Small molecule QF84139 ameliorates cardiac hypertrophy via activating the AMPK signaling pathway

Article abstract of DOI:10.1038/s41401-021-00678-5

Cardiac hypertrophy is a common adaptive response to a variety of stimuli, but prolonged hypertrophy leads to heart failure. Hence, discovery of agents treating cardiac hypertrophy is urgently needed. In the present study, we investigated the effects of QF84139, a newly synthesized pyrazine derivative, on cardiac hypertrophy and the underlying mechanisms. In neonatal rat cardiomyocytes (NRCMs), pretreatment with QF84139 (1–10 μM) concentration-dependently inhibited phenylephrine-induced hypertrophic responses characterized by fetal genes reactivation, increased ANP protein level and enlarged cardiomyocytes. In adult male mice, administration of QF84139 (5–90 mg·kg^?1·d^?1, i.p., for 2 weeks) dose-dependently reversed transverse aortic constriction (TAC)-induced cardiac hypertrophy displayed by cardiomyocyte size, left ventricular mass, heart weights, and reactivation of fetal genes. We further revealed that QF84139 selectively activated the AMPK signaling pathway without affecting the phosphorylation of CaMKIIδ, ERK1/2, AKT, PKCε, and P38 kinases in phenylephrine-treated NRCMs and in the hearts of TAC-treated mice. In NRCMs, QF84139 did not show additive effects with metformin on the AMPK activation, whereas the anti-hypertrophic effect of QF84139 was abolished by an AMPK inhibitor Compound C or knockdown of AMPKα2. In AMPKα2-deficient mice, the anti-hypertrophic effect of QF84139 was also vanished. These results demonstrate that QF84139 attenuates the PE- and TAC-induced cardiac hypertrophy via activating the AMPK signaling. This structurally novel compound would be a promising lead compound for developing effective agents for the treatment of cardiac hypertrophy.

Full text of DOI:10.1038/s41401-021-00678-5

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ARTICLE
Small molecule QF84139 ameliorates cardiac hypertrophy
via activating the AMPK signaling pathway
1
1
2
3
1
1
1
2
3
Xu-xia Li , Peng Zhang , Yang Yang , Jing-jing Wang , Yan-jun Zheng , Ji-liang Tan , Shen-yan Liu , Yong-ming Yan , You-yi Zhang ,
2,4
1,5,6
Yong-xian Cheng and Huang-tian Yang
Cardiac hypertrophy is a common adaptive response to a variety of stimuli, but prolonged hypertrophy leads to heart failure.
Hence, discovery of agents treating cardiac hypertrophy is urgently needed. In the present study, we investigated the effects of
QF84139, a newly synthesized pyrazine derivative, on cardiac hypertrophy and the underlying mechanisms. In neonatal rat
cardiomyocytes (NRCMs), pretreatment with QF84139 (1–10 μM) concentration-dependently inhibited phenylephrine-induced
hypertrophic responses characterized by fetal genes reactivation, increased ANP protein level and enlarged cardiomyocytes. In
−
1
−1
adult male mice, administration of QF84139 (5–90 mg·kg ·d , i.p., for 2 weeks) dose-dependently reversed transverse aortic
constriction (TAC)-induced cardiac hypertrophy displayed by cardiomyocyte size, left ventricular mass, heart weights, and
reactivation of fetal genes. We further revealed that QF84139 selectively activated the AMPK signaling pathway without affecting
the phosphorylation of CaMKIIδ, ERK1/2, AKT, PKCε, and P38 kinases in phenylephrine-treated NRCMs and in the hearts of TAC-
treated mice. In NRCMs, QF84139 did not show additive effects with metformin on the AMPK activation, whereas the anti-
hypertrophic effect of QF84139 was abolished by an AMPK inhibitor Compound C or knockdown of AMPKα2. In AMPKα2-deficient
mice, the anti-hypertrophic effect of QF84139 was also vanished. These results demonstrate that QF84139 attenuates the PE- and
TAC-induced cardiac hypertrophy via activating the AMPK signaling. This structurally novel compound would be a promising lead
compound for developing effective agents for the treatment of cardiac hypertrophy.
Keywords: cardiac hypertrophy; pyrazine derivative; QF84139; phenylephrine; transverse aortic constriction; AMPK signaling
pathway
Acta Pharmacologica Sinica (2021) 0:1–14; https://doi.org/10.1038/s41401-021-00678-5
INTRODUCTION
Development of cardiac hypertrophy to maladaptive is regu-
lated by various signaling pathways, such as the calcium/
calmodulin-dependent protein kinase type II delta (CaMKIIδ),
mitogen-activated protein kinase (MAPK) cascade including P38
kinase and extracellular signal-regulated kinase 1/2 (ERK1/2),
protein kinase B (PKB/AKT), and protein kinase C epsilon (PKCε)
[1, 6–8]. One of the most important changes that promote cardiac
hypertrophy is cardiomyocyte growth. Since the energy status of
cells can dictate cell growth, it is likely that adenosine 5′-
monophosphate-activated protein kinase (AMPK), a key regulator
of cellular energy homeostasis, is importantly involved in the
regulation of cell growth and hypertrophy [9, 10]. AMPK is a
serine-threonine kinase consisting of a catalytic subunit (α) and 2
regulatory subunits (β, γ). The α2 isoform of AMPK (AMPKα2) is
abundantly expressed in cardiomyocytes [11]. It participates in a
variety of cellular processes to protect against cardiac hypertrophy
[1, 10]. In pressure overload-induced cardiac hypertrophy, AMPK is
Cardiac hypertrophy is a response to the increased hemody-
namic workload that arises from various physiological stimuli and
pathological insults [1, 2]. It is characterized by an enlargement
of myocardium with increases in cardiac mass and cardiomyo-
cyte size, protein synthesis, and reactivation of fetal genes [1, 3].
Although it initially appears to be adaptive, the long-term cardiac
hypertrophy is associated with a significantly increased risk of
heart failure and ultimately leads to high rates of mortality and
morbidity [4, 5]. Although much progress has been made in
understanding the molecular mechanisms underlying cardiac
hypertrophy, small molecules specifically targeting these path-
ways need to be discovered, which would allow to determine
whether selectively regulating certain signaling pathways could
reverse pathological hypertrophy [1] and help to develop
new molecules for the treatment of patients with cardiac
hypertrophy.
1
CAS Key Laboratory of Tissue Microenvironment and Tumor, Laboratory of Molecular Cardiology, Shanghai Institute of Nutrition and Health, University of Chinese Academy of
2
Sciences, CAS, Shanghai 200031, China; State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, CAS, Kunming 650201, China;
3
Department of Cardiology and Institute of Vascular Medicine, Peking University Third Hospital; NHC Key Laboratory of Cardiovascular Molecular Biology and Regulatory
4
Peptides; Key Laboratory of Molecular Cardiovascular Science, Ministry of Education, Beijing Key Laboratory of Cardiovascular Receptors Research, Beijing 100191, China; School
of Pharmaceutical Sciences, Health Science Center, Shenzhen University, Shenzhen 518060, China; Department of Cardiology, Shanghai Jiao Tong University Affiliated Sixth
People’s Hospital, Shanghai 200233, China and Institute for Stem Cell and Regeneration, CAS, Beijing 100101, China
5
6
Correspondence: Yong-xian Cheng (yxcheng@szu.edu.cn) or Huang-tian Yang (htyang@sibs.ac.cn)
These authors contributed equally: Xu-xia Li, Peng Zhang, Yang Yang
Received: 19 November 2020 Accepted: 2 April 2021
©
The Author(s), under exclusive licence to CPS and SIMM 2021

