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The transient receptor potential vanilloid 4 channel modulates uterine tone during pregnancy

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Science Translational Medicine  23 Dec 2015:
Vol. 7, Issue 319, pp. 319ra204
DOI: 10.1126/scitranslmed.aad0376
  • Fig. 1. TRPV4 expression and cell membrane localization increase during late gestation.

    (A) Relative Trpv4 gene expression was determined by qPCR in mSMC isolated from nonpregnant rats and rats at various stages of pregnancy or postpartum (PP). n = 6 samples per group, with each sample containing cells from at least three animals; **P = 0.0094 for D14, **P = 0.0087 for D17, **P = 0.0075 for D19, and **P = 0.01 D21 versus nonpregnant (NP) via one-way analysis of variance (ANOVA). (B and C) Western immunoblot to detect TRPV4 protein in (B) whole-cell lysates and (C) membrane fractions of myometrial SMC isolated from nonpregnant and pregnant (D21) rats normalized to Coomassie staining. Quantification shown as bar graphs on the right, expressed as fold increase in expression versus nonpregnant; n = 4 samples containing cells obtained from a total of 12 animals; *P = 0.0286 for (B) and *P = 0.0265 for (C) via Mann-Whitney U test. (D) Immunocytochemistry of rat mSMC obtained from nonpregnant and D21 rats, shown with the CellMask (Invitrogen) plasma membrane stain (red), 4′,6-diamidino-2-phenylindole (DAPI) (blue), and a primary antibody to detect TRPV4 (green), with the presence of TRPV4 on the plasma membrane evidenced by the colocalization signal (yellow). Scale bar, 50 μm.

  • Fig. 2. TRPV channel activation increases cytosolic calcium in pregnant more than nonpregnant mSMC.

    (A) mSMCs from nonpregnant (gray) and D21 (black) rats were loaded with fura-2, and the apparent cytosolic calcium (R/R0) was measured at baseline and after the addition of the TRPV channel agonist, 4αPDD (arrow). (B) Peak cytosolic calcium (R/R0) in nonpregnant and D21 mSMC; n = 52 to 86 cells and **P = 0.0012 via Mann-Whitney U test. (C) mSMCs from D21 pregnant (P) rats were loaded with fura-2, and the R/R0 induced by 4αPDD in cells pretreated with the TRPV blocker ruthenium red (RR) was compared to cells treated with 4αPDD alone. (D) Peak cytosolic calcium (R/R0) in D21 mSMC treated with 4αPDD and either vehicle or ruthenium red; n = 52 to 76 cells and ***P = 0.004 via Mann-Whitney U test. (E) mSMCs from nonpregnant rats were loaded with fura-2, and the R/R0 induced by 4αPDD in cells pretreated with the TRPV blocker ruthenium red was compared to cells treated with 4αPDD alone. (F) Peak cytosolic calcium (R/R0) in nonpregnant mSMC treated with 4αPDD and either vehicle or ruthenium red; n = 86 cells.

  • Fig. 3. The expression of β-arrestin proteins and their physical interaction with TRPV4 decrease during pregnancy.

    (A) Confocal microscopy was performed on frozen myometrial tissue from both pregnant and nonpregnant mice immunostained to detect TRPV4 (red). Scale bars, 100 μm. (B and C) Western blotting of myometrial tissue from pregnant and nonpregnant mice to detect (B) β-arrestin-1 (ARRB1) and (C) β-arrestin-2 (ARRB2), with β-actin as a loading control; n = 3 and P = 0.10 for both (B) and (C) via Mann-Whitney U test. (D) In situ proximity ligation assay (PLA) was performed on nonpregnant and pregnant uterine tissue from wild-type (WT) and Arrb2−/− mice, with physical interaction of TRPV4 and ARRB2 visualized by pink staining. Tissue from Arrb2−/− mice was used as a negative control. Scale bar, 100 μm.

  • Fig. 4. Depletion of TRPV4 attenuates human mSMC contraction in response to oxytocin.

    (A and B) Human mSMCs were transfected with either NTC or TRPV4 siRNA, and TRPV4 gene (A) and protein (B) expression were determined. (C and D) Collagen gel contraction assays were performed with human mSMC treated with NTC or TRPV4 siRNA and stimulated with vehicle, oxytocin, or LPS. The collagen-covered area in each well was compared to the area of the well containing collagen alone and expressed as a percentage. §P = 0.022 for oxytocin-stimulated and P = 0.1216 for LPS-stimulated NTC siRNA versus vehicle, and ****P < 0.0001 for both oxytocin- and LPS-stimulated TRPV4 siRNA gels versus NTC siRNA gels, via two-way ANOVA; n = 3 to 6 for all groups.

