Regulation of Cardiac Autophagic Flux In Vivo by the Ubiquitin Ligase Muscle Ring Finger‐1 (MuRF1)

Abstract

The ubiquitin‐proteasome and autophagy systems are comdplementary protein degradation pathways that play a critical role in protein quality control in cardiomyocytes. Our laboratory studies the muscle specific ubiquitin ligase MuRF1 and have identified that mice with cardiac‐specific increased MuRF1 expression is cardioprotective in cardiac I/R injury, while also more susceptible to heart failure when challenged with pressure overload‐induced cardiac hypertrophy. Since these phenotypes are consistent with increased autophagic flux, we tested the hypothesis that MuRF1 positively regulated cardiac autophagy. We first crossed GFP‐RFP‐LC3 Tg+ mice created to track autophagy with our MuRF1 Tg+ mice to create MuRF1/GFP‐RFP‐LC3 dual Tg+ mice. Using confocal analysis of early (dual green and red) and late (red) punta representing the autophagosome‐autolysosome transition, we identified that MuRF1 Tg+ mice had significantly increased cardiac autophagic flux. MuRF1 Tg+ hearts also had significantly increased LC3II levels by immunoblot post‐bafilomycin treatment indicative of increased autophagic flux. Conversely, MuRF1−/− hearts had significantly decreased LC3II levels by immunoblot analysis, consistent with decreased autophagic flux. We next hypothesized that MuRF1 supported autophagy by post‐translational modification of FoxO1 and FoxO3a, two major transcription factors previously described to regulate autophagy. Immunoblot analysis of the inactive p‐FoxO1 and p‐FoxO3a proteins in MuRF1Tg+ hearts demonstrated decreased p‐FoxO3a (p<0.05), while MuRF1−/− hearts had increased levels (p<0.05), consistent with MuRF1's support of Foxo1/3 activity. To determine if MuRF1 regulated FoxO activity transcriptionally, FoxO3a activity assays were performed by in vitro luciferase assays. Increasing MuRF1 levels were shown to enhance FoxO3a transcriptional activity (p<0.05), offering one potential mechanism by which MuRF1 supports autophagy in vivo. Lastly, we determined if MuRF1‐induced autophagy was cardioprotective in doxorubicin‐induced cardiomyocyte death, a model where increased autophagy has been reported to be protective. When MuRF1 expression was increased in the H9C2 cardiomyocyte‐derived cell line and challenged with 1 microM doxorubicin for 24 hours, a 66% reduction in Caspase 3/7 activity was seen compared to control cells, indicating that MuRF1's cardioprotection may be due, in part, to enhanced autophagy. These findings establish MuRF1 as the first ubiquitin ligase to regulate cardiac autophagy, which occurs via the FoxO3a transcription factor. Understanding MuRF1's regulation of autophagy provides insight for the development of therapies targeting autophagy in heart disease.