Systems-level prediction of candidate drugs with potential to induce transcriptional responses as observed in vivo during heart regeneration in the zebrafish. We predicted candidate compounds for repositioning based on a new computational strategy that matches multiple heart regeneration expression signatures with drug-induced expression profiles in the Connectivity Map (CMap).

Fisetin enhanced the viability of H9c2 cardiomyocytes. (A) Survival tests were performed in normoxia, HS or HS/R experiments. Different concentrations of fisetin (from 0 to 100 μM) were tested. In normoxia experiments cells were cultured during 24 h in normal conditions, while in HS experiments cells were cultured in serum free DMEM at 0.5% O₂, both in presence of the drug. In the HS/R experiments, cells were treated with fisetin following HS, during the whole reoxygenation phase. Cell survival was monitored by CyQuant assay. Results are expressed as the mean of three independent experiments performed in triplicate (error bars: SEM). (B) Effect of fisetin on cardiomyocytes viability upon HS/R treatment. Cell viability was measured in FACS by annexin V/PI staining: annexin V−/PI− cells were considered as viable. Normoxia: cells cultured under normoxia without fisetin (control group). HS/R: cells subjected to HS/R, treated with DMSO as vehicle control. HS/R + F: cells subjected to HS/R, treated with 15 μM fisetin. Results are expressed as the mean of three independent experiments. One-way ANOVA. Post-hoc analysis by Tukey. *P ≤ 0.05, ****P ≤ 0.0001.

Effect of fisetin on cardiomyocytes proliferation and apoptosis. Flow cytometry experiments were carried out to measure the amount of proliferating and apoptotic cells in each experimental group. (A) Proliferation of H9c2 cells assessed by Ki67 staining. Ki67+ cells are proliferating. Results are expressed as the mean of three independent experiments performed in triplicate. One-way ANOVA. Post-hoc analysis by Tukey. No significant difference: P > 0.05. (B) Dot plot representation of H9c2 apoptotic ratio measured by annexin V/PI staining. Annexin V−/PI− cells were considered as viable, annexin V+/PI− cells were considered as early apoptotic and annexin V+/PI+ cells were considered as late apoptotic. Normoxia: control group, cells cultured under normoxia. HS/R: cells subjected to HS/R, treated with DMSO as vehicle control. HS/R + F: cells subjected to HS/R, treated with 15 μM fisetin. Results are expressed as the mean of three independent experiments performed in triplicate. Two-way ANOVA. Post-hoc analysis by Tukey. ***P ≤ 0.001.

Fisetin regulated a panel of genes related to cardioprotection, proliferation and maturation. Gene expression was assessed by qRT-PCR in cells cultured under normoxia, subjected to HS/R and treated with DMSO as vehicle control (HS/R) or subjected to HS/R and treated with 15 μM fisetin (HS/R + F) during 24 h. Results are expressed as the mean of four independent experiments. Statistical significance was determined using a one-way ANOVA corrected for multiple testing with a Tukey-Kramer as post-test (corrected p-value < 0.05). Statistically significant results are indicated in bold with a star. Fold change < 1: genes down-regulated compared to normoxia (yellow). Fold change > 1: genes up-regulated compared to normoxia (blue).

Fisetin decreased ROS expression level in cardiomyocytes. The cell permeable, nonfluorescent dihydrorhodamine 123 probe enters the cell where it is oxidized by ROS to fluorescent rhodamine 123. Fluorescence intensity, proportional to ROS expression level, was measured by flow cytometry in each experimental group. Normoxia: control group; HS/R: cells subjected to HS/R, treated with DMSO as vehicle control; HS/R + F: cells subjected to HS/R, treated with 15 μM fisetin. Results are expressed as the mean of three independent experiments. One-way ANOVA. Post-hoc analysis by Tukey. **P ≤ 0.01.

Fisetin inhibited activation of caspases 8, 9 and 3 in H9c2 cells. Caspases activity was assessed by flow cytometry using active caspase staining kits specific to caspase 8, 9 and 3, respectively. Dot plots represent the percentage of cardiomyocytes expressing activated caspase 8, 9 or 3. Normoxia: control group. HS/R: cells subjected to HS/R, treated with DMSO as vehicle control. HS/R + F: cells subjected to HS/R, treated with 15 μM fisetin. Results are expressed as the mean of three independent experiments. Two-way ANOVA. Post-hoc analysis by Tukey. **P ≤ 0.01, *P ≤ 0.05.

Fisetin protected cardiomyocytes cultured under HS from DNA damage. The proportion of damaged cells in each experimental group was measured by flow cytometry using the anti-8 Hydroxyguanosine antibody as DNA damage marker. Normoxia: control group. HS: cells in HS, treated with DMSO as vehicle control. HS + F: cells in HS, treated with 15 μM fisetin. Results are expressed as the mean of three independent experiments. ANOVA two factors (1: biological replicates/random; 2: Treatments). Post-hoc analysis by Tukey. ***P ≤ 0.001, **P ≤ 0.01.

Schematic representation of fisetin cardioprotective effects on neonatal rat cardiomyocytes. Fisetin protects cells from apoptosis by targeting both intrinsic and extrinsic apoptotic pathways. Fisetin also enhances proliferation by activating expression of cell cycle activators.