Anti-hypertrophic effects of a new compound QF84139
XX Li et al.
2
activated to preserve energy homeostasis [1, 10]. In addition, it can
suppress protein synthesis via the inhibition of eukaryotic
elongation and factor-2 (eEF2) kinase/eEF2 axis and mammalian
target of rapamycin (mTOR)/p70 ribosomal protein S6 kinase (p70
S6K) pathway that are activated during hypertrophy [1, 10, 12, 13].
AMPKα2 was reported to protect mouse hearts against transverse
aortic constriction (TAC)-induced ventricular hypertrophy and
dysfunction, in part, by repressing mTOR signaling [14]. AMPK has
also been reported to inhibit cardiac hypertrophy through
regulation of microtubule densification, autophagy, transcriptional
activity, endoplasmic reticulum (ER) stress, and microRNA expres-
sion [1, 10, 15–17]. Pharmacological activation of AMPK, either by
direct or indirect activators, can attenuate cardiac hypertrophy
temperature was back to 10 °C. The pH value of the resulting
reaction mixture was adjusted to 3 at –10 °C with diluted
hydrochloric acid, then filtrated and washed by water, dried in
air to afford a yellow solid as QYY000 (5-(furan-2-yl)pyrazin-2(1H)-
−
1
one (430 mg, 19.5% yield over 3 steps). ESIMS m/z 161 [M – H] ; H
NMR (CDCl , 600 MHz) δ: 8.27 (1H, d, J = 1.5 Hz, H-2), 7.62 (1H, d,
J = 1.5 Hz, H-4), 7.43 (1H, d, J = 1.8 Hz, H-8), 6.79 (1H, d, J = 3.3 Hz,
3
13
3
H-6), 6.50 (1H, dd, J = 3.4, 1.8 Hz, H-7); C NMR (CDCl , 150 MHz) δ:
157.0 (C-1), 150.5 (C-5), 148.7 (C-2), 142.5 (C-8), 129.1 (C-3), 120.4
(C-4), 112.1 (C-7), 107.0 (C-6). Its planar structure was further
1
identified by spectroscopic methods including H NMR (Supple-
13
mentary Fig. S3a), C NMR (Supplementary Fig. S3b).
Second step: the synthesis of QF84139 (Supplementary Fig. S2b):
To a solution of (−)-borneol (3) (5.0 g, 32.5 mmol) as (1 S,2 R,4 S)-
1,7,7-trimethylbicyclo[2.2.1]heptan-2-ol, 2-bromoacetic acid (4)
(9.0 g, 64.9 mmol) and DCC (13.4 g, 64.9 mmol) in dichloro-
methane at room temperature was slowly added 4-(dimethyla-
mino)-pyridine (1.9 g, 15.6 mmol) dissolved in dichloromethane,
stirred for 1 h. The resulting reaction mixture was filtrated and
[
8, 12, 18, 19]. These findings indicate crucial roles of the AMPK
signaling pathway in the intervention of cardiac hypertrophy and
suggest that AMPK activators might act as novel therapeutic
reagents for cardiac hypertrophy. Therefore, the discovery of new
AMPK activators would be valuable for the drug development
against cardiac hypertrophy.
1
Natural products play a pivotal role for drug discovery [20]. Over
concentrated to afford 5 (4.2 g, 0.36 mmol, 47%). H NMR (400
6
6% of new drugs based on small molecules approved by the US
3
MHz, CDCl ) δ: 4.88 (ddd, J = 10.0, 3.5, 2.1 Hz), 3.78 (s, 2H), 2.36 −
Food and Drug Administration between 1981 and 2019 are natural
products and their direct derivatives or inspired by natural
products [20]. Although the number of natural products is limited,
millions of natural product hybrids (NPHs) as combinations of
parts of different natural products can be developed and severed
as new leads for drug discovery [21].
Bearing this emerging concept in mind, we have purposely
synthesized several NPHs aiming to discover new lead compounds
for the treatment of heart associated disorders, including cardiac
hypertrophy. In this study, using phenylephrine (PE)-induced
cardiomyocyte hypertrophic cell model, we identified QF84139 as
a candidate against cardiac hypertrophy. Then, we used the
in vitro PE- and in vivo TAC-induced cardiac hypertrophy models,
combining with signaling pathways screening and manipulation,
to investigate (i) the effect of QF84139 in cardiac hypertrophy; and
2.23 (m, 1H), 1.89 (m, 1H), 1.76 − 1.59 (m, 2H), 1.32 − 1.12 (m, 2H),
0.95 (dd, J = 14.0, 3.6 Hz, 1H), 0.84 (s, 3H), 0.81 (s, 3H), 0.79 (s, 3H);
13
C NMR (100 MHz, CDCl
26.9, 26.3, 19.6, 18.8, 13.4.
To a solution of 2 (4.0 g, 14.6 mmol, QYY000) and K CO (2.6 g,
3
) δ 167.5, 82.0, 49.0, 47.9, 44.7, 36.4, 27.9,
2
3
19 mmol) in acetone was added into a solution of 5 (1 S,2 R,4 S)-
1,7,7-trimethylbicyclo[2.2.1]heptan-2-yl 2-bromoacetate (1.6 g, 9.7
mmol) dissolved in acetone, stirred at room temperature for 5 h,
then filtrated and concentrated the solution to afford a yellow
solid, after recrystallization with ethyl acetate and petroleum ether
afforded 630 mg (18%) yellow solid as 6 (QF84139). QF84139: [α]
–33 (c 0.20, CHCl ); UV λ
(MeOH) (log ε): 287 (3.71), 233 (3.54)
3
max
nm; IR (KBr) νmax 3427, 2956, 2881, 1741, 1679, 1622, 1455, 1386,
−
1
–
1360, 1306, 1216, 1116, 1019, 995 cm ; ESIMS m/z [M – H] 355;
–
23 2 4
HRESIMS, calcd for C20H N O : [M – H] 355.1663; found:
355.1656; H NMR (CDCl , 600 MHz) δ: 8.23 (1H, brs, H-2), 7.47
1
(
ii) the signaling pathways involved in the anti-hypertrophic
3
effects of QF84139. Our results demonstrated that QF84139 can
attenuate cardiac hypertrophy via selectively activating the AMPK
signaling pathway without affecting the CaMKIIδ, ERK1/2, AKT,
PKCε, and P38 signaling pathways. Thus, QF84139 may act as a
new activator of the AMPK signaling pathway used in the study of
AMPK activation-related disorders and may have potential
application value in the development of drugs for the treatment
of cardiac hypertrophy.
(1H, brs, H-4), 7.38 (1H, brs, H-8), 6.75 (1H, d, J = 3.2 Hz, H-6), 6.47
(1H, dd, J = 3.2, 1.7 Hz, H-7), 4.98 (1H, ddd, J = 9.9, 3.1, 2.2 Hz, H-1’),
4.67 (2H, s, H-9), 2.35 (1H, m, H-6’a), 1.75 (1H, overlap, H-3’a), 1.75
(1H, overlap, H-4’a), 1.68 (1H, m, H-5’), 1.28 (1H, m, H-3’b), 1.20 (1H,
m, H-4’b), 1.03 (1H, dd, J = 13.9, 3.3 Hz, H-6’b), 0.87 (3H, s, H-9’),
13
3
0.85 (3H, s, H-10’), 0.82 (3H, s, H-8’); C NMR (CDCl , 150 MHz)
δ: 166.6 (C-10), 155.0 (C-1), 150.3 (C-5), 149.1 (C-2), 142.2 (C-8),
127.4 (C-3), 123.5 (C-4), 112.1 (C-7), 106.8 (C-6), 82.6 (C-1’), 50.5 (C-
9
3
), 49.1 (C-7’), 48.0 (C-2’), 44.8 (C-5’), 36.6 (C-6’), 28.0 (C-4’), 27.0 (C-
’), 19.7 (C-10’), 18.9 (C-9’), 13.6 (C-8’).
MATERIALS AND METHODS
Preparation of QF84139
Isolation of primary neonatal rat cardiomyocytes (NRCMs)
The small molecule used in this study (Code: QF84139) is a new
compound, which was synthesized by a natural product derivative
NRCMs were isolated as previously described [23, 24]. Briefly, the
hearts were removed from 1-day-old Sprague Dawley (SD) rats,
the atria was trimmed off, and then the tissue was minced by
sequential digestion with 1 mg/mL collagenase II (Worthington,
Lakewood, NJ, USA) and 0.125% trypsin. By differential plating, the
ventricular cardiomyocytes were separated from fibroblasts and
then cultured in gelatin-coated tissue culture plates in media
containing Dulbecco’s modified Eagle’s medium (DMEM, Thermo
Fisher Scientific, Waltham, MA, USA), 10% fetal bovine serum
(Thermo Fisher Scientific, Waltham, MA, USA), 100 μM bromodeox-
yuridine (Sigma, Carlsbad, CA, USA), 1% penicillin-streptomycin
(Thermo Fisher Scientific, Waltham, MA, USA), and 1% L-glutamine
(Thermo Fisher Scientific, Waltham, MA, USA) with 95% air and 5%
2
and (−)-borneol (3) via a concise linker (-CH CO-). The target
2
compound QF84139 was accomplished by three-step reactions
with a 1.65% yield. The detailed reaction processes were as
follows:
First step: the synthesis of 2 (QYY000, 5-(furan-2-yl)pyrazin-2
(
2
(
1H)-one) (Supplementary Fig. S2a): To a solution of SeO
0.5 mmol) in 30 mL of dioxane was added 1-(furan-2-yl)ethanone
1.5 g, 13.6 mmol) dissolved in dioxane and then refluxed for 8 h
2
(2.2 g,
according to Sato’s procedures [22]. The reaction mixture was
filtrated and concentrated under reduced pressure to afford
a mixture containing 1.3 g of 2-(furan-2-yl)-2-oxoacetaldehyde
without purification. To a solution of 2-(furan-2-yl)-2-oxoacetalde-
hyde (1.3 g, 10.6 mmol) in 15 mL of methanol was added 2-
aminoacetamide chloride (973 mg, 8.9 mmol) dissolved in 20 mL
CO at 37 °C for 24 h.
2
Cardiomyocyte hypertrophic cell model and screening of NPHs
The isolated NRCMs were cultured in the serum-free DMEM
(Thermo Fisher Scientific, Waltham, MA, USA) for 12 h. Then the
cells were treated with 10 μM NPHs, 50 μM resveratrol (RSV), or
o
of methanol/water (2:1; at −30 C), and then the sodium
hydroxide (885 mg, 22.1 mmol) in 5 mL of water was dropped
slowly into the solution. The reaction was kept for 90 min until the
Acta Pharmacologica Sinica (2021) 0:1 – 14

