Expression Of Cardiac Transcription Factors Research Articles

Smad4 mediated BMP2 signal is essential for the regulation of GATA4 and Nkx2.5 by affecting the histone H3 acetylation in H9c2 cells

BMP2 signaling pathway plays critical roles during heart development, Smad4 encodes the only common Smad protein in mammals, which is a pivotal nuclear mediator. Our previous studies showed that BMP2 enhanced the expression of cardiac transcription factors in part by increasing histone H3 acetylation. In the present study, we tested the hypothesis that Smad4 mediated BMP2 signaling pathway is essential for the expression of cardiac core transcription factors by affecting the histone H3 acetylation. We successfully constructed a lentivirus-mediated short hairpin RNA interference vector targeting Smad4 (Lv-Smad4) in rat H9c2 embryonic cardiac myocytes (H9c2 cells) and demonstrated that it suppressed the expression of the Smad4 gene. Cultured H9c2 cells were transfected with recombinant adenoviruses expressing human BMP2 (AdBMP2) with or without Lv-Smad4. Quantitative real-time RT-PCR analysis showed that knocking down of Smad4 substantially inhibited both AdBMP2-induced and basal expression levels of cardiac transcription factors GATA4 and Nkx2.5, but not MEF2c and Tbx5. Similarly, chromatin immunoprecipitation (ChIP) analysis showed that knocking down of Smad4 inhibited both AdBMP2-induced and basal histone H3 acetylation levels in the promoter regions of GATA4 and Nkx2.5, but not of Tbx5 and MEF2c. In addition, Lv-Smad4 selectively suppressed AdBMP2-induced expression of HAT p300, but not of HAT GCN5 in H9c2 cells. The data indicated that inhibition of Smad4 diminished both AdBMP2 induced and basal histone acetylation levels in the promoter regions of GATA4 and Nkx2.5, suggesting that Smad4 mediated BMP2 signaling pathway was essential for the regulation of GATA4 and Nkx2.5 by affecting the histone H3 acetylation in H9c2 cells.

Curcumin-Mediated Cardiac Defects in Mouse is Associated with a Reduced Histone H3 Acetylation and Reduced Expression of Cardiac Transcription Factors

Histone acetylation plays an important role in heart development. However, the mechanism(s) remains unclear. This study was designed to evaluate the effect of curcumin-caused histone hypo-acetylation on the development of mouse embryonic heart and the expression of cardiac transcription factors in vivo. The results showed that curcumin treatment significantly decreased histone acetylase activity and histone acetylation level in mouse embryonic heart. In curcumin-treated mice, the hearts on E11.5 were smaller with thinner ventricular wall and a delayed development of trabeculae and ventricular septum compared with the controls. The ventricular septum was complete on E14.5; however, the ventricular wall and septum were thinner with fewer trabeculae than those in the controls. On E17.5, the cardiac structure appeared normal, but the ventricular wall and septum were thinner. The expression of GATA4, Nkx2.5 and Mef2c in the heart on E11.5 and E14.5 was decreased significantly as compared to the controls. There was no significant difference in Mef2c expression on E17.5 between curcumin-treated group and the controls, while GATA4 and Nkx2.5 expression remained significantly reduced. These results indicate that inhibition of histone acetylation by curcumin can reduce the expression of the cardiac transcription factors resulting in an abnormal heart development in mice.

P300-Mediated Histone Acetylation is Essential for the Regulation of GATA4 and MEF2C by BMP2 in H9c2 Cells

