Autophagy and mitophagy in the context of doxorubicin-induced cardiotoxicity

Abstract

Doxorubicin (Dox) is a cytotoxic drug widely incorporated in various chemotherapy protocols. Severe side effects such as cardiotoxicity, however, limit Dox application. Mechanisms by which Dox promotes cardiac damage and cardiomyocyte cell death have been investigated extensively, but a definitive picture has yet to emerge. Autophagy, regarded generally as a protective mechanism that maintains cell viability by recycling unwanted and damaged cellular constituents, is nevertheless subject to dysregulation having detrimental effects for the cell. Autophagic cell death has been described, and has been proposed to contribute to Dox-cardiotoxicity. Additionally, mitophagy, autophagic removal of damaged mitochondria, is affected by Dox in a manner contributing to toxicity. Here we will review Dox-induced cardiotoxicity and cell death in the broad context of the autophagy and mitophagy processes.

Keywords: anthracyclines; heart failure; impaired autophagy and mitophagy; lysosomal dysfunction; oncocardiology.

Figure 1. Subcellular events associated with Doxorubicin-induced cardiotoxicity

The image highlights acute, intermediate, and end-point (cell death) events resulting from exposure to Doxorubicin (Dox). The acute section illustrates the direct interaction of Dox with subcellular entities; the intermediate section illustrates direct consequences of these interactions. Events within the acute, or intermediate sections are likely to occur simultaneously and cross-talk with each other. The end-point section is meant to show that the preceding events lead to apoptotic, necrotic, and/or dysregulated autophagy-associated cell death. Acute Events: Dox, upon entering the cell, interacts directly with molecules and organelles: interaction with topoisomerase-IIβ (TOPIIβ) leads to DNA damage. Interaction with nitric oxide synthase (NOS), nicotinamide adenine dinucleotide phosphate-oxidase (NOX) and Fe2+, promotes reactive oxygen or nitrogen species stress (ROS or RNS, respectively), contributing to further DNA damage, oxidation and nitrosylation of proteins and peroxidation of lipids. Dox binds to mitochondrial DNA and impairs the electron transport chain resulting in production of ROS and decreased ATP. Fe2+-Dox complexes are toxic to mitochondria and the endoplasmic reticulum (ER), by causing, for example, lipid (including cardiolipin) peroxidation. The DNA damage response activates the ataxia telangiectasia mutated (ATM) protein which upregulates and activates the tumor suppressor p53. P53 upregulates expression of pro-apoptotic members of the Bcl-2 family such as Bax and Bad; it also increases expression of Bcl2/adenovirus E1B 19 kDa protein-interacting protein 3 (Bnip3), which can cause mitochondrial damage and necrotic cell death, as well as initiate mitophagy. DNA damage and increased levels of ROS lead to downregulation of the transcription factor GATA-4 which decreases expression of the anti-apoptotic, and anti-autophagy-initiation protein, Bcl-2. P53 can also inhibit the activity of mammalian target of Rapamycin (mTOR) signaling, thus dis-inhibiting autophagy initiation. Some studies have indicated that Dox can elicit AMP-activated kinase (AMPK) activation. Activation of AMPK, resulting from reduced ATP levels, can inactivate mTOR and initiate autophagy. Dox-induced effects on ROS and RNS production, mitochondrial and ER damage, DNA and gene expression, culminate in the promotion of apoptotic and necrotic cell death. Dysregulation of the autophagy/mitophagy processes are also linked to Dox-induced cell death. Signals/events associated mostly, although not exclusively, with apoptotic or necrotic cell death are included, respectively, in pink or pale-blue boxes. Pale-purple boxes contain signals/pathways associated with various types of subcellular dysfunction, including autophagic dysregulation.

Figure 2. The autophagy process

The figure illustrates major steps constituting the process of autophagy, aiming at recycling targeted cargo. Vertical blue arrows point to a progression from autophagy-initiation via the activated unc-51-like autophagy activating kinase 1 (ULK-1) complex, to the formation/elongation of membranous entities such as the phagophore; to the formation of the autophagosome around the cargo, followed by fusion with the lysosomes and formation of autolysosomes where cargo is degraded. Major signaling pathways regulating the initiation of autophagy are included in the upper portion, separated from the rest of the figure by the broken line, and include the mammalian target of rapamycin (mTOR) and AMP activated kinase (AMPK) pathways. Activators of mTOR, include growth factors and high nutrient status, while activation of AMPK occurs when AMP/ATP ratio increases, and also upon increased calcium activating calcium/calmodulin-dependent protein kinase kinase 2 (CaMKK). The figure shows the antithetical action of mTOR versus AMPK on autophagy initiation, as manifested by ULK-1 activation. ULK-1 mediated activation of Beclin-1 and vesicle-mediated vacuolar protein sorting 34 (VPS34) complex initiates a cascade of events leading to phagophore formation. A few representative proteins associated with the autophagy process are included for each step. Autophagy related gene (Atg) proteins 5, 12, and 16 incorporation elongates the formed phagophore. LC3-II is formed by proteolytic cleavage and lipidation of microtubule-associated proteins light chain 3B (LC3-II) which anchors it to the phagophore. Ubiquitination tags damaged cargo which can then interact with the ubiquitin binding protein, p62/SQSTM1, and, via interaction of the latter with LC3-II, cargo is engulfed within the autophagosome. For completion of the process, fusion of autophagosomes with lysosomes is required, to form autolysosomes. In the autolysosomes cargo and associated proteins are digested by the various degradative lysosomal enzymes, symbolized by scissor images.

Figure 3. Mitophagy receptors

Four pathways leading to targeting/ recognition of mitochondria for autophagic elimination have been described. The Parkin/PINK1 pathway, operating on depolarized mitochondria, consists of PINK1 stabilization, enabling interaction with Parkin and its translocation to the outer mitochondrial membrane, OMM. Parkin can ubiquitinate various OMM proteins enabling recognition and interaction with p62/SQSTM1. LC3-II can interact with p62/SQSTM1 allowing autophagosomal engulfment of mitochondria, and subsequent degradation via fusion with the lysosome. In the second pathway, Bcl2/adenovirus E1B 19 kDa protein-interacting protein 3 (Bnip3) can also act as mitophagy receptor, as it possesses the LC3-II recognition motif, as well as a transmembrane domain which anchors it to mitochondria. Nix/Bnip3L (the other member of Bnip3 family) acts in a similar fashion. Translocation of Bnip3 to mitochondria does not require loss of membrane potential. Additional molecules possessing LC3-II-interacting ability include FUN14 Domain Containing 1 (FUNDC1), and peroxidized cardiolipin.

Figure 4. Proposed mechanisms of Dox-induced dysregulation of autophagy and mitophagy resulting in cell death

Dox stimulates autophagy initiation (by upregulating Atg genes, for example) and at the same time, by decreasing expression of the master transcription factor EB (TFEB), impairs function of available lysosomes and prevents their biogenesis resulting in autophagosome accumulation and inhibition of flux. Autophagosome accumulation contributes to the accumulation of reactive oxygen species (ROS). Dox induced changes in gene expression include the downregulation of Parkin and via p53, in impairment of its translocation to mitochondria, thus inhibiting Parkin/PINK1 mediated mitophagy. Parkin-independent mitophagy, such as Bcl-2-like 19 KDa-interacting protein-3 (Bnip3) - mediated mitophagy can be upregulated. Overall, the dysregulated autophagy and impaired mitophagy can induce cardiomyocyte death.