Neurotoxic Effect of Doxorubicin Treatment on Cardiac Sympathetic Neurons

Abstract

Doxorubicin (DOXO) remains amongst the most commonly used anti-cancer agents for the treatment of solid tumors, lymphomas, and leukemias. However, its clinical use is hampered by cardiotoxicity, characterized by heart failure and arrhythmias, which may require chemotherapy interruption, with devastating consequences on patient survival and quality of life. Although the adverse cardiac effects of DOXO are consolidated, the underlying mechanisms are still incompletely understood. It was previously shown that DOXO leads to proteotoxic cardiomyocyte (CM) death and myocardial fibrosis, both mechanisms leading to mechanical and electrical dysfunction. While several works focused on CMs as the culprits of DOXO-induced arrhythmias and heart failure, recent studies suggest that DOXO may also affect cardiac sympathetic neurons (cSNs), which would thus represent additional cells targeted in DOXO-cardiotoxicity. Confocal immunofluorescence and morphometric analyses revealed alterations in SN innervation density and topology in hearts from DOXO-treated mice, which was consistent with the reduced cardiotropic effect of adrenergic neurons in vivo. Ex vivo analyses suggested that DOXO-induced denervation may be linked to reduced neurotrophic input, which we have shown to rely on nerve growth factor, released from innervated CMs. Notably, similar alterations were observed in explanted hearts from DOXO-treated patients. Our data demonstrate that chemotherapy cardiotoxicity includes alterations in cardiac innervation, unveiling a previously unrecognized effect of DOXO on cardiac autonomic regulation, which is involved in both cardiac physiology and pathology, including heart failure and arrhythmias.

Full text

Citation: Moro, N.; Dokshokova, L.;
Perumal Vanaja, I.; Prando, V.;
Cnudde, S.J.A.; Di Bona, A.; Bariani,
R.; Schirone, L.; Bauce, B.; Angelini,
A.; et al. Neurotoxic Effect of
Doxorubicin Treatment on Cardiac
Sympathetic Neurons. Int. J. Mol. Sci.
2022,23, 11098. https://doi.org/
10.3390/ijms231911098
Academic Editors: Alice Bonomi,
Erica Rurali and Maria
Cristina Vinci
Received: 10 August 2022
Accepted: 17 September 2022
Published: 21 September 2022
Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
published maps and institutional affil-
iations.
Copyright: © 2022 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
International Journal of
Molecular Sciences
Article
Neurotoxic Effect of Doxorubicin Treatment on Cardiac
Sympathetic Neurons
Nicola Moro 1, Lolita Dokshokova 1, Induja Perumal Vanaja 2, Valentina Prando 1, Sophie Julie A Cnudde 3,
Anna Di Bona 2, Riccardo Bariani 2, Leonardo Schirone 4, Barbara Bauce 2, Annalisa Angelini 2,
Sebastiano Sciarretta 4, Alessandra Ghigo 3, Marco Mongillo 1, * and Tania Zaglia 1 ,*
1Department of Biomedical Sciences, University of Padova, Via Ugo Bassi 58/B, 35131 Padova, Italy
2Department of Cardiac, Thoracic, Vascular Sciences and Public Health, University of Padova,
Via Giustiniani 2, 35128 Padova, Italy
3
Molecular Biotechnology Center, Department of Molecular Biotechnology and Health Sciences, University of
Torino, 10126 Torino, Italy
4Department of Medical and Surgical Sciences and Biotechnologies, Sapienza, University of Rome,
04100 Latina, Italy
*
Correspondence: marco.mongillo@unipd.it (M.M.); tania.zaglia@unipd.it (T.Z.); Tel.: +39-0497923229 (M.M.);
+39-0497923294 (T.Z.); Fax: +39-0497923250 (M.M.); +39-0497923250 (T.Z.)
Abstract:
Doxorubicin (DOXO) remains amongst the most commonly used anti-cancer agents for
the treatment of solid tumors, lymphomas, and leukemias. However, its clinical use is hampered
by cardiotoxicity, characterized by heart failure and arrhythmias, which may require chemotherapy
interruption, with devastating consequences on patient survival and quality of life. Although the
adverse cardiac effects of DOXO are consolidated, the underlying mechanisms are still incompletely
understood. It was previously shown that DOXO leads to proteotoxic cardiomyocyte (CM) death
and myocardial fibrosis, both mechanisms leading to mechanical and electrical dysfunction. While
several works focused on CMs as the culprits of DOXO-induced arrhythmias and heart failure,
recent studies suggest that DOXO may also affect cardiac sympathetic neurons (cSNs), which would
thus represent additional cells targeted in DOXO-cardiotoxicity. Confocal immunofluorescence and
morphometric analyses revealed alterations in SN innervation density and topology in hearts from
DOXO-treated mice, which was consistent with the reduced cardiotropic effect of adrenergic neurons
in vivo
. Ex vivo analyses suggested that DOXO-induced denervation may be linked to reduced
neurotrophic input, which we have shown to rely on nerve growth factor, released from innervated
CMs. Notably, similar alterations were observed in explanted hearts from DOXO-treated patients.
Our data demonstrate that chemotherapy cardiotoxicity includes alterations in cardiac innervation,
unveiling a previously unrecognized effect of DOXO on cardiac autonomic regulation, which is
involved in both cardiac physiology and pathology, including heart failure and arrhythmias.
Keywords:
Doxorubicin; cardiotoxicity; sympathetic neurons; cardiac innervation; nerve growth factor
1. Introduction
Cancer accounts for more than 8 million deaths per year. Doxorubicin (DOXO) is one
of the most common anti-cancer agents, used for the treatment of several solid tumors,
lymphomas, and leukemias. Unfortunately, a major complication of DOXO regimens
is represented by cardiotoxicity [
1
–
5
], characterized by heart failure (HF) and increased
arrhythmic vulnerability, which may require chemotherapy interruption, with devastating
consequences on tumor progression and increased mortality [
4
,
6
]. Remarkably, HF is
the first cause of mortality in cancer survivors [
7
–
11
]. Despite intense research efforts,
the mechanisms underlying DOXO-induced cardiotoxicity are still largely nebulous and
strategies to treat the adverse consequences of the drug are lacking. Thus, cancer patients
are in urgent need of effective responses to prevent a life-saving therapy from marking
Int. J. Mol. Sci. 2022,23, 11098. https://doi.org/10.3390/ijms231911098 https://www.mdpi.com/journal/ijms

Int. J. Mol. Sci. 2022,23, 11098 2 of 19
the onset of a new deadly disease. In addition, although the link between DOXO and
malignant arrhythmias is consolidated [
12
,
13
], the underlying mechanisms are still obscure.
We and others recently demonstrated that DOXO leads to heart dysfunction [
14
–
17
].
In addition, a set of studies demonstrated that DOXO impinges on cardiomyocyte (CM)
signaling pathways that are linked to arrhythmogenesis [
18
–
20
]. While these and other
reports attribute a primary role of CM injury in DOXO-induced arrhythmias, it has to be
kept in mind that alterations in the function and topology of cardiac sympathetic neurons
(cSNs) may play a relevant role in arrhythmic vulnerability [
21
–
25
]. Interestingly,
in vitro
studies evidenced a direct effect of DOXO on several neuronal populations, including
cSNs, through mechanisms that are similar to those that were described for CMs, such as
autophagy impairment [
26
,
27
], increased oxidative stress [
28
–
32
], and DNA damage [
33
],
culminating in neurotoxicity and cell death [30,31,34–36].
Although such studies undoubtedly demonstrate that DOXO has direct toxic effects
on neurons, the
in vitro
approach disregards that survival and function of peripheral neu-
rons
in vivo
depends on intercellular signaling and cell–cell interactions taking place in
the innervated microenvironment [
37
,
38
]. On this trail, in the effort to understand the
interactions between innervating sympathetic neurons (SNs) and cardiac cellular compo-
nents, we have recently studied in detail the functional and structural interactions between
SNs and CMs [
39
–
41
]. This has led us to demonstrate emerging aspects of neuro-cardiac
physiology, reflecting on neuro-cardiac regulation and on the effect of CMs on neuronal
health [
39
–
41
], which laid the hypothesis of the current study. In detail, SNs highly inner-
vate mammalian hearts, including human, with a precise species-specific topology, which,
if altered, may cause uneven modification of electrophysiology in discrete heart regions,
favoring arrhythmias [
25
,
41
]. SN cell bodies mainly organize in cervical ganglia, while their
processes invade the myocardial interstitium, with the typical ‘pearl-necklace’ morphol-
ogy, characterized by regularly distributed varicosities, i.e., the neurotransmitter-releasing
sites, which establish, with CMs, a synaptic contact, recently named the neuro-cardiac
junction (NCJ) [
39
–
41
]. At the contact site, a tight intercellular interaction occurs between
the neuron and the targeted CM, which underlays both anterograde (SN-to-CM) and
retrograde (CM–SN) communication. Through the former, SNs stimulate the heart via
noradrenaline which, by activating
β
-adrenoceptors (
β
-ARs), enhances heart rate and
contractility during stresses [
25
,
39
,
41
], and modulates CM size and electrophysiology, by
regulating proteostasis [
41
–
43
]. In parallel, reverse cardio-neuronal signaling, enacted
through nerve growth factor (NGF), provides the continuing trophic input that is required
to maintain the functioning and correctly patterned innervation in the developed heart [
40
].
Indeed, NGF, released from CMs, binds to specific receptors that are expressed on SNs (i.e.,
TrkA and p75), and the neurotrophin/receptor complexes are retrogradely transported to
the neuronal soma, where NGF activates differentiation, survival, and functional signaling
pathways [
40
]. Thus, the refined intercellular interaction, at the NCJ, ties neurons and CMs
in a double-stranded bond, which implies that dysfunction of one of such post-mitotic cells
negatively impacts on the other.
On these bases, we tested here the hypothesis whereby the effect of DOXO on CMs
may reflect on SN health and, as such, that DOXO negatively impinges on multiple cardiac
cell types, including SNs. Confocal immunofluorescence and morphometric analyses
in murine and human heart sections have thus been used to assess the state of cardiac
sympathetic innervation upon DOXO treatment. Our results show that cardiac innervation
is severely compromised by DOXO, which causes a significant decrease in the neuronal
density and alterations in innervation topology. In line with our surmised working model,
these effects were attributed to the impairment of cardiac neurotrophic signaling by NGF.
2. Results
2.1. Doxorubicin Alters Cardiac Sympathetic Neuron Function
To assess whether DOXO treatment affects the function of cSNs, we treated normal
adult C57BL/6J male mice with DOXO, following the protocol that was described in the

