The serotonin receptor 2b (5-HT2B) modulates heart remodeling following myocardial infarction via regulation of Hippo pathway

Figures

• 

  Figure 1. Regulation of the Hippo pathway by 5-HT2B in cardiomyocytes (A) Western blot confirmation that Ad-FLAG-5-HT2B infection in NRCMs resulted in the expression of FLAG- 5-HT2B that was absent in Ad-LacZ-infected controls (representative image of n = 3 independent experiments). (B) YAP transcriptional activity was enhanced in NRCMs following 5-HT2B overexpression (n = 5 independent experiments with five technical replications in each experiment). (C) Representative images of GFP-YAP nuclear translocation in NRCMs overexpressing 5-HT2B or control LacZ (scale bars, 100 μm). (D) Quantification of NRCMs with nuclear GFP-YAP showed that cells overexpressing 5-HT2B exhibited a higher proportion of nuclear YAP (n = 5 independent experiments with three technical replicates in each prep). (E and F) (E) Western blots and (F) quantification of band density showing a reduction in YAP phosphorylation in NRCMs overexpressing 5-HT2B (n = 3 independent experiments). (G and H) (G) Western blots and (H) quantification of band density showing a significant reduction in LATS1 phosphorylation in NRCMs overexpressing 5-HT2B (n = 3 independent experiments). (I and J) (I) Western blots and (J) quantification…

• 

  Figure 2. Effects of 5-HT2B overexpression on cardiomyocytes in vitro (A and B) (A) Representative immunofluorescence images detecting the proliferative marker Ki67 (scale bars, 100 μm) and (B) quantification of Ki67-positive cardiomyocyte nuclei suggests 5-HT2B overexpression increased proliferation (n = 5 independent experiments with three technical replicates in each experiment). (C and D) (C) Representative immunofluorescence images detecting the mitosis marker PHH3 (scale bars, 100 μm) and (D) quantification of PHH3-positive cardiomyocyte nuclei suggests 5-HT2B overexpression increased mitosis events (n = 5 independent experiments with three technical replicates in each experiment). (E and F) (E) Representative immunofluorescence images detecting the thymidine analog EdU (scale bars, 100 μm) and (F) quantification of EdU-positive cardiomyocyte nuclei suggests 5-HT2B overexpression increased EdU incorporation and therefore increased DNA replication (n = 5 independent experiments with three technical replicates in each experiment). (G and H) (G) Representative immunofluorescence images detecting TUNEL-labelled DNA breaks (scale bars, 50 μm), and (H) quantification of TUNEL-positive…

• 

  Figure 3. 5-HT2B regulates the Hippo pathway in human iPSC-CMs (A) Western blot confirmation that Ad-FLAG-5-HT2B infection in hiPSC-CMs resulted in expression of FLAG-5-HT2B that was absent in Ad-LacZ infected controls (n = 3 independent experiments). (B) YAP transcriptional activity was enhanced in hiPSC-CMs following 5-HT2B overexpression (n = 6 independent experiments). (C and D) (C) Western blots and (D) quantification of band density showing a reduction in YAP phosphorylation in hiPSC-CMs overexpressing 5-HT2B (n = 9 independent experiments). (E and F) (E) Representative immunofluorescence images detecting the thymidine analog EdU (scale bars, 50 μm) and (F) quantification of EdU-positive cardiomyocyte nuclei suggests 5-HT2B overexpression increased EdU incorporation and therefore increased DNA replication (n = 3 independent experiments). Data are presented as mean ± SEM. Statistical tests used: (B, D, and F) Student’s t test.

• 

  Figure 4. Effects of transgenic overexpression of 5-HT2B on cardiac function and remodeling 4 weeks following myocardial infarction (A and B) (A) Western blot analysis of protein lysates from 12-week-old 5-HT2BcTG (TG) and WT mice and (B) quantification of 5-HT2B overexpression (n = 4 in each group). (C) TG mice survival to 4 weeks was markedly lower than that of WT mice subjected to MI (WT-sham, n = 9; WT-MI, n = 11; TG-sham, n = 11; TG-MI, n = 18 at the beginning of experiments). (D) Serum cTnI titer 24 h post-MI was significantly elevated in TG mice compared to WT mice (WT-MI, n = 13; TG-MI, n = 19). (E) The LV mass/body weight ratio was increased 1 week post-MI in TG mice only (WT-sham, n = 8; WT-MI, n = 9; TG-sham, n = 10; TG-MI, n = 11). (F) The LV mass/body weight ratio was elevated in both WT and TG mice by 4 weeks post-MI and was significantly higher in TG mice (WT-sham, n = 9; WT-MI, n = 9; TG-sham, n = 9; TG-MI, n = 8). (G) Cardiac function as indicated by EF was decreased 1 week post-MI in TG mice only (WT-sham, n = 7; WT-MI, n = 8; TG-sham, n = 11; TG-MI, n = 8). (H) By 4 weeks post-MI, EF was not significantly different between WT and TG mice (WT-sham, n = 9; WT-MI, n = 5;…

