ANTs are cytostatic antibiotics that are extracted from Streptomyces bacterium (Fujiwara et al., 1985; Octavia et al., 2012). ANTs include daunorubicin (DAU), doxorubicin (DOX), epirubicin (EPI), idarubicin (IDA), and valrubicin (VAL) (Figure 1) (Dimarco et al., 1963; Capelôa et al., 2020). DAU was the first ANT used to treat acute pediatric leukemia in 1964 (Dimarco et al., 1963). DOX was later isolated from a mutant of Streptomyces peucetius in 1969 (Arcamone et al., 1969). The structural formula of DAU and DOX is very similar, except for the substitution of a hydroxyl group at the carbon 14 position in DOX (Escherich et al., 2013). DAU is utilized against acute lymphoblastic and myeloblastic leukemias, whereas DOX is more effective in lymphomas, sarcomas, and a broad spectrum of solid tumors, such as breast, lung, bladder, and bone cancers (Minotti et al., 2004; Arcamone, 2009; Tacar et al., 2013). Later on, several newer ANTs have been developed to treat multiple types of cancers (Bonfante et al., 1980; McGowan et al., 2017). Although ANTs have been successful in treating a variety of cancers, they have been associated with both acute and chronic cardiotoxicity, depending on the cumulative dose of each agent (Table 1) (Venkatesh and Kasi, 2019; Rocca et al., 2020). For example, early adverse effects of DOX have been reported to reduce the left ventricular ejection fraction within months post-treatment with a cumulative dose ≥350 mg/g² (Buzdar et al., 1985). Studies with 630 breast and lung cancer patients have revealed that 32 of those 630 patients (5.1%) had DOX-induced congestive heart failure (Swain et al., 2003). Most patients with congestive heart failure were treated with a cumulative dose of ≥500 mg/m². The estimated cumulative percentages of patients with DOX-induced congestive heart failure were 5%, 16% and 48% at a cumulative doses of 400 mg/g², 500 mg/m², and 700 mg/m², respectively (Swain et al., 2003). Therefore, the dosage adjustments of ANTs are required to prevent the effects of cardiotoxicity and maximize the therapeutic effects.

The activity of ANTs results in strong inhibitory effects on nucleic acid synthesis (Gewirtz, 1999; Shandilya et al., 2020). Nuclear DNA has been recognized as the primary target of ANTs (Karadurmus et al., 2021). ANTs consist of flat aromatic moieties that intercalate between DNA base pairs (Figure 2) (Frederick et al., 1990). The intercalation inhibits DNA and RNA synthesis, subsequently blocking the transcription and replication in highly replicating cells (Gewirtz, 1999). The specificity, binding affinity, and the binding mode of each ANT depend on differences in the sequence of the DNA base (Shandilya et al., 2020). The intercalation of ANTs can distort DNA and interfere with the nuclear functions in cancer cells (Wang et al., 1987; Chaires, 1990). ANTs also intercalate mitochondrial DNA (Lebrecht and Walker) to induce single or double-strand mtDNA breaks and quantitative defects in mtDNA copy number (Tewey et al., 1984; Lawrence et al., 1993; Lebrecht and Walker, 2007). Both the mutation and deletion of mtDNA-lesion compromise the synthesis of mtDNA-encoded respiratory chain subunits in the mitochondrial inner membrane, contributing to the marked mitochondrial toxicity (Lebrecht and Walker, 2007; Ashley and Poulton, 2009). Consequently, ANT-induced mitochondrial toxicity causes increased production of mitochondrial reactive oxygen species (Priya et al., 2017), which is one of the mechanisms of ANT-induced cardiotoxicity (Lebrecht and Walker, 2007).

Along with DNA intercalation, topoisomerase II is also considered as one of the primary targets of ANT-induced cytotoxic activity in cancer cells. Topoisomerase II is a nuclear enzyme that manages DNA tangles and supercoils by cutting both strands of the DNA helix during replication and transcription (Deweese and Osheroff, 2009). ANTs intercalated into DNA form a stable ANT-DNA-topoisomerase II ternary complex, thereby poisoning the enzyme activity. This ternary complex impends the relegation of breaks in the double-stranded DNA (Shandilya et al., 2020). As a result, ANTs induce irreversible DNA damage, leading to genomic instability and ultimately apoptotic cell death in rapidly dividing cancer cells (Li and Liu, 2001; Minotti et al., 2004; Minev, 2011). This ANT-induced topoisomerase II poisoning is also the molecular basis of cardiotoxicity. Since topoisomerase II β is present in cardiomyocytes (Capranico et al., 1992; Vejpongsa and Yeh, 2014), the inhibition of its isoform has been shown to induce long-term side effects of ANTs in cardiac muscle, resulting in cardiomyopathy (Cornarotti et al., 1996; Austin and Marsh, 1998).