Anti-hypertrophic effects of a new compound QF84139
XX Li et al.
3
vehicle and 1 h later were stimulated by 10 μM PE (Sigma,
Carlsbad, CA, USA) for 48 h in the presence of NPHs, RSV, or
vehicle. The same protocol was used for the QF84139 treatment
with various concentrations.
and scrambled siRNA, 5ʹ-AUUGUAUGCGAUCGCAGAC-3ʹ. Scrambled
siRNA was used as a control in all experiments. NRCMs were
transfected with Scrambled siRNA (4 μg) or AMPKɑ2 siRNA (2 μg)
using DharmaFECT 1 transfection reagent (Thermo Fisher Scientific,
Waltham, MA, USA) according to the manufacturer’s instruction for
24 h and then with QF84139 for another 1 h prior to PE treatment.
Cell viability assay
The Cell Counting Kit-8 (CCK-8, Sigma, Carlsbad, CA, USA) was
used to determine cell viability as previously reported [24]. Briefly,
the NRCMs were plated into 96 well plates for 24 h, and incubated
with the medium containing indicated dosages of QF84139 or
vehicle for 48 h. After treatment, the CCK-8 containing medium
was added into each well for 4 h. The values were detected and
analyzed by Synergy platereader (BioTek, Winooski, VT, USA).
Echocardiography
Cardiac function and structure were performed as previously
described [24]. Briefly, the Vevo2100 imaging system (Visual
Sonics, Toronto, Canada) with 30 MHz linear transducer probe
(MS400D) was used for two-dimensional B-mode and M-mode
analyses. The heart rate was maintained at 450-550 bpm via
isoflurane anesthesia (1.5%–2%, 2 L/min oxygen flow rate). Normal
body temperature was maintained. The mitral valve leaflet was
visualized and cardiac function was assessed at long axis B-mode
view by placing the transducer on the left lateral chest wall.
Measurements were recorded as the mean of at least three
consecutive cardiac cycles. Left ventricular (LV) internal dimension
(LVID;s) in systole and left ventricular internal dimension diastole
(LVID;d), thickness of the interventricular septum in systole (IVS;s)
and interventricular septum in diastole (IVS;d) and thickness of the
LV posterior wall in systole and diastole (LVPW;s and LVPW;d,
respectively) were obtained from the echocardiograms. Using the
following formula: EF (%) = [(LVvol;d- LVvol;s)/LVvol;d]×100, the LV
ejection fraction (EF) was calculated; and the LV fractional
shortening (FS) was calculated using FS (%) = [(LVID;d - LVID;s)/
LVID;d]×100.
Animals
Animals were cared complied with the Guidelines for Care and
Use of Laboratory Animals published by the US National Institutes
of Health (NIH Publication, 8th Edition, 2011). Experimental
procedures involving animals were approved by the Institutional
Animal Care and Use Committee of Shanghai Institutes for
Biological Sciences (recently, it was named to Shanghai Institute
of Nutrition and Health, Shanghai, China). Adult male C57BL/6
mice (10 weeks old) and neonatal SD rats (1 days) were purchased
-
/-
from Shanghai Slac Laboratory Animal Co. Ltd. AMPKα2 male
mice (10 weeks) were kindly provided by Dr. Benoit Viollet
(
Institute National de la Santé et de la Recherche Médicale U567,
Paris, France). The animals were allowed free access to food and
water and were maintained on a 12 h light/dark cycle in a
controlled temperature (20–22 °C) and humidity (50% ± 5%)
environment.
Histological analysis
Histological examination was performed as previously described
[23, 25]. Briefly, the mice hearts from each group were quickly
harvested, and washed with 5 mL cold cardiac arresting buffer
(10% KCl), phosphate buffered saline (PBS), fixed with 4%
paraformaldehyde for 48 h, and then embedded in paraffin. The
LV cross sections at 5 μm thick were deparaffinized by immersing
in xylene, and rehydrated and then stained with hematoxylin-
eosin (H&E) and FITC-labeled wheat germ agglutinin (WGA,
Thermo Fisher Scientific, Waltham, MA, USA). All of the immuno-
fluorescence images were blindly taken with a Zeiss microscope.
The Image-processing software (ImageJ) was used to analyze the
cross-sectional area of cardiomyocytes.
Mice TAC surgical procedure and study design
The mice TAC model was used to induce cardiac hypertrophy as
described previously [23]. Briefly, male mice (10 weeks) were
anaesthetized with pentobarbital sodium (50 mg/kg) by intraper-
itoneal (i.p.) injection and placed on a temperature (37 °C)-
controlled surgical table. Volume-regulated respirator (SAR830,
Cwe Incorporated, Ardmore, PA, USA) at a frequency of 90 bpm
and the tidal volume ranged 200–300 mL/min was used to
ventilate. The mice were divided into Sham and TAC groups. A
longitudinal cut was made in the proximal portion of the sternum.
A 7-0 silk suture was placed around the aorta between the right
innominate artery and left common carotid artery. The suture was
tied around a 27-gauge needle and the aorta. After ligation in
place, the needle was promptly removed. Sham-operated mice
underwent the same procedure but without ligation around the
aorta. At the same day after aortic constriction, TAC-operated wild-
type (WT) mice were randomly divided into five groups: TAC +
vehicle and four TAC + QF84139 groups with the administration of
Immunofluorescence analysis
For immunofluorescence, cardiomyocytes grown in 6-well plates
were washed three times with phosphate-buffered saline (PBS)
after PE treatment for 48 h and fixed with 4% (w/v) paraformal-
dehyde for 30 min at room temperature. Then the cells were
permeabilized in 0.3% Triton X-100, blocked in 10% normal goat
serum (Vector Laboratories, Burlingame, CA, USA) and then
incubated with primary antibodies α-actinin (1:200, Abcam,
Cambridge, UK) and atrial natriuretic peptide (ANP) (1:200,
Bioworld, Dublin, OH, USA) at 4 °C overnight before the
secondary antibodies (1:500, Thermo Fisher Scientific, Waltham,
MA, USA). Negative controls were performed with secondary
antibody-only staining. Nuclei were stained with DAPI (Sigma-
Aldrich, Carlsbad, CA, USA). Images were taken using a Zeiss Cell
Observer confocal laser scanning microscope 710 (Zeiss,
Germany) and processed using ZEN software. Fluorescence
images were analyzed with ImageJ.
-
1
−1
−1 −1
−1 −1
QF84139 at 5 mg·kg ·d , 15 mg·kg ·d , 45 mg·kg ·d , and
9
−
1
−1
0 mg·kg ·d , respectively (i.p.). Sham-operated WT mice were
randomly divided into two groups: Sham + vehicle and Sham +
−/−
−
1
−1
QF84139 (90 mg·kg ·d , i.p.). The TAC-operated AMPKα2
mice were randomly divided into two groups: TAC + vehicle,
−
1
−1
−/−
TAC + QF84139 (15 mg·kg ·d , i.p.). Sham-operated AMPKα2
−
/−
mice were randomly divided into two groups: AMPKα2 Sham
−1 −1
−
/−
+
vehicle and AMPKα2
Sham + QF84139 (15 mg·kg ·d , i.p.).
The vehicle and QF84139 were administered once per day, from
day 1 to day 14 post-surgery at the same volume. At the end of
the study, animals were anaesthetized with an overdose of
sodium pentobarbital (200 mg/kg, i.p.) as previously described [9],
and the hearts, lungs, livers, spleens, kidneys, and tibia were
collected.
Quantitative reverse transcription polymerase chain reaction
(qRT-PCR)
Total RNA was extracted from mouse LVs or NRCMs using Trizol
(Invitrogen, Carlsbad, CA, USA) or RNeasy Mini kit (QIAGE, Hilden,
Germany) following the manufacturer’s instruction and analyzed
by qRT-PCR as previously described [24]. Briefly, cDNA was
generated by reverse-transcribed total RNA (1 μg) using oligo
Small interfering RNA for AMPKα2 (si-AMPKα2)
Oligonucleotide sequences were purchased from Ribobio to
prepare small interfering RNA for AMPKα2. The siRNA sequences
were as follows: AMPKα2 siRNA, 5ʹ-CGTCATTGATGATGAGGCT-3ʹ,
Acta Pharmacologica Sinica (2021) 0:1 – 14

