Molecular and Cellular Biochemistry

https://doi.org/10.1007/s11010-023-04783-3

Novel molecular mechanisms of doxorubicin cardiotoxicity: latest

leading‑edge advances and clinical implications
Y. Robert Li1 · Kassim Traore2 · Hong Zhu3

Received: 11 April 2023 / Accepted: 4 June 2023

© The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature 2023

Abstract
Doxorubicin (Dox) is among the most widely used cancer chemotherapeutic drugs. The clinical use of Dox is, however,
limited due to its cardiotoxicity. Studies over the past several decades have suggested various mechanisms of Dox-induced
cardiotoxicity (DIC). Among them are oxidative stress, topoisomerase inhibition, and mitochondrial damage. Several novel
molecular targets and signaling pathways underlying DIC have emerged over the past few years. The most notable advances
include discovery of ferroptosis as a major form of cell death in Dox cytotoxicity, and elucidation of the involvement of
cardiogenetics and regulatory RNAs as well as multiple other targets in DIC. In this review, we discuss these advances,
focusing on latest cutting-edge research discoveries from mechanistic studies reported in influential journals rather than
surveying all research studies available in the literature.

Keywords  Cardiogenetics · Cardiomyopathy · Cardiotoxicity · Doxorubicin · Ferroptosis · Heme · Hemopexin ·

Macrophage · Mitochondria · Nrf1 · Regulatory RNA

Abbreviations RCD Regulated cell death

circRNA Circular RNA SLC Solute carrier
DIC Doxorubicin-induced cardiotoxicity SNP Single nucleotide polymorphisms
Dox Doxorubicin TOP2B Topoisomerase 2β
ERK Extracellular regulated kinase
GPx4 Glutathione peroxidase-4
hiPSC-CMs Human induced pluripotent stem cell- Introduction
derived cardiomyocytes
HO-1 Heme oxygenase-1 Doxorubicin is a member of the anthracycline anticancer
lncRNA Long non-coding RNA drug class; other members of this drug class include dauno-
miRNA MicroRNA rubicin and epirubicin. Dox was initially isolated in 1967 in
Nrf1 Nuclear factor E2-related factor 1 the Farmitalia Research Laboratories in Italy from cultures
Nrf2 Nuclear factor E2-related factor 2 of a mutant Streptomyces peucetius (Streptomyces peucetius
OCT3 Cation transporter 3 caesius) [1]. The drug was first approved in 1974 by the
RARG​ Retinoic acid receptor gamma U.S. Food and Drug Administration (FDA) for clinical use.
Presently, Dox remains one of the most widely used cancer
chemotherapeutic agents. It is effective for a range of human
* Y. Robert Li cancers, including leukemias, Wilms tumor, neuroblastoma,
yli@campbell.edu
soft tissue and bone sarcomas, breast carcinoma, ovarian
1
Department of Pharmacology, Campbell University Jerry carcinoma, transitional cell bladder carcinoma, thyroid car-
Wallace School of Osteopathic Medicine, Buies Creek, cinoma, gastric carcinoma, Hodgkin’s disease, malignant
NC 27560, USA lymphoma, and lung cancer [2]. Despite its well-established
2
Department of Biochemistry, Duquesne University College anticancer efficacy, the clinical use of Dox is limited due to
of Osteopathic Medicine, Pittsburgh, PA 15282, USA its cardiotoxicity. In many cases, Dox-induced cardiotox-
3
Department of Physiology and Pathophysiology, Campbell icity (DIC) has been linked to the development of dilated
University Jerry Wallace School of Osteopathic Medicine, cardiomyopathy and heart failure. In fact, Dox-induced
Buies Creek, NC 27560, USA

