An Original Aspirin-Containing Carbonic Anhydrase 9 Inhibitor Overcomes Hypoxia-Induced Drug Resistance to Enhance the Efficacy of Myocardial Protection

Article abstract of DOI:10.1007/s10557-021-07182-2

Purpose: Hypoxic microenvironment plays a vital role in myocardial ischemia injury, generally leading to the resistance of chemotherapeutic drugs. This induces an intriguing study on mechanism exploration and prodrug design to overcome the hypoxia-induced drug resistance. Methods: In this study, we hypothesized that the overexpression of carbonic anhydrase 9 (CAIX) in myocardial cells is closely related to the drug resistance. Herein, bioinformatics analysis, gene knockdown, and overexpression assay certificated the correlation between CAIX overexpression and hypoxia. An original aspirin-containing CAIX inhibitor AcAs has been developed. Results: Based on the downregulation of CAIX level, both in vitro and in vivo, AcAs can overcome the acquired resistance and more effectively attenuate myocardial ischemia and hypoxia injury than that of aspirin. CAIX inhibitor is believed to recover the extracellular pH value so as to ensure the stable effect of aspirin. Conclusion: Results indicate great potential of CAIX inhibitor for further application in myocardial hypoxia injury therapy.

Full text of DOI:10.1007/s10557-021-07182-2

Cardiovascular Drugs and Therapy
https://doi.org/10.1007/s10557-021-07182-2
ORIGINAL ARTICLE
An Original Aspirin-Containing Carbonic Anhydrase 9 Inhibitor
Overcomes Hypoxia-Induced Drug Resistance to Enhance the Efficacy
of Myocardial Protection
Wen Zhou1,2 & Bin Zhang2 & Keyu Fan2 & Xiaojian Yin3 & Jinfeng Liu4 & Shaohua Gou1,2
Accepted: 31 March 2021
#
Springer Science+Business Media, LLC, part of Springer Nature 2021
Abstract
Purpose Hypoxic microenvironment plays a vital role in myocardial ischemia injury, generally leading to the resistance of
chemotherapeutic drugs. This induces an intriguing study on mechanism exploration and prodrug design to overcome the
hypoxia-induced drug resistance.
Methods In this study, we hypothesized that the overexpression of carbonic anhydrase 9 (CAIX) in myocardial cells is closely
related to the drug resistance. Herein, bioinformatics analysis, gene knockdown, and overexpression assay certificated the
correlation between CAIX overexpression and hypoxia. An original aspirin-containing CAIX inhibitor AcAs has been
developed.
Results Based on the downregulation of CAIX level, both in vitro and in vivo, AcAs can overcome the acquired resistance and
more effectively attenuate myocardial ischemia and hypoxia injury than that of aspirin. CAIX inhibitor is believed to recover the
extracellular pH value so as to ensure the stable effect of aspirin.
Conclusion Results indicate great potential of CAIX inhibitor for further application in myocardial hypoxia injury therapy.
.
.
.
Keywords Myocardial hypoxia injury Drug resistance Carbonic anhydrase 9 Aspirin derivative
Introduction
fact, hypoxia-induced resistance to the conventional antitumor
chemotherapy has been confirmed, and great efforts have been
made to develop hypoxia-specific theranostics [6, 7]. However,
this problem has not attracted much attention in the study of
anti-myocardial hypoxia injury. Therefore, original target ex-
ploration and drug discovery will be of great requirement to
overcome the resistance to ensure the drug efficacy.
Myocardial ischemia injury is a pressing issue which denotes
the presence of heart diseases, such as coronary artery disease
(CAD) and myocardial infarction (MI) [1]. Unfortunately, cel-
lular hypoxic microenvironment significantly limits the effica-
cy of most chemotherapeutic drugs, such as aspirin [2–5]. In
In detail, aspirin is a classic and practical drug to prevent
the occurrence of heart disease. It can inhibit platelet adhesion
and aggregation and prevent the formation of thrombus by
inhibiting the generation of cyclooxygenase-1 (Cox-1) and
cyclooxygenase-2 (Cox-2) coenzymes [8, 9]. However, latest
clinical statistics and studies have challenged the role of aspi-
rin in preventing and attenuating myocardial injury. Aspirin
cannot attenuate myocardial injury when ischemia and hyp-
oxia occur [10]. It also has no efficacy given before blood
reperfusion surgery to attenuate myocardial ischemia reperfu-
sion injury [4, 5]. Evidently, recent studies have called into
question about the efficacy of traditional drugs under hypoxic
microenvironment. So there is an urgent need to optimize
traditional drugs or develop new drugs to attenuate myocardial
ischemia injury.
* Shaohua Gou
sgou@seu.edu.cn
1
Jiangsu Province Hi-Tech Key Laboratory for Biomedical Research,
Southeast University, Nanjing 211189, Jiangsu, People’s Republic of
China
2
Pharmaceutical Research Center and School of Chemistry and
Chemical Engineering, Southeast University,
Nanjing 211189, Jiangsu, People’s Republic of China
3
State Key Laboratory of Natural Medicines, China Pharmaceutical
University, Nanjing 210009, Jiangsu, People’s Republic of China
4
School of Basic Medical Sciences and Clinical Pharmacy, China
Pharmaceutical University, Nanjing 211198, Jiangsu, People’s
Republic of China