Anti-hypertrophic effects of a new compound QF84139
XX Li et al.
4
(
dT) primer and ReverTra Ace reverse transcriptase (TOYOBO,
NRCMs. Resveratrol (RSV) was used as positive control. After 10 μM
of PE treatment for 48 h, the mRNA expression of ANP, a
hypertrophy marker gene, was significantly increased (Supplemen-
tary Fig. S1), indicating a hypertrophic response in NRCMs. Among
the 24 compounds examined, QF84139 and RSV significantly
attenuated the expression of ANP induced by PE, suggesting the
anti-hypertrophic effect of QF84139.
Japan). Taq DNA Polymerase (Takara, Japan) was used to carry out
PCR. qRT-PCR was performed using an ABI VII7 system (Applied
Biosystems, Foster City, CA, USA) with the SYBR Green Real-time
PCR Master Mix plus (TOYOBO, Japan) for relative quantification of
the indicated genes. The amplification program was performed as
follows: 94 °C for 10 min and 30 cycles at 94 °C for 30 s, 55 °C for
3
0 s, and 72 °C for 30 s. The transcript of GAPDH was used for
internal normalization. The sequences of primer pairs are listed in
Supplementary Table S1.
Preparation of QF84139 by total synthesis
QF84139 is a small-molecular compound totally synthesized via
three steps starting respectively from 1-(furan-2-yl) ethanone and
(−)-borneol (for details, see “Materials and methods”). The chemical
structure is shown in Fig. 1a. Its planar structure was further
Western blot analysis
Mice LV were homogenized and the proteins were extracted as
previously described [26]. Briefly, about 50 mg of left ventricular
tissues were homogenized on ice with a homogenizer in 10 vols of
immunoprecipitation assay (RIPA) lysis buffer containing 1% Triton
X-100, 5 mmol/L 2-mercaptoethanol, and 1% deoxycholate, 10%
glycerin, 150 mM NaCl, 2.5 mM EDTA and 1 mg/mL protease
inhibitor cocktail. For cultured cells, the NRCMs were scraped into
RIPA lysis buffer containing protease inhibitor cocktail (Roche,
Mannheim, Germany), 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1%
NP-40, 0.5% deoxycholate and 1 mM EDTA, followed by vigorous
vortexing and cooling on ice for 15 min before centrifugation at
1
identified by spectroscopic methods including H NMR (Supple-
13
1
1
mentary Fig. S4a), C NMR (Supplementary Fig. S4b), H- H COSY
(Supplementary Fig. S5a), HSQC (Supplementary Fig. S5b), HMBC
(Supplementary Fig. S5c), ROESY (Supplementary Fig. S5d), and
HRESIMS spectrum (Supplementary Fig. S6a). The absolute config-
uration of QF84139 was assigned as 1 S, 2 R, 4 S because that
(−)-borneol which possesses a known configuration was used as a
building block. The purity of QF84139 was verified by HPLC analysis
(Supplementary Fig. S6b and c).
1
2,000 × g for 15 min. The concentration of proteins was
QF84139 inhibits PE-induced hypertrophic responses in NRCMs
To evaluate the cytotoxicity of QF84139 in cardiomyocytes, a
standard CCK-8 assay was performed. Compared with the control
group, the cell viability did not differ after QF84139 treatments at
the concentrations from 0.3 μM to 100 μM for 2 days (Supple-
mentary Fig. S7). The anti-hypertrophic effect was firstly detected
using the in vitro PE-induced NRCM hypertrophic model with
various concentrations of QF84139 given 1 h before PE stimulation
as shown in the schematic of the cell model and treatments
(Fig. 1b). The mRNA expression level of ANP, brain natriuretic
peptide (BNP), and β-myosin heavy chain (β-MHC) was signifi-
cantly increased by 10 μM of PE treatment for 48 h. However, the
PE-increased gene expression was inhibited by QF84139 in a
concentration-dependent manner and was reversed to the basal
level at 3 μM of QF84139 (Fig. 1c–e). We thus used 3 μM of
QF84139 for the following experiments. The immunostaining
analysis further validated that the PE-enhanced protein level of
ANP was significantly inhibited by QF84139 (Fig. 1f). Moreover, the
PE stimulation-increased cell size was reduced by QF84139
(Fig. 1g). QF84139 alone did not affect the protein level of ANP
(Fig. 1f) and the cell size (Fig. 1g). These data demonstrate that
QF84139 attenuates hypertrophic response in NRCMs.
determined by the bicinchoninic acid assay (BCA) using the Pierce
BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA,
USA). The homogenates/lysates were stored at −80 °C. Tissue
homogenates and whole cell lysates were analyzed by standard
immunoblotting. The proteins (20 μg) were loaded and detected
by specific antibodies against ANP (1:2000, Bioworld, Dublin,
OH, USA), GAPDH (1:8000, CST, Danvers, MA, USA), PKCε
(
1:1000, Millipore, Burlington, MA, USA), phospho-PKCε (1:1000,
Abcam, Cambridge, UK), CaMKIIδ (1:1000, Abcam, Cambridge, UK),
phospho-CaMKIIδ (1:1000, Sigma, Carlsbad, CA, USA), ERK (1:1000,
CST, Danvers, MA, USA), phospho-ERK (1:1000, CST, Danvers, MA,
USA), AKT (1:1000, CST, Danvers, MA, USA), phospho-AKT (1:1000,
CST, Danvers, MA, USA), P38 (1:1000, CST, Danvers, MA, USA),
phospho-P38 (1:1000, CST, Danvers, MA, USA), AMPK (1:1000,
Abcam, ab3769), phospho-AMPK (T172, 1:1000, CST, Danvers, MA,
USA), AMPKα2 (1:1000, CST, Danvers, MA, USA), ACC (1:1000, CST,
Danvers, MA, USA), phospho-ACC (Ser79, 1:1000, CST, Danvers,
MA, USA), mTOR (1:2,000, CST, Danvers, MA, USA), phospho-mTOR
(
Ser2448, 1:2000, CST, Danvers, MA, USA), p70 ribosomal protein
S6 kinase (P70 S6K) (1:1000, CST, Danvers, MA, USA), phospho-P70
S6K (Thr389, 1:1000, CST, Danvers, MA, USA), eukaryotic translation
initiation factor 4E-binding protein 1 (4E-BP1) (1:1000, CST,
Danvers, MA, USA), and phosphor-4E-BP1 (Thr37/46, 1:1000, CST,
Danvers, MA, USA). The immunoreaction was visualized with an
enhanced ECL detection kit (PerkinElmer Life, Waltham, MA, USA)
and then exposed to film and quantified with Quantity One. The
densitometry analysis of immunoblots was conducted in a double-
blinded fashion.
QF84139 alleviates pressure overload-induced cardiac
hypertrophy in WT mice
To further evaluate the effect of QF84139 on the process of
cardiac hypertrophy, we used TAC-induced cardiac hypertrophy
model in WT adult male C57BL/6 mice. The vehicle or QF84139
was administered right after the Sham or TAC operation via
intraperitoneal injection at different doses daily for 2 weeks
(Fig. 2a). The mice under vehicle and QF84139 treatments
exhibited comparable appearance (Supplementary Fig. S8a), organ
shape and color (Supplementary Fig. S8b), and organ weight (lung
(Supplementary Fig. S10e, f), liver (Supplementary Fig. S8c, f),
spleen (Supplementary Fig. S8d, g) and kidney (Supplementary
Fig. S8e, h)). The body weight (Supplementary Fig. S9a) and heart
rate (Supplementary Fig. S9b) were comparable between the mice
with or without the QF84139 treatment either in Sham and TAC
groups. The echocardiographic analysis showed that TAC-induced
increases in LVPW;d, LVPW;s, LVS;d, LVS;s, and LV mass and
decrease in LVID;d were significantly attenuated by QF84139 in a
dose-dependent manner and these parameters were not affected
by QF84139 in the Sham mice (Fig. 2b–h) even though the LVID;s,
EF, and FS (Supplementary Fig. S10a–c) remained unchanged.
Consistently, HE and WGA staining demonstrated that the
Statistical analysis
Data were expressed as means ± SEM. Statistical analysis for
comparison between two groups was performed using two-tailed
unpaired t test. Statistical analysis for comparison in multiple
groups was performed using one-way or two-way ANOVA
followed by Dunnett’s multiple comparisons post hoc test.
Analyses were performed with GraphPad Prism v 8.0. P value <
0.05 was considered statistically significant.
RESULTS
Screening of anti-hypertrophic compounds from NPHs
We screened anti-hypertrophy compounds from a library of 24
compounds derived from insects and plants or synthesized
derivates using an in vitro PE-induced hypertrophic model in
Acta Pharmacologica Sinica (2021) 0:1 – 14

