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Articles in PresS. Am J Physiol Heart Circ Physiol (November 20, 2015). doi:10.1152/ajpheart.00372.2015
Sphingosine 1-phosphate signaling contributes to cardiac inflammation, dysfunction, and
remodeling following myocardial infarction.
Running title: S1P links cardiac inflammation, dysfunction, and remodeling.
Fuyang Zhang1#; Yunlong Xia1#; Wenjuan Yan1#; Haoqiang Zhang2; Fen Zhou1; Shihao Zhao1;
Wei Wang1; Di Zhu1; Chao Xin1; Yan Lee1; Ling Zhang1; Yuan He1; Erhe Gao3; Ling Tao1*.
#Fuyang Zhang, Yunlong Xia, and Wenjun Yan contributed equally to this work.
Department of Cardiology, 2Department of Orthopedics, Xijing hospital, Fourth military medical
1
university, Xi’an, Shannxi 710032, China. 3Center for Translational Medicine, Temple university,
Philadelphia, PA, 19107, USA.
*Corresponding author:
Dr. Ling Tao
Department of Cardiology, Xijing hospital, Fourth military medical university
169 West Changle Road, Xi’an, Shannxi 710032, China.
E-mail: [email protected] and [email protected]
Tel: +86-29-84771024, +86-29-84775183
Fax: +86-29-84771024
1
Copyright © 2015 by the American Physiological Society.
Abstract:
1
Sphingosine 1-phosphate (S1P) mediates multiple pathophysiological effects in the
2
cardiovascular system. However, the role of S1P signaling in pathological cardiac remodeling
3
following myocardial infarction (MI) remains controversial. In this study, we found that cardiac
4
S1P greatly increased post-MI, accompanied with a significant upregulation of cardiac
5
sphingosine kinase-1 (SphK1) and S1P receptor 1 (S1PR1) expression. In MI-operated mice,
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inhibition of S1P production by using PF543 (the SphK1 inhibitor) ameliorated cardiac
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remodeling and dysfunction. Conversely, interruption of S1P degradation by inhibiting S1P lyase
8
augmented cardiac S1P accumulation and exacerbated cardiac remodeling and dysfunction. In the
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cardiomyocyte, S1P directly activated proinflammatory responses via a S1PR1-dependent manner.
10
Furthermore, activation of SphK1/S1P/S1PR1 signaling attributed to β1-adrenergic receptor
11
stimulation-induced proinflammatory responses in the cardiomyocyte. Administration of FTY720,
12
a functional S1PR1 antagonist, obviously blocked cardiac SphK1/S1P/S1PR1 signaling,
13
ameliorated chronic cardiac inflammation, and then improved cardiac remodeling and dysfunction
14
in vivo post-MI. In conclusion, our results demonstrate that cardiac SphK1/S1P/S1PR1 signaling
15
plays an important role in the regulation of proinflammatory responses in the cardiomyocyte and
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targeting cardiac S1P signaling is a novel therapeutic strategy to improve post-MI cardiac
17
remodeling and dysfunction.
18
Key words: sphingosine 1-phosphate, myocardial infarction, inflammation, cardiac remodeling.
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2
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New & Noteworthy:
The present study for the first time demonstrates that (1) cardiac S1P signaling is upregulated
22
within 4 weeks post-MI; (2) cardiac S1P signaling plays a critical role in the regulation of
23
proinflammatory responses in the cardiomyocyte; (3) interruption of cardiac S1P signaling
24
ameliorates post-MI cardiac inflammation, dysfunction, and remodeling.
3
25
26
Introduction
Myocardial infarction (MI) is the most common cause of heart failure (HF) in the world1.
27
Despite of significant advances achieved in HF therapies in recent decades, the incidence of HF
28
remains extremely high. It has been reported that there are 5.1 million American adults suffering
29
from HF in 2013 and the prevalence of HF will rapidly increase 25% by 20301. Therefore, there is
30
an urgent need for identification of novel preventive and therapeutic strategies to improve life and
31
survival quality of HF individuals.
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Chronic inflammation is a hallmark of the failing heart. Abundant animal and human
33
investigations have revealed that chronic cardiac inflammation contributes to post-MI left
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ventricular remodeling and HF progression7. Targeting chronic cardiac inflammation has been
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well-recognized as a highly promising and effective strategy for HF treatment. However, the
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molecular and signaling mechanisms underlying chronic cardiac inflammation are not totally
37
clear.
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Sphingosine 1-phosphate (S1P) is a bio-active sphingolipid metabolite that produced by two
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sphingosine kinases (SphKs), namely SphK1 and SphK222. Most of the biological effects of S1P
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are mediated through its ligation to five known G protein-coupled S1P receptors (S1PRs) called
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S1PR1 to S1PR53, 22. S1PR1 and S1PR3 are both expressed on the surface of the cardiomyocyte
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and cardiac SphK1/S1P/S1PR1 signaling has been emerged as an important cardioprotective
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signaling against acute myocardial ischemia/reperfusion (MI/R) injury15, 19. In the setting of MI/R
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injury, activation of cardiac SphK1/S1P/S1PR1 signaling reduces cardiomyocyte apoptosis and
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preserves cardiac function33. Our previous work also demonstrates that S1P signaling mediates
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adiponectin cardioprotection against MI/R injury via improving calcium recycling in the
4
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cardiomyocyte31. Although the role of cardiac S1P signaling in MI/R injury has been clarified,
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there is little direct evidence to verify the role of S1P signaling in chronic cardiac remodeling
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processes in response to long-term ischemic injuries, such as permanent MI. Due to rapidly
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increasing hospitalization and mortality caused by HF post-MI, it is of great significance to test
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whether cardiac S1P signaling contributes to chronic cardiac remodeling and HF progression or
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not. Therefore, we designed the present study to evaluate the role of S1P signaling in pathological
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cardiac remodeling using murine MI models, which are the most common animal models used to
54
investigate myocardial remodeling and HF.