Int. J. Mol. Sci. 2022,23, 11098 3 of 19
Method section and in Figure 1a. In line with published data, this DOXO regimen caused a
progressive decline in body weight during drug administration (Figure 1b), which returned
to normal weight within a week from DOXO interruption. In addition, cardiac function,
assessed by echocardiography (ECHO) showed declined cardiac contractility six weeks after
the end of DOXO treatment (ejection fraction, vehicle: 64.79
±
5.32 vs. DOXO: 51.43
±
6.61;
fractional shortening, vehicle: 34.40
±
3.74 vs. DOXO: 25.84
±
4.16, in %) (Figure 1c,d),
which was thus the time point that was chosen for all subsequent histologic and functional
heart analyses.
Int. J. Mol. Sci. 2022, 23, x FOR PEER REVIEW 3 of 19
2. Results
2.1. Doxorubicin Alters Cardiac Sympathetic Neuron Function
To assess whether DOXO treatment affects the function of cSNs, we treated normal
adult C57BL/6J male mice with DOXO, following the protocol that was described in the
Method section and in Figure 1a. In line with published data, this DOXO regimen caused
a progressive decline in body weight during drug administration (Figure 1b), which re-
turned to normal weight within a week from DOXO interruption. In addition, cardiac
function, assessed by echocardiography (ECHO) showed declined cardiac contractility six
weeks after the end of DOXO treatment (ejection fraction, vehicle: 64.79 ± 5.32 vs. DOXO:
51.43 ± 6.61; fractional shortening, vehicle: 34.40 ± 3.74 vs. DOXO: 25.84 ± 4.16, in %) (Fig-
ure 1c,d), which was thus the time point that was chosen for all subsequent histologic and
functional heart analyses.
Figure 1. DOXO treatment leads to cardiac contractile dysfunction. (a) DOXO administration and
ex vivo analyses protocol: study design. (b) Body weight evaluation during mouse treatment with
DOXO. The nonlinear fitting line is shown in red. (c) Echocardiographic short-axis (top) and B-mode
(bottom) views of hearts from vehicle- vs. DOXO-treated mice. These analyses were performed six
weeks after completion of drug administration regimen. Blue/red lines and arrows represent left
ventricle (LV) systolic and diastolic diameters in vehicle- and DOXO-treated mice, respectively. (d)
Echocardiographic assessment of LV ejection fraction in vehicle- and DOXO-treated mice, at differ-
ent time points after completion of DOXO administration. Bars represent the standard deviation
(s.d.). Differences among the groups were determined using one way ANOVA with Dunnett’s test
for multiple comparisons. (n = 5 mice for each group; *, p < 0.05). DOXO, doxorubicin; Veh, vehicle.
At this time point, in which the cardiotoxic effects of DOXO were previously reported
to manifest [44–46], harvested hearts were grossly normal, with slightly expanded inter-
stitial spaces, and moderately infiltrated foci and collagen deposition (Figure 2a,b). Mor-
phometrically, the hearts were moderately atrophic, with a mean decrease in the CM
cross-sectional area of 25.40 ± 37.90% (Figure 2c,d).
Figure 1.
DOXO treatment leads to cardiac contractile dysfunction. (
a
) DOXO administration and
ex vivo analyses protocol: study design. (
b
) Body weight evaluation during mouse treatment with
DOXO. The nonlinear fitting line is shown in red. (
c
) Echocardiographic short-axis (top) and B-mode
(bottom) views of hearts from vehicle- vs. DOXO-treated mice. These analyses were performed
six weeks after completion of drug administration regimen. Blue/red lines and arrows represent
left ventricle (LV) systolic and diastolic diameters in vehicle- and DOXO-treated mice, respectively.
(
d
) Echocardiographic assessment of LV ejection fraction in vehicle- and DOXO-treated mice, at
different time points after completion of DOXO administration. Bars represent the standard deviation
(s.d.). Differences among the groups were determined using one way ANOVA with Dunnett’s test for
multiple comparisons. (n= 5 mice for each group; *, p< 0.05). DOXO, doxorubicin; Veh, vehicle.
At this time point, in which the cardiotoxic effects of DOXO were previously re-
ported to manifest [
44
–
46
], harvested hearts were grossly normal, with slightly expanded
interstitial spaces, and moderately infiltrated foci and collagen deposition (Figure 2a,b).
Morphometrically, the hearts were moderately atrophic, with a mean decrease in the CM
cross-sectional area of 25.40 ±37.90% (Figure 2c,d).

Int. J. Mol. Sci. 2022,23, 11098 4 of 19
Int. J. Mol. Sci. 2022, 23, x FOR PEER REVIEW 4 of 19
Figure 2. DOXO treatment leads to cardiac atrophic remodeling. (a,b) Hematoxylin-eosin staining
in heart sections from the vehicle- (a) and DOXO- (b) treated mice, six weeks after the completion
of DOXO administration. Right panels in (a,b) are high magnification of the black boxed areas in
the left panels. (c) Confocal immunofluorescence on heart sections from the mid-portion of the ven-
tricles of vehicle- vs. DOXO-treated mice, stained with Alexa Fluor™-555-conjugated wheat germ
agglutinin (WGA). The nuclei were counterstained with 4',6-Diamidino-2-Phenylindole (DAPI). Im-
ages were used to calculate the cardiomyocyte (CM) cross-sectional areas (CSA) (d). Differences
among the groups were determined using a Mann–Whitney test. (n > 2500 CMs from four different
hearts for each group; ****, p < 0.0001). The solid line on violin plot represents the median, dashed
lines are 25/75 percentiles. RV, right ventricle; IVS, interventricular septum; LV, left ventricle;
DOXO, doxorubicin; Veh, vehicle.
Electrocardiographic (ECG) recording, performed at different time points before sac-
rifice, did not evidence differences in the heart rate and QRS duration in anesthetized
DOXO- vs. vehicle-treated mice (Figure 3a,b). To determine the functional effects of
DOXO on cSNs, the resting SN activity was estimated at the end of experimental protocol
by acute administration of atropine, which antagonizes the effects of the parasympathetic
branch of the autonomic nervous system on pacemaker cells [42]. While atropine caused
the expected effect on heart rate, which increased by 22.43 ± 9.27% in the vehicle-treated
Figure 2.
DOXO treatment leads to cardiac atrophic remodeling. (
a
,
b
) Hematoxylin-eosin staining in
heart sections from the vehicle- (
a
) and DOXO- (
b
) treated mice, six weeks after the completion of
DOXO administration. Right panels in (
a
,
b
) are high magnification of the black boxed areas in the left
panels. (
c
) Confocal immunofluorescence on heart sections from the mid-portion of the ventricles of
vehicle- vs. DOXO-treated mice, stained with Alexa Fluor
™
-555-conjugated wheat germ agglutinin
(WGA). The nuclei were counterstained with 4
0
,6-Diamidino-2-Phenylindole (DAPI). Images were
used to calculate the cardiomyocyte (CM) cross-sectional areas (CSA) (
d
). Differences among the
groups were determined using a Mann–Whitney test. (n > 2500 CMs from four different hearts
for each group; ****, p< 0.0001). The solid line on violin plot represents the median, dashed lines
are 25/75 percentiles. RV, right ventricle; IVS, interventricular septum; LV, left ventricle; DOXO,
doxorubicin; Veh, vehicle.
Electrocardiographic (ECG) recording, performed at different time points before sac-
rifice, did not evidence differences in the heart rate and QRS duration in anesthetized
DOXO- vs. vehicle-treated mice (Figure 3a,b). To determine the functional effects of
DOXO on cSNs, the resting SN activity was estimated at the end of experimental protocol
by acute administration of atropine, which antagonizes the effects of the parasympa-