Cardiovasc Drugs Ther
Moreover, myocardial ischemia injury is closely related to
hypoxia-inducible factor 1ɑ (HIF-1α) pathway abnormality
[11, 12]. Coincidentally, carbonic anhydrase 9 (CAIX), a
transmembrane protein composed of acidic amino acids, has
a HIF-responsive element in its promoter region. More signif-
icantly, CAIX is also involved in regulation of in vitro pH.
Existing researches have shown that high expression of HIF-
1α can upregulate the expression of downstream target gene
product CAIX in tumor cells [13–16]. However, little atten-
tion has been paid to whether overexpression of HIF-1α in-
duces high expression of CAIX under conditions of cardiac
ischemia and cardiomyocyte hypoxia as well. Up till now, the
association between myocardial ischemia injury and abnormal
CAIX expression is rarely reported. In fact, CAIX is
expressed not only in tumor cells, but also in the mammalian
heart [17, 18], localizing to the t-tubules of cardiomyocytes
[19]. In view of the similar hypoxic microenvironment be-
tween tumor cells and hypoxic cardiomyocytes, the role of
CAIX in myocardial hypoxia injury will be a meaningful ex-
ploration. Inhibiting CAIX overexpression to regulate the ab-
normal acidic microenvironment in vivo will probably be a
new and promising strategy to overcome the hypoxia-induced
drug resistance and can effectively attenuate myocardial is-
chemia injury.
In this study, the correlation between myocardial hypoxia
injury and CAIX overexpression was studied. A novel com-
pound AcAs was designed and synthesized, which combines
aspirin (As) with the CAIX inhibitor acetazolamide (Ac) [20].
In order to verify the effect of AcAs and the combined use of
Ac and As (Ac&As) on attenuating myocardial hypoxia inju-
ry, the cardiac myocytes hypoxia model and the mice ische-
mia model were set up. The molecular regulations on cell
proliferation, ROS level, ATP level, CAIX overexpression,
extracellular pH values, and myocardial tissue histopathology
were profoundly studied.
Bgee is a database to retrieve and compare gene expression
patterns in multiple animal species based exclusively on cu-
rated “normal,” healthy, expression data. This allows compar-
isons of expression patterns between species. Pathway
Commons is an access to biological pathway information col-
lected from public pathway databases. String is a database of
known and predicted protein-protein interactions. The interac-
tions include direct (physical) and indirect (functional) asso-
ciations; they stem from computational prediction, from
knowledge transfer between organisms, and from interactions
aggregated from other (primary) databases [21].
Palmitic Acid (PA)-Simulated Hypoxia Modeling
The variation trend of HIF-1α and CAIX protein expression
was studied by western blot (WB). The correlation between
myocardial CAIX and HIF-1α could be found. In detail, H9c2
cell hypoxia model was simulated by palmitic acid (PA) with
different concentrations (0−100 μM). Protein was extracted
from cells with ice-cold radioimmunoprecipitation assay
(RIPA) buffer containing 1 mmol/L phenylmethanesulfonyl
fluoride (PMSF). The lysates were centrifuged at 12,000 g for
15 min at 4°C. The protein concentration was quantified by
Bicinchoninic Acid Protein Assay kit (Biosky Biotechnology
Corporation, Nanjing, China). Western blot assay was used
for protein expression analysis with primary antibody (anti-
HIF-1α, proteintech, 1:500, 20960-1-AP; anti-CAIX,
proteintech, 1:500, 11071-1-AP; anti-GAPDH, Bioworld
Technology, 1:500, AP0063) and HRP-conjugated secondary
antibody (Goat Anti-Rabbit IgG (H+L) HRP, 1:3000,
Bioworld Technology, BS13278, or Rabbit Anti-Goat IgG
(H+L)-HRP, 1:3000, Bioworld Technology, BS30503).
Equal loading was verified by incubation with anti-GAPDH.
The blots were visualized using ECL, and the signals were
quantified by densitometry (Image-Pro Plus 6.0).
HIF-1α siRNA Transfection
Experimental Section
H9c2 cells were transfected with HIF-1α siRNA (sc-45919)
(Santa Cruz, CA, USA) in 6-well plates using siRNA trans-
fection reagent (sc-29528) (Santa Cruz). One-milliliter pre-
diluted mixture containing siRNA (HIF-1α) and transfection
reagent was incubated at 37°C for 5−7h, and the culture was
continued for 48h with twice liquid exchange. Some transfec-
tion groups were subjected to 100 μmol/L palmitic acid (PA)
for additional 2h. The cell proteins were obtained for WB
analysis to analyze HIF-1α and CAIX expressions. The de-
tailed WB method was the same as in 2.2.
Protein Distribution of Carbonic Anhydrase
Isoenzymes in the Heart and Protein-Protein
Interaction Analysis
CAIX is one of the carbonic anhydrase isoenzymes.
Isoenzymes have the same catalytic function, but there are
differences in immunological properties, tissue, and organ
distribution among them. We searched and compared with
these isoenzymes by various databases. The databases include
UniProt database (https://www.uniprot.org), Bgee database
(https://bgee.org), Pathway Commons database (http://apps.
pathwaycommons.org), and String database (https://string-
db.org). The Universal Protein Resource (UniProt) is a com-
prehensive resource for protein sequence and annotation data.
Overexpression Plasmid Construction
H9c2 cells were transfected with pcDNA3.1-HIF-1α
(GenePharma, Shanghai, China) in 6-well plates using Hieff

Cardiovasc Drugs Ther
Trans™ Liposomal Transfection Reagent (Yeasen, Shanghai,
China). pcDNA3.1-HIF-1α and transfection reagent were
mixed and incubated at r.t. for 20 min to form a complex of
DNA-liposomes. The mixed solution was added to each well
of the cell culture plate, and after continuous culture for 48h,
the cell proteins in each group were extracted. WB assay was
performed to determine the protein expression level and the
correlation between HIF-1α and CAIX. The detailed WB
method was the same as in the “Palmitic Acid (PA)-
Simulated Hypoxia Modeling” section.
3. 2-Bromoethyl 2-acetoxybenzoate (Intermediate 5):
Aspirin (360 mg), 2 eq oxaloyl chloride, and a drop of
DMF were mixed in DCM (6 mL) in an ice bath. The
mixture was kept stirring for 6–8 h, and then DCM was
removed by a rota-vapour. To a solution of 2-
bromoethanol (250 mg) and trimethylamine (303 mg) in
DCM (6 mL) in an ice bath, the above solution of
aspirinyl chloride was added. The reaction continued stir-
ring at 0°C for 6 h, DCM was used for extraction, the
resulting organic phase was concentrated in vacuum,
and the residue was isolated by silica gel column chroma-
tography (petroleum ether:ethyl acetate = 8:1, V:V) to
obtain yellow oil. Yield: 0.40 g, 70.2 %.
Synthesis of Compound AcAs
Reagents for compound synthesis such as acetazolamide (Ac),
aspirin (As), 6-heptyl acetylenic acid, 2-bromine ethanol,
triethylamine, sodium azide, copper acetate, ascorbic acid,
concentrated hydrochloric acid, and sodium bicarbonate were
of analytical grade and used without further purification. 1H-
NMR spectra were performed on a Bruker 600 MHz Ultra
shield spectrometer (Avance Av-500) and reported as parts
per million (ppm) from TMS. The final product AcAs was
also characterized by 150-MHz 13C-NMR spectra. High-
resolution mass spectrum was measured by an Agilent 6224
ESI/TOF MS instrument.
4. 2-Azidoethyl 2-acetoxybenzoate (Intermediate 6): A mix-
ture of intermediate 5 and NaN3 (130 mg) in DMF (6 mL)
was heated to 50°C and kept stirring overnight. The solu-
tion was concentrated and the resulting residue was iso-
lated by silica gel column chromatography to get yellow
oil. Yield: 190 mg, 76.3%. 1H NMR (600 MHz, CDCl3) δ
8.04 (d, J = 7.8 Hz, 1H), 7.58 (t, J = 7.8 Hz, 1H), 7.33 (t, J
= 7.8 Hz, 1H), 7.12 (d, J = 7.8 Hz, 1H), 4.43 (d, J = 4.8
Hz, 1H), 3.59 (d, J = 4.8 Hz, 1H), 2.37 (s, 3H).
5. Compound AcAs: To a stirring solution of intermediate 3
(60 mg) in methanol (8 mL), copper acetate (8 mg) and
intermediate 6 (52 mg) were subsequently added. Fifteen
milligrams of ascorbic acid was added 5 min later. The
reaction maintained stirring for 4h, and then the solution
was concentrated, and white solid powder was obtained
by silica gel column chromatography. Yield: 80 mg,
The compound was prepared as follows.
1. 5-Amino-1,3,4-thiadiazole-2-sulfonamide (Intermediate
2): To a solution of acetazolamide (22.5 mM, 5.0 g) in
ethanol (30 mL), concentrated hydrochloric acid (5 mL)
was added at room temperature, and then the mixture was
kept stirring and refluxing until the reaction completed.
After the solution was concentrated in vacuum, a small
amount of saturated sodium bicarbonate solution was
added to adjust pH=9. The resulting solution was extract-
ed by ethyl acetate, and the organic phase was concentrat-
1
71.4%. Purity is 98.9% detected by HPLC. H NMR
(600 MHz, DMSO-d6) δ 13.00 (s, 1H), 8.32 (s, 2H),
7.93 (s, 1H), 7.82 (d, J = 6.9 Hz, 1H), 7.69–7.60 (m,
1H), 7.42–7.32 (m, 1H), 7.21 (d, J = 7.6 Hz, 1H), 4.69
(s, 2H), 4.61 (s, 2H), 2.69–2.59 (m, 2H), 2.59–2.50 (m,
2H), 2.20 (s, 3H), 1.70–1.54 (m, 4H). 13C NMR (150
MHz, DMSO-d6) δ 172.19, 169.10, 164.26, 163.40,
161.13, 150.16, 146.66, 134.52, 131.16, 126.24, 124.10,
122.50, 122.34, 63.27, 48.28, 34.55, 28.35, 24.67, 23.92,
20.65. Ms: Found m/z: 538.11157 (calculated for
C20H23N7O7S2, 537.1).
1
ed to produce white powder. Yield: 3.5g, 86.4 %. H
NMR (600 MHz, DMSO-d6) δ 8.06 (s, 2H), 7.81 (s, 2H).
2. N-(5-Sulfamoyl-1,3,4-thiadiazol-2-yl)hept-6-ynamide
(Intermediate 3): Under ice bath, 2 eq oxaloyl chloride
and a drop of DMF was added to a solution of 6-
heptylic acid (277 mg) in dichloromethane (DCM) (6
mL). After stirring for 6–8 h, it was concentrated to re-
move DCM by a rota-vapour. Intermediate 2 (360 mg)
and pyridine (316 mg) were dissolved in DMF (5 mL)
under ice bath, and then the above 6-heptylenyl chloride
in DMF was added for reaction which was monitored by
TCL. The solution was concentrated in vacuum and the
residue was isolated by silica gel column chromatography
(DCM: MeOH= 40:1, V:V). The product was obtained as
white powder. Yield: 0.42g, 73.7%. 1H NMR (600 MHz,
DMSO-d6) δ 13.0 (s, 1H), 8.32 (s, 2H), 2.78 (t, J = 2.4
Hz, 1H), 2.55 (t, J = 7.2 Hz, 2H), 2.20–2.17 (m, 2H),
1.73–1.68 (m, 2H), 1.50–1.45 (m, 2H).
Cell Culture and Modeling
Cardiac myoblasts H9c2 were cultured in Dulbecco’s
Minimum Essential Medium (DMEM) containing 10%
fetal bovine serum (FBS). Meanwhile, primary cultured
neonatal rat myocardial cells were used in some experi-
ments. These cells were extracted from the hearts of 10
neonatal rats born 1–2 days old and cultured with DMEM
containing 10% FBS. Blank group cells were common
cultured under normal oxygen. Model group cells were
cultured under hypoxia or given simulated hypoxia

