Cited4 is related to cardiogenic induction and maintenance of proliferation capacity of embryonic stem cell-derived cardiomyocytes during in vitro cardiogenesis

Cardiac progenitor cells have a limited proliferative capacity. The CREB-binding protein/p300-interacting transactivator, with the Glu/Asp-rich carboxy-terminal domain (Cited) gene family, regulates gene transcription. Increased expression of the Cited4 gene in an adult mouse is associated with exercise-induced cardiomyocyte hypertrophy and proliferation. However, the expression patterns and functional roles of the Cited4 gene during cardiogenesis are largely unknown. Therefore, in the present study, we investigated the expression patterns and functional roles of the Cited4 gene during in vitro cardiogenesis. Using embryoid bodies formed from mouse embryonic stem cells, we evaluated the expression patterns of the Cited4 gene by quantitative reverse transcriptase-polymerase chain reaction. Cited4 gene expression levels increased and decreased during the early and late phases of cardiogenesis, respectively. Moreover, Cited4 gene levels were significantly high in the cardiac progenitor cell population. A functional assay of the Cited4 gene in cardiac progenitor cells using flow cytometry indicated that overexpression of the Cited4 gene significantly increased the cardiac progenitor cell population compared with the control and knockdown groups. A cell proliferation assay, with 5-ethynyl-2′-deoxyuridine incorporation and Ki67 expression during the late phase of cardiogenesis, indicated that the number of troponin T-positive embryonic stem cell-direived cardiomyocytes with proliferative capacity was significantly greater in the overexpression group than in the control and knockdown groups. Our study results suggest that the Cited4 gene is related to cardiac differentiation and maintenance of proliferation capacity of embryonic stem cell-derived cardiomyocytes during in vitro cardiogenesis. Therefore, manipulation of Cited4 gene expression may be of great interest for cardiac regeneration.


Introduction
Cited4 is a gene of the CREB-binding protein/p300-interacting transactivator, with Glu/Asprich carboxy-terminal domain (Cited) family and regulates gene transcription [1]. The Cited4 gene is expressed in the developing heart and the expression is restricted to the endocardium [1]. In the adult mouse, the increased expression of the Cited4 with exercise is associated with cardiomyocyte hypertrophy and proliferation [2].
Embryonic stem (ES) cell-derived cardiogenesis using embryoid bodies (EBs) formed from mouse ES cells is a useful in vitro system to assess the molecular mechanisms of cardiogenesis [3,4]. There is an increased need to understand the biological properties of cardiac progenitor cells for their application in regenerative medicine. Studies of in vitro cardiogenesis suggest that the proliferative capacity of ES cell-derived cardiomyocytes is markedly decreased after cardiogenic induction [5].
In this study, we aimed to investigate the time-dependent expression patterns of the Cited4 in the EBs, compare the lineage-specific expressions of the Cited4, and investigate whether the Cited4 is associated with cardiogenic induction and proliferation capacity of ES cell-derived cardiomyocytes during in vitro cardiogenesis.

