MicroRNA-363 negatively regulates the left ventricular determining transcription factor HAND1 in human embryonic stem cell-derived cardiomyocytes
© Wagh et al.; licensee BioMed Central Ltd. 2014
Received: 20 January 2014
Accepted: 15 May 2014
Published: 6 June 2014
Posttranscriptional control of mRNA by microRNA (miRNA) has been implicated in the regulation of diverse biologic processes from directed differentiation of stem cells through organism development. We describe a unique pathway by which miRNA regulates the specialized differentiation of cardiomyocyte (CM) subtypes.
We differentiated human embryonic stem cells (hESCs) to cardiac progenitor cells and functional CMs, and characterized the regulated expression of specific miRNAs that target transcriptional regulators of left/right ventricular-subtype specification.
From >900 known human miRNAs in hESC-derived cardiac progenitor cells and functional CMs, a subset of differentially expressed cardiac miRNAs was identified, and in silico analysis predicted highly conserved binding sites in the 3′-untranslated regions (3′UTRs) of Hand-and-neural-crest-derivative-expressed (HAND) genes 1 and 2 that are involved in left and right ventricular development. We studied the temporal and spatial expression patterns of four miRNAs in differentiating hESCs, and found that expression of miRNA (miR)-363, miR-367, miR-181a, and miR-181c was specific for stage and site. Further analysis showed that miR-363 overexpression resulted in downregulation of HAND1 mRNA and protein levels. A dual luciferase reporter assay demonstrated functional interaction of miR-363 with the full-length 3′UTR of HAND1. Expression of anti-miR-363 in-vitro resulted in enrichment for HAND1-expressing CM subtype populations. We also showed that BMP4 treatment induced the expression of HAND2 with less effect on HAND1, whereas miR-363 overexpression selectively inhibited HAND1.
These data show that miR-363 negatively regulates the expression of HAND1 and suggest that suppression of miR-363 could provide a novel strategy for generating functional left-ventricular CMs.
Heart cells are unable to repair after damage, which ultimately leads to more than 5 million deaths per year worldwide due to heart failure. The past few decades have witnessed new therapeutic options for treating diseases that are caused by cell damage; however, the prevalence of heart failure continues to increase. Repair and regeneration of lost cardiac cells either with endogenous cells (direct reprogramming) or by using cell-based therapies (cell transplant) holds great promise, but obstacles must be addressed before widespread clinical use is adopted. For example, the cardiac cells obtained after differentiation of pluripotent stem cells comprise mixed cardiomyocyte (CM) populations with heterogeneous mechanical and electrical properties that may be more or less useful for transplantation.
The Hand-And-Neural-crest-Derivative-expressed (HAND) superfamily of class B basic helix-loop-helix factors consists of two members, HAND1 and HAND2, both of which are dynamically expressed in embryologically distinct lineages during development, and have been shown to play essential roles in the developing heart. To date, the precise mode of function for either HAND protein remains unknown, although many studies collectively suggest that these factors play roles in activating multiple genes and that the mechanism of their regulation is complex[4, 5]. Although the progenitor cells and general timing of myocardial differentiation have been determined, little is known about the mechanisms controlling commitment of progenitors and the maturation of myogenic cells that give rise to atrial, ventricular, and specialized-conduction CMs. Although it is clear that heterogeneous CM populations arise during stem cell differentiation, mechanisms that control the cellular subspecification of cardiogenic mesoderm remain obscure.
Small, noncoding microRNAs (miRNAs) are emerging as important posttranscriptional regulators of gene expression, with each miRNA predicted to regulate hundreds of mRNA target genes[7, 8]. miRNAs have tissue-specific distributions that play key roles in cellular physiology, such as cell proliferation, differentiation, and death. miRNAs also are known to influence biologic and metabolic processes that are dysregulated in various diseases. To explore their role in normal development and disease, we and others have described miRNA expression profiles that are informative about cardiogenesis and skeletal muscle differentiation[11–13].
As miRNAs function mainly through the inhibition of target genes, we analyzed the expression of these oligomers in hESC-derived cardiac cell populations with the intention of identifying miRNAs involved in CM-subtype specification, in particular left ventricular CMs. We identified four miRNAs (miR-363, -367, -181a, and -181c) that putatively target HAND1 and/or HAND2. Of these miRNAs, we showed that miR-363 specifically targets HAND1 during CM-subtype specification in hESCs.
