MiR-133 promotes cardiac reprogramming by directly repressing Snai1 and silencing fibroblast signatures

Abstract

Fibroblasts can be directly reprogrammed into cardiomyocyte-like cells (iCMs) by overexpression of cardiac transcription factors or microRNAs. However, induction of functional cardiomyocytes is inefficient, and molecular mechanisms of direct reprogramming remain undefined. Here, we demonstrate that addition of miR-133a (miR-133) to Gata4, Mef2c, and Tbx5 (GMT) or GMT plus Mesp1 and Myocd improved cardiac reprogramming from mouse or human fibroblasts by directly repressing Snai1, a master regulator of epithelial-to-mesenchymal transition. MiR-133 overexpression with GMT generated sevenfold more beating iCMs from mouse embryonic fibroblasts and shortened the duration to induce beating cells from 30 to 10 days, compared to GMT alone. Snai1 knockdown suppressed fibroblast genes, upregulated cardiac gene expression, and induced more contracting iCMs with GMT transduction, recapitulating the effects of miR-133 overexpression. In contrast, overexpression of Snai1 in GMT/miR-133-transduced cells maintained fibroblast signatures and inhibited generation of beating iCMs. MiR-133-mediated Snai1 repression was also critical for cardiac reprogramming in adult mouse and human cardiac fibroblasts. Thus, silencing fibroblast signatures, mediated by miR-133/Snai1, is a key molecular roadblock during cardiac reprogramming.

Keywords: Snai1; cardiomyocyte; microRNA; reprogramming; transcription factor.

Figure 1. MiR-133 promotes Gata4/Mef2c/Tbx5-induced cardiac reprogramming

A, B FACS analyses for αMHC-GFP⁺ and cTnT⁺ cells 1 week after GMT transduction or miRNA transfection. Quantitative data are shown in (B) (n = 3). C, D FACS analyses for αMHC-GFP⁺ and cTnT⁺ cells 1 week after GMT and miRNA transduction. Quantitative data are shown in (D) (n = 3). E, F Dose dependency of miR-133-mediated cardiac induction with GMT. Quantitative data are shown in (F) (n = 3). G FACS analyses for α-actinin⁺ cells 1 week after transduction. H Immunocytochemisty for αMHC-GFP, α-actinin, and DAPI. GMT/miR-133 induced abundant αMHC-GFP and α-actinin expression 2 weeks after transduction. High-magnification views in insets show sarcomeric organization. GMT/miR-133 induced cTnT and ANP expression 2 weeks after transduction. Insets are high-magnification views. Data information: All data are presented as means ± SEM. *P < 0.05, **P < 0.01 versus relevant control. Scale bars represent 100 μm.

Figure 2. MiR-133 enhances generation of functional iCMs

A Time course of αMHC-GFP and cTnT expression by GMT or GMT/miR-133 transduction in MEFs. See also Supplementary Fig S2A. B qRT-PCR for cardiac and fibroblast gene expression in αMHC-GFP⁺ cells by GMT or GMT/miR-133 transduction (n = 4). Data were normalized against the values of GMT-iCMs at day 7 (Actn2,Myh6,Ryr2,Tnni3) or MEFs at day 0 (Col1a1,Fn1). See also Supplementary Fig S2B. C GMT/miR-133 induced expression of α-actinin with sarcomeric organization 1 week after transduction. D, E Spontaneous Ca²⁺ oscillations observed in MEF-derived iCMs (arrows) after 4 weeks of induction, corresponding to Supplementary Movie S1. Rhod-3 images at Ca²⁺ max and min are shown in the upper panels and Rhod-3 intensity trace is shown in the lower panel (D). Total number of Ca²⁺ oscillation⁺ cells in 10 randomly selected fields per well is shown in (E) (n = 3). F Spontaneously beating GMT/miR-133 iCMs 4 weeks after transduction (arrows), corresponding to Supplementary Movie S2. See also Supplementary Fig S2E and Movie S3. G Number of spontaneously beating cells in each well after transduction of mock, GMT, or GMT/miR-133 at the indicated time. H, I Mesp1-GFP⁻/Thy1⁺ MEFs were sorted and transduced with GMT or GMT/miR-133 (H). All cTnT⁺ cells were negative for Mesp1-GFP (I). J qRT-PCR for isl1 expression in the cells transduced with GMT or GMT/miR-133 (n = 3). Data information: All data are presented as means ± SEM. *P < 0.05, **P < 0.01 versus relevant control. Scale bars: 100 μm (C, F); 5 s (D).

