THE MULTIFACETED ROLE OF ANGIOTENSIN II IN SKELETAL MUSCLE AUTOPHAGY SUPPRESSION IN HEART FAILURE

Abstract

Chronic skeletal muscle (SKM) atrophy, or cachexia, occurs in diseases such as congestive heart failure (CHF), and are often associated with high levels of angiotensin II (ANG-II). We have shown that ANG-II infusion reproduces major aspects of CHF-induced SKM atrophy and causes mitochondrial damage and dysfunction in SKM. ANG-II also inhibits autophagy, and a combination of these effects likely causes accumulation of damaged mitochondria, leading to SKM atrophy. However, the precise molecular mechanisms whereby ANG-II inhibits autophagy are unknown. We hypothesize that ANG-II infusion suppresses autophagy in SKM via activation of mTOR and inhibition of FOXO3a. To test this hypothesis, we infused ANG-II into twelve-week-old FVB mice via osmotic minipumps for 7 days. Expression of autophagic markers and FOXO3a target genes were analyzed in SKM by western blotting and RT-qPCR, respectively. Colchicine and 24-hours STR treatment were included to assess autophagic flux in vivo. We found that in the presence of colchicine, LC3B lipidation was decreased in the ANG-II group, indicating that ANG-II inhibits autophagy at the APS formation step. Furthermore, ANG-II increased p62 and PINK1 and decreased BNIP3, suggesting that ANG-II-mediated reduction of autophagy causes disruption of the normal process of damaged mitochondrial clearance via mitophagy. To quantify the number and size of the APS and ALS were quantified in SKM frozen sections with a combination of fluorescent imaging and machine-leaning based image segmentation. We observed reduced number of APS and ALS in ANG-II group compared to control, suggesting that ANG-II suppresses APS and ALS formation. Mechanistically, ANG-II likely inhibits APS and ALS formation via mTOR and FOXO3a pathways: our data indicate that ANG-II increased mTOR phosphorylation (i.e. mTOR activation) and its downstream targets. ANG-II increased FOXO3a phosphorylation (i.e. FOXO3a inhibition), which was associated with reduced mRNA expression of FOXO3a target genes, such as LC3B and BNIP3. In summary, our study suggests that ANG-II-mediated activation of mTOR and inhibition of FoxO3a leads to suppression of APS and ALS formation in SKM. It is suggested that suppression of autophagy via these pathways likely disrupts clearance of damaged mitochondria, leading to SKM atrophy. These results provide the rationale for future development of therapies to treat CHF-induced cachexia via targeting the mTOR-FOXO3a axis to restore normal levels of autophagy.