Fig. 5.

Activation of cyclic AMP/protein kinase A pathway inhibits diclofenac-induced cardiac reprogramming. a, b Immunocytochemistry for αMHC-GFP, cTnT, and DAPI. PGE2 treatment inhibited GHMT/diclofenac-induced cardiac protein expression in postnatal TTFs after 4 weeks. High-magnification views, insets, show the sarcomeric organization. Quantitative data are shown in b; n = 5 biologically independent experiments. See also Supplementary Figure 4. c, d Immunocytochemistry for αMHC-GFP, α-actinin, and DAPI. EP4 antagonist (ant) and diclofenac treatment increased generation of iCMs, while EP4 agonist (ago) inhibited GHMT/diclofenac-induced cardiac reprogramming in TTFs after 4 weeks. High-magnification views, insets, show the sarcomeric organization. Quantitative data are shown in d; n = 5 biologically independent experiments. e qRT-PCR analyses for cardiac gene expression in postnatal TTFs transduced with GHMT and treated with or without indicated reagents for 1 week; n = 3 biologically independent experiments. f, g FACS analyses for αMHC-GFP⁺ and cTnT⁺ cells. GHMT-transduced TTFs were cultured with diclofenac with or without forskolin or dibutyryl-cAMP (Db-cAMP) for 1 week. Quantitative data are shown in g; n = 3 biologically independent experiments. h qRT-PCR analyses for cardiac gene expression in postnatal TTFs treated as indicated for 1 week; n = 3 biologically independent experiments. i Concentration of intracellular cAMP in GHMT-transduced TTFs treated with the indicated EP antagonists (ant); n = 4 biologically independent experiments. One-way ANOVA with Tukey’s post hoc test was performed for b, d, e, g–i. All data are presented as mean ±SEM. *P < 0.05, **P < 0.01 vs. the relevant control. NS, not significant. Diclo, diclofenac. Scale bars represent 100 μm