FUDNC1 ameliorates doxorubicin-induced cardiotoxicity by restoring MERCs structure and blocked autophagic flux

Abstract Background Doxorubicin (DOX)-induced cardiotoxicity (DIC) is frequently observed in clinical practice. Autophagy dysregulation is known to be a mechanism of DIC. Mitochondrial-Endoplasmic Reticulum Contact sites (MERCs) is where autophagy initiates and autophagosomes form. However, the role of MERCs in autophagy dysregulation in DIC remains elusive. FUNDC1 is a mitochondria outer membrane protein that serves as a mitophagy receptor as well as a tethering protein of MERCs. Purpose The aim of our study was to investigate the effect of DOX on MERCs and explore whether it is involved in the dysregulation of autophagy. Our hypothesis is that FUNDC1 overexpression ameliorates DIC by restoring MERCs structure and dysregulated autophagic flux. Method We use confocal microscopy and transmission electron microscopy to assess MERCs structure. Overall autophagic flux was analyzed by RFP-EGFP-LC3B fluorescence assay and western blotting for LC3BII. A total dose of 15 mg/kg of doxorubicin was administrated in C57BL/6J mice to construct a DIC model in vivo. Adeno-associated virus (AAV) was used to cardiac-specifically overexpress FUNDC1. Cardiac remodeling was evaluated by Masson’s trichrome staining. Cardiac function (left ventricle ejection fraction, LVEF; left ventricle fraction shortening, LVFS) was measured by echocardiography. Results DOX treatment led to a significant downregulation of multiple tethering proteins (including FUNDC1) of MERCs and inhibition of MERCs formation, as demonstrated by decreased colocalization coefficients of mitochondria and endoplasmic reticulum in cardiomyocytes. The overexpression of FUNDC1 abolished DOX-induced inhibition on MERCs formation. In addition, DOX treatment blocked the normal autophagic flux, as demonstrated by the decreased autophagosome synthesis and impaired autophagosome turnover. Maintaining the MERCs structure by FUNDC1 overexpression was able to restore the blocked autophagic flux mentioned above. Moreover, FUNDC1 overexpression restored the blocked autophagic flux in vivo, as demonstrated by the increased LC3B turnover compared to the DOX treatment group. Importantly, global heart function of FUNDC1 overexpressed mice was significantly improved post DOX treatment compared to the Wildtype DOX treatment group, as demonstrated by the increased LVEF and LVFS. In addition, LV fibrosis level was found to be significantly decreased in FUNDC1 overexpressed mice treated with DOX compared to the Wildtype DOX treatment group. Conclusion We reported for the first time that the impaired MERCs formation in DOX-treated cardiomyocytes and revealed that restoring the disrupted MERCs structure by FUNDC1 overexpression was able to revert the blocked autophagic flux induced by DOX treatment. Furthermore, cardiac-specific overexpression of FUNDC1 protected DOX-treated mice against adverse cardiac remodeling and improved cardiac function. These findings may shed light on the prevention and treatment of DIC.Figure