Figure 3. MiR-133 silences fibroblast signatures and activates cardiac programs

A Heat-map image of microarray data illustrating the global gene expression pattern of MEFs, iCMs, and hearts. The iCMs were sorted as αMHC-GFP⁺ cells after 3 (D3), 7 (D7), and 18 (D18) days of GMT or GMT/miR-133 transduction. Differentially expressed genes between MEFs and hearts are shown (n = 1). B Differentially expressed genes between GMT-iCMs and GMT/miR-iCMs are shown (n = 1). See also Supplementaray Table S1. C, D GO analyses of the upregulated (C) and downregulated (D) genes in GMT/miR-iCMs at all stages. Top 10 GO categories are shown. Cardiac (C) and fibroblast-related (D) GO terms are shown in red. E The upregulated and downregulated genes in GMT/miR-iCMs at day 3 were analyzed by scatter plots. F The relative mRNA expression of cardiomyocyte, fibroblast, and epithelial cell genes in D7 GMT/miR-iCMs compared to D7 GMT-iCMs by microarray. G The relative mRNA expression of D7 GMT/miR-iCMs compared to D7 GMT-iCMs was determined by qRT-PCR (n = 3). Data information: Data were normalized by the values of GMT-iCMs. All data are presented as means ± SEM (G). *P < 0.05, **P < 0.01 versus relevant control.

Figure 4. Repression of Snai1 silences fibroblast profile and promotes cardiac reprogramming

A The relative mRNA expression of potential direct targets of miR-133 in D7 GMT/miR-iCMs compared to D7 GMT-iCMs by microarray. B Snai1 3′UTR contains two predicted miR-133a binding sites. Both are conserved among species, shown in red. C MiR-133a directly repressed WT Snai1 3′UTR in luciferase assay, and the repression was abolished when both of binding sites were mutated (n = 3). D Relative miR-133a expression in MEFs, GMT-iCMs, GMT/miR133-iCMs, and hearts (n = 3). E Relative mRNA expression of Snai1 in MEFs, GMT-iCMs, GMT/miR133-iCMs, and hearts (n = 3). F Western blot analyses for Snai1 expression in MEFs, MEFs transfected with miR-133 alone, and MEFs transduced with GMT and GMT/miR-133. G Relative mRNA expression of Snai1 in MEFs and MEFs transfected with siRNA against Snai1 (5, 15, 100 nM) (n = 3). H Relative mRNA expression of cardiac (Actn2,Ttn,Gja1,Nppa) and fibroblast genes (Fn1,Postn,Snai2) in MEFs transduced with GMT, GMT/si-Snai1, or GMT/miR-133 (n = 3). See also Supplementary Fig S3A. I, J FACS analyses for αMHC-GFP⁺ and cTnT⁺ cells 1 week after GMT transduction with si-Snai1 or miR-133 transfection. Quantitative data are shown in (J) (n = 3). K–M Immunocytochemisty for αMHC-GFP, α-actinin, cTnT, and DAPI. Snai1 suppression increased cardiac protein expression in GMT-transduced cells (M, n = 5). See also Supplementary Fig S3C. N Total number of Ca²⁺ oscillation⁺ cells in 10 randomly selected fields per well is shown (the left panel, n = 8). Spontaneously beating cells were counted in each well after 4 weeks of infection (the right panel, n = 3). See also Supplementary Fig S3D and Movie S4. Data information: All data are presented as means ± SEM. *P < 0.05, **P < 0.01 versus relevant control. Scale bars, 100 μm.

Figure 5. Overexpression of Snai1 inhibits cardiac reprogramming

A Western blot analyses for Snai1 expression in MEFs and MEFs transduced with GMT, GMT/miR-133, and GMT/miR-133 with Snai1 overexpression. B Heat-map image of microarray data for GMT-, GMT/miR-, and GMT/miR/Snai1-iCMs sorted as αMHC-GFP⁺ cells after 1 week of transduction (left panel, n = 1). Differentially expressed genes between GMT-iCMs and GMT/miR-iCMs are shown (see also Fig3B). Thirty-nine genes out of 46 upregulated genes were suppressed by Snai1 overexpression, while 105 genes out of 129 downregulated genes were increased with Snai1 transduction (right panel). C The relative mRNA expression of D7 GMT/miR-iCMs compared to D7 GMT-iCMs was determined by qRT-PCR (n = 3). Relative mRNA expression of cardiac (Actn2,Myh6,Ryr2) and fibroblast genes (Col1a1,Fn1,Postn) in MEFs transduced with GMT and GMT/miR-133 with or without Snai1 overexpression (n = 3). D, E FACS analyses for αMHC-GFP ⁺ and cTnT⁺ cells 1 week after GMT and GMT/miR-133 transduction with or without Snai1 overexpression. Quantitative data are shown in (E) (n = 3). F–H Immunocytochemisty for αMHC-GFP, α-actinin, cTnT, and DAPI. Snai1 overexpression suppressed cardiac protein expression in GMT/miR-133-transduced cells (H, n = 5). I Numbers of Ca²⁺ oscillation⁺ cells in 10 randomly selected fields per well are shown (left panel, n = 8). Number of spontaneously beating cells in each well after 4 weeks of infection (right panel, n = 3). Data information: All data are presented as means ± SEM. *P < 0.05, **P < 0.01 versus relevant control. Scale bars, 100 μm.

