Cardiomyocyte‐Specific Overexpression of Activated Yes‐Associated Protein Modified‐RNA Promotes Cardiomyocyte Proliferation and Myocardial Regeneration

Figures

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  Figure 1. CM‐SMRTs drives cardiomyocyte‐specific YAP5SA and GFP expression. A, The sequences of the modRNA constructs composing the YAP5SACM‐SMRTs and GFPCM‐SMRTs are displayed as a schematic. B, hiPSC‐CMs were transfected with YAP5SACM‐SMRTs at the indicated concentration of ktm‐YAP5SA, and the abundance of total YAP, p‐YAP, and GAPDH was evaluated via Western blot 48 hours later. C, YAP and p‐YAP measurements were normalized to GAPDH to control for unequal loading. D and E, hiPSC‐CMs were transfected with YAP5SACM‐SMRTs (2.5 μg ktm‐YAP5SA and 1.25 μg miR1/208RE‐L7Ae) or GFPCM‐SMRTs (2.5 μg ktm‐GFP and 1.25 μg miR1/208RE‐L7Ae), and the abundance of (D) total YAP, p‐YAP, and cTnT or (E) GFP was evaluated via Western blot at the indicated time points. GAPDH abundance was also evaluated to control for unequal loading. F, GFP fluorescence (green) was monitored in hiPSC‐CMs at the indicated time points after GFPCM‐SMRTs transfection. G, GFP fluorescence was monitored in HUVECs and hiPSC‐CFs 24 hours and 3 days after transfection with GFP modRNA or GFPCM‐SMRTs. Scale bar=200 μm. N=3 biological replicates per experimental condition. *P<0.05, **P<0.01, ***P<0.001. Statistical significance was…

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  Figure 2. Treatment with YAP5SACM‐SMRTs promotes a more proliferative, less mature phenotype in cultured hiPSC‐CMs. A through F, hiPSC‐CMs were transfected with YAP5SACM‐SMRTs or GFPCM‐SMRTs. A, Forty‐eight hours later, the abundance of mRNA transcripts for YAP and 2 downstream targets of YAP signaling, CTGF and CYR61, were measured via RT‐qPCR. B, Cells were stained for the expression of YAP and cTnT, nuclei were counterstained with DAPI, and the percentage of hiPSC‐CMs that were YAP‐positive was calculated. C, Abundance of mRNA transcripts for the proliferation/cell‐cycle–activity markers Ki67 (Ki67), PCNA, and CCND2 was measured via RT‐qPCR. D through F, Cells were stained for the expression of the proliferation markers (D) Ki67, (E) PH3, or (F) AuroraB, and for cTnT, nuclei were counterstained with DAPI, and the percentage of cells that were positive for each proliferation marker was calculated. G, hiPSC‐CMs cell counts were determined before (0 day) and 14 days after transfection with YAP5SACM‐SMRTs or CM‐SMRTs driving luciferase expression (LucCM‐SMRTs). H, hiPSC‐CMs were transfected with YAP5SACM‐SMRTs or GFPCM‐SMRTs; 48 hours later, the abundance of mRNA transcripts for the…

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  Figure 3. Treatment with YAP5SACM‐SMRTs improved recovery from MI in mice. A, Experimental timeline is displayed as a schematic. B through F, MI was experimentally induced in mice, and then the animals were treated with (B) GFPCM‐SMRTs or (C through F) YAP5SACM‐SMRTs (MI+YAP5SACM‐SMRTs) or the delivery vehicle (MI+vehicle). A third group of animals (the Sham group) underwent all surgical procedures for MI induction except the ligation step and recovered without YAP5SACM‐SMRTs or vehicle administration. B, Two days after MI and treatment administration, GFP fluorescence (green) was visualized in heart sections stained for the expression cTnT (red); nuclei were counterstained with DAPI (blue). Scale bar=50 μm. C through E, Echocardiographic images were collected before MI induction (day 0) and 1, 7, 14, 21, and 28 days afterward. C, Representative images are displayed for each animal group at the indicated time points. D and E, Left ventricular (D) ejection fraction, (E) fractional shortening, and (F) radial strain were determined from images collected at the indicated time points. Hearts were harvested 28 days after Sham surgery or MI induction and treatment. G, Representative images of…

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  Figure 4. Treatment with YAP5SACM‐SMRTs promoted cardiomyocyte proliferation in infarcted mouse hearts. A through C, Sections were collected from the border zone of the infarct in the hearts of mice from the MI+YAP5SACM‐SMRTs and MI+vehicle groups on day 28 after MI induction and then stained for the expression of (A) YAP or the proliferation markers (B) Ki67 or (C) PH3 and cTnT; nuclei were counterstained with DAPI. Scale bar=50 μm. D, Sections were collected from the border zone of the infarct in the hearts of mice from the MI+YAP5SACM‐SMRTs and MI+vehicle groups, and from the corresponding region of hearts from the Sham group on day 28 and then stained with WGA (light blue) to identify cell borders and for the expression of cTnT. nuclei were counterstained with DAPI. Scale bar=20 μm. N=5 biological replicates per experimental condition. **P<0.01 and ***P<0.001. Statistical significance was tested via unpaired t test in (A) through (C) and 1‐way ANOVA with Tukey test in (D). CM indcates cardiomyocyte; CM‐SMRTS, cardiomyocyte‐specific modRNA translation system; DAPI, 4′,6‐diamidino‐2‐phenylindole; cTnT, cardiac troponin T; Ki67, activity markers Ki67; GFP, green fluorescent protein; MI,…