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Previous studies suggest that activation of cardiac SphK1/S1P/S1PR1 signaling plays a
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deleterious role in chronic cardiac remodeling. The reasons are as the follows: first,
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SphK1/S1P/S1PR1 signaling is a major mediator of transforming growth factor-β
58
(TGF-β)-stimulated myofibroblast activation and collagen deposition, which contributes to cardiac
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fibrosis and remodeling13; second, activation of SphK1/S1P/S1PR1 signaling results in cardiac
60
hypertrophic responses induced by several harmful stimuli, such as pressure overload24, 26. The
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conclusion is also supported by the observation that upregulation of cardiac S1P production by
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SphK1 overexpression in rodent hearts causes progressive myocardial degeneration and interstitial
63
fibrosis29. Moreover, in post-MI HF animal models, increased vascular S1P signaling triggers
64
peripheral vascular resistance and contributes to HF development21. Taken together, these above
65
investigations conclude that interruption of cardiac SphK1/S1P/S1PR1 signaling should be a
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promising therapeutic strategy for chronic cardiac remodeling and HF development. However,
67
there are recent studies demonstrating that pharmacological or genetic activation of cardiac
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SphK1/S1P/S1PR1 signaling ameliorates post-MI cardiac remodeling and improves cardiac
5
69
performance in mice after coronary artery ligation9, 32. These results show that activation of S1PR1
70
signaling downregulates β1-adrenergic receptor (β1-AR) expression and reduces β1-AR
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hyperactivation-induced cardiomyocyte apoptosis8, 32.
72
Therefore, the aim of this study was to determine whether cardiac S1P signaling plays a
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protective or a detrimental role in pathological cardiac remodeling post-MI. In this study, we
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found that (1) pharmacological inhibition of cardiac S1P production ameliorated post-MI cardiac
75
remodeling and dysfunction; (2) activation of cardiac SphK1/S1P/S1PR1 signaling was required
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for MI or β1-AR stimulation-induced proinflammatory responses in the cardiomyocyte; and (3)
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administration of FTY720, a functional S1PR1 antagonist, downregulated cardiac
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SphK1/S1P/S1PR1 signaling, blocked chronic cardiac inflammation, and ameliorated cardiac
79
remodeling post-MI. Collectively, our data provide the evidence that targeting cardiac
80
SphK1/S1P/S1PR1 signaling is a novel and promising therapeutic strategy to prevent chronic
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cardiac inflammation and pathological ventricular remodeling following MI.
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Materials and Methods
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Mice and experimental protocol
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All of the experimental protocols in this study were approved by the Animal Care and Use
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Committee of Fourth Military Medical University (FMMU). Male C57bl6 mice (aged 10 to 12
86
weeks) were obtained from the Laboratory Animal Center of FMMU. To induce MI models, the
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mice were anesthetized by inhaling 1-2% isoflurane, and the left anterior descending coronary
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artery ligation surgery was performed as described in our previous work12. The mice were injected
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intraperitoneally with vehicle (2% DMSO in saline), FTY720 (1mg/kg body weight, Cayman
90
Chemical, US14), or PF543 (1mg/kg body weight, Merck Millipore, US35) at 24 hours after MI
6
91
operation. The drugs were administrated once daily for the following 28 days or until the animal
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died. THI (25mg/L, Avanti Polar Lipids, US4) was fed to the mice in the drinking water. The mice
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were sacrificed by cutting the carotid artery at the day 28 after MI operation and blood samples
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were collected. Hearts were washed with the ice-cold phosphate buffer (PBS). The portions of left
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ventricles (LV) were either frozen in the liquid nitrogen for biochemical analysis, or fixed in 4%
96
paraformaldehyde for histological analysis. The D-erythro-sphingosine-1-phosphate (Avanti Polar
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Lipids, US) and the specific S1PR1 antagonist W146 (Cayman Chemical, US) were commercially
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obtained.
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Echocardiography
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The echocardiography was conducted as described previously31. Briefly, after the mice were
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anesthetized by 1-2% isoflurane inhalation, the transthoracic 2-dimensional motion-mode
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echocardiography (VisualSonics, Canada) was performed. The left ventricular (LV) end-systolic
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dimension, end-diastolic dimension, and ejection fraction values were analyzed using the Vevo770
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program software (VisualSonics, Canada).
105
Histological analysis
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Ten sections (7-10 μM thick) per heart were prepared for the histological analysis. The
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masson trichrome staining and the picrosirius red staining were used to evaluate interstitial
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fibrosis and structural changes of the heart. High-magnification light micrographs were obtained
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using a light microscopy. The size of interstitial fibrosis was measured using the software
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Image-Pro plus 6.0 (Media Cybernetics, US).
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Sphingosine 1-phosphate measurement
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LV tissues were sonicated in the ice-cold 50mM Tris buffer (pH 7.4) containing 0.25M
7
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sucrose, 25mM KCl, 0.5mM EDTA, and 1% phosphatase inhibitor cocktail (Beyotime, China).
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Then LV homogenates or serum samples were centrifuged at 2,500g for 10min and S1P
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concentrations were quantified using a specific S1P enzyme-linked immunosorbent assay (ELISA)
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kit (Echelon Biosciences, US) as the manufacturer instructed.