Histone acetylase (HAT) p300 plays an important role in the regulation of cardiac gene expression. During cardiac development, bone morphogenetic protein (BMP)-2 induces the expression of cardiac transcription factors. However, the underlying molecular mechanism(s) is not clear. In the present study, we tested the hypothesis that p300-mediated histone acetylation was essential for the regulation of cardiac transcription factors by BMP2. Cultured rat H9c2 embryonic cardiac myocytes (H9c2 cells) were transfected with recombinant adenoviruses expressing human BMP2 (AdBMP2) with or without curcumin, a specific p300-HAT inhibitor. Quantitative real-time RT-PCR analysis showed that curcumin substantially inhibited both AdBMP2-induced and basal expression levels of cardiac transcription factors GATA4 and MEF2C, but not Tbx5. Similarly, chromatin immunoprecipitation (ChIP) analysis showed that curcumin inhibited both AdBMP2-induced and basal histone H3 acetylation levels in the promoter regions of GATA4 and MEF2C, but not of Tbx5. In addition, curcumin selectively suppressed AdBMP2-induced expression of HAT p300, but not HAT GCN5 in H9c2 cells. The data indicated that inhibition of histone H3 acetylation with curcumin diminished BMP2-induced expression of GATA4 and MEF2C, suggesting that p300-mediated histone acetylation was essential for the regulation of GATA4 and MEF2C by BMP2 in H9c2 cells.

Reprogramming of human fibroblasts toward a cardiac fate

Reprogramming of mouse fibroblasts toward a myocardial cell fate by forced expression of cardiac transcription factors or microRNAs has recently been demonstrated. The potential clinical applicability of these findings is based on the minimal regenerative potential of the adult human heart and the limited availability of human heart tissue. An initial but mandatory step toward clinical application of this approach is to establish conditions for conversion of adult human fibroblasts to a cardiac phenotype. Toward this goal, we sought to determine the optimal combination of factors necessary and sufficient for direct myocardial reprogramming of human fibroblasts. Here we show that four human cardiac transcription factors, including GATA binding protein 4, Hand2, T-box5, and myocardin, and two microRNAs, miR-1 and miR-133, activated cardiac marker expression in neonatal and adult human fibroblasts. After maintenance in culture for 4-11 wk, human fibroblasts reprogrammed with these proteins and microRNAs displayed sarcomere-like structures and calcium transients, and a small subset of such cells exhibited spontaneous contractility. These phenotypic changes were accompanied by expression of a broad range of cardiac genes and suppression of nonmyocyte genes. These findings indicate that human fibroblasts can be reprogrammed to cardiac-like myocytes by forced expression of cardiac transcription factors with muscle-specific microRNAs and represent a step toward possible therapeutic application of this reprogramming approach.

Bone morphogenetic protein-2 enhances the expression of cardiac transcription factors by increasing histone H3 acetylation in H9c2 cells

Bone morphogenetic protein (BMP)‑2 induces the expression of cardiac transcription factors during early heart development, however, the underlying mechanisms for this are not clear. Our previous studies indicated that histone acetylation is critical in the regulation of cardiac gene expression. In the present study, the hypothesis that BMP2 enhances the expression of cardiac transcription factors by increasing histoneH3 acetylation was tested. Cultured H9c2 rat embryonic cardiac myocytes were transfected with adenoviruses expressing human BMP2 (AdBMP2). Real‑time RT‑PCR, western blotting, chromatin immunoprecipitation (ChIP) and colorimetric assays were employed to determine gene expression, histoneH3 acetylation levels and histone acetylase (HAT) activities. The mRNA expression levels of BMP2, GATA4, MEF2C and p300, but not of Tbx5 and GCN5, were significantly upregulated following transfection with AdBMP2. Similarly, the histoneH3 acetylation levels were enhanced in the whole chromatin and in the promoter regions of GATA4 and MEF2C, but not Tbx5, in the transfected cells. The HAT activities were also enhanced. These results indicate that BMP2 is able to upregulate the expression of the cardiac transcription factors GATA4 and MEF2C, in part by increasing histoneH3 acetylation in the promoter regions of these genes.

Directed cardiomyocyte differentiation from human pluripotent stem cells by modulating Wnt/β-catenin signaling under fully defined conditions

The protocol described here efficiently directs human pluripotent stem cells (hPSCs) to functional cardiomyocytes in a completely defined, growth factor- and serum-free system by temporal modulation of regulators of canonical Wnt signaling. Appropriate temporal application of a glycogen synthase kinase 3 (GSK3) inhibitor combined with the expression of β-catenin shRNA or a chemical Wnt inhibitor is sufficient to produce a high yield (0.8-1.3 million cardiomyocytes per cm(2)) of virtually pure (80-98%) functional cardiomyocytes in 14 d from multiple hPSC lines without cell sorting or selection. Qualitative (immunostaining) and quantitative (flow cytometry) characterization of differentiated cells is described to assess the expression of cardiac transcription factors and myofilament proteins. Flow cytometry of BrdU incorporation or Ki67 expression in conjunction with cardiac sarcomere myosin protein expression can be used to determine the proliferative capacity of hPSC-derived cardiomyocytes. Functional human cardiomyocytes differentiated via these protocols may constitute a potential cell source for heart disease modeling, drug screening and cell-based therapeutic applications.