13
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cardiomyopathy is among the most common etiologies of
cancer chemotherapy-associated heart failure [3, 4].
Although Dox has been used in the treatment of human
cancers for over five decades, the molecular mechanisms
underlying its cardiotoxicity remain partially understood.
Multiple theories have been proposed to explain DIC, which
include redox cycling in mitochondria, topoisomerase inhi-
bition, and oxidative stress [2, 5]. Studies over the past few
years have identified several novel molecular targets and
signaling pathways underlying DIC. This mini-review sur-
veys latest cutting-edge mechanistic findings on DIC and
discusses emerging concepts that may lead to the develop-
ment of more effective strategies for cardioprotection in
Dox-treated cancer patients. To this end, the article focuses
on latest advances in areas related to ferroptosis, cardioge-
netics, regulatory RNAs, and other emerging targets under-
lying DIC.
Ferroptosis in doxorubicin cardiotoxicity
Overview of regulated cell death
It is well known that Dox causes apoptosis in cardiomyo-
cytes, and cardiac apoptosis is the most commonly described
form of cell death induced by Dox in both in  vitro and
in vivo models [6]. Apoptosis is the prototypical form of
regulated cell death (RCD); the newly discovered other
forms of RCD include necroptosis, pyroptosis, ferroptosis,
entotic cell death, netotic cell death, parthanatos, lysosome-
dependent cell death, autophagy-dependent cell death, alka-
liptosis, and oxeiptosis [7], as well as cuproptosis, a most
recently identified form of RCD underlying copper-induced
cytotoxicity [8].
Ferroptosis as a major form of cell death
in doxorubicin cardiotoxicity
In addition to inducing apoptosis, Dox also causes ferrop-
tosis, which appears to be a major form of cell death under-
lying Dox-induced cardiomyopathy in mice [9]. Ferropto-
sis differs from other forms of cell death; it results from
iron-dependent lipid peroxide accumulation triggered by
insufficiency of glutathione peroxidase-4 (GPx4) [10–12].
Dysregulated ferroptosis has been implicated in various dis-
ease processes, including oxidative tissue injury and tumo-
rigenesis [13]. The notion of ferroptosis as a major form of
Dox-induced cell death is in line with the well-established
causal role of iron in DIC [14]. The sections below discuss
latest novel findings on the molecular events underlying
Dox-induced cardiac ferroptosis.
13
Molecular and Cellular Biochemistry
Molecular pathways underlying
doxorubicin‑induced myocardial ferroptosis
HO‑1 upregulation
A study reported in 2019 by Fang et  al. [9], for the
first time, demonstrated that ferroptosis mediates Dox-
induced cardiomyopathy in mice. Using canonical apop-
tosis and/or necroptosis-defective ­Ripk3−/−, ­Mlkl−/−, or
­Fadd−/−Mlkl−/−mice, the authors found that Dox-induced
myocardial ferroptosis drives cardiomyopathy and mortal-
ity following Dox exposure. They further showed that inhi-
bition of ferroptosis by ferrostatin-1 markedly ameliorates
Dox cardiomyopathy. RNA-sequencing analysis revealed
upregulation of heme oxygenase-1 (HO-1) by Dox as a
major mechanism of ferroptotic cardiomyopathy. HO-1
degrades heme and releases free iron in cardiomyocytes,
which in turn leads to the generation of oxidized lipids
in the mitochondrial membrane. Notably, iron chelation
therapy with dexrazoxane (the only FDA-approved drug
for protecting against DIC) alleviates Dox cardiomyopathy
in mice. Moreover, mitochondrially targeted antioxidant
MitoTEMPO rescues Dox cardiomyopathy, supporting
oxidative damage of mitochondria as a major mechanism
underlying Dox-induced ferroptotic cardiac injury [9].
Another important finding of the study is that upregula-
tion of HO-1 by Dox occurs via an Nrf2 (nuclear factor
E2-related factor 2)-dependent mechanism. Consequently,
Dox-induced accumulation of free iron (non-heme iron) in
cardiomyocytes is abolished in Nrf2-null mice [9].
The study by Fang et al., however, did not report the
effect of Nrf2 knockout on Dox-induced cardiomyopa-
thy. Nrf2 is the central regulator of a wide range of genes
whose products are involved in diverse cellular processes,
including antioxidative cytoprotection, antiinflammation,
and drug metabolism, as well as regulation of many other
metabolic pathways [15]. As such, the overall impact of
Nrf2 signaling on Dox cardiomyopathy would be dictated
by the balanced effects of both detrimental factors (in this
case, upregulation of HO-1 and the consequent accumula-
tion of free iron) and protective factors, such as antioxidant
enzymes that have been shown to be protective against
DIC. In this context, a protective role for Nrf2 signaling
and HO-1 expression in doxorubicin-induced cardiotoxic-
ity has been reported previously in other models of DIC
[16, 17]. The exact reasons for the discrepancy between
the findings by Fang et al. and those by others remain
unknown. Nevertheless, the study by Fang et al. conclu-
sively demonstrates a causal role of iron-dependent ferrop-
tosis in Dox cardiomyopathy and the involvement of mito-
chondrial oxidative stress in initiating the cardiac injury.
Molecular and Cellular Biochemistry

