Gremlin enhances the determined path to cardiomyogenesis

Abstract

Background: The critical event in heart formation is commitment of mesodermal cells to a cardiomyogenic fate, and cardiac fate determination is regulated by a series of cytokines. Bone morphogenetic proteins (BMPs) and fibroblast growth factors have been shown to be involved in this process, however additional factors needs to be identified for the fate determination, especially at the early stage of cardiomyogenic development.

Methodology/principal findings: Global gene expression analysis using a series of human cells with a cardiomyogenic potential suggested Gremlin (Grem1) is a candidate gene responsible for in vitro cardiomyogenic differentiation. Grem1, a known BMP antagonist, enhanced DMSO-induced cardiomyogenesis of P19CL6 embryonal carcinoma cells (CL6 cells) 10-35 fold in an area of beating differentiated cardiomyocytes. The Grem1 action was most effective at the early differentiation stage when CL6 cells were destined to cardiomyogenesis, and was mediated through inhibition of BMP2. Furthermore, BMP2 inhibited Wnt/beta-catenin signaling that promoted CL6 cardiomyogenesis.

Conclusions/significance: Grem1 enhances the determined path to cardiomyogenesis in a stage-specific manner, and inhibition of the BMP signaling pathway is involved in initial determination of Grem1-promoted cardiomyogenesis. Our results shed new light on renewal of the cardiovascular system using Grem1 in human.

Figure 1. Hierarchical clustering analysis on cultured human cells.

(A) Hierarchical clustering analyzed by GeneSpring. Based on gene expression pattern, 69 human cells were clustered into 30 sub-groups. The raw data from the GeneChip analysis are available at the GEO database with accession number GSE8481, GSM41342- GSM41344, and GSM201137- GSM201145. (B) Hierarchical clustering analysis was performed by NIA array (http://lgsun.grc.nia.nih.gov/ANOVA/), using averaged values of 30 sub-groups. Among the 30 groups, 21 groups included cells with a cardiomyogenic potential. To identify genes specific for these groups, hierarchical clustering was employed. Grem1 was nominated as a cluster-specific cardiomyocyte-promoting gene in cells that could differentiate into cardiomyocytes.

Figure 2. Grem1 enhanced cardiomyogenic differentiation in DMSO-induced CL6 cells.

(A) Phase contrast micrograph of CL6 cells with exposure to DMSO alone (a), Grem1 (125 ng/ml) and DMSO (b) for 14 days. The medium, including Grem1 and DMSO, was changed every day. CL6 cells exhibited apparent spontaneous beating between days 9–11. Beating CL6 cell colonies are outlined by white lines. (B) Percentage of beating area in differentiated CL6 cells. CL6 cell treated with Grem1 (125 ng/ml) and DMSO exhibited the strongest contraction. (C) RT-PCR analysis of the genes encoding cardiac-specific transcriptional factors (Csx/Nkx2.5, Gata4, Mef2c, Hand2), circulating hormone (ANP, BNP), cardiac-specific proteins (MyLC-2a, MyLC-2v, β-MyHC), cytokines (Bmp2, Bmp4, Fgf8, Grem1, Wnt1, Wnt3a, Wnt5a, Wnt7a, Wnt11), SM-MyHC, and Gapdh (From top to bottom). Mouse total heart RNA for the Csx/Nkx2.5, Gata4, Mef2c, Hand2, ANP, BNP, MyLC-2a, MyLC-2v, β-MyHC, Bmp2, Bmp4, Grem1, Wnt11, SM-MyHC, and Gapdh genes, mouse embryonic stem cell RNA for the Fgf8 gene, and mouse total skeletal muscle RNA for the Wnt1, Wnt3a, Wnt5a, and Wnt7a genes were used for positive controls. H₂O (without RNA) served as a negative control. (D) Immunocytochemistry of CL6 cells 14 days after exposure to Grem1 (125 ng/ml) and DMSO with MF20 and cTnT (a), and α-actinin (b). Cell nuclei are stained with DAPI. Clear striations are evident. (E) Immunocytochemistry of CL6 cells 14 days after exposure to Grem1 and DMSO with cardiac troponin T (cTnT) and sarcomeric myosin (MF20). CL6 cells treated with Grem1 (125 ng/ml) and DMSO (a), and DMSO alone (b) stained positive for cTnT and MF20. Untreated CL6 cells, i.e. not exposed to Grem1(125 ng/ml) or DMSO, stained negative for cTnT and MF20. Cell nuclei were stained with DAPI. (F) Percentage of MF20- and cTnT-double positive area.