Cardiovasc Drugs Ther
modeling reagent palmitic acid (PA). For hypoxia treat-
ment, cells were starved 2h in DMEM without FBS and
glucose in cell incubator (37°C, 21% O2, 5% CO2), treat-
ed with or without drugs (10 μm) for 2h, and finally
subjected to hypoxia conditions (37°C, 1% O2, 5% CO2)
for 4h, and then cells were placed on ice to stop the cell
activities. It has been reported that PA oxidation leads to
local hypoxia in myocardial cells, so PA can be used as a
simulated hypoxia modeling agent [22]. 0.0513g PA was
dissolved in 1 ml anhydrous ethanol to get 200 mM moth-
er liquor, which was diluted at 1:19 (10% BSA) to 10 mM
working liquor and further diluted 100 times to 100 μM
as modeling reagent.
ROS Level
DCFH-DA was used as a fluorescent probe to detect the con-
tent of ROS in cells. DCFH-DA is not fluorescent. It easily
penetrates into the cell and is hydrolyzed to DCFH by the
esterase. Nonfluorescent DCFH can no longer penetrate the
cell membrane, so DCFH-DA is easily load fluorescence
probe. ROS in cells can oxidize DCFH to fluorescent DCF,
and the fluorescence intensity can indicate the level of ROS
content in cells. Cell grouping and drug administration
methods were the same as described in the “Cell Membrane
Integrity Assay” section. DCFH-DA was added into DMEM
(containing 10% FBS) to incubate for at least 30 min. ROS
level was then detected by fluorescence spectrophotometer
and flow cytometry.
Cell Proliferation Assay
ATP Level
After H9c2 cells cultured with aspirin (As), acetazolamide
(Ac), AcAs, or a combined use of Ac and As (Ac&As) under
hypoxia or normoxia treatment, cell viability was detected
with Cell Counting Kit-8 (CCK-8, Beyotime Institute of
Biotechnology, Shanghai, China). The CCK-8 kit contains
2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-
disulfophenyl)-2H-tetrazoliumsodiumsalt (WST-8). In the
presence of the electron carrier 1-methoxy phenazine
methosulfate (1-methoxy PMS), WST-8 is reduced by intra-
cellular dehydrogenase to generate water-soluble orange-yel-
low formazan which can be dissolved in the tissue culture
medium, and the amount of formazan is proportional to the
number of living cells [23]. Cells were harvested and stained
in a microplate with 96 wells with working solution WST-8.
The absorbance intensity at 450 nm was measured with a
microplate reader. We calculated the ratio of the absorbance
value between the administration group and the blank control
group for graphical analysis.
ATP production was measured using ATP Assay Kit
(Beyotime Institute of Biotechnology, Shanghai, China), ac-
cording to the manufacturer’s instructions. The kit is based on
the fact that firefly luciferase catalyzes the fluorescence pro-
duction of fluorescein, which requires ATP to provide energy,
and the fluorescence is proportional to the concentration of
ATP, thereby detecting ATP concentration [23]. After drug
delivery (10 μmol/L AcAs or 10 μmol/L each for Ac and As)
and hypoxia treatment, the culture dish was placed on ice
rapidly, and all metabolic activities of the cells were stopped.
The culture solution was aspirated and the cell lysate was
added, cell lysis solution was collected and centrifuged at
12,000 g for 5 min under 4°C, and the supernatant was taken
for detection. The RLU value (the fluorescence intensity) was
measured by fluorescence spectrophotometer. Solutions were
evaluated in triplicate.
Western Blot to Study the Interaction Between
Compounds and CAIX
Cell Membrane Integrity Assay
After hypoxia or normoxia cell culture with aspirin (As), ac-
etazolamide (Ac), or AcAs, CAIX expression level in myo-
cardial cells was studied by WB assay. The detailed WB
method was the same as in the “Palmitic Acid (PA)-
Simulated Hypoxia Modeling” section.
The integrity of cell membrane can be used to distinguish
normal cells from necrosis. In this study, apoptosis and necro-
sis assay kit was used to detect the improvement effect of
aspirin (As), acetazolamide (Ac), AcAs, or a combined use
of Ac and As (Ac&As) on apoptosis or necrosis caused by
hypoxia. H9c2 cells were cultured in a six-well plate over-
night. After the cells adhered, As, Ac, AcAs (10 μmol/L), or
Ac&As (10 μmol/L each for Ac and As) were added, respec-
tively, and then incubated for 4h under hypoxia condition.
Cells were washed with PBS for 3 times and resuspended in
1-mL cell staining buffer, added Hoechst 33342 (5 μL) and PI
(5 μL) and incubated at 4°C for 30 minutes, and then detected
by flow cytometry. The results of cell double staining test
were analyzed with FCS Express V3 software.
Extracellular pH Regulation by Compounds
The cells were inoculated at the density of 1×105 cells/well in
a 24-well plate. After cells were attached to the well, the su-
pernatant was replaced with DMED containing compounds
and incubated in a normoxic (21% O2) or hypoxic (1% O2)
incubator. At the beginning and end of each experiment, we
inserted the pH probe into the medium to measure the