PARM-1 promotes cardiomyogenic differentiation through regulating the BMP/Smad signaling pathway

PARM-1, prostatic androgen repressed message-1, is an endoplasmic reticulum (ER) molecule that is involved in ER stress-induced apoptosis in cardiomyocytes. In this study, we assessed whether PARM-1 plays a role in the differentiation of stem cells into cardiomyocytes. While PARM-1 was not expressed in undifferentiated P19CL6 embryonic carcinoma cells, PARM-1 expression was induced during cardiomyogenic differentiation. This expression followed expression of mesodermal markers, and preceded expression of cardiac transcription factors. PARM-1 overexpression did not alter the expression of undifferentiated markers and the proliferative property in undifferentiated P19CL6 cells. Expression of cardiac transcription factors during cardiomyogenesis was markedly enhanced by overexpression of PARM-1, while expression of mesodermal markers was not altered, suggesting that PARM-1 is involved in the differentiation from the mesodermal lineage to cardiomyocytes. Furthermore, overexpression of PARM-1 induced BMP2 mRNA expression in undifferentiated P19CL6 cells and enhanced both BMP2 and BMP4 mRNA expression in the early phase of cardiomyogenesis. PARM-1 overexpression also enhanced phosphorylation of Smads1/5/8. Thus, PARM-1 plays an important role in the cardiomyogenic differentiation of P19CL6 cells through regulating BMP/Smad signaling pathways, demonstrating a novel role of PARM-1 in the cardiomyogenic differentiation of stem cells.

Tyrosine hydroxylase is expressed during early heart development and is required for cardiac chamber formation

Tyrosine hydroxylase (TH) is the first and rate-limiting enzyme in catecholamine biosynthesis. Whereas the neuroendocrine roles of cathecolamines postnatally are well known, the presence and function of TH in organogenesis is unclear. The aim of this study was to define the expression of TH during cardiac development and to unravel the role it may play in heart formation. We studied TH expression in chick embryos by whole mount in situ hybridization and by quantitative reverse transcription-polymerase chain reaction and analysed TH activity by high-performance liquid chromatography. We used gain- and loss-of-function models to characterize the role of TH in early cardiogenesis. We found that TH expression was enriched in the cardiac field of gastrulating chick embryos. By stage 8, TH mRNA was restricted to the splanchnic mesoderm of both endocardial tubes and was subsequently expressed predominantly in the myocardial layer of the atrial segment. Overexpression of TH led to increased atrial myosin heavy chain (AMHC1) and T-box 5 gene (Tbx5) expression in the ventricular region and induced bradyarrhythmia. Similarly, addition of l-3,4-dihydroxyphenylalanine (l-DOPA) or dopamine induced ectopic expression of cardiac transcription factors (cNkx2.5, Tbx5) and AMHC1 as well as sarcomere formation. Conversely, blockage of dopamine biosynthesis and loss of TH activity decreased AMHC1 and Tbx5 expression, whereas exposure to retinoic acid (RA) induced TH expression in parallel to that of AMHC1 and Tbx5. Concordantly, inhibition of endogenous RA synthesis decreased TH expression as well as that of AMHC1 and Tbx5. TH is expressed in a dynamic pattern during the primitive heart tube formation. TH induces cardiac differentiation in vivo and it is a key regulator of the heart patterning, conferring atriogenic identity.