Figure 6. MiR-133-mediated Snai1 repression is critical for cardiac reprogramming in adult cardiac fibroblasts

A, B FACS analyses for αMHC-GFP ⁺ and cTnT⁺ cells in adult CFs 1 week after GMT and GMT/miR-133 transduction with or without Snai1 overexpression. Quantitative data are shown in (B) (n = 3). C Relative mRNA expression of cardiac (Actn2,Myh6,Gja1,Scn5a) and fibroblast genes (Col1a1,Col3a1,Fn1) in adult CFs transduced with GMT and GMT/miR-133 with or without Snai1 overexpression (n = 3). D, E Immunocytochemisty for αMHC-GFP and α-actinin with DAPI staining in GMT, GMT/miR-133, or GMT/miR-133/Snai1-transduced adult CFs 4 weeks after transduction. High-magnification views in insets show sarcomeric organization. Quantitative data are shown in (E) (n = 5). F, G Total number of Ca²⁺ oscillation⁺ cells in 10 randomly selected fields per well is shown in (F) (n = 3). Spontaneous Ca²⁺ oscillations observed in adult CF-derived GMT/miR-133-iCMs (arrows in G), corresponding to Supplementary Movie S5. The Rhod-3 images and intensity trace are shown in (G). H Relative Snai1 mRNA and miR-133a expression in adult CFs, GMT-iCMs, and GMT/miR133-iCMs (n = 3). I Relative mRNA expression of cardiac (Actn2,Actc1,Slc8a1,Nppa) and fibroblast genes (Col1a2,Col5a2,Snai2) in adult CFs transduced with GMT, GMT/si-Snai1, or GMT/miR-133 (n = 3). J, K Immunocytochemisty for αMHC-GFP and α-actinin in GMT or GMT/si-Snai1 transduced adult CFs 4 weeks after transduction. High-magnification views in insets show sarcomeric organization. Quantitative data are shown in (K) (n = 5). Data information: All data are presented as means ± SEM. *P < 0.05, **P < 0.01 versus relevant control. Scale bars, 100 μm.

Figure 7. MiR-133/Snai1 pathway is critical for human cardiac reprogramming

A, B FACS analyses for cTnT⁺ cells in HCFs 1 week after GMTMM, GMTMM/miR-133, and GMTMM/miR-133/Snai1 transduction. Quantitative data are shown in (B) (n = 4). C Heat-map image of microarray data illustrating the global gene expression pattern of HCFs, GMTMM-HCFs, and GMTMM/miR-133-HCFs after 1 week of transduction (n = 1, left panel). The differentially expressed genes between GMTMM- or GMTMM/miR-133-HCFs and HCFs are shown. Cardiac genes were upregulated and fibroblast genes were downregulated by transduction of the reprogramming factors, as shown in the scatter plot analyses (right panel). D 399 genes were upregulated and 264 genes were downregulated by the addition of miR-133 to GMTMM. E GO term analyses of the upregulated and downregulated genes, shown in (D). Cardiac- and fibroblast-related GO terms are shown. F The mRNA expression of Snai1 in HCFs, GMTMM-HCFs, and GMTMM/miR-133-HCFs (n = 3). G Snai1 restoration counteracted the effects of miR-133. 309 out of 399 upregulated genes were suppressed by Snai1 overexpression, while 214 out of 264 downregulated genes were increased with Snai1. See also Fig7D. H Relative mRNA expression of cardiac (Myh6, Actn2,Ttn,Nppa) and fibroblast genes (Col1a1,Fn1,Postn) in GMTMM-, GMTMM/miR-133-, and GMTMM/miR-133/Snai1-HCFs after 1 week of transduction (n = 3). I, J Immunocytochemisty for α-actinin and DAPI. Snai1 overexpression suppressed cardiac protein expression in GMTMM/miR-133-transduced HCFs (J, n = 10). High-magnification view in inset shows sarcomeric organization. Data information: All data are presented as means ± SEM. *P < 0.05, **P < 0.01 versus relevant control. Scale bars, 100 μm.