Cathy J. Hatcher

Mutations in the protein kinase A R1α regulatory subunit cause familial cardiac myxomas and Carney complex

Cardiac myxomas are benign mesenchymal tumors that can present as components of the human autosomal dominant disorder Carney complex. Syndromic cardiac myxomas are associated with spotty pigmentation of the skin and endocrinopathy. Our linkage analysis mapped a Carney complex gene defect to chromosome 17q24. We now demonstrate that the PRKAR1alpha gene encoding the R1alpha regulatory subunit of cAMP-dependent protein kinase A (PKA) maps to this chromosome 17q24 locus. Furthermore, we show that PRKAR1alpha frameshift mutations in three unrelated families result in haploinsufficiency of R1alpha and cause Carney complex. We did not detect any truncated R1alpha protein encoded by mutant PRKAR1alpha. Although cardiac tumorigenesis may require a second somatic mutation, DNA and protein analyses of an atrial myxoma resected from a Carney complex patient with a PRKAR1alpha deletion revealed that the myxoma cells retain both the wild-type and the mutant PRKAR1alpha alleles and that wild-type R1alpha protein is stably expressed. However, in this atrial myxoma, we did observe a reversal of the ratio of R1alpha to R2beta regulatory subunit protein, which may contribute to tumorigenesