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The ubiquitin-proteasome system (UPS) plays an important role in protein quality control, cellular signalings, and cell differentiation through the regulated turnover of key transcription factors in cardiac tissue. However, the molecular mechanism underlying Fbxo25-mediated ubiquitination of cardiac transcription factors remains elusive. We report that an Fbxo25-mediated SCF ubiquitination pathway regulates the protein levels and activities of Tbx5 and Nkx2-5 based on our studies using MG132, proteasome inhibitor, and the temperature sensitive ubiquitin system in ts20 cells. Our data indicate that Fbxo25 directly interacts with Tbx5 and Nkx2-5 in vitro and in vivo. In support of our findings, a dominant-negative mutant of Fbxo25, Fbxo251-236, prevents Tbx5 degradation and increases Tbx5 transcriptional activity in a Tbx5 responsive luciferase assay. Therefore, Fbxo25 facilitates Tbx5 degradation in an SCF-dependent manner. In addition, the silencing of endogenous Fbxo25 increases Tbx5 and Nkx2-5 mRNA levels and suppresses mESC-derived cardiomyocyte differentiation. Likewise, the exogenous expression of FBXO25 downregulates NKX2-5 level in human ESC-derived cardiomyocytes. In myocardial infarction model, Fbxo25 mRNA decreases, whereas the mRNA and protein levels of Tbx5 and Nkx2-5 increase. The protein levels of Tbx5 and Nkx2-5 are regulated negatively by Fbxo25-mediated SCF ubiquitination pathway. Thus, our findings reveal a novel mechanism for regulation of SCFFbox25-dependent Nkx2-5 and Tbx5 ubiquitination in cardiac development and provide a new insight into the regulatory mechanism of Nkx2-5 and Tbx5 transcriptional activity., (Copyright © 2015 Elsevier B.V. All rights reserved.)

The transcriptional profiles of cardiac cells derived from murine embryos and from mouse embryonic stem cells (mESCs) have primarily been studied within a cell population. However, the characterization of gene expression in these cells at a single-cell level might demonstrate unique variations that cannot be appreciated within a cell pool. In this study, we aimed to establish a single-cell quantitative PCR platform and perform side-by-side comparison between cardiac progenitor cells (CPCs) and cardiomyocytes (CMs) derived from mESCs and mouse embryos. We first generated a reference map for cardiovascular single cells through quantifying lineage-defining genes for CPCs, CMs, smooth muscle cells (SMCs), endothelial cells (EDCs), fibroblasts and mESCs. This panel was then applied against single embryonic day 10.5 heart cells to demonstrate its ability to identify each endocardial cell and chamber-specific CM. In addition, we compared the gene expression profile of embryo- and mESC-derived CPCs and CMs at different developmental stages and showed that mESC-derived CMs are phenotypically similar to embryo-derived CMs up to the neonatal stage. Furthermore, we showed that single-cell expression assays coupled with time-lapse microscopy can resolve the identity and the lineage relationships between progenies of single cultured CPCs. With this approach, we found that mESC-derived Nkx2-5(+) CPCs preferentially become SMCs or CMs, whereas single embryo-derived Nkx2-5(+) CPCs represent two phenotypically distinct subpopulations that can become either EDCs or CMs. These results demonstrate that multiplex gene expression analysis in single cells is a powerful tool for examining the unique behaviors of individual embryo- or mESC-derived cardiac cells., (© 2015. Published by The Company of Biologists Ltd.)

Understanding early differentiation events leading to cardiogenesis is crucial for controlling fate of human pluripotent stem cells and developing protocols that yield sufficient cell numbers for use in regenerative medicine and drug screening. Here, we develop a new tool to visualize patterning of early cardiac mesoderm and cardiomyocyte development in vitro by generating a dual MESP1(mCherry/w)-NKX2-5(eGFP/w) reporter line in human embryonic stem cells (hESCs) and using it to examine signals that lead to formation of cardiac progenitors and subsequent differentiation. MESP1 is a pivotal transcription factor for precardiac mesoderm in the embryo, from which the majority of cardiovascular cells arise. Transcription factor NKX2-5 is expressed upon cardiac crescent formation. Induction of cardiac differentiation in this reporter line resulted in transient expression of MESP1-mCherry, followed by continuous expression of NKX2-5-eGFP. MESP1-mCherry cells showed increased expression of mesodermal and epithelial-mesenchymal-transition markers confirming their mesodermal identity. Whole-genome microarray profiling and fluorescence-activated cell sorting analysis of MESP1-mCherry cells showed enrichment for mesodermal progenitor cell surface markers PDGFR-α, CD13, and ROR-2. No enrichment was found for the previously described KDR+PDGFR-α+ progenitors. MESP1-mCherry derivatives contained an enriched percentage of NKX2-5-eGFP and Troponin T expressing cells, indicating preferential cardiac differentiation; this was enhanced by inhibition of the Wnt-pathway. Furthermore, MESP1-mCherry derivatives harbored smooth muscle cells and endothelial cells, demonstrating their cardiac and vascular differentiation potential under appropriate conditions. The MESP1-NKX2-5 hESC reporter line allows us to identify molecular cues crucial for specification and expansion of human cardiac mesoderm and early progenitors and their differentiation to specific cardiovascular derivatives., (© 2014 AlphaMed Press.)

