Effects of doxorubicin‐induced cardiotoxicity on cardiac mitochondrial dynamics and mitochondrial function: Insights for future interventions

Abstract Anthracyclines is an effective chemotherapeutic treatment used for many types of cancer. However, high cumulative dosage of anthracyclines leads to cardiac toxicity and heart failure. Dysregulation of mitochondrial dynamics and function are major pathways driving this toxicity. Several pharmacological and non‐pharmacological interventions aiming to attenuate cardiac toxicity by targeting mitochondrial dynamics and function have shown beneficial effects in cell and animal models. However, in clinical practice, there is currently no standard therapy for the prevention of anthracycline‐induced cardiotoxicity. This review summarizes current reports on the impact of anthracyclines on cardiac mitochondrial dynamics and mitochondrial function and potential interventions targeting these pathways. The roles of mitochondrial dynamics and mitochondrial function in the development of anthracycline‐induced cardiotoxicity should provide insights in devising novel strategies to attenuate the cardiac toxicity induced by anthracyclines.

9% and the majority of the cases (98%) occurring within the first year of treatment. It has been shown that the risk of heart failure could be cumulative for up to 30 years after exposure to anthracyclines. 6 Currently, there is no standard effective therapy for the prevention of anthracycline-induced cardiotoxicity.
There are several proposed mechanisms for doxorubicin-induced cardiotoxicity. Doxorubicin primarily targets topoisomerase IIβ (Top2β) and induces DNA double-strand breaks. 7 It also disrupts cardiac pro-survival pathway, the neuregulin/ErbB signalling which results in mitochondrial dysfunction and apoptosis. 8,9 Moreover, the most pronounce mechanism responsible for doxorubicin-induced cardiotoxicity is the formation of reactive oxygen species (ROS), leading to oxidative stress. 10 The generation of ROS occurs via multiple pathways. 10 Mitochondria are subcellular organelle, which are the main source and target of ROS. 11 Several studies indicated that doxorubicin-induced mitochondrial dysfunction, increased production of ROS which subsequently led to the development of cardiac muscle dysfunction and heart failure. 12,13 Mitochondria occupy about 40% of each cardiomyocyte volume, and the majority of the energy produced in the cardiomyocyte is from mitochondrial respiration. 14,15 Doxorubicin can target to the mitochondria mainly through ROS production. 16 ROS induces mitochondrial DNA damage and decreases mitochondrial transmembrane potential. 12,17 In addition, doxorubicin also directly interferes with mitochondrial function by inhibiting electron transport chain proteins expression and promotes mPTP opening. 12,18 Inhibition of mitochondrial function by doxorubicin contributes to cardiac energy starvation and cell death. 12 All of these data suggest that mitochondria are one of the main regulators in the development of doxorubicin-induced cardiotoxicity.
Mitochondria are dynamic organelles whose primary function is ATP production. 19 Mitochondria protect cells against cellular stress by several processes. Mitochondrial transcription factors such as peroxisome proliferator-activated receptor-γ coactivator 1α (PGC-1α) and mitochondrial transcription factor A (TFAM) mediate an increase in mitochondrial number in response to cellular damage. 20,21 This process is called mitochondrial biogenesis which could regulate mitochondrial dynamics. Mitochondrial dynamics consists of mitochondrial fusion and fission. Maintaining balance of mitochondrial dynamics is a key for achieving normal mitochondrial function by controlling several aspects including mitochondrial respiration, mitochondrial metabolism and ROS production. 22 Mitochondrial dynamics are regulated by guanosine triphosphatases (GTPases) in the dynamin family. Fusion is mediated by mitofusin-1 (MFN1) and mitofusin-2 (MFN2) proteins at the outer mitochondrial membrane and optic atrophy 1 (OPA1) at the inner mitochondrial membrane. 19 Mitofusins initiate fusion by the process of mitochondrial tethering of two adjacent mitochondria and create homodimeric or heterodimeric complexes of MFN1 and MFN2 at the outer mitochondrial membrane. 23 After fusion of the outer mitochondrial membrane, OPA1 mediates the inner mitochondrial membrane fusion resulting in a more interconnected mitochondrial network. With regards to mitochondrial fission, DRP1 is recruited from the cytosol and binds to DRP1 receptor proteins at the outer mitochondrial membrane including mitochondrial fission protein1 (MTFP1) and mitochondrial fission factor (MFF). 24 Fission leads to mitochondrial fragmentation and enhances the generation of ROS. There is growing evidence that doxorubicin disrupts mitochondrial dynamics and mitochondrial function. 14,25 In this review, the effects of doxorubicin on mitochondrial dynamics and mitochondrial function have been comprehensively summarized. Consistent and controversial reports from in vitro and in vivo models have been presented. The proposed pharmacological interventions to ameliorate doxorubicin-induced cardiotoxicity have been presented and discussed.