One of the mechanisms responsible for ANT-induced cytotoxicity is the generation of excessive reactive oxygen species (Priya et al., 2017). ROS, including superoxide radical (O₂ ^•−), hydrogen peroxide (H₂O₂), and hydroxyl radical (HO^•), are byproducts of the normal metabolisms and play roles in homeostasis in normal cells (Rocca et al., 2020). However, excessively high and persistent levels of ROS result in an imbalance between the production of free radicals and antioxidant defense systems, triggering oxidative stress and cellular damage (Devasagayam et al., 2004). It has been demonstrated that mitochondria are one of the major sites for ANT-induced oxidative stress and cellular damage (Min et al., 2015). One electron is transferred from NADPH to the flavoprotein in the mitochondrial electron transport chain. The quinone ring of ANTs acts as an electron acceptor to form semiquinone, which produces superoxide anion (Berthiaume and Wallace, 2007). This reaction is catalyzed by NADH reductase at complex I in the inner mitochondrial membrane (Berthiaume and Wallace, 2007; Murabito et al., 2020). The superoxide dismutase neutralizes superoxide anion into hydrogen peroxide (Stěrba et al., 2013). The production of superoxide anion and hydrogen peroxide stimulates enzyme-mediated reduction-oxidation cycles, producing reactive and destructive hydroxyl radicals, which cause nucleic acid damage, protein alkylation, and lipid peroxidation, followed by apoptosis (Figure 3) (Simůnek et al., 2009; Angsutararux et al., 2015). In addition to mitochondrial ROS, other enzymes including NADPH oxidase (NOX) and nitric oxide synthase (NOS) also contribute to DOX-induced cardiotoxicity (Gilleron et al., 2009; Lin et al., 2019). NOX has been identified as one of the most important sources of ANT-induced ROS (Gilleron et al., 2009; Priya et al., 2017; Lin et al., 2019). NOX is a transmembrane enzyme that is located in intracellular organelles and consists of serval isoforms (Panday et al., 2015). Specifically, NOX2 has been shown to contribute to ANT-induced cardiotoxicity (Wojnowski et al., 2005; Zhao et al., 2010). Growing evidence shows that NOX2 deficiency attenuates superoxide production, preventing cardiomyocytes cell death, myocardial fibrosis, and leukocyte infiltration following DOX administration (Zhao et al., 2010; McLaughlin et al., 2017). NOS is also one of the contributing enzymes to oxidative stress and damage to cardiac muscle following DOX treatment. NOS catalyzes the conversion of L-arginine to nitric oxide (Mukai et al.) (Knowles and Moncada, 1994). Three NOS isoforms have been identified in mammals, including neuronal NOS (nNOS), cytokine-inducible NOS (iNOS) and endothelial NOS (eNOS) (Knowles and Moncada, 1994). DOX administration increases the levels of NO through the activation of eNOS and iNOS (Vásquez-Vivar et al., 1997). eNOS has been shown to catalyze NADPH-dependent superoxide formation following DOX treatment by directly binding the reductase domain of eNOS (Vásquez-Vivar et al., 1997). Eventually, the overproduction of ROS and NO generates reactive nitrogen species (RNS), which lead to cardiotoxicity following ANT treatment (Fogli et al., 2004; Štěrba et al., 2013).

The Ubiquitin proteasome system (UPS) is an ATP-dependent proteolytic system composed of numerous ubiquitin ligase enzymes and a large proteolytic complex called the proteasome (Grune et al., 2003; Liu et al., 2016; Hyatt et al., 2019). The UPS plays a role in the protein breakdown that occurs during muscle damage (Murton et al., 2008; Powers et al., 2011). The UPS requires polyubiquitination of proteins through ubiquitin ligase enzymes, including E1 (ubiquitin-activating enzyme), E2, and E3 (Ichimura et al., 2000; Bodine et al., 2001). The polyubiquitinated proteins that are damaged or deemed unnecessary are degraded by the proteasome. Specifically, two muscle-specific E3 ligases, Muscle Atrophy F-box (MAFbx)/atrogin-1 and Muscle-Ring Finger-1 (MuRF-1), contribute to the UPS-mediated protein degradation in cardiac muscle. Numerous studies have indicated that DOX treatment stimulates UPS in cardiac muscle, leading to cardiac muscle damage (Powers et al., 2011; Derouiche et al., 2014; Min et al., 2015; Montalvo et al., 2020). Specifically, DOX treatment activates UPS through mitochondrial ROS production in cardiac muscle (Montalvo et al., 2020). Indeed, DOX administration significantly increases both mitochondrial H₂O₂ production and MAFbx, whereas mitochondria-targeted antioxidant protects mitochondria against DOX-induced oxidative stress and attenuates the expression of atrogin-1/MAFbx in cardiac muscle (Montalvo et al., 2020). Another study also showed that exercise preconditioning improves mitochondrial biogenesis and prevents gene expression of MuRF-1 in DOX-administrated cardiac muscle (Kavazis et al., 2014). A recent study demonstrated that a DOX dose-dependent (1–25 mg/kg) increases MuRF-1 mRNA and protein levels in myocardial tissues, accompanied by decreases in cardiac mass and cardiomyocyte cross-sectional area (Willis et al., 2019). However, mice lacking MuRF-1 were protected against DOX-induced cardiac atrophy and contractile dysfunction (Willis et al., 2019). These findings suggest that DOX administration induces pathological protein degradation through the activation of UPS in cardiac muscle.

