypoxia via EM, the representative ultra-structures of the autophagosome under EM microphotography were found in H9C2 cells under hypoxia, rather than in H9C2 cells under normoxia (Figure 1B)
We analysed the expression of ATG and autophagyregulated genes, such as LC3, mammalian target of rapamycin (mTOR) and Atg7 and the expression of hypoxia-inducible factor (HIF)-1α, which is up-regulated by hypoxia [21]

Summary

Introduction

Ischaemic/hypoxic (I/R) heart disease is widely conceived as the single leading cause of death worldwide. The percentage keeps in high of patients suffering from acute myocardial infarction (MI) and experiencing sudden cardiac death [1]. We are far from clear understanding the mechanisms underlining the myocardial ischaemia/infarction, though there are accumulating and massive studies focusing on the subject. Autophagy is a dynamic self-degradation process for cellular components by cellular lysosome under a stringent regulation [3,4], and has been recognized to involve in the ischaemic heart disease [5]. Autophagy normally maintains at a low level in heart, and is sharply promoted as a response to such environmental stress conditions as ATP depletion, excessive reactive oxygen species (ROS) and mitochondrial dysfunction [6,7]

Methods

Results

Conclusion