Culture of mouse embryonic stem cells and in vitro cardiogenesis
The 129/Ola-derived ES cell lines used in this study are ht7, which was provided by Hitoshi Niwa, Kumamoto University, Japan, and its derivatives. The ht7 carries a hygromycin resistance gene in one of the Oct-3/4 loci, which allows selection of Oct-3/4-positive undifferentiated stem cells [11]. Hcgp7 has been previously reported: hcgp7 cells are derived from ht7 cells and carry a GFP reporter gene in one of the Nkx2.5 loci [12]. ES cells were maintained on gelatin-coated dishes without feeder cells in Glasgow Minimum Essential Medium (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10% fetal bovine serum (JRH Bioscience, Lenexa, KS, USA), non-essential amino acids (Gibco-BRL; Life Technologies, Carlsbad, CA, USA), 1 mmol/L sodium pyruvate (Sigma-Aldrich), penicillin-streptomycinglutamine (Gibco-BRL), 0.1 mmol/L 2-mercaptoethanol (Sigma-Aldrich), 1000 units/mL leukaemia inhibitory factor (LIF, Chemicon; Millipore, Billerica, MA, USA), and 0.1 mg/mL Hygromycin B (Gibco-BRL). For cardiac differentiation, 500 ES cells in 20 μL aliquots of differentiation medium (maintenance medium without LIF and Hygromycin) were cultured in hanging drops for 3 days. After that, the EBs were further cultured in a floating condition. To evaluate the cardiac differentiation efficiency of ES cells, some of the resultant EBs were transferred to individual wells of gelatin-coated 24-well culture plates on the fifth day, and the 24-well culture plates were monitored every day under a microscope to detect the appearance of spontaneously contracting cardiomyocytes. The percentage of EBs exhibiting spontaneous contraction was calculated as the cardiac differentiation efficiency. The medium was changed every other day. The day when hanging drop culture was initiated was defined as day 1.

Quantitative reverse transcriptase-polymerase chain reaction
Total RNA extraction from ES cells before differentiation, and EBs on day 3.5, 4.5, 5.5, 6.5, 7.5, 8.5, 9.5, and 10.5 after differentiation, was performed using an RNeasy Mini Kit (Qiagen, Valencia, CA, USA). First-strand cDNA synthesis was performed using SuperScript II reverse transcriptase (Gibco-BRL). Quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) was performed with a LightCycler SYBR Green I Kit (Roche Diagnostics, Mannheim, Germany) according to the manufacturer's protocol. Gene expression levels were determined by a LightCycler 1.2 (Roche, Basel, Switzerland). The qRT-PCR data were analyzed using the second derivative maximum method available with LightCycler Software Version 3.5.3. The sequences of the qRT-PCR primers for Rex1, Brachyury (Bra), Flk1, Nkx2.5, Cited4, and β-actin genes are listed in Table 1. The β-actin gene was used as a reference molecule.

Plasmid construction and transfection
Enhanced green fluorescent protein (EGFP) was used as a reporter of expression. The Rex1-promoter EGFP construct was a gift from Dr. Yasuaki Shirayoshi. Briefly, the Rex1-promoter EGFP construct was generated by cloning a promoter fragment of the mouse Rex1 gene ranging from −270 to +51 of the 5 0 region of Rex1 into pd2EGFP-1 (Clontech, Mountain View, CA, USA). The Bra-promoter EGFP construct was generated by cloning a promoter fragment of the mouse Bra gene ranging from −484 to +134 of the 5 0 region of Bra into the promoter-less EGFP reporter vector, pEGFP-1 (Invitrogen; Life Technologies). The Bra promoter region of the plasmid TpGL3 (a gift from Dr. Rolf Kemler) was digested by Sac I and isolated by electrophoresis, and then ligated into the pEGFP-1 plasmid.
The Cited4 cDNA fragment was obtained by RT-PCR with total RNA extracted from the ht7 cell-derived EBs on day 7.5. Primers for cloning the Cited4 gene are presented in Table 2. Amplified Cited4 gene fragments were ligated into a pGEM-T vector (Promega, Madison, WI, USA). The Cited4 gene fragments were then digested from the plasmid by Eco RI and Spe I, and isolated by electrophoresis. To create p3xFLAG-CMV-10-Cited4 plasmid (pCited4), the digested Cited4 gene fragments were cloned into a p3xFLAG-CMV-10 expression vector (Sigma-Aldrich) digested by Eco RI and Xba I.
