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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, 6 inhibition of S1P production by using PF543 (the SphK1 inhibitor) ameliorated cardiac 7 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 9 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 16 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. 19 2 20 21 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. 32 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 34 ventricular remodeling and HF progression7. Targeting chronic cardiac inflammation has been 35 well-recognized as a highly promising and effective strategy for HF treatment. However, the 36 molecular and signaling mechanisms underlying chronic cardiac inflammation are not totally 37 clear. 38 Sphingosine 1-phosphate (S1P) is a bio-active sphingolipid metabolite that produced by two 39 sphingosine kinases (SphKs), namely SphK1 and SphK222. Most of the biological effects of S1P 40 are mediated through its ligation to five known G protein-coupled S1P receptors (S1PRs) called 41 S1PR1 to S1PR53, 22. S1PR1 and S1PR3 are both expressed on the surface of the cardiomyocyte 42 and cardiac SphK1/S1P/S1PR1 signaling has been emerged as an important cardioprotective 43 signaling against acute myocardial ischemia/reperfusion (MI/R) injury15, 19. In the setting of MI/R 44 injury, activation of cardiac SphK1/S1P/S1PR1 signaling reduces cardiomyocyte apoptosis and 45 preserves cardiac function33. Our previous work also demonstrates that S1P signaling mediates 46 adiponectin cardioprotection against MI/R injury via improving calcium recycling in the 4 47 cardiomyocyte31. Although the role of cardiac S1P signaling in MI/R injury has been clarified, 48 there is little direct evidence to verify the role of S1P signaling in chronic cardiac remodeling 49 processes in response to long-term ischemic injuries, such as permanent MI. Due to rapidly 50 increasing hospitalization and mortality caused by HF post-MI, it is of great significance to test 51 whether cardiac S1P signaling contributes to chronic cardiac remodeling and HF progression or 52 not. Therefore, we designed the present study to evaluate the role of S1P signaling in pathological 53 cardiac remodeling using murine MI models, which are the most common animal models used to 54 investigate myocardial remodeling and HF. 55 Previous studies suggest that activation of cardiac SphK1/S1P/S1PR1 signaling plays a 56 deleterious role in chronic cardiac remodeling. The reasons are as the follows: first, 57 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 59 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 61 conclusion is also supported by the observation that upregulation of cardiac S1P production by 62 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 66 promising therapeutic strategy for chronic cardiac remodeling and HF development. However, 67 there are recent studies demonstrating that pharmacological or genetic activation of cardiac 68 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 71 hyperactivation-induced cardiomyocyte apoptosis8, 32. 72 Therefore, the aim of this study was to determine whether cardiac S1P signaling plays a 73 protective or a detrimental role in pathological cardiac remodeling post-MI. In this study, we 74 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 76 for MI or β1-AR stimulation-induced proinflammatory responses in the cardiomyocyte; and (3) 77 administration of FTY720, a functional S1PR1 antagonist, downregulated cardiac 78 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 81 cardiac inflammation and pathological ventricular remodeling following MI. 82 Materials and Methods 83 Mice and experimental protocol 84 All of the experimental protocols in this study were approved by the Animal Care and Use 85 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 87 mice were anesthetized by inhaling 1-2% isoflurane, and the