Sfrp3

NFAT-luciferase mice _ENREF_9^{9 (link)}, kindly provided by Dr. Jeffery D. Molkentin (Cincinnati Children’s Hospital Medical Center, Cincinnati, OH), carry nine copies of NFAT-binding sites from the interleukin-4 promoter, upstream of the luciferase gene. Primary ventricular cardiomyocyte cultures from neonatal (one-three days old) NFAT-luciferase reporter mice were prepared as described^{54 (link)}. Cell cultures from three separate isolations were used for experiments. Cardiomyocytes were maintained in serum-free medium 24 hours prior to treatment with recombinant Wnt5a, sFRP3 (R&D Systems, Minneapolis, MN, USA) or endothelin-1 (used as positive control, Sigma-Aldrich, St. Louis, MO, USA) for 24 hours, washed twice with PBS and harvested for luciferase activity quantification according to the luciferase assay protocol (Promega, Madison, WI, USA). Luminescence from duplicates was quantified on a Victor 3 1420 Multilabel Counter (PerkinElmer, MA). Cell culture medium was collected for cell viability analyses (ToxiLight, Lonza Group Ltd, Basel, Switzerland) and measurements of sFRP3 release measured by EIAs (R&D Systems; Stillwater, MN, USA).

LTP was induced in 13–14 DIV hippocampal cultures using an NMDAR mediated chemical LTP (cLTP) protocol as previously described (Fortin et al., 2010 (link); McLeod et al., 2018 (link)). Briefly, hippocampal neurons were kept at room temperature (RT) for 20–30 min in control solution (125 mM NaCl, 2.5 mM KCl, 1 mM MgCl₂, 2 mM CaCl₂, 33 mM D-glucose, 5 mM HEPES, 20 μM D-APV, 3 μM strychnine, 20 μM bicuculline and 0.5 μM TTX; pH 7.4). After cLTP induction (addition of glycine 200 μM for 10 min in the absence of Mg²⁺, D-APV and TTX) cultures were returned to control solution for 1 h prior to fixation (all performed at RT). To block endogenous Wnt proteins, recombinant Sfrp3 (250 ng/mL; R&D Systems) was used only throughout the induction of cLTP and after.

LTP was induced in cultured hippocampal neurons with a glycine-mediated form of cLTP as previously described (Fortin et al., 2010 (link), Stamatakou et al., 2013 (link)). To block Wnts, a combination of recombinant Sfrp1 (2.5 μg/mL; R&D Systems, Abingdon, UK) and Sfrp3 (250 ng/mL; R&D Systems) was used throughout the induction of cLTP and during the incubation with control solution. See Supplemental Experimental Procedures for more information.