For the RNA interference assay, a plasmid expressing double-stranded, small interfering RNA against the mouse Cited4 gene was generated using the siLentGene U6 Hairpin Cloning System (Promega). To generate the Cited4 knockdown vector (siCited4), the DNA cassette containing the hairpin structure to target the Cited4 gene was amplified by PCR and inserted into the psiLentGene vector. To create the control vector (scrambled Cited4-siRNA), a nonspecific siRNA duplex containing the same nucleotides but in irregular sequence was prepared. The siRNA and scrambled siRNA sequences are provided in Table 3. The constructed plasmids were transfected into the ht7 or hcgp7 cells after linearizing the constructs with Lipofectamine and Plus Reagent. Approximately 24 h after transfection, the transfected cells were transferred to medium containing G418 sodium salt at a concentration of 400 μg/mL for more than 1 week to select stable transfectants. The Rex1-GFP-transfected ht7, Bra-GFP-transfected ht7, pCited4-transfected ht7 and pCited4-transfected hcgp7 cells were designated as Rex1-ht7, Bra-ht7, Cited4-ht7 and Cited4-hcgp7, respectively. The siCited4 vector was transfected into ht7 and hcgp7 cells, and designated as siCited4-ht7 and siCi-ted4-hcgp7, respectively. Downregulation of Cited4 gene expression by functional siRNA, but not by the control, was also confirmed by qRT-PCR.

Flow cytometry and cell sorting
ES cells and EBs were dissociated with trypsin (0.25%)-EDTA (1 mmol/L) on the indicated differentiation day. The dissociated cells were suspended in Hank's Balanced Salt Solution (Cambrex; Lonza, Basel, Switzerland) with 1% bovine serum albumin (BSA, Sigma-Aldrich). Phycoerythrin (PE) and GFP fluorescence was detected using a 488-nm argon laser with an EPICS 1 ELITE ESP (Beckman Coulter, Brea, CA, USA). For sorting Rex1-positive cells, the dissociated Rex1-ht7 cells on day 0 were incubated with an anti-mouse E-cadherin antibody followed by a PE-conjugated anti-mouse IgG antibody. For sorting Bra-positive and Flk1-positive cells, the dissociated Bra-ht7 cells on day 4.5 were incubated with a PE-conjugated anti-mouse Flk1 antibody. For sorting Nkx2.5-positive cells, the dissociated hcgp7 cells on day 7.5 were incubated with the PE-conjugated anti-mouse Flk1 antibody. Propidium iodide detected dead cells. Antibodies are listed in Table 4.

Cell proliferation assay for cardiac progenitors
The cell proliferation assay was performed using 5-ethynyl-2 0 -deoxyuridine (EdU) incorporation and Ki67 expression. EdU incorporation was performed with a Click-iT EdU Alexa  Table 5.

Statistical analyses
Continuous variables are presented as the mean ± standard error of the mean. Categorical variables were analyzed by chi-square analysis followed by Bonferroni's post-hoc comparison tests. All statistical analyzes were performed with the software R (The R Foundation for Statistical Computing, Vienna, Austria; version 3.1.1). P < 0.05 was considered statistically significant.

Cited4 gene expression increases transiently during the early phase of cardiogenesis
To explore time-dependent expression patterns of the Cited4 gene during in vitro cardiogenesis, we studied the time course of expression levels of the Cited4 gene and lineage marker genes. We used the EBs as an in vitro differentiation system, which comprised cell aggregates formed with mouse ES cells to recapitulate in vivo cardiac differentiation. We studied the expression patterns of lineage marker genes with qRT-PCR. Consequently, Rex1 gene expression was high at differentiation day 0 and decreased during differentiation (Fig 1A). Bra gene expression was transient during differentiation with a peak expression level at day 4.5 ( Fig 1B). Subsequently, Flk1 gene expression increased at day 4.5-5.5 ( Fig 1C). Nkx2.5 gene expression increased at day 6.5-7.5 (Fig 1D), which was consistent with the initiation of cellular beating ( Fig 1E). Cited4 gene expression was transient at the early phase of cardiogenesis; i.e. Cited4 gene expression increased when the expression of the Bra and the Nkx2.5 genes decreased and increased, respectively, and the EBs started beating (Fig 1F).