Cell culture and differentiation
The UCSF Stem Cell Research Oversight Committee approved all experiments with hESCs. H9 hESC (WA09, WiCell) and BCiPSP16 human induced pluripotent stem cells (hiPSCs, a gift from B. Conklin, Gladstone Institutes, UCSF) were passaged as undifferentiated cells on irradiated mouse embryonic feeder cells (MEFs) in Hes medium (Knockout-DMEM-F12, 20% Knockout-Serum-Replacement, 1% nonessential amino acids, 0.1 mM β-mercaptoethanol (all purchased from Invitrogen, Carlsbad, CA, USA) supplemented with 15 ng/ml Basic fibroblast growth factor-2 (FGF-2; R & D Systems, Minneapolis, MN, USA)) as previously described. Alternatively, cells were cultured in feeder-independent conditions on plates coated with Matrigel (BD Biosciences, Bedford, MA, USA), in mTeSR medium (Stem Cell Technologies, Vancouver BC, Canada), according to manufacturer’s instructions.
Differentiation was initiated by passaging hESCs or hiPSCs onto low-attachment plates (Corning Inc., Acton, MA, USA) in differentiation medium consisting of Knockout-DMEM-F12, 20% Fetal Bovine Serum, 1% nonessential amino acids, and 0.1 mM β-mercaptoethanol. Medium was replenished every second day.
Isolation of differentiated cell populations
hESC- and hIPSC-derived cells were isolated on alternate days (2, 4, 6, 8, 10, 12, and 14 days) after differentiation. Additionally, all beating foci from 12 and 14 days were microdissected and collected as representative samples of CM subtypes.
miRNA inhibition and overexpression
Pre- and anti-miRNAs were purchased from Ambion (Austin, TX, USA). Transfections were performed with Lipofectamine (RNAimax; Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. Transfection complexes were prepared with 50 to 100 nM pre- or anti-miRNA, and cells were transfected 24 hours after plating.
Fluorescent in situ hybridization
Fluorescent in situ hybridization (F-ISH) with double-Dig-labeled miR-363 miRCURY LNA probes (Exiqon, Copenhagen, Denmark) on E 10.5 mouse embryos was performed as described in the manufacturer’s protocol. In brief, slides were treated with PCR-grade proteinase-K (Roche Diagnostics, Mannheim, Germany) after fixation. The hybridization mix was prepared with 20 pmol of miR-363 double-labeled LNA probes in hybridization solution. The hybridization temperature used was 15°C below the melting temperature of the miR-363 probe.
Quantitative real-time PCR
Total RNA was extracted by using the mirVana RNA isolation kit (Ambion, Austin, TX, USA). miRNA expression was analyzed by mirVana reverse quantitative transcription-PCR miRNA detection assay according to the manufacturer’s protocol. In brief, cDNA was converted from 10 ng of total RNA by using miRNA-specific primers with the multiScribe reverse transcription kit (Ambion, Austin, TX, USA). cDNA was diluted 15 times with nuclease-free water, and 5 μl was used as a template for PCR. Quantitative real-time reverse transcriptase PCR (qPCR) was carried out on an Applied Biosystems 7300 cycler. Relative miRNA levels were calculated by using the ∆∆Ct method and represented relative to housekeeping miRNA (RNU48) expression. For mRNA expression analysis, the Taqman assay was carried out per the manufacturer’s instructions. The data were represented as relative quantitation with GAPDH as an internal control.
3′UTR reporter assay
The full-length 3′UTRs of human HAND1, HAND2, and NKX2.5 were each inserted downstream of the Firefly luciferase gene in pEZX-MT01 (GeneCopoeia, Rockville, MD, USA). Renilla luciferase encoded by the same vector was used as transfection control in the Dual-Luciferase Assay (Promega, Madison, WI, USA). hESCs were plated in a 96-well plate and co-transfected with 50 ng of luciferase vector and 50 nM precursor by using lipofectamine. Dual-Luciferase assays were performed according to the manufacturer’s instructions on a Wallac-Trilux luminometer. Luciferase assays also were performed in HEK293 cells. The luciferase assay kit (Promega Inc., Madison, WI, USA) was used to measure the reporter activity according to the manufacturer’s instructions.
Immunoblot analysis was performed as previously described. Cell lysates were separated with SDS-PAGE in 10% polyacrylamide gels and transferred onto nitrocellulose membranes. After blocking of nonspecific binding sites for 2 hours with 5% nonfat milk in PBS with 0.1% Tween-20, we incubated the membranes with 1:1,000 anti-HAND1 polyclonal antibody or 1:1,000 anti-HAND2 monoclonal antibody (Abcam, Cambridge, MA, USA); anti-β-actin antibody (Sigma-Aldrich, St Louis, MO, USA; dilution 1:100,000) was used as protein loading control. The proteins were detected by using Enhanced Chemiluminescent detection reagent (Amersham, Piscataway, NJ, USA), according to the manufacturer’s instructions.