GPx4 downregulation

In line with findings by Fang et al. [9], a subsequent study

by Tadokoro et al. [18] demonstrated that mitochondria-
dependent ferroptosis plays a pivotal role in Dox cardiomyo-
pathy in mice. They showed that Dox deceases expression
of GPx4 (a cellular suppressor of ferroptosis) and induces
excessive lipid peroxidation through accumulation of Dox-
Fe2+ complex in mitochondria, leading to mitochondria-
dependent ferroptosis. They also demonstrated that mito-
chondria-dependent ferroptosis is a major cause of DOX
cardiomyopathy. Notably, compared with wild-type mice,
Dox-induced cardiomyopathy and left ventricular dysfunc-
tion are ameliorated in GPx4-overexpressing mice and exac-
erbated in GPx4-heteroknockout ­(GPx4±) mice, indicating
a causal involvement of GPx4 downregulation in Dox car-
diomyopathy [18]. Moreover, the authors found that chela-
tion of intramitochondrial ­Fe2+ by Mito-FerroGreen, but not
­ e3+ by deferoxamine or dexrazoxane, prevents
chelation of F
Dox-induced lipid peroxidation and ferroptosis in cultured
cardiomyocytes, suggesting that pharmacological chelation
of mitochondrial F ­ e2+ may be a more effective approach
to preventing DIC than F ­ e3+ chelation (such as by dexra-
zoxane) [18]. Indeed, the clinical efficacy of dexrazoxane
in preventing DIC in cancer patients has been shown to be
rather limited [19, 20].
Suppression of heme biosynthesis
More recently, Abe et al. further investigated the molecu-
lar events leading to mitochondria-dependent ferroptosis
underlying DIC [21]. Using cultured cardiomyocytes, they
showed that Dox accumulates in mitochondria by intercalat-
ing into mitochondrial DNA (mtDNA), inducing ferroptosis
in an mtDNA content-dependent manner. Moreover, Dox
suppresses the synthesis of heme by decreasing the level
of 5′-aminolevulinate synthase 1 (Alas1), the rate-limiting
enzyme in heme biosynthesis, thereby impairing iron utili-
zation, resulting in iron overload and mitochondria-depend-
ent ferroptosis. As a result, either Alas1 overexpression or
supplementation with 5-aminolevulinic acid (5-ALA), the
product of Alas1, prevents Dox-induced iron overload and
ferroptosis in cardiomyocytes. Notably, administration of
5-ALA to mice also suppresses Dox-induced iron overload
and ferroptotic cardiomyopathy [21]. Hence, the study iden-
tifies Alas1 as a novel molecular target of Dox and suggests
a therapeutic potential of 5-ALA in DIC.
In summary, as illustrated in Fig. 1, induction of myo-
cardial iron-dependent ferroptosis is a major cellular event
underlying DIC. Dox may cause mitochondrial iron accumu-
lation via two mechanisms: (1) upregulation of HO-1, lead-
ing to degradation of heme to release free iron; and (2) inhi-
bition of heme synthesis, resulting in decreased utilization
Fig. 1  Novel mechanisms by which doxorubicin induces iron over-
load, lipid peroxidation, and ferroptosis. As illustrated, both Nrf2/
HO-1 and Alas1/heme synthesis pathways as well as GPx4 downreg-
ulation may be involved in doxorubicin-induced iron overload, lipid
peroxidative, and mitochondrial ferroptosis, leading to cardiomyopa-
thy. See text for detailed description. (+) denotes increase or activa-
tion; (−) denotes decrease or inhibition. This diagram is based pri-
marily on novel findings reported by Fang et al. [9], Tadokoro et al.
[18], and Abe et al. [21]
of free iron and consequent iron overload. On the other hand,
Dox downregulates GPx4, an enzyme responsible for elimi-
nating lipid hydroperoxides and acting as an endogenous
suppressor of ferroptosis [22]. Iron accumulation in mito-
chondria promotes oxidative stress, lipid peroxidation, and
ferroptosis, leading to cardiomyopathy and left ventricular
dysfunction. Hence, selective chelation of mitochondrial
iron may be a promising approach to suppressing ferropto-
sis and thereby protecting against cardiomyopathy and heart
failure in Dox-treated cancer patients. It, however, should
be noted that cardioprotective modalities must not hinder
the anticancer activity of Dox. In this context, iron chela-
tion, including mitochondrial iron chelation has been shown
to suppress tumor growth and metastasis in animal models
[23]. It is, thus, imperative to determine the effect of iron
chelation on Dox’s anticancer activity.
Cardiogenetics of doxorubicin cardiotoxicity
Overview of cardiogenetics
Cardiogenetics is the study of the genetic causes of heart
diseases and how genetic variations affect cardiac patholo-
gies and their response to drug therapy. As an important
aspect of cardiogenetics, studying the effects of genetic
variations on patients’ response to cardiovascular drugs has