Mesp1 Acts as a Master Regulator of Multipotent Cardiovascular Progenitor Specification

During embryonic development, multipotent cardiovascular progenitor cells are specified from early mesoderm. Using mouse ESCs in which gene expression can be temporally regulated, we have found that transient expression of Mesp1 dramatically accelerates and enhances multipotent cardiovascular progenitor specification through an intrinsic and cell autonomous mechanism. Genome-wide transcriptional analysis indicates that Mesp1 rapidly activates and represses a discrete set of genes, and chromatin immunoprecipitation shows that Mesp1 directly binds to regulatory DNA sequences located in the promoter of many key genes in the core cardiac transcriptional machinery, resulting in their rapid upregulation. Mesp1 also directly represses the expression of key genes regulating other early mesoderm and endoderm cell fates. Our results demonstrate that Mesp1 acts as a key regulatory switch during cardiovascular specification, residing at the top of the hierarchy of the gene network responsible for cardiovascular cell-fate determination.

Pre-Treatment of Mesenchymal Stem Cells With a Combination of Growth Factors Enhances Gap Junction Formation, Cytoprotective Effect on Cardiomyocytes, and Therapeutic Efficacy for Myocardial Infarction

The goal of this study was to investigate the effect of pre-treatment of mesenchymal stem cells (MSCs) with growth factors (GFs) on cardiomyogenic differentiation, cytoprotective action on cardiomyocytes (CMCs), and their therapeutic efficacy in myocardial infarction. Mechanisms of myocardial repair with MSC transplantation have not been fully elucidated, and therapeutic efficacy needs to be enhanced. The MSCs obtained from the bone marrow of Fisher344 rats were treated with fibroblast growth factor-2, insulin-like growth factor-1, and bone morphogenetic protein-2. The expression of cardiac specific markers and the cytoprotective effect of MSCs with its mechanism were evaluated. Efficacy of MSCs transplantation was studied in rat myocardial infarction model. Treatment of MSCs with cocktails of GFs enhanced expression of cardiac transcription factors and survival. Induction of cardiac specific markers by coculture with CMCs and gap junctional communication with CMCs was more active in GF-treated MSCs than untreated MSCs. The GF-treated MSCs reduced apoptosis of neighboring CMCs in a hypoxic condition and enhanced the phosphorylated Akt and phosphorylated c-AMP response element binding protein expression of CMCs, which was markedly reduced by gap junction blockade. In a rat myocardial infarction model, transplantation of GF-treated MSCs resulted in smaller infarct size and better cardiac function than transplantation of untreated MSCs. Additionally, GF treatment enhanced gap junction formation of transplanted MSCs, which did not aggravate arrhythmia. Pre-treatment of MSCs with GFs enhanced cytoprotective effects on neighboring CMCs through gap junction and improved the therapeutic efficacy of MSC transplantation for myocardial repair. "Priming of MSCs with GFs" before transplantation might improve the therapeutic efficacy of cell therapy.

Differentially expressed transcription factors in the cardiac outflow tract tissue of connexin43 knockout mice

To investigate the changes in the expression of cardiac transcription factors in the cardiac outflow tract (OFT) tissues in the connexin43 knockout homozygotes (Cx43 KO), connexin43 heterozygotes, and connexin43 wild-type mice (Cx43 WT). The cDNA was retrotranscribed from the RNA extracted from the OFT tissues of 6 Cx43 KO, 6 Cx43 WT, and 6 Cx43 heterozygotes genotyped by PCR method on the embryonic day (ED) 13.5 and ED 14.5. The biotin-labeled cRNA derived from the transcription of cDNA was fragmented as probes. The probes were hybridized with Affymetrix Mouse Genome 430 2.0 Array. Gene Array Scanner was used to screen the signals of hybridization and detect the expression of genes. The mRNA expression levels of 3 cardiac transcription factors: Sox11, Foxp1, and Tbx20 were measured by real time quantitative RT-PCR. The ratios of the expression of the 6 genes, all cardiac transcription factors: Gata4, Mef2C, Sox4, Sox11, Foxp1, and Tbx20 between the Cx43 KO and Cx43 WT groups were 1:1.41, 1:2.30, 1:3.25, 1:0.71, 1:0.66, and 1:0.54. The expression levels of Sox11 and Foxp1 on ED13.5 in the Cx43 K group were 4.76 +/- 0.19 and 5.08 +/- 0.28 respectively, both significantly lower than those of the Cx43 WT group (5.34 +/- 0.25 and 5.64 +/- 0.15 respectively, both P < 0.01), and expression level of Tbx20 on ED 13.5 in the Cx43 K group was 7.18 +/- 0.16, not significantly different from that of the Cx43 WT group (7.47 +/- 0.27, P > 0.05). The expression levels of the genes Sox11, Foxp1, Tbx20 on ED 14,5 were 4.71 +/- 0.27, 5.25 +/- 0.31, and 7.05 +/- 0.17 respectively, all significantly lower than those of the Cx43 WT group (5.00 +/- 0.19, 5.77 +/- 0.16,) and 7.43 +/- 0.25, all P < 0.05). The results of the expression of these genes by real time PCR analysis showed an excellent concordance with those indicated by the microarray analysis. The cardiac transcription factors such as Sox11, Foxp1, and Tbx20 that are differently expressed in the Cx43 KO OFT tissue may be involved in the pathogenesis of the OFT defects.