| DOXORUB ICIN -INDUCED C ARD I OTOXI CIT Y AND C ARD IAC MITOCHONDRIAL DYNAMIC S: E VIDEN CE FROM IN VITRO AND IN VIVO S TUD IE S
Interference with mitochondrial dynamics is associated with the development of cardiovascular and metabolic diseases including ischaemia-reperfusion injury, metabolic syndrome and also in doxorubicin-induced cardiotoxicity. [26][27][28][29] Doxorubicin increased mitochondrial fragmentation followed by ROS generation and apoptosis. 14,25,30 These effects of doxorubicin could be explained by an imbalance of mitochondrial dynamics. In vitro studies in neonatal rat cardiomyocytes (NRCMs) treated with doxorubicin dosage between 0.86 and 1.72 µmol/L for 1-24 hours showed a significant decrease in mitochondrial fusion proteins, MFN1, MFN2, OPA1 and increase in phosphorylation of DRP1 at serine 616. 14,30 Using H9c2 cell and postnatal rat cardiomyocytes also showed consistent result that phosphorylation of DRP1 at serine 616 was increased. 29 Differences between dosage and analytical methods may explain the discrepancy in results found concerning the mitochondrial fusion process.
The precise mechanism of how doxorubicin regulates mitochondrial dynamics proteins is unclear. It has been shown that DRP1 translocation is controlled by MTFP1. 28 Transfection of HL-1 cardiac cell with Mtfp1-shRNA inhibited mitochondrial fission and apoptosis. 28 Interestingly, recent evidence showed that sirtuin-3 (SIRT3) could regulate mitochondrial dynamics. SIRT3 is a protein that possess a deacetylase activity and is responsible for deacetylating of several mitochondrial proteins including OPA1. 25

| DOXORUB ICIN -INDUCED C ARD I OTOXI CIT Y AND C ARD IAC MITOCHONDRIAL DYS FUN C TION: E VIDEN CE FROM IN VITRO AND IN VIVO S TUD IE S
Molecular mechanisms associated with doxorubicin-induced cardiotoxicity are multifactorial and complex. 34 After several decades of investigation, doxorubicin-induced cardiotoxicity is still a necessary focus in the field of cardio-oncology research as the precise mechanism associated with its cardiotoxicity remains unclear.
Increased oxidative stress has been shown as a key feature of doxorubicin-induced heart failure. 35,36 Cardiomyocytes are known to be more susceptible to oxidative damage than other cells due to their lower capacity for antioxidant defence and their high density of mitochondria, which are the main source of ROS generation. 37,38 The chemical structure of doxorubicin is susceptible to redox cycling. As it is a quinone compound, doxorubicin is reduced by oxidoreductases within the cell including NADPH oxidase, xanthine oxidase and mitochondrial electron transport chain enzymes, especially complex I. 39 Redox cycling leads to the formation of a semiquinone compound which interacts with oxygen to form the superoxide anion. 40 The generation of ROS induces lipid peroxidation at the cellular membrane and has impact on other subcellular organelles including the nucleus and mitochondria. 16  These could be the effect of ROS production and dysregulation of calcium channels or transporters, which are susceptible to redox cycling. 63 Furthermore, doxorubicin interferes with mitochondrial respiration at electron transport chain (ETC) level by inhibiting complex I, II, IV proteins 18 and complex I activity. 12,31,43,55,64,65 These ETC complexes are necessary for the process of oxidative phosphorylation which is the major source of ATP production.
Alteration in mitochondrial respiration was observed in doxorubicin models as shown by an increase in state 4, and a decrease in state 3 respiration and respiratory control ratio. 13 Suppression of Sirt3 in hiPSC-CMs also increased the toxicity of doxorubicin 69 which suggested the protective role of SIRT3, and this finding is also consistent with previous reports using NRCM and H9C2 cells. 18,25 A summary of these reports is shown in Table 2.
In addition to increasing ROS production, doxorubicin can also suppress cardiac antioxidant defence system. This has been postulated to be partially mediated by alteration in the sirtuins family proteins particularly SIRT1 and SIRT3. 18 Suppression of sirtuins expression inhibited various endogenous antioxidant enzyme. 18 Doxorubicin reduced endogenous antioxidant enzyme activity including that of haem oxygenase 1 (HO-1), superoxide dismutase (SOD) and glutathione peroxidase (GPx), the levels being measured in both in vitro and in vivo models. 18,47,49,57,[70][71][72][73] Transcription and translation of mitochondrial antioxidant enzymes required the activation of nuclear respiratory factor-1 and 2 (NRF1, NRF2). 20 Several studies indicated that doxorubicin decreased NRF1, nuclear NRF2, TFAM and HO1 expression. 42,49,52 These findings suggested that doxorubicin altered the cardiac antioxidant defence system and could potentially disrupt mitochondrial biogenesis. Taken together, these underlies the importance of mitochondria as the mediator of doxorubicin-induced cardiotoxicity. A summary of reports on cardiac oxidative stress due to doxorubicin is shown in Table 2.
Although oxidative stress is the most notable of mechanisms involved in doxorubicin-induced cardiotoxicity, there is accumulating evidence to suggest that doxorubicin can induce apoptosis through mechanisms that are independent of the ROS production. damage. 74 Consistently, an in vivo study in rats treated with subchronic doxorubicin (2 mg/kg/wk/ip/7 wk) showed a significant increase in PINK1. 32 These findings suggested that doxorubicin-induced mitophagy and contributed to mitochondrial dysfunction.
However, there is evidence to suggest that doxorubicin inhibited mitophagy. 75,76 An in vivo study in rats received doxorubicin (4 mg/kg/ dose/ip/every 5 d/3 doses) indicated that doxorubicin suppressed Parkin-mediated mitophagy. Inhibition of mitophagy resulted in accumulation of damaged mitochondria, mitochondrial dysfunction and impairment in heart function. 76 With regard to the role of autophagy, the autophagic proteins were affected by doxorubicin. 32,77,78 Doxorubicin altered LC3-II and increased p62 level. 32,77,78 There is an emerging consensus that doxorubicin blocked the lysosomal degradation process and increased accumulation of autophagosome and autolysosome; thus, the autophagic process cannot be completed. 79,80 These findings suggested that doxorubicin dysregulated autophagy and inhibited autophagic clearance which accompanied by ROS production, myocardial dysfunction and apoptosis. Reports on these findings are summarized in Table 2.