left anterior descending coronary 88 artery ligation surgery was performed as described in our previous work12. The mice were injected 89 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 92 died. THI (25mg/L, Avanti Polar Lipids, US4) was fed to the mice in the drinking water. The mice 93 were sacrificed by cutting the carotid artery at the day 28 after MI operation and blood samples 94 were collected. Hearts were washed with the ice-cold phosphate buffer (PBS). The portions of left 95 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 97 Lipids, US) and the specific S1PR1 antagonist W146 (Cayman Chemical, US) were commercially 98 obtained. 99 Echocardiography 100 The echocardiography was conducted as described previously31. Briefly, after the mice were 101 anesthetized by 1-2% isoflurane inhalation, the transthoracic 2-dimensional motion-mode 102 echocardiography (VisualSonics, Canada) was performed. The left ventricular (LV) end-systolic 103 dimension, end-diastolic dimension, and ejection fraction values were analyzed using the Vevo770 104 program software (VisualSonics, Canada). 105 Histological analysis 106 Ten sections (7-10 μM thick) per heart were prepared for the histological analysis. The 107 masson trichrome staining and the picrosirius red staining were used to evaluate interstitial 108 fibrosis and structural changes of the heart. High-magnification light micrographs were obtained 109 using a light microscopy. The size of interstitial fibrosis was measured using the software 110 Image-Pro plus 6.0 (Media Cybernetics, US). 111 Sphingosine 1-phosphate measurement 112 LV tissues were sonicated in the ice-cold 50mM Tris buffer (pH 7.4) containing 0.25M 7 113 sucrose, 25mM KCl, 0.5mM EDTA, and 1% phosphatase inhibitor cocktail (Beyotime, China). 114 Then LV homogenates or serum samples were centrifuged at 2,500g for 10min and S1P 115 concentrations were quantified using a specific S1P enzyme-linked immunosorbent assay (ELISA) 116 kit (Echelon Biosciences, US) as the manufacturer instructed. 117 Sphingosine kinase activity assay 118 Sphingosine kinase (SphK) activity was determined as previously described18. Briefly, the 119 heart was rapidly excised from the anesthetized mouse, and was clearly washed in the ice-cold 120 PBS. Then the LV tissue was isolated and was homogenized in the ice-cold isolation buffer 121 containing 0.13M KCl, 20mM HEPES, 1mM EDTA, 1μg/L leupeptin, 0.25μg/L chymostatin, and 122 0.25μg/L pepstatin A. Each sample was assayed for SphK activity by incubation with 123 D-erythro-sphingosine (Avanti Polar Lipids, US) and [ɣ-32P] ATP in the 96-well flashplate. Then 124 the product [ɣ-32P] S1P was quantified by the scintillation counting. The background values were 125 determined by the negative controls which were the reaction mixture without 126 D-erythro-sphingosine. 127 Small interfering RNA transfection and neonatal rat ventricular cardiomyocyte culture. 128 Primary ventricular cardiomyocytes were isolated from Sprague-Dawley rats (aged 1 to 2 129 days) as described previously in our work31. Briefly, the isolated cardiomyocytes of the rats were 130 seeded in six-well plates in the culture medium, which consisted of Dulbecco’s Modified Eagle’s 131 Medium (DMEM), 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin. The specific 132 rat S1PR1 small interfering RNA (siRNA) was purchased (GenePharma, China). The sequences of 133 S1PR1 siRNA were 5’-GCUGCUUCAUCAUCCUAGATT-3’ and 134 5’-UCUAGGAUGAUGAAGCAGCTT-3’. Cultured ventricular cardiomyocytes were transfected 8 135 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 137 cardiomyocytes were serum-starved for 1 hour, and then were treated with 100nM S1PR1 siRNA 138 or scrambled RNA for another 6 hours in the FBS-free culture medium. Next, 10% 139 FBS-containing culture medium was added to the cells for the following 72 hours, and then the 140 biochemical experiments were conducted. 