Abstract 13219: ER-Selective Autophagy Alleviates ER Stress-Mediated Myocardial Injury

Abstract

Introduction: Increasing lines of evidence suggest endoplasmic reticulum-selective autophagy (ER-phagy) as a cell-protective pathway to mediate degradation of damaged ER. However, the role of ER-phagy in cardiomyocytes and its molecular mechanisms remain unsolved. Hypothesis: Considering that various cardiac loads cause ER stress which contributes to the development of heart failure, we hypothesized that ER-phagy would be a compensatory system for protecting cardiomyocytes against ER stress. Methods and Results: To evaluate ER-phagy in cardiomyocytes, we generated lentivirus (LV) and cardiac-specific transgenic mice (Tg-mice) harboring ss-RFP-GFP-KDEL sequence, an ER-phagy reporter possessing an N-terminal ER signal sequence followed by tandem fluorescence sequences and ER retention signal. Fluorescent microscopic analyses and immunoblotting demonstrated that amino acid deprivation of LV-ss-RFP-GFP-KDEL-tranduced H9c2 (tfH9c2) cells markedly elevated the amount of RFP fragments, suggesting that ER-phagy is increased in tfH9c2 cells. 48 hours of food starvation markedly induced ER-phagy in Tg-mice hearts, as well. Treatment with Doxorubicin (Dox), a cardiotoxic compound inducing ER stress, activated ER-phagy in tfH9c2 cells as evidenced by the increase in RFP fragments detected by immunoblotting (Control vs. Dox: 1.0±0.2a.u. vs. 31.6±6.3a.u.; p<0.05). Consistently, treatment with Dox (20 mg/kg) significantly induced ER-phagy in myocardium of Tg-mice compared with those of saline-treated ones (Saline vs. Dox: 1.0±0.05a.u. vs. 2.5±0.2a.u.; p<0.05). qRT-PCR analyses revealed that treatment with Dox enhanced the expression of CCPG1, an ER-phagy receptor, in tfH9c2 cells (Control vs. Dox: 1.0±0.05a.u. vs. 2.1±0.2a.u.; p<0.05). Furthermore, LV-CCPG1 transduction-mediated silencing CCPG1 of tfH9c2 cells resulted in the reduction of ER-phagy activity, accumulation of pro-apoptotic proteins including cleaved caspase 3 and deterioration of cell survival (Cell viability: Control vs. Knockdown: 79.0±3.3% vs. 62.4±2.7%; p<0.05) in response to Dox treatment. Conclusions: These results suggest that ER-phagy plays a protective role in cardiomyocytes against Dox toxicity, possibly through CCPG1-mediated signaling.