Pharmacological Research 170 (2021) 105714

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Pharmacological Research
journal homepage: www.elsevier.com/locate/yphrs

Second-generation antipsychotics induce cardiotoxicity by disrupting

spliceosome signaling: Implications from proteomic and
transcriptomic analyses
Jing Wang a, 1, Xiaoqing Li a, 1, Zheng Liu a, Xinyi Lin a, Fan Zhong b, c, Shuhao Li a, Xinru Tang a,
Yang Zhang c, Liliang Li a, *
a
Department of Forensic Medicine, School of Basic Medical Sciences, Fudan University, Shanghai 200032, China
b
Department of Systems Biology for Medicine, Shanghai Medical College, Fudan University, Shanghai 200032, China
c
Institutes of Biomedical Sciences, Fudan University, Shanghai 200032, China

A R T I C L E I N F O A B S T R A C T

Keywords: Second-generation antipsychotics (SGAs) are first-line drugs that are prescribed for mental disorders in clinic.
Antipsychotics Severe cardiotoxicity has been widely reported and thus limits their clinical application. This study aimed to
Spliceosome identify the common mechanism underlying SGAs-induced cardiotoxicity using dual-omics analyses. Balb/C
Cardiotoxicity
mice were intraperitoneally injected with two representative SGAs, olanzapine (2.5 mg/kg) and clozapine (25
Proteome
mg/kg), at clinically comparable doses for 0, 7, 14 and 21 days. Our results showed that both SGAs induced
Transcriptome
cardiomyocyte degeneration, inflammation infiltration, and cardiac fibrosis, all of which worsened with time.
Chemical compounds studied in this article:
Olanzapine (Compound CID: 135398745)
Proteomic analysis revelaed that 22 differentially expressed (DE) proteins overlapped in olanzapine and
Clozapine (Compound CID: 135398737) clozapine-treated hearts. These proteins were significantly enriched in muscle contraction, amino acid meta­
Pladienolide B (Compound CID: 16202130) bolism and spliceosomal assembly by GO term analysis and spliceosome signaling was among the top enriched
2-Pyridylethylamine (Compound CID: 201148) pathways by KEGG analysis. Among the 22 DE proteins, three spliceosome signal proteins were validated in a
dynamic detection, and their expression significantly correlated with the extent of SGAs-induced cardiac fibrosis.
Following the spliceosome signaling dysregulation, RNA sequencing revealed that alternative splicing events in
the mouse hearts were markedly enhanced by SGAs treatments, and the production of vast transcript variants
resulted in dysregulation of multiple pathways that are critical for cardiomyocytes adaptation and cardiac
remodeling. Pladienolide B, a specific inhibitor of mRNA splicing, successfully corrected SGAs-induced alter­
native splicing and significantly attenuated the secretion of pro-inflammatory factors and cell deaths induced by
SGAs exposure. Our study concluded that the spliceosome signaling was a common pathway driving SGAs
cardiotoxicity. Pharmacological inhibition of the spliceosome signaling represents a novel therapeutic strategy
against SGAs cardiotoxicity.

1. Introduction major depressive disorders. The prescription of SGAs has increased more
than 18-fold in the past decades, helping 10 million people worldwide
Second-generation antipsychotics (SGAs) are the cornerstone of [1,2]. With increasingly wider clinical application, the toxic effects of
treating mental disorders such as schizophrenia, bipolar disorder and SGAs become a critical concern. These toxic effects range from minor

Abbreviations: AS, alternative splicing; A3SS, alternative 3′ splice site; A5SS, alternative 5′ splice site; BP, biological process; CC, cellular component; Clz, clo­
zapine; CK, creatine kinase; DE, differentially expressed; GO, gene ontology; HE, haematoxylin and eosin; IHC, immunohistochemistry; KEGG, Kyoto Encyclopedia of
Genes and Genomes; LD50, half lethal dose; LFQ, label-free quantification; MF, molecular function; MXE, mutually exclusive exons; Olz, olanzapine; PB, pladienolide
B; RI, retained intron; RNA-seq, RNA sequencing; RT-PCR, reverse transcription polymerase chain reaction; SE, skipped exon; SGA, second-generation antipsychotics;
snRNA, small nuclear RNAs; TEM, Transmission electron microscope; 2-PAE, 2-Pyridylethylamine.
* Correspondence to: Department of Forensic Medicine, School of Basic Medical Sciences, Fudan University, 131 Dongan Road, Shanghai 200032, China.
E-mail addresses: wangj16@fudan.edu.cn (J. Wang), lixq15@fudan.edu.cn (X. Li), liuz18@fudan.edu.cn (Z. Liu), linxy16@fudan.edu.cn (X. Lin), zonefan@163.
com (F. Zhong), shli15@fudan.edu.cn (S. Li), xrtang18@fudan.edu.cn (X. Tang), zhangyang@fudan.edu.cn (Y. Zhang), liliangli11@fudan.edu.cn (L. Li).
1
These authors contributed equally to this study