Figure 3. Percentage of myogenic differentiation by period of treatment with Grem1 in CL6 cells.

(A) Protocol for treatment of Grem1 and DMSO. CL6 cells were passaged at 1.8×10⁵ cells in 6-well plate on Day 0. CL6 cells were exposed to Grem1 (125 ng/ml) and/or DMSO on the indicated day. Day when the cells were exposed to the inducers is shown by “+” (in gray cells for clarity). The medium including Grem1 and DMSO was changed every day. On day 14, the cells were immunocytochemically stained with MF20 antibody. (B) Myogenic differentiation of CL6 cells was estimated by sarcomeric myosin (MF20)-positive area. CL6 cells were treated with Grem1 (125 ng/ml) and DMSO for the indicated days. (C) Myogenic differentiation of CL6 cells was estimated by beating area. CL6 cells treated with DMSO and Grem1 (125 ng/ml) were incubated at indicated days.

Figure 4. Cardiomyogenic differentiation in CL6 cells (days 1–3) is inhibited by BMP2.

(A) RT-PCR analysis of the gene encoding BrachyuryT, Tbx6, cardiac-specific transcriptional factor (Csx/Nkx2.5), cardiac-specific protein (β-MyHC), and Gapdh (From top to bottom) of CL6 cells treated with DMSO alone, or DMSO and BMP2 (100 ng/ml) for the first 3 days (days 1–3). The medium, including BMP2 and DMSO, was changed every day. (B) Percentage of MF20-positive area. Immunocytochemistry was carried out on CL6 cells 14 days after cells had been exposed to DMSO and BMP2 (100 ng/ml) for the first 3 days (days 1–3). The asterisk indicates a significant statistical difference (P<0.05). (C) Quantitative real-time RT-PCR analysis of the gene encoding Bmp2 (left), and Bmp4 (right) in CL6 cells treated with DMSO alone (open square), or DMSO and BMP2 (100 ng/ml) (closed square) for the first 3 days (days 1–3). (D) BMP signaling activity of CL6 cells treated with DMSO alone (open square), or DMSO and BMP2 (100 ng/ml) (closed square) for the first 3 days (days 1–3) were determined by luciferase activity analysis using Id1 promoter-Lux (a firefly luciferase reporter plasmid driven by the Id1 binding sites), pRL-CMV as co-transfected control, and Dual luciferase reporter assay system. Relative luciferase unit of the CL6 cells untreated with inducers at day 3 is regarded as 0.1 (data not shown). (E) Quantitative real-time RT-PCR analysis of the gene encoding Wnt3a in CL6 cells treated with DMSO alone (open square), or DMSO and BMP2 (100 ng/ml) (closed square) for the first 3 days (days 1–3). (F) Wnt/β-catenin signaling activity of CL6 cells 48 h after exposure to DMSO, or DMSO and BMP2 (100 ng/ml) was determined by luciferase activity analysis using TOPflash (a firefly luciferase reporter plasmid driven by two sets of three copies of the TCF binding site and herpes simple virus thymidine kinase minimal promoter), pRL-CMV as co-transfected control, and Dual luciferase reporter assay system. Relative luciferase unit of the CL6 cells untreated with inducers is regarded as 0.1. The asterisk indicates a significant statistical difference (P<0.05). (G) Timeframe of Wnt/β-catenin signaling activity in CL6 cells treated with DMSO alone (open square), or DMSO and BMP2 (100 ng/ml) (closed square) for the first 3 days (days 1–3). Relative luciferase unit of the CL6 cells untreated with inducers at day 3 is regarded as 0.1 (data not shown).

Figure 5. Grem1-accelerated CL6 cardiomyogenesis through regulation of BMP- and Wnt/β-catenin-signaling pathways.

CL6 embryonic cells start to differentiate into mesodermal cells through Wnt/β-catenin signaling pathway at the early stage (days 1–3), and mesodermal CL6 cells differentiate into mature cardiomyocytes by BMP pathway at the late stage (days 4–14). Grem1 accelerates DMSO-induced cardiomyogenesis through inhibition of the BMP-signaling pathway.