Int. J. Mol. Sci. 2022,23, 11098 5 of 19
thetic branch of the autonomic nervous system on pacemaker cells [
42
]. While atropine
caused the expected effect on heart rate, which increased by 22.43
±
9.27% in the vehicle-
treated mice, DOXO almost completely ablated the atropine-induced heart rate increase
(5.68
±
4.80%), suggesting dysfunction in the neurogenic control of heart rhythm, consistent
with heart sympathetic denervation (Figure 3c,d).
Int. J. Mol. Sci. 2022, 23, x FOR PEER REVIEW 5 of 19
mice, DOXO almost completely ablated the atropine-induced heart rate increase (5.68 ±
4.80%), suggesting dysfunction in the neurogenic control of heart rhythm, consistent with
heart sympathetic denervation (Figure 3c,d).
Figure 3. DOXO treatment affects cardiac sympathetic neuron function. (a,b) Electrocardiographic
evaluation of heart rate (HR, (a)) and QRS duration (b) in the DOXO- vs. vehicle-treated mice, at
different time points after the completion of DOXO administration. (c) Representative trace of HR
increase in a vehicle- vs. a DOXO-treated mouse upon Atropine injection. (d) Evaluation of the frac-
tional HR increase 5 min after atropine administration. Bars represent the s.d. Differences among
the groups were determined using an unpaired t-test. (n = 4 mice for each group; *, p < 0.05). DOXO,
doxorubicin; Veh, vehicle; BPM, beats per minute.
2.2. Doxorubicin Compromises the State of Sympathetic Innervation in Murine Hearts
The evidence of decreased chronotropic effect of SNs in DOXO-treated mice
prompted us to assess whether such functional failure was underscored by sympathetic
neuropathology. To this purpose, non-consecutive heart sections from the mid-portion of
the ventricles were analyzed by confocal immunofluorescence, and SN processes were
identified using an anti-tyrosine hydroxylase antibody. Normal mouse hearts are highly
innervated by SNs, which distribute in the myocardial interstitium showing regularly dis-
placed varicosities along the neuronal process [40]. Qualitative analysis of the heart from
the DOXO-treated mice suggested that the density of cardiac sympathetic innervation was
profoundly decreased in both the right and left ventricles (Figure 4a). This evidence was
confirmed by morphometric analysis of the innervation density in the left ventricle (LV),
demonstrating an 82.22 ± 13.93% decrease in the fraction of myocardial area that was oc-
cupied by tyrosine hydroxylase-positive fibers (Figure 4b,c). Such denervation was of a
similar degree in both the sub-epicardial and sub-endocardial regions (sub-epicardium,
vehicle: 3.11 ± 0.76 vs. DOXO: 0.58 ± 0.45; sub-endocardium, vehicle: 2.15 ± 0.45 vs. DOXO:
0.35 ± 0.26, in %) (Figure 4c), which we have recently demonstrated to be innervated at
Figure 3.
DOXO treatment affects cardiac sympathetic neuron function. (
a
,
b
) Electrocardiographic
evaluation of heart rate (HR, (
a
)) and QRS duration (
b
) in the DOXO- vs. vehicle-treated mice, at
different time points after the completion of DOXO administration. (
c
) Representative trace of HR
increase in a vehicle- vs. a DOXO-treated mouse upon Atropine injection. (
d
) Evaluation of the
fractional HR increase 5 min after atropine administration. Bars represent the s.d. Differences among
the groups were determined using an unpaired t-test. (n = 4 mice for each group; *, p< 0.05). DOXO,
doxorubicin; Veh, vehicle; BPM, beats per minute.
2.2. Doxorubicin Compromises the State of Sympathetic Innervation in Murine Hearts
The evidence of decreased chronotropic effect of SNs in DOXO-treated mice prompted
us to assess whether such functional failure was underscored by sympathetic neuropathol-
ogy. To this purpose, non-consecutive heart sections from the mid-portion of the ventricles
were analyzed by confocal immunofluorescence, and SN processes were identified using
an anti-tyrosine hydroxylase antibody. Normal mouse hearts are highly innervated by SNs,
which distribute in the myocardial interstitium showing regularly displaced varicosities
along the neuronal process [
40
]. Qualitative analysis of the heart from the DOXO-treated
mice suggested that the density of cardiac sympathetic innervation was profoundly de-
creased in both the right and left ventricles (Figure 4a). This evidence was confirmed by
morphometric analysis of the innervation density in the left ventricle (LV), demonstrating
an 82.22
±
13.93% decrease in the fraction of myocardial area that was occupied by tyrosine
hydroxylase-positive fibers (Figure 4b,c). Such denervation was of a similar degree in both

Int. J. Mol. Sci. 2022,23, 11098 6 of 19
the sub-epicardial and sub-endocardial regions (sub-epicardium, vehicle: 3.11
±
0.76 vs.
DOXO: 0.58
±
0.45; sub-endocardium, vehicle: 2.15
±
0.45 vs. DOXO: 0.35
±
0.26, in %)
(Figure 4c), which we have recently demonstrated to be innervated at different density,
reflecting the peculiar electrophysiologic and trophic effects of neuronal activity in the two
regions [41].
Int. J. Mol. Sci. 2022, 23, x FOR PEER REVIEW 6 of 19
different density, reflecting the peculiar electrophysiologic and trophic effects of neuronal
activity in the two regions [41].
In both the sub-endocardial and sub-epicardial regions, SN processes of DOXO-
treated hearts appeared thinner and fragmented, with a significant reduction in the area
of varicosities (Figure 4d,e) and immunoreactivity to anti-tyrosine hydroxylase (fluores-
cence intensity, vehicle: 45.31 ± 4.71 vs. DOXO: 13.89 ± 8.18, a.u.), all features that are sug-
gestive of sympathetic neurodegeneration.
Figure 4. DOXO treatment causes cardiac sympathetic denervation in mice. (a) Confocal immuno-
fluorescence on heart sections from the ventricular mid-portion of vehicle- vs. DOXO-treated mice.
Sections were stained with an antibody to tyrosine hydroxylase (TH). (b) Images are high magnifi-
cation of the white boxed areas in (a). (c) Quantification of the percentage of TH-positive area in the
sub-epicardial (Epi) and sub-endocardial (Endo) LV myocardium of the control (Veh) and DOXO
hearts. Each point represents the average tyrosine hydroxylase positive (TH+) area of 20 independ-
ent images from the Epi and Endo regions in five hearts analyzed/group. Bars represent the s.d.
Differences among the groups were determined using an unpaired t-test (*, p < 0.05; ***, p < 0.001;
****, p < 0.0001; ns, no significant). (d) High magnifications of SN processes in the LV of control and
DOXO hearts. (e) Quantification of sympathetic neuron (SN) varicosity area. Differences among the
groups were determined using a Mann–Whitney test. (n > 1000 varicosities from five different hearts
Figure 4.
DOXO treatment causes cardiac sympathetic denervation in mice. (
a
) Confocal immunoflu-
orescence on heart sections from the ventricular mid-portion of vehicle- vs. DOXO-treated mice.
Sections were stained with an antibody to tyrosine hydroxylase (TH). (
b
) Images are high magnifica-
tion of the white boxed areas in (
a
). (
c
) Quantification of the percentage of TH-positive area in the
sub-epicardial (Epi) and sub-endocardial (Endo) LV myocardium of the control (Veh) and DOXO
hearts. Each point represents the average tyrosine hydroxylase positive (TH+) area of 20 indepen-
dent images from the Epi and Endo regions in five hearts analyzed/group. Bars represent the s.d.
Differences among the groups were determined using an unpaired t-test (*, p< 0.05; ***, p< 0.001;
****, p< 0.0001; ns, no significant). (
d
) High magnifications of SN processes in the LV of control and
DOXO hearts. (
e
) Quantification of sympathetic neuron (SN) varicosity area. Differences among the
groups were determined using a Mann–Whitney test. (n > 1000 varicosities from five different hearts