https://doi.org/10.1016/j.phrs.2021.105714
Received 29 January 2021; Received in revised form 18 May 2021; Accepted 2 June 2021
Available online 5 June 2021
1043-6618/© 2021 Elsevier Ltd. All rights reserved.
J. Wang et al.
issues (e.g., mild sedation or dry mouth) to troubling issues (e.g., weight
gain or metabolic disturbances) to even life threatening issues (e.g.,
cardiotoxicity or agranulocytosis) [3–6]. For example, cardiac electrical
activity can be altered by the application of antipsychotics, resulting in
QTc prolongation which increases the risk of torsades de pointes and
eventually sudden cardiac deaths [7]. Other serious cardiovascular
adverse effects include myocardial infarction, myocarditis and Brugada
syndrome phenotype [8]. The cardiotoxicity of SGAs strongly limits
their utility in clinic, even resulting in discontinuation of potentially
successful regimens.
Among SGAs, olanzapine (olz) and clozapine (clz) are the two most
prescribed regimens in clinic. Unfortunately, both of them have been
reported to associate with cardiotoxicity. According to a population-
based study, current users of SGAs had higher rates of sudden cardiac
deaths than non-users, with an adjusted incidence-rate ratio of 2.26 [9].
SGAs enjoy a wide pharmacological profile including D2 dopaminergic,
5-HT1A, 5-HT2A, 5-HT2C serotonergic, and histaminergic receptors, all
of which are responsible for their pharmacological actions and adverse
effects [10]. Olanzapine, a first-line SGA, has been increasingly pre­
scribed for nausea and vomiting caused by cancer and chemotherapy.
Current literature has reported that olz can lead to QTc changes [11],
with the blockade of Human Ether-ago-go Related Gene (hERG) potas­
sium channel being the predisposing factor [12]. Acute olz administra­
tion interfered with the cardiac energy metabolism and elevated
acetyl-CoA carboxylase phosphorylation and tissue ATP levels in iso­
lated rat hearts [13]. Though rarely presented, cases with dilated car­
diomyopathy after long-term exposure of olz have also been reported
[14]. Similar to olz in binding and functional profile [15,16], clz is the
most powerful antipsychotic in treating refractory schizophrenia [17,
18]. Over two thirds of the patients who fail to respond to other anti­
psychotics may benefit from clz [19], making clz another potent
regimen. However, the high risk of cardiotoxicity is preventing clz from
becoming a first-line treatment [20]. We have demonstrated that clz is
the most likely drug to trigger sudden cardiac death among all anti­
psychotics [21]. Analog to olz, clz also blocks the hERG potassium
channel, leading to torsadogenic cardiotoxicity [22]. Myocarditis and
cardiomyopathy may also occur after clz administration, potentially
leading to heart failure [23]. Therefore, olz and clz are two powerful
remedies with similar chemical structures and reported cardiotoxicity.
Although the toxicological effects of olz and clz in hearts have been
reported [13,24,25], these studies were largely clinical reports. The
cardiotoxic mechanisms of olz and clz have yet to be fully elucidated. To
the best of our knowledge, previous studies investigating the car­
diotoxicity of SGAs focused on single drug at a time or were mainly
observations of the consequences after cardiotoxicity [26]. Based on the
cardiopathology (cardiomyopathy and myocarditis), arrhythmogenic
changes, and similar chemical structure shared by these antipsychotics
[15], we presumed that olz and clz share common molecular mecha­
nisms when inducing cardiotoxicity. The shared ground for the car­
diotoxicity by both drugs, however, are still largely unexplored [27],
despite the notion that histamine 1 receptor (HRH1) and histamine 3
receptor (HRH3) are believed to mediate SGAs-induced weight gain and
cardiometabolic disturbance [28,29]. Thereby, the potential toxicolog­
ical effects shared by the representative SGAs and the causal mecha­
nisms accounting for their cardiotoxicity remain unknown.
In the present study, we performed dual-omics analyses integrating
proteomics and transcriptomics to obtain systemic information on the
common molecular alterations in representative SGAs (olz and clz)-
exposed mouse hearts. We aimed to uncover the primary mechanism
underlying cardiac toxicity shared by olz and clz which may aid in
providing venues for developing wide-spectrum cardioprotectants. Our
results revealed signatures of spliceosome signaling that underlined the
common causal mechanisms of SGAs cardiotoxicity. This study, to the
best of our knowledge, represents the first to depict the molecular re­
sponses to chronic SGAs exposure in the mouse heart.
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Pharmacological Research 170 (2021) 105714
2. Material and methods
2.1. Chemicals and reagents
Antipsychotic clz was purchased from Sigma-Aldrich (St. Louis, MO,
USA). Clozapine was dissolved in 0.1 M HCl and pH balanced in PBS to
make a stock solution of clz (25 mg/mL, equal to 80 mM). The stock
solution was then diluted on the basis of use dosage. Antipsychotic olz
was purchased from Selleck Chemicals (Houston, TX, USA) and prepared
as 5 mg/mL (equal to 16 mM) stock solution in solvents consisting of
DMSO and PBS. The highest final concentration of DMSO in external
solution was ≤ 1%, a concentration that had no effect on the survival of
mice. Primary antibodies against Sf3b3 (Catalog: ab209402) and Snrpf
(Catalog: ab154870) were purchased from Abcam (Cambridge, United
Kingdom). A rabbit polyclonal antibody against Snrpd1 (Catalog:
10352-1-AP) was purchased from Proteintech Inc. (Rosemont, IL, USA).
A mouse monoclonal antibody against β-actin (Catalog: sc-47778) was
purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). A
rabbit polyclonal antibody against HRH1 (Catalog: DF4980) was pur­
chased from Affinity Biosciences Inc. (Cincinnati, OH, USA). All the
primary antibodies were validated by commercial companies and re-
validated by us before use. HRP-linked secondary antibodies were pur­
chased from Jackson laboratories Inc. (West Grove, PA, USA). Propi­
dium Iodide (PI, Catalog: ST512) and 4′ ,6-diamidino-2-phenylindole
(DAPI, Catalog: C1006) dyes were purchased from Beyotime Biotech­
nology (Nantong, Jiangsu, China). Mouse IL-1β (Catalog: BP-E93105)
and mouse IL-18 (Catalog: BP-E00001) detection kits were commer­
cially purchased from Boyun Biotechnology, Shanghai, China. The
specific inhibitor of spliceosome signaling (pladienolide B, PB) was a
generous gift from Dr. Yongbo Wang at the Department of Cellular and
Genetic Medicine, School of Basic Medical Sciences, Fudan University at
a stock solution of 1 mM. A specific agonist of HRH1 (2-Pyridylethyl­
amine, 2-PAE, Catalog: GC11001) was purchased from GLPBio (Mon­
tclair, CA, USA) and prepared as 10 mM stock solution in deionized
water.
2.2. Animal study
All experimental procedures were conducted with the protocols for
animal experiments in compliance with the regulations of the Institu­
tional Animal Care Committee at the School of Basic Medical Sciences,
Fudan University (Approval No.: 20170223-004). The sample size for
animal studies was set as ≥ 5 unless otherwise stated. Specific pathogen-
free (SPF) male Balb/C mice (approximately 6 weeks old at delivery)
were purchased from Shanghai Laboratory Animal Center (Shanghai,
China). The animals were group-housed in cages and acclimated to the
new environment for one week. A 12-h light and dark cycle and
continuous food and water supply were offered.
To mimic human therapeutic regimens, a daily dose of 2.5 mg/kg
body weight of olz or 25 mg/kg body weight of clz were intraperito­
neally administered for 0, 7, 14 and 21 days, respectively. Mice were
randomly distributed into the 4 time periods (n = 5–7 mice per drug per
time period). Mice weights were recorded on a daily basis before each
injection. Mice were sacrificed at each indicated time period by cervical
dislocation after euthanasia using 2.5% isoflurane. Hearts were trans­
versely dissected and part of the heart tissue was fixed in formalin and
embedded in paraffin for sectioning and histology. The remaining heart
tissues were stored at − 80 ℃ for subsequent analysis.
2.3. Histological examination
Heart tissues were fixed in a 10% neutral formalin solution,
embedded in paraffin, sectioned at a thickness of 4 µm, and stained with
haematoxylin and eosin (HE) to examine inflammatory infiltrates.
Fibrotic tissues within the hearts were examined by Masson’s trichrome
staining. After Masson’s trichrome staining, five fields were randomly
J. Wang et al.
selected for each slide and the area of fibrosis was measured by Image J
software (National Institutes of Health, Bethesda, MD, USA). The
percent of fibrosis in each slide was then averaged over the 5 fields.
2.4. Immunohistochemistry (IHC) analysis
IHC staining protocols were in accordance with our previous publi­
cation [5]. Briefly, slides were antigen-retrieved by heating in a micro­
wave at 100 ℃ for 10 min in 0.1 M citric acid buffer (pH= 6.0) and were
then rinsed in 3% hydrogen peroxidase for 10 min. After that, slides
were incubated with corresponding antibodies at 4 ℃ overnight. After
secondary antibody incubation at room temperature for 1 h, the
immunoreactivity was developed in 0.05% diaminobenzidine. Haema­
toxylin was then co-stained for 3 min to visualize nuclei. For the
quantification of protein positivity in nuclei, five fields were randomly
selected for each slide and the nucleus with brown signal was counted as
positive expression. Then the protein-positive rate was calculated as the
number of positive nuclei in proportion to the total number of nuclei
within the whole-scale field. The results from the five fields were then
averaged.
2.5. Determination of creatine kinase (CK) levels and pro-inflammatory
factor contents
To determine the activity of a myocardial injury marker CK, cell
supernatants were collected from 5 independent assays for detection
using a diagnostic kit (Catalog: YB-CK-Mu, YuBo Bioengineering Insti­
tute, Shanghai, China). The detection protocol was in accordance with
the manufacturers’ instructions. The absorbance at a wavelength of 450
nm was measured spectrophotometrically by a microplate spectropho­
tometer (BioTek Epoch, VT, USA) to calculate CK levels. To detect the
contents of pro-inflammatory factors IL-1β and IL-18, cell supernatants
were collected after 24-hour exposure to SGAs and subject to enzyme-
linked immunosorbent assays (ELISA) using commercial kits. The pro­
tocol for each ELISA test was in accordance with the manufacturers’
instructions.
2.6. Transmission electron microscope (TEM)
Balb/C mice were exposed to saline (ctrl), olz (2.5 mg/kg) or clz (25
mg/kg) for 21 days. Immediately after sacrifice, the heart tissues were
immersed in 2.5% glutaraldehyde for overnight fixation at 4 ℃. Then
samples were postfixed in 2% osmium tetraoxide, dehydrated through
graded concentrations of ethanol, infiltrated and embedded in Epon 618
at 60 ℃ for 48 h. Ultrathin sections were cut with a diamond knife,
mounted on formvar-coated slot grids, and subsequently stained with
1% uranyl acetate and lead citrate. The images were taken with a CM-
120 model FEI/PHILIPS TEM (Philips Electron Optics B.V., Eindhoven,
The Netherlands).
2.7. Cell viability assessment
To assess the effects of olz and clz on mouse cardiomyocyte viability,
a cardiac-derived HL-1 cell line (Millipore, MA, USA, Cat# SCC065;
RRID: CVCL_0303) was seeded into 96-well plates in quadruplicates. The
HL-1 cells were derived from atrial cardiac muscle cells obtained from
transgenic mice [30]. Previous studies indicate that HL-1 cells retain
phenotypic characteristics of the adult cardiomyocyte, exhibit adult
cardiomyocyte-like gene expression profiles, possess intercalated discs,
maintain the ability to contract, retain differentiated cardiac morpho­
logical, electro-physiological characteristics and display the pharmaco­
logical properties of primary cardiac myocytes [31,32]. Therefore, the
cells were utilized and treated with either DMSO (ctrl), olz (64 μM), or
clz (20 μM) for consecutive days. To assess the effects of a HRH1 agonist
on SGAs-induced cellular toxicity, HL-1 cells were primed with 2-PAE at
the indicated dose 1 h prior to SGA treatments. Cell viability was
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Pharmacological Research 170 (2021) 105714
monitored on a daily basis. On each monitored day, an aliquot of 10 μL
CCK-8 solution (Beyotime, Nantong, China) was added into the culture
medium. Cells were then re-incubated at 37 ℃ for additional 2 h before
measurement of the optical density at a wavelength of 450 nm on a
microplate reader (BioTek, Winooski, VT, USA). All assays were inde­
pendently repeated for three times.
2.8. Label-free proteome analysis
At the time of sacrifice, the mouse heart tissues after 21-day drug
treatment (n = 3/group) were individually homogenized in lysis buffer
(4% SDS, 0.1 M DTT, 100 mM Tris/HCl, pH7.6) and boiled for 15 min.
Label-free proteome analysis was performed at the Shanghai Applied
Protein Technology Co. Ltd. (Shanghai, China). Protease and phospha­
tase inhibitors were used to limit potential protein degradation. The
lysed protein samples were then quantified using a BCA kit (Thermo
Scientific, CA, USA). Filter-aided sample preparation (FASP) was used to
prepare the protein samples with mild modification. Briefly, each pro­
tein sample was mixed with 200 μL of urea (8 M) in 150 mM Tris-HCl
(pH 8.0) (UA buffer) and centrifuged at 14,000 g for 15 min at 20 ℃
to remove SDS. A second washing step with UA buffer was taken to
exchange the remaining SDS with urea. The proteins were alkylated with
100 μL of 50 mM iodoacetamide for 39 min at room temperature in the
dark, followed by repeated washing with 100 μL of UA buffer and 100 μL
aliquots of 50 mM NH4HCO3. Then, trypsin was added to proteins at a
substrate/enzyme ratio of 50:1 (w/w) at 37 ℃ overnight in a thermo­
mixer (Eppendorf, Mittelsachsen, Saxony, Germany). After digestion,
the peptides were eluted and collected by centrifugation. The acidified
tryptic peptides were sampled on a SC001 traps 20 mm × 150 µm
reverse phase C18 trap column and then separated on a SC200 100 mm
× 150 µm reverse phase C18 nanocolumn (Easy-nLC1000 system,
Thermo Scientific) for 120 min 0.1% formic acid in 2% acetonitrile and
0.1% formic acid in 84% acetonitrile was used for mobile phases A and
B, respectively, with the flow rate set at 300 nL/min. Orbitrap Q Exac­
tive mass spectrometry (Thermo Scientific) was coupled to the liquid
chromatography system (Thermo Scientific). The range of the precursor
ion in the MS scan was from 300 to 1800 m/z. After a full scan, the top 20
MS/MS events were acquired. Then, MaxQuant (Ver. 1.5.3.17) was used
for analyzing all MS/MS data from the mouse heart samples. Main
search was set to 6 ppm, MS/MS tolerance was set to 20 ppm, and two
missed trypsin cleavages were allowed. Cysteine carbamidomethylation
and methionine oxidation were specified as the fixed and variable
modifications, respectively. The false discovery rate of the protein level
and peptide level were set to 0.05. Label-free quantification (LFQ) in­
tensity was performed in MaxQuant, as described previously [33].
Proteins identified more than twice in the three biological replicates
were defined as quantifiable proteins. The LFQ intensity of each protein
was then statistically compared between olz and ctrl groups, or between
clz and ctrl groups using Microsoft Excel 2016 software (Microsoft,
Seatle, WA, USA). In our study, proteins with p values of less than 0.05
and fold differences greater than 1.5 were classified as significantly
differential proteins. The proteins that were undetectable in specific
group but quantifiable in their counterparts were considered as specif­
ically expressed proteins.
Both the significantly differential proteins and specific proteins were
termed as differentially expressed (DE) proteins which were clustered
for bioinformatical analysis. Briefly, Gene Ontology (GO) and annota­
tion for proteins were performed using Blast2GO (Version 2.8.0) (https:
//www.blast2go.com/), while the Kyoto Encyclopedia of Genes and
Genomes (KEGG) analysis was performed using KEGG Automatic
Annotation Server (KAAS). All proteins were matched to the Swiss-Prot
database (http://www.ebi.ac.uk/swissprot/). p value of pathway
enrichment was calculated using the following formula:
J. Wang et al.
( )( )
M N− M
∑
m− 1
i n− i
p = 1− ( )
N BCA kit (Thermo Scientific, CA, USA) according to the manufacturer’s
i=0
n protocol. An equal amount of 20 ng protein was loaded on a 12% SDS-
N is the number of all identified proteins that can be connected with
GO or KEGG Pathway analysis information.
n is the number of differential proteins in N.
M is the number of proteins that can be connected with a certain GO
term or pathway.
m is the number of differential proteins with certain GO term or
KEGG pathway.
If a p value was less than 0.05, this GO term or pathway was regarded
as a significant enrichment.
2.9. Protein-protein interaction analysis
The protein-protein interaction network analysis was performed by
using the STRING tool (version 11.0) (https://string-db.org/) to identify
interactions between the DE proteins shared by olz-treated and clz-
treated groups.
2.10. Transcriptome analysis
Total RNAs were isolated from the hearts of ctrl, olz, and clz at the
21-day treatment groups (n = 3/group) using Trizol solution (Vazyme,
Nanjing, China) following the manufacturer’s instructions. The purity
was assessed by an Agilent 2100 Bioanalyzer using the RNA 6000 Nano
Chip (Agilent Technologies, Santa Clara, CA, USA). RNA quantification
was performed using Thermo scientific Multiskan GO microplate reader
with μDrop™ Plate (Waltham, MA, USA).
To construct the RNA-seq library, 2 μg of total RNA per sample was
exposed to poly-T oligo attached magnetic beads to isolate poly-A mRNA
following mRNA fragmentation. The cleaved RNA fragments were
transcribed into double-stranded cDNAs which were then purified using
AMPure XP beads to remove all reaction components. Reads containing
ploy-N, reads containing adapters, and low-quality reads from the raw
data were removed to obtain clean high-quality data for subsequent
analysis.
Transcript quantification of RNA-seq reads was performed with
Genomic Alignments (ver.1.20.1) by reads aligned to Ensemble Mus
musculus transcriptome annotation (GRCm.38. p5). The FPKM values
were calculated using ‘fpkm’ function from DESeq2 (ver. 1.24.0) that
were processed with the robust median ratio method and transcript
reads were normalized by the ‘voom’ function from Limma (ver. 3.40.6).
To assess whether a transcript was differentially expressed, EdgeR (ver.
3.24.3) calculates the results based on the normalized counts from entire
sequence alignments. Significantly differential transcripts were defined
as a fold change of raw FPKM value > 2 and adjusted p value < 0.01 and
specifically expressed transcripts were defined as the transcripts that
were undetectable in specific group but quantifiable in their counter­
parts. Both the significantly differential transcripts and specific tran­
scripts were pooled as DE transcripts which were clustered for GO term
and KEGG pathway enrichment analyses on the free online MajorBio i-
Sanger Cloud Platform (www.i-sanger.com). The cutoff p value of
pathway enrichment was set as 0.05 and the formula for calculating
pathway enrichment p values was as stated in the Label-free proteomics.
2.11. Western blot analysis
To validate the results of proteomic analysis, total proteins were
extracted from SGAs-exposed cardiomyocytes with RIPA buffer (Beyo­
time, Nantong, China) mixed with protease/phosphatase inhibitor
cocktail (Cell Signal Technology, Boston, MA, USA). Briefly, the cardio-
derived HL-1 cells were treated with olz or clz for 0 h, 2 h, 4 h, 8 h, 12 h
and 24 h before harvest. To assess the effects of a HRH1 agonist on
4
Pharmacological Research 170 (2021) 105714
spliceosome protein expression, HL-1 cells were primed with indicated
doses of 2-PAE 1 h prior to SGA treatments. Protein was quantified by a
PAGE gel and transferred onto a polyvinylidene fluoride membrane.
The blots were blocked by 5% skim milk for 1 h at room temperature and
incubated with primary antibodies at 4 ℃ overnight. The primary an­
tibodies were used at a final dilution of 1:1000 with tris-buffered saline
containing 0.1% Tween-80. Membranes were then incubated with cor­
responding secondary antibodies for 1 h at room temperature. The
immunoreactivity was detected using an enhanced chemiluminescent
autoradiography kit (Amersham, Pittsburgh, PA, USA). β-actin was
synchronously developed as an internal control.
2.12. Reverse transcription polymerase chain reaction (RT-PCR)
Total RNA was extracted from the mouse cardiac HL-1 cells using the
Trizol solution (Vazyme Biotechnology, Nanjing, China) according to
the manufacturer’s instructions. An equal amount of total RNA (500 ng)
was reverse-transcribed and amplified using the RT-PCR kit (Vazyme)
according to the manufacturer’s instructions. The specificity of primers
was assessed using the NCBI BLAST tool (https://blast.ncbi.nlm.nih.
gov/Blast.cgi) and validated by electrophoresis. Primer sequences are
listed as follows:
Npr2, forward 5′ -CAAGAGAATGGGCAGCCCTA-3′ .
reverse 5′ -TACTTGGGTATGAGTGGGAGGT-3′ ;.
Ubp1, forward 5′ - TCAACAGGCCGTGGAAATGAA-3′ ,
reverse 5′ - GATGCACAATTTGAGCCTGGT-3′ .
PCR products were separated by electrophoresis using 2% agarose
gels containing nucleic acid red stain (Beyotime).
2.13. Assessment of cell death rate
Mouse cardiac HL-1 cells were seeded on the coverslips in a 24-well
plate and treated with DMSO (ctrl), olz, or clz with or without PB co-
treatment in distinct concentrations (0.1 nM, 1 nM and 10 nM). After
48 h incubation, PI dye was added to the culture medium, and cells were
further incubated for 30 min under 37 ℃. The culture medium was then
replaced by washing three times with cold PBS, and cells were fixed with
pure acetone for 10 min, followed by co-staining with DAPI for 10 min.
The coverslips were then mounted and photographed under a fluores­
cence microscope (Zeiss, Oberkochen, Germany). Five fields were
randomly imaged for each group. Thereafter, the PI-positive cell
numbers and DAPI-positive cell numbers were counted, respectively.
The cell death rate (%) was defined as the PI-positive cell numbers in
proportion to the DAPI-positive cell numbers.
2.14. Statistical analysis
Data are presented as mean ± standard error of mean (SEM). A
Student’s t-test was used for comparisons between 2 groups. A one-way
or two-way analysis of variance (ANOVA), when necessary, was used to
compare 3 or more groups, followed by Bonferroni post-hoc tests. A
linear regression analysis was performed to analyze the correlation of
protein expression with extents of cardiac fibrosis. Unless elsewhere
stated, all statistical analysis was performed with the GraphPad Prism 8
(GraphPad Software Inc., San Diego, CA, USA). A p value of less than
0.05 was considered statistically significant.
3. Results
3.1. Second-generation antipsychotics (SGAs) induced chronic
cardiotoxicity in mice
To mimic the clinical scenario, the maintenance doses of olz and clz
used in clinic were converted to appropriate doses in mice. Therefore,
J. Wang et al. Pharmacological Research 170 (2021) 105714