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Sphingosine kinase activity assay
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Sphingosine kinase (SphK) activity was determined as previously described18. Briefly, the
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heart was rapidly excised from the anesthetized mouse, and was clearly washed in the ice-cold
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PBS. Then the LV tissue was isolated and was homogenized in the ice-cold isolation buffer
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containing 0.13M KCl, 20mM HEPES, 1mM EDTA, 1μg/L leupeptin, 0.25μg/L chymostatin, and
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0.25μg/L pepstatin A. Each sample was assayed for SphK activity by incubation with
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D-erythro-sphingosine (Avanti Polar Lipids, US) and [ɣ-32P] ATP in the 96-well flashplate. Then
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the product [ɣ-32P] S1P was quantified by the scintillation counting. The background values were
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determined by the negative controls which were the reaction mixture without
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D-erythro-sphingosine.
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Small interfering RNA transfection and neonatal rat ventricular cardiomyocyte culture.
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Primary ventricular cardiomyocytes were isolated from Sprague-Dawley rats (aged 1 to 2
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days) as described previously in our work31. Briefly, the isolated cardiomyocytes of the rats were
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seeded in six-well plates in the culture medium, which consisted of Dulbecco’s Modified Eagle’s
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Medium (DMEM), 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin. The specific
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rat S1PR1 small interfering RNA (siRNA) was purchased (GenePharma, China). The sequences of
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S1PR1 siRNA were 5’-GCUGCUUCAUCAUCCUAGATT-3’ and
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5’-UCUAGGAUGAUGAAGCAGCTT-3’. Cultured ventricular cardiomyocytes were transfected
8
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with scramble RNA or S1PR1 siRNA using the lipofextamine2000 transfection reagent kit (Life
136
technologies, China) according to the manufacturer's protocol. In brief, the cultured
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cardiomyocytes were serum-starved for 1 hour, and then were treated with 100nM S1PR1 siRNA
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or scrambled RNA for another 6 hours in the FBS-free culture medium. Next, 10%
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FBS-containing culture medium was added to the cells for the following 72 hours, and then the
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biochemical experiments were conducted.
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Western blot
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Total protein was extracted from the infarction boarder zone of frozen LV tissues or cultured
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cardiomyocytes. Then the protein samples were separated by SDS-PAGE and were transferred to
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nitrocellulose membranes (Merck Millipore, US). After incubation overnight with primary
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antibodies, the membranes were washed using PBS with 0.1% tween-20, and were incubated with
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secondary antibodies conjugated to horseradish peroxidase (HRP) for 1 hour at room temperature.
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The membranes were scanned using the Immobilon Western Chemiluminescent HRP Substrate
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(Merck Millipore, US) and the ChemiDOC XRS system (Bio-Rad Laboratories, US). The blots
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were quantified by the Quantity-one analysis software (Bio-Rad Laboratories, US). The primary
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antibodies were anti-SphK1, anti-SphK2, anti-S1PR1, anti-S1PR3, and anti-β-actin (1:500, Santa
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Cruz, US), anti-p-p65, anti-p65, anti-p-STAT3, and anti-STAT3 (1:1,000, Cell Signaling
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Technology, US).
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Immunohistochemistry
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Immunohistochemistry was performed by using an immunohistochemistry detection kit
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(Cowin, China) according to the manufacturer’s instructions. Mouse CD45 primary antibody was
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purchased (Abcam, US) and images were visualized by a light microscopy.
9
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Real time-PCR
Total RNA was extracted from frozen LV tissues or cultured cardiomyocytes using the RNA
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isolation kit (Tiandz, China). The cDNA was synthesized from 2μg RNA per sample using the
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PrimeScript cDNA synthesis kit (Takara Biotechnology, China). To examine the mRNA levels of
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SphK1, SphK2, S1PR1, S1PR3, TNF-α, IL-6, atrial natriuretic peptide (ANP), brain natriuretic
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peptide (BNP), β-myosin heavy chain (β-MHC), transforming growth factor-β (TGF-β),
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connective tissue growth factor (CTGF), fibronectin, collagen I, and collagen III, real time-PCR
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(RT-PCR) was performed using the UltraSYBR Mixture (CoWin Biotechnology, China) and the
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CFX96 quantitative real time-PCR detection system (Bio-Rad Laboratories, US). All the primer
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used for RT-PCR were synthesized and validated by Takara biotechnology.
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Statistical analyses
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All the data are presented as means+standard error of the mean (SEM). Student’s t-test with a
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two-tail distribution was performed to test for statistical significance for two groups. For more
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than two groups, two-way ANOVA followed by a post-hoc analysis was performed for analyzing
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two parameters. Statistical significance was analyzed by using the GraphPad Prism software and a
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P <0.05 was considered as statistically significant.
10
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Results
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Cardiac SphK1/S1P/S1PR1 signaling is upregulated post-MI.
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The previous investigation demonstrated that cardiac SphK1/S1P/S1PR1 signaling was
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downregulated in advanced HF stage9. However, the dynamic changes of circulating and cardiac
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S1P levels after MI have not been clearly illustrated before. Thus, we detected serum and cardiac
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S1P levels of sham and MI mice at different time points after coronary artery ligation. Intriguingly,
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in our experimental settings, we found that cardiac S1P levels and cardiac SphK activities
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significantly elevated at 24 hours after MI, peaking at day 7 and remaining remarkably increased
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within 4 weeks post-MI (Figure 1B and 1C), whereas there were no significant changes of serum
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S1P levels (Figure 1A). As shown in Figure 1D, cardiac SphK1 and S1PR1 expression was
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increased within 4 weeks post-MI, while cardiac SphK2 and S1PR3 expression were unchanged
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(Figure 1D). These results illustrate that cardiac SphK1/S1P/S1PR1 signaling is upregulated
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within 4 weeks post-MI. Upregulated cardiac SphK1/S1P/S1PR1 signaling should play a role in
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the pathogenesis of post-MI cardiac remodeling and HF.
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Pharmacological inhibition of SphK1 prevents cardiac dysfunction and remodeling induced
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by MI.