Stimulation with bone morphogenetic protein-4 (BMP4)
Cells were incubated with 5 ng/ml recombinant BMP4 (Peprotech, Rocky Hill, NJ, USA), followed by 100 nM pre-miR-363 precursors. Cardiac mesoderm induction was measured by comparing HAND1 and HAND2 expression with cells transfected with scrambled miR precursors. Additionally, other growth factors, such as TGF-β, Activin A, and DKK1, were tested for their ability to induce HAND1 and HAND2 expression.
All experiments were performed in biological replicates, as indicated, and significance was tested by using the Student t test. A P value of <0.05 was considered significant.
Identification of miRNAs differentially expressed during hESC differentiation and cardiomyocyte-subtype specification
Differentially expressed miRNAs and predicted targets
Predicted mRNA targets (from TargetScan)
Total number of mRNA targets
mRNA targets specific to cardiac lineage (miRNAs targeting HAND1, HAND2, or NKX2.5)
Day 8 versu s Undiff.
12 (miR-363, miR-367)
Day 14 versu s Undiff.
10 (miR-363, miR-367)
Day 8 versu s Day 14
5 (miR-181a, miR-181c)
miRNA seed-pairing in cardiac-specific mRNAs
Regulate terminal differentiation/proliferation
Commitment to myocardial lineage
Left ventricular cardiac morphogenesis, giant cell differentiation
Cardiac morphogenesis, particularly right ventricle and aortic arch
In silico prediction and validation of miRNAs with predicted binding sites in the 3′UTRs of HAND1 and HAND2
Overexpression of miR-363 specifically downregulates HAND1 expression in differentiating hESCs
miR-363 inhibits HAND1 expression through 3′UTR binding
miR-363 regulates BMP4-mediated HAND gene expression during cardiomyocyte differentiation
Anti-miR-363 directs the differentiation of HAND1-enriched cardiomyocytes
MiRNAs are known to regulate gene expression in various organs and are recognized as important regulators of cardiac development and function[16, 17]. The cardiac-specific transcription factors, HAND1 and HAND2, play important roles in left versus right ventricular determination. We tested a subset of differentially expressed miRNAs predicated to target CM subtype-specifying transcription factors, such as HAND1 and HAND2. In vivo regional expression, in silico predictions, and experimental validation demonstrated that miR-363 is an upstream regulator of HAND1 translation, leading to a role in left ventricular CM differentiation.
To our knowledge, regulation of HAND1 or HAND2 by miRNAs has not previously been reported. These two genes are closely related and display complementary and overlapping expression patterns in the developing heart[18, 19]. During development, HAND1 is expressed in the inflow segment of the linear heart tube destined to become the left ventricle, whereas HAND2 is expressed much earlier throughout the linear heart tube, and then later expressed in the outflow portion of the heart tube destined to become the right ventricle and atria. However, little is known about the posttranscriptional control of HAND gene expression, although conserved miRNA seed-pairing sequences in the 3′UTRs of both HAND1 and HAND2 suggest control by miRNAs. In this study, we sought to identify specific miRNAs that target specifically ventricular CM-determining genes. The expression profiles of known human miRNAs were analyzed to identify potential miRNA-mRNA interactions that effect hESC differentiation into CMs.
miRNAs are known to target mRNAs by imperfect base pairing with their 3′UTR. This in turn inhibits translation and/or destabilizes the targeted mRNA, ultimately controlling its expression. Here, we identified subsets of 137, 100, and 47 miRNAs that were highly expressed in day 8 CM precursors versus undifferentiated cells, day 14 CMs versus undifferentiated cells, and Day 8 CM precursors versus Day 14 CMs, respectively (Table 1). The left/right ventricle transcriptional determinants, HAND1 and HAND2, were identified as targets of four miRNAs, (miR-363, -367, -181a, and -181c).
In a previous report, we showed that miR-125b regulated the development of hESC-derived early mesoderm and was highly expressed in cardiac precursors. We showed that miR-125b targets Lin28, indirectly inhibits Nanog and Oct4, and promotes onset of Brachyury, GATA4, and NKX2.5 expression to induce cardiac mesoderm formation. In both this and the current study, we predicted the target genes based on conserved pairing regions. We are aware that the majority of these predictions rely on extensive complementarity, while accounting for other features that contribute to miRNA 3′UTR recognition[23–26].
hESC differentiation in vitro leads to multiple cell lineages arising from the three embryonic germ layers, including CMs. The spontaneous differentiation of hESCs into CMs, however, leads to a heterogeneous mixture of CMs[3, 10]. Although several protocols are used for inducing CM differentiation, we used a method that allows ongoing interaction between many CM subtypes. In addition, we used a previously described aMHC-EGFP reporter hESC line that allowed us to sort specifically cardiac precursors and embryonic CMs.