mice were treated with the clinically comparable doses of olz (2.5 mg/
kg) and clz (25 mg/kg) for respective 0, 7, 14 and 21 days. The histo­
logical analyses (HE staining) showed that, compared to the untreated
control groups, the continuous olz and clz treatments resulted in sig­
nificant inflammatory infiltration in a time-dependent manner (Fig. 1a,
Fig. 1b). Besides inflammation, antipsychotic-induced fibrosis was also
detected in both perivascular and interstitial areas by Masson’s tri­
chrome staining (Fig. 1a, Fig. 1b). Quantification of the fibrotic area
showed that it increased with prolonged duration of olz (Fig. 1c) and clz
administration (Fig. 1d). In the in vitro cell viability assays, it was found
that both olz and clz dose-dependently inhibited cell proliferation with
the calculated LD50 being 83.437 μM for olz (Supplementary Fig. 1a)
and being 26.660 μM for clz (Supplementary Fig. 1b). Furthermore,
when olz and clz were added at a dose proximal to their respective LD50,
they consistently inhibited cell proliferation completely since day 3
onwards (Fig. 1e). Cell supernatant from HL-1 cells were collected on the
48 h to test levels of the myocardial injury marker CK in different groups
(Fig. 1f). After olz or clz treatment, CK levels in antipsychotic groups
were significantly higher than that of the control group. We further
assessed the ultrastructure of cardiomyocytes in heart sections using
transmission electron microscopy (Fig. 1g). It was observed that olz or
clz-treated hearts consistently presented with swollen cytoplasm, dis­
arrayed myofibril, and disrupted intercalated disc, which were in great
contrast with control heart. Moreover, both olz and clz-treated car­
diomyocytes displayed sparse vacuoles (asterisks, Fig. 1g), electron-
lucent spaces (arrowheads, Fig. 1g), and accumulation of autophagic
vacuoles (arrows, Fig. 1g) in the cytoplasm. Altogether, these results
confirmed that both olz and clz have a time-dependent cardiotoxic effect