13
 Molecular and Cellular Biochemistry

Fig. 2  Role of SNPs in
doxorubicin cardiotoxicity. As
illustrated, multiple SNPs can
downregulate SLC28A3, lead-
ing to reduced transport of Dox
into cardiomyocytes and thereby
decreased Dox cardiotoxicity
(upper panel). On the other
hand, certain SNPs may affect
various cellular signaling path-
ways to augment Dox cardio-
toxicity (lower panel). See text
for detailed discussion of the
original findings on which this
diagram is primarily based

critical implications in precision medicine. Indeed, personal-
ized therapies based on pharmacogenetic data have greatly
improved the clinical management of various cardiovascular
diseases, especially atherosclerotic coronary artery disease
[24–26]. On the other hand, studies of the effects of genetic
variations on drug-induced cardiac injury, including Dox
cardiomyopathy have also advanced our understanding of
the mechanisms of drug-induced cardiotoxicity and may
contribute to the development of strategies to identify sus-
ceptible patients so as to minimize the risk of cardiotoxicity.
The following sections discuss latest mechanistic studies on
major genetic variations affecting DIC.
Genetic variations in doxorubicin cardiotoxicity:
recent mechanistic studies
While there have been many reports on the association of
diverse single nucleotide polymorphisms (SNPs) with Dox
cardiotoxicity [27–29], conclusive, mechanistic studies to
establish the causality are relatively few. Described below
are notable mechanistic studies on genetic variations in drug
transporters and retinoic acid receptor gamma (RARG), as
well as non-coding SNPs underlying DIC (Fig. 2).
Drug transporters
Multiple pharmacogenomic studies have identified the
synonymous genomic (exonic) variant rs7853758 (G > A,
L461L) and the intronic variant rs885004 in SLC28A3
(solute carrier family 28 member 3) to be associated with a
lower incidence of DIC [30–32]. However, the true causal
variant(s), the cardioprotective mechanism of this locus, the
role of SLC28A3 in DIC, and the suitability of SLC28A3
as a target for cardioprotective drugs had remained unde-
termined until a very recent study by Magdy et al. [33].
SLC28A3, also known as concentrative nucleoside trans-
porter 3 (CNT3), plays an important role in mediating the
cellular entry of a broad array of physiological nucleosides
and synthetic anticancer nucleoside analog drugs [34].
The association of the above genetic variations of
SLC28A3 with DIC suggests that this membrane trans-
porter may also mediate Dox uptake into cardiac cells.
To define the role of SCL28A3 genetic variants in
DIC, Magdy et  al. [33] recruited 6 well-phenotyped,