5' Adenosine monophosphate-activated protein kinase (AMPK) is
a key energy sensor and is activated in response to cellular energy depletion. 81 Phosphorylation of both AMPK and its downstream regulator acetyl-CoA carboxylase (ACC) is observed after ATP depletion or an increase in the AMP/ATP ratio. 82 From in vitro studies, NRCMs treated with doxorubicin (1 µmol/L) showed an increase in the phosphorylation of AMPK (p-AMPK) at 24 hours resulting in activation of the apoptotic pathway and cell death. 54,56 However, another in vitro study using the same model and dosage of doxorubicin showed that p-AMPK measured at 2 hours after doxorubicin treatment was increased. 59 This result suggested that p-AMPK was increased in response to acute stress. Nevertheless, another model using H9c2 cell with doxorubicin 10 nmol/L found that p-AMPK measured at 72 hours afterdoxorubicin therapy was unchanged. 83 In vivo reports also indicated the same results that p-AMPK level could possibly depend on the doxorubicin dosage and exposure time. 54,56,59 Targeting AMPK signalling could be another molecular pathway to mitigate in addressing doxorubicin cardiotoxicity. A summary of these reports is shown in Table 2.

| THE ROLE OF IRON S I G NALLING ON DOXORUB ICIN -INDUCED C ARDIOTOXICIT Y
Iron signalling plays an important role in the mechanism of doxorubicin-induced cardiotoxicity. Specifically, doxorubicin is able to chelate free iron forming doxorubicin-iron complexes which promotes oxidative stress. 84 In addition to the ROS pathway, evidence suggests that doxorubicin cardiotoxicity is also mediated through the oxidative-independent mechanism which is considered to be a key process. 85 87 However, several studies indicated that FHC gene and protein expression are increased following doxorubicin exposure. 43,71,88 This finding is considered to be a defensive mechanism against cytotoxicity of ROS production. Moreover, a recent in vitro study demonstrated that doxorubicin induced the accumulation of free iron, and this was aggravated by haem degradation process. 89 Accumulation of iron within mitochondria caused lipid peroxidation on its membrane which triggered a specific type of programmed cell death called 'ferroptosis'. 89,90 The ferroptosis is a distinct form of regulated cell death which is an iron-dependent process. Blocking of ferroptosis showed a reduction of mortality in mice treated with doxorubicin (20 mg/kg). 89 These reports indicated that doxorubicin can induce cell death through ferroptosis apart from the notable apoptosis. Due to this evidence, prevention of cardiac iron overload by targeting the iron signalling pathway could be an effective strategy in the amelioration of doxorubicin-induced cardiotoxicity. Moreover, inhibition of ferroptosis could be a potential intervention to prevent doxorubicin-induced cardiotoxicity. Future studies are needed to elucidate this hypothesis.

| EFFEC TS OF PHARMACOLOG I C AL AND NON -PHARMACOLOG IC AL INTERVENTIONS ON MITOCHONDRIAL DYNAMIC S IN DOXORUB ICIN -INDUCED C ARD I OTOXI CIT Y: E VIDEN CE FROM IN VITRO AND IN VIVO REP ORTS
Since doxorubicin has been shown to alter the level of mitochondrial  Table 3.