Fig. 1. Time effects of SGAs cardiotoxicity. a, b Mice were treated with olz at 2.5 mg/kg or clz at 25 mg/kg for 0, 7, 14 and 21 consecutive days. Then mouse hearts
were harvested for haematoxylin and eosin (HE) staining and Masson’s trichrome staining. Magnification: 400 × . c, d The percentage of fibrotic lesion was
calculated by Image J software, n = 7/group. e Mouse cardiac HL-1 cells were incubated with olz (64 μM) or clz (20 μM) for serial days (1, 2, 3, 4, and 5 days) and
cell viability was determined using a CCK-8 kit. Each value represented the average of quadruplicates. f The supernatants from mouse cardiac HL-1 cells under ctrl
(dmso) or antipsychotic treatments were collected after 48 h′ exposure and were determined of the myocardial injury marker creatine kinase (CK) activity, n = 5/
group. g Transmission electron microscopy (TEM) revealed that cardiomyocytes from the SGAs-treated mice hearts displayed degeneration and myofibril disarray.
Asterisks indicate vacuoles in the cytoplasm. Arrowheads indicate electron-lucent spaces. Arrows indicate accumulation of autophagic vacuoles at various stages of
autophagy flux. *p < 0.05, **p < 0.01, ***p < 0.001 vs. Ctrl. Ctrl, control. CK, creatine kinase.

5
J. Wang et al. Pharmacological Research 170 (2021) 105714

and these SGAs shared common pathological changes, indicating po­ Table 1
tential common molecular basis behind their cardiotoxicity. Summary of differentially expressed (DE) proteins by proteomic analysis.
Comparisons Upregulated proteins Downregulated proteins Total
3.2. Proteomic profiling of the SGAs-exposed hearts Significant Specific Significant Specific

olz vs. ctrl 10 21 21 62 114

Since the continuous administration for 21 days was sufficient to clz vs. ctrl 17 32 13 28 90
induce cardiotoxicity, heart tissues at this time point were used for Overlapped 1 7 1 11 22
subsequent dual-omics analyses to better explore the molecular alter­ Olz, olanzapine. Clz, clozapine. Ctrl, control (Saline).
ations during SGAs exposure. In the proteomic profiling, LC-ESI-QqTOF
MS/MS identified a total of 2217, 2190, and 2215 proteins in the saline
line with the proteomic data, the HRH1 was observed to be
(ctrl), olz-exposed and clz-exposed mouse hearts, respectively. The de­
membrane-positive at mouse hearts from control, but remarkably
tails of comparative proteomics were documented in the Supplementary
decreased after different days of SGAs exposure (Supplementary
Data 1. In the olz-exposed group, compared to the control group, 114
Fig. 2a). The time-dependent decrease of HRH1 was also identified in
differentially expressed (DE) proteins were detected, out of which 31
the western blotting analysis (Supplementary Fig. 2b), further rein­
were up-regulated and 83 were down-regulated. In the DE proteins
forcing the reliability of our proteomic data. Thereafter, to better un­
induced by the clz treatment, there were 49 up-regulated proteins and
derstand the molecular mechanism shared by olz/clz-induced
41 down-regulated proteins (Fig. 2a). Some of the DE proteins were
cardiotoxicity, we focused on the overlapped DE proteins by olz and clz
quantifiable with certified fold-changes as compared with the control
treatments. Twenty-two protein (10 up-regulated and 12
group; others were detectable in only one group (control or olz/clz-
down-regulated proteins) were found to be shared by both drug treat­
exposed group), which were defined as specifically expressed proteins
ments (Table 1 and Fig. 2d). Details of the 22 proteins were documented
(Table 1). Volcano plots showed the global distribution of proteins in olz
in Table 2.
vs. ctrl and clz vs. ctrl comparisons (Fig. 2b). A hierarchical clustering
heat-map further depicted these significantly altered proteins in detail
(Fig. 2c). Specifically, HRH1, a histamine receptor reported to associate 3.3. Spliceosome signaling was significantly enriched in the proteomic
with antipsychotics-induced metabolic disorders [34,35], was also analysis
identified in the mouse hearts of control group (Supplementary Data 1),
suggesting the reliability of our proteomic data. Of note, this receptor Based on the 22 DE proteins, GO and KEGG pathway analyses were
was undetectable in clz-treated mouse hearts and only minimally conducted. The cellular roles of identified proteins were classified in
expressed in one olz-treated mouse heart (Supplementary Data 1). In terms of cellular component (CC), biological process (BP) and molecular