The Cited4 gene is expressed specifically in a cardiac progenitor cell population during in vitro cardiogenesis To study the lineage-specific expression of the Cited4 gene during in vitro cardiogenesis, we evaluated Cited4 gene expression in the different lineage marker-positive cell populations isolated by fluorescence-activated cell sorting. First, we confirmed that the expression patterns of GFP under the control of Rex1 (Fig 2A) and Bra (Fig 2B) promoters were consistent with the expression patterns of Rex1 and Bra genes as shown in Fig 1A and 1B, respectively. Then, we isolated the Rex1-GFP-positive and E-cadherin-positive undifferentiated stem cell population from the Rex1-ht7 cell line at day 0 (Fig 2C), the Bra-positive and Flk1-negative primitive mesodermal cell population from the Bra-ht7 cell line at day 4.5 (Fig 2D), the Bra-negative and Flk1-positive early lateral mesodermal cell population from the Bra-ht7 cell line at day 4.5 ( Fig  2D), and the Nkx2.5-positive and Flk1-negative early cardiac progenitor cell population from the hcgp7 cell line at day 7.5 ( Fig 2E). Consequently, the level of Cited4 gene expression was specifically high in the early cardiac progenitor cell population (Fig 2F). Taken together with the results shown in Fig 1F, these results indicated that Cited4 expression was spatiotemporally specific to the early cardiac progenitor cell population, suggesting that the Cited4 gene is an important cardiogenesis-related factor. Therefore, we subsequently investigated the functional roles of the Cited4 gene in cardiogenesis. The Cited4 gene increases the early cardiac progenitor cell population To study the possibility that Cited4 gene expression levels relate to the amounts of early cardiac progenitor cell population, we created Cited4-overexpressing hcgp7 (Cited4-hcgp7) and Cited4-knockdown hcgp7 (siCited4-hcgp7) cells. Levels of the Cited4 gene expression at day 6.5 were increased 2.0-fold and decreased 0.27-fold in the overexpressing and knockdown cells, respectively. The protein expression levels of Cited4 at day 6.5 were increased 1.7-fold and decreased 0.3-fold in the overexpressing and knockdown cells, respectively, compared with the control cells (S1 Protocol, S1 Table and S1 Fig). We then compared the amounts of the early cardiac progenitor cell populations in the EBs at day 6.5 formed from the control hcgp7 cells, the Cited4-hcgp7 cells, and the siCited4-hcgp7 cells (Fig 3A-3C). Consequently, without changing the timing of cellular beating, the Nkx2.5-positive cardiac progenitor cell population was significantly increased about 4-fold in the Cited4-hcgp7 group compared with the control group ( Fig 3D); meanwhile, the Nkx2.5-positive cardiac progenitor cell population was decreased in the siCited4-hcgp7 group compared with the control group, although this did not reach statistical significance (Fig 3D). These results suggest that the Cited4 gene is associated with the cardiogenic induction.
The Cited4 gene maintains the proliferation capacity of TnT-positive ES cell-derived cardiomyocytes To further confirm the functional roles of Cited4 gene in the proliferation capacity of differrentiated ES cell-derived cardiomyocytes expressing the myogenic marker of TnT, we evaluated EdU incorporation as a marker of DNA synthesis (Fig 4A, 4E, 4G and 4I) and Ki67 expression  as an indicator of mitotically active cells (Fig 4B, 4F, 4H and 4J). The proliferation capacities of TnT-positive ES cell-derived cardiomyocytes derived from control ht7, Cited4-overexpressing ht7 (Cited4-ht7), and Cited4-knockdown ht7 (siCited4-ht7) cells were compared at day 10.5, at which time ES cell-derived cardiomyocytes start to decrease proliferation capacity. To do this, we first studied the proliferation capacity of TnT-positive ES cell-derived cardiomyocytes at day 7.5 from control ht7 cells (Fig 4A and 4B). As a result, 73.3% and 91.4% of the cells were EdU-positive and Ki67-positive, respectively. In the TnT-positive ES cell-derived cardiomyocytes at day 10.5, 17.0% and 9.3% of the TnT-positive ES cell-derived cardiomyocytes from control ht7 cells were EdU-positive and Ki67-positive, respectively (Fig 4E and 4F); 55.0% and 43.6% of those from Cited4-ht7 cells were EdU-positive and Ki67-positive, respectively ( Fig  4G and 4H); 11.9% and 10.1% of those from siCited4-ht7 cells were EdU-positive and Ki67-positive, respectively (Fig 4I and 4J). In the TnT-positive ES cell-derived cardiomyocyte derived from Cited4-ht7 cells, the EdU-positive or Ki67-positve cells were significantly increased compared with those derived from control ht7 and siCited4-ht7 cells (Fig 4K and  4L).