Doxorubicin inhibits mitochondrial fusion and promotes
mitochondrial fragmentation which is accompanied by an increased susceptibility to mPTP opening and ROS generation.
Co-treatment of cyclosporin A, an mPTP inhibitor, with a dose of 1 mg/kg/alternate day in mice treated with doxorubicin (10 mg/

| EFFEC TS OF PHARMACOLOG I C AL AND NON -PHARMACOLOG IC AL INTERVENTIONS ON C ARDIAC MITOCHONDRIAL FUN C TION IN DOXORUB ICIN -INDUCED C ARD I OTOXI CIT Y: E VIDEN CE FROM IN VITRO AND IN VIVO S TUD IE S
The oxidative stress hypothesis is the most widely accepted mechanism for the cause of doxorubicin-induced cardiotoxicity.

Several antioxidant agents have been studied in both in vitro and
animal models. 35  Choosing the interventions that have been widely used in clinical practice could be a favourable way due to the less concern of their adverse reactions. In this regard, beta blocker, statin and metformin have been studied in doxorubicin model. For statin reports, the proposed cardioprotective mechanism of lovastatin is associated with the inhibition of RAC1 signalling with subsequent reduction in apoptosis. 99 Both aerobic exercise training and RESV reduced oxidative stress, promoted expression of mitochondrial electron transport chain proteins and improved heart function attenuated mitochondrial dysfunction by reducing mitochondrial proliferation. 101 Atorvastatin and rosuvastatin also have been shown to prevent doxorubicin-induced cardiotoxicity by reducing oxidative stress and inhibition of apoptosis. 102,103 In addition to statin, beta blocker has also been investigated in doxorubicin model. For example, carvedilol possesses a distinct cardioprotective properties due to its antioxidant effects and the ability to inhibit lipid peroxidation within myocardial cells. 104,105 The studies in animal models indicated that co-administration of carvedilol with doxorubicin prevented the inhibitory effects of doxorubicin on mitochondrial respiration. 106 Carvedilol also prevented mitochondrial damage and the decrease in mitochondrial calcium loading capacity in rats treated with doxorubicin. 104,106 These findings suggested that carvedilol could prevent cardiac mitochondrial dysfunction in doxorubicin model.  (Table 4). It has been shown that inhibition of adiponectin receptor1 (adipoR1) and adiponectin receptor2 (adi-poR2) abrogated the protective effects of metformin in HL-1 cell. 70 Since adiponectin could activate AMPK, this finding indicated the regulation of AMPK as the mechanism responsible for the atten- Evidence from in vivo studies also supports the protective effect of metformin in doxorubicin models (Table 4) Table 4 With regard to non-pharmacological intervention, various physical exercises have been shown to be the effective interventions against doxorubicin-induced cardiotoxicity. Treadmill training in mice treated with doxorubicin showed a reduction in oxidative stress, increased expression of mitochondrial electron transport chain proteins and an improved heart function. 91 The beneficial effects of swim training was involved in an increase in heat shock proteins of the 60 kD family (HSP60). 108 Furthermore, both treadmill training (TM) and free wheel activity (FW) could normalize the increase in the autophagic initiation protein, beclin-1/bcl2 ratio and reduced apoptosis. 32 This finding suggested that lower intensity and longer duration (FW) may be as protective against doxorubicin toxicity as higher intensity and shorter duration exercise (TM). These data strengthen the role of physical exercise in attenuating doxorubicin-induced cardiotoxicity apart from the regulation in mitochondrial dynamics. The comprehensive summary of these findings is shown in Table 4.

Various pharmacological and non-pharmacological interventions
have been studied in both in vitro and animal model as we have discussed. Whether these interventions contribute to beneficial outcome in clinical trials are inconsistent and controversial. [109][110][111] Most of the clinical trials evaluated only heart function and did not demonstrate the possible involved cardioprotective mechanism. [109][110][111] In addition, there is lack of clinical study which investigate the effects of the intervention drugs on mitochondrial dynamics and function. Further clinical studies in this field are needed to improve outcome in these patients.

| CON CLUS ION
The

CO N FLI C T O F I NTE R E S T
None.