Selective coupling of the S1P3 receptor subtype to S1P-mediated RhoA activation and cardioprotection

Abstract

Sphingosine-1-phosphate (S1P), a bioactive lysophospholipid, is generated and released at sites of tissue injury in the heart and can act on S1P₁, S1P₂, and S1P₃ receptor subtypes to affect cardiovascular responses. We established that S1P causes little phosphoinositide hydrolysis and does not induce hypertrophy indicating that it does not cause receptor coupling to G_q. We previously demonstrated that S1P confers cardioprotection against ischemia/reperfusion by activating RhoA and its downstream effector PKD. The S1P receptor subtypes and G proteins that regulate RhoA activation and downstream responses in the heart have not been determined. Using siRNA or pertussis toxin to inhibit different G proteins in NRVMs we established that S1P regulates RhoA activation through Gα₁₃ but not Gα₁₂, Gα_q, or Gα_i. Knockdown of the three major S1P receptors using siRNA demonstrated a requirement for S1P₃ in RhoA activation and subsequent phosphorylation of PKD, and this was confirmed in studies using isolated hearts from S1P₃ knockout (KO) mice. S1P treatment reduced infarct size induced by ischemia/reperfusion in Langendorff perfused wild-type (WT) hearts and this protection was abolished in the S1P₃ KO mouse heart. CYM-51736, an S1P₃-specific agonist, also decreased infarct size after ischemia/reperfusion to a degree similar to that achieved by S1P. The finding that S1P₃ receptor- and Gα₁₃-mediated RhoA activation is responsible for protection against ischemia/reperfusion suggests that selective targeting of S1P₃ receptors could provide therapeutic benefits in ischemic heart disease.

Keywords: Cardioprotection; G protein-coupled receptor (GPCR); Ischemia/reperfusion (I/R); Phospholipase C (PLC); Protein kinase D (PKD); Ras homolog gene family member A (RhoA); Sphingosine-1-phosphate (S1P).

Fig. 1. S1P receptor signaling stimulates modest increases in PI hydrolysis and does not mediate in vitro or in vivo cardiac hypertrophy

(A) Time course of S1P- and PE-induced phosphatidylinositol hydrolysis. NRVMs were serum-starved overnight in the presence of [³H] inositol. Cells were then treated with agonists for 1, 5, 10, 30, and 60 minutes in the presence of LiCl before isolation of [³H] inositol phosphates (InsPs). The data displayed are the mean ± SEM. **P < 0.01 vs. vehicle (Veh), ##P < 0.01 vs. S1P (n = 5). (B) NRVMs were pretreated with 2.0 μg/mL C3 exoenzyme (Rho inhibitor) for 6 hours or (C) transfected with Control siRNA (siCtrl) or siRNA against PLCε for 48 hours before challenge with agonists for 60 minutes and assessed for InsPs production. **P < 0.01 vs. vehicle + Control or siCtrl, ##P < 0.01 vs. S1P + Control or siCtrl (n = 5). (D) Representative immunofluorescent images depicting NRVMs stained for α-actinin, atrial natriuretic factor (ANF), and nuclei with DAPI after treatment with either vehicle, 0.3 μM S1P, or 50 μM PE for 24 hours. Scale bar: 20 μm. (E) Quantified relative cell area and (F) quantified ANF positive cells (n = 400 cells). (G) mRNA expression of brain natriuretic peptide (BNP) and (H) skeletal muscle α-actin (ACTA1). **P < 0.01 vs. vehicle (n = 4). (I) heart weight (HW) to body weight (BW) ratio of WT, S1P₂ KO, and S1P₃ KO mice following transverse aortic constriction (TAC) to induce pressure overload hypertrophy for one week. **P < 0.01 vs. Sham (n ≥ 5).

Fig. 2. S1P activates RhoA through Gα₁₃ in NRVMs

(A) NRVMs were treated with 0.3 μM S1P or 50 μM PE for 5 minutes and RhoA activation was measured by a GTP-RhoA pull-down assay. The data displayed are the mean ± SEM (n = 5), **P < 0.01 vs. vehicle (Veh). (B) NRVMs were pretreated with 0.1 μg/mL pertussis toxin (PTX) overnight and then stimulated with 0.3 μM S1P for 5 minutes. RhoA activation was assessed by GTP-RhoA pull-down assay. NS indicates not significant (n = 4). (C) NRVMs were transfected with either Control siRNA (siCtrl) or siRNA against Gα_q, (D) Gα₁₂, or (E) Gα₁₃. G protein expression levels were assessed by Western blotting after 72 hour- knockdown, **P < 0.01 (n = 3). Cytoskeletal protein α-actinin was blotted as a loading control. (F) NRVMs were transfected with either siCtrl or siRNA against Gα_q, Gα₁₂, or Gα₁₃ for 72 hours and then stimulated with 0.3 μM S1P for 5 minutes. RhoA activation was assessed by GTP-RhoA pull-down assay. *, **P < 0.05, 0.01 vs. siCtrl vehicle (Veh), #P < 0.05 vs. siCtrl + S1P (n = 7). (G) Dual-Luciferase Reporter Assay was performed as described in Materials and Methods to assess RhoA activation. **P < 0.01 vs. siCtrl, ##P < 0.01 vs. siCtrl + S1P (n = 6).