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In order to determine whether upregulated cardiac SphK1/S1P/S1PR1 signaling plays a
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protective role or a harmful role in post-MI cardiac remodeling, we evaluated the effects of PF543,
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a specific SphK1 inhibitor, on the mice subjected to predominant MI operation. We found that
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PF543 significantly inhibited cardiac SphK activity and reduced both serum and cardiac S1P
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levels after MI (Figure 2A to 2C). Administration of PF543 in sham-operated mice did not
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obviously affect cardiac function assessed by echocardiography (Figure 2D) and had no obvious
11
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effects on cardiac structure evidenced by histological analysis (Figure 2G). However, PF543
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treatment significantly improved post-MI cardiac dysfunction, as assessed by left ventricular
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ejection fraction (LVEF, Figure 2D). Furthermore, SphK1 inhibition reduced heart weight to body
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weight ratios (Figure 2E) and lung weight to body weight ratios (Figure 2F) and attenuated
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adverse cardiac structural changes (Figure 2G) following MI. Next, we assessed transcriptional
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changes in post-MI hearts and found that PF543 markedly reduced remodeling genes expression,
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including atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), and β-myosin heavy
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chain (β-MHC) (Figure 2H). These results indicate that attenuation of cardiac S1P accumulation,
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via inhibiting cardiac SphK1 activity, plays a beneficial role in post-MI cardiac remodeling and
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dysfunction.
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Amplification of cardiac S1P signaling by inhibiting S1P lyase activity exacerbates post-MI
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cardiac remodeling and dysfunction.
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In the mammalian tissues, S1P is irreversibly degraded by S1P lyase (SPL)6. In order to
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determine whether inhibition of SPL activity, resulting in an elevation of cardiac S1P levels,
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aggravated pathological cardiac remodeling after MI or not, we examined the effects of THI, a
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specific SPL inhibitor, in both sham and MI-operated mice. The THI-fed mice exhibited higher
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S1P levels in the blood and in myocardial tissues (Figure 3A and 3B). Our results showed that
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SPL inhibition aggravated MI-induced cardiac systolic dysfunction in terms of LVEF determined
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by echocardiography, compared with vehicle group (Figure 3C). In addition, the THI-fed mice
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showed higher heart weight to body weight ratios (Figure 3D), higher lung weight to body weight
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ratios (Figure 3E), and severely adverse cardiac structural changes at day 28 after MI (Figure 3F).
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SPL inhibition also markedly increased mRNA expression levels of remodeling genes, including
12
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ANP, BNP, and β-MHC (Figure 3G). These data demonstrate that augment of cardiac S1P
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signaling exacerbates cardiac dysfunction and remodeling after MI. Taken together with the results
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obtained from the study of PF543 treatment group, these data suggest that upregulated cardiac
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SphK1/S1P signaling plays a harmful role in post-MI cardiac remodeling and HF progression.
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S1P promotes the transcription of proinflammatory cytokines in the cardiomyocyte via its
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ligation to S1PR1.
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Chronic induction of proinflammatory cytokines in the cardiomyocyte plays a harmful role in
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post-MI pathological cardiac remodeling and HF progression10. To investigate the role of S1P in
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the regulation of proinflammatory responses in the cardiomyocyte, we treated isolated neonatal rat
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ventricular myocytes (NRVMs) with different concentrations of S1P. Direct stimulation of S1P in
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the NRVMs for 24 hours activated NF-κB (indicated by phosphorylated p65 subunit at ser311) and
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STAT3 (indicated by phosphorylated STAT3 at ser727) (Figure 4A) and promoted
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proinflammatory cytokines (TNF-ɑ and IL-6) expression in a dose-dependent manner (Figure 4B).
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These data show that S1P directly regulates the transcription of proinflammatory cytokines in the
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cardiomyocyte. S1PR1 and S1PR3 are both expressed on the cardiomyocyte surface, while S1PR1
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is the predominant S1P receptor subtype expressed in the cardiomyocyte26. The former data
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demonstrated that S1PR1 protein expression were upregulated after MI, but not S1PR3 (Figure
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1D). Given the observation that S1PR1 expression was upregulated in post-MI cardiac myocytes,
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we investigated whether the proinflammatory effects of S1P were mediated via its ligation to
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S1PR1 or not. We found that specific knockdown of S1PR1 expression by siRNA transfection in
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NRVMs obviously inhibited S1P-mediated activation of proinflammatory transcription factors
238
(NF-κB and STAT3, Figure 4D) and production of proinflammatory cytokines (TNF-α and IL-6,
13
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Figure 4E). Similarly, blocking S1PR1 signaling by W146, a specific S1PR1 antagonist, also
240
reduced S1P-induced NF-κB and STAT3 activation (Figure 4G) and proinflammatory cytokines
241
expression (Figure 4I) in the cardiomyocyte. These in vitro results illustrate that S1P directly
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activates proinflammatory responses in the cardiomyocyte in a S1PR1-dependent manner.
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Intriguingly, some S1PR1 agonists potently induce irreversible downregulation of S1PR1
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expression by promoting its ubiquitinylation and proteasomal degradation14. These S1PR1
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agonists, referred as functional S1PR1 antagonists, block S1P/S1PR1 signaling and exert effects
246
similar to those observed with other S1PR1 antagonists14. FTY720, a clinical drug has been
247
approved by US Food and Drug Administration for multiple sclerosis treatment, is one of the most
248
effective functional S1PR1 antagonists due to its high binding affinity to S1PR18. Here raises the
249
following question. Can FTY720 block the proinflammatory effects of S1P in the cardiomyocyte?