Int. J. Mol. Sci. 2022,23, 11098 7 of 19
in each group; ****, p< 0.0001). RV, right ventricle; IVS, interventricular septum; LV, left ventricle.
DOXO, doxorubicin; Veh, vehicle; DAPI, 40,6-Diamidino-2-Phenylindole.
In both the sub-endocardial and sub-epicardial regions, SN processes of DOXO-treated
hearts appeared thinner and fragmented, with a significant reduction in the area of varicosi-
ties (Figure 4d,e) and immunoreactivity to anti-tyrosine hydroxylase (fluorescence intensity,
vehicle: 45.31
±
4.71 vs. DOXO: 13.89
±
8.18, a.u.), all features that are suggestive of
sympathetic neurodegeneration.
Based on these results, we harvested and analyzed superior cervical and stellate
ganglia, which contain the cell soma of most cSNs, from both the control and DOXO-
treated mice. Confocal immunofluorescence analysis demonstrated that the DOXO-treated
ganglia had a significant reduction in the density of neuronal soma (Figure 5a,b) and a
decrease in the average size of the remainder, which were atrophic (Figure 5c).
Int. J. Mol. Sci. 2022, 23, x FOR PEER REVIEW 7 of 19
in each group; ****, p < 0.0001). RV, right ventricle; IVS, interventricular septum; LV, left ventricle.
DOXO, doxorubicin; Veh, vehicle; DAPI, 4',6-Diamidino-2-Phenylindole.
Based on these results, we harvested and analyzed superior cervical and stellate gan-
glia, which contain the cell soma of most cSNs, from both the control and DOXO-treated
mice. Confocal immunofluorescence analysis demonstrated that the DOXO-treated gan-
glia had a significant reduction in the density of neuronal soma (Figure 5a,b) and a de-
crease in the average size of the remainder, which were atrophic (Figure 5c).
Figure 5. DOXO causes cardiac sympathetic neuron degeneration. (a) Immunofluorescence on stel-
late ganglia sections from the vehicle- vs. DOXO-treated mice. Slices were stained with an antibody
to tyrosine hydroxylase (TH). Nuclei were counterstained with 4',6-Diamidino-2-Phenylindole
(DAPI). (b,c) Quantification of the fraction of neuronal soma/total cell number (b) and the mean
area of the sympathetic neuron (SN) cell bodies (c) in the stellate ganglia of the DOXO- vs. vehicle-
treated mice. Bars represent the s.d. Differences among the groups were determined using a Mann–
Whitney test. (n > 500 cells from nine independent images/group from three different mice; **, p <
0.01; ****, p < 0.0001). DOXO, doxorubicin; Veh, vehicle.
2.3. Doxorubicin Treatment Reduces Heart-Derived NGF Input to Neurons
The evidence that, in DOXO-treated mice, cSN varicosities were smaller in size and
SN cell bodies were atrophic are all features that were previously linked to reduced neu-
rotrophic input, which we have shown to rely on NGF that is released from innervated
CMs [40]. This hypothesis is in line with the previous demonstration that DOXO treatment
severely compromises NGF production in rat hearts [47]. In line with this study, our re-
sults showed that the NGF protein content in hearts trended to decrease, since three days
after initiating DOXO treatment, and become significantly lower at three days after com-
pletion of the drug regimen up to six weeks (Figure 6a,b). Consistently, immunofluores-
cence with an anti-NGF antibody in SN of cervical ganglia sections, revealed decreased
Figure 5.
DOXO causes cardiac sympathetic neuron degeneration. (
a
) Immunofluorescence on stellate
ganglia sections from the vehicle- vs. DOXO-treated mice. Slices were stained with an antibody to
tyrosine hydroxylase (TH). Nuclei were counterstained with 4
0
,6-Diamidino-2-Phenylindole (DAPI).
(
b
,
c
) Quantification of the fraction of neuronal soma/total cell number (
b
) and the mean area of
the sympathetic neuron (SN) cell bodies (
c
) in the stellate ganglia of the DOXO- vs. vehicle-treated
mice. Bars represent the s.d. Differences among the groups were determined using a Mann–Whitney
test. (n > 500 cells from nine independent images/group from three different mice; **, p< 0.01;
****, p< 0.0001). DOXO, doxorubicin; Veh, vehicle.
2.3. Doxorubicin Treatment Reduces Heart-Derived NGF Input to Neurons
The evidence that, in DOXO-treated mice, cSN varicosities were smaller in size and
SN cell bodies were atrophic are all features that were previously linked to reduced neu-
rotrophic input, which we have shown to rely on NGF that is released from innervated

Int. J. Mol. Sci. 2022,23, 11098 8 of 19
CMs [
40
]. This hypothesis is in line with the previous demonstration that DOXO treatment
severely compromises NGF production in rat hearts [
47
]. In line with this study, our results
showed that the NGF protein content in hearts trended to decrease, since three days after
initiating DOXO treatment, and become significantly lower at three days after completion of
the drug regimen up to six weeks (Figure 6a,b). Consistently, immunofluorescence with an
anti-NGF antibody in SN of cervical ganglia sections, revealed decreased immunoreactivity
in the DOXO-treated samples, supporting defective retrograde CM–SN signaling via NGF
(Figure 6c).
Int. J. Mol. Sci. 2022, 23, x FOR PEER REVIEW 8 of 19
immunoreactivity in the DOXO-treated samples, supporting defective retrograde CM–SN
signaling via NGF (Figure 6c).
Figure 6. DOXO treatment is accompanied by reduced cardiac NGF content. (a) Western blotting
on the protein extracts of hearts that were explanted from: control mice (vehicle-treated); mice that
were treated with DOXO for three days (group#1); mice that were analyzed three days after the
completion of DOXO treatment (group#2); mice that were analyzed 6 weeks after the completion of
DOXO administration (group#3). (b) The relative densitometry is shown in the right graph. Bars
represent the s.d. Differences among the groups were determined using a one-way ANOVA with
Dunnett’s test for multiple comparisons. (n = 6 hearts for each group; *, p < 0.05; **, p < 0.01, ns,
statistically non-significant). (c) Immunofluorescence on stellate ganglia sections from vehicle- vs.
DOXO-treated mice, six weeks after completion of DOXO treatment. Sections were stained with an
antibody to Nerve Growth Factor (NGF). DOXO, doxorubicin; Veh, vehicle.
2.4. Doxorubicin Compromises Sympathetic Neurons in Human Hearts
The results that we acquired in the experimental models suggest that the broad spec-
trum of manifestations of DOXO cardiotoxicity may include the degeneration of cSNs, an
aspect that is potentially linked to heart dysfunction (e.g., arrhythmias), that has never
addressed in detail thus far. To ascertain whether neuropathologic features, similar to
those that were observed in mouse hearts could be detected in cSN processes of patients
that had undergone DOXO treatment, we analyzed sections of explanted hearts from
three patients who developed HF upon chemotherapy cardiotoxicity (Figure 7a). In these
Figure 6.
DOXO treatment is accompanied by reduced cardiac NGF content. (
a
) Western blotting
on the protein extracts of hearts that were explanted from: control mice (vehicle-treated); mice that
were treated with DOXO for three days (group#1); mice that were analyzed three days after the
completion of DOXO treatment (group#2); mice that were analyzed 6 weeks after the completion
of DOXO administration (group#3). (
b
) The relative densitometry is shown in the right graph. Bars
represent the s.d. Differences among the groups were determined using a one-way ANOVA with
Dunnett’s test for multiple comparisons. (n = 6 hearts for each group; *, p< 0.05; **, p< 0.01, ns,
statistically non-significant). (
c
) Immunofluorescence on stellate ganglia sections from vehicle- vs.
DOXO-treated mice, six weeks after completion of DOXO treatment. Sections were stained with an
antibody to Nerve Growth Factor (NGF). DOXO, doxorubicin; Veh, vehicle.