Cardiovasc Drugs Ther
extracellular pH and △pH was calculated. Three replicate
wells were set for each group.
staining, or stored at −80°C for subsequent western blot ex-
periments, or placed in 4% paraformaldehyde for immunohis-
tochemical (IHC) detection. Myocardial tissue protein was
attracted for WB detection. Blood was taken for the detection
of myocardial enzymes.
Docking
Molecular modeling was operated to exhibit the detail binding
mode between the compounds and CAIX by docking program
AutoDock 4.2.6 [24, 25]. The 3D structure of CAIX (PDB ID:
6FE2) was downloaded from NCBI database (http://www.
ncbi.nlm.nih.gov/). The structure of compounds AcAs and
acetazolamide was extracted from Chem3D Ultra Pro 14.0
(Table S3). The size of the docking box was 60 Å × 60 Å ×
60 Å, which was centered at the enzyme active pocket. The
dimension grid box with a certain grid spacing (0.375 Å) was
large enough to enclose the active pocket. The protein
structure was kept fixed during molecular docking. For each
complex, the top-ranked docking pose was optimized in the
binding pocket and then used as the geometry for the binding
mode analysis. All the water molecules except coordinated
water were removed and charges were assigned to the mole-
cule file. The remaining genetic algorithm (GA) parameters
were set as default values, except the genetic algorithm adjust-
ed to run at 100 [26].
TTC Staining of the Mouse Heart
The mice hearts were isolated followed by 1% triphenyltetra-
zolium chloride (TTC) staining to test infarct size. TTC is a
proton receptor of the pyridine-nucleoside structure enzyme
system in the respiratory chain. It reacts with dehydrogenases
in normal tissues and turns red, while dehydrogenase activity
in ischemic tissues decreases and turns white. The heart was
washed by PBS and cut into 5 pieces and then stained in TTC
solution with a water bath at 37°C for 15−30 min. Pictures
were taken under the microscope to calculate the percentage
of myocardial infarction area.
HE Staining of the Mouse Heart
Hematoxylin-eosin (HE) staining was used to observe the
pathological changes of myocardial tissue. The hearts of mice
were fixed by soaking in 4% paraformaldehyde, and paraffin
sections were prepared for staining. They were observed by
light microscope and images were collected for analysis.
Myocardial Ischemia Injury Mouse Modeling and
Evaluation of Drug Efficacy
Kunming male mice (4–6 weeks) were purchased from
Shanghai Jiesijie Laboratory Animal Co., LTD. The animal
care and experimental procedures were approved by Animal
Ethics Committee of Southeast University (0990200).
IHC Assay of the Mouse Heart
The mouse hearts of each group were isolated and fixed with
4% paraformaldehyde. After dehydration, the hearts were em-
bedded in paraffin and sliced. The slices were pasted on glass
slides and put in the fixative. After blocking with 3% peroxy
methanol for 25 min at r.t., the primary antibody (anti-HIF-1-
inhibitor, proteintech, 1:50, 20960-1-AP; anti-CAIX,
proteintech, 1:50, 11071-1-AP) was added to incubate over-
night at 4 °C, then incubated with HRP-labeled broad-spec-
trum secondary antibody for 1h at r.t.. The nucleus was stained
with DAPI. After dehydration stabilized, the staining was de-
tected with NanoZoomer 2.0-RS (Hamamatsu, Japan).
Male Kunming mice were randomly divided into four
groups, six in each group, respectively, blank group (normal
feeding), model group (ISO modeling), and two medicated
groups (20 mg/kg As or AcAs and ISO). The hypoxia model-
ing method was as follows: in the pre-experimental stage, both
high and low concentrations were used for modeling, results
showed that 20 mg/kg continuous administration for 3 days
could not cause significant myocardial hypoxia injury, and
85mg/kg showed obvious effect. For three consecutive days,
85 mg/kg isoproterenol (ISO) was injected subcutaneously
once a day. ISO is a β-adrenergic agonist that could induces
severe stress to cardiomyocyte, which can lead to the loss of
myocardial integrity through oxygen deficit [27]. ISO-
induced myocardial injury serves as a well-standardized mod-
el to study the beneficial effects of drugs and cardiac function
[28]. In the blank group, an equal amount of 0.5% sodium
carboxymethyl cellulose was injected. In the medicated
groups, drugs were given 20 mg/kg 2h before the injection
of ISO. After the ISO injection on the third day for 1h, the
mice were sacrificed by cervical dislocation method. The heart
was quickly removed after the euthanasia, detected by triphe-
nyltetrazolium chloride (TTC) and hematoxylin-eosin (HE)
WB Assay of the Myocardial Tissue Protein
Myocardial tissue protein preparation: After the end of the
perfusion experiment, quickly remove the heart, cut out the
ventricular tissue, weigh a certain amount of tissue and cut it,
and add the lysate containing the protease inhibitor at a ratio of
1:9 (mg:μL). Grind on ice for 40 times using a glass homog-
enizer. The homogenate was collected, centrifuged at
12,000 rpm for 15 min at 4°C, and the supernatant was col-
lected. A small amount of the supernatant was collected for
protein quantification according to the BCA protein content
determination kit. Add the remaining supernatant to 1/2