Calpain is an intracellular calcium-dependent cysteine protease (Khorchid and Ikura, 2002; Vickers, 2017). Calpain exists as an inactive proenzyme in the cytosol. When intracellular calcium levels increase, the proenzyme form of calpain is converted to its active form, which cleaves cytoplasmic and nuclear substrates, leading to apoptosis (Momeni, 2011). Calpain activation has been implicated in myocardial injuries, including ischemia/reperfusion myocardial injury, pressure overload-induced cardiomyopathy, and heart failure (Li et al., 2009; Wang et al., 2018b; Wang et al., 2020). It has been demonstrated that DOX treatment causes calcium overload, which increases calpain activity (Szenczi et al., 2005; Emanuelov et al., 2010). Campos et al. established that the expression of active calpain increased in cardiomyocytes isolated from DOX-treated rats that showed dystrophin disruption in cardiac muscle (Campos et al., 2011). However, a calcium-blocking agent prevented calpain activation and preserved cardiac function. Another study also investigated the calpain-induced cardiomyopathy in rats injected with DOX (Min et al., 2015). Echocardiography analysis showed that DOX administration resulted in impaired cardiac function with decreased fractional shortening and thinning of the septal and left ventricular posterior wall. In contrast, rats treated with a calpain inhibitor prior to DOX injection attenuated cardiac dysfunction. In vitro experiments have also shown that DOX induces calpain activation in cardiomyocytes (Lim et al., 2004). Calpain activation in cardiomyocytes treated with DOX resulted in myofilament protein degradation and necrosis, while calpain inhibitors preserved the myofilament protein degradation (Lim et al., 2004). These studies indicate that calpain activation is one of the contributors that cause DOX-induced cardiomyopathy.

Caspases are a family of cysteine protease enzymes that contribute to programmed cell death (Shi, 2004). In healthy cells, the caspases exist as dormant pro-enzymes. The caspases undergo cleavage events in response to a death-inducing signal to release a large subunit and a small subunit that heterodimerize into the active enzyme (Roy, 2000). It has been well known that caspase-3 is a crucial mediator of apoptosis by efficiently cleaving many key cellular proteins (Porter and Jänicke, 1999). In fact, caspase-3 is highly activated during the progression of multiple forms of heart diseases (Philipp et al., 2004; Putinski et al., 2013; Hashmi and Al-Salam, 2015; Min et al., 2015). Caspase-3 activation is capable of promoting the degradation of cardiac myofibrillar proteins, such as α-actin, α-actinin, and cTnT (Communal et al., 2002). Numerous studies have established that DOX treatment induces apoptosis through the activation of caspase-3 in cardiac muscle (Michihiko et al., 2006; Pointon et al., 2010; Min et al., 2015; Sun et al., 2016) and the inhibition of caspase-3 activity attenuates DOX-induced cardiotoxicity (Wang et al., 2001; Zhang et al., 2009a; Ma et al., 2013). Although the regulation of caspase-3 activity is complex and involves several interconnected signaling pathways, both extrinsic and intrinsic pathways have been postulated to activate caspase-3 in cardiac muscle treated with DOX. DOX treatment can induce the extrinsic apoptotic pathway via the upregulation of death receptors (Nakamura et al., 2000). Death ligands, including FasL and TNFα, bind to their receptors, leading to caspase-8 activation. Activated caspase-8 increases caspase-3 activity, resulting in cardiomyocyte apoptosis (Zhao and Zhang, 2017; Pan et al., 2021). It is also feasible that DOX contributes to caspase-3 activation through the intrinsic apoptotic pathways by stimulating ROS production in cardiac muscle. Indeed, increased cellular levels of ROS have been reported to activate caspase-3 in a variety of cell types, including cardiomyocytes (Powers et al., 2011; Yeh et al., 2019). DOX can activate the core apoptosis regulators, such as Bax and Bak in the cytosol. The activated Bax/Bak are translocated from the cytosol to the outer membrane of mitochondria, increasing mitochondrial membrane permeability. Cytochrome c in the inner membrane of mitochondria is released into the cytoplasm (An et al., 2009). Subsequently, cytochrome c activates caspase-9, resulting in the activation of caspase-3 in DOX-treated cardiomyocytes (Wei et al., 2022). These findings many explain how DOX treatment can induce cardiomyocyte apoptosis, which causes cardiotoxicity.

