In Vivo Direct Reprogramming by AAV Improves Cardiac Function after Myocardial Infarction. [RNA-seq]
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ABSTRACT: The overexpression of cardiac transcription factors, Gata4/Hand2/Tbx5, and Mef2c (GHT/M) has been indicated that directly reprogram cardiac fibroblasts (CFs) into induced cardiomyocytes (iCMs) in vivo, and improve cardiac function after MI in mice. Previous studies demonstrated in vivo reprogramming by using Sendai virus vectors. Here we show that in vivo reprogramming by using Adeno-associated virus (AAV) vectors. Additionally, we use Mef2cM3 (M3), a fusion of Mef2c with a strong MyoD transcriptional activation domain. RNA-seq revealed that directly cardiac reprogramming of GHT/M3 by AAV vector activated the cardiac program and concomitantly suppressed fibroblast and inflammatory signatures.
Project description:Fibrosis is important pathogenesis in heart failure with preserved ejection fraction (HFpEF). We previously reported that the overexpression of cardiac transcription factors, Mef2c/Gata4/Tbx5/Hand2 (MGTH) could directly reprogram cardiac fibroblasts (CFs) into induced CMs (iCMs) and reduce fibrosis. Here we show that in vivo cardiac reprogramming generated iCMs from resident CFs, improved cardiac function, and reversed fibrosis in HFpEF model using a novel transgenic mouse system. scRNA-seq of non-cardiomyocytes revealed that cardiac reprogramming suppressed fibroblastic gene expression via conversion of profibrotic profile to a quiescent state. Thus, in vivo cardiac reprogramming may be a promising approach for HFpEF.
Project description:Direct cardiac reprogramming converts fibroblasts into induced cardiomyocytes (iCMs) with the minimal combination of transcription factors, Gata4 (G), Mef2c (M), and Tbx5 (T). However, the induction of functional mature iCMs is inefficient and the mechanisms remain elusive. Mef2c is a central transcription factor in direct cardiac reprogramming. We investigated the effect of Mef2c isoforms(M1, M2, M6) and transcriptional activity (M2TAD) on cardiac reprogramming on cardiac reprogramming. Then, we found that the active form of Mef2c evoked epigenetic remodeling cooperating with p300 and promoted the maturation of iCMs.
Project description:Fibrosis is important pathogenesis in heart failure with preserved ejection fraction (HFpEF). We previously reported that the overexpression of cardiac transcription factors, Mef2c/Gata4/Tbx5/Hand2 (MGTH) could directly reprogram cardiac fibroblasts (CFs) into induced CMs (iCMs) and reduce fibrosis. Here we show that in vivo cardiac reprogramming generated iCMs from resident CFs, improved cardiac function, and reversed fibrosis in HFpEF model using a novel transgenic mouse system. Visium revealed spatial information on changes in gene expression in HFpEF.
Project description:Fibrosis is important pathogenesis in heart failure with preserved ejection fraction (HFpEF). We previously reported that the overexpression of cardiac transcription factors, Mef2c/Gata4/Tbx5/Hand2 (MGTH) could directly reprogram cardiac fibroblasts (CFs) into induced CMs (iCMs) and reduce fibrosis. Here we show that in vivo cardiac reprogramming generated iCMs from resident CFs, improved cardiac function, and reversed fibrosis in HFpEF model using a novel transgenic mouse system. RNA-seq revealed that the MGTH activated the cardiac program and concomitantly suppressed fibroblast and inflammatory signatures. Thus, cardiac reprogramming improves HFpEF via myocardial regeneration and anti-fibrosis.
Project description:FGF2, FGF10, and VEGF greatly promote cardiac reprogramming under defined serum-free conditions by enhancing the conversion of partially reprogrammed cells into fully reprogrammed functional iCMs. Fibroblasts can be directly reprogrammed into cardiomyocyte-like cells (iCMs) by overexpression of cardiac transcription factors, including Gata4, Mef2c, and Tbx5; however, this process is inefficient under serum-based culture conditions, in which the conversion of partially reprogrammed cells into fully reprogrammed functional iCMs has been a major hurdle. Here, we report that a combination of fibroblast growth factor (FGF) 2, FGF10, and vascular endothelial growth factor (VEGF), termed FFV, promoted cardiac reprogramming under defined serum-free conditions, increasing spontaneously beating iCMs by 100-fold compared with those under conventional serum-based conditions. Mechanistically, FFV activated multiple cardiac transcriptional regulators and converted partially reprogrammed cells into functional iCMs through the p38 mitogen-activated protein kinase and phosphoinositol 3-kinase/AKT pathways. Moreover, FFV enabled cardiac reprogramming with only Mef2c and Tbx5 through the induction of cardiac reprogramming factors, including Gata4. Thus, defined culture conditions promoted the quality of cardiac reprogramming, and this finding provides new insights into the mechanism of cardiac reprogramming.