13
Molecular and Cellular Biochemistry
Dox-treated pediatric patients from the original associa-
tion study cohort, and generated human induced pluri-
potent stem cell-derived cardiomyocytes (hiPSC-CMs)
from them. With this approach, the authors showed that
the patient-specific hiPSC-CMs recapitulate the cardio-
protective effect of the SNP rs7853758. SLC28A3 gene
knockdown and overexpression using CRISPR/Cas9
reduces and increases Dox uptake into the cardiomyocytes,
respectively, altering their sensitivity to Dox cytotoxic-
ity. These observations confirm the role of this locus in
DIC. Notably, upon fine mapping of the SLC28A3 locus,
the authors discovered that a linked SNP rs11140490,
rather than the originally identified rs7853758, is actu-
ally the causal cardioprotective variant within that locus.
Mechanistically, rs11140490 exerts its action by upregu-
lating a SLC28A3-overlapping antisense long non-cod-
ing RNA (lncRNA) SLC28A3-AS1. Overexpression of
SLC28A3-AS1 downregulates SCL28A3, reducing Dox
uptake into the cardiomyocytes. Moreover, via screening a
drug library using hiPSC-CMs followed by in vivo valida-
tion in a mouse model of DIC, the authors demonstrated
that the SLC competitive inhibitor, desipramine protects
against DIC without compromising Dox anticancer effi-
cacy. Hence, these novel findings establish SLC28A3 as
a transporter for Dox uptake into cardiomyocyte and pro-
vide a basis for genetic testing of rs11140490 to identify
patients who are protected from DIC. The findings also
point to two potential therapeutic options: either using
the lncRNA SLC28A3-AS1 or developing a desipramine
analog to protect against DIC [33]. The work by Magdy
et al. represents an excellent example of a mechanistic
cardiogenetic study to establish a causal role of a genetic
variant in DIC. The study also demonstrates the power of
patient-specific hiPSC-CMs in mechanistic investigation
of cardiogenetics of DIC as well as cardioprotective drug
discovery (see below).
Indeed, in a recent study, using hiPSC-CMs from patients
receiving Dox, Huang et al. probed the transcriptomic land-
scape of solute carriers and identified another transporter
SLC22A3 (also known as organic cation transporter 3 or
OCT3) as a critical player regulating cardiac accumulation
of Dox [35]. They showed that OCT3 deficiency markedly
reduces Dox accumulation in cardiac tissue and protects
mice from Dox-induced cardiac injury and left ventricular
dysfunction. They demonstrated that pharmacological inhi-
bition of OCT3 (e.g., by nilotinib) preserves cardiac function
following treatment with Dox without affecting its plasma
levels or antitumor effects in multiple models of leukemia
and breast cancer. Hence, the findings identify OCT3 as a
previously unrecognized Dox uptake transporter in cardio-
myocytes that can be targeted pharmacologically to aug-
ment the therapeutic index of Dox [35]. As SNPs in OCT3
gene have been identified in human populations and to be
associated with cardiovascular risk [36, 37], it is important
to determine if such genetic variations also affect patients’
susceptibility to DIC.
Although both SLC28A3 and SLC22A3 act as Dox
uptake transporters in the heart, the relative importance of
these transporters in modulating DIC in a particular patient
remains unclear. In addition, other transporter such as the
organic cation transporters SLC22A4 and SLC22A17 are
also implicated in Dox uptake into cardiomyocytes [33].
Hence, it is likely that the eventual impact would be deter-
mined by the relative cardiac abundance and activity of these
transporters in a particular patient. Indeed, genetic variations
in genes encoding membrane transporters are common in
human populations [38, 39].
RARG​
In addition to modulating Dox transport, genetic variations
may also affect the intracellular machinery underlying Dox
cytotoxicity. For example, a missense variant rs2229774
(S427L) in the RARG gene is associated with increased
susceptibility to DIC in pediatric cancer patients [40].
Recently, Burridge and associates showed that hiPSC-CMs
from patients who had rs2229774 and suffered DIC are more
sensitive to Dox cytotoxicity [41]. Mechanistically, this
RARG variant effect is mediated via increased expression of
topoisomerase 2β (TOP2B; a critical mediator of DIC [42])
and decreased activation of the cardioprotective extracellu-
lar regulated kinase (ERK) pathway. The authors also used
patient-specific hiPSC-CMs as a drug discovery platform
and demonstrated that the RARG agonist CD1530 attenu-
ates DIC in mice. The findings hence provide a rationale for
clinical prechemotherapy genetic screening for rs2229774
and a foundation for the clinical use of RARG agonist
treatment to protect cancer patients from DIC [41]. More
recently, using CRISPR-Cas9-genome-edited hiPSC-CMs,
Huang et al. reported that the RARG variant rs2229774 has
impaired ability to activate its target genes in response to
Dox, including gene pathways involved in DNA repair and
consequently an inability to mediate DNA repair after Dox
treatment. This results in enhanced susceptivity to Dox-
induced cardio-cytotoxicity [43]. The findings by Huang
et al. thus suggest another novel cellular pathway mediated
by RARG that could be targeted for protecting against DIC.
Non‑coding SNPs
Genetic variations in the non-coding sequences may also
affect DIC. In this regard, a genome-wide association study
(GWAS) identified an association of a non-coding SNP
rs28714259 (G/A) with an increased risk of developing Dox-
induced heart failure [44]. Using hiPSC-CMs with either
intrinsic polymorphism or CRISPR-Cas9-mediated deletion
13