141 Western blot 142 Total protein was extracted from the infarction boarder zone of frozen LV tissues or cultured 143 cardiomyocytes. Then the protein samples were separated by SDS-PAGE and were transferred to 144 nitrocellulose membranes (Merck Millipore, US). After incubation overnight with primary 145 antibodies, the membranes were washed using PBS with 0.1% tween-20, and were incubated with 146 secondary antibodies conjugated to horseradish peroxidase (HRP) for 1 hour at room temperature. 147 The membranes were scanned using the Immobilon Western Chemiluminescent HRP Substrate 148 (Merck Millipore, US) and the ChemiDOC XRS system (Bio-Rad Laboratories, US). The blots 149 were quantified by the Quantity-one analysis software (Bio-Rad Laboratories, US). The primary 150 antibodies were anti-SphK1, anti-SphK2, anti-S1PR1, anti-S1PR3, and anti-β-actin (1:500, Santa 151 Cruz, US), anti-p-p65, anti-p65, anti-p-STAT3, and anti-STAT3 (1:1,000, Cell Signaling 152 Technology, US). 153 Immunohistochemistry 154 Immunohistochemistry was performed by using an immunohistochemistry detection kit 155 (Cowin, China) according to the manufacturer’s instructions. Mouse CD45 primary antibody was 156 purchased (Abcam, US) and images were visualized by a light microscopy. 9 157 158 Real time-PCR Total RNA was extracted from frozen LV tissues or cultured cardiomyocytes using the RNA 159 isolation kit (Tiandz, China). The cDNA was synthesized from 2μg RNA per sample using the 160 PrimeScript cDNA synthesis kit (Takara Biotechnology, China). To examine the mRNA levels of 161 SphK1, SphK2, S1PR1, S1PR3, TNF-α, IL-6, atrial natriuretic peptide (ANP), brain natriuretic 162 peptide (BNP), β-myosin heavy chain (β-MHC), transforming growth factor-β (TGF-β), 163 connective tissue growth factor (CTGF), fibronectin, collagen I, and collagen III, real time-PCR 164 (RT-PCR) was performed using the UltraSYBR Mixture (CoWin Biotechnology, China) and the 165 CFX96 quantitative real time-PCR detection system (Bio-Rad Laboratories, US). All the primer 166 used for RT-PCR were synthesized and validated by Takara biotechnology. 167 Statistical analyses 168 All the data are presented as means+standard error of the mean (SEM). Student’s t-test with a 169 two-tail distribution was performed to test for statistical significance for two groups. For more 170 than two groups, two-way ANOVA followed by a post-hoc analysis was performed for analyzing 171 two parameters. Statistical significance was analyzed by using the GraphPad Prism software and a 172 P <0.05 was considered as statistically significant. 10 173 Results 174 Cardiac SphK1/S1P/S1PR1 signaling is upregulated post-MI. 175 The previous investigation demonstrated that cardiac SphK1/S1P/S1PR1 signaling was 176 downregulated in advanced HF stage9. However, the dynamic changes of circulating and cardiac 177 S1P levels after MI have not been clearly illustrated before. Thus, we detected serum and cardiac 178 S1P levels of sham and MI mice at different time points after coronary artery ligation. Intriguingly, 179 in our experimental settings, we found that cardiac S1P levels and cardiac SphK activities 180 significantly elevated at 24 hours after MI, peaking at day 7 and remaining remarkably increased 181 within 4 weeks post-MI (Figure 1B and 1C), whereas there were no significant changes of serum 182 S1P levels (Figure 1A). As shown in Figure 1D, cardiac SphK1 and S1PR1 expression was 183 increased within 4 weeks post-MI, while cardiac SphK2 and S1PR3 expression were unchanged 184 (Figure 1D). These results illustrate that cardiac SphK1/S1P/S1PR1 signaling is upregulated 185 within 4 weeks post-MI. Upregulated cardiac SphK1/S1P/S1PR1 signaling should play a role in 186 the pathogenesis of post-MI cardiac remodeling and HF. 187 Pharmacological inhibition of SphK1 prevents cardiac dysfunction and remodeling induced 188 by MI. 189 In order to determine whether upregulated cardiac SphK1/S1P/S1PR1 signaling