miR-182-5p is an evolutionarily conserved Tbx5 effector that impacts cardiac development and electrical activity in zebrafish.

To dissect the TBX5 regulatory circuit, we focused on microRNAs (miRNAs) that collectively contribute to make TBX5 a pivotal cardiac regulator. We profiled miRNAs in hearts isolated from wild-type, CRE, Tbx5lox/+and Tbx5del/+ mice using a Next Generation Sequencing (NGS) approach. TBX5 deficiency in cardiomyocytes increased the expression of the miR-183 cluster family that is controlled by Kruppel-like factor 4, a transcription factor repressed by TBX5. MiR-182-5p, the most highly expressed miRNA of this family, was functionally analyzed in zebrafish. Transient overexpression of miR-182-5p affected heart morphology, calcium handling and the onset of arrhythmias as detected by ECG tracings. Accordingly, several calcium channel proteins identified as putative miR-182-5p targets were downregulated in miR-182-5p overexpressing hearts. In stable zebrafish transgenic lines, we demonstrated that selective miRNA-182-5p upregulation contributes to arrhythmias. Moreover, cardiac-specific down-regulation of miR-182-5p rescued cardiac defects in a zebrafish model of Holt-Oram syndrome. In conclusion, miR-182-5p exerts an evolutionarily conserved role as a TBX5 effector in the onset of cardiac propensity for arrhythmia, and constitutes a relevant target for mediating the relationship between TBX5, arrhythmia and heart development.

Tbx5 is required for avian and Mammalian epicardial formation and coronary vasculogenesis.

RATIONALE: Holt-Oram syndrome is an autosomal dominant heart-hand syndrome caused by mutations in the TBX5 gene. Overexpression of Tbx5 in the chick proepicardial organ impaired coronary blood vessel formation. However, the potential activity of Tbx5 in the epicardium itself, and the role of Tbx5 in mammalian coronary vasculogenesis, remains largely unknown. OBJECTIVE: To evaluate the consequences of altered Tbx5 gene dosage during proepicardial organ and epicardial development in the embryonic chick and mouse. METHODS AND RESULTS: Retroviral-mediated knockdown or upregulation of Tbx5 expression in the embryonic chick proepicardial organ and proepicardial-specific deletion of Tbx5 in the embryonic mouse (Tbx5(epi-/)) impaired normal proepicardial organ cell development, inhibited epicardial and coronary blood vessel formation, and altered developmental gene expression. The generation of epicardial-derived cells and their migration into the myocardium were impaired between embryonic day (E) 13.5 to 15.5 in mutant hearts because of delayed epicardial attachment to the myocardium and subepicardial accumulation of epicardial-derived cells. This caused defective coronary vasculogenesis associated with impaired vascular smooth muscle cell recruitment and reduced invasion of cardiac fibroblasts and endothelial cells into myocardium. In contrast to wild-type hearts that exhibited an elaborate ventricular vascular network, Tbx5(epi-/-) hearts displayed a marked decrease in vascular density that was associated with myocardial hypoxia as exemplified by hypoxia inducible factor-1α upregulation and increased binding of hypoxyprobe-1. Tbx5(epi-/-) mice with such myocardial hypoxia exhibited reduced exercise capacity when compared with wild-type mice. CONCLUSIONS: Our findings support a conserved Tbx5 dose-dependent requirement for both proepicardial and epicardial progenitor cell development in chick and in mouse coronary vascular formation.

Keratin gene expression profiles after digit amputation in C57BL/6 vs. regenerative MRL mice imply an early regenerative keratinocyte activated-like state.