Cardiovasc Drugs Ther
volume of 6× loading buffer, mix well, boil in boiling water
for 10 min, cool it for WB detection, or put it in −80°C refrig-
erator for future use.
2a shows, with the increase of PA concentration (0−100 μM),
the expression of HIF-1α gradually increased, indicating the
feasibility of PA to simulate hypoxia modeling. At the same
time, the expression of CAIX showed the same increasing
trend as HIF-1α had, which showed the correlation between
CAIX and HIF-1α. Furthermore, specific siRNA was used to
silence HIF-1α expression of H9c2 cells. In Fig. 2b, overex-
pression of HIF-1α in H9c2 cells transfected with plasmid
pcDNA3.1-HIF-1α enhanced CAIX expression, indicating
that myocardial CAIX protein expression was regulated by
HIF-1α. In contrast, as shown in Fig. 2c, HIF-1α knockdown
diminished CAIX expression, and PA stimulation did not
cause upregulation of CAIX expression. Moreover, the extra-
cellular pH value of H9c2 cells transfected with HIF-1α
siRNA was closer to normal value (Fig. 2d), ROS level of
them was reduced when compared with H9c2 cells under
hypoxia condition (Fig. 2e), and cell viability of them was
also enhanced (Fig. 2f).
Myocardial Enzyme Level in Mouse Serum
Creatine kinase MB isoenzyme (CK-MB) is one of the most
important indicators of diagnosing myocardial injury through
detecting enzyme levels in serum. Cardiac troponin I (cTNI) is
a specific antigen of cardiomyocytes, which is degraded from
the myocardial fiber when the cardiomyocytes are injured, and
the content significantly increases. cTNI is another marker of
myocardial injury. The contents of CK-MB and cTNI were
detected by the corresponding kit and the automatic biochem-
ical instrument.
Statistical Analysis
The results were expressed as the mean SD. The significance
of differences was analyzed by one-way ANOVA followed by
the Bonferroni correction. A value of P < 0.05 was considered
statistically significant.
Synthesis of the Target Compound Containing Aspirin
and Acetazolamide Units
The target compound AcAs containing aspirin (As) and acet-
azolamide (Ac) was prepared by the synthetic way illustrated
in Fig. 3, with purity > 95% as determined by HPLC. The
molecular structure of AcAs was characterized by 1H-NMR,
13C-NMR, and ESI/TOF MS. All characterization results in-
dicate that the target compound AcAs coincides with the
structure as designed (Fig. S2–S8, Table S1).
Results
CAIX Distributes in the Heart and Interacts with HIF-
1α
From the UniProt database, we found that carbonic anhydrase
(CA) has 15 isoenzymes (Homo sapiens). They have a com-
mon catalytic function, reversible hydration of carbon diox-
ide, and participation in extracellular pH adjustment.
However, there are differences in the molecular structure,
physical and chemical properties, immunological properties,
and tissue and organ distribution of isoenzymes. We have
further analyzed them through Bgee, String, and Pathway
Commons. Data from Bgee database show that all isoenzymes
of CA except CA7 have different levels of distribution in the
heart. The CAIX (CA9) gene expression score is 74.4, and the
gene expression level is moderate (Fig. 1a). In the Pathway
Commons analysis, among the 15 CA isozymes, only CAIX
(CA9) interacts with HIF-1α (Fig. 1b). In the analysis of
String protein interaction, only CAIX (CA9) interacts with
HIF-1α. As an upstream gene, HIF-1α can regulate the ex-
pression level of CAIX (CA9) (Fig. 1c).
AcAs Attenuates Hypoxia Injury of Cardiomyocytes
More Effectively Than Aspirin
With CCK8 assay, the cell proliferation level was calculated as
a ratio of the experiment hole absorbance at 450 nm to that of
the control hole. It has been proved by UV spectrophotometer
that the compound has no absorbance at 450 nm (Fig. S1).
After dosing and hypoxia treatment, CCK8 detection showed
cell proliferation changed with varying degrees. Under normal
oxygen condition (21% O2), AcAs and Ac&As (a combined
use of Ac and As) could enhance the cell proliferation and
improve the activity of cardiomyocytes as well as As, while
Ac did not (Fig. 4a). Intriguingly, under hypoxic condition (1%
O2), the effect of As was weakened remarkably, while AcAs
and Ac&As had a better effect, and AcAs even showed increas-
ing cell proliferation obviously at low concentrations (Fig. 4,),
indicating that cell viabilities of AcAs and Ac&As groups were
enhanced under hypoxic condition. AcAs and Ac&As also
increased the cell viability of primary cultured neonatal rat
myocardial cells under hypoxia condition (Fig. S9).
Upstream Protein HIF-1α Regulates CAIX Expression
in Myocardial Cells
To analyze the correlation between HIF-1α and CAIX expres-
sion in myocardial cells, we used palmitic acid (PA) in differ-
ent concentrations to simulate hypoxia in H9c2 cells. As Fig.
The cell membrane integrities of different groups were ex-
amined by flow cytometry using Hoechst 33342 and
propidium Iodide (PI) double staining. Hoechst 33342

Cardiovasc Drugs Ther
Fig. 1 Protein distribution of carbonic anhydrase isoenzymes in the heart (A) and protein-protein interaction between CAIX and HIF-1α (a, Bgee
database; b, Pathway Commons database; c, String database)
(bisBenzimide H33342) is a blue fluorescent dye which can
penetrate cell membrane. When the cell undergoes apoptosis,
the chromatin shrinks, and the fluorescence of the apoptotic
cell will be significantly enhanced after staining. PI cannot
penetrate cell membranes and cannot stain normal cells or
viable apoptotic cells with intact cell membranes. For necrotic
cells and late apoptotic cells, the integrity of the cell mem-
brane is lost, and PI can stain the necrotic cells. This distin-
guishes normal cells, apoptotic cells, and necrotic cells. As
shown in Fig. 4c, cells in the blank group were almost
completely normal cells (located at Hoechst 33342−/PI−),
and the necrotic cells and late apoptotic cells in the model
group were significantly increased to 14.02% (located at
Hoechst 33342+/PI+), indicating that hypoxia stimulation
could easily lead to late apoptosis and programmed cell ne-
crosis. In the medicated group, necrotic cells and late apopto-
tic cells were reduced at different levels, 10.64, 7.87, 5.13, and
3.51% of As, Ac, Ac&As, and AcAs medicated groups, re-
spectively. The results indicate that AcAs can most effectively
resist cell apoptosis and necrosis caused by hypoxia.
ROS, while As had no effect on ROS reduction at the same
concentration. In this study, ROS level is proportional to
dihydroethidium (DHE) oxide’s fluorescence intensity. The
fluorescence emission spectra of DHE oxide can be detected
by fluorescence spectrophotometer (Fs, Fig. 4e), and the peak
intensity variation trend of DHE oxide is consistent with the
flow cytometry data.
The comparison of ATP levels in different groups is shown
in Fig. 4f. Results indicate that hypoxia treatment by tri-gas
incubator can reduce ATP level. Ten-micrometer AcAs is ca-
pable of restoring ATP level, more distinctly than that of Ac,
As, or Ac&As.
In brief, under hypoxia conditions, compared with Ac&As,
Ac, and As, AcAs can best inhibit cell apoptosis and necrosis,
restore cell vitality, and regulate abnormal ROS and ATP levels.
AcAs Works by Acting on CAIX
As shown in Fig. 5a, hypoxia modeling enhances CAIX pro-
tein expression in H9c2 cells. Both Ac and AcAs (10 μM)
reduced CAIX expression, but As did not. Further study
showed that the regulation effect of AcAs (0.01−10 μM) on
CAIX expression was concentration dependent (Fig. 5b).
As Fig. 5c shows, pH value of H9c2 cells under hypoxia
(1% O2) is lower when compared with normoxia (21% O2)-
cultured H9c2 cells, indicating that there is a positive correla-
tion between extracellular acidification and O2 levels. In Ac
Under hypoxic conditions, perturbation in electron trans-
port is associated with leakage of electrons from the respira-
tory chain, resulting in increased ROS that could lead to myo-
cardial hypoxia oxidative injury [29]. Results from the flow
cytometry (Fig. 4d) indicate that hypoxic treatment can lead to
a significant increase of ROS level. Distinctly, 10 μM of
AcAs and Ac&As could effectively reduce the production of