Discussion
In the present study, we identified that the Cited4 gene expression was transient during in vitro cardiogenesis and specific to Nkx2.5-positive cardiac lineage cells. Furthermore, we confirmed that the Cited4 gene was associated with cardiogenic induction and proliferation capacity of ES cell-derived cardiomyocytes expressing TnT during in vitro cardiogenesis. We studied the in vitro expression patterns of the Cited4 gene with those of the lineage marker genes, Rex1, Bra, Flk1 and Nkx2.5. The strong Cited4 gene expression has been observed in developing embryonic heart with in situ hybridization, compared with other organs [1]. In agreement with the previous in vivo study, the expression of the Cited4 gene during in vitro cardiogenesis in this study was specifically high in Nkx2.5-positive cardiac progenitor cells. Moreover, the expression patterns of Rex1, Bra, Flk1 and Nkx2.5 during in vitro cardiogenesis in this study were considered to recapitulate those during in vivo development [6][7][8][9][10]. Therefore, the in vitro expression pattern of the Cited4 gene together with the lineage marker genes in this study was considered to be consistent to in vivo mouse embryonic differentiation process. However, further investigation and comparison of the time-dependent Cited4 expression levels in in vitro and in vivo differentiation processes are necessary.
Although, in the adult mouse, the expression of the Cited4 is associated with exerciseinduced cardiomyocyte proliferation [2], functional roles of the Cited4 gene during cardiogenesis are largely unknown. In this study, the Cited4 expression was spatiotemporally specific to the early cardiac progenitor cell population. Furthermore, the Cited4 gene increased the early cardiac progenitor cell population. Moreover, we have observed that the expression levels of Nkx2.5 and Gata4 gene were increased and decreased in the Cited4 overexpression and downregulation, respectively, compared with the control cells. Therefore, although the precise regulatory relationship between Cited4 and Nkx2.5 gene and between Cited4 and Gata4 gene, Cited4 gene was suggested to be associated with the cardiogenic induction.
So far, knockouts of individual or a combination of positive cell cycle regulators, such as cyclins and cyclin-dependent kinases, failed to show a significant defect of cell proliferation in early cardiogenesis [13][14][15][16][17]. Cardiac progenitor cells at the early stage of in vitro cardiogenesis are lacking of TnT expression, and TnT has been considered to be a marker for differentiated ES cell-derived cardiomyocytes at the stage of cardiomyogenesis [4], at which stage the ES cellderived cardyomyocytes start to lose the proliferation capacity [5]. In agreement with these reports, as shown in this study, the proliferation capacity of TnT-positive ES cell-derived cardiomyocytes dropped dramatically within a brief period from day 7.5 to day 10.5 of in vitro differentiation, which resulted in the small number of EdU-positive/Ki67-positive ES cell-derived cardiomyocytes in the control group at day 10.5. In this study, although we could not detect statistically significant differences between the Cited4 inhibition group and the control group due to this, the overexpression of the Cited4 gene maintained the proliferation capacity of ES cell-derived cardiomyocytes expressing the myogenic marker of TnT. Moreover, at day 7.5, at which stage the majority of troponin T-positive ES cell-derived cardiomyocytes maintained the proliferation capacity as shown in this study, we confirmed that there was significant decrease of proliferation-competent TnT-positive ES cell-derived cardiomyocytes in the Cited4 inhibition group than in the control group. Furthermore, we confirmed no significant changes of the expression level of Nav1.5 gene coding voltage-gated sodium channel, another marker for differentiation ES cell-derived cardiomyocytes [4], in the Cited4 overexpression group, compared with the control group. Taken together, it was suggested that the function of the Cited4 gene was associated with the maintenance of proliferation capacity of TnT-positive ES cell-derived cardiac progenitor cells without inhibiting cardiomyocyte maturation. However, further studies are necessary to determine the possibility of the inhibitory funciton of the Cited4 on cardiomyocyte maturation.