Fig. 2. Proteomic profiling of mouse hearts after 21-day SGA treatments. a 114 and 90 differentially expressed (DE) proteins were detected in olz and clz-exposed
mouse hearts, respectively. b Volcano plot depicted the DE proteins in olz or clz-treated group as compared to the control. Red and green dots represented significant
upregulation and downregulation, respectively (fold change ≥1.5 and p value <0.05). c Hierarchically clustered heatmap of proteins. d Interactive analysis of the
proteomic changes after olz and clz exposure. Twenty-two proteins overlapped by olz and clz treatments.

6
 J. Wang et al. Pharmacological Research 170 (2021) 105714

function (MF). As shown in Fig. 3 and Supplementary Data 2, the

1.57 (0.042)

0.63 (0.011)
enriched cellular components were mainly U12-type spliceosomal
olz vs. ctrl complex, precatalytic spliceosome, U2-type precatalyctic spliceosome,
U2-type spliceosomal complex and U1 snRNP, all of which are closely

NA
NA

NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
Fold-change (p value)

related to activities of RNA splicing. Changes in biological processes

highlighted the spliceosomal snRNP assembly process and fundamental

1.67 (0.0049)
1.63 (0.0066)
processes such as muscle contraction and metabolic/catabolic processes.

1.56 (0.001)
0.60 (0.018)
clz vs. ctrl

Changes in molecular function were annotated to the activity of de­

hydrogenases and transaminases, which are fundamental adaptive re­

NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA

sponses to drug toxicity. KEGG analysis was performed to determine the

significantly enriched biological pathways in the 22 overlapped DE
proteins. Consistent with the GO analysis, spliceosome signaling was the

28,770.33 ± 4429.10
most enriched pathway (Fig. 4a and Supplementary Data 3). Other

272.19 ± 58.72

500.70 ± 79.78
240.66 ± 67.61
312.18 ± 91.42

160.49 ± 10.37
enriched pathways were beta-alanine metabolism, vitamin B6 meta­

286.39 ± 45.6
176.80 ± 6.58

244.07 ± 2.98
bolism, valine, leucine and isoleucine degradation, and ascorbate and
olz-treated

aldarate metabolism (Fig. 4a and Supplementary Data 3). A map of the

spliceosome pathway was shown in Fig. 4b, where the expression levels

ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND

of several proteins that play core roles in RNA splicing were altered
31,051.33 ± 12,676.53 under SGAs exposure. Moreover, three of the 22 DE proteins (Sf3b3,
Snrpf, and Snrpd1) were from the spliceosome signaling and they were
shown to play hub roles in linking other proteins as per protein-protein
407.48 ± 166.35
469.74 ± 191.77
483.48 ± 197.38
187.79 ± 12.81

242.07 ± 13.84
285.97 ± 57.17
305.15 ± 51.83
300.21 ± 41.64
218.43 ± 3.28
213.23 ± 9.08

interaction (PPI) network analysis using the STRING tool (https://strin

LFQ intensity (Mean ± SEM, ×105)

clz-treated

g-db.org/) (Fig. 5). These data indicated that SGAs exposure disrupted
the RNA splicing signaling in mouse heart.
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND

3.4. Expression validation of the spliceosome signal proteins and their

pathological relevance
18,615 ± 2032.54
844.32 ± 222.29

250.48 ± 15.81

797.95 ± 48.76
229.71 ± 14.98
246.34 ± 96.75
120.73 ± 33.08
116.57 ± 34.46
210.75 ± 72.79
269.56 ± 12.86
233.25 ± 59.88

300.84 ± 2.65
191.74 ± 21.9
135.46 ± 2.93

To validate the spliceosome signal proteins, we detected the

88.7 ± 2.92

expression of these proteins in situ on heart sections by IHC staining.

Dynamic detection showed that Sf3b3 and Snrpf were maximally nuclei-
ctrl

ND
ND
ND
ND
ND
ND
ND

resided in drug-free heart but gradually decreased in olz or clz-treated

hearts (Fig. 6a). Unlike Sf3b3 and Snrpf, Snrpd1 was barely detected
Swiss-Prot ID

in drug-free hearts but increased dramatically while extending drug

Details of the 22 differentially expressed (DE) proteins that overlapped in olz and clz-treated mouse hearts.

exposure periods (Fig. 6a). Indeed, quantification of the IHC staining

Q9DCG9
Q921M3

Q9D1R1
Q9CZR2

Q9DBF1

Q9CR63

Q921U8
Q9CR26
Q91YR9

Q9D958

Q9ER88
Q8K274

Q6Y685

Q91XF0

Q6P1F6
O09110
O35127

P61922

P09528
P62315
P62307
P97379

showed that the nuclei-positive rates of Sf3b3 dropped from 6% in SGAs-

free hearts to approximate 3% after 21 days’ SGAs exposure (Fig. 6b,
upper panels). Similarly, the nuclei-positive rates of Snrpf decreased
ND, not detectable. NA, not applicable. LFQ, label-free quantification. Ctrl, control (Saline).
Tmem126b

linearly from 9% to approximate 3% by the treatment end, making up of

Trmt112
Aldh7a1

Ppp2r2a
Naalad2

Map2k3

over 60% drop after 21 days’ SGAs treatments (Fig. 6b, middle panels).
Symbol

Snrpd1
Fn3krp
Grcc10

G3bp2

Cox16
Tacc1
Sf3b3

Spcs1
Ptgr1
Snrpf

Dap3
Smtn
Pnpo

Abat
Vta1

Fth1

Snrpd1, the elevated spliceosome signal protein in the proteomic

profiling, was indeed increased by continuous SGAs treatments, partic­
ularly on the time window of 21 days (Fig. 6b, lower panels). To further
Serine/threonine-protein phosphatase2A 55 kDa regulatory subunit B alpha isoform

validate the expression dynamics of the spliceosome signal proteins, we

cultured HL-1 cells (a mouse cardiac-derived cell line) with extended
SGAs exposure hours. Western blot analysis showed that Sf3b3 and Snrpf
time-dependently decreased while Snrpd1 increased after 24-hour
exposure (Fig. 6c). Quantification of the blot intensity further sup­
ported that all of the three spliceosome signal proteins significantly
Multifunctional methyltransferase subunit TRM112-like protein

altered in response to SGAs treatments (Fig. 6d). These in vivo and in

Vacuolar protein sorting-associated protein VTA1 homolog

Dual specificity mitogen-activated protein kinase kinase 3

vitro data validated the expression of spliceosome signal proteins.