Cardiovasc Drugs Ther
Fig. 2 a HIF-1α and CAIX protein expression in H9c2 cells treated with
palmitic acid (PA) (0–100 μM) for 4 h; b HIF-1α and CAIX expression
in H9c2 cells transfected with pcDNA3.1-HIF-1α or vehicle. The results
were expressed as the mean SD of three independent experiments; c
HIF-1α and CAIX expression in H9c2 cells transfected with HIF-1α or
control scrambled siRNA; d extracellular pH values of H9c2 cells and
cells transfected with HIF-1α or control scrambled siRNA.; e cell distri-
bution of H9c2 cells under different fluorescence intensities of DCF in
blank group, model group (1% O2) of H9c2 cells and cells transfected
with HIF-1α or control scrambled siRNA; f cell viability of H9c2 cells
and cells transfected with HIF-1α or control scrambled siRNA
and AcAs medicated group, the pH values are increased by
about 0.15, similar to the case of a CAIX inhibitor SLC-0111
(50 μM, 1h) treatment, indicating that both Ac and AcAs can
weaken extracellular acidification via the inhibition of CAIX
overexpression. The pH values are unaffected by As with the
same condition.
To predict the interaction between CAIX and molecules,
computer simulation was preliminarily operated. As Fig. 5d
shows, AcAs inserts in the pocket surrounded by four amino
acid residues Thr200, Gln92, Thr69, and Gln67 with hydro-
gen bonds. The binding energy is –5.76 kcal/mol. Meanwhile,
Ac inserts in the same pocket surrounded by three amino acid
residues Thr200, Thr199, and His119 with hydrogen bonds.
The binding energy is –6.0 kcal/mol. The results indicate that
the binding sites and modes of AcAs and Ac to CAIX are
similar and both AcAs and Ac bind to the same amino acid
residue Thr200. The original compound AcAs can bind to
CAIX as stable as that of Ac. The PDB files of homology
(docking) models were listed in Table S3.
AcAs Attenuates Mouse Myocardial Ischemia Injury
More Efficiently Than Aspirin
In Fig. 6a, macroscopic enzyme mapping assay (triphenyltet-
razolium chloride, TTC test) is depicted to detect the myocar-
dial changes in the mice heart of blank and medicated groups.
A high percentage of mean infarct size with increased staining
was observed in isoproterenol (ISO)-administered mice when
compared to blank group. When compared to 85 mg/kg ISO-
administered mice, 20 mg/kg AcAs-pretreated mice showed a

Cardiovasc Drugs Ther
Fig. 3 Synthetic way of preparing the target compound AcAs
moderately lower infarct size with reduced staining.
Administration of 20 mg/kg As had less conspicuous effect
on heart tissue.
without drugs were detected. The CK-MB and cTNI levels
in the model group were significantly increased greater than
that of the blank group. As checked by two commonly used
markers of myocardial injury, the myocardial injury happened
in the model group tissue, whereas in the medicated group,
AcAs was found to effectively attenuate myocardial injury
when compared with As (Table 1).
In Fig. 6b, the approximately normal morphology and
structure of myocardial cells and tissues are shown in the
blank group by hematoxylin-eosin (HE) staining.
Myocardial tissues from ISO-induced mice show the distur-
bance of myocardial structure, extensive edema, and multiple
cell boundaries blurred. The pathological injury of myocardi-
um was alleviated in As-treated and AcAs-treated group to
varying degrees. In comparison with the effect of As group,
the disordered structure in AcAs group was greatly reduced,
and the cell edema was much significantly improved with
clearer cell boundaries. Moreover, in the preliminary experi-
ment, both AcAs and a combined use of Ac&As could atten-
uate myocardial pathological injury (Fig. S10).
The CAIX protein expression in myocardial tissues of dif-
ferent groups was measured using immunohistochemistry as-
say (IHC). The percentage of positive cells under the micro-
scope and the positive staining intensity were scored and com-
pared, respectively. The positive grade was obtained by mul-
tiplying them. The higher the score is, the stronger the positive
is. Compared with the blank group, the CAIX level in ISO-
induced model group was enhanced nearly three times. The
overexpression of CAIX was not affected by As but substan-
tially suppressed after AcAs treatment (Fig. 6c).
Discussion and Conclusion
In this research, we have made original discoveries about the
CAIX expression in myocardial ischemia and hypoxia injury.
By searching the protein databases, we found that CAIX is
normally expressed in the heart (Fig. 1a). Among the protein
isoforms of carbonic anhydrase (CA), only CAIX has a
protein-protein interaction with HIF-1α (Fig. 1b, c).
Although there is an interaction between them, due to the
common hypoxic microenvironment in tumors, CAIX is cur-
rently only considered to be highly expressed in hypoxic tu-
mor cells. Despite CAIX is a confirmed specific marker of
tumorigenesis, there is a lack of systematic research on
CAIX-related mechanism of myocardial hypoxic injury. In
view of the similar hypoxic microenvironment between tumor
cells and myocardial cells, the relevant mechanism of CAIX
in myocardial hypoxia injury is worthy of further
investigation.
As shown in Fig. 6d, ISO modeling can enhance CAIX
protein expression in myocardial tissues. AcAs (20 mg/kg)
reduced CAIX expression, while As did not. These are in
consistent with the results at cellular level.
The creatine kinase MB (CK-MB) and cardiac troponin
(cTNI) level of the blank and ISO-treated groups with or
The correlation between CAIX and HIF-1α in hypoxia
cardiomyocytes was studied in detail. By gradient modeling
of simulated hypoxia (Fig. 2a), HIF-1α overexpression (Fig.
2b), and knockdown assay (Fig. 2c), the results jointly re-
vealed that CAIX is overexpressed in hypoxic

Cardiovasc Drugs Ther
Fig. 4 Cell proliferation influenced by As, Ac, AcAs, or a combined use
of Ac and As (Ac&As) (0.001–10 μM), respectively, under 21 or 1%
oxygen conditions. Data were expressed as the mean SD from three
independent experiments. *p<0.05, **<0.01 versus the group without
drug (a 1% O2 model group, b 21% O2 blank group). c H9c2 cell mem-
brane integrities of blank group, model group (1% O2), and medicated
groups (10 μmol/L As, Ac, AcAs, or Ac&As under 1% O2, respectively).
d Cell distribution of H9c2 cells under different fluorescence intensities
of DCF in blank group, model group (1% O2), and medicated groups (1%
O2 and 10 μM As, Ac, AcAs, or Ac&As, respectively). e ROS regulatory
effect of As, Ac, AcAs, or Ac&As (10 μM) for hypoxia-treated H9c2
cells (DHE oxide, λex=488 nm, λem=520 nm). f ATP regulation effect of
As, Ac, AcAs, or Ac&As for hypoxia-treated H9c2 cells. Data were
expressed as the mean SD from three independent experiments.
**p<0.01, model group vs blank group; #p<0.05, AcAs medicated group
vs model group
cardiomyocytes, and CAIX expression is depended on HIF-
1α-related pathways. Myocardial hypoxia injury of H9c2
cells transfected with HIF-1α siRNA could be attenuated
when compared with normal H9c2 cells (Fig. 2d, e, f). The
inhibition of CAIX overexpression is expected to be an effec-
tive strategy to overcome the drug resistance due to hypoxia
and better attenuate myocardial hypoxic injury. In order to
verify whether the regulation of CAIX overexpression could
attenuate myocardial injury, we not only selected well-known
CAIX inhibitor acetazolamide (Ac) for verification but also
designed a brand-new compound AcAs for exploration (Fig.
3). We connected the CAIX inhibitor Ac to the classic

