Global gene expression in adult cardiac fibroblasts transduced with cardiac transcription factor
ABSTRACT: Four transcription factors, GATA4, Hand2, MEF2C, Tbx5 (GHMT) activated cardiac gene expression in cardiac fibroblasts, suggesting that these factors are able to reprogram fibroblasts toward a cardaic cell fate. Total RNA isolated from adult cardiac fibroblasts transduced with empty retroviral vector or GHMT-retroviruses for 2, and 4 weeks.
Project description:During reprogramming of fibroblasts into cardiomyocyte-like cells by overexpression of transcription factors, GATA4, Hand2, Mef2C and Tbx5 (GHMT), H3K4Me2, an active histone code, shifts from fibroblast-exclusive peaks to cardiomyocyte-exclusive peaks. Important cardiac genes are gradually marked by this active histone marker. Mouse embryonic fibroblasts (MEFs) and neonatal mouse ventricular cardiomyocytes (NMVMs) represent fibroblasts and cardiomyocytes, respectively. Chromatins harvested from MEFs infected with retroviruses carrying GHMT at day 3, day 5, day 7 post-viral infection were prepared for immunoprecipitation.
Project description:The purpose of this experiment is to anlyze the transcriptomic changes associated with Notch inhibition, by DAPT treatment, during cardiac reprogramming mediated by GHMT (Gata4, Hand2, Mef2c anf Tbx5). Overall design: RNA-seq was performed on MEFs infected with GHMT (Gata4, Hand2, Mef2c anf Tbx5) and treated for 15 days with DMSO (vehicle) or DAPT.
Project description:Fibroblasts can be reprogrammed into cardiomyocyte-like cells by overexpressing transcription factors, GATA4, Hand2, Mef2C and Tbx5 (GHMT). A83-01, an inhibitor of ALK4, ALK5 and ALK7 and two microRNA, miR-1 and miR-133 increase the efficiency of cardiac reprogramming. RNA_Seq was performed to anyalyze effects of these factors on gene expression. Total RNAs were prepared from mouse embryonic fibroblasts (MEFs); Reprogramming fibroblasts including MEFs transduced with retroviruses encoding GHMT, MEFs transduced with with retroviruses encoding GHMT plus miR-1 and miR-133 (GHMT2m), MEFs transduced with with retroviruses encoding GHMT2m treated with A83-01, at day 7 after viral transduction; and neonatal mouse cardiomyocytes (NMCMs).
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/Hand2 (GHMT) transduction at 2 and 4 weeks after transduction (2W, 4W). Hand2 upregulated a panel of cardiac genes and suppressed cell cylce genes during cardiac reprogramming. Overall design: MEFs were used for negative control, mouse heart tissue for positive control. Gene expression profiles were compared among MEFs, iCMs and heart.
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: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. Overall design: In this study, we compared the global gene expression patterns of MEFs, iCMs cultured under the indicated conditions (FBS, SF, FFV), and the hearts. The iCMs were sorted as aMHC-GFP+ cells at 2W and 4W weeks after GMT transduction. We also analyzed the iCMs induced by MT transduction, and iCMs induced from TTFs by GMTMM.
Project description:Conversion of fibroblasts to functional cardiomyocytes represents a potential approach for restoring cardiac function following myocardial injury, but the technique thus far has been slow and inefficient. To improve the efficiency of reprogramming fibroblasts to cardiac-like myocytes (iCMs) by cardiac transcription factors (Gata4, Hand2, Mef2c, and Tbx5=GHMT), we screened 192 protein kinases and discovered that Akt/protein kinase B dramatically accelerates and amplifies this process. Approximately 50% of reprogrammed fibroblasts displayed spontaneous beating after three weeks of induction by Akt plus GHMT. Furthermore, addition of Akt1 to GHMT evoked a more mature cardiac phenotype for iCMs, as seen by enhanced polynucleation, cellular hypertrophy, gene expression, and metabolic reprogramming. Igf1 and Pi3 kinase acted upstream of Akt, whereas mTORC1 and Foxo3a acted downstream of Akt to influence fibroblast-to-cardiomyocyte reprogramming. These findings provide new insights into the molecular basis of cardiac reprogramming and represent an important step toward further application of this technique. Overall design: We performed RNA-Seq using either isolated adult mouse ventricular cardiomyocytes (CMs) or MEFs treated for three weeks with empty vector, GHMT (iCMs cell sorted using αMHC-GFP before RNA-Seq), or AGHMT (iCMs cell sorted using αMHC-GFP before RNA-Seq).
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. Overall design: Examine RNA expression profile in iCMs (mouse in vitro, mouse in vivo and human in vitro)
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:Background: Cardiac transcription factors are master regulators during heart development. Recently, some were shown to transdifferentiate noncardiac mesoderm cells and cardiac fibroblasts into cardiomyocytes. However, the individual roles of each transcription factors in activating cardiac gene program have not been elucidated. We examined cardiac-specific and genome-wide gene expression in fibroblasts induced with cardiac transcription factors Nkx2.5 (N), Tbx5 (T), Gata4 (G), Myocardin (M) alone or different combinations. Methodology/Principal Findings: We applied different combinations of human Nkx2.5 (N), Tbx5 (T), Gata4 (G) and Myocardin (M) lentiviruses into 10T1/2 fibroblasts. Immunostaining and quantitative reverse transcription polymerase chain reaction (qRT-PCR) showed that N, T, G or M alone did not induce expression of cardiac marker genes α-myosin heavy chain (αMHC) and cardiac troponin T (cTnT). Only T+M and T+G+M combinations induced αMHC and cTnT expression. Microarray-based gene ontology analysis revealed that T alone inhibited most genes involved in cardiac-related processes and activated genes involved in Wnt receptor signaling pathway and in aberrant processes. M alone inhibited genes involved in Wnt receptor signaling pathway and activated genes involved in cardiac-related processes and in aberrant processes. G alone inhibited genes involved in ectoderm development. T+G+M combination was the most effective activator of genes associated with cardiac-related processes including muscle cell differentiation, sarcomere, striated muscle contraction, regulation of heart contraction, and glucose metabolism and fatty acid oxidation (two significant forms of cardiomyocyte energy metabolism). And unlike T, M, G alone or T+M, T+G+M did not activate genes associated with aberrant processes. Conclusions: Tbx5, Gata4 and Myocardin play different roles in activating cardiac gene program and in avoiding aberrant gene program activation. The combination of T+G+M activated cardiac gene program and avoided aberrant gene program activation. Two weeks after doxycline induction, total RNA was isolated from 10T1/2-tTA cells infected with different combinations of Tbx5, Gata4, and Myocardin lentiviruses. Biological triplicated.