plays a 190 protective role or a harmful role in post-MI cardiac remodeling, we evaluated the effects of PF543, 191 a specific SphK1 inhibitor, on the mice subjected to predominant MI operation. We found that 192 PF543 significantly inhibited cardiac SphK activity and reduced both serum and cardiac S1P 193 levels after MI (Figure 2A to 2C). Administration of PF543 in sham-operated mice did not 194 obviously affect cardiac function assessed by echocardiography (Figure 2D) and had no obvious 11 195 effects on cardiac structure evidenced by histological analysis (Figure 2G). However, PF543 196 treatment significantly improved post-MI cardiac dysfunction, as assessed by left ventricular 197 ejection fraction (LVEF, Figure 2D). Furthermore, SphK1 inhibition reduced heart weight to body 198 weight ratios (Figure 2E) and lung weight to body weight ratios (Figure 2F) and attenuated 199 adverse cardiac structural changes (Figure 2G) following MI. Next, we assessed transcriptional 200 changes in post-MI hearts and found that PF543 markedly reduced remodeling genes expression, 201 including atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), and β-myosin heavy 202 chain (β-MHC) (Figure 2H). These results indicate that attenuation of cardiac S1P accumulation, 203 via inhibiting cardiac SphK1 activity, plays a beneficial role in post-MI cardiac remodeling and 204 dysfunction. 205 Amplification of cardiac S1P signaling by inhibiting S1P lyase activity exacerbates post-MI 206 cardiac remodeling and dysfunction. 207 In the mammalian tissues, S1P is irreversibly degraded by S1P lyase (SPL)6. In order to 208 determine whether inhibition of SPL activity, resulting in an elevation of cardiac S1P levels, 209 aggravated pathological cardiac remodeling after MI or not, we examined the effects of THI, a 210 specific SPL inhibitor, in both sham and MI-operated mice. The THI-fed mice exhibited higher 211 S1P levels in the blood and in myocardial tissues (Figure 3A and 3B). Our results showed that 212 SPL inhibition aggravated MI-induced cardiac systolic dysfunction in terms of LVEF determined 213 by echocardiography, compared with vehicle group (Figure 3C). In addition, the THI-fed mice 214 showed higher heart weight to body weight ratios (Figure 3D), higher lung weight to body weight 215 ratios (Figure 3E), and severely adverse cardiac structural changes at day 28 after MI (Figure 3F). 216 SPL inhibition also markedly increased mRNA expression levels of remodeling genes, including 12 217 ANP, BNP, and β-MHC (Figure 3G). These data demonstrate that augment of cardiac S1P 218 signaling exacerbates cardiac dysfunction and remodeling after MI. Taken together with the results 219 obtained from the study of PF543 treatment group, these data suggest that upregulated cardiac 220 SphK1/S1P signaling plays a harmful role in post-MI cardiac remodeling and HF progression. 221 S1P promotes the transcription of proinflammatory cytokines in the cardiomyocyte via its 222 ligation to S1PR1. 223 Chronic induction of proinflammatory cytokines in the cardiomyocyte plays a harmful role in 224 post-MI pathological cardiac remodeling and HF progression10. To investigate the role of S1P in 225 the regulation of proinflammatory responses in the cardiomyocyte, we treated isolated neonatal rat 226 ventricular myocytes (NRVMs) with different concentrations of S1P. Direct stimulation of S1P in 227 the NRVMs for 24 hours activated NF-κB (indicated by phosphorylated p65 subunit at ser311) and 228 STAT3 (indicated by phosphorylated STAT3 at ser727) (Figure 4A) and promoted 229 proinflammatory cytokines (TNF-ɑ and IL-6) expression in a dose-dependent manner (Figure 4B). 