Cardiovasc Drugs Ther
Fig. 5 a CAIX protein expression in H9c2 cells in response to hypoxia
modeling with or without As, Ac, and AcAs, respectively. b CAIX
protein expression in H9c2 cells in response to hypoxia modeling with
or without 0.01−10 μM AcAs, respectively. c Impact of Ac, As, AcAs,
and SLC-0111 on extracellular pH values of H9c2 cells. d The best
docking mode between Ac or AcAs and amino acid residues around the
active site of CAIX (PDB ID: 6FE2)
cardioprotective drug aspirin (As) in a molecular entity so as
to obtain a kind of potential drug with enhancing efficacy.
Our study indicates that AcAs has obvious advantages in
improving cell viability, especially under hypoxic microenvi-
ronment (Fig. 4a, b), reducing cell necrosis (Fig. 4c), decreas-
ing ROS level (Fig. 4d, e), and increasing ATP level (Fig. 4f).
By comparing the efficacy between As and AcAs, we found
that at cellular level, AcAs can better downregulate CAIX
overexpression (Fig. 5a, b). Both Ac and AcAs could bind
with CAIX around the active site (Fig. 5d). In mouse experi-
ments, both TTC (Fig. 6a) and HE (Fig. 6b) assay showed that
AcAs could better attenuate ISO-induced myocardial ische-
mia injury than As. In IHC (Fig. 6c) and WB (Fig. 6d) assay,
we found that CAIX level in mice heart can be downregulated
by AcAs. Moreover, creatine kinase MB isoenzyme (CK-
MB) and cardiac troponin I (cTNI) are two important indica-
tors to diagnose myocardial injury through detecting enzyme
levels in serum. The effect of AcAs to reduce CK-MB activity
and cTNI level was greater than that of As (Table 1). The
results have verified our idea of CAIX inhibitors for myocar-
dial injury protection and provided a promise for the research
and development of new myocardial injury protection drugs
targeting CAIX.
In view of the role of CAIX in cells, we explored the mech-
anism by which compounds optimized by CAIX inhibitor
could effectively attenuate myocardial injury under hypoxic
microenvironment. CAIX catalyzes the reversible hydration
of CO2 in cells and makes conversion of CO2 into bicarbon-
ate, resulting in promoting the transfer and discharge of CO2,
and maintaining the acid-base balance, the reversible reaction

Cardiovasc Drugs Ther
Fig. 6 a Macroscopic enzyme
mapping assays (TTC test) of
mouse heart tissues in blank,
model (85 mg/kg ISO), and med-
icated groups (20 mg/kg As+85
mg/kg ISO, 20 mg/kg AcAs+85
mg/kg ISO). Six mice in each
group were used. b Effect of pre-
treatment with As or AcAs on
pathological changes in ISO-
induced myocardial ischemia in-
jury mice model. c
Immunohistochemistry examina-
tions of CAIX accumulation in
mice heart. d CAIX protein ex-
pression in myocardial tissue in
response to ISO treatment with or
without As and AcAs,
respectively
is like this, CO2+H2O ↔ H++HCO3 . When the CAIX protein
-
Table 1 Effect of AcAs and As on CK-MB activity and cTNI level in
ISO-treated myocardial ischemia injury mice
is overexpressed, CO2 will be excessively excreted and H+
-
and HCO3 will increase significantly, resulting in an en-
CK-MB (U/L)
cTNI (pg/mL)
hanced extracellular acidic environment. Aspirin has an ester
bond that is prone to hydrolysis when the acidity is increased.
The inhibition of CAIX overexpression is expected to recover
the normal extracellular pH value and ensure that aspirin ex-
erts a stable effect. By testing and comparing the extracellular
pH variations of cardiomyocytes in the blank, hypoxic model,
Blank
Model
AcAs
As
97.0 10.1
313.4 32.8
116.9 23.3
267.2 37.1
28.9 5.2
1453.6 376.6
516.5 90.4
871.7 102.9