of rs28714259 locus, Schneider and coworkers [45] recently
found that rs28714259 disrupts glucocorticoid receptor (GR)
binding and thus blocks GR-activated expression of genes
involved in adaptive survival responses to Dox-induced
cardio-cytotoxicity. As a result, rs28714259 risk allele or
loss of rs28714259 locus decreases dexamethasone-induced
protection of cardiomyocyte viability and contractility after
doxorubicin exposure [45]. Glucocorticoids remain a stand-
ard therapy in the concurrent treatment with Dox to prevent
nausea and allergic reactions [46]. Hence, the above findings
highlight additional benefits of adjunctive glucocorticoid
therapy in reducing DIC in cancer patients [45].
Regulatory RNAs in doxorubicin
cardiotoxicity
Overview of regulatory RNAs
Regulatory RNAs are composed mostly of non-coding
RNAs (ncRNAs). Among the regulatory RNAs, microRNAs
(miRNAs), long non-coding RNAs (lncRNAs), and circular
RNAs (circRNAs) have received the most attention in recent
years for their ability to modulate diverse physiological and
pathophysiological processes [47–49], including cardiovas-
cular homeostasis and pathologies [50–52]. Over the past
few years, the potential involvement of a number of regula-
tory RNAs, including miRNAs, lncRNAs, and circRNAs, in
DIC has been reported in the literature though high-quality
mechanistic studies published in influential journals are rela-
tively few. Hence, in the sections below, we focus only on
discussing the novel findings from these few mechanistic
studies.
Regulatory RNAs in doxorubicin cardiotoxicity:
recent mechanistic studies
lncRNA SLC28A3‑AS1
As discussed earlier, the SLC28A3-overlapping antisense
lncRNA SLC28A3-AS1 is a critical mediator of the associa-
tion of SNP rs11140490 with decreased SLC28A3 expres-
sion (and thus decreased Dox uptake into cardiomyocytes)
[33]. This illustrates a scenario where a lncRNA mediates
an SNP-induced downregulation of a membrane transporter
for Dox.
Circular RNA CircITCH
In an exhaustive mechanistic study, Han et al. discovered
that the tumor-suppressive human circular RNA CircITCH
sequesters miR-330-5p to ameliorate DIC, revealing an inti-
mate interaction between a circRNA and an miRNA in DIC
13
Molecular and Cellular Biochemistry
[53]. CircITCH (circular RNA ITCH [E3 ubiquitin-protein
ligase]) is a broad-spectrum tumor-suppressive circRNA.
Early studies suggested that the host gene of CircITCH,
ITCH (E3 ubiquitin-protein ligase), is involved in DIC [54].
Using hiPSC-CMs and specimens from cancer patients who
suffered from Dox-induced cardiomyopathy, Han et al. found
that CircITCH is expressed in both human cardiac tissue
and hiPSC-CMs, and its expression is significantly down-
regulated in DIC. Overexpression of CircITCH in hiPSC-
CMs protects against Dox cytotoxicity. Mechanistically,
CircITCH functions as a competing endogenous RNA to
sequester miR-330-5p, resulting in activation of cytopro-
tective SIRT6, BIRC5, and ATP2A2 and the consequently
increased resistance of cardiomyocytes to Dox-induced
mitochondrial oxidative stress and DNA damage. In this
context, miR-330-5p interacts with the 3′-UTRs of SIRT6,
BIRC5, and ATP2A2 mRNAs to cause their downregulation;
accordingly, sequestering miR-330-5p by CircITCH pre-
vents this downregulation, thereby augmenting the expres-
sion of these cytoprotective factors. Notably, overexpression
of CircITCH in mice represses the levels of miR-330-5p,
upregulates SIRT6, BIRC5, and ATP2A2, and attenuates
Dox-induced cardiomyopathy and left ventricular dysfunc-
tion. Collectively, these findings reveal a novel role of the
“CircITCH/miR-330-5p/SIRT6-BIRC5-ATP2A2” axis as a
competing endogenous RNA network governing DIC [53].
The findings also point to the feasibility of using a circu-
lar RNA (i.e., CircITCH)-based modality to protect against
DIC in cancer patients. In this regard, circRNAs exhibit high
druggability due to their high stability arising from special
closed loop structure [55].
Circular RNA CircINSR
A circular RNA derived from the insulin receptor locus,
known as CircINSR, has recently been shown to also
protect against DIC [56]. While insulin receptor signal-
ing plays an important role in cardiac homeostasis [57,
58], the role of CircINSR, a highly species-conserved
circRNA derived from the insulin receptor gene, in car-
diac physiology and disease remains unclear. Lu et al.
[56] recently showed that CircINSR is downregulated in
heart failure in both animal models and humans, including
heart failure induced by Dox. CircINSP downregulation
leads to mitochondrial damage and cardiomyocyte death,
and cardiac dysfunction. Conversely, overexpression of