250
We found that FTY720 effectively decreased the expression of S1PR1 in the cardiomyocyte in a
251
dose-dependent manner (Figure 4F). Then FTY720 significantly inhibited S1P-mediated
252
proinflammatory transcription factors activation (Figure 4H) and proinflammatory cytokines
253
production (Figure 4J), which mimicked the effects of specific S1PR1 antagonist W146. The
254
above data support that FTY720 serves as an effective antagonist of S1PR1, instead of an agonist
255
of S1PR1, in the cardiomyocyte.
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Activation of SphK1/S1P/S1PR1 signaling contributes to β1-adrenergic receptor
257
stimulation-induced proinflammatory responses in the cardiomyocyte.
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In the development of MI-induced cardiac remodeling, sustained β1-AR stimulation
259
contributes to chronic production of proinflammatory cytokines, including TNF-α and IL-623.
260
According to the previous study, S1PR1 signaling downregulates β1-AR expression via
14
261
upregulating G protein coupled receptor 2 (GRK2) signaling in cultured cardiomyocyte 9. GRK2,
262
the primary GRK isoform expressed in the cardiomyocyte, is a very important negative regulator
263
of β1-AR signaling in the failing heart34. However, whether S1PR1 antagonism affects cardiac
264
β1-AR signaling in vivo or not has not been previously investigated. Here we found that FTY720
265
obviously downregulated cardiac S1PR1 expression, suggesting that FTY720 is an effective agent
266
to inhibit cardiac S1PR1 signaling in vivo (Figure 6D). However, we did not observe that
267
inhibition of S1PR1 signaling by FTY720 treatment had remarkable effects on cardiac β1-AR
268
expression post-MI (Figure 5A). Cardiac GRK2 expression was also not significantly changed by
269
FTY720 administration (Figure 5A). Moreover, we found that cardiac cyclic adenosine
270
monophosphate (cAMP), a critical second messenger of β1-AR signaling, was also not obviously
271
affected by FTY720 administration (Figure 5B). Collectively, these data demonstrate that S1PR1
272
signaling does not affect cardiac β1-AR signaling in vivo post-MI, which could be explained by
273
the phenomenon that S1PR1 antagonism has extremely limited effects on the expression of GRK2
274
in postischemic cardiac myocytes.
275
Having demonstrated that S1PR1 inhibition failed to alter cardiac GRK2 expression and
276
ß1-AR signaling, we further determined whether S1PR1 signaling functions as a downstream
277
molecule, mediating ß1-AR activation-induced proinflammatory responses in the cardiomyocyte.
278
After 12 hours exposure to β1-AR agonist isoprenaline (ISO), the expression of SphK1, not
279
SphK2, was markedly upregulated in the cardiomyocyte (Figure 5C), resulting in higher S1P
280
production in the culture medium (Figure 5D). After prolonged exposure to ISO, the expression
281
levels of proinflammatory cytokines (TNF-α and IL-6) were significantly upregulated in the
282
cardiomyocyte. Notably, β1-AR stimulation-induced proinflammatory responses were obviously
15
283
reduced, but not totally abrogated by pharmacological SphK1 inhibition or S1PR1 antagonism
284
(Figure 5E and 5F). These data demonstrate that SphK1/S1P/S1PR1 signaling contributes to
285
chronic β1-AR stimulation-induced proinflammatory responses in the cardiomyocyte. Given the
286
fact that sustained β1-AR activation contributes to chronic cardiac inflammation and cardiac
287
remodeling under multiple pathological conditions, our results suggest that interruption of cardiac
288
SphK1/S1P/S1PR1 signaling is a novel approach to control chronic cardiac inflammation and
289
remodeling under the conditions that cardiac β1-AR signaling is hyperactive, such as ischemic
290
heart diseases.
291
In vivo FTY720 administration interferes with cardiac SphK1/S1P/S1PR1 signaling and
292
prevents chronic proinflammatory cytokines production following MI.
293
The above results have indicated that S1PR1 plays a crucial role in the regulation of
294
proinflammatory responses in the cardiomyocyte, a major mechanism underlying post-MI cardiac
295
remodeling. We have doubted whether S1PR1 antagonism exerts beneficial effects on post-MI
296
cardiac inflammation and remodeling in vivo or not. FTY720, also known as fingolimod, is one of
297
the most effective functional S1PR1 antagonists and has been approved in the clinical use for
298
other indications. Therefore, the drug has provided us an available and safe means to interrupt
299
S1PR1 signaling in vivo. Here we found that in vivo FTY720 administration significantly reduced
300
S1PR1 expression in post-MI myocardial tissues (Figure 6D). The previous study has
301
demonstrated that FTY720 induces proteasomal degradation of SphK116. Consistent with the
302
previous observation, we also observed that FTY720 treatment obviously decreased cardiac
303
SphK1 expression, but not SphK2 (Figure 6D). Accordingly, FTY720 treatment dramatically
304
inhibited cardiac SphK activity and reversed cardiac S1P accumulation after MI (Figure 6B and
16
305
6C), whereas serum S1P levels were unchanged. These data demonstrate that FTY720
306
administration is an effective and an accessible approach to block cardiac SphK1/S1P/S1PR1
307
signaling in vivo.
308
Sustained activation of NF-κB and STAT3 in post-MI myocardium has been recognized as a
309
dominant contributor to the chronic induction of proinflammatory cytokines in the cardiomyocyte,
310
resulting in pathological cardiac remodeling and HF development10. In the study, we examined the
311
effects of FTY720 on chronic induction of proinflammatory cytokines in post-MI hearts.