Anti-hypertrophic effects of a new compound QF84139
XX Li et al.
5
a
c
b
O
QF84139
CSA, protein and
O
N
Serum free
Phenylephrine (PE) mRNA detection
O
N
H
O
-
12
0
1
48 (h)
d
n.s.
n.s.
*
**
*
**
*
* n.s.**
*
*
**
n.s.
2
.5
.0
2
.5
2
2
.0
1
1
.5
.0
1
1
0
0
.5
.0
.5
.0
0.5
0.0
PE ( M)
0
0
10 10 10 10
PE ( M)
QF ( M)
0
0
10 10 10 10
10
QF ( M)
0
1
3
10
0
1
3
e
f
n.s.
MW(kDa)
*
**
1
7
2
.5
.0
** n.s. **
ANP
GAPDH
35
2
1
1
0
0
.5
.0
.5
.0
6
4
2
***
***
PE ( M)
QF ( M)
0
0
10 10 10 10
10
0
1
3
0
g
Control
QF
3
2
1
0
***
***
PE
PE+QF
-actinin ANP DAPI
Fig. 1 QF84139 (QF) inhibits phenylephrine (PE)-induced cardiac hypertrophic responses in vitro. Neonatal cardiac myocytes (NRCMs)
treated with various concentrations of QF or vehicle for 1 h before the cells were treated with PE (10 μM) for 48 h. a The chemical
structure of QF84139. b Schematic of PE and QF treatment. qRT-PCR analysis of mRNA expression of ANP (c), BNP (d) and β-MHC (e). n = 5
each. f Immunoblot analysis of ANP protein with or without QF (3 μM) and PE (10 μM) treatments n = 5. g Representative images (left panels)
and averaged cell size (right panels) for immunofluorescence staining with anti-α-actinin antibody (red), anti-ANP antibody (green), and DAPI
(
blue) in NRCMs treated with vehicle or QF (3 μM) in the absence or presence of PE (10 μM). Scale bar = 10 μm. n = 200 from 3 individual
experiments. **P < 0.01, ***P < 0.001. n.s., nonsignificant
enlargement of cardiomyocytes in TAC mice was significantly
inhibited by the QF84139 treatment (Fig. 3a). The quantified cross-
sectional area further confirmed the dose-dependent anti-
hypertrophic effect of QF84139 (Fig. 3b). Moreover, the enhanced
expression of hypertrophic marker genes ANP, BNP, and β-MHC in
hearts from TAC mice were suppressed by QF84139 treatment,
whereas these parameters remained unchanged in the Sham-
operated mice treated with QF84139 when compared with
Acta Pharmacologica Sinica (2021) 0:1 – 14

Anti-hypertrophic effects of a new compound QF84139
XX Li et al.
6
a
b
Echocardiography
Tissue collection
Histology
Sham/TAC
0
Vehicle/QF84139
i.p. daily
14 Days
Sham
TAC
15
QF (mg/kg)
0
90
0
5
45
90
c
Sham
TAC
d
Sham
TAC
e
Sham
TAC
*
**
***
*
**
*
*
**
*
2
.0
***
**
***
n.s*. *
*
**
*
n.s.
n.s.
2.5
2.5
2.0
1.5
1.0
0.5
0.0
*
*
*** n.s.
n.s.
2
1
1
0
.0
n.s.
1
1
.5
.0
.5
.0
.5
0
0
.5
.0
0.0
0
90
0
5
15 45 90
0
90
0
5
15 45 90
0
90 0
5 15 45 90
QF84139 (mg/kg)
QF84139 (mg/kg)
QF84139 (mg/kg)
f
Sham
TAC
g
Sham
TAC
h
Sham
TAC
*
*
*
*
**
*
*
*
**
***
**
*
n.s.***
**
n.s.
*** n.s*. *
*
*
**
n.s.
4.5
*** n.s.
200
150
2.5
2.0
1.5
1.0
0.5
0.0
n.s.
3
1
.0
.5
100
5
0
0
0.0
0
90
0
5
15 45 90
0
90
0
5
15 45 90
0
90 0
5 15 45 90
QF84139 (mg/kg)
QF84139 (mg/kg)
QF84139 (mg/kg)
Fig. 2 QF84139 (QF) dose-dependently attenuates transverse aortic constriction (TAC)-induced cardiac dysfunction in wild-type (WT)
mice at 2 weeks post-TAC or Sham surgery. a Schematic diagram of QF84139 treatment and analysis. b Representative images of
echocardiograms. c LVPW;d, left ventricle posterior wall thickness diastole. d LVPW;s, left ventricle posterior wall thickness systole. e IVS;d
interventricular septum diastolic dimension. f IVS;s, interventricular septum systolic dimension. g LVID;d left ventricular internal dimension in
diastole. h LV Mass, left ventricle mass. n = 8–11 per group. *P < 0.05, **P < 0.01, ***P < 0.001. n.s., nonsignificant
the vehicle group (Fig. 3c–e). Moreover, the TAC mice exhibited
markedly increased ratios of heart weight/tibia length (HW/TL,
Fig. 3f), heart weight/body weight (HW/BW, Supplementary
Fig. S10d), lung weight/tibia length (LW/TL, Supplementary
Fig. S10e), and lung weight/body weight (LW/BW, Supplementary
Fig. S10f) compared with the Sham-operated group. However,
these alterations were reversed by the administration of QF84139
in a dose-dependent manner and a significant inhibition was
heart size induced by TAC (Fig. 3g). Taken together, these data
demonstrate that QF84139 prevents the TAC-induced develop-
ment of ventricular hypertrophy in the WT mice.
QF84139 activates AMPK in the hypertrophic NRCMs and mice
hearts
To identify signaling pathways involved in the anti-hypertrophic
effect of QF84139, we then screened protein kinases which may
regulate cardiac hypertrophy reported previously [1, 6–8]. In
NRCMs, the phosphorylation levels of CaMKIIδ, ERK, AKT, PKCε,
and P38 were significantly increased after PE treatment, while
−
1
−1
observed at 15 mg·kg ·d of QF84139 (Fig. 3f). Furthermore,
the gross hearts and whole hearts with H&E staining confirmed
−
1
−1
that QF84139 treatment at 15 mg·kg ·d inhibited the larger
Acta Pharmacologica Sinica (2021) 0:1 – 14

Anti-hypertrophic effects of a new compound QF84139
XX Li et al.
7
a
b
Sham
TAC
15
QF (mg/kg)
H&E
0
90
0
5
45
90
WGA
Sham
TAC
c
Sham
TAC
*
d
Sham
TAC
**
**
*** ***
***
*
**
**
***
*
**
*
*
*
*
** ***
*
**
n.s.
5
4
3
00
00
00
n.s.
15
n.s.
12
8
*** n.s.
** n.s.
*
n.s.
1
0
2
1
00
00
5
0
4
0
0
0
90
0
5 15 45 90
0
90 0 5 15 45 90
QF84139 (mg/kg)
0
90 0 5 15 45 90
QF84139 (mg/kg)
QF84139 (mg/kg)
e
f
Sham
TAC
Sham
TAC
*
**
*
**
*
**
*
**
8
6
***
***
n.s. n.s.
*** n.s
*
.
1
2
*
n.s.
n.s.
8
4
4
2
0
0
0
90 0
QF84139 (mg/kg)
5
15 45 90
0
90 0 5 15 45 90
QF84139 (mg/kg)
g
Sham
TAC
QF
Veh
QF
Veh
Gross
Whole
Fig. 3 QF84139 (QF) dose-dependently attenuates TAC-induced cardiac hypertrophy in WT mice at 2 weeks post-TAC or sham surgery.
a H&E staining and WGA staining analysis of hypertrophic growth of cardiomyocytes. Scale bar = 20 μm. b Quantification of the size of
cardiomyocytes by measurement of the cross-sectional area (200 random cells/heart, n = 3 hearts per groups). qRT-PCR analysis of mRNA
expression levels of ANP (c), BNP (d), and β-MHC (e). n = 8–11. f Statistical results of heart weight/tibia length ratio (HW/TL). n = 8–11.
g Representative gross hearts (top) and whole hearts sections stained by H&E (bottom) of adult hearts from WT mice with or without vehicle
Veh) or 15 mg·kg− ·d of QF. Scale bars = 1 mm. *P < 0.05, ***P < 0.001. n.s., nonsignificant
1
−1
(
their total protein levels remained unchanged compared with
the control ones (Supplementary Fig. S11). Intriguingly, the PE-
enhanced phosphorylation levels of these protein kinases were
not significantly affected by QF84139 treatment (Supplementary
Fig. S11). Notably, the total protein and phosphorylation levels of
AMPK remained unchanged in the PE-treated NRCMs, whereas the
phosphorylation levels of AMPK were significantly increased after
QF84139 treatment in both groups with or without PE stimulation
Acta Pharmacologica Sinica (2021) 0:1 – 14