  • Fig. 5. TRPV4 channel activity modulates uterine contractility.

    (A) Myography of uterine muscle strips from pregnant WT mice at D18 was performed while incubating the strips with increasing doses of oxytocin in the presence of vehicle (black circles), 200 nM HC 067047 (dark gray squares), or 1 μM HC 067047 (light gray triangles). ****P < 0.0001 versus 1 × 10−10 M oxytocin, §§P = 0.0086 for 200 nM HC 067047, and P = 0.022 for 1 μM HC 067047 versus 1 × 10−8 M oxytocin, §§P = 0.0235 for 200 nM HC 067047 and P = 0.0034 for 1 μM HC 067047 versus 1 × 10−7 M oxytocin, §§§P = 0.0005 for 200 nM HC 067047, and P = 0.0002 for 1 μM HC 067047 versus 1 × 10−6 M oxytocin via two-way ANOVA with n = 4 to 7. (B) Representative myography tracings for each treatment group at baseline (left) and in response to 1 × 10−6 M oxytocin (right). (C) Myography of uterine muscle strips from pregnant WT mice at D18 was performed while incubating the strips with increasing doses of the TRPV4 activator GSK1016790A in the presence of vehicle (black circles), 200 nM HC 067047 (dark gray squares), or 1 μM HC 067047 (light gray triangles). **P = 0.0023 and ****P < 0.0001 versus 1 × 10−10 M GSK1016790A, §§P = 0.0056 for 200 nM HC 067047 and P = 0.0076 for 1 μM HC 067047 versus 1 × 10−7 M GSK1016790A, and §§§§P < 0.0001 for 200 nM and 1 μM HC 067047 versus 1 × 10−6 M GSK1016790A via two-way ANOVA with n = 6 to 8. (D) Representative myography tracings for each treatment group at baseline (left) and in response to 1 × 10−6 M GSK1016790A (right). (E) Myography of uterine muscle strips from pregnant WT mice (black circles) and Trpv4−/− mice (dark gray squares) at D18 was performed while incubating strips with increasing doses of oxytocin. ****P < 0.0001 versus 1 × 10−10 M oxytocin, §P = 0.012 for Trpv4−/− versus WT at 1 × 10−8 M oxytocin, §§§§P < 0.0001 for Trpv4−/− versus WT at 1 × 10−7 M and 1 × 10−6 M oxytocin, and ## versusTrp4−/− mice at 1 × 10−10 M oxytocin via two-way ANOVA with n = 4. (F) Representative myography tracings for each treatment group at baseline (left) and in response to 1 × 10−6 M oxytocin (right).

  • Fig. 6. TRPV4 inhibition protects against preterm labor, whereas loss of β-arrestin-1 or β-arrestin-2 increases susceptibility to preterm labor.

    (A) Pregnant WT mice at D15 were treated with RU-486 (150 μg) intraperitoneally, and after 8 hours, groups were treated with vehicle, HC 067047 as a single dose (20 mg/kg), or multiple doses of HC 067047 (10 mg/kg) every 8 hours. The time until delivery of the first pup was recorded. ***P = 0.0002 and ****P < 0.0001 versus vehicle, and §§§P = 0.008 versus RU-486 + HC 067047 (single) via one-way ANOVA with n = 3 to 5 per group. (B) Pregnant WT mice at D15 were treated with LPS (3 mg/kg) intraperitoneally, and after 8 hours, groups were treated with vehicle, HC 067047 as a single dose (20 mg/kg), or multiple doses of HC 067047 (10 mg/kg) every 8 hours. The time until delivery of the first pup was recorded. ****P < 0.0001 versus vehicle via one-way ANOVA with n = 3 to 6 per group. (C) Pregnant WT, Arrb1−/−, or Arrb2−/− mice were treated with LPS (1 mg/kg) intraperitoneally, and the time until delivery of the first pup was recorded. ****P < 0.0001 versus vehicle for each genotype, and §§P = 0.0018 and §§§§P < 0.0001 versus LPS WT, by two-way ANOVA with n = 4 per group.

Supplementary Materials

  • Supplementary Material for:

    The transient receptor potential vanilloid 4 channel modulates uterine tone during pregnancy

    Lihua Ying, Margaux Becard, Deirdre Lyell, Xiaoyuan Han, Linda Shortliffe, Cristiana Iosef Husted, Cristina M. Alvira, David N. Cornfield*

    *Corresponding author. E-mail: cornfield{at}stanford.edu

    Published 23 December 2015, Sci. Transl. Med. 7, 319ra204 (2015)
    DOI: 10.1126/scitranslmed.aad0376

    This PDF file includes:

    • Fig. S1. TRPV4 expression is increased in pregnant human uterine tissue.

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