The expression of miR-363 was detected with fluorescence in situ hybridization in E 10.5 mouse embryos. miR-363 was expressed in the developing limb bud, notochord, ectoderm, and brain. These results are consistent with the miR-363 expression in chick embryo reported previously, in which miR-363 was observed in ectoderm, pharyngeal arches, notochord, and limb bud, suggestive of wide function in limb development, patterning, and central nervous system development. Tissue-specific miRNA expression implies a negative regulatory role in expression f their targets. However, many transcription factors demonstrate more-complicated, stoichiometric expression during the course of tissue development. Relevant to this study, HAND2 is initially expressed throughout the developing heart tube, but then is restricted to second heart-field structures, with HAND1 expression restricted to the developing left ventricle. The mechanisms responsible for this developmentally regulated expression of HAND genes has until now been elusive.
Two evolutionarily conserved miR-363 seed-pairing sites in human HAND1 3′UTR suggested that the miRNA pairing sequences predicted in silico contribute to HAND1 regulation. HAND1 3′UTR reporter activity was completely abolished by miR-363 in contrast to HAND2 3′UTR activity. NKX2.5 also has been implicated in left/right asymmetric expression of HAND1 and HAND2; however, a role for miR-363 in regulating NKX2.5 expression was not suggested by our results.
BMP signaling controls the differentiation of CMs in multiple ways. In this work, BMP4 or BMP2 was shown to elicit induction of CM differentiation not only from precardiac mesoderm but also from tissue that is normally not cardiogenic. We tested various signaling molecules that have been implicated in cardiac induction[30–32]. TGF-β family members such as Activin A, bFGF, and BMPs, have been identified as promoting the terminal differentiation of precardiac mesoderm; however, when used at concentrations reported to be pharmacologically effective, neither Activin A nor bFGF displayed any discernible HAND1/2-inducing effect. BMP4 is closely related to the TGF-β family member BMP2, is expressed in anterior lateral ectoderm, and is indistinguishable from BMP2 in cardiac-inducing activity. We observed similar induction of HAND1 and HAND2 with BMP4 and BMP2. Taken together, these findings suggested a model for BMP-mediated cardiac induction and CM-subtype specification through miR-363 and differential expression of HAND1/2 (Figure 8B).
CM commitment after differentiation of hESCs was studied in the presence of a miR-363 antagomir. The isolated cardiogenic cells overexpressing anti-miR-363 expressed the left-specifying factor HAND1 at levels significantly higher than control. Cardiac progenitors that differentiate in vitro accumulate muscle-specific proteins but do not necessarily exhibit conspicuous beating or cross-striations; thus, these may be overlooked with less-sensitive assays with histology or spontaneous contractility.
We used qPCR to assess the differences in miRNA expression between beating foci and evaluated the ratio of HAND1 to HAND2 genes. miRNAs are known to act as transcriptional repressors of their target RNAs, thereby downregulating gene expression. However, it is possible that critical regulatory proteins may compensate for posttranscriptional downregulation by other mechanisms (for example, increased protein stability, decreased protein turnover). Therefore, our analysis may not identify every regulatory feature of the miRNA-CM subtype-specification pathway.
Right-ventricle CMs were devoid of HAND1 expression, which allowed us to distinguish between left and the right ventricular CMs. One explanation could be that the emergence of CMs from committed progenitors is influenced by cell density. High densities of committed progenitor cells show distinct populations of atrial and ventricular CMs. It appears that differentiation in the presence of anti-miR-363 causes progenitor cells to differentiate into a HAND1-rich population, and left-ventricular CMs constitute the greatest percentage of cells expressing HAND1.
Our results demonstrate for the first time that miR-363 plays an important role in posttranscriptional regulation of CM differentiation by targeting HAND1. These findings elucidate the mechanism by which differential HAND gene expression is achieved during cardiac development at the cellular level, and may be a valuable strategy for generating left ventricular CMs for further basic study and cell-therapy applications.
Bone morphogenetic protein 4
false discovery rate
fibroblast growth factor 2
fluorescence in situ hybridization
glyceraldyde 3-phosphate dehydrogenase
hand and neural crest derivative expressed 1
human embryonic stem cell
human induced pluripotent stem cell
locked nucleic acid
real-time quantitative polymerase chain reaction
Tris-buffered saline and 10% Tween 20
transforming growth factor-β
The authors thank members of the Bernstein Laboratory for helpful discussions, Kyle G. Richards for technical assistance, and Suzanne Chen for editorial assistance during the preparation of this manuscript. This work was supported by a grant from the California Institute of Regenerative Medicine (RC1-00104) to H.S.B.
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