CircINSR prevents Dox-induced cytotoxicity in cultured
human cardiomyocytes and dramatically protects against
Dox-induced cardiomyopathy and ventricular dysfunction
in mice. Mechanistically, CircINSR physically interacts
with the single-stranded DNA-binding protein 1 (SSBP1)
to maintain mitochondrial function by stabilizing mito-
chondrial DNA during Dox exposure, thereby preventing
Molecular and Cellular Biochemistry
Dox-induced mitochondrial damage and cardiomyocyte
death [56]. Notably, a human CircINSR mimics, synthe-
sized through in vitro transcription and subsequent circu-
larization via a DNA splint and enzymatic ligation, also
prevents Dox-induced cardiac cytotoxicity, highlighting
the feasibility of using CircINSR-based therapeutics for
the intervention of DIC in cancer patients [56].
In summary, multiple regulatory RNAs have been impli-
cated in DIC, either as mediators or protectors of DIC,
depending on the mechanistic pathways involved (Fig. 3).
As regulatory RNAs play important roles in diverse physi-
ological and pathophysiological processes, targeting these
molecules by Dox likely also causes diverse consequences.
Hence, future studies should determine not only the role of
regulatory RNAs in DIC, but also their impact on Dox’s
anticancer efficacy. Likewise, future investigation is war-
ranted to assess the potential adverse effects of regula-
tory RNA-based therapeutics on body systems. This is
particularly relevant in view of the current high enthu-
siasm in developing regulatory RNA-based therapies for
human diseases [55, 59]. On the other hand, mechanistic
studies on regulatory RNAs in DIC not only deepen our
understanding of the molecular mechanisms by which Dox
causes cardiomyopathy and heart failure, but also advance
our knowledge of cardiac disease pathogenesis.
Fig. 3  Role of circRNAs in doxorubicin cardiotoxicity. As illustrated,
circRNAs may modulate doxorubicin cardiotoxicity via either alter-
ing endogenous cardioprotective defenses or regulating mitochon-
drial DNA (mitoDNA) stability. See text for detailed description of
the original findings on which this diagram is primarily based. (+)
denotes increase or activation; (−) denotes decrease or inhibition
Other emerging targets of doxorubicin
cardiotoxicity
In addition to the novel molecular targets and signaling
pathways described in the preceding sections, several addi-
tional molecular and cellular targets have emerged over the
past few years to either underly DIC or serve as endogenous
defenses against DIC. The sections below highlight some
notable studies.
Cardiac macrophages
Macrophages are found in all tissues and exhibit great func-
tional diversities, including roles in physiological homeosta-
sis and pathophysiological processes. Tissue macrophages
include: (1) those derived from blood monocytes, which
in turn are derived from bone marrow progenitors and (2)
resident macrophages, which are derived from embryonic
progenitors that populate all tissues, and persist through-
out life [60]. Cardiac macrophages play important roles
in cardiac homeostasis and disease [61–64]. For example,
cardiac resident macrophages prevent fibrosis and stimulate
angiogenesis in response to cardiac pressure overload in
mice [64]. This is in line with the notion that tissue resident
macrophages help resolve inflammation and tissue damage,
whereas recruited blood monocyte-derived macrophages
possess a proinflammatory phenotype, contributing to
inflammatory stress and tissue injury [60, 65, 66].
Cardiac macrophages are also involved in Dox-induced
cardiomyopathy [67]. In this regard, recruited blood-borne
macrophages with a proinflammatory phenotype constitute
the primary source of accumulated cardiac macrophages,
dominating the progression of Dox-induced cardiomyopathy
in mice. In contrast, proliferation of cardiac resident mac-
rophages driven by SR-A1-c-Myc signaling axis attenuates
Dox-induced cardiomyopathy by helping resolving inflam-
mation and retarding cardiomyopathy progression. Hence,
the SR-A1-c-Myc axis may represent a promising target to
treat Dox cardiomyopathy through augmentation of cardiac
resident reparative macrophage proliferation [67].
Circulating hemopexin
As aforementioned, iron plays a critical role in DIC, espe-
cially myocardial ferroptosis, and chelation of iron ame-
liorates Dox-induced cardiomyopathy and heart failure
in cancer patients [68, 69]. A recent study demonstrated
that hemopexin (a circulating heme-binding protein [70])
modulates Dox-induced cardiomyopathy in patients and
mice [71]. Specifically, via proteomic analysis of plasma
samples from Dox-treated patients, the authors showed that
13