Homeodomain factor Nkx2-5 is a central component of the transcription factor network that guides cardiac development; in humans, mutations in NKX2.5 lead to congenital heart disease (CHD). We have genetically defined a novel conserved tyrosine-rich domain (YRD) within Nkx2-5 that has co-evolved with its homeodomain. Mutation of the YRD did not affect DNA binding and only slightly diminished transcriptional activity of Nkx2-5 in a context-specific manner in vitro. However, the YRD was absolutely essential for the function of Nkx2-5 in cardiogenesis during ES cell differentiation and in the developing embryo. Furthermore, heterozygous mutation of all nine tyrosines to alanine created an allele with a strong dominant-negative-like activity in vivo: ES cellembryo chimaeras bearing the heterozygous mutation died before term with cardiac malformations similar to the more severe anomalies seen in NKX2.5 mutant families. These studies suggest a functional interdependence between the NK2 class homeodomain and YRD in cardiac development and evolution, and establish a new model for analysis of Nkx2-5 function in CHD. [ABSTRACT FROM AUTHOR]

Atrial fibrillation (AF) is the most common form of sustained cardiac arrhythmia in humans and is responsible for substantial morbidity and mortality worldwide. Emerging evidence indicates that abnormal cardiovascular development is involved in the pathogenesis of AF. In this study, the coding exons and splice sites of the NKX2-5 gene, which encodes a homeodomain-containing transcription factor essential for cardiovascular genesis, were sequenced in 146 unrelated patients with lone AF as well as the available relatives of the mutation carriers. A total of 700 unrelated ethnically matched healthy individuals used as controls were genotyped. The disease-causing potential of the identified NKX2-5 variations was predicted by MutationTaster and PolyPhen-2. The functional characteristics of the mutant NKX2-5 proteins were analyzed using a dual-luciferase reporter assay system. As a result, two heterozygous NKX2-5 mutations, including a previously reported p.E21Q and a novel p.T180A mutation, were identified in two families with AF transmitted in an autosomal dominant pattern. The mutations co-segregated with AF in the families with complete penetrance. The detected substitutions, which altered the amino acids highly conserved evolutionarily across species, were absent in 700 control individuals and were both predicted to be causative. Functional analyses demonstrated that the NKX2-5 mutants were associated with significantly decreased transcriptional activity compared with their wild-type counterpart. The findings expand the spectrum of NKX2-5 mutations linked to AF and provide additional evidence that dysfunctional NKX2-5 may confer vulnerability to AF, suggesting the potential benefit for the early prophylaxis and personalized treatment of AF.

Rationale: Tight control of cardiomyocyte proliferation is essential for the formation of four-chambered heart. Although human mutation of NKX2-5 is linked to septal defects and atrioventricular conduction abnormalities, early lethality and hemodynamic alteration in the mutant models have caused controversy as to whether Nkx2-5 regulates cardiomyocyte proliferation., Objective: In this study, we circumvented these limitations by atrial-restricted deletion of Nkx2-5., Method and Results: Atrial-specific Nkx2-5 mutants died shortly after birth with hyperplastic working myocytes and conduction system including two nodes and internodal tracts. Multicolor reporter analysis revealed that Nkx2-5-null cardiomyocytes displayed clonal proliferative activity throughout the atria, indicating the suppressive role of Nkx2-5 in cardiomyocyte proliferation after chamber ballooning stages. Transcriptome analysis revealed that aberrant activation of Notch signaling underlies hyperproliferation of mutant cardiomyocytes, and forced activation of Notch signaling recapitulates hyperproliferation of working myocytes but not the conduction system., Conclusions: Collectively, these data suggest that Nkx2-5 regulates the proliferation of atrial working and conduction myocardium in coordination with Notch pathway.

A critical but molecularly uncharacterized step in heart formation and regeneration is the process that commits progenitor cells to differentiate into cardiomyocytes. Here, we show that the endoderm-derived dual Nodal/bone morphogenetic protein (BMP) antagonist Cerberus-1 (Cer1) in embryonic stem cell cultures orchestrates two signaling pathways that direct the SWI/SNF chromatin remodeling complex to cardiomyogenic loci in multipotent (KDR/Flk1+) progenitors, activating lineage-specific transcription. Transient inhibition of Nodal by Cer1 induces Brahma-associated factor 60c (Baf60c), one of three Baf60 variants (a, b, and c) that are mutually exclusively assembled into SWI/SNF. Blocking Nodal and BMP also induces lineage-specific transcription factors Gata4 and Tbx5, which interact with Baf60c. siRNA to Cer1, Baf60c, or the catalytic SWI/SNF subunit Brg1 prevented the developmental opening of chromatin surrounding the Nkx2.5 early cardiac enhancer and cardiomyocyte differentiation. Overexpression of Baf60c fully rescued these deficits, positioning Baf60c and SWI/SNF function downstream from Cer1. Thus, antagonism of Nodal and BMP coordinates induction of the myogenic Baf60c variant and interacting transcription factors to program the developmental opening of cardiomyocyte-specific loci in chromatin. This is the first demonstration that cues from the progenitor cell environment direct the subunit variant composition of SWI/SNF to remodel the transcriptional landscape for lineage-specific differentiation.

Congenital Heart Disease (CHD) is the most frequent and deadly birth defect. Patients with CHD that survive the neonatal period often progress to develop advanced heart failure requiring specialized treatment including cardiac transplantation. A full understanding of the transcriptional networks that direct cardiac progenitors during heart development will enhance our understanding of both normal cardiac function and pathological states. These findings will also have important applications for emerging therapies and the treatment of congenital heart disease. Furthermore, a number of shared transcriptional pathways or networks have been proposed to regulate the development and regeneration of tissues such as the heart. We have utilized transgenic technology to isolate and characterize cardiac progenitor cells from the developing mouse heart and have begun to define specific transcriptional networks of cardiovascular development. Initial studies identified Tdgf1 as a potential target of Nkx2-5. To mechanistically dissect the regulation of this molecular program, we utilized an array of molecular biological techniques to confirm that Nkx2-5 is an upstream regulator of the Tdgf1 gene in early cardiac development. These studies further define Nkx2-5 mediated transcriptional networks and enhance our understanding of cardiac morphogenesis.