Autophagy is a homeostatic process by which cytoplasmic components are degraded and recycled under normal and stress conditions through lysosomal pathways (Hansen et al., 2018). Autophagy has emerged as a major regulator of cardiac homeostasis and function. The level of autophagy in cardiac muscle is low under normal conditions, whereas it is upregulated in response to pathological stress (Nishida et al., 2009). Under physiological conditions, autophagy is essential for optimal cellular function and survival as it removes damaged or unwanted proteins and organelles. Under pathological conditions, autophagy may be stimulated to induce toxic effects (Smuder et al., 2013; Cui et al., 2021). Excessive autophagy activation can cause damage to organelles such as the mitochondria and endoplasmic reticulum, releasing compounds into the cytoplasm (e.g., cytochrome c and calcium) that can induce cell death (Nishida et al., 2008; Zhang et al., 2009b; Nishida et al., 2009). The activation of autophagy begins with the formation of a phagophore through a system of autophagy proteins (Atg proteins) (Smuder et al., 2013). The phagophore, also known as a double-membrane structure, sequesters bulk cytoplasmic components, such as abnormal intracellular proteins, excess or damaged organelles, and invading microorganisms. The phagophore expands to a sealed, double-membrane vesicle called the autophagosome (Hollenstein and Kraft, 2020). Beclin-1 plays an important role in the initial steps of autophagosome formation by mediating the localization of other Atg proteins to the phagophore (Gustafsson and Gottlieb, 2008). Elongation of the autophagosome requires the interaction of several Atg proteins (Mizushima et al., 1999; Smuder et al., 2013). Specifically, Atg4 is responsible for the cleavage of microtubule-associated protein 1A/1B-light chain 3 (LC3) to LC3-I (Yang et al., 2015). Cleaved LC3-I is conjugated by Atg7, Atg3, and Atg12-Atg5-Atg16L complex, leading to LC3-II synthesis, which is recruited to the autophagosomal membrane for the elongation (Ichimura et al., 2000). P62 is an autophagosome cargo protein that is used as a reporter of autophagy activity (Liu et al., 2016). After the formation of the autophagosome, cytoplasmic components are delivered to the lysosomes (Mizushima et al., 2002). The outer membrane of the autophagosome fuses with the lysosome to form an autolysosome (Mizushima et al., 2002). Hydrolases in lysosomes degrade the autophagosome-delivered components (Mizushima et al., 2002). Much evidence shows that the activation of autophagic signaling is associated with various forms of heart disease, including heart failure, ischemia-reperfusion injury, and metabolic cardiomyopathies (Shimomura et al., 2001; Kostin et al., 2003; Kanamori et al., 2015). This suggests that autophagy emerges as a new therapeutic target for heart disease. DOX treatment also induces autophagic signaling in cardiac muscle. Indeed, DOX-induced autophagosome and autolysosome accumulation were confirmed in vivo by using GFP-LC3 and mRFP-GFP-LC3 transgenic mice (Abdullah et al., 2019). In this study, both acute DOX treatment (20 mg/kg) and chronic DOX treatment (5 mg/kg every week for 4 weeks) exhibited time-dependent accumulation of LC3B II levels in cardiac muscle. Conversely, it has been reported that inhibition of autophagy via 3-methyladenine (3-MA) is sufficient to protect against DOX-induced autophagy, mitochondrial dysfunction, and cardiac contractile dysfunction (Lu et al., 2009; Xu et al., 2012). A proposed mechanism responsible for DOX-induced autophagy is that DOX administration results in damage to the mitochondria and induction of Beclin-1 expression, leading to accelerated autophagy and cardiomyopathy (Lu et al., 2009). Other groups also demonstrated that anti-apoptotic protein Bcl-2 can form a complex with Beclin-1 to inhibit autophagic apoptosis and autophagosome formation in cardiomyocyte (Wang et al., 2018a; Yu et al., 2022). However, DOX treatment increases the expression of Beclin-1 and decreases Bcl-2 protein expression, increasing the Beclin-1/Bcl-2 ratio, which indicates the activation of autophagic signaling and apoptosis (Smuder et al., 2013). These findings implicate that autophagy antagonists may represent a therapeutic approach for the preservation and/or maintenance of cardiac muscle function during and/or doxorubicin treatment.