• 

  Figure 5. Effects of modRNA-mediated overexpression of 5-HT2B on cardiac function and remodeling 4 weeks following myocardial infarction (A) Initial analysis was performed to validate modRNA-mediated overexpression of Htr2b. Wild-type mice were injected with either modRNA-Htr2b or modRNA-luciferase intracardially (n = 1 each). Western blot analysis showed expression of 5-HT2B protein in mouse injected with modRNA-Htr2b. (B) Overall survival of mice did not significantly differ between modRNA groups at 4 weeks post MI (sham, n = 8; modRNA-Luc, n = 17; modRNA-Htr2b, n = 17 at the beginning of experiment). (C) Serum cTnI titer 24 h post-MI was similar in both modRNA groups (modRNA-Luc, n = 9; modRNA-Htr2b, n = 10). (D) The LV mass/body weight ratio was unchanged 1 week post-MI (sham, n = 8; modRNA-Luc, n = 15; modRNA-Htr2b, n = 16). (E) The LV mass/body weight ratio was significantly higher 4 weeks post-MI in modRNA-Luc mice but not in modRNA-Htr2b mice (sham, n = 8; modRNA-Luc, n = 13; modRNA-Htr2b, n = 16). (F–G) Cardiac function indicated by EF was similar at 1 week (sham, n = 8; modRNA-Luc, n = 12; modRNA-Htr2b, n = 14) and 4 weeks (sham, n = 7; modRNA-Luc, n = 11; modRNA-Htr2b, n = 15)…

• 

  Figure 6. Serotonergic screening for YAP-activating compounds (A and B) (A) First-round screening data identified possible YAP modulating compounds in the H9C2 cardiomyoblast cell line (n = 3 independent experiments) and (B) the NIH/3T3 fibroblast cell line (n = 3 independent experiments). (C and D) 18 lead compounds were selected on the basis of differential YAP activity in H9C2 cells compared with NIH3T3cells. (E and F) Second-round screening data in isolated primary NRCMs and primary NRCFs (n = 3 independent experiments). (G–J) (G) Western blot and (H) quantification of active YAP expression in primary cardiomyocytes (NRCMs) treated with 1 μM SB204741 or 1 μM ipsapirone for 24 h (n = 3 independent experiments), and (I) western blot and (J) quantification of active YAP expression in primary fibroblasts (NRCFs) treated with 1 μM SB204741 or 1 μM ipsapirone for 24 h (n = 3 independent experiments) suggests that SB204741 modulates the Hippo pathway in cardiomyocytes but not in cardiac fibroblasts. Data are presented as mean ± SEM. Statistical tests used: (C–F) one-way analysis of variance followed by post hoc test for multiple pairwise comparisons. (H and J) Kruskal-Wallis test followed by…

• 

  Figure 7. The effects of treatment with Ipsapirone in a model of IR (A and B) (A) Representative immunofluorescence images of Ki67-stained NRCMs (scale bars = 100 μm) and (B) quantification of Ki67 detection showed ipsapirone increased cell proliferation in vitro (n = 3 independent experiments). (C) The LV mass/body weight ratio was significantly increased 2 weeks post-IR in ipsapirone-treated mice only. (D) Cardiac function indicated by EF was unchanged 2 weeks post-IR. (E) Heart weight normalized to body weight was similarly elevated in both IR groups. (F and G) (F) Representative Picrosirius Red-stained heart sections (scale bar, 500 μm) and (G) quantification of infarct size suggests that ipsapirone did not affect infarct size compared to vehicle control (n number for C,D,E,G, sham, n = 3; IR-DMSO, n = 5; IR-Ipsapirone, n = 5). Data are presented as mean ± SEM. Statistical tests used: (B), Student’s t test. (C, D, E, and G) one-way ANOVA followed by post hoc test for multiple pairwise comparisons. Numbers above the bars indicate p values.