Int. J. Mol. Sci. 2022,23, 11098 9 of 19
2.4. Doxorubicin Compromises Sympathetic Neurons in Human Hearts
The results that we acquired in the experimental models suggest that the broad
spectrum of manifestations of DOXO cardiotoxicity may include the degeneration of cSNs,
an aspect that is potentially linked to heart dysfunction (e.g., arrhythmias), that has never
addressed in detail thus far. To ascertain whether neuropathologic features, similar to
those that were observed in mouse hearts could be detected in cSN processes of patients
that had undergone DOXO treatment, we analyzed sections of explanted hearts from
three patients who developed HF upon chemotherapy cardiotoxicity (Figure 7a). In these
samples, confocal immunofluorescence demonstrated a global decrease of myocardial
innervation density and fragmentation of neuronal processes (Figure 7b,c). Our results
surmise that morphologic and functional alterations in cSNs, resulting in denervation and
loss of neuronal inputs to cardiac cells, are additional and previously neglected effects of
DOXO treatment.
Int. J. Mol. Sci. 2022, 23, x FOR PEER REVIEW 9 of 19
samples, confocal immunofluorescence demonstrated a global decrease of myocardial in-
nervation density and fragmentation of neuronal processes (Figure 7b,c). Our results sur-
mise that morphologic and functional alterations in cSNs, resulting in denervation and
loss of neuronal inputs to cardiac cells, are additional and previously neglected effects of
DOXO treatment.
Figure 7. Patients receiving DOXO treatment show cardiac sympathetic denervation. (a) Character-
istics of patients that were analyzed in the study. D, DOXO; C, cytarabine; V, vincristine. The per-
centage of fibrosis was calculated as described in the Methods section. Heart weight was assessed
at the time of explant. (b) Confocal immunofluorescence on human heart sections from the mid-
portion of the LV in healthy vs. DOXO-treated hearts. Sections were stained with an antibody to
tyrosine hydroxylase (TH). The images were used to quantify the percentage of myocardial area
that was occupied by TH-positive (TH+) fibers (c). Bars represent s.d. Differences among groups
were determined using unpaired t-test with Welch’s correction. (Four (Ctrl) and three (DOXO) in-
dependently processed heart slices from three different hearts/groups were analyzed; *, p < 0.05).
3. Discussion
In this study, by combining in vivo assessment of cardiac function and analyses of
heart samples from mice that had undergone DOXO administration, with that of hearts
that were explanted from cancer-bearing patients that were suffering post-chemotherapy
HF, we show that cSNs are affected by DOXO treatment. Our results suggest that cSN
degeneration is accompanied with reduced cardiac NGF production, likely compromising
‘CM-neuron’ neurotrophic input, which we previously showed to be essential to maintain
correct cardiac sympathetic innervation [40]. These results identify a previously neglected
mechanism of DOXO-induced neurodegeneration, potentially synergizing with other di-
rect effects of the drug on neurons, including autophagy impairment, DNA damage, and
increased oxidative stress [26,28,29,31–33]. In addition, we uncover the neuropathologic
effect of DOXO on SNs regulating heart function, which may be involved in HF and ar-
rhythmias, both distinctive features of DOXO cardiotoxicity [4,13,48–51].
Figure 7.
Patients receiving DOXO treatment show cardiac sympathetic denervation. (
a
) Characteris-
tics of patients that were analyzed in the study. D, DOXO; C, cytarabine; V, vincristine. The percentage
of fibrosis was calculated as described in the Methods section. Heart weight was assessed at the time
of explant. (
b
) Confocal immunofluorescence on human heart sections from the mid-portion of the LV
in healthy vs. DOXO-treated hearts. Sections were stained with an antibody to tyrosine hydroxylase
(TH). The images were used to quantify the percentage of myocardial area that was occupied by
TH-positive (TH+) fibers (
c
). Bars represent s.d. Differences among groups were determined using
unpaired t-test with Welch’s correction. (Four (Ctrl) and three (DOXO) independently processed
heart slices from three different hearts/groups were analyzed; *, p< 0.05).
3. Discussion
In this study, by combining
in vivo
assessment of cardiac function and analyses of
heart samples from mice that had undergone DOXO administration, with that of hearts that
were explanted from cancer-bearing patients that were suffering post-chemotherapy HF, we
show that cSNs are affected by DOXO treatment. Our results suggest that cSN degeneration

Int. J. Mol. Sci. 2022,23, 11098 10 of 19
is accompanied with reduced cardiac NGF production, likely compromising ‘CM-neuron’
neurotrophic input, which we previously showed to be essential to maintain correct cardiac
sympathetic innervation [40]. These results identify a previously neglected mechanism of
DOXO-induced neurodegeneration, potentially synergizing with other direct effects of the
drug on neurons, including autophagy impairment, DNA damage, and increased oxidative
stress [
26
,
28
,
29
,
31
–
33
]. In addition, we uncover the neuropathologic effect of DOXO on SNs
regulating heart function, which may be involved in HF and arrhythmias, both distinctive
features of DOXO cardiotoxicity [4,13,48–51].
Although DOXO is the most common chemotherapeutic agent in several malignancies,
its use is associated with important cardiac complications, enclosed in the broad definition
of “DOXO cardiotoxicity” [
52
–
55
]. Based on the time of onset, cardiotoxicity may be acute,
manifesting within two weeks from the end of treatment; chronic, developing within one
year; or late-onset, developing even several years after chemotherapy completion. In
addition, subclinical DOXO cardiotoxicity has increasingly been described, and prompts
the identification of criteria and biomarkers for early diagnosis and treatment [
51
,
56
–
60
].
Cardiac complications of DOXO represent a high socio-economic burden, as they may lead
to HF, the first cause of death/transplant in cancer survivors, myocardial infarction, and
arrhythmic episodes (i.e., tachycardia, atrial and ventricular arrhythmias) [
12
,
50
,
61
–
65
].
In addition, such complications, when occurring acutely during treatment, may require
chemotherapy interruption with inauspicious outcomes for patients, while, in the long-
term, they may severely compromise patient life quality and expectancy, as in the case of
heart transplant (for HF) or implantable cardioverter-defibrillator (ICD) implantation (for
arrhythmias) [
4
,
61
–
63
,
65
–
67
]. Thus, preventing/reducing the risks of cardiotoxicity and its
progression, without reducing the efficacy of anti-cancer treatment, is a primary goal of
cardio-oncology.
Up to now, the term ‘cardiotoxicity’ has been used as synonym of ‘cardiomyocyte (CM)
toxicity’ and, as such, cardio-oncology has mainly focused on CMs, which are undoubtedly
affected by DOXO [
53
–
55
,
68
–
73
]. Abundant research has attributed the adverse cardiac
effects of DOXO to a plethora of mechanisms affecting cell electrophysiology, signaling,
mechanics, and cell survival, which subsequently lead to myocardial remodeling, con-
tractile dysfunction, or arrhythmias [
4
,
18
,
54
,
55
,
74
,
75
]. However, some manifestations of
cardiotoxicity (e.g., arrhythmic episodes, alterations in ECG parameters) may involve the
impairment of cardiac extrinsic regulatory systems, including e.g., the altered function or
distribution (i.e., myocardial topology) of cSNs [76–80].
The heart is highly innervated by SNs, whose activity has conventionally been as-
sociated with heart adaptation to acute stresses, during the ‘fight-or-flight’ reaction [
25
].
Recently, we have uncovered additional long-term effects of cSNs which, by regulating
proteolytic machineries via
β
-ARs, determine CM size and electrophysiology [
25
,
41
,
42
].
The microscopic interactions between neurons and target cardiac cells have recently been
characterized in higher detail, and the concept of direct neuro-cardiac communication, as
underscored by specific intercellular synaptic contacts has been appraised [
39
,
40
,
81
]. Such
contact sites have been identified as a hub of adrenergic signaling ‘from-neuron–CMs’,
shaping and controlling target cell trophism and function, on the one hand [
39
,
41
], and, on
the other, of neurotrophic signaling ‘from-CMs–neurons’, determining and maintaining
the topology of the myocardial innervation network [
40
]. Such local and spatially deter-
mined neuro-cardiac interactions allow the activation of cSNs to result in adequate cardiac
responses, while avoiding arrhythmias. Bidirectional SN-CM coupling implies that failure
of CMs to support the innervating neurons with NGF would result in neurodegeneration,
local cardiac denervation, and aberrant innervation topology, the latter potentially ensuing
in arrhythmias [
76
–
80
]. Neurodegeneration may, in turn, compromise cardiac adrenergic
signaling, which reflects on CM structure and function, jeopardizing NGF production, and
in a vicious cycle, worsen sympathetic denervation.
On these bases, we here approached DOXO cardiotoxicity as a condition affecting the
entire myocardial cell network and focused on the effects of DOXO on cardiac innervation,

Int. J. Mol. Sci. 2022,23, 11098 11 of 19
as explored in the hearts of mice that were exposed to DOXO in a chemotherapy regimen
that was similar to that which is used in humans [
14
,
82
]. Our data demonstrate that DOXO
causes severe cardiac sympathetic denervation, accompanied with alterations in neuronal
distribution pattern in both ventricles, as shown by immunofluorescence, and function, as
indicated by the reduced heart rate increase, upon atropine administration in DOXO mice.
Interestingly, the reduced SN inputs to target CMs is predicted to impinge on signaling
pathways and cell homeostatic systems (e.g., autophagy, oxidative stress, Ca
2+
dynamics)
which have been described to be independently affected by DOXO [
14
,
42
,
53
,
82
,
83
]. It
can thus be inferred that the toxicity of DOXO on CMs may be amplified by its adverse
effects on cSNs. As an example, the atrophic remodeling of DOXO-treated CMs may be
the consequence of activated intracellular proteolysis, by the ubiquitin proteasome system,
combined with the indirect effect of SN degeneration, further promoting CM induction of
atrogenes through decreased
β
2-AR stimulation [
42
]. In a similar manner, SN degeneration
may result from a cell-directed effect of DOXO on neurons, the secondary effect of toxicity
on CMs, or a combination of both mechanisms. In line with recently published data [
47
],
we observed that hearts from DOXO-treated mice have reduced NGF content, already
in the initial phases of treatment, which is expected to compromise the ability of CMs
to feed innervating neurons. Such mechanisms, previously not taken into account, may
overlap the neurotoxic effects of DOXO, including the induction of oxidative stress and
autophagy impairment, given that NGF, in addition to activating pro-survival and trophic
signaling [
84
–
87
], has antioxidant effects and is an autophagy activator in both CMs and
SNs [88–91].
Our data identifies new cellular targets and systems that are affected by DOXO.
While this finding seemingly increases the complexity of the pathogenetic framework
underlying DOXO cardiotoxicity, it also directs therapeutic strategies to comprehensively
treat CMs, cSNs, and the neuro-cardiac axis. The inference of our results would suggest
approaches that are based on NGF targeting, i.e., to increase cardiac NGF availability. Such
an approach, however, is complicated by the multitude of effects that are exerted by the
neurotrophin on different cell types, including cancer cells, whose treatment with a growth
factor is in obvious contrast with the general goal of chemotherapy [
92
–
96
]. Interestingly,
what emerges from our results and consolidated published data, is that most mechanisms
leading to cardiotoxicity, including the alteration of neuro-cardiac regulation, converge on
autophagy impairment and the activation of oxidative stress. This is in line with the lead
concept supporting current research efforts, which target autophagy and oxidative stress in
DOXO cardio-protection, bringing into the scenario the idea that therapeutic approaches
need to be directed to specific regulatory mechanisms in different cell types, not to the
sole CMs.
The finding that cSNs are targeted by DOXO is in line with existing clinical evidence
and has implications at different levels, spanning from basic knowledge on disease mech-
anisms, research pursuing the identification of therapeutic strategies in DOXO-treated
patients, to the definition of improved diagnostic tools for the management of cancer
patients.
From the preclinical standpoint, our evidence of cardiac denervation, obtained in experi-
mental rodents and human heart samples, confirmed previous case reports in which nuclear
imaging revealed cSN atrophy and denervation in patients with DOXO-cardiomyopathy.
Interestingly, both in humans and in mice, it was surmised that SN degeneration is a
precocious event preceding cardiac adverse symptoms [
97
,
98
], suggesting that hearts are
exposed to progressively variable, and possibly heterogeneous or erratic SN activity, until
denervation. Although with the obvious limitation of a proof-of-concept study and those
of individual case reports, this data broadens the spectrum of cells and signaling pathways
that are affected by DOXO treatment, increasing the knowledge on the basic mechanisms
of DOXO cardiotoxicity. In addition, these results laid the ground for further research that
is aimed at assessing the state of sympathetic innervation of other organs and districts, e.g.,