230 These data show that S1P directly regulates the transcription of proinflammatory cytokines in the 231 cardiomyocyte. S1PR1 and S1PR3 are both expressed on the cardiomyocyte surface, while S1PR1 232 is the predominant S1P receptor subtype expressed in the cardiomyocyte26. The former data 233 demonstrated that S1PR1 protein expression were upregulated after MI, but not S1PR3 (Figure 234 1D). Given the observation that S1PR1 expression was upregulated in post-MI cardiac myocytes, 235 we investigated whether the proinflammatory effects of S1P were mediated via its ligation to 236 S1PR1 or not. We found that specific knockdown of S1PR1 expression by siRNA transfection in 237 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, 13 239 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 242 activates proinflammatory responses in the cardiomyocyte in a S1PR1-dependent manner. 243 Intriguingly, some S1PR1 agonists potently induce irreversible downregulation of S1PR1 244 expression by promoting its ubiquitinylation and proteasomal degradation14. These S1PR1 245 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. 256 Activation of SphK1/S1P/S1PR1 signaling contributes to β1-adrenergic receptor 257 stimulation-induced proinflammatory responses in the cardiomyocyte. 258 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 447 448 449 450 451 452 453 454 455 456 457 458 459 460 461 462 463 464 465 466 467 468 469 470 471 472 473 474 475 476 477 478 479 480 481 482 483 484 485 486 487 488 1. Alan S. Go, Dariush Mozaffarian, Véronique L. Roger, Emelia J. Benjamin, Jarett D. Berry, William B. Borden, Dawn M. Bravata, Shifan Dai, Earl S. Ford, Caroline S. Fox, Sheila Franco, Heather J. Fullerton, Cathleen Gillespie, Susan M. Hailpern, John A. Heit, Virginia J. Howard, Mark D. Huffman, Brett M. Kissela, Steven J. Kittner, Daniel T. Lackland, Judith H. Lichtman, Lynda D. Lisabeth, David Magid, Gregory M. Marcus, Ariane Marelli, David B. Matchar, Darren K. McGuire, Emile R. Mohler, Claudia S. Moy, Michael E. Mussolino, Graham Nichol, Nina P. Paynter, Pamela J. Schreiner, Paul D. Sorlie, Joel Stein, Tanya N. Turan, Salim S.Virani, Nathan D. Wong, Daniel Woo and Melanie B. Turner. Heart Disease and Stroke Statistics -- 2013 Update: A Report From the American Heart Association. Circulation. 2013 Jan 1;127(1):e6-e245. 2. Allende ML, Yamashita T, Proia RL. G-protein-coupled receptor S1P1 acts within endothelial cells to regulate vascular maturation. Blood 2003 Nov 15;102(10):3665-7. 3. Alvarez SE1, Milstien S, Spiegel S. Autocrine and paracrine roles of sphingosine-1-phosphate. Trends Endocrinol Metab. 2007 Oct;18(8):300-7. 4. Bandhuvula P, Honbo N, Wang GY, Jin ZQ, Fyrst H, Zhang M, Borowsky AD, Dillard L, Karliner JS, Saba JD. S1P lyase: a novel therapeutic target for ischemia-reperfusion injury of the heart. Am J Physiol Heart Circ Physiol 2011 May;300(5):H1753-61. 5. Ben Shoham A, Malkinson G, Krief S, Shwartz Y, Ely Y, Ferrara N, Yaniv K, Zelzer E. S1P1 inhibits sprouting angiogenesis during vascular development. Development. 2012 Oct;139(20):3859-69. Epub 2012 Sep 5. 6. Borowsky AD, Bandhuvula P, Kumar A, Yoshinaga Y, Nefedov M, Fong LG, Zhang M, Baridon B, Dillard L, de Jong P, Young SG, West DB, Saba JD. Sphingosine-1-phosphate lyase expression in embryonic and adult murine tissues. J Lipid Res 2012;53(9):1920-31. 7. Bouras G, Giannopoulos G, Hatzis G, Alexopoulos D, Leventopoulos G, Deftereos S. Inflammation and chronic heart failure: from biomarkers to novel anti-inflammatory therapeutic strategies. Med Chem. 2014;10(7):682-99. 8. Brinkmann V, Billich A, Baumruker T, Heining P, Schmouder R, Francis G, Aradhye S, Burtin P. Fingolimod (FTY720): discovery and development of an oral drug to treat multiple sclerosis. Nat Rev Drug Discov 2010;9(11):883-97. 9. Cannavo A, Rengo G, Liccardo D, Pagano G, Zincarelli C, De Angelis MC, Puglia R, Di Pietro E, Rabinowitz JE, Barone MV, Cirillo P, Trimarco B, Palmer TM, Ferrara N, Koch WJ, Leosco D, Rapacciuolo A. β1-adrenergic receptor and sphingosine-1-phosphate receptor 1 (S1PR1) reciprocal downregulation influences cardiac hypertrophic response and progression to heart failure: protective role of S1PR1 cardiac gene therapy. Circulation 2013;128(15):1612-22. 