312
Compared with vehicle group, FTY720 administration significantly inhibited the activation of p65
313
NF-κB subunit (Figure 6E) and decreased phosphorylated STAT3 levels (Figure 6E) in post-MI
314
cardiac myocytes. Moreover, FTY720 administration practically normalized proinflammatory
315
cytokines levels in the heart (Figure 6F). Proinflammatory cell infiltration is also an important
316
indication of chronic cardiac inflammation after MI10. It has been well-known that S1P is a critical
317
molecule in the regulation of immune cell migration3. Thus, we further tested leukocyte
318
infiltration (CD45 positive cells) in the infarction border and remote zone. As shown in Figure 6G,
319
FTY720 treatment significantly reduced proinflammatory cell infiltration post-MI, which supports
320
the conclusion that interruption of S1P/S1PR1 signaling by FTY720 treatment is an effective
321
strategy to control chronic cardiac inflammation post-MI.
322
FTY720 suppresses MI-induced cardiac remodeling and dysfunction.
323
In the following, we investigated whether inhibition of cardiac SphK1/S1P/S1PR1 signaling
324
by using FTY720 ameliorated post-MI cardiac remodeling and dysfunction or not. We found that
325
administration of FTY720 significantly preserved LVEF at day 28 after MI operation (Figure 7A
326
and 7B), whereas the infarct size was not obviously influenced by FTY720 treatment
17
327
(43.6%+4.1% versus 40.7%+3.8%). Then we examined the indexes of pathological cardiac
328
remodeling evidenced by echocardiography and histological analysis. Compared with vehicle
329
group, FTY720 administration obviously reduced heart weight to body weight ratios (Figure 7G),
330
attenuated lung edema (Figure 7H), and lowered LV end-systolic diameters (LVESD, Figure 7C)
331
and LV end-diastolic diameters (LVEDD, Figure 7D). Moreover, FTY720 treatment significantly
332
ameliorated adverse cardiac structural changes (Figure 7E), reduced interstitial fibrosis (Figure
333
7F), and decreased TGF-β family genes (Figure 7J) and collagen genes (Figure 7K) expression.
334
Expression levels of remodeling marker genes, including ANP, BNP, and β-MHC, were also
335
evidently decreased in FTY720 treatment group (Figure 7I). Taken together, these in vivo
336
experiments suggest that interruption of cardiac SphK1/S1P/S1PR1 signaling by FTY720
337
treatment is a promising therapeutic approach to improve post-MI cardiac dysfunction and
338
remodeling.
339
Discussion
340
HF is a major public health burden in the modern world1. Numerous human and animal
341
studies have provided the evidence that chronic cardiac inflammation, including the activation of
342
proinflammatory transcription factors and the production of proinflammatory cytokines,
343
contributes to pathological cardiac remodeling and HF development7, 10, 28. However, the
344
mechanisms underlying post-MI chronic cardiac inflammation are not well understood. S1P
345
signaling has been linked with proinflammatory disorders. SphK1 and S1P are crucial mediators
346
of TNF-ɑ/NF-κB signaling25. Moreover, S1P/S1PR1 signaling functions as the nodule point in the
347
IL-6-STAT3 positive feedback loop and promotes tumorigenesis17, 18. However, whether S1P
348
signaling plays a role in the regulation of proinflammatory responses in the cardiomyocyte
18
349
remains totally unknown. Here we first identify that upregulated cardiac SphK1/S1P/S1PR1
350
signaling promotes the activation of NF-κB and STAT3 in the cardiomyocyte, contributing to the
351
overproduction of proinflammatory cytokines and pathological cardiac remodeling induced by MI.
352
Chronic β1-AR stimulation is a well-recognized mechanism of chronic cardiac inflammation
353
following MI. In this study, we confirm that β1-AR stimulation upregulates SphK1 expression and
354
increases S1P production in the cardiomyocyte. Furthermore, our findings demonstrate that
355
SphK1/S1P/S1PR1 signaling mediates β1-AR stimulation-induced activation of proinflammatory
356
responses in the cardiomyocyte. Taken together, these findings for the first time provide the
357
evidence that cardiac SphK1/S1P/S1PR1 signaling plays a crucial role in the regulation of
358
proinflammatory responses in the cardiomyocyte and contributes to post-MI chronic cardiac
359
inflammation.
360
In addition to regulating proinflammatory responses, S1P signaling has been reported to
361
mediate multiple important biological functions in the cardiovascular system. For instance, it is
362
notable that S1P/S1PR1 signaling acts as a critical signaling pathway in the regulation of vascular
363
development, which is evidenced by the fact that deletion of endothelial S1PR1 leads to
364
embryonic lethality due to vascular immature in mice2. Numerous investigations have clearly
365
demonstrated that activation of S1PR1 signaling exerts anti-angiogenic effects in vitro and in
366
vivo5, 11, 20, 27. Given the fact that induction of cardiac angiogenesis protects against postischemic
367
HF, interruption of cardiac S1P/S1PR1 signaling may be a novel approach to promote cardiac
368
angiogenesis and to improve cardiac function following MI. Moreover, S1P signaling has also
369
been identified as a critical regulator of immune cell migration. In this study, we observed that
370
systemic inhibition of S1PR1 signaling by FTY720 treatment reduced cardiac leukocyte
19
371
infiltration post-MI. These immunosuppressive effects may also contribute to S1PR1
372
antagonism-mediated cardioprotection against cardiac dysfunction and remodeling post-MI. Taken
373
together, inhibition of SphK1/S1P/S1PR1 signaling improves post-MI cardiac performance and
374
remodeling probably via multiple potential mechanisms. However, based on the fact that chronic
375
cardiac inflammation is a major cause of pathological cardiac remodeling and HF,
376
S1P/S1PR1-induced cardiac inflammation contribute to the deleterious effects of S1P after MI.
377
Further studies are needed to uncover the potential mechanisms of S1P/S1PR1 signaling-induced
378
harmful effects on cardiac function and structure post-MI.
