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. 2020 Dec 29;10(1):e019685. doi: 10.1161/JAHA.120.019685

Figure 4. ExoCDNF treatment increases the level of phosphorylated AKT (p‐AKT) in rat isolated hearts and in mouse and human cardiomyocytes.

Figure 4

Cultures of (A) hiPSC‐dCM or (B) mouse cardiac myocytes were treated with CDNF (1 µmol/L/20 hours) or with wortmannin (0.3 µmol/L/15 minutes) before CDNF addition. In (C), rat isolated hearts were perfused with CDNF (1 µmol/L/5 minutes, preconditioning) before I/R either alone or in combination with wortmannin (0.3 µmol/L/5 minutes) before CDNF treatment. D, The peptide THPKTEL that binds to the KDEL‐R blocks CDNF‐induced PI3K/AKT activation. Isolated hearts were perfused with CDNF (1 µmol/L/5 minutes, preconditioning) before I/R either alone or in combination with THPKTEL or with the scrambled peptide DRATSAL (peptides added 5 minutes before CDNF treatment and during the 5 minutes CDNF treatment). E, CDNF‐ΔKTEL was unable to activate p‐AKT in isolated perfused hearts. Isolated hearts were perfused with CDNF‐ΔKTEL (1 µmol/L/5 minutes) before I/R. The intensities of the p‐AKT bands were normalized to total‐AKT levels. F, The STAT3 inhibitor (AG490, 10 µmol/L/5 minutes) and the PKC inhibitor (chelerythrine, 10 µmol/L/5 minutes) not abolished the protection generated by CDNF. Isolated hearts were perfused with CDNF (1 µmol/L/5 minutes, preconditioning) before I/R either alone or in combination with AG490, 10 µmol/L/5 minutes or chelerythrine, 10 µmol/L/5 minutes before CDNF treatment. The number of experiments is equal to the number of lanes. The data were expressed as means±SEM, with 1‐way ANOVA followed by Bonferroni post hoc tests. AKT indicates protein kinase B; CDNF, cerebral dopamine neurotrophic factor; and iPSC‐dCM, iPSC‐dCM, induced pluripotent stem cells differentiated into cardiomyocytes.