10. Fujiu K, Nagai R. Contributions of cardiomyocyte-cardiac fibroblast-immune cell interactions in heart failure development. Basic Res Cardiol 2013;108(4):357. 11. Gaengel K, Niaudet C, Hagikura K, Laviña B, Muhl L, Hofmann JJ, Ebarasi L, Nyström S, Rymo S, Chen LL, Pang MF, Jin Y, Raschperger E, Roswall P, Schulte D, Benedito R, Larsson J, Hellström M, Fuxe J, Uhlén P, Adams R, Jakobsson L, Majumdar A, Vestweber D, Uv A, Betsholtz C. The sphingosine-1-phosphate receptor S1PR1 restricts sprouting angiogenesis by regulating the interplay between VE-cadherin and VEGFR2. Dev Cell. 2012 24 489 490 491 492 493 494 495 496 497 498 499 500 501 502 503 504 505 506 507 508 509 510 511 512 513 514 515 516 517 518 519 520 521 522 523 524 525 526 527 528 529 530 531 532 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. Sep 11;23(3):587-99. Gao E, Lei YH, Shang X, Huang ZM, Zuo L, Boucher M, Fan Q, Chuprun JK, Ma XL, Koch WJ. A novel and efficient model of coronary artery ligation and myocardial infarction in the mouse. Circ Res 2010;107(12):1445-53. Gellings Lowe N, Swaney JS, Moreno KM, Sabbadini RA. Sphingosine-1-phosphate and sphingosine kinase are critical for transforming growth factor-beta-stimulated collagen production by cardiac fibroblasts. Cardiovasc Res 2009;82(2):303-12. Janes K, Little JW, Li C, Bryant L, Chen C, Chen Z, Kamocki K, Doyle T, Snider A, Esposito E, Cuzzocrea S, Bieberich E, Obeid L, Petrache I, Nicol G, Neumann WL, Salvemini D. The Development and Maintenance of Paclitaxel-induced Neuropathic Pain Require Activation of the Sphingosine 1-Phosphate Receptor Subtype 1. J Biol Chem 2014;25;289(30):21082-21097. Karliner JS. Sphingosine kinase and sphingosine 1-phosphate in the heart: a decade of progress. Biochim Biophys Acta 2013;1831(1):203-12. K.G. Lim, F. Tonelli, Z. Li, X. Lu, R. Bittman, S. Pyne, N.J. Pyne. FTY720 analogues as sphingosine kinase 1 inhibitors: enzyme inhibition kinetics, allosterism, proteasomal degradation, and actin rearrangement in MCF-7 breast cancer cells. J Biol Chem 2011; 286: 18633–18640. Lee H, Deng J, Kujawski M, Yang C, Liu Y, Herrmann A, Kortylewski M, Horne D, Somlo G, Forman S, Jove R, Yu H. STAT3-induced S1PR1 expression is crucial for persistent STAT3 activation in tumors. Nat Med 2010;16(12):1421-8. Liang J, Nagahashi M, Kim EY, Harikumar KB, Yamada A, Huang WC, Hait NC, Allegood JC, Price MM, Avni D, Takabe K, Kordula T, Milstien S, Spiegel S. Sphingosine-1-phosphate links persistent STAT3 activation, chronic intestinal inflammation, and development of colitis-associated cancer. Cancer Cell 2013;23(1):107-20. Mann DL. Stress-activated cytokines and the heart: from adaptation to maladaptation. Annu Rev Physiol 2003;65:81-101.Means CK, Xiao CY, Li Z, Zhang T, Omens JH, Ishii I, Chun J, Brown JH. Sphingosine 1-phosphate S1P2 and S1P3 receptor-mediated Akt activation protects against in vivo myocardial ischemia-reperfusion injury. Am J Physiol Heart Circ Physiol. 2007;292(6):H2944-51. Mascall KS, Small GR, Gibson G, Nixon GF. Sphingosine-1-phosphate-induced release of TIMP-2 from vascular smooth muscle cells inhibits angiogenesis. J Cell Sci. 2012 May 1;125(Pt 9):2267-75. Meissner A, Yang J, Kroetsch JT, Sauvé M, Dax H, Momen A, Noyan-Ashraf MH, Heximer S, Husain M, Lidington D, Bolz SS. Tumor necrosis factor-α-mediated downregulation of the cystic fibrosis transmembrane conductance regulator drives pathological sphingosine-1-phosphate signaling in a mouse model of heart failure. Circulation 2012;125(22):2739-50. Michael Maceyka, Kuzhuvelil B. Harikumar, Sheldon Milstien and Sarah Spiegel. Sphingosine-1-phosphate signaling and its role in disease. Trends Cell Biol 2012;22(1):50-60. Murray DR, Prabhu SD, Chandrasekar B. Chronic beta-adrenergic stimulation induces myocardial proinflammatory cytokine expression. Circulation 2000;23;101(20):2338-41. Pchejetski D, Foussal C, Alfarano C, Lairez O, Calise