Fig 2: Immunofluorescence (IF) staining of selected candidates in control and HCM hiPSC-cardiomyocytes. The same amount of control and HCM hiPSC-cardiomyocytes (20,000 cells per well) were seeded to the plate and cultured for 27 days prior to the staining. a Representative IF staining showing the expression of KLF15 in HCM and control cardiomyocytes (KLF15: red; ACTN2: green; blue: cell nuclei). b Representative IF staining showing disrupted ACTN2 in HCM cardiomyocytes compared to controls (ACTN2: green; blue: cell nuclei). c Representative IF staining showing suppressed ATP2A2 in HCM cardiomyocytes compared to controls (ATP2A2: red; blue: cell nuclei). Nuclei were stained by Hoechst. All overview images were taken at 20 × magnification, scale bar = 100 µm. All zoomed regions were taken under Zoom Factor = 1.5

Fig 3: Stress Influences Cellular Uptake of Anti-miRs in Neonatal Rat Ventricular Myocytes(A) NRVMs stained for ACTN2 after treatment with or without isoproterenol or phenylephrine for 24 hr. (B) Quantification of cross-sectional area (CSA) of NRVMs in the presence or absence of ISO or PE (per condition, five to six biological samples were generated, and per samples, ±22–41 cells were quantified). (C) Real-time PCR analysis of stress marker expression in NRVMs in the presence or absence of ISO or PE (n = 6 per condition). (D) Schematic representation of miR-208a and the Cy3-labeled anti-miR-208a. (E) Fluorescent images of NRVMs treated with increasing doses of Cy3-anti-miR-208a for 24 hr. (F) Quantification of Cy3 signal at different concentrations of anti-miR-208a. (G) Fluorescent images of NRVMs treated with 1 μM Cy3-anti-miR-208a for different time periods. (H) Quantification of Cy3 signal at different time points after 1 μM Cy3-anti-miR-208a. (I) Fluorescent images of NRVMs that were either left untreated or stimulated with ISO or PE for 8 hr, after which they received 1 μM Cy3-anti-miR-208a. (J) Quantification of total fluorescence (fluorescence intensity corrected for cell size, n = 8–12). Data are represented as mean fold change ± SEM. *p < 0.05 for anti-miR-208a treatment versus control treatment. Scale bars represent 50 μM.

Fig 4: Reprogrammed MEFs express cardiac specific proteins and organize them in a cross-striated manner A–I. Induction of TF overexpression for 7 days in MEFs transduced with only FUW.M2rtTA or the four listed combinations of TF modules. The reprogrammed cells were cultured on gelatin-coated plastic in low serum growth medium. Using double-antibody immunofluorescence analysis (Actn2/Red, Tnnt2/Green) we detected cells expressing both cardiac proteins and organizing them in a cross-striated manner resembling cardiomyocytes (B, D, F, H). We detected significantly more double-positive cells in MEFs transduced with G4T5MCMDSF. For each of the transcriptional module combinations we also detected double-positive cells without any obvious cross-striated cytoskeletal organization (C, E, G, I). No Actn2, or Tnnt2 cross-striated expression was detected in the negative control cells. J–N. The Tnnt2 expressing cells also stained positive for the atrial protein marker Nppa. O. Quantification of the fraction of cells staining positive for the Tnnt2 cardiac protein (low serum growth medium) as compared to the total number of cells (black columns) and measurement of the fraction of Tnnt2-expressing cells per square millimeter (red line). Results are based on biological triplicates. Error bars represent calculated standard deviation. All four cell groups had a significant increase in the number of Tnnt2(+) cells as compared to the negative control group (P<0.01). Cells transduced with either G4T5MCMDSF (P<0.01), G4T5MCMDSFM1S3 (P<0.05), G4T5MCMDSFM1S3 (P<0.01) also had a significant increase in the number of Tnnt2(+) cells as compared to cells transduced with G4T5MC.

Fig 5: miR combo reprograms pig and dog cardiac fibroblasts into cardiomyocyte-like cells.(A) Pig cardiac fibroblasts from the left ventricle were transfected with miR combo or the non-targeting miR negmiR. Fourteen days post-transfection, RNA was extracted and analyzed for the expression of the indicated cardiomyocyte-specific genes by qPCR. Expression values are shown relative to the house keeping gene Gapdh. N = 3. *P < 0.05, **P < 0.01, ***P < 0.001.(B) Pig cells were fixed fourteen days post-transfection and incubated with Actn2 binding antibodies to determine the number of cardiomyocyte-like cells. Representative images are shown (scale bar 100 μm) with quantification provided. Cardiomyocyte-like cell counts were determined from individual wells (N = 5). ***P < 0.001.(C) Dog cardiac fibroblasts from the left ventricle were transfected with miR combo or the non-targeting miR negmiR. Fourteen days post-transfection, RNA was extracted and analyzed for the expression of the indicated cardiomyocyte-specific genes by qPCR. Expression values are shown relative to the house keeping gene Gapdh. N = 3. *P < 0.05, **P < 0.01, ***P < 0.001.(D) Dog cells were fixed fourteen days post-transfection and incubated with Actn2 binding antibodies to determine the number of cardiomyocyte-like cells. Representative images are shown (scale bar 100 μm) with quantification provided. There were two populations of Actn2+ cells: low Actn2 expression and high Actn2 expression. Cell counts were determined for the high Actn2 expression population (N = 7). ***P < 0.001.