Early stage-specific inhibitions of cardiomyocyte differentiation and expression of Csx/Nkx-2.5 and GATA-4 by phosphatidylinositol 3-kinase inhibitor LY294002

Inhibition of phosphatidylinositol 3-kinase (PI3-kinase) has been reported to block cardiomyocyte differentiation. However, at which stage PI3-kinase plays this important role and what its molecular targets are remain unknown. To answer these questions, we induced cardiomyocyte differentiation of P19CL6 mouse embryonal carcinoma cells and investigated the activation of PI3-kinase by analyzing phospho-Akt. We also treated P19CL6 cells with the PI3-kinase-specific inhibitor LY294002 either continuously or at various time points and monitored the expression of cardiac contractile proteins and transcription factors. Most cells differentiated into sarcomeric myosin heavy chain (MHC)-positive cardiomyocytes on day 16 after induction. An increase in phospho-Akt was observed after induction and was maintained throughout the differentiation. LY294002 treatment restricted to the phase from days 0 to 4 was sufficient to inhibit cardiomyocyte differentiation in a dose-dependent manner. In contrast, LY294002 treatment either from days 4 to 8 or from days 8 to 12 did not cause significant changes in sarcomeric MHC expression. LY294002 treatment from days 0 to 4 also suppressed Csx/Nkx-2.5 and GATA-4 expression. These results demonstrate that PI3-kinase becomes activated and plays a pivotal role at a very early stage of cardiomyocyte differentiation, possibly by modulating the expression of the cardiac transcription factors.

Phosphatidylinositol 3-Kinase Regulates Bone Morphogenetic Protein-2 (BMP-2)-induced Myocyte Enhancer Factor 2A-dependent Transcription of BMP-2 Gene in Cardiomyocyte Precursor Cells

The growth and differentiation factor bone morphogenetic protein-2 (BMP-2) regulates cardiac development during vertebrate embryogenesis. In cardiac precursor cells, BMP-2 has recently been shown to induce expression of cardiac transcription factors, including myocyte enhancer factor 2A (MEF-2A). The specific signal transduction mechanism by which BMP-2 regulates these actions is not known. We investigated the role of phosphatidylinositol (PI) 3-kinase in regulating these processes in cardiomyocyte precursor CL6 cells. BMP-2 increased PI 3-kinase activity in these cells in a time-dependent manner, resulting in increased expression of sarcomeric myosin heavy chain (MHC) and MEF-2A. Inhibition of PI 3-kinase abolished these actions of BMP-2, indicating the involvement of PI 3-kinase in these processes. Furthermore, BMP-2 stimulated specific protein.DNA complex formation when an MEF-2 DNA recognition element was used as probe. Antibody supershift assay confirmed the presence of MEF-2A in this protein.DNA complex. Inhibition of PI 3-kinase activity completely prevented the MEF-2A.DNA complex formation. BMP-2 also increased transcription of a reporter gene driven by an MEF-2-specific DNA element in a PI 3-kinase-dependent manner. Ectopic expression of MEF-2A increased BMP-2 transcription to the same extent induced by BMP-2, indicating that MEF-2A may participate in BMP-2 autoregulation in CL6 cells. Expression of dominant negative PI 3-kinase completely abolished BMP-2-induced as well as MEF-2A-mediated BMP-2 transcription. Furthermore expression of MEF-2A increased MHC expression in a PI 3-kinase-dependent manner. Together these data provide the first evidence that BMP-2-induced PI 3-kinase signaling regulates MEF-2A expression and define a mechanism of MEF-2A-dependent BMP-2 transcription.

