Volume 21, Issue 1, Pages 246-258 (October 2017) Omega-3 Fatty Acids Modulate TRPV4 Function through Plasma Membrane Remodeling  Rebeca Caires, Francisco J. Sierra-Valdez, Jonathan R.M. Millet, Joshua D. Herwig, Esra Roan, Valeria Vásquez, Julio F. Cordero- Morales  Cell Reports  Volume 21, Issue 1, Pages 246-258 (October 2017) DOI: 10.1016/j.celrep.2017.09.029 Copyright © 2017 The Authors Terms and Conditions

Cell Reports 2017 21, 246-258DOI: (10.1016/j.celrep.2017.09.029) Copyright © 2017 The Authors Terms and Conditions

Figure 1 GSK101 Elicits Withdrawal Responses in Rat TRPV4-Expressing Worms (A) Schematic representation of the withdrawal responses after addition of GSK101 drop in front of freely moving worms. (B) GSK101 dose-response profile for wild-type (WT [N2]) and TRPV4-expressing worms. (C) Inhibition of GSK101-mediated withdrawal responses in TRPV4 worms by HC067047 (2 μM). (D) Withdrawal responses elicited by 4α-phorbol in WT and TRPV4 worms. (E) Withdrawal responses elicited by 1 M glycerol and nose touch in WT, osm9, and TRPV4; osm9 strains. Bars represent mean ± SEM; the number of worms tested during 3 assays sessions is indicated inside the bars. The asterisks indicate values significantly different from control (∗∗∗p < 0.001, ∗∗p < 0.01, and ∗p < 0.05). See also Figure S1. Cell Reports 2017 21, 246-258DOI: (10.1016/j.celrep.2017.09.029) Copyright © 2017 The Authors Terms and Conditions

Figure 2 PUFAs Are Required for TRPV4 Function in C. elegans (A) Fatty acid desaturase (FAT) and elongase enzymes (ELO) synthesize long PUFAs, and cytochrome P450 (CYPs) generates eicosanoid derivatives, adapted from (Watts, 2009). LA, linolenic acid; γLA, γ-linolenic acid; DγLA, dihomo-linolenic acid; ω-6 AA, arachidonic acid; EET, epoxy-eicosatrienoic acid; ALA, α-linolenic acid; STA, stearidonic acid; ω-3 AA; EPA, eicosapentaenoic acid; EEQ, 17′18′-epoxy eicosatetraenoic acid. (B) Withdrawal responses elicited by GSK101 in WT, TRPV4, TRPV4; fat-3, and TRPV4; fat-3 worms supplemented with PUFAs. (C) Withdrawal responses elicited by 4α-Phorbol in WT, TRPV4, and TRPV4; fat-3 worms. (D) Withdrawal responses elicited by GSK101 in TRPV4 and TRPV4; fat-4 worms. (E) Withdrawal responses elicited by 1 M glycerol and nose touch in TRPV4; osm9 and TRPV4; osm9fat-3 strains. (F) Representative micrographs of TRPV4::GFP and TRPV4::GFP; fat-3 ASH neurons. (G) Boxplots show the mean, median, and the 75th to 25th percentiles of the fluorescence intensity analysis from images in (F). The number of neurons imaged during 2 sessions is indicated below the boxes. (H) Schematic representation of the phospholipid synthesis. (I) GSK101 withdrawal responses after knocking down the expression of mboa-6 in TRPV4 worms. Bars represent mean ± SEM; the number of worms tested during 3 assays sessions is indicated inside the bars. The asterisks indicate values significantly different from control (∗∗∗p < 0.001; ns, not significant). See also Figure S2. Cell Reports 2017 21, 246-258DOI: (10.1016/j.celrep.2017.09.029) Copyright © 2017 The Authors Terms and Conditions

Figure 3 EPA and 17,18-EEQ Fully Restore TRPV4 Function in C. elegans (A) GSK101-mediated withdrawal responses of TRPV4 and TRPV4; fat 3 mutants after worms were fed with specified PUFAs and eicosanoid derivatives (200 μM). Dotted red and blue lines represent the 20% and 45% thresholds for positive and intermediate responses, respectively. (B) Schematic representation of the effect of ETYA (non-metabolizable analog of ω-6 AA) in worms. (C) Top inset, ω-6 PUFAs present in fat-1 and fat-1fat-4. Bottom, withdrawal responses elicited by GSK101 in TRPV4; fat-3, TRPV4; fat-3 supplemented with ETYA, TRPV4; fat-1, TRPV4; fat-1fat-4, and TRPV4; fat-1fat-4 supplemented with ω-6 AA. (D) Withdrawal responses elicited by 1 M glycerol and nose touch in TRPV4; osm9fat-3 mutants after being fed with EPA (200 μM). Bars represent mean ± SEM; the number of worms tested during 3 assays sessions is indicated inside the bars. The asterisks indicate values significantly different from control (∗∗∗p < 0.001, ∗∗p < 0.01, and ∗p < 0.05). See also Figure S3. Cell Reports 2017 21, 246-258DOI: (10.1016/j.celrep.2017.09.029) Copyright © 2017 The Authors Terms and Conditions