Fig. 3. S1P activates RhoA through S1P₃ in NRVMs and in isolated perfused hearts

NRVMs were transfected with either siCtrl or siRNA against (A) S1P₁, (B) S1P₂, (C) or S1P₃. S1P receptor levels were assessed by qPCR analysis after 48-hour siRNA transfection. Data shown represent the mean ± SEM, **P < 0.01 vs. siCtrl (n = 3) (D) NRVMs were transfected with siRNA to knockdown S1P receptor subtypes for 48 hours and then stimulated with 0.3 μM S1P for 5 minutes. RhoA activation was assessed by GTP-RhoA pull-down assay. *, **P < 0.05, 0.01 vs. siCtrl, ##P < 0.01 vs. siCtrl + S1P, (n = 4). (E) NRVMs were transfected with siRNA to knock down S1P₃ receptor and then stimulated with 0.3μM S1P or 10μM CYM-51736 for 5 minutes. RhoA activation was assessed by the GTP-RhoA pulldown assay. **P < 0.01 vs. Veh (n = 4). (F) NRVMs were pretreated for 30 minutes with 1 μM of JTE-013, an S1P₂ receptor antagonist, and then stimulated with 0.3 μM S1P for 5 minutes. RhoA activation was assessed by the GTP-RhoA pulldown assay. *P < 0.05 vs. Veh (n = 3). (G) Isolated wild-type (WT), S1P₂ KO, and S1P₃ KO mouse hearts were perfused with either vehicle or 0.3 μM S1P in Krebs-Henseleit buffer for 5 minutes in Langendorff mode and RhoA activation was assessed by GTP-RhoA pull-down assay. **P < 0.01 vs. vehicle, ##P < 0.01 vs. WT + S1P (n = 3 to 5).

Fig. 4. S1P protects against ex vivo I/R through S1P₃ receptor

Representative images of TTC-stained cross sections of isolated perfused mouse hearts after I/R injury (top) and quantification of infarct size (bottom). White areas are infarcted tissue and red areas are viable tissue. (A) Isolated WT and S1P₃ KO hearts were perfused with either Veh or 0.3 μM S1P for 10 minutes and subjected to 22 minute global ischemia followed by 60 minute reperfusion. *P < 0.05 vs. WT + Veh (n ≥ 5). The data shown are the mean ± SEM of each group’s animals. (B) WT hearts were perfused with either vehicle or 10 μM CYM-51736 (S1P₃ specific agonist) for 10 minutes and subjected to I/R. Infarct size was assessed by TTC staining. **P < 0.01 vs Vehicle (n = 5).

Fig. 5. The S1P₃ receptor mediates the activation of PKD by S1P in NRVMs

Representative Western blots of NRVMs that were transfected with siRNA against S1P receptor subtypes for 48 hours followed by treatment with either (A) 0.3 μM S1P for 5 minutes or (B) 10 μM CYM-51736 for 20 minutes. The data displayed are the mean ± SEM. *P < 0.05 vs. siCtrl vehicle, #P < 0.05 vs. siCtrl + S1P or CYM-51736 (n = 4 to 5). (C) NRVMs were transfected with siRNA against Gα₁₃ for 72 hours or treated with 2 μg/ml C3 Rho inhibitor 12 hours, followed by treatment with 10 μM CYM-51736 for 15 minutes. Data displayed are the experimental means ± SEM. **P < 0.01 vs. siCtrl + Veh, #P < 0.05 vs. siCtrl + CYM-51736, ##P < 0.01 vs. siCtrl + CYM-51736 (n = 5). (D) NRVMs were pretreated for 30 minutes with 1 μM of S1P₂ receptor antagonist JTE-013, then stimulated with 0.3 μM S1P. Data displayed are the experimental means +/− SEM. **P < 0.01 vs. vehicle, #P < 0.05 vs. S1P stimulation (n = 3).