Cardiovasc Drugs Ther
2. Sharma A, Arambula JF, Koo S, Kumar R, Singh H, Sessler JL,
et al. Hypoxia-targeted drug delivery. Chem Soc Rev. 2019;48(3):
771–813.
3. Lan GX, Ni KY, Xu ZW, Veroneau SS, Song Y, Lin WB.
Nanoscale metal-organic framework overcomes hypoxia for pho-
todynamic therapy primed cancer immunotherapy. J Am Chem
Soc. 2018;140(17):5670–3.
4. Raber I, McCarthy CP, Vaduganathan M, Bhatt DL, Wood DA,
Cleland JGF, et al. The rise and fall of aspirin in the primary pre-
vention of cardiovascular disease. Lancet. 2019;393(10186):2155–
67.
5. Ridker PM. Should aspirin be used for primary prevention in the
post-statin era? N Engl J Med. 2018;379(16):1572–4.
6. Chen J, Luo HL, Liu Y, Zhang W, Li HX, Luo T, et al. Oxygen-
self-produced nanoplatform for relieving hypoxia and breaking re-
sistance to sonodynamic treatment of pancreatic cancer. ACS Nano.
2017;11(12):12849–62.
7. Liu JN, Bu WB, Shi JL. Chemical design and synthesis of func-
tionalized probes for imaging and treating tumor hypoxia. Chem
Rev. 2017;117(9):6160–224.
8. Berk M, Dean D, Drexhage H, McNeil JJ, Moylan S, O’Neil A,
et al. Aspirin: a review of its neurobiological properties and thera-
peutic potential for mental illness. BMC Med. 2013;11(1):74.
9. Guirguis-Blake JM, Evans CV, Senger CA, O’Connor EA,
Whitlock EP. Aspirin for the primary prevention of cardiovascular
events: a systematic evidence review for the US preventive services
task force. Ann Intern Med. 2016;164(12):804–13.
and medicated groups (Fig. 5c), we found that the pH value in
the hypoxic model group was significantly reduced, which
was consistent with the result of CAIX overexpression in the
model group. The AcAs medicated group was found to effec-
tively regulate abnormal acidity, proving that CAIX inhibition
regulates the extracellular pH values to overcome the drug
resistance due to hypoxia.
In summary, the association between myocardial ischemia
injury and CAIX overexpression has been confirmed. An
original aspirin-containing CAIX inhibitor was designed and
synthesized to validate our hypothesis. The experimental re-
sults of the drug efficacy at cell and animal levels confirmed
that downregulation of CAIX overexpression is an absolutely
new strategy to effectively attenuate myocardial ischemia in-
jury. Such an approach is able to overcome the drug resistance
due to hypoxia. More intriguingly, we have explicated that
CAIX inhibitors can regulate the abnormal extracellular acid-
ity to ensure the drug stability. In all, our work on the appli-
cation of CAIX offers a promising strategy for drug design
aiming at myocardial ischemia and hypoxia injury treatment.
Supplementary Information The online version contains supplementary
material available at https://doi.org/10.1007/s10557-021-07182-2.
10. McNeil JJ, Wolfe R, Woods RL, Tonkin AM, Donnan GA, Nelson
MR, et al. Effect of aspirin on cardiovascular events and bleeding in
the healthy elderly. N Engl J Med. 2018;379(16):1509–18.
11. Packer M. Critical examination of mechanisms underlying the re-
duction in heart failure events with SGLT2 inhibitors: identification
of a molecular link between their actions to stimulate erythrocytosis
and to alleviate cellular stress. Cardiovasc Res. 2020;117(1):74–84.
12. Semenza GL. Mechanisms of disease oxygen sensing, homeostasis,
and disease. N Engl J Med. 2011;365(10):537–47.
Authors’ Contributions GS conceived and designed the project. ZW per-
formed the experiments with the help of ZB and FK, and ZW was a major
contributor in writing the manuscript. YX provided guidance on the bio-
informatics analysis. LJ performed the docking work. All authors read
and approved the final manuscript.
13. Cao Q, Zhou DJ, Pan ZY, Yang GG, Zhang H, Ji LN, et al.
CAIXplatins: highly potent Pt(IV) prodrugs selectively against
hypoxic tumors via microenvironment and metabolism regulation.
Angew Chem Int Edit. 2020;59(42):18556–62.
Funding This work was supported by the Natural Science Foundation of
Jiangsu Province [Grant number BK20180570]. We are grateful to
Jiangsu Province Hi-Tech Key Laboratory for Biomedical Research for
the support of this work.
14. Cui J, Zhang Q, Song Q, Wang HR, Dmitriev P, Sun MY, et al.
Targeting hypoxia downstream signaling protein, CAIX, for CAR
T-cell therapy against glioblastoma. Neuro-Oncology.
2019;21(11):1436–46.
15. John A, Sivashanmugam M, Natarajan SK, Umashankar V.
Computational modeling of novel inhibitory peptides targeting pro-
teoglycan like region of carbonic anhydrase IX and in vitro valida-
tion in HeLa cells. J Biomol Struct Dyn. 2020;38(7):1995–2006.
16. Jung HS, Han J, Shi H, Koo S, Singh H, Kim HJ, et al. Overcoming
the limits of hypoxia in photodynamic therapy: a carbonic
anhydrase IX-targeted approach. J Am Chem Soc. 2017;139(22):
7595–602.
Data and Materials Availability The isoenzymes and functions of carbon-
ic anhydrase were found in UniProt database (https://www.uniprot.org).
The gene expression score in Fig. 1a was found in Bgee database (https://
bgee.org). The protein-protein interaction in Fig. 1b and Fig. 1c was
analyzed in Pathway Commons database (http://www.
pathwaycommons.org/) and String database (https://string-db.org/),
respectively.
Declarations
Ethics Approval All applicable international, national, and/or institu-
tional guidelines for the care and use of animals were followed.
17. Scheibe RJ, Gros G, Parkkila S, Waheed A, Grubb JH, Shah GN,
et al. Expression of membrane-bound carbonic anhydrases IV, IX,
and XIV in the mouse heart. J Histochem Cytochem. 2006;54(12):
1379–91.
Conflict of Interest The authors declare no competing interests.
18. Vargas LA, Pinilla OA, Diaz RG, Sepulveda DE, Swenson ER,
Perez NG, et al. Carbonic anhydrase inhibitors reduce cardiac dys-
function after sustained coronary artery ligation in rats. Cardiovasc
Pathol. 2016;25(6):468–77.
References
19. Orlowski A, De Giusti VC, Morgan PE, Aiello EA, Alvarez BV.
Binding of carbonic anhydrase IX to extracellular loop 4 of the
-
1. Thygesen K, Alpert JS, Jaffe AS, Chaitman BR, Bax JJ, Morrow
DA, et al. Fourth universal definition of myocardial infarction
(2018). J Am Coll Cardiol. 2019;72(18):2231–64.
NBCe1 Na+/HCO3 cotransporter enhances NBCe1-mediated
HCO3- influx in the rat heart. Am J Physiol-Cell Ph. 2012;303(1):
C69–80.

Cardiovasc Drugs Ther
20. Robertson N, Potter C, Harris AL. Role of carbonic anhydrase IX in
human tumor cell growth, survival, and invasion. Cancer Res.
2004;64(17):6160–5.
algorithm and an empirical binding fee energy function. J Comput
Chem. 1998;19(14):1639–62.
26. Wan F, Peng QQ, Liu JF, Alolga RN, Zhou W. A novel ferulic acid
derivative attenuates myocardial cell hypoxia reoxygenation injury
through a succinate dehydrogenase dependent antioxidant mecha-
nism. Eur J Pharmacol. 2019;856:172417.
27. Osadchii OE. Cardiac hypertrophy induced by sustained beta-
adrenoreceptor activation: pathophysiological aspects. Heart Fail
Rev. 2007;12:66–86.
28. Allawadhi P, Khurana A, Sayed N, Kumari P, Godugu C.
Isoproterenol-induced cardiac ischemia and fibrosis: Plant-based
approaches for intervention. Phytother Res. 2018;32(10):1908–32.
29. Kim JW, Tchernyshyov I, Semenza GL, Dang CV. HIF-1-mediated
expression of pyruvate dehydrogenase kinase: A metabolic switch
required for cellular adaptation to hypoxia. Cell Metab. 2006;3(3):
177–85.
21. Takov K, He ZH, Johnston HE, Timms JF, Guillot PV, Yellon DM,
et al. Small extracellular vesicles secreted from human amniotic
fluid mesenchymal stromal cells possess cardioprotective and
promigratory potential. Basic Res Cardiol. 2020;115(3):26.
22. He Y, Zhou LY, Fan ZQ, Liu SK, Fang WJ. Palmitic acid, but not
high-glucose, induced myocardial apoptosis is alleviated by N-
acetylcysteine due to attenuated mitochondrial-derived ROS
accumulation-induced endoplasmic reticulum stress. Cell Death
Dis. 2018;9(5):568.
23. Zhao YP, Wang F, Jiang W, Liu JF, Liu BL, Qi LW, et al. A
mitochondrion-targeting tanshinone IIA derivative attenuates myo-
cardial hypoxia reoxygenation injury through a SDH-dependent
antioxidant mechanism. J Drug Target. 2019;27(8):896–902.
24. Levey AS, Stevens LA, Schmid CH, Zhang YP, Castro AF,
Feldman HI, et al. A new equation to estimate glomerular filtration
rate. Ann Intern Med. 2009;150(9):604–12.
Publisher’s Note Springer Nature remains neutral with regard to jurisdic-
tional claims in published maps and institutional affiliations.
25. Morris GM, Goodsell DS, Halliday RS, Huey R, Hart WE, Belew
RK, et al. Automated docking using a Lamarckian genetic