Fig 2: HO-1 does not influence the efficiency of hiPSCs.2 differentiation to cardiomyocytes. (A) Immunofluorescence analysis of markers of three germ-layers (Vimentin, α-SMA, GATA4, AFP, and NFH) in spontaneously differentiated WT (upper panel) and HO-1 KO (bottom panel) hiPSC.3 via embryoid bodies. Bar indicates 100 µm. qRT-PCR analysis of expression of cardiac mesoderm markers: (B) ISL1, (C) GATA6, (D) TNNT2 and (E) MIXL1. Expression was normalized to EEF-2 levels. (F) Flow cytometric analysis of direct cardiomyocyte differentiation efficiency (based on TNNT2 expression) of WT hiPSC.2 treated with tin protoporphyrin IX (SnPP), cobalt protoporphyrin IX (CoPP) and hemin. Ctr1-DMSO control for SnPP and CoPP, ctr2-2 µM NaOH control for hemin. Bars represent mean ± SD of N = 3 experiments. Dots, squares and triangles represent each replicate for corresponding groups. * p < 0.05, one-way ANOVA test.

Fig 3: Transgene Expression from the miRNA-122- and miRNA-206-Regulated Vectors Is Inhibited in Tissues Expressing the Corresponding miRNAs(A–G) Female mice of C57BL/6J × FVB strain 4 weeks after intravenous scAAV9 vectors administration (n = 3 mice/group): (A) qPCR quantification of scAAV genomes based on copies of ITR region detected with qPCR using TaqMan probe, normalized to 18S gene copies; (B) qPCR analysis of human HO-1 (HMOX1) transcript level; (C) ELISA for human HO-1 protein; (D) qPCR analysis of relative miRNA-122 expression in hearts and livers of female and male mice of C57BL/6J × FVB strain, normalized to U6 snRNA level. Male mice of C57BL/6J × FVB strain 4 weeks after intravenous scAAV9 vectors administration (n = 3 mice/group): (E) qPCR quantification of scAAV genomes based on copies of ITR region detected with qPCR using TaqMan probe, normalized to 18S gene copies; (F) qPCR analysis of human HO-1 (HMOX1 gene) transcript level; and (G) ELISA for human HO-1 protein. Bars represent mean ± SEM in all graphs.

Fig 4: Transgene Expression from the miRNA-122- and miRNA-206-Regulated Vectors Is Inhibited in Cell Lines Expressing the Corresponding miRNAs(A) qPCR analysis of relative miRNA-122 expression in different cell lines, normalized to U6 snRNA. Bars represent mean +/- SEM (n = 3-5) Representative western blot analysis of HO-1 protein level in: (B) HEK293 cells 7 days after transduction with scAAV9-HO1 (positive control) or scAAV9-HO1-iTS, and hypoxic conditions (0.5% O2) were applied 24 h before protein isolation; (C) HEK293 cells 7 days after transduction with scAAV9-HO1 (positive control) or scAAV9-HO1-TS, and hypoxic conditions (0.5% O2) were applied 24 h before protein isolation; (D) AML12 cells 72 h after transfection with pdAAV-HO-TS or pdAAV-HO1-iTS plasmid; (E) differentiated C2C12 cells 7 days after transduction with scAAV9-HO-TS or scAAV9-HO1-iTS vectors; (F) undifferentiated C2C12 cells 72 h after transfection with pdAAV-HO-TS or pdAAV-HO1-iTS plasmid; and (G) HL-1 cells 72 h after transfection with pdAAV-HO-TS or pdAAV-HO1-iTS plasmid. All experiments were performed in duplicate and were repeated at least three times. In all western blot analyses, a-tubulin served as a loading control.

Fig 5: Stimulation of HO-1 expression by cobalt protoporphyrin IX (CoPP) does not influence the metabolic activity of hiPSC-CMs. Seahorse analysis of hiPSC.3-CMs treated with CoPP (A–D). (A) Graphical illustration of Seahorse results. (B) Basal respiration, (C) maximal respiration and (D) ATP production. Tetramethylrhodamine (TMRM) assay of (E) hiPSC.2-CMs and (F) hiPSC.3-CMs treated with CoPP. Bars represent median ± SD of N = 2 experiments. Dots, squares and triangles represent each replicate for corresponding groups, one-way ANOVA test.