Loss of Rubicon ameliorates doxorubicin-induced cardiotoxicity through enhancement of mitochondrial quality
Introduction
Doxorubicin (DOX) is one of the most potent chemotherapy agents. However, its clinical use is limited by potential development of cardiotoxicity. Oxidative stress caused by increased reactive oxygen species (ROS) has been considered as one of the major mediators of DOX cardiotoxicity [[1], [2], [3]]. As an electron acceptor, DOX induces mitochondrial ROS generation. Increased ROS causes mitochondrial damage, which in turn, amplifies ROS production, forming a vicious cycle. This ROS-induced ROS release ultimately leads to cardiac damage and heart failure [4]. Therefore, DOX cardiotoxicity may be ameliorated through enhancement of mitochondrial quality, which is mainly governed by mitophagy and mitochondrial dynamics [5].
Macroautophagy (hereafter referred to as autophagy) is an evolutionally conserved pathway delivering cytoplasmic contents to lysosome for degradation [6]. Mitophagy is a type of autophagy that selectively removes damaged mitochondria. Mitophagy occurs under baseline conditions and stress conditions such as oxidative stress to maintain cellular functions [7]. Mitophagy is regulated by PTEN-induced kinase 1 (PINK1) and Parkin proteins [8,9]. PINK1 controls Parkin translocation from cytoplasm to mitochondria [10]. Mitochondrial Parkin ubiquitinates target proteins localized on the outer mitochondrial membrane, which mediate mitochondrial degradation by autophagy [11]. Animals deficient for Parkin show reduced mitophagy and accumulation of damaged mitochondria, leading to cardiac dysfunction under physiological and stress conditions [12,13].
Mitochondrial dynamics including mitochondrial fusion and fission are also critical for mitochondrial quality control [14]. Mitochondrial fusion is regulated by Mitofusin1 (MFN1), Mitofusin2 (MFN2), and optic atrophy factor 1 (OPA1) proteins [14]. Mitochondrial fission is mainly regulated by a large GTPase dynamin-related protein 1 (Drp1) [15]. A number of proteins including mitochondrial fission 1 protein (Fis1), mitochondrial fission factor (Mff), mitochondrial division factors 49 and 51 kDa proteins (Mid49 and Mid51) interact with Drp1. For mitochondrial fission, these proteins coordinate to recruit Drp1 from cytoplasm to mitochondria [15]. Under physiological conditions, mitochondrial fusion and fission are delicately balanced. Disturbance in the balance between mitochondrial fusion and fission causes heart disease [[15], [16], [17]].
Ultraviolet irradiation resistance-associated gene (UVRAG), an autophagy-related gene, regulates autophagosome maturation [18,19]. Rubicon (Run domain Beclin-1-interacting and cysteine-rich domain-containing protein) binds to UVRAG and inhibits UVRAG-mediated autophagosome maturation [20,21]. Previously we and others have shown that DOX disrupts multiple steps of autophagy in the heart including autophagosome formation, maturation and degradation [[22], [23], [24], [25]]. It remains unknown whether Rubicon plays a role in autophagy impairment in DOX cardiotoxicity. In this study, we test the hypothesis that loss of Rubicon ameliorates DOX-induced DOX cardiotoxicity through improving autophagy and mitochondrial quality.
Section snippets
Animal experiments
All mice used were on the FVB/NJ background. Animal protocols were approved by the Institutional Animal Care and Use Committee of Shanghai, China [SYXK (SH) 2011-0112]. Seven-week-old male mice were used for experiments unless stated otherwise. Generation of Rubicon-deficient mice was described previously [26,27]. A single dose of DOX (BBI, Canada) at 20 mg/kg was injected intraperitoneally into mice to establish acute DOX cardiotoxicity. For assessment of cardiac morphometry and function, mice
Rubicon expression in the heart after DOX treatment
We first assessed Rubicon expression in the hearts from DOX-treated wild type (WT) mice. Rubicon protein levels were significantly reduced 16 h after DOX treatment (Fig. 1A and B). However, Rubicon protein levels remained unaltered 3 and 5 days after DOX treatment (Fig. 1A and B). The absence of Rubicon protein in the hearts from Rubicon-deficient mice was confirmed by Western blot (Fig. 1C).
Loss of Rubicon improves survival in DOX-treated animals
WT mice began to die 4 days after DOX treatment (Fig. 1D). All male and female WT mice died 8 and 6 days
Discussion
The major findings of the present study are (i) loss of Rubicon ameliorates DOX-induced cardiotoxicity; (ii) DOX-induced mitochondrial damage in the heart is attenuated by Rubicon deficiency; and (iii) loss of Rubicon mitigates DOX-induced impairment of autophagic flux, mitophagy and mitochondrial dynamics in the heart.
We observed a significant reduction in Rubicon expression 16 h, but not 3 and 5 days after DOX treatment. Given that DOX impaired autophagic flux in the heart and induced cardiac
Declaration of Competing Interest
The authors declare that they have no conflicts of interest.
Acknowledgements
This work was supported by research grant from Natural Science Foundation of Shanghai (16ZR1418200); National Natural Science Foundation of China (81974020). We acknowledge Institute of Developmental Biology and Molecular Medicine of Fudan University for providing Rubicon-deficient mice generated by piggyBac transposition.
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