Some reports indicate that the Cited4 gene contains domains that interact with several transcriptional factors or cofactors [1,18]. Therefore, the Cited4 gene may have a number of mechanisms to control cell cycle-regulatory gene expression during in vitro cardiogenesis. Although the C/EBPβ gene is reported to relate to Cited4 gene expression in adult mice [2], the precise regulatory mechanisms by which the Cited4 gene controls cell cycle of cardiac progenitor cells are unknown. Further studies are needed to elucidate the functional roles of the Cited4 gene in cell cycle of cardiac progenitor cells.
Cardiac regeneration is hampered by the low renewal rate of cardiomyocytes [19,20] or by the limited number of donor cardiomyocytes derived from ES cells [12]. Although the exogenous expression of Myc gene has been recently reported to promote the proliferation of cardiac progenitor cells, the intrinsic genes to control the proliferation capacity of cardiac progenitor cells are unknown [21]. Therefore, the intrinsic genes that enable cardiac progenitor cells to maintain proliferative capacity are of great interest. The results in this study indicated that the Cited4 gene should be a candidate molecule for cardiac regeneration.
Supporting information S1 Protocol. Western blot analysis of endogenous and exogenous Cited4 expression. The ht7 cell line without modification of the Cited4 gene expression was used as a control. The Cited4-ht7 cell line comprised cells with the overexpression of the FLAG-tagged Cited4 gene. The siCited4-ht7 cell line comprised cells with the knockdown of the Cited4 gene. Whole-cell lysates were collected using RIPA buffer from ES cells before differentiation at day 0 and EBs on day 6.5 after differentiation, and electrophoresed on 12% SDS-PAGE gel. The electrophoresed gels were transferred onto polyvinylidene fluoride membranes (Merck Millipore), and processed for Western blotting with an anti-Cited4, anti-FLAG tag, and anti-β-actin antibody in the same membrane: The membranes were blocked with 5% nonfat milk in TBST, incubated with diluted primary antibody overnight at 4˚C, and then incubated with diluted secondary antibody for 1 h at room temperature. Antibodies are listed in S1 Table. Bands were visualized by chemiluminescent method using ECL plus system (Thermo Fisher Scientific). (PDF) S1 Table. List of antibodies for Western blot analysis. (PDF) S1 Fig. Western blot analysis of endogenous and exogenous Cited4 expression. A. Analysis of endogenous and exogenous Cited4 expression levels with an anti-Cited4 antibody at day 0 and day 6.5. At day 0 of differentiation, the Cited4 expression was detected in the overexpression group, while it was hardly detected in the control and knockdown group. At day 6.5, the Cited4 expression level was increased 1.7-fold in the overexpression group and decreased 0.3-fold in the knockdown group, compared to the control group. B. Analysis of exogenous Cited4 expression levels with an anti-FLAG antibody at day 0 and day 6.5. Both at day 0 and day 6.5, the exogenous Cited4 expression was detected only in the overexpression group, but not in the control and knockdown group. C. Internal control for Western blotting. β-actin was used as an internal control for Western blotting. (PDF)