Bone Morphogenetic Protein-2 Inhibits Serum Deprivation-induced Apoptosis of Neonatal Cardiac Myocytes through Activation of the Smad1 Pathway

Bone morphogenetic protein (BMP)-2 has been shown to induce ectopic expression of cardiac transcription factors and beating cardiomyocytes in non-precardiac mesodermal cells, suggesting that BMP-2 is an inductive signaling molecule that participates in cardiac development. However, direct evidence of the effects of BMP-2 on cardiac myocytes has not been reported. To examine the role of BMP-2 and its receptors, we studied the ability of BMP-2 to promote survival of isolated neonatal rat cardiac myocytes. BMP receptors IA, IB, and II and activin receptor I were found to be expressed in myocytes, and BMP-2 phosphorylated Smad1 and p38 MAPK. Interestingly, BMP-2 promoted survival and inhibited apoptosis of serum-deprived myocytes, although it did not strongly induce hypertrophic growth. To explore the mechanisms for this protective effect, an adenovirus-based vector system was used. Similar to BMP-2, Smad1 promoted survival that was repressed by Smad6. Moreover, BMP-2 and Smad1 enhanced the expression of the anti-apoptotic molecule Bcl-x(L). Antisense oligonucleotides to bcl-x(L) attenuated the survival effected by BMP-2. Overall, our findings suggest that BMP-2 prevents apoptosis of myocytes by induction of Bcl-x(L) via a Smad1 pathway and might be a novel survival factor without any hypertrophic effect on myocytes.

Bone morphogenetic proteins induce cardiomyocyte differentiation through the mitogen-activated protein kinase kinase kinase TAK1 and cardiac transcription factors Csx/Nkx-2.5 and GATA-4.

Bone morphogenetic proteins (BMPs) have been shown to induce ectopic expression of cardiac transcription factors and beating cardiomyocytes in nonprecardiac mesodermal cells in chicks, suggesting that BMPs are inductive signaling molecules that participate in the development of the heart. However, the precise molecular mechanisms by which BMPs regulate cardiac development are largely unknown. In the present study, we examined the molecular mechanisms by which BMPs induce cardiac differentiation by using the P19CL6 in vitro cardiomyocyte differentiation system, a clonal derivative of P19 embryonic teratocarcinoma cells. We established a permanent P19CL6 cell line, P19CL6noggin, which constitutively overexpresses the BMP antagonist noggin. Although almost all parental P19CL6 cells differentiate into beating cardiomyocytes when treated with 1% dimethyl sulfoxide, P19CL6noggin cells did not differentiate into beating cardiomyocytes nor did they express cardiac transcription factors or contractile protein genes. The failure of differentiation was rescued by overexpression of BMP-2 or addition of BMP protein to the culture media, indicating that BMPs were indispensable for cardiomyocyte differentiation in this system. Overexpression of TAK1, a member of the mitogen-activated protein kinase kinase kinase superfamily which transduces BMP signaling, restored the ability of P19CL6noggin cells to differentiate into cardiomyocytes and concomitantly express cardiac genes, whereas overexpression of the dominant negative form of TAK1 in parental P19CL6 cells inhibited cardiomyocyte differentiation. Overexpression of both cardiac transcription factors Csx/Nkx-2.5 and GATA-4 but not of Csx/Nkx-2.5 or GATA-4 alone also induced differentiation of P19CL6noggin cells into cardiomyocytes. These results suggest that TAK1, Csx/Nkx-2.5, and GATA-4 play a pivotal role in the cardiogenic BMP signaling pathway.