Zacopride

Isoproterenol (Iso, Sigma) was administered by intraperitoneal injection (i.p.) once a day for 3, 10, and 30 days, respectively, to establish temporal cardiac remodeling. An experimental protocol scheme including grouping and treatments is shown in Figure 1, and more information about the experiments including treatments and animal numbers is shown in Table S1. Pharmacological treatments were as follows: Iso (3 mg/kg/day, i.p.), zacopride (I_K1 agonist, 15 µg/kg/day, i.p.) (Tocris, England), chloroquine (I_K1 antagonist, 7.5 µg/kg/day, i.p.) (Sigma, USA), RS23597-190 (5-HT₄ receptor antagonist, 0.27 mg/kg/day, i.p.) (Tocris, England), and m-chlorophenylbiguanide (m-CPBG, 5-HT₃ receptor agonist, 0.19 mg/kg/day, i.p.) (Tocris, England). Age-matched control rats were administered with the same volume of saline. The dose of zacopride and chloroquine were applied according to our previous study (Liu et al., 2016 (link)) and preliminary experiment.

Intact Htr4 (WT) and mutant Htr4^delE5 (delE5) cDNA coding sequences (CDSs) obtained from the DG of cKO and WT mice were cloned into a mammalian expression plasmid as described in Supplementary Methods. HEK293T cells were transfected with the delE5, WT, or EGFP plasmids. Forty-eight hrs after the transfection, cells were incubated in stimulation buffer consisting of DMEM + 0.5 mM IBMX (Sigma Aldrich, St. Louis, MO) for 30 min. For cAMP induction, cells were further incubated in either stimulation buffer or 100 µM Zacopride (Tocris Bioscience, Bio-Techne, Minneapolis, MN) in stimulation buffer for 45 min. cAMP levels in the supernatants were measured by monoclonal anti-cAMP antibody-based direct cAMP ELISA kit according to the manufacturer’s guidelines (NewEast Biosciences, King of Prussia, PA). Four biological replicates and two technical replicates were performed for each experimental group. The cAMP level of each biological replicate was normalized to its mean protein concentration measured using Qubit protein assay kit (Thermo Fisher Scientific, Waltham, MA). See Supplementary Methods for detailed description.