increased hemopexin is associated with cardiac toxicity and
dysfunction assessed by echocardiography. Administration
of hemopexin to wild-type mice treated with Dox improves
cardiac function. Conversely, hemopexin-knockout mice
show increased Dox cardiac toxicity compared to wild-type
mice. Mechanistically, hemopexin is likely transported to
the heart by circulating monocytes/macrophages and that
hemopexin mitigates Dox-induced ferroptosis to confer
cardioprotection. Together, these observations suggest that
hemopexin induction represents a compensatory response
during Dox treatment [71]. The findings may motivate addi-
tional research to determine whether the hemopexin path-
way could be leveraged for therapeutic benefit in patients
at risk of Dox cardiomyopathy. In this context, based on
its beneficial effects in reverting heme-induced damage in
animal models of sick cell disease (SCD) [72], hemopexin
is currently under clinical trial to determine its efficacy in
treating SCD patients (https://​sickl​ecell​socie​ty.​org/​csl889-​
clini​cal-​trial/).
Nrf1
The nuclear factor E2-related factor 1 (Nrf1) belongs to the
Cap’N’Collar (CNC) family of leucine zipper transcription
factors, which include also Nrf2 (nuclear factor E2-related
factor 2), Nrf3 (nuclear factor E2-related factor 3), and Nfe2
(nuclear factor E2) [73, 74]. While Nrf2 is the most exten-
sively studied transcriptional activator of cytoprotective
genes, studies on Nrf1 are relatively fewer. Like Nrf2, Nrf1
also binds to the antioxidant response element (ARE), but
Nrf1 and Nrf2 play distinct roles in the activation of ARE-
dependent genes. For example, while both Nrf1 and Nrf2
bind with comparable affinity to the ARE in the metallothio-
nein-1 (MT1) gene promoter, only Nrf1 is able to upregulate
reporter gene expression driven by the MT1 promoter [75].
On the other hand, the inducible expression of many other
antioxidant and cytoprotective genes is largely dependent on
Nrf2. In contrast to Nrf2, Nrf1 is an essential gene during
development. Homozygous deletion of Nrf1 gene in mice
results in late gestational embryonic lethality [76].
Nrf1 is a critical regulator of multiple essential cellular
processes, including cholesterol homeostasis [77], ferropto-
sis [78], and proteostasis [79, 80]. More recently, Cui et al.
showed that among the CNC family transcription factors,
Nrf1 is the most highly expressed member in the heart and
promotes heart regeneration and repair by regulating pro-
teostasis and redox balance. [81]. Specifically, using a neo-
natal mouse heart regeneration model, the authors demon-
strated that Nrf1 is activated in regenerating cardiomyocytes.
Genetic deletion of Nrf1 prevents regenerating cardiomyo-
cytes from activating a transcriptional program required for
heart regeneration. Conversely, Nrf1 overexpression protects
the adult mouse heart from ischemia/reperfusion injury.
13
Molecular and Cellular Biochemistry
Notably, using hiPSC-CMs, the authors also found that
overexpression of Nrf1 markedly attenuates Dox-induced
cardio-cytotoxicity, as evidenced by improved cell viability
and preserved contractility [81]. This finding suggests that
Nrf1 (a highly expressed CNC transcription factor in car-
diomyocytes) may function as an important cytoprotector
against DIC. Further studies are warranted to determine the
cardioprotective function of Nrf1 in animal models of DIC.
Conclusion and perspectives
While oxidative stress, mitochondrial damage, and topoi-
somerase inhibition remain the key mechanistic scheme of
DIC, the past few years have witnessed a rapid growth in
the number of high-quality mechanistic studies reported in
influential journals. These studies have uncovered a range
of novel molecular and cellular targets/signaling pathways
underlying DIC in animal models as well as patient-specific
hiPSC-CMs. These include mitochondrial iron-dependent
ferroptosis, genetic variations and regulatory RNAs, as well
as cardiac resident macrophages, circulating hemopexin,
and Nrf1 signaling. These novel findings not only broaden
our understanding of the molecular mechanisms of DIC,
but also provide opportunities to develop mechanistically
based modalities for DIC intervention in cancer patients. In
addition, these advances may also benefit the management
of cardiomyopathy caused by other drugs or pathological
conditions. It should be noted, however, that the primary
goal of cancer chemotherapy is to eradicate cancer cells, and
as such, cardioprotective approach in such patients should
not hinder the cancer-killing activity of the chemotherapy. It
is thus imperative that experimental studies on cardioprotec-
tion of DIC should also simultaneously assess the impact on
Dox’s anticancer activity.
As discussed in this review, multiple underlying mecha-
nisms and numerous molecular targets are involved in DIC,
and each of them is concluded to be a significant cause of
DIC by the individual high-quality study. An immediate
question is which one really plays a dominant role. While
there is no easy answer to this question, it is likely that the
significance of a particular finding made in a specific experi-
mental model might not be applicable to other experimental
models or conditions. In this context, while studies using
animal models are critical for advancing knowledge, their
validation and translatability into clinical medicine remain
a pressing challenge. It is hoped that continued systematic
mechanistic studies would eventually lead to a clear pic-
ture of the predominant mechanism of DIC or elucidation
of a final common pathway at which distinct mechanisms
converge to cause cardiomyopathy and heart failure in Dox-
treated patients.
Molecular and Cellular Biochemistry
Author contributions  All authors contributed to the review article topic
conception, literature searching and analysis, and the writing of the
manuscript. All authors read and approved the final manuscript.
Funding  This work was supported in part by an IIG Grant (09A084)
from the American Institute for Cancer Research and a Grant
(CA192936) from the National Institutes of Health/National Cancer
Institute.
Data availability  Not applicable as this is a review article.
Declarations  (2017) Oxidized arachidonic and adrenic PEs navigate cells to
Conflict of interest  The author has no relevant financial or non-finan-
cial interests to disclose.
Ethical approval  Not applicable. This work does not involve animals
or human subjects.
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