Int. J. Mol. Sci. 2022,23, 11098 12 of 19
gut, skeletal muscles, which may explain extracardiac manifestations of DOXO toxicity
(e.g., modification of gut microbiota, tissue inflammation) [99–101].
Our results may also guide towards the identification of mechanism-driven therapies in
DOXO-treated patients. Indeed, given that cardiac sympathetic dysfunction has previously
been linked to arrhythmias and HF, both of which are frequently experienced by patients,
understanding the time- and dose-dependency of DOXO effects on cSNs is key for refining
therapy (including
β
-AR blockers and neuromodulation) to prevent such adverse conse-
quences. Prospectively, future implications may include use of neuro-protective approaches
to alleviate cardiac and the systemic side effects of DOXO.
Finally, this study may stimulate research on additional criteria and biomarkers to be
considered for the early detection and optimal management of DOXO cardiotoxicity, in-
cluding functional (e.g., autonomic function assessment), molecular (e.g., biochemical:
noradrenaline, neuropeptide-Y plasma levels), and histopathologic (e.g., sympathetic in-
nervation of skin biopsies) autonomic tests.
4. Materials and Methods
4.1. Human Samples
Here, we analyzed sections from human subjects who died for non-cardiac causes
(n = 3) and subjects who underwent a chemotherapy regimen including DOXO and de-
veloped cardiomyopathy (n = 3). Ventricular samples were acquired during routine post-
mortem investigations or during post-transplant evaluation. Then, the samples were
archived in the anatomical collection of the Institute of Pathological Anatomy of the Univer-
sity of Padova. The samples were anonymous to the investigators and used in accordance
with the “Recommendation CM/Rec (2016) of the Committee of Ministers of member States
on research on biological materials of human origin”, released by the Council of Europe, as
received by the Italian National Council of Bioethics. The samples were analyzed using
protocols previously described [102].
4.2. Animal Models
In this study, we used adult C57BL/6J male mice (Charles River, Milan, Italy). The
animals were maintained in authorized animal facilities (authorization number 175/2002A),
at controlled temperature, with a 12-on/12-off light cycle and had access to water and
food ad libitum. All the experimental procedures that were performed on rodents were
approved by the local ethical committee and the Ministry of Health (Authorization numbers
408/2018PR, 129/2018PR and 738/2016PR), in compliance with Italian Animal Welfare Law
(D.Leg 4/3/2014 and subsequent modifications). All procedures were performed by trained
personnel with documented formal training and previous experience in experimental
animal handling and care. All protocols were refined prior to starting the study, and the
number of animals was calculated to use the least number of animals that was sufficient to
achieve statistical significance according to sample power calculation.
4.3. In Vivo DOXO Treatment
Two months (mo.) old C57BL/6J male mice were injected with either DOXO (3mg/kg,
i.p., Tocris, Bristol, UK) or vehicle solution (sterile water) on alternate days for 14 days, as
described in (Figure 1a). This regimen was associated to an index of mortality that was
lower than 5%. At the end of treatment, mice underwent electrocardiographic (ECG) and
echocardiographic (ECHO) analyses at the time points indicated in Figure 1a. At six weeks
after the completion of the DOXO treatment, the mice were sacrificed by cervical disloca-
tion. The hearts and superior cervical/stellate ganglia were harvested, washed in 1X PBS,
and processed for molecular and IF/histological analyses as previously reported [
40
,
103
].
Samples to be analyzed by IF were fixed in 1% paraformaldehyde for 30 min, dehydrated
in sucrose gradient, and frozen in liquid nitrogen.

Int. J. Mol. Sci. 2022,23, 11098 13 of 19
4.4. Echocardiographic Analysis
ECHO was performed as previously described [
103
], in mice that were anesthetized
with isoflurane (2.5% v:vin O
2
) during constant monitoring of temperature, heart, and
respiration rates and ECG parameters. The animals were imaged using a Vevo 2100 system
(Fujifilm VisualSonics, Toronto, Canada), that was equipped with a 30-MHz transducer.
Briefly, two-dimensional cine loops with frame rates of 200 frames per second of a long-axis
view and a short-axis view at proximal, mid, and apical level of the LV were recorded.
The ejection fraction (EF) was determined by the following formula, based on the Simple
method (Simp): %EF = 100 ×systolic LV volume/diastolic LV volume.
4.5. Electrocardiographic Analysis
The mice were anesthetized with isofluorane (2.5% v:vin O
2
) and ECG was recorded
by Powerlab 8/30, Bioamp (from AD Instruments, Dunedin, New Zeland) both at baseline
and upon atropine administration (2mg/kg, i.p.), as described in [
104
]. Heart Rate and
standard ECG parameters were calculated using the software LabChart 8 (AD Instruments,
Dunedin, New Zealand).
4.6. Confocal Immunofluorescence in Murine Samples
Ten-
µ
m heart or stellate/cervical ganglia cryosections were obtained with a cryo-
stat (CM1860; Leica, Wetzlar, Germany) and processed for IF analysis as previously re-
ported [
102
]. Briefly, heart or ganglia cryosections were incubated O/N at 4
◦
C with
primary antibodies that were diluted in PBS, supplemented with 1% Bovine Serum Albu-
min and 0.5% Triton-X100 (all from Sigma-Aldrich, St. Louis, MO, USA). Cryosections were
then incubated with secondary antibodies for 30 min at 37
◦
C. The primary and secondary
antibodies that were used in this study are listed in Table 1. The images were acquired
with a confocal microscope that was equipped with a 63x objective (1.4 NA) (Zeiss LSM900,
Carl Zeiss, Oberkochen, Germany), and used for the morphometric analyses that were
described in 4.8.
Table 1.
List of primary and secondary antibodies that were used for immunofluorescence analysis.
Target Host Company Ref. Number Dilution
Tyrosine Hydroxylase Rabbit Millipore Ab152 1:400
Nerve Growth Factor Rabbit Alomone AN-240 1:100
WGA-Alexa Fluor™-555 None Invitrogen W32464 1:400
Anti-Rabbit-488 Goat Jackson Lab. 111-545-144 1:200
4.7. Confocal Immunofluorescence in Human Samples
The samples were analyzed using the protocol that was previously described in [
102
].
Briefly, 3
µ
m thick heart sections were unmasked using microwave irradiation. The sections
were permeabilized with 10% Triton X-100 (Sigma-Aldrich, St. Louis, MO, USA) for 2 h at
37 ◦C, and incubated O/N with the appropriate primary antibody (Table 1).
4.8. Morphometric Evaluation of Cardiac Innervation Density
Neuronal density and CM cross-sectional areas were calculated in six non-consecutive
cryosections from the mid-portion of the ventricles of vehicle- and DOXO-treated mice. For
each section, six images from both the subepicardial and subendocardial regions, identified
as described in [
41
], were acquired and analyzed using the software ImageJ (version 1.53q,
National Institutes of Health, Bethesda, MD, USA). In detail, a z-projection of the stack was
obtained, and neuronal density was calculated as the percentage of tyrosine hydroxylase-
positive area over the area of epi- or endo- regions that were analyzed. CM cross-sectional
areas were calculated as previously described [41].