Figure 4 EPA Supplementation Enhances TRPV4 Activity in HMVECs (A) Representative whole-cell patch-clamp recordings (+80 mV) of control and EPA (100 μM)-treated HMVECs challenged with GSK101 (100 nM) and HC067047 (10 μM). (B) Boxplots show the mean, median, SD, and SEM from TRPV4 currents (IGSK101 − IHC/pF) obtained by whole-cell patch-clamp recordings (+80 mV) of control, EPA-, and ω-6 AA-treated HMVECs. (C) Left: representative current-voltage relationships determined by whole-cell patch-clamp recording of control and EPA (100 μM)-treated HMVECs challenged with GSK101 (100 nM) in the presence of 5 mM Ca2+. Right: bar graph of peak currents (at +80 mV) relative to the currents after 5 min of exposure to GSK101 (I max/I 5 min). (D) HMVECs were challenged with isosmotic (IB; 320 mOsm), hyposmotic (HB; 240 mOsm), and GSK101 (100 nM) solutions and analyzed for their responses using Ca2+ imaging (Fluo-4 AM); color bar indicates relative change in fluorescence intensity. Control and EPA (100–300 μM)-treated HMVECs were analyzed from 5 independent preparations. (E) Representative traces corresponding to normalized (ΔF/F) intensity changes of individual cells shown in (D). (F) Area under the curve of control and EPA-treated HMVECs challenged with hyposmotic buffer. Bars represent mean ± SEM. The number of endothelial cells measured is indicated below the boxes and inside the bars. The asterisks indicate values significantly different from control (∗∗∗p < 0.001 and ∗∗p < 0.01). See also Figure S4. Cell Reports 2017 21, 246-258DOI: (10.1016/j.celrep.2017.09.029) Copyright © 2017 The Authors Terms and Conditions

Figure 5 EPA Supplementation Increases ω-3 Fatty Acid Eicosanoid Derivatives in HMVECs and Does Not Affect TRPV4 Expression and Trafficking (A) EPA and ω-6 AA content in control and EPA (100 μM)-treated HMVECs, as determined by LC-MS. (B) ω-6 AA, EPA, and DHA eicosanoid derivatives content in control and EPA-treated HMVECs, as determined by LC-MS. (C) TRPV4 expression levels detected in control and EPA-treated HMVECs by immunostaining. (D and E) Western blots with anti-TRPV4 antibody in control and EPA-treated HMVECs from total protein extracts (D) and membrane protein fractions (E). Normalized relative intensities (RI) against total protein present in the PVDF membranes (Figure S5) are denoted. Similar results were observed in at least five independent western blots. See also Figure S5. Cell Reports 2017 21, 246-258DOI: (10.1016/j.celrep.2017.09.029) Copyright © 2017 The Authors Terms and Conditions

Figure 6 ω-3 PUFAs Contribute to Membrane Fluidity and Bending Stiffness (A) Thermotropic characterization of the DPPC/PUFA systems using DSC: control (Tm = 41.68°C), ω-6 AA (41.04°C), EPA (40.85°C), 5,6-EET (39.36°C), and 17,18-EEQ (37.85°C). (B) Effect of DPPC/PUFAs on melting temperatures (ΔTm) with respect to DPPC membranes. We plotted ΔTm absolute magnitude to better illustrate the effect. Experiments were performed from two independent preparations. Bars are mean obtained during two liposome assay sessions. (C) Top: schematic representation of the AFM setup on HMVECs. Bottom: representative force distance traces acquired at 10 μm/s and magnification of the force step for control and EPA (300 μM)-treated cells. (D) Boxplots show the mean, median, and the 75th to 25th percentile analysis from tether forces of control and EPA-treated cells. The number of endothelial cells measured during two sessions is indicated below the boxes. The asterisks indicate values significantly different from control (∗∗∗p < 0.001). See also Figure S6. Cell Reports 2017 21, 246-258DOI: (10.1016/j.celrep.2017.09.029) Copyright © 2017 The Authors Terms and Conditions

Figure 7 Proposed Model by Which ω-3 Fatty Acids Enhance Human Endothelial Cell Responses (A and B) Plasma membranes with reduced levels of ω-3 fatty acid displays low number of channels available for activation (A) as well as a reduced number of active channels after stimulation (B). (C and D) ω-3 fatty acids enriched plasma membranes increase the number of channels available for activation (C) as well as the number of active channels after stimulation (D). Cell Reports 2017 21, 246-258DOI: (10.1016/j.celrep.2017.09.029) Copyright © 2017 The Authors Terms and Conditions