Anti-hypertrophic effects of a new compound QF84139
XX Li et al.
8
a
MW (kDa)
62
62
265
265
289
289
35
4
**n.s.
**
3
2
1
0
* n.s.
*
3
2
1
0
*** ***
p-AMPK
AMPK
p-ACC
ACC
p-mTOR
mTOR
3
2
1
0
GAPDH
b
Sham
TAC
Sham
TAC
Sham
TAC
Sham
TAC
MW (kDa)
62
62
*
*n.s. **
**
**
*n.s.
*
4
4
4
3
1
0
.5
p-AMPK
AMPK
p-ACC
ACC
p-mTOR
mTOR
GAPDH
3
2
1
0
3
2
.0
.5
.0
265
265
289
289
35
1
0
Fig. 4 QF84139 (QF) activates AMPK in NRCMs and the hearts from TAC mice. a Representative images (left panels) and averaged data
right panels) of immunoblot analysis for the total and phosphorylation level of AMPK, ACC, and mTOR in NRCMs exposed to PE with or
without QF treatment. b Immunoblot analysis of total and phosphorylation level of AMPK, ACC, and mTOR in WT mice subjected to TAC or
(
Sham surgery in the presence or absence of QF (15 mg·kg− ·d ). GAPDH was used as loading control. n = 5 each. *P < 0.05, **P < 0.01, ***P <
1
−1
0
.001. n.s., nonsignificant
(
Fig. 4a). It was further confirmed in the hearts from Sham
metformin- and QF84139-treated groups, while these anti-
hypertrophic effects were attenuated by CpC (Fig. 5b–d). Similar
changes were observed in the protein level of ANP (Fig. 5e). The
decreased cell size by QF84139 and metformin treatments under
PE stimulation was also blocked by CpC (Fig. 5f, g). These results
indicate that QF84139 attenuates PE-induced hypertrophic
response in NRCMs via activating AMPK.
and TAC mice (Fig. 4b). Acetyl-CoA carboxylase (ACC) is a direct
downstream target of AMPK [27], we found that the QF84139
treatment elevated the phosphorylation level of ACC in the
NRCMs with or without PE stimulation (Fig. 4a) and in the hearts
from Sham and TAC mice (Fig. 4b). mTOR is a downstream target
of AMPK [28]. We then examined the phosphorylation of mTOR
and observed
a significant decrease in the PE-enhanced
phosphorylation level of mTOR after QF84139 treatment in the
NRCMs, with the total protein level of mTOR was unchanged
AMPKα2 knockdown cancels the protective effects of QF84139
against PE-induced cardiomyocyte hypertrophy
(
Fig. 4a). The alterations were further confirmed in the hearts of
As mentioned above, AMPKα2 is the dominant form of AMPK in
cardiomyocytes [11]. Thus, to further validate the contribution of
AMPK, we knocked down AMPKα2 in NRCMs. The AMPKα2
mRNA level was significantly reduced by the AMPKα2 siRNA
(Supplementary Fig. S14a), while the expression of AMPKα1 was
not affected (Supplementary Fig. S14b). The downregulation of
AMPKα2 completely blocked the anti-hypertrophic effect of
QF84139 as indicated by the expression of cardiomyocyte
hypertrophic marker genes (Fig. 6a-c), enlarged cell size (Fig. 6d,
e), and ANP protein level (Fig. 6f) in the PE-stimulated NRCMs.
These results suggest the key role of AMPKα2 in the anti-
hypertrophic effect of QF84139.
Sham and TAC mice with QF84139 treatment (Fig. 4b). Moreover,
the PE- and TAC-induced phosphorylation of 4E-BP1 and S6K was
significant inhibited after the QF84139 treatment (Supplementary
Fig. S12), while the PE-induced phosphorylation of AKT was not
affected by QF84139 (Supplementary Fig. S11). Therefore,
QF84139 can activate AMPK signaling pathway in basal and
hypertrophic conditions via an AKT-independent mechanism.
Inhibition of AMPK blocks effects of QF84139 against PE-induced
cardiomyocyte hypertrophy
To verify the hypothesis that the protection of QF84139 against
cardiac hypertrophy is mediated by the activation of AMPK, we
treated NRCMs with a selective AMPK inhibitor Compound C (CpC)
and an AMPK activator metformin. Indeed, compared with the
control group, QF84139 increased the phosphorylation levels of
AMPK and ACC as did by metformin, but this effect was blocked
by CpC (Supplementary Fig. S13). Moreover, the role of AMPK in
QF84139-mediated protection against hypertrophy was deter-
mined in the PE-induced cardiomyocyte hypertrophy model
AMPKα2 depletion abolishes the anti-hypertrophic effect of
QF84139 in pressure overload-induced cardiac hypertrophy
The contribution of AMPKα2 to the cardioprotective role of
QF84139 was further determined in mice with TAC. Vehicle or
−
1
−1
QF84139 (15 mg·kg ·d ) was intraperitoneally injected once
daily for 2 weeks after Sham or TAC operation in the WT and
−
/−
AMPKα2 deficient (AMPKα2 ) littermates. There were no
−
/−
(
Fig. 5a). As expected, the up-regulation of the mRNA of ANP,
differences between WT and AMPKα2
mice in basal condition
BNP and β-MHC stimulated by PE were significantly blocked in
(Supplementary Fig. S15). Western blot analysis demonstrated
Acta Pharmacologica Sinica (2021) 0:1 – 14

Anti-hypertrophic effects of a new compound QF84139
XX Li et al.
9
a
b
Compoud C
Phenylephrine (PE)
CSA, protein and
Serum free
12
mRNA detection
-
0
1
2
48 (hours)
QF84139/MET
n.s.
n.s.
c
n.s.
n.s.
*
**
**
3
2
1
3
2
1
*
** ***
*
**
*
*
** **
*
0
PE
0
PE
-
+
-
+
+
-
+
+
-
+
-
-
-
-
-
+
-
+
+
-
+
-
+
+
-
+
-
MET
-
-
-
MET
+
-
QF
CpC
-
-
+
+
QF
CpC
-
-
+
+
-
-
+
+
-
-
+
+
d
e
-
PE
+
-
+
+
-
+
+
+
n.s.
n.s.
MET
-
-
-
-
+
-
QF
CpC
-
-
+
-
+
+
3
*
*
-
-
+
MW (kDa)
17
*
** **
*
ANP
2
1
0
GAPDH
35
n.s.
n.s.
4
3
2
1
0
*
*
*
*
*
**
*
PE
MET
QF
CpC
-
+
-
+
+
-
+
-
+
+
-
-
-
+
-
-
-
+
-
+
-
-
+
+
f
g
Control
PE+QF
PE
PE+MET
4
3
2
1
0
*
**
***
*
** *** ***
PE+CpC+MET
PE+CpC+QF
PE
-
-
-
-
+
-
+
+
-
+
-
+
+
+
-
+
-
MET
QF
CpC
-
-
+
-
-
+
+
Fig. 5 Effects of QF84139 (QF) against PE-induced myocyte hypertrophy are blocked by compound C (CpC) in vitro. The NRCMs were
treated with CpC for 1 h before addition with metformin (MET, 1 mM) or QF (3 μM), and then stimulated with PE (10 μM) for 48 h. a Schematic
diagram of different treatments and analysis. qRT-PCR analysis of the mRNA expression of ANP (b), BNP (c) and β-MHC (d), n = 6.
e Representative images (upper panels) and averaged data (lower panels) for immunoblot analysis of ANP in NRCMs. n = 5. Representative
images (f) and averaged cell size (g) for immunofluorescence staining with anti-α-actinin antibody (red), anti-ANP antibody (green), and DAPI
(blue) in NRCMs (200 random cells/heart, n = 3 hearts per group). Scale bar = 10 μm. *P < 0.05, **P < 0.01, ***P < 0.001. n.s., nonsignificant
−
/−
the depletion of AMPKα2 in the hearts of AMPKα2
(
mice
echocardiographic analysis demonstrated that QF84139 failed
to reverse TAC-induced cardiac dysfunction and remodeling
(Fig. 7d), as indicated by the parameters such as EF (Fig. 7e), FS
(Fig. 7f), LVPW;d (Fig. 7g), LVPW;s (Fig. 7h), IVS;d (Fig. 7i), IVS;s
(Fig. 7g), and LV mass (Fig. 7k). In addition, the TAC-induced
-
/-
Fig. 7a). Sustained treatment with QF84139 in the AMPKα2
littermates did not inhibit TAC-induced increases in the
mRNA level of ANP and BNP (Fig. 7b) or the protein level
−
/−
of ANP (Fig. 7c). Moreover, in the AMPKα2
mice, the
Acta Pharmacologica Sinica (2021) 0:1 – 14