Int. J. Mol. Sci. 2022,23, 11098 14 of 19
4.9. Morphometric Evaluation of Cardiac Fibrosis
The percentage of fibrosis in human heart samples was calculated automatically on
a virtual-colour-based system, and reported as the mean value of 10 different randomly
chosen fields [105].
4.10. Protein Extraction and Western Blotting Analysis
The analysis was performed as previously described [
103
]. In detail, the hearts were
lysed on ice for 15 min in 120 mM NaCl, 50 mM Tris-HCl (pH 8.0), 1% Triton X-100,
protease inhibitor Complete (Roche Applied Science, Penzberg, Germany), and phosphatase
inhibitors (50 mM sodium fluoride, 1 mM sodium orthovanadate, and 10 mM sodium
pyrophosphate). The lysates were cleared by centrifugation at 13,000 rpm for 15 min at
4
◦
C. The protein concentration was determined by the Bradford method. The proteins from
hearts or cellular lysates were separated by SDS-polyacrylamide gel electrophoresis (SDS-
PAGE) and transferred to methanol-activated polyvinylidene difluoride (PVDF) membranes
(Millipore Corporation, Billerica, MA, USA). The membranes were incubated for 1 h with
5% bovine serum albumin (BSA)-TBST [tris-buffered saline (TBS)-0.3% Tween 20] at room
temperature and overnight incubated with primary antibodies at 4
◦
C. Appropriate host
species horseradish peroxidase-conjugated secondary antibodies were added and signals
were detected with enhanced chemiluminescence (Millipore Corporation, Billerica, MA,
USA). The antibodies that were used for this analysis are listed in Table 2.
Table 2. List of primary antibodies used for biochemical analyses.
Target Host Company Ref. Number Dilution
Nerve Growth Factor Rabbit Alomone AN-240 1:200
Vinculin Rabbit Cell Signaling 4650 1:1000
4.11. Statistics
Statistical analysis was performed using GraphPad Prism 8. The normality of data
distribution was assessed with a Shapiro–Wilk test. An unpaired t-test (for two groups) or
one-way ANOVA (for three or more groups) were used for normally distributed data. An
unpaired t-test with Welch’s correction was used to compare two groups with normally
distributed data and unequal variance. A Mann–Whitney test was used to compare two
groups with non-normally distributed data. A p-value < 0.05 was considered statistically
significant.
Author Contributions:
N.M. analyzed the ganglia and hearts from control and DOXO-treated mice,
as well as human heart samples. He discussed data and contributed to manuscript preparation and
revision; L.D. performed mouse treatment with DOXO, and ECHO/ECG analyses in control and
DOXO-treated mice; I.P.V. performed morphometric analyses in murine and human heart samples,
and contributed to manuscript revision; V.P. performed preliminary
in vivo
experiments; S.J.A.C.
performed WB in heart extracts; A.D.B. performed preliminary experiments in human heart samples;
R.B. analyzed the ECHO data; L.S. shared reagents, protocols, and discussed data; B.B. contributed
to ECHO and ECG analyses; A.A. provided human heart samples from DOXO-treated patients and
control subjects; S.S. and A.G. shared reagents and expertise and contributed to data interpretation
and discussion; M.M. and T.Z. designed the study, analyzed and interpreted data, and wrote and
revised the manuscript. M.M. and T.Z. supervised the work of N.M., L.D., I.P.V., V.P. and A.D.B.
All authors approved the final version of the manuscript and agree to be accountable for all aspects
of the work, in ensuring that questions related to the accuracy or integrity of any part of the work
are appropriately investigated and resolved, and that all persons designated as authors qualify for
authorship and have been listed. All authors have read and agreed to the published version of the
manuscript.
Funding: This research was funded by STARS-SKoOP to Tania Zaglia.

Int. J. Mol. Sci. 2022,23, 11098 15 of 19
Institutional Review Board Statement:
In compliance with the Italian and European legislation, this
research was approved by the Ufficio VI, Ministry of Health, with authorization numbers listed in
the method section.
Informed Consent Statement:
Ventricular samples were acquired during routine post-mortem in-
vestigations or during post-transplant evaluation. Then, the samples were archived in the anatomical
collection of the Institute of Pathological Anatomy of the University of Padova. The samples were
anonymous to the investigators and used in accordance with the “Recommendation CM/Rec (2016)
of the Committee of Ministers of member States on research on biological materials of human origin”,
released by the Council of Europe, as received by the Italian National Council of Bioethics.
Data Availability Statement: Data available upon request.
Conflicts of Interest:
The authors declare no conflict of interest. A.G. is co-founder and shareholder
of Kither Biotech, a pharmaceutical company developing PI3K inhibitors for respiratory diseases, not
in conflict with the content of this article.
References
1.
Swain, S.M.; Whaley, F.S.; Ewer, M.S. Congestive Heart Failure in Patients Treated with Doxorubicin: A Retrospective Analysis of
Three Trials. Cancer 2003,97, 2869–2879. [CrossRef] [PubMed]
2.
Sawyer, D.B.; Peng, X.; Chen, B.; Pentassuglia, L.; Lim, C.C. Mechanisms of Anthracycline Cardiac Injury: Can We Identify
Strategies for Cardioprotection? Prog. Cardiovasc. Dis. 2010,53, 105–113. [CrossRef] [PubMed]
3.
Vejpongsa, P.; Yeh, E.T.H. Prevention of Anthracycline-Induced Cardiotoxicity: Challenges and Opportunities. J. Am. Coll. Cardiol.
2014,64, 938–945. [CrossRef]
4.
Zamorano, J.L.; Lancellotti, P.; Rodriguez Muñoz, D.; Aboyans, V.; Asteggiano, R.; Galderisi, M.; Habib, G.; Lenihan, D.J.; Lip,
G.Y.H.; Lyon, A.R.; et al. 2016 ESC Position Paper on Cancer Treatments and Cardiovascular Toxicity Developed under the
Auspices of the ESC Committee for Practice Guidelines: The Task Force for Cancer Treatments and Cardiovascular Toxicity of the
European Society of Cardiology (ESC). Eur. Heart J. 2016,37, 2768–2801. [CrossRef]
5.
Carver, J.R.; Shapiro, C.L.; Ng, A.; Jacobs, L.; Schwartz, C.; Virgo, K.S.; Hagerty, K.L.; Somerfield, M.R.; Vaughn, D.J. American
Society of Clinical Oncology Clinical Evidence Review on the Ongoing Care of Adult Cancer Survivors: Cardiac and Pulmonary
Late Effects. J. Clin. Oncol. 2007,25, 3991–4008. [CrossRef] [PubMed]
6.
Armenian, S.H.; Lacchetti, C.; Barac, A.; Carver, J.; Constine, L.S.; Denduluri, N.; Dent, S.; Douglas, P.S.; Durand, J.B.; Ewer, M.;
et al. Prevention and Monitoring of Cardiac Dysfunction in Survivors of Adult Cancers: American Society of Clinical Oncology
Clinical Practice Guideline. J. Clin. Oncol. 2017,35, 893–911. [CrossRef]
7.
Henson, K.E.; Reulen, R.C.; Winter, D.L.; Bright, C.J.; Fidler, M.M.; Frobisher, C.; Guha, J.; Wong, K.F.; Kelly, J.; Edgar, A.B.; et al.
Cardiac Mortality among 200 000 Five-Year Survivors of Cancer Diagnosed at 15 to 39 Years of Age: The Teenage and Young
Adult Cancer Survivor Study. Circulation 2016,134, 1519–1531. [CrossRef]
8.
Zaorsky, N.G.; Churilla, T.M.; Egleston, B.L.; Fisher, S.G.; Ridge, J.A.; Horwitz, E.M.; Meyer, J.E. Causes of Death among Cancer
Patients. Ann. Oncol. 2017,28, 400–407. [CrossRef]
9.
Mulrooney, D.A.; Yeazel, M.W.; Kawashima, T.; Mertens, A.C.; Mitby, P.; Stovall, M.; Donaldson, S.S.; Green, D.M.; Sklar, C.A.;
Robison, L.L.; et al. Cardiac Outcomes in a Cohort of Adult Survivors of Childhood and Adolescent Cancer: Retrospective
Analysis of the Childhood Cancer Survivor Study Cohort. BMJ 2009,339, 34. [CrossRef]
10.
Stoltzfus, K.C.; Zhang, Y.; Sturgeon, K.; Sinoway, L.I.; Trifiletti, D.M.; Chinchilli, V.M.; Zaorsky, N.G. Fatal Heart Disease among
Cancer Patients. Nat. Commun. 2020,11, 2011. [CrossRef]
11.
Greenlee, H.; Iribarren, C.; Rana, J.S.; Cheng, R.; Nguyen-Huynh, M.; Rillamas-Sun, E.; Shi, Z.; Laurent, C.A.; Lee, V.S.; Roh, J.M.;
et al. Risk of Cardiovascular Disease in Women with and without Breast Cancer: The Pathways Heart Study. J. Clin. Oncol.
2022
,
40, 1647–1658. [CrossRef]
12.
Benjanuwattra, J.; Siri-Angkul, N.; Chattipakorn, S.C.; Chattipakorn, N. Doxorubicin and Its Proarrhythmic Effects: A Com-
prehensive Review of the Evidence from Experimental and Clinical Studies. Pharmacol. Res.
2020
,151, 104542. [CrossRef]
[PubMed]
13.
Pai, V.B.; Nahata, M.C. Cardiotoxicity of Chemotherapeutic Agents: Incidence, Treatment and Prevention. Drug Saf.
2000
,22,
263–302. [CrossRef] [PubMed]
14.
Li, M.; Sala, V.; de Santis, M.C.; Cimino, J.; Cappello, P.; Pianca, N.; di Bona, A.; Margaria, J.P.; Martini, M.; Lazzarini, E.; et al.
Phosphoinositide 3-Kinase Gamma Inhibition Protects from Anthracycline Cardiotoxicity and Reduces Tumor Growth. Circulation
2018,138, 696–711. [CrossRef] [PubMed]
15.
Ikeda, S.; Zablocki, D.; Sadoshima, J. The Role of Autophagy in Death of Cardiomyocytes. J. Mol. Cell Cardiol.
2022
,165, 1–8.
[CrossRef]
16.
Li, D.L.; Wang, Z.V.; Ding, G.; Tan, W.; Luo, X.; Criollo, A.; Xie, M.; Jiang, N.; May, H.; Kyrychenko, V.; et al. Doxorubicin Blocks
Cardiomyocyte Autophagic Flux by Inhibiting Lysosome Acidification. Circulation 2016,133, 1668–1687. [CrossRef]