379
Another notable point is that we observed that cardiac SphK1/S1P/S1PR1 signaling was
380
upregulated within 4 weeks after MI, opposite to the changes of cardiac S1P signaling in advanced
381
HF stage9. It is well-known that major remodeling events, including fibrotic and proinflammatory
382
responses in the heart, peaks within 1 week after coronary artery ligation and early therapeutic
383
approaches against remodeling events preserve cardiac function and improve the prognosis of
384
individuals with MI28, 30. Therefore, it is of great importance to clarify the dynamic changes of
385
cardiac SphK1/S1P/S1PR1 signaling and to identify whether cardiac S1P signaling plays a
386
protective or a detrimental role in a relatively early period of time post-MI. In the settings of our
387
study, we observed that cardiac SphK1/S1P/S1PR1 signaling was upregulated within 4 weeks
388
after MI and interruption of cardiac S1P signaling benefited the infarcted heart. According to
389
previous studies, SphK1-derived S1P promotes S1PR1 expression, forming a SphK1/S1P/S1PR1
390
amplification feedback loop17. Here we found that, in postischemic myocardial tissues, the
391
SphK1/S1P/S1PR1 positive feedback loop was activated within 4 weeks post-MI, which caused
392
cardiac S1P accumulation. FTY720 is a functional S1PR1 antagonist which is able to interrupt the
20
393
SphK1/S1P/S1PR1 feedback loop18. Our data showed that, by using FTY720, cardiac
394
SphK1/S1P/S1PR1 feedback loop was interrupted and cardiac S1P accumulation was reversed,
395
contributing to S1PR1 antagonism-mediated cardioprotection against post-MI cardiac dysfunction
396
and remodeling. Taken together, these data demonstrate that interruption of cardiac
397
SphK1/S1P/S1PR1 feedback loop is a novel therapeutic strategy for pathological cardiac
398
remodeling and dysfunction during an early period of time post-MI. Our data also suggest that
399
FTY720, a clinical drug for other indications, is a potential therapeutic candidate for post-MI HF
400
treatment.
401
The limitation of the present study is that we did not observe the effects of SphK1 inhibition
402
or S1PR1 antagonism on pathological cardiac remodeling and dysfunction during the period
403
beyond 4 weeks after MI. Thus, the role of cardiac SphK1/S1P/S1PR1 signaling in pathological
404
cardiac remodeling and HF development needs to be systemically investigated during a longer
405
period of time post-MI in additional studies. Furthermore, we focused on S1PR1, which represents
406
S1PR with the highest expression in the cardiomyocyte. However, S1PR3 is another S1PR
407
subtype expressed in the cardiomyocyte and the role of S1PR3 in the pathogenesis of post-MI HF
408
remains largely unknown. More studies are deeply needed to further investigate the relevant role
409
of S1PR3 in post-MI cardiac dysfunction and remodeling.
410
In conclusion, the present study provides the evidence demonstrating that: (1) cardiac
411
SphK1/S1P/S1PR1 signaling increases within 4 weeks after MI; (2) cardiac SphK1/S1P/S1PR1
412
signaling contributes to proinflammatory responses in the cardiomyocyte; (3) Inhibition of cardiac
413
S1P signaling ameliorates chronic cardiac inflammation, remodeling, and dysfunction post-MI.
414
Therefore, targeting cardiac SphK1/S1P/S1PR1 signaling is a promising and an effective
21
415
therapeutic strategy for post-MI HF treatment.
416
Acknowledgement
417
We thank Dr. Jie Liang of the Department of Gastroenterology, State Key Laboratory of
418
Cancer Biology, Fourth Military Medical University, for her helpful discussion and technical
419
support.
420
Source of funding
421
This work was supported by Program for Chinese National Science Fund for Distinguished
422
Young Scholars (Grant No.81225001), National Key Basic Research Program of China (973
423
Program, 2013CB531204), New Century Excellent Talents in University (Grant
424
No.NCET-11-0870), Chinese National Science Funds(Grants No. 81070676, 81170186, and
425
81441015), Innovation Team Development Grant by China Department of Education
426
(2010CXTD01) and Major Science, Technology Projects of China “Significant New Drug
427
Development” (Grant No. 2012ZX09J12108-06B) and Xijing Hospital Supporting Program
428
(Grant No. XJZT14M01).
429
Disclosures
430
None.
431
List of abbreviations:
432
ANP, atrial natriuretic peptide;
433
BNP, brain natriuretic peptide;
434
CTGF, collective tissue growth factor;
435
GRK2, G protein coupled receptor kinase 2;
436
LVEF, left ventricular ejection fraction;
22
437
LVEDD, left ventricular end-diastolic diameter;
438
LVESD, left ventricular end-systolic diameter;
439
PCR, polymerase chain reaction;
440
S1P, sphingosine 1-phosphate;
441
SphK1, sphingosine kinase 1;
442
SphK2, sphingosine kinase 2;
443
S1PR1, S1P receptor 1;
444
S1PR3, S1P receptor 3;
445
TGF-β, Transforming growth factor-β.
23
446
References
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Figure legends
575
Figure 1. Cardiac SphK1/S1P/S1PR1 signaling is upregulated post-MI.
576
Animals were subjected to MI by left coronary artery ligation. Serum S1P (A), cardiac S1P (B),
577
and cardiac SphK activity (C) were determined at different time points after the operation. The
578
expression of SphK1, SphK2, S1PR1, and S1PR3 were determined by western blot at different
579
time points after MI (D). Data are presented as mean+SEM, n=8 per group. #P<0.05 vs. Sham.
580
Figure 2. SphK1 inhibition ameliorates cardiac dysfunction and remodeling induced by MI.
581
Animals were subjected to MI operation. The mice were randomized to receive vehicle or PF543
582
(1mg/kg body weight i.p.) for the following observation period. Cardiac SphK activity (A) was
583
determined at 4 weeks post-MI. Serum (B) and cardiac (C) S1P levels were determined by ELISA.