Mouse strains C57BL/6 (B6) and MRL were studied by whole mouse genome chip microarray analyses of RNA isolated from amputation sites at different times pre- and postamputation at the midsecond phalange of the middle digit. Many keratin genes were highly differentially expressed. All keratin genes were placed into three temporal response classes determined by injury/preinjury ratios. One class, containing only Krt6 and Krt16, were uniquely expressed relative to the other two classes and exhibited different temporal responses in MRL vs. B6. Immunohistochemical staining for Krt6 and Krt16 in tissue sections, including normal digit, flank skin, and small intestine, and from normal and injured ear pinna tissue exhibited staining differences in B6 (low) and MRL (high) that were consistent with the microarray results. Krt10 staining showed no injury-induced differences, consistent with microarray expression. We analyzed Krt6 and Krt16 gene association networks and observed in uninjured tissue several genes with higher expression levels in MRL, but not B6, that were associated with the keratinocyte activated state: Krt6, Krt16, S100a8, S100a9, and Il1b; these data suggest that keratinocytes in the MRL strain, but not in B6, are in an activated state prior to wounding. These expression levels decreased in MRL at all times postwounding but rose in the B6, peaking at day 3. Other keratins significantly expressed in the normal basal keratinocyte state showed no significant strain differences. These data suggest that normal MRL skin is in a keratinocyte activated state, which may provide it with superior responses to wounding.

Specification of the cardiac conduction system by transcription factors.

Diseases of the cardiovascular system that cause sudden cardiac deaths are often caused by lethal arrhythmias that originate from defects in the cardiac conduction system. Development of the cardiac conduction system is a complex biological process that can be wrought with problems. Although several genes involved in mature conduction system function have been identified, their association with development of specific subcomponents of the cardiac conduction system remains challenging. Several transcription factors, including homeodomain proteins and T-box proteins, are essential for cardiac conduction system morphogenesis and activation or repression of key regulatory genes. In addition, several transcription factors modify expression of genes encoding the ion channel proteins that contribute to the electrophysiological properties of the conduction system and govern contraction of the surrounding myocardium. Loss of transcriptional regulation during cardiac development has detrimental effects on cardiogenesis that may lead to arrhythmias. Human genetic mutations in some of these transcription factors have been identified and are known to cause congenital heart diseases that include cardiac conduction system malformations. In this review, we summarize the contributions of several key transcription factors to specification, patterning, maturation, and function of the cardiac conduction system. Further analysis of the molecular programs involved in this process should lead to improved diagnosis and therapy of conduction system disease.

Taking a bite out of hypertrophic cardiomyopathy: soy diet and disease.

Some forms of hypertrophic cardiomyopathy (HCM) are caused by mutations in cardiac sarcomeric genes, but environmental factors are believed to influence the hypertrophic response. A highly variable but potentially significant environmental factor is diet. Since soy-rich diets have been speculated to confer protection against cardiovascular disease, Stauffer et al. have explored the influence of a soy diet on cardiac growth and function in a transgenic mouse model of HCM. They report that mice fed a soy diet exhibited significantly worse HCM than mice fed a soy-free (milk protein) diet. This study provides the first evidence of an environmental modifier--diet--on the hypertrophic phenotype and has implications for the way in which disease phenotypes are assessed in genetically altered murine models of disease.

Genetics of cardiac septation defects and their pre-implantation diagnosis.

Cardiac septation defects are among the most common birth defects in humans. The frequency of these defects reflects the complexity of cardiogenesis, which involves such processes as cell proliferation, migration, differentiation, and morphogenetic interactions. Major advances in the understanding of the underlying genetic etiologies of cardiac septation defects have provided insight into the genetic pathways involved. These genetic factors are most often transcription factors involved in the early stages of cardiogenesis. The ability to modify these genes in animal models is providing a better understanding of the role of these genes in common pathways leading to diverse forms of cardiac defects. Ultimately, our understanding of these basic processes should lead to molecular-based treatment and prevention options for those individuals most at risk for such birth defects.

A role for Tbx5 in proepicardial cell migration during cardiogenesis.