Int. J. Mol. Sci. 2022,23, 11098 16 of 19
17.
Abdullah, C.S.; Alam, S.; Aishwarya, R.; Miriyala, S.; Bhuiyan, M.A.N.; Panchatcharam, M.; Pattillo, C.B.; Orr, A.W.; Sadoshima,
J.; Hill, J.A.; et al. Doxorubicin-Induced Cardiomyopathy Associated with Inhibition of Autophagic Degradation Process and
Defects in Mitochondrial Respiration. Sci. Rep. 2019,9, 2002. [CrossRef]
18.
Shinlapawittayatorn, K.; Chattipakorn, S.C.; Chattipakorn, N. The Effects of Doxorubicin on Cardiac Calcium Homeostasis and
Contractile Function. J. Cardiol. 2022,80, 125–132. [CrossRef]
19.
Llach, A.; Mazevet, M.; Mateo, P.; Villejouvert, O.; Ridoux, A.; Rucker-Martin, C.; Ribeiro, M.; Fischmeister, R.; Crozatier, B.;
Benitah, J.P.; et al. Progression of Excitation-Contraction Coupling Defects in Doxorubicin Cardiotoxicity. J. Mol. Cell Cardiol.
2019,126, 129–139. [CrossRef]
20.
Kim, S.Y.; Kim, S.J.; Kim, B.J.; Rah, S.Y.; Sung, M.C.; Im, M.J.; Kim, U.H. Doxorubicin-Induced Reactive Oxygen Species Generation
and Intracellular Ca2+increase Are Reciprocally Modulated in Rat Cardiomyocytes. Exp. Mol. Med.
2006
,38, 535–545. [CrossRef]
21.
Wichter, T.; Schäfers, M.; Rhodes, C.G.; Borggrefe, M.; Lerch, H.; Lammertsma, A.A.; Hermansen, F.; Schober, O.; Breithardt, G.;
Camici, P.G. Abnormalities of Cardiac Sympathetic Innervation in Arrhythmogenic Right Ventricular Cardiomyopathy. Circulation
2000,101, 1552–1558. [CrossRef] [PubMed]
22.
Chen, P.S.; Chen, L.S.; Cao, J.M.; Sharifi, B.; Karagueuzian, H.S.; Fishbein, M.C. Sympathetic Nerve Sprouting, Electrical
Remodeling and the Mechanisms of Sudden Cardiac Death. Cardiovasc. Res. 2001,50, 409–416. [CrossRef]
23.
Cao, J.M.; Fishbein, M.C.; Han, J.B.; Lai, W.W.; Lai, A.C.; Wu, T.J.; Czer, L.; Wolf, P.L.; Denton, T.A.; Shintaku, I.P.; et al. Relationship
Between Regional Cardiac Hyperinnervation and Ventricular Arrhythmia. Circulation
2000
,101, 1960–1969. [CrossRef] [PubMed]
24.
Vaseghi, M.; Lux, R.L.; Mahajan, A.; Shivkumar, K. Sympathetic Stimulation Increases Dispersion of Repolarization in Humans
with Myocardial Infarction. Am. J. Physiol. Heart Circ. Physiol. 2012,302, 1838–1846. [CrossRef]
25.
Scalco, A.; Moro, N.; Mongillo, M.; Zaglia, T. Neurohumoral Cardiac Regulation: Optogenetics Gets Into the Groove. Front.
Physiol. 2021,12, 726895. [CrossRef] [PubMed]
26.
Moruno-Manchon, J.F.; Uzor, N.E.; Kesler, S.R.; Wefel, J.S.; Townley, D.M.; Nagaraja, A.S.; Pradeep, S.; Mangala, L.S.; Sood, A.K.;
Tsvetkov, A.S. TFEB Ameliorates the Impairment of the Autophagy-Lysosome Pathway in Neurons Induced by Doxorubicin.
Aging 2016,8, 3507–3519. [CrossRef]
27.
Zhou, X.; Xu, P.; Dang, R.; Guo, Y.; Li, G.; Qiao, Y.; Xie, R.; Liu, Y.; Jiang, P. The Involvement of Autophagic Flux in the
Development and Recovery of Doxorubicin-Induced Neurotoxicity. Free Radic. Biol. Med. 2018,129, 440–445. [CrossRef]
28.
Mahmoodazdeh, A.; Shafiee, S.M.; Sisakht, M.; Khoshdel, Z.; Takhshid, M.A. Adrenomedullin Protects Rat Dorsal Root Ganglion
Neurons against Doxorubicin-Induced Toxicity by Ameliorating Oxidative Stress. Iran. J. Basic Med. Sci.
2020
,23, 1197. [CrossRef]
29.
Moruno-Manchon, J.F.; Uzor, N.E.; Kesler, S.R.; Wefel, J.S.; Townley, D.M.; Nagaraja, A.S.; Pradeep, S.; Mangala, L.S.; Sood,
A.K.; Tsvetkov, A.S. Peroxisomes Contribute to Oxidative Stress in Neurons during Doxorubicin-Based Chemotherapy. Mol. Cell
Neurosci. 2018,86, 65–71. [CrossRef]
30.
Alhowail, A.H.; Bloemer, J.; Majrashi, M.; Pinky, P.D.; Bhattacharya, S.; Yongli, Z.; Bhattacharya, D.; Eggert, M.; Woodie, L.;
Buabeid, M.A.; et al. Doxorubicin-Induced Neurotoxicity Is Associated with Acute Alterations in Synaptic Plasticity, Apoptosis,
and Lipid Peroxidation. Toxicol. Mech. Methods 2019,29, 457–466. [CrossRef]
31.
Lopes, M.Â.; Meisel, A.; Carvalho, F.D.; de Lourdes Bastos, M. Neuronal Nitric Oxide Synthase Is a Key Factor in Doxorubicin-
Induced Toxicity to Rat-Isolated Cortical Neurons. Neurotox. Res. 2011,19, 14–22. [CrossRef] [PubMed]
32.
Shokoohinia, Y.; Hosseinzadeh, L.; Moieni-Arya, M.; Mostafaie, A.; Mohammadi-Motlagh, H.R. Osthole Attenuates Doxorubicin-
Induced Apoptosis in PC12 Cells through Inhibition of Mitochondrial Dysfunction and ROS Production. Biomed. Res. Int.
2014
,
2014, 156848. [CrossRef] [PubMed]
33.
Manchon, J.F.M.; Dabaghian, Y.; Uzor, N.E.; Kesler, S.R.; Wefel, J.S.; Tsvetkov, A.S. Levetiracetam Mitigates Doxorubicin-Induced
DNA and Synaptic Damage in Neurons. Sci. Rep. 2016,6, 25705. [CrossRef] [PubMed]
34.
Lekakis, J.; Prassopoulos, V.; Athanassiadis, P.; Kostamis, P.; Moulopoulos, S. Doxorubicin-Induced Cardiac Neurotoxicity: Study
with Iodine 123-Labeled Metaiodobenzylguanidine Scintigraphy. J. Nucl. Cardiol. 1996,3, 37–41. [CrossRef]
35.
Jeon, T.J.; Jong Doo, L.; Jong-Won, H.; Yang, W.I.; Sang Ho, C. Evaluation of Cardiac Adrenergic Neuronal Damage in