Essential roles of the bHLH transcription factor Hrt2 in repression of atrial gene expression and maintenance of postnatal cardiac function

Xin, Mei; Small, Eric M.; Rooij, Eva van; Qi, Xiaoxia; Richardson, James A.; Srivastava, Deepak; Nakagawa, Osamu; Olson, Eric N.

doi:10.1073/pnas.0702447104

Cited by 104 publications

(145 citation statements)

References 53 publications

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“…1). However, deletion of hrt2/hey2 in murine cardiomyocytes causes ectopic atrial gene expression in the ventricle (30). This might be due to the difference of expression patterns of hrt2/hey2 and grl in mice versus fish.…”

Section: Discussionmentioning

confidence: 97%

Vertebrate heart growth is regulated by functional antagonism between Gridlock and Gata5

Jia

King²,

Chopra³

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

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Embryonic organs attain their final dimensions through the generation of proper cell number and size, but the control mechanisms remain obscure. Here, we establish Gridlock (Grl), a Hairy-related basic helix-loop-helix (bHLH) transcription factor, as a negative regulator of cardiomyocyte proliferative growth in zebrafish embryos. Mutations in grl cause an increase in expression of a group of immediate-early growth genes, myocardial genes, and development of hyperplastic hearts. Conversely, cardiomyocytes with augmented Grl activity have diminished cell volume and fail to divide, resulting in a marked reduction in heart size. Both bHLH domain and carboxyl region are required for Grl negative control of myocardial proliferative growth. These Grl-induced cardiac effects are counterbalanced by the transcriptional activator Gata5 but not Gata4, which promotes cardiomyocyte expansion in the embryo. Biochemical analyses show that Grl forms a complex with Gata5 through the carboxyl region and can repress Gata5-mediated transcription via the bHLH domain. Hence, our studies suggest that Grl regulates embryonic heart growth via opposing Gata5, at least in part through their protein interactions in modulating gene expression.cardiomyocyte ͉ proliferation ͉ size control ͉ transcription T he embryonic heart grows through a combination of cardiomyocyte proliferation and an increase in cell mass due to the generation of myofibrillar arrays (1). Cardiac proliferation diminishes progressively after birth, and postmitotic hypertrophy in the adult heart provides most of the adaptive responses necessary to supply increased cardiac output (2). Despite recent progress that has been made in the regulation of postnatal cardiac hypertrophy, the mechanisms and pathways that control embryonic heart growth are poorly delineated. It is not known what molecular signals restrain cardiomyocyte proliferative growth in the embryonic heart. The GATA zinc-finger transcription factors promote myocardial differentiation and expansion. Among the three gata genes (gata4, gata5, and gata6) in zebrafish, gata5 plays the most prominent role in heart growth and development (3). Mutations in gata5 in zebrafish cause a reduction in expression of early and late myocardial genes and a decrease in cardiac progenitor cells and proliferative cardiomyocytes, resulting in small hearts. Forced gata5 expression in the zebrafish embryo increases heart size and occasionally produces ectopically beating myocardial tissue (4). The phenotype of zebrafish gata5 mutants closely resembles the cardiac phenotypes in gata4 mutant mice. Myocardium-restricted deletion of murine gata4 or gata4/gata6 double heterozygote causes a marked reduction in cardiomyocyte proliferation and results in hypoplastic hearts (5-7). Although it seems to be clear that certain levels of GATA activity are required to drive myocardial proliferative growth, opposing signals might also be necessary to constrain the excessive cardiac growth during development.grl encodes a hairy-related basic helix-loop-h...

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Section: Discussionmentioning

confidence: 97%

Vertebrate heart growth is regulated by functional antagonism between Gridlock and Gata5

Jia

King²,

Chopra³

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

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“…Therefore, it is expected that implementing standardized protocols for antibody labeling and analysis should better permit reliable comparison of the efficiency of cardiomyogenesis and subtype identity among protocols. As the field continues to establish robust protocols for efficient cardiomyogenesis, future efforts to more fully define the cells generated from such protocols will include the analysis of maturation stage and chamber-restricted markers such as hairy-related transcription factors 1 and 2 (HRT1, HRT2 38,39 ), gap junction alpha-5 protein (GJA5, CX40 40 ), sarcolipin (SLN 41 ) natriuretic peptides A (NPPA 42,43 ), potassium voltage-gated channel subfamily E member 1 (KCNE1 44 ), T-box transcription factor TBX3 (TBX3 45 ),heart-and neural crest derivatives-expressed protein(HAND1, HAND2 [46][47][48] ), and potassium/sodium hyperpolarization-activated cyclic nucleotide-gated channel 4 (HCN4 [49][50][51] ), among others. For marker analysis, it is recommended that both mRNA and protein levels be measured when possible, as protein modifications, stability and turnover can affect the relationship between gene expression and protein abundance (e.g., TNNT2 in Figures 1B, 2E) and, during development, patterns of expression differ between mRNA and protein for some markers (reviewed in Franco et al 20 ).…”