Cytochrome c oxidase assembly protein COX16 homolog
Complex I assembly factor TMEM126B, mitochondrial

To evaluate whether the spliceosome signal dysregulation was

Transforming acidic coiled-coil-containing protein 1

4-aminobutyrate aminotransferase, mitochondrial

receptor-dependent, we used a specific agonist 2-PAE to re-activate

Alpha-aminoadipic semialdehyde dehydrogenase
Ras GTPase-activating protein-binding protein 2

N-acetylated-alpha-linked acidic dipeptidase 2

HRH1. Our results showed that, when 2-PAE was pre-treated with HL-
28S ribosomal protein S29, mitochondrial

1 cells, the antipsychotic-induced decrease of Sf3b3 was successfully

Small nuclear ribonucleoprotein Sm D1

blunted (Supplementary Fig. 3a). However, the pre-agonizing of HRH1

Signal peptidase complex subunit 1

failed to rescue olz or clz-disturbed Snrpf and Snrpd1 expression, even at

Small nuclear ribonucleoprotein F

Pyridoxine-5-phosphate oxidase

higher doses of 2-PAE (Supplementary Fig. 3a). Consistently, olz or clz-

Splicing factor 3B subunit 3

inhibited cell viability was significantly recovered in early periods of 2-

Prostaglandin reductase 1

PAE pretreatment (289% increase, olz+2-PAE 100 μM vs. olz on day 3;

Ketosamine-3-kinase

Ferritin heavy chain

228% increase, clz+2-PAE 100 μM vs. clz on day 3). 2-PAE, again, failed
to maintain a continuous promotion on cell viability at even high doses,
Protein name

evidenced by the less minimal recovery of cell viability on day 4 (only

Protein C10

Smoothelin

148% increase, olz+2-PAE 100 μM vs. olz; only 110% increase, clz+2-
Table 2

PAE 100 μM vs. clz) when comparing with day 3 (Supplementary

Fig. 3b). These data implied that SGAs might dysregulate the

7
J. Wang et al.
Fig. 3. Gene ontology (GO) analysis of the 22 overlapped proteins. The color intensity of bar charts corresponded to the statistical significance of the GO Term. BP,
biological process. MF, molecular function. CC, cellular component.
spliceosome signaling in a HRH1 receptor-independent manner.
To further study the spliceosome signal relevance with cardiac pa­
thology, we quantified the extent of cardiac fibrosis after 7, 14, and 21
days’ SGAs exposure and correlated the cardiac fibrosis rate with the
spliceosome protein expression (Fig. 6e). In the olz treatments, it was
shown that the expression of Sf3b3, Snrpf, and Snrpd1 correlated line­
arly with the fibrosis rate (goodness of fit: R = 0.5726 and p val­
ue = 0.0054 for Sf3b3, R = 0.6180 and p = 0.0022 for Snrpf,
R = 0.5061 and p = 0.0162 for Snrpd1, respectively, Fig. 6e, upper
panels). Similar results were also observed in the clz treatments, where
Sf3b3 and Snrpf negatively correlated and Snrpd1 positively correlated
with the extent of cardiac fibrosis (goodness of fit: R = 0.4286 and p
value = 0.0466 for Sf3b3, R=0.4357 and p = 0.0427 for Snrpf,
R = 0.7056 and p = 0.0002 for Snrpd1, respectively, Fig. 6e, lower
panels). Altogether, these results suggested that SGAs exposure
disturbed the spliceosome signaling.
3.5. Alternative splicing events were markedly enhanced in the SGAs-
exposed hearts
To assess whether spliceosome signaling dysregulation results in
biological outcomes, we performed RNA sequencing (RNA-seq) to
analyze alternative splicing (AS) events and transcript variants, two
indices lying directly downstream of the spliceosome signaling. Total
RNAs were isolated from saline (ctrl), olz, and clz-treated hearts. After
filtering the RNA-seq data, a comprehensive list of transcripts was
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Pharmacological Research 170 (2021) 105714
obtained (Supplementary Data 4). A total of 69,734 transcripts were
identified, including 52,795, 53,495, and 54,428 transcripts for ctrl, olz,
and clz groups, respectively. The principal component analysis (PCA)
showed that there was a large variance between control and
antipsychotic-treated groups (Fig. 7a). Volcano plots were constructed
by integrating the p value and fold change of each transcript (p <0.01
and fold change ≥2), to filter the DE transcripts for the olz and clz
exposure groups compared to the control group (Figs. 7b and 7c). A total
number of 26,152 transcripts were differentially expressed in olz groups
as compared with the control hearts, of which 13,869 were up-regulated
and 12,283 were down-regulated (Fig. 7b and Supplementary Data 4).
Similarly, a total of 21,985 transcripts were differentially expressed in
the clz vs. ctrl comparisons, of which 11,768 were upregulated and
10,217 were down-regulated (Fig. 7c and Supplementary Data 4). After
interactive analysis, a total of 12,124 DE transcripts overlapped by olz
and clz groups which made up of 46.4% of the total DE transcripts after
olz treatment, and 55.1% of the DE transcripts after clz treatments
(Fig. 7d).
After analysis of the AS events, it turned out that five major types of
AS events, termed retained intron (RI), skipped exon (SE), alternative 5′
splice site (A5SS), alternative 3′ splice site (A3SS), and mutually
exclusive exons (MXE) were all observed after olz or clz treatments
(Fig. 8a). Skipped exon (SE) was the most common AS event in both
treatments (Figs. 8a and 8b), and substantial retained intron (RI) events
were also observed for both treatments (Figs. 8a and 8b). In olz-treated
hearts, 14,720 (67.36%) SE events and 2069 (9.47%) RI events were
J. Wang et al. Pharmacological Research 170 (2021) 105714

Fig. 4. Spliceosome was significantly enriched by the KEGG pathway analysis. a KEGG pathway analysis of the 22 overlapped proteins was performed and the top
KEGG pathways were shown. The p value of each enriched pathway was reflected by the intensity of bar color. b A map of the spliceosome. Red boxes represent the
altered molecules in the pathway. Sm, short for Sm-D1 (or Snrpd1).

observed (Fig. 8b). Similar to the olz-exposed group, 19,214 (70.36%)
SE events and 2092 (7.66%) RI events were detected after clz exposure
(Fig. 8b). Interestingly, 8 of the 22 DE proteins (36.4%) from the pro­
teomic screening were detected to have transcript variants and partic­
ularly, to show high proportions of novel AS after either olz or clz
treatments (Fig. 8c). In particular, multiple functional genes were
observed to be alternatively spliced. Malate dehydrogenase 1 (Mdh1),
an enzyme in the citric acid cycle whose activity may increase during
myocardial injury [36], and lysine (K)-specific methyltransferase 2B
(Kmt2b), an enzyme involved in mRNA production [37], were detected
of multiple transcript variants that consistently altered by both olz and
clz treatments (Fig. 8d). Natriuretic peptide receptor 2 (Npr2), a critical
gene for cardiac hypertrophic growth and cardiac remodeling [38],
displayed intron retain after SGAs treatment (Fig. 8e). To investigate the
dose and time effects of AS events, Npr2 and upstream binding protein 1
(Ubp1), a gene controlling nuclear pre-mRNA maturation [39], were
amplified using primers targeting the retained introns (Figs. 8f and 8g).
At the increasing SGAs doses, it was observed that the intron inbetween
the exons 10 and 11 of Npr2 was retained dose-dependently. The intron
between exons 5 and 6 of Ubp1 was barely detectable in drug-free

9
J. Wang et al. Pharmacological Research 170 (2021) 105714

Fig. 5. Protein-protein interactions network of the 22 proteins overlapped by olz- and clz-treated mouse hearts using the STRING tool (https://string-db.org/). The
three spliceosome signal proteins were boxed in red for highlight.

cardiomyocytes but was intensively retained after SGAs exposure
(Fig. 8f). With different time windows, it was also observed that the RI
events of Npr2 and Ubp1 were markedly enhanced at extended hours of
SGAs treatments (Fig. 8g).
Subsequently, the 12,124 DE transcripts that shared by both SGAs
treatments were subject to bioinformatics analysis. The GO analysis of
biological processes showed significant overrepresentation of tran­
scripts attributed to ribosome biogenesis, ribonucleoprotein complex
assembly, and cell differentiation. The molecular function analysis
showed overrepresentation of transcripts attributed to transmembrane
transporter activity, cytoskeletal protein binding, and RNA binding.
Ribosome and other subcellular components such as the plasma mem­
brane, extracellular region, and nucleus were also significantly enriched
in the cellular component analysis (Fig. 9a and Supplementary Data 5).
In addition, KEGG pathway analysis showed that RNA transport, mRNA
surveillance, and spliceosome were significantly enriched (Fig. 9b and
Supplementary Data 6). The maladaptive pathways such as neuroactive
ligand-receptor interaction, cytokine-cytokine receptor interaction,
linoleic acid metabolism, steroid hormone biosynthesis, and cAMP
signaling were also enriched (Fig. 9b and Supplementary Data 6), mir­
roring the cell fate toward pathological cardiac remodeling. Taken
together, the transcriptomic analysis showed direct evidence of
enhanced alterative splicing in response to SGAs-induced dysregulation
of spliceosome signaling.
3.6. Pharmacological blockade of spliceosome pathway rescued SGAs-
induced cardiotoxicity
In view that dysregulated spliceosome signaling resulted in vast
alternative splicing and consequently led to multiple biological path­
ways imbalance, we next blocked the spliceosome pathway using a
specific pharmacologic inhibitor pladienolide B (PB) to assess whether