Anti-hypertrophic effects of a new compound QF84139
XX Li et al.
1
0
a
*
b
*
** n.s.
***
*** n.s.
4
3
2
1
*
**
***
*
**
***
3
2
1
0
NC
0
NC
+
-
+
-
-
-
+
+
-
+
-
-
-
+
-
+
-
-
+
+
-
+
+
+
-
-
-
-
-
+
+
-
+
+
+
+
-
PE
-
+
+
+
+
-
PE
QF
-
+
-
+
+
QF
-
+
-
-
+
+
siAMPK 2
-
-
+
-
+
siAMPK 2
-
-
+
-
+
c
4
3
2
1
d
NC
NC+QF
siAMPK 2
NC+PE
*
*
n.s.
*
*
**
PE+QF+
PE+
QF+
NC+PE+QF siAMPK 2 siAMPK 2 siAMPK 2
0
NC
+
+
-
-
+
+
+
+
-
-
-
-
f
PE
-
-
-
-
+
-
+
+
+
+
-
NC
PE
QF
+
-
+
-
-
-
+
+
-
+
+
+
-
-
-
-
QF
+
-
-
+
+
+
+
+
+
-
siAMPK 2
-
+
-
+
-
+
-
-
+
+
siAMPK 2
-
-
+
-
+
MW (kDa)
17
e
ANP
*
***
**
AMPK 2
62
*
*
n.s.
3
2
1
*
**
3
5
GAPDH
4
*
*
n.s.
*
*
3
2
1
0
0
NC
+
-
+
-
+
-
-
-
+
+
-
+
+
+
-
-
-
-
PE
+
+
+
+
-
QF
-
-
+
+
siAMPK 2
-
-
+
-
+
Fig. 6 QF84139 (QF) fails to inhibit PE-induced cardiac hypertrophy in AMPKα2 knockdown NRCMs. a–c qRT-PCR analysis of mRNA
expression levels of ANP, BNP, and β-MHC. n=5. Representative cardiomyocyte images (d) and averaged cell size (e) analyzed by
immunofluorescence staining with anti-α-actinin antibody (red), anti-ANP antibody (green) and DAPI (blue) in NRCMs (200 random cells/heart,
n = 3 hearts per group). Scale bar = 20 μm. f Representative images (upper panels) and averaged data (lower panels) for immunoblot analysis
of ANP protein level in NRCMs. n = 3. NC, negative control. *P < 0.05, **P < 0.01, ***P < 0.001. n.s., nonsignificant
hypertrophic responses characterized by the enlarged heart sizes
Fig. 7l), increased ratios of HW/TL (Fig. 7m), HW/BW (Supple-
mentary Fig. S16a), LW/TL (Supplementary Fig. S16b), LW/BW
DISCUSSION
(
In the present study, we demonstrated for the first time that (i) a
synthesized natural product hybrid QF84139 inhibits the progres-
sion of PE-induced cardiomyocyte hypertrophy in vitro and the
TAC-induced myocardial hypertrophy in vivo; (ii) QF84139 does
not affect the activation of CaMKIIδ, ERK, AKT, PKCε, and P38
occurred in the hypertrophic cardiomyocytes, whereas it activates
(
(
Supplementary Fig. S16c), and augmented cross-sectional area
−/−
Fig. 7n) were still observed in the AMPKα2
mice even treated
with QF84139. These data further confirmed AMPK activation is
responsible for the anti-hypertrophic effect of QF84139.
Acta Pharmacologica Sinica (2021) 0:1 – 14

Anti-hypertrophic effects of a new compound QF84139
XX Li et al.
1
1
a
b
AMPK 2-/-
AMPK 2-/-
AMPK 2-/-
Sham
TAC
Sham
TAC
WT
*
**
Sham TAC Sham TAC
***n.s.
n.s.
***
1
1
5
0
*
**
6
4
2
0
Veh QF Veh QFVehQF Veh QF MW (kDa)
AMPK 2
GAPDH
62
35
5
0
Veh QF Veh QF
Veh QF Veh QF
c
d
AMPK 2-/-
e
AMPK 2-/-
AMPK 2-/-
Sham
TAC
*n.s.
AMPK 2-/-
Sham
TAC
Veh
QF
*
Sham
TAC
***n.s.
80
60
*
**
1
2
9
6
3
0
VehQF Veh QF
***
MW (kDa)
62
ANP
4
0
0
0
GAPDH
35
2
Veh QF Veh QF
AMPK 2-/-
Veh QF Veh QF
i
f
AMPK 2-/-
g
AMPK 2-/-
h
AMPK 2-/-
Sham
TAC
Sham
TAC
Sham
TAC
Sham
TAC
n.s.
*
**n.s.
*
**n.s.
1.5
50
40
30
20
10
0
***n.s.
***
*
*
1.5
1.5
*
*
*
**
1
0
0
.0
.5
.0
1
0
.0
.5
1.0
0.5
0.0
0
.0
Veh QF Veh QF
TAC
Veh QF Veh QF
AMPK 2-/-
Veh QF Veh QF
AMPK 2-/-
Veh QF Veh QF
l
Sham
Veh
j
k
n
QF
Veh
QF
Sham
TAC
Sham
TAC
*
**
***n.s.
n.s.
1
1
0
0
.5
.0
.5
.0
***
200
*
**
1
1
50
00
5
0
0
Veh QF Veh QF
AMPK 2-/-
Veh QF Veh QF
Sham
AMPK 2-/-
Sham TAC
m
TAC
QF
Sham
TAC
***n.s.
Veh
QF
Veh
500
400
*
**
1
5
0
***n.s.
*
**
3
2
00
00
1
5
0
100
0
Veh QF Veh QF
Veh QF Veh QF
−
/−
Fig. 7 QF84139 (QF) fails to inhibit pressure overload-induced cardiac hypertrophy in AMPKα2
mice. a Representative images of
−
/−
−1 −1
immunoblot analysis of AMPKα2 in WT and AMPKα2
mice in the presence or absence of QF (15 mg·kg ·d ). b qRT-PCR analysis of
mRNA expression levels of ANP and BNP. n = 8. c Representative images and averaged data of immunoblot analysis of ANP. n = 5.
−
/−
−1 −1
d Representative images of echocardiograms of AMPKα2
mice in the presence or absence of QF (15 mg·kg ·d ). e EF%, ejection
fraction (%). f FS%, fractional shortening (%). g LVPW;d, left ventricle posterior wall thickness diastole. h LVPW;s, left ventricle posterior
wall thickness systole. i IVS;d, interventricular septum diastolic dimension. j IVS;s, interventricular septum systolic dimension. k LV Mass,
left ventricle mass. l Representative Gross morphology (top) and whole heart sections stained by H&E staining (bottom) of adult hearts
−
/−
from AMPKα2
mice. Scale bars = 1 mm. m Statistical results of heart weight/tibia length ratio (HW/TL). n = 8. n Left panels,
representative H&E staining (top) and WGA staining (bottom) at 2 weeks post-TAC or Sham surgery. Scale bars = 20 μm; right panel,
quantification of cardiomyocyte size based on the cross-sectional area (200 random cells/heart, n = 3 hearts per group). **P < 0.01, ***P <
0
.001. n.s., nonsignificant
Acta Pharmacologica Sinica (2021) 0:1 – 14