Transcriptional regulatory cascades during epicardial and coronary vascular development from proepicardial progenitor cells remain to be defined. We have used immunohistochemistry of human embryonic tissues to demonstrate that the TBX5 transcription factor is expressed not only in the myocardium, but also throughout the embryonic epicardium and coronary vasculature. TBX5 is not expressed in other human fetal vascular beds. Furthermore, immunohistochemical analyses of human embryonic tissues reveals that unlike their epicardial counterparts, delaminating epicardial-derived cells do not express TBX5 as they migrate through the subepicardium before undergoing epithelial-mesenchymal transformation required for coronary vasculogenesis. In the chick, Tbx5 is expressed in the embryonic proepicardial organ (PEO), which is composed of the epicardial and coronary vascular progenitor cells. Retrovirus-mediated overexpression of human TBX5 inhibits cell incorporation of infected proepicardial cells into the nascent chick epicardium and coronary vasculature. TBX5 overexpression as well as antisense-mediated knockdown of chick Tbx5 produce a cell-autonomous defect in the PEO that prevents proepicardial cell migration. Thus, both increasing and decreasing Tbx5 dosage impairs development of the proepicardium. Culture of explanted PEOs demonstrates that untreated chick proepicardial cells downregulate Tbx5 expression during cell migration. Therefore, we propose that Tbx5 participates in regulation of proepicardial cell migration, a critical event in the establishment of the epicardium and coronary vasculature.

TGFßRIIb mutations trigger aortic aneurysm pathogenesis by altering transforming growth factor ß2 signal transduction.

BACKGROUND: Thoracic aortic aneurysm (TAA) is a common progressive disorder involving gradual dilation of the ascending and/or descending thoracic aorta that eventually leads to dissection or rupture. Nonsydromic TAA can occur as a genetically triggered, familial disorder that is usually transmitted in a monogenic autosomal dominant fashion and is known as familial TAA. Genetic analyses of families affected with TAA have identified several chromosomal loci, and further mapping of familial TAA genes has highlighted disease-causing mutations in at least 4 genes: myosin heavy chain 11 (MYH11), α-smooth muscle actin (ACTA2), and transforming growth factor ß receptors I and II (TGFßRI and TGFßRII). METHODS AND RESULTS: We evaluated 100 probands to determine the mutation frequency in MYH11, ACTA2, TGFßRI, and TGFßRII in an unbiased population of individuals with genetically mediated TAA. In this study, 9% of patients had a mutation in one of the genes analyzed, 3% of patients had mutations in ACTA2, 3% in MYH11, 1% in TGFßRII, and no mutations were found in TGFßRI. Additionally, we identified mutations in a 75 base pair alternatively spliced TGFßRII exon, exon 1a that produces the TGFßRIIb isoform and accounted for 2% of patients with mutations. Our in vitro analyses indicate that the TGFßRIIb activating mutations alter receptor function on TGFß2 signaling. CONCLUSIONS: We propose that TGFßRIIb expression is a regulatory mechanism for TGFß2 signal transduction. Dysregulation of the TGFß2 signaling pathway, as a consequence of TGFßRIIb mutations, results in aortic aneurysm pathogenesis.

Using the TBX5 transcription factor to grow and sculpt the heart.

TBX5 mutations cause the cardiac and limb defects of the autosomal dominant Holt-Oram syndrome (HOS). We have explored the role of the TBX5 transcription factor during cardiogenesis and have elucidated some of its functions in regulating myocardial cell proliferation and proepicardial cell migration. Our identification of TBX5 mutations has enabled us to offer genetic testing for diagnosis of HOS in patients and also to perform preimplantation genetic diagnosis on blastocysts for couples desiring to have a child unaffected by HOS. We hope that our genetic testing approach will serve as a paradigm for mutation screening in other inherited syndromes.

TBX5 nuclear localization is mediated by dual cooperative intramolecular signals.

The TBX5 transcription factor is required for normal cardiogenesis, and human TBX5 mutations cause congenital heart defects. Previous studies have shown that TBX5 can localize to cellular nuclei during embryogenesis and have suggested that altered nuclear localization may contribute to disease pathogenesis. Current analyses suggest that TBX5 nuclear localization is not uniform during organogenesis. To determine the biochemical mechanisms underlying TBX5 nuclear import, we performed site-directed mutagenesis of human TBX5. We identified two distinct nuclear localization signals in TBX5, one monopartite and one bipartite. While each is insufficient to promote complete TBX5 nuclear localization, they act cooperatively to do so. These sequences are evolutionarily conserved and have cognates in other T-box gene family members.