Abstract P045: Two Forms of Autophagy in the Heart: Constitutive Autophagy via an LC3-Dependent Pathway and Starvation-Induced Autophagy via a Rab9-Dependent Pathway

Abstract

Autophagy is an intracellular bulk degradation process in which cytosolic proteins/organelles are sequestered into double-membrane vesicles termed autophagosomes to be fused with lysosomes for degradation. Recent evidence suggests that an alternative form of autophagy, which utilizes distinct sources of membrane for autophagosome formation, may exist. Whether alternative autophagy exists in the heart is unknown. Cardiac-specific Atg7 -KO ( Atg7 -CKO) and systemic Ulk1 -KO mice were subjected to starvation for 48 hours. LC3-II expression and p62 degradation were increased in wild-type (WT) mice after starvation (ST), indicating increased autophagy. In Atg7 -CKO mice, LC3-II expression was reduced and p62 was markedly increased at baseline and in response to ST, indicating inhibition of LC3-dependent autophagy. In contrast, in Ulk1 -KO mice, neither LC3-II nor p62 was affected at baseline or in response to ST. However, autolysosome formation evaluated by Lamp2/Rab9 staining, indicators of unconventional autophagy, was abolished. Furthermore, the level of polyubiquitinated protein was increased after ST. Echocardiographic analyses showed that fractional shortening (FS) was maintained after ST in WT mice (38%). However, in Atg7 -CKO mice, FS deteriorated at baseline (22%) but was not further affected by ST (20%). In contrast, in Ulk1 -KO mice, FS was relatively preserved at baseline (32%) but remarkably decreased during ST (19%). After glucose deprivation (GD) GFP-LC3 was co-localized with calnexin (ER marker) in Ad-sh-Control- and Ad-sh-Ulk1-transduced, but not in Ad-sh-Atg7-transduced, cultured cardiomyocytes (CMs) in vitro. In contrast, Lamp2 was co-localized with TGN46 (Golgi marker) in Ad-sh-Control and Ad-sh-Atg7-transduced, but not in Ad-sh-Ulk1-transduced, CMs after GD. These results suggest that the autophagosomes are derived from ER in LC3-dependent autophagy and Golgi in Rab9-dependent autophagy in CMs. Atg7 is required for maintaining cardiac function at baseline in an LC3-dependent manner, whereas Ulk1 is required for preserving cardiac function during ST, possibly through stimulation of Rab9-dependent autophagy. These results support the presence of alternative autophagy in the heart.