10
J. Wang et al. Pharmacological Research 170 (2021) 105714

Fig. 6. Expression validation of the spli­

ceosome signal proteins in mouse hearts
and cardiac cells. a Dynamic detection of
Sf3b3, Snrpf, and Snrpd1 in mouse heart
sections using immunohistochemistry
(IHC) staining. b Quantitative analysis of
the IHC staining results, n = 5–6/group. c
Representative western blotting analysis of
Sf3b3, Snrpf, and Snrpd1 in a cardiac cell
line (HL-1 cells) under olz or clz treat­
ments with distinct hours. d Quantitative
analysis of the western blotting analysis,
n = 3. e Cardiac fibrosis was quantified
and the extent of fibrosis was correlated
with the expression levels of the three
spliceosome signal proteins (Sf3b3, Snrpf,
and Snrpd1) at different drug exposure
time windows. Black dots represent mouse
hearts receiving saline treatments (n = 6);
Red, green, and blue dots represent hearts
receiving olz or clz treatments for 7 days
(n = 6), 14 days (n = 5), and 21 days
(n = 5), respectively. The linear correla­
tion coefficiency was plotted. For consis­
tency, we used positive numbers of R value
for all the regression analysis, while it
should be the negative number when
yielding negative correlations. ns, not sig­
nificant; *p < 0.05, **p < 0.01,
***p < 0.001 as indicated.

11
J. Wang et al. Pharmacological Research 170 (2021) 105714

Fig. 7. Transcriptome profiling of mouse hearts after 21-day antipsychotic treatments. a Principle component analysis of RNA-seq data. The ctrl, olz, and clz
treatment groups were marked as indicated. b, c Volcano plot showed differentially expressed (DE) transcripts in olz vs. ctrl and clz vs. ctrl comparisons (p<0.01 and
fold change ≥2). Red and blue dots represent significantly up-regulated and down-regulated transcripts, respectively. The number of DE transcripts, including
significantly differential transcripts (left) and specific transcripts (right), were denoted in the corresponding quadrants. d Overlapping transcripts after olz and clz
exposure. A total of 12,124 DE transcripts were overlapped by both drugs.

the SGAs-induced cellular toxicity could be rescued. At a molecular
level, we observed that the retained intron of Npr2 or Ubp1 by SGAs
exposure was corrected with PB co-treatment, particularly at a relatively
higher dose of PB (10 nM) (Fig. 10a). Further, we detected the pro-
inflammatory factors in a mouse cardiac HL-1 cell line. It was found
that both SGAs significantly promoted the secretion of IL-18 and IL-1β
(Fig. 10b), a supportive evidence of the inflammatory infiltration after
SGAs exposure in mouse hearts. More importantly, with the co-
treatment of PB ranging from 0.1 nM to 10 nM, the contents of IL-18
and IL-1β significantly declined as compared with SGA single treat­
ment. In particular, when PB was co-treated at 10 nM, the olz or clz-
induced production of pro-inflammatory factors dramatically dropped
to levels comparable to the control group (Fig. 10b). Moreover, at the
cellular levels, it was observed that mouse cardiomyocytes after
exposure to olz (Fig. 10c) or clz (Fig. 10d) displayed marked PI uptake,
an indicator of cytomembrane rupture and cell death. However, when
cells were co-treated with PB, the PI uptake induced by SGAs exposure
markedly attenuated (Figs. 10c and 10d). Cell death rates, calculated as
the PI-ingested cells in proportion to the DAPI-stained cells, reached
approximate 50% for both SGAs, but dose-dependently declined with PB
co-treatments, particularly at a dose of 10 nM (Fig. 10e). These data
suggested that spliceosome signaling was the causal mechanisms of both
SGAs cardiotoxicity. Spliceosome signaling has functional significance
with regard to SGAs cardiotoxicity.
4. Discussion
As classical SGAs, olz and clz are commonly prescribed drugs for

12
J. Wang et al. Pharmacological Research 170 (2021) 105714

Fig. 8. Alternative splicing (AS) in the

heart was enhanced by SGAs exposure.
a Schematic illustration of the five
types of AS events. The number of AS
events in each comparison was anno­
tated at the right side. b Frequencies of
different AS events were shown. c
Novel AS events for the 22 differen­
tially expressed (DE) proteins that
were identified from the proteomic
analysis were tabulated. d Effects of
SGAs exposure on the splicing of
Kmt2b and Mdh1, two representative
genes controlling pathophysiological
processes. Red line: significantly up-
regulated transcripts in olz/clz-
exposed groups; blue line: signifi­
cantly down-regulated transcripts in
olz/clz-treated groups; black line: no
significant changes. e A schematic di­
agram of RI event of Npr2 in SGAs-
treated mouse hearts. f, g Assessment
of the dose and time effects of SGAs
exposure on the intron retain of Npr2
and Ubp1.

13
J. Wang et al. Pharmacological Research 170 (2021) 105714

Fig. 9. Bioinformatics analysis of the differentially expressed (DE) transcripts revealed biological impact by SGAs exposure. a Enriched GO terms of the 12,124 DE
transcripts shared by olz and clz were depicted. Multiple critical biological processes, molecular functions, and cellular components were disrupted. b Enriched KEGG
pathways of the 12,124 transcripts shared by olz and clz were depicted. Multiple pathways that control cell adaptation and cardiac remodeling were imbalanced.

mental disorders. Pathologically, antipsychotic-induced cardiomyopa­
thy presented with cell hypertrophy, myocardial inflammatory infiltra­
tion, and fibrotic remodeling. With long term or high dose exposure,
myocardial remodeling and fibrosis can be detected, clinically diag­
nosed as myocarditis and cardiac hypertrophy [40–42]. The onset of
severe cardiotoxicity may develop as early as 8 days after starting
clozapine [43]. It was also reported that during treatment windows of 7,
14, and 28 days, antipsychotic-induced cardiotoxicity was significantly
higher among those with short-term use [44]. These reports indicated
the time effect of SGA cardiotoxicity. Consistent with previous clinical
reports, our data showed that both olz and clz induced time-dependent
inflammatory infiltration and cardiac fibrosis, as well as increases in the

14
J. Wang et al. Pharmacological Research 170 (2021) 105714

Fig. 10. Pharmacological blockade of the spliceosome pathway rescued SGAs-induced cardiotoxicity. a Effects of pladienolide B (PB, a specific mRNA splicing
inhibitor) on SGAs-induced Npr2 and Ubp1 alternative splicing (intron retain) were assessed using RT-PCR. b Effects of pladienolide B on SGAs-induced secretion of
pro-inflammatory factors were assessed using ELISA kits targeting IL-18 and IL-1β in cell supernatants, n = 3/group. c, d Immunofluorescence analysis of the PI
uptake in mouse cardiac cells under SGAs exposure with or without PB co-treatments. The dye PI is used for visualization of dead cells that only ingested PI when
membrane ruptured. DAPI was co-stained to reveal nuclei. Magnification: 400× . e Quantification of cell death rates in the immunofluorescence assays, n = 5/group.
*p < 0.05, **p < 0.01, ***p < 0.001 as indicated.