584
Representative M-mode echocardiography images were presented and left ventricular ejection
585
fractions (LVEF) were calculated (D). Heart weight to body weight ratios (E) and lung weight to
586
body weight ratios (F) were calculated. Representative masson trichrome-stained images were
587
obtained from heart sections (G). Remodeling marker genes ANP, BNP, and β-MHC mRNA
588
expression levels were determined by RT-PCR (H). Data are presented as mean+SEM, n=8 per
589
group. ^P<0.05 and #P<0.05 vs. Sham, *P<0.05 vs. MI.
590
Figure 3. SPL inhibition exacerbates post-MI cardiac dysfunction and remodeling.
591
Animals were subjected to MI operation. The mice were randomized to receive vehicle or THI
592
(25mg/L in drinking water) for the following 4 weeks post-MI. Serum (A) and cardiac (B) S1P
593
levels were determined by ELISA. Left ventricular ejection fractions (LVEF) were calculated by
594
echocardiography (C). Heart weight to body weight ratios (D) and lung weight to body weight
595
ratios (E) were calculated. Representative images of masson trichrome stained-heart sections were
27
596
presented (F). The expression of remodeling related genes were determined by RT-PCR (F). Data
597
are presented as mean+SEM, n=8 per group. ^P<0.05 and #P<0.05 vs. Sham. *P<0.05 vs. MI
598
group.
599
Figure 4. S1P directly stimulates proinflammatory transcription factors activation and
600
proinflammatory cytokines production in the cardiomyocyte via its ligation to S1PR1.
601
Cardiomyocytes were isolated as described in the methods. After treatment with different
602
concentrations of S1P for 24 hours, the phosphorylation of p65 NF-κB and STAT3 were
603
determined by western blot (A) and the expression of proinflammatory cytokines were determined
604
by RT-PCR (B). After transfection with S1PR1 siRNA, the expression of S1PR1 were determined
605
(C). After S1PR1 knockdown (S1PR1 KD) by siRNA transfection, cardiomyocytes were treated
606
with vehicle or S1P (1μM) for 24 hours. The phosphorylation of p65 NF-κB and STAT3 were
607
determined by western blot (D) and the expression of TNF-α and IL-6 were determined by
608
RT-PCR (E). After treated with different concentrations of FTY720 for 12 hours, the S1PR1
609
expression in the cardiomyocyte was determined (F). After treated with vehicle, S1P (1μM), S1P
610
(1μM) plus FTY720 (10μM), and S1P (1μM) plus W146 (10μM) for 12 hours, the
611
phosphorylation of p65 NF-κB and STAT3 were determined by western blot (G and H) and the
612
expression of TNF-α and IL-6 were determined by RT-PCR (I and J). Results are presented as
613
mean+SEM, n=6 per group. #P<0.05 vs. Control. &P<0.05 vs. S1P treatment group.
614
Figure 5. ISO-stimulated proinflammatory cytokines production involves the enlargement of
615
SphK1/S1P/S1PR1 signaling in the cardiomyocyte.
616
Cardiac β1-AR and GRK2 expression levels were determined (A) and cardiac cAMP levels were
617
assayed (B) in MI and MI+FTY720 group at 4 weeks post-MI. Cardiomyocytes were isolated and
28
618
cultured as methods described. Cells were treated with isoprenaline (ISO, 10μM) for 12 hours.
619
The expression of SphK1 and SphK2 were determined (C). Medium S1P levels were quantified by
620
ELISA (D). ISO-stimulated cardiomyocytes were treated with vehicle, PF543 (10μM), and W146
621
(10μM) for 12 hours. The mRNA expression levels of TNF-ɑ and IL-6 in the cardiomyocyte were
622
determined by RT-PCR (E and F). Data are presented as mean+SEM, n=6 per group. #P<0.05 vs.
623
Control or Sham group. &P<0.05 vs. ISO group.
624
Figure 6. In vivo administration of FTY720 blocks cardiac SphK1/S1P/S1PR1 signaling and
625
inhibits activation of proinflammatory responses after MI.
626
Mice were subjected to MI operation as methods described. Animals were randomized to receive
627
vehicle or FTY720 for the following 4 weeks post-MI. Serum S1P (A), cardiac S1P (B), and
628
cardiac SphK activity (C) were determined. The SphK1, SphK2, and S1PR1 expression were
629
determined by western blot (D). The phosphorylation levels of p65 NF-κB and STAT3 were
630
determined (E). Cardiac TNF-ɑ and cardiac IL-6 were quantified by ELISA (F). Cardiac leukocyte
631
infiltration was determined by CD45 immunohistochemistry staining (G). Data are presented as
632
mean+SEM, n=12-15 per group. #P<0.05 vs. Sham. *P<0.05 vs. MI.
633
Figure 7. FTY720 treatment improves MI-induced cardiac dysfunction and remodeling.
634
Mice were subjected to MI via left coronary artery ligation. Mice were randomized to receive
635
vehicle or FTY720 treatment for 4 weeks post-MI. Representative images of echocardiography
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were shown (A). Left ventricular ejection fraction (LVEF, B), left ventricular end-systolic
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diameter (LVESD, C), and left ventricular end-diastolic diameters (LVEDD, D) were determined
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by echocardiography at day 28 post-MI. Representative images of masson trichrome-stained and
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picrosirius red-stained heart sections were presented (E). The size of interstitial fibrosis was
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calculated (F). Heart weight to body weight ratios (G) and lung weight to body weight ratios (H)
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were calculated. Relative expression levels of remodeling-related genes (I), TGF-β family genes
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(J), and collagen genes (K) were determined by RT-PCR. Data are presented as mean+SEM,
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n=12-15 per group. #p<0.05 vs. Sham. *p<0.05 vs. MI group.
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