Project description:Global gene expression profile of total 24460 probes in the iCMs. The gene expression shifts from a fibroblast state toward a cardiac-like phenotype by Gata4/Mef2c/Tbx5/Mesp1/Myocd (GMTMM) or GMTMM/miR-133 transduction at 7 days after transduction. MiR-133 silenced fibroblast signatures in parallel with cardiac gene activation, and Snai1 overexpression inhibited the effects of miR-133-mediated cardiac reprogramming. HCFs were used for negative control, human heart tissue for positive control. Gene expression profiles were compared among HCFs, iCMs and heart. 24460 probes were analyzed in each experiment.
Project description:Global gene expression patterns of the iCMs shift from a MEF state toward a cardiac-like phenotype by Gata4/Mef2c/Tbx5 (GMT) or GMT/miR-133 transduction at 3, 7 and 18 days after transduction (D3, D7 and D18) MiR-133 silenced fibroblast signatures in parallel with cardiac gene activation, and Snai1 overexpression inhibited the effects of miR-133-mediated cardiac reprogramming. MEFs were used for negative control, mouse heart tissue for positive control. Gene expression profiles were compared among MEFs, iCMs and heart. 23474 probes were analyzed in each experiment.
Project description:Cardiac transcription factors (TFs) directly reprogram fibroblasts into induced cardiomyocytes (iCMs), where Mef2c acts as a pioneer factor with Gata4 and Tbx5 (GT). However, generation of functional and mature iCMs is inefficient and molecular mechanisms underlying this process remains largely unknown. Here we found that transduction of transcriptionally activated Mef2c via fusion of the powerful MyoD transactivation domain increased generation of beating iCMs by 30-fold in combination with GT.
Project description:Recent studies have been successful at utilizing ectopic expression of transcription factors to generate induced cardiomyocytes (iCMs) from fibroblasts, albeit at a low frequency in vitro. This work investigates the influence of small molecules that have been previously reported to improve differentiation to cardiomyocytes as well as reprogramming to iPSCs in conjunction with ectopic expression of the transcription factors Hand2, Nkx2.5, Gata4, Mef2C, and Tbx5 on the conversion to functional iCMs. We utilized a reporter system in which the calcium indicator GCaMP is driven by the cardiac Troponin T promoter to quantify iCM yield. The TGFβ inhibitor, SB431542 (SB), was identified as a small molecule capable of increasing the conversion of both mouse embryonic fibroblasts and adult cardiac fibroblasts to iCMs up to ~5 fold. Further characterization revealed that inhibition of TGFβ by SB early in the reprogramming process led to the greatest increase in conversion of fibroblasts to iCMs in a dose-responsive manner. Global transcriptional analysis at Day 3 post-induction of the transcription factors revealed an increased expression of genes associated with the development of cardiac muscle in the presence of SB compared to the vehicle control. Incorporation of SB in the reprogramming process increases the efficiency of iCM generation, one of the major goals necessary to enable the use of iCMs for discovery-based applications and for the clinic. Mouse embryonic fibroblasts (MEFs) and adult mouse cardiac fibroblasts (CFs) were transfected with an empty vector (0F) or the combination of Hand2, Nkx2.5, Gata4, Mef2C, and Tbx5 (5F). Samples were exposed to the vehicle control (D, DMSO), SB431542 (SB, 0.5 uM MEF, 5 uM CF), or TGFb1 (T, 2 ng/mL) during culture. Transcription factor expression was induced at Day 0 and samples were isolated at Day 3 post-induction.
Project description:Reprogramming of cardiac fibroblasts into induced cardiomyocyte-like cells (iCMs) in situ represents a promising strategy for cardiac regeneration. A combination of three cardiac transcription factors, Gata4, Mef2c and Tbx5 (GMT), can convert fibroblasts into iCMs, albeit with low efficiency in vitro. Here, we screened 5,500 compounds in primary cardiac fibroblasts and found that a combination of the transforming growth factor (TGF)-β inhibitor SB431542 and the WNT inhibitor XAV939 increased reprogramming efficiency eight-fold when added to GMT-overexpressing cardiac fibroblasts. The small-molecules also enhanced the speed and the quality of cell conversion, as we observed beating cells as early as 1 week after reprogramming compared to 6–8 weeks with GMT alone. In vivo, mice exposed to GMT, SB431542, and XAV939 for 2 weeks after myocardial infarction showed significantly improved reprogramming and cardiac function compared to those exposed to only GMT. Human cardiac reprogramming was similarly enhanced upon TGF-b and WNT inhibition and was achieved most efficiently with GMT plus Myocardin. Thus, TGF-β and WNT inhibitors jointly enhance GMT-induced direct cardiac reprogramming from cardiac fibroblasts in vitro and in vivo and provide a more robust platform for cardiac regeneration.