15
J. Wang et al.
CK activity of the SGAs-insulted cardiac cells. At the ultrastructure
levels, both olz and clz treatment led to cytoplasmic edema, autopha­
gosome and autolysosome formation, and rupture of intercalated discs
as evidenced by electron microscopy. This comparative evidence not
only suggested the time-dependent cardiotoxic effects of SGAs, but also
implicated there might be shared mechanisms underlying SGAs
cardiotoxicity.
To our knowledge, most of the previous studies focused on single
antipsychotic toxicity and those reports were mainly observations of the
consequences after cardiotoxicity. The causal mechanisms accounting
for the cardiotoxicity of clz and olz remain to be elucidated. Therefore,
the present study aimed to probe the common causal mechanisms that
drive SGAs cardiotoxicity. To this end, olz and clz, two SGAs that share
similar chemical structures and receptor affinity, were selected as
representative SGAs. To better mimic clinical scenario, the clinical
comparable dose of each drug was adopted when establishing mouse
models. After dual-omics analyses, our study provided multiple lines of
evidence that the spliceosome signaling is a previously unappreciated
mechanism that underlined the similar cardiotoxicity induced by the
representative SGAs. In the proteomic analysis, RNA splicing was among
the top enriched pathways in mouse hearts following olz or clz exposure.
Sf3b3, Snrpf, and Snrpd1, the three spliceosome signal proteins identi­
fied by proteomic profiling, were validated to be time-dependently
altered by olz and clz exposure in mouse hearts and in the cultured
cardiomyocytes. Importantly, the alteration of these proteins signifi­
cantly correlated with the extent of cardiac fibrosis, a supportive evi­
dence of the biological importance of the spliceosome in SGAs
cardiotoxicity. As a direct evidence of spliceosome pathway dysregula­
tion, RNA sequencing showed that a vast of AS events occurred. As a
consequence, various transcript variants yielded and thereby disrupting
multiple pathways that are critical for maintaining cell adaptation and
cardiac remodeling. Particular examples of the unwelcome splicing were
the Npr2 and Ubp1 genes, which control cardiac remodeling [38] and
pre-mRNA maturation [39] under physiological conditions but were
incorrectly spliced by olz and clz exposure in a dose and time-dependent
manner. The involvement of spliceosome signaling in SGAs cardiotox­
icity was further reinforced when PB, a specific inhibitor of mRNA
splicing [45], corrected olz or clz-induced intron retain of Npr2 and
Ubp1 and significantly attenuated the drugs-induced secretion of
pro-inflammatory factors and cell death rates. These lines of evidence
therefore strongly suggested that the spliceosome signaling was a crit­
ical pathway driving the SGAs cardiotoxic effects.
In particular, the three spliceosome signal proteins (Sf3b3, Snrpf, and
Snrpd1) have been previously characterized in cancer biology but
received minor attention in heart diseases. Spliceosome, the key ma­
chinery in RNA splicing, is a large and dynamic ribonucleoprotein
complex made up of proteins and small nuclear RNAs (snRNAs) [46].
Ribonucleoproteins are composed of 1 or 2 small nuclear RNAs (snRNPs;
U1, U2, U4, U5, U6) and various complex-specific proteins [47].
Changes in expression levels or mutations of snRNP and splice factors
can produce aberrant mRNA splicing patterns on a genome-wide scale.
Generally, Snrpd1 and Snrpf are small nuclear ribonucleoproteins
participating in the whole process of splicing and the splicing factor
Sf3b3 is part of the U2 snRNP and the minor U12-type spliceosome.
According to previous studies, Snrpd1 and Snrpf were reported to be
biomarkers for breast cancer [48] and systemic lupus erythematosus
[49]. Differential expression of Sf3b3 was detected in diffuse malignant
peritoneal mesothelioma [50] and chronic lymphocyte leukemia [51].
The data obtained from our omics analyses suggested that in addition to
cancer biology, these proteins might also critically dominate the car­
diotoxic effects by SGAs or other pharmacological compounds. Of note,
these proteins displayed different expression patterns, mirroring func­
tional differences when executing SGAs cardiotoxicity. Hence, our
findings broadened the functional importance of these spliceosome
signal proteins and it merits further investigation of the detailed roles
played by individual protein.
16
Pharmacological Research 170 (2021) 105714
Interestingly, among the wide range of SGAs-targeted pharmaco­
logical receptors, HRH1 was the only identified receptor in the present
cardiac proteomic study. This might be due to technical limitation of the
proteomic profiling as it only identifies proteins of considerable abun­
dance. HRH1, a receptor believed to be responsible for SGAs-induced
metabolic disturbance [28], was indeed decreased by olz and clz treat­
ments in validations. However, the selective agonist of HRH1, 2-PAE,
only rescued the antipsychotics-dampened Sf3b3 expression, leaving
Snrpf and Snrpd1 expression unchanged even at its higher doses. Olz or
clz-inhibited cell viability was also only partially recovered by 2-PAE at
early periods, suggesting that SGAs dysregulated the spliceosome
signaling in a H1 receptor-independent manner. Further genetic ap­
proaches that edit the Hrh1 gene expression might aid in supporting our
pharmacological results. Another possible explanation might be that
aside from the H1 receptor, other pharmacological targets, such as
histamine H3 receptor which protects hearts by promoting the expres­
sion of anti-inflammatory genes in renal-heart syndrome [52] and other
potential G protein-coupled receptors (i.e. cannabinoid CB1 and CB2
receptors that oppositely regulated clz and quetiapine cardiotoxicity [5,
41]) may involve in the regulation of the spliceosome signal proteins.
Investigation of other potential receptors would deserve further study.
The identification of the spliceosome signaling as a common driver of
SGAs cardiotoxicity is of paramount importance. First, the mechanisms
of SGAs-induced life-threatening cardiac effects remain largely un­
known. A recent report demonstrated that acute olz administration
interfered with the cardiac energy metabolism and elevated acetyl-CoA
carboxylase phosphorylation and tissue ATP levels [13]. The car­
diotoxicity of clz was associated with an increase in myocardial oxida­
tive stress, inflammatory cytokines, DNA damage and apoptosis with
attenuation in antioxidant defenses [40,42,53]. Elevated catechol­
amines were also responsible for cardiac damage after clz exposure [54].
These reports, however, were mainly observations of the consequences
of drug toxicity. As we have provided solid data that blockade of the
spliceosome signaling rescued SGAs toxicity in multiple perspectives,
including the incorrect splicing of critical genes, secretion of
pro-inflammatory factors, and the cell deaths, we believe the spliceo­
some signaling represents the primary and causal mechanisms under­
lying the SGAs cardiotoxicity. Second, previous studies have shown that
PB, an inhibitor of the spliceosome signaling, evokes apoptosis and cell
death in several carcinoma cell lines [50,55]. We tested various con­
centrations of PB and found that under a low dose ranging from 0.1 to
10 nM, the PB has resulted in beneficial effects, suggesting that low
doses of spliceosome signaling inhibitors might confer therapeutic,
instead of additional toxic, outcomes. Third and more importantly, we
aimed to identify the common pathways that shared by representative
SGAs. The identification of the spliceosome signaling as a shared
mechanism driving SGAs cardiotoxicity would shed insights into the
development of wide-spectrum cardioprotectants that may avoid
discontinuation of on-the-market potent drug.
One limitation of the present study is that the number of replicates in
omics studies is relatively restricted (n = 3). In further studies, more
biological replicates can be applied to validate the altered spliceosome
signaling after SGAs exposure.
In all, this study investigated the primary mechanism of cardiotox­
icity induced by both olz, a first-line SGA regimen, and clz, another
powerful SGA for mental disorders. The dual-omics study integrating
proteome and transcriptome analyses identified the spliceosome
signaling as the common mechanism behind SGAs cardiotoxicity.
Pharmacological blockade of the spliceosome signaling under low doses
successfully rescued SGAs-induced cardiotoxic effects. Our study
depicted the molecular responses to chronic SGAs exposure and may
provide venues for developing wide-spectrum cardioprotectants against
SGAs cardiotoxicity.
J. Wang et al.
Funding
This work was financially supported by the National Key Research
and Development Program of China (No. 2018YFC0910700 and
2016YFB0201702), the National Natural Science Foundation of China
(No. 81701861, 81871527, and 82070285), the Shanghai Health Com­
mittee Research Foundation (20194Y0066), the Wang-Dao Research
Program of Fudan University (No. 20017), The Qing-Feng Scholar
Foundation of Shanghai Medical College, Fudan University (No.
QF2007), the Zhengyi Scholar Foundation of School of Basic Medical
Sciences, Fudan University (No. S24-4).
Authors contribution
JW and XL performed animal experiments and analyzed data. ZL and
XL helped in animal experiments. SL and XT performed cell culture. FZ
and YZ analyzed the dual-omics data. JW drafted the manuscript. LL
conceived, designed the study and edited the manuscript. All authors
read and approved the final version of the manuscript.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Acknowledgment
The authors would like to thank professor Yongbo Wang from the
Department of Cellular and Genetic Medicine, School of Basic Medical
Sciences, Fudan University for his generous gift of the spliceosome
signaling inhibitor (pladienolide B) and professor L. D. Timmie Top­
oleski from the Mechanical Engineering Department at the University of
Maryland, Baltimore County, MD, USA, for his thoughtful comments and
careful editing.
Appendix A. Supporting information
Supplementary data associated with this article can be found in the
online version at doi:10.1016/j.phrs.2021.105714.
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