D, Guilbeau-Frugier C, Schaak S, 25 533 534 535 536 537 538 539 540 541 542 543 544 545 546 547 548 549 550 551 552 553 554 555 556 557 558 559 560 561 562 563 564 565 566 567 568 569 570 571 572 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. Seguelas MH, Wanecq E, Valet P, Parini A, Kunduzova O. Apelin prevents cardiac fibroblast activation and collagen production through inhibition of sphingosine kinase 1. Eur Heart J 2012;33(18):2360-9 Pettus BJ, Bielawski J, Porcelli AM, Reames DL, Johnson KR, Morrow J, Chalfant CE, Obeid LM, Hannun YA. The sphingosine kinase 1/sphingosine-1-phosphate pathway mediates COX-2 induction and PGE2 production in response to TNF-alpha. FASEB J. 2003;17:1411-21. Robert P, Tsui P, Laville MP, Livi GP, Sarau HM, Bril A, Berrebi-Bertrand I. EDG1 receptor stimulation leads to cardiac hypertrophy in rat neonatal myocytes. J Mol Cell Cardiol 2001;33(9):1589-606. Sarkisyan G, Gay LJ, Nguyen N, Felding BH, Rosen H. Host endothelial S1PR1 regulation of vascular permeability modulates tumor growth. Am J Physiol Cell Physiol. 2014 Jul 1;307(1):C14-24. Sutton MG, Sharpe N. Left ventricular remodeling after myocardial infarction: pathophysiology and therapy. Circulation 2000; 101:2981-8. Takuwa N, Ohkura S, Takashima S, Ohtani K, Okamoto Y, Tanaka T, Hirano K, Usui S, Wang F, Du W, Yoshioka K, Banno Y, Sasaki M, Ichi I, Okamura M,Sugimoto N, Mizugishi K, Nakanuma Y, Ishii I, Takamura M, Kaneko S, Kojo S, Satouchi K, Mitumori K, Chun J, Takuwa Y. S1P3-mediated cardiac fibrosis in sphingosine kinase 1 transgenic mice involves reactive oxygen species. Cardiovasc Res 2010;85(3):484-93. Yang F, Liu YH, Yang XP, Xu J, Kapke A, Carretero OA. Myocardial infarction and cardiac remodeling in mice. Exp Physiol. 2002;87(5):547-55. Yan W, Zhang F, Zhang R, Zhang X, Wang Y, Zhou F, Xia Y, Liu P, Gao C, Wang H, Zhang L, Zhou J, Gao F, Gao E, Koch WJ, Wang H, Cheng H, Qu Y, Tao L. Adiponectin regulates SR Ca(2+) cycling following ischemia/reperfusion via sphingosine 1-phosphate-CaMKII signaling in mice. J Mol Cell Cardiol 2014;74:183-92. Yeh CC, Li H, Malhotra D, Huang MC, Zhu BQ, Goetzl EJ, Vessey DA, Karliner JS, Mann MJ. Sphingolipid signaling and treatment during remodeling of the uninfarcted ventricular wall after myocardial infarction. Am J Physiol Heart Circ Physiol 2009;296(4):H1193-9. Yoh Takuwa, Yasuo Okamoto, Kazuaki Yoshioka, Noriko Takuwa. Sphingosine-1-phosphate signaling and biological activities in the cardiovascular system. Biochimica et Biophysica Acta 2008;1781:483-488. Yu X, Zhang M, Kyker K, Patterson E, Benovic JL, Kem DC. Ischemic inactivation of G protein-coupled receptor kinase and altered desensitization of canine cardiac beta-adrenergic receptors. Circulation. 2000; 102(20):2535-40. Zhang Y, Berka V, Song A, Sun K, Wang W, Zhang W, Ning C, Li C, Zhang Q, Bogdanov M, Alexander DC, Milburn MV, Ahmed MH, Lin H, Idowu M, Zhang J,Kato GJ, Abdulmalik OY, Zhang W, Dowhan W, Kellems RE, Zhang P, Jin J, Safo M, Tsai AL, Juneja HS, Xia Y. Elevated sphingosine-1-phosphate promotes sickling and sickle cell disease progression. J Clin Invest. 2014 Jun;124(6):2750-61. 573 26 574 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 636 were shown (A). Left ventricular ejection fraction (LVEF, B), left ventricular end-systolic 637 diameter (LVESD, C), and left ventricular end-diastolic diameters (LVEDD, D) were determined 638 by echocardiography at day 28 post-MI. Representative images of masson trichrome-stained and 639 picrosirius red-stained heart sections were presented (E). The size of interstitial fibrosis was 29 640 calculated (F). Heart weight to body weight ratios (G) and lung weight to body weight ratios (H) 641 were calculated. Relative expression levels of remodeling-related genes (I), TGF-β family genes 642 (J), and collagen genes (K) were determined by RT-PCR. Data are presented as mean+SEM, 643 n=12-15 per group. #p<0.05 vs. Sham. *p<0.05 vs. MI group. 30