Section: Discussionmentioning

confidence: 99%

High Efficiency Differentiation of Human Pluripotent Stem Cells to Cardiomyocytes and Characterization by Flow Cytometry

Bhattacharya

Burridge

Kropp

et al. 2014

JoVE

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There is an urgent need to develop approaches for repairing the damaged heart, discovering new therapeutic drugs that do not have toxic effects on the heart, and improving strategies to accurately model heart disease. The potential of exploiting human induced pluripotent stem cell (hiPSC) technology to generate cardiac muscle "in a dish" for these applications continues to generate high enthusiasm. In recent years, the ability to efficiently generate cardiomyogenic cells from human pluripotent stem cells (hPSCs) has greatly improved, offering us new opportunities to model very early stages of human cardiac development not otherwise accessible. In contrast to many previous methods, the cardiomyocyte differentiation protocol described here does not require cell aggregation or the addition of Activin A or BMP4 and robustly generates cultures of cells that are highly positive for cardiac troponin I and T (TNNI3, TNNT2), iroquois-class homeodomain protein IRX-4 (IRX4), myosin regulatory light chain 2, ventricular/cardiac muscle isoform (MLC2v) and myosin regulatory light chain 2, atrial isoform (MLC2a) by day 10 across all human embryonic stem cell (hESC) and hiPSC lines tested to date. Cells can be passaged and maintained for more than 90 days in culture. The strategy is technically simple to implement and cost-effective. Characterization of cardiomyocytes derived from pluripotent cells often includes the analysis of reference markers, both at the mRNA and protein level. For protein analysis, flow cytometry is a powerful analytical tool for assessing quality of cells in culture and determining subpopulation homogeneity. However, technical variation in sample preparation can significantly affect quality of flow cytometry data. Thus, standardization of staining protocols should facilitate comparisons among various differentiation strategies. Accordingly, optimized staining protocols for the analysis of IRX4, MLC2v, MLC2a, TNNI3, and TNNT2 by flow cytometry are described. Video LinkThe video component of this article can be found at

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“…Furthermore, HEY2-deficient embryos displayed a change in the expression patterns of GJA5, NPPA, and TBX5. 43,45,46 These genes have all previously been associated with AF. [47][48][49][50] Thus, several studies support our finding that BrS-associated genetic variants protect against AF.…”

Section: Discussionmentioning

confidence: 99%

“…[41][42][43][44] Bezzina et al 27 found homozygous HEY2 knock-out embryos to have increased Na V 1.5 expression and a flattened transmural expression gradient of the channel in the cardiac ventricles. In HEY2 þ / À knock-out mice, the conduction velocity was increased in the right ventricle outflow tract (RVOT), and the action potential upstroke velocity was increased, indicating an increase in sodium channel peak current.…”

Section: Discussionmentioning

confidence: 99%

Brugada syndrome risk loci seem protective against atrial fibrillation

et al. 2014

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Several studies have shown an overlap between genes involved in the pathophysiological mechanisms of atrial fibrillation (AF) and Brugada Syndrome (BrS). We investigated whether three single-nucleotide polymorphisms (SNPs) (rs11708996; G4C located intronic to SCN5A, rs10428132; T4G located in SCN10A, and rs9388451; T4C located downstream to HEY2) at loci associated with BrS in a recent genome-wide association study (GWAS) also were associated with AF. A total of 657 patients diagnosed with AF and a control group comprising 741 individuals free of AF were included. The three SNPs were genotyped using TaqMan assays. The frequencies of risk alleles in the AF population and the control population were compared in two-by-two models. One variant, rs10428132 at SCN10A, was associated with a statistically significant decreased risk of AF (odds ratio (OR) ¼ 0.77, P ¼ 0.001). A meta-analysis was performed by enriching the control population with allele frequencies from controls in the recently published BrS GWAS (2230 alleles). In this meta-analysis, both rs10428132 at SCN10A (OR ¼ 0.73, P ¼ 5.7 Â 10 À6 ) and rs11708996 at SCN5A (OR ¼ 0.80, P ¼ 0.02) showed a statistically significant decreased risk of AF. When assessing the additive effect of the three loci, we found that the risk of AF decreased in a dose-responsive manner with increasing numbers of risk alleles (OR ¼ 0.50, P ¼ 0.001 for individuals carrying Z4 risk alleles vs r1 allele).In conclusion, the prevalence of three risk alleles previously associated with BrS was lower in AF patients than in patients free of AF, suggesting a protective role of these loci in developing AF.

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Essential roles of the bHLH transcription factor Hrt2 in repression of atrial gene expression and maintenance of postnatal cardiac function

Abstract: congenital heart disease ͉ heart development ͉ heart failure

Cited by 104 publications

References 53 publications

Vertebrate heart growth is regulated by functional antagonism between Gridlock and Gata5

Vertebrate heart growth is regulated by functional antagonism between Gridlock and Gata5

High Efficiency Differentiation of Human Pluripotent Stem Cells to Cardiomyocytes and Characterization by Flow Cytometry

Brugada syndrome risk loci seem protective against atrial fibrillation

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