Anti-hypertrophic effects of a new compound QF84139
XX Li et al.
1
2
the AMPK signaling pathway in cardiomyocytes and hearts; and
These data reveal that QF84139 exerts the anti-hypertrophic effect
by activating the AMPK signaling pathway and suggest that
QF84139 might act as a potent AMPK activator. The AMPK
pathway is the primary mechanism in detecting and responding
to changes in the cellular energy level [48]. It has been shown that
AMPK activation can protect the heart from ischemic injury and
pressure overload induced cardiac remodeling [49, 50], type 2
diabetes as well as other metabolic diseases and cancer [48]. The
AMPK activation represents a potential target for the treatment of
these diseases. Over the last few years, a significant development
in the identifying of small molecule AMPK activators has been
made, from which we understand the mechanism of these drugs
to activate the kinases. Exploiting a new AMPK activator means
opening up an exciting new therapeutic opportunity to the
diseases that are related to AMPK signaling pathway. Up to now,
AMPK activators can be classified as direct or indirect activators,
based on the interaction of AMPK and the molecules [51]. The
indirect activator activates AMPK by an increase of the AMP: ATP
ratio [27], which is represented by metformin. Metformin is also a
natural product-derivative originated from the plant Galega
officinalis [52]. The activation of AMPK by metformin is based on
the inhibition of complex I and thus leads to an increased AMP:
ATP ratio [53]. The AMPK direct activator is binding directly to
activate AMPK, which is represented by 5-aminoimidazole-4-
carboxamide riboside (AICAR) that mimics the 5’-AMP and
activates AMPK [51]. AICAR and metformin activate AMPK with
different efficiency and mechanisms [54, 55]. They also have
AMPK-independent functions. AICAR can inhibit P38 activation
through AMPK independent pathway [56], while metformin can
inhibit high-mobility group box 1-induced P38 activation and the
activation of ERK without altering the phosphorylation level of
AMPK [57]. In our results, QF84139 did not alter the phosphoryla-
tion level of p-P38, p-ERK or p-AKT (Supplementary Fig. S11),
indicating that QF84139 can activate AMPK without affecting the
activation of P38, ERK, and AKT. The different properties of these
molecules may offer them to meet better application demands of
patients with various conditions. Compared with the structure of
AMPK activators based on patent literatures [51], QF84139 is
different from them in the structure and represents a new type of
structure scaffold of AMPK activators. Therefore, the finding of
QF84139 may offer a new choice with clinical application
potential. By further modification, QF84139-derivatives might be
created with more power and safety. In addition, the QF84139 is a
small molecule synthesized from tradition Chinese medicine,
which would promote the modernization of Chinese traditional
medicine.
(
iii) the anti-hypertrophic effect of QF84139 is canceled by AMPK
inhibitor and knockdown of AMPKα2 in the PE-treated cardio-
myocytes and in the AMPKα2
−/−
mice. These data demonstrate
that QF84139 attenuates cardiac hypertrophy via the activation of
the AMPK signaling pathway and suggest that the activation
of the AMPK signaling pathway can effectively reverses the
cardiac hypertrophy even if the CaMKIIδ, ERK, AKT, PKCε, and
P38 signaling pathways remain activated.
Cardiac hypertrophy is an important hallmark of pressure
overload and a main cause of heart failure [1]. Thus, novel
therapeutics for the cardiac hypertrophy is viewed as a most
important goal for the treatment of heart diseases and is urgently
needed [1, 29]. Ethnomedicine knowledge based drug discovery
has been proven to be of great success in history [30, 31].
Traditional Chinese medicine (TCM) has been practiced for
thousands of years and provided a vast source of pharmaceutical
materials. Accumulating evidence indicate that traditional Chinese
herbs are beneficial for cardiac hypertrophy exemplified by Panax
notoginse [32, 33], Ligusticum chuanxiong [34], and Scutellaria
baicalensis [35]. In this regard, natural products in these herbs
responsible for cardiac hypertrophy might bring new hopes for
the patients suffering from cardiac hypertrophy. The rhizomes of
Acorus tatarinowii are commonly used to treat brain related
disorders as well as heart diseases [36–38]. We have characterized
pyrazine derivatives namely 2-(3’,4’-dihydroxy-1’-butylenyl)-
5
-(2”,3”,4’-trihydroxybutyl)-pyrazine [39] and crotonine from A.
tatarinowii. For the latter, it has also been isolated from the leaves
of Croton tiglium with pronounced analgesic activity [40]. It is
evident that these compounds possess a pyrazine core in the
structure. Although several drugs such as glipizide, pyrazinamide,
and zopiclone embrace this structure motif, we speculated that
crotonine from Acorus tatarinowii might be a useful compound for
the protection of heart. Indeed such structures like ligustrazine, a
molecule from Ligusticum chuanxiong, have been proven to be
beneficial for heart [41]. With this hypothesis, we have synthesized
several crotonine-based scaffolds (data not shown) by using an
oriented synthesis strategy inspired by the concept of powerful
NPHs [42]. Among these, we found QF84139, a small molecule
consisting of (−)-borneol, 5-(furan-2-yl)pyrazin-2(1H)-one which is
a partial structure of natural product crotonine and a simple linker,
shows potential protective roles in cardiac hypertrophy.
The TAC-induced cardiac hypertrophy model is broadly used to
study anti-hypertrophic effect [2, 3]. In our study, the changes in
LVPW;d, LVPW;s, LVS;d, LVS;s, LVID;d, and LV mass in TAC mice
without QF84139 treatment are consistent with other reports
[
3, 43]. It is noticed that in our experiments the EF and FS are not
significantly affected in WT mice after 2 weeks of TAC surgery
Supplementary Fig. S10b, c). The unchanged EF and FS are also
Another novel finding here is that activation of the AMPK
signaling pathway by QF84139 exerts powerful anti-hypertrophic
effects in hearts and cardiomyocytes without affecting signaling
pathways involved in the development of cardiac hypertrophy.
Cardiac hypertrophy is usually associated with complex spectrum
(
observed by others [2, 3, 44, 45], though the decreased EF and FS
are also reported [9]. The inconsistent observation might be
caused by the degree of hypertrophy induced by the performance
of the TAC operation. Supportively, the EF and FS were not altered
in the first 2 weeks after TAC, but they significantly decreased at 5
and 8 weeks after TAC [2], demonstrating the negative correlation
of the EF and FS with the severity of TAC model. In addition, we
2
of pathophysiological alterations, such as cell death, fibrosis, Ca
+
-handling protein dysregulation, mitochondrial dysfunction,
metabolic reprogramming, etc. The signaling pathways of ERK,
CaMKIIδ, AKT, P38, and PKCε mechanisms involved in these
responses promote maladaptive cardiac hypertrophy and ulti-
mately induce heart failure [1, 6, 8]. These signaling pathways are
also activated in the PE-stimulated cardiomyocytes in the present
study (Supplementary Fig. S11), whereas the phosphorylation level
of AMPK remains unchanged in the cardiac hypertrophic models
either induced by PE in the cardiomyocytes or by TAC in the mice.
These observations are consistent with previous reports [3, 58].
Notably, QF84139 blocks the PE-induced cardiomyocyte hyper-
trophy without affecting these signaling pathways (Fig. 1b–g and
Supplementary Fig. S11). These observations suggest that the
kinases (ERK, CaMKIIδ, AKT, P38, and PKCε) might transduce
signals to the same “molecular hub” that could be regulated by
AMPK. mTOR might be a candidate for the regulation as the
−
/−
did observe the decreased EF and FS in AMPKα2
mice 2 weeks
after TAC (Fig. 7e, f), suggesting the sensitivity of the mice hearts
is subjected to hypertrophic stimuli after the deletion of AMPKα2,
an anti-hypertrophic factor [46, 47].
One important finding in this study is that QF84139 acts as a
potent AMPK activator. We found that the AMPK signaling is
significantly activated in both basic and hypertrophic conditions
after QF84139 treatment (Fig. 4a, b), whereas the inhibition of
AMPK activation by CpC (Fig. 5) or specific siRNA (Fig. 6) can block
the anti-hypertrophic effect of QF84139 in the PE-stimulated
cardiomyocytes. Moreover, the anti-hypertrophic effect of
−/−
QF84139 is totally reversed in the AMPKα2
TAC mice (Fig. 7).
Acta Pharmacologica Sinica (2021) 0:1 – 14

Anti-hypertrophic effects of a new compound QF84139
XX Li et al.
1
3
phosphorylation of mTOR is significantly increased in the response
to PE or TAC stimulation, while its activation is totally suppressed
by the QF84139 treatment (Fig. 4a, b). mTOR is a well-known
downstream target of AMPK and can be inhibited by the
activation of AMPK through TSC complex [28]. The multiple faces
of mTOR, including regulating protein synthesis, cell growth and
proliferation, cell metabolism and stress responses [28], making it
acts as a crucial protein in connecting the upstream stimuli and
the downstream hypertrophic process [59, 60]. Supportively,
mTOR could be inhibited by metformin [61] and no additive
effects on AMPK activation are observed when use of QF84139
and metformin together (Supplementary Fig. S9), suggesting a
similar effect of QF84139 with metformin on AMPK activation.
Although mTOR-S2448 can be phosphorylated by AKT, in our
results, the phosphorylation levels of AKT induced by PE and TAC
were not affected by QF84139 (Supplementary Fig. S11). Taken
together, the activation of the AMPK signaling pathway plays a
crucial role in the attenuation of cardiac hypertrophy even if the
signaling involved in the development of cardiac hypertrophy
remain activated. The inhibition of mTOR might be responsible for
the anti-cardiac hypertrophic effect of AMPK activation by
QF84139. This possibility needs to be tested further. Moreover,
how the QF84139-mediated AMPK activation results in the
attenuation of mTOR phosphorylation at Ser2448 remains elusive
and needs to be elucidated.
In conclusion, we reported a new type of natural product
derivative QF84139 which attenuates cardiac hypertrophy induced
by hypertrophic stimuli either in vitro or in vivo via the activation of
AMPK. Moreover, we demonstrated that the activation of the AMPK
signaling pathway by the QF84139 can significantly attenuate
cardiac hypertrophy even if the hypertrophy-related signaling
pathways are activated. Our findings also emphasize the value of
natural product derivatives in the development of pharmacological
therapy for cardiac hypertrophy and QF84139 would provide a
promising lead compound for developing effective agents for the
treatment of cardiac hypertrophy.
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ACKNOWLEDGEMENTS
This work was supported by the National Natural Science Foundation of China
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(81770402; 81520108004 to H-tY), Strategic Priority Research Program of the CAS (No.
XDA16010201 to HTY), National Key R&D Program of China (2017YFA0103700 to
HTY), the research funding from Shanghai Jiao Tong University Affiliated Sixth
People’s Hospital to HTY and National Science Fund for Distinguished Young Scholars
(81525026 to YXC). We thank Prof. Yong Ji (Nanjing Medical University, Nanjing,
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AUTHOR CONTRIBUTIONS
Conception or design of the study: HTY, YXC, XXL, PZ, YY, YYZ; data collection: XXL,
YY, PZ, YJZ, JJW, JLT, SYL, YMY; data analysis and interpretation: XXL, PZ, HTY, YXC,
YYZ, YMY; drafting the manuscript: XXL, HTY, YXC, PZ, YY; financial support and final
approval for the version to be published: HTY, YXC.
2
016;22:1131–9.
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contributes to the impairment of the autophagic flux via unopposed activation of
mTORC1 signaling during myocardial ischemia/reperfusion injury. Circ Res.
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020;127:e148–e165.
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cardiac hypertrophy involving the augmentation of ubiquitination-mediated
degradation of TLR4 and TLR9. Basic Res Cardiol. 2020;115:19.
ADDITIONAL INFORMATION
Supplementary information The online version contains supplementary material
available at https://doi.org/10.1038/s41401-021-00678-5.
26. Wang J, Liu M, Wu Q, Li Q, Gao L, Jiang Y, et al. Human embryonic stem cell-
derived cardiovascular progenitors repair infarcted hearts through modulation of
macrophages via activation of signal transducer and activator of transcription 6.
Antioxid Redox Signal. 2019;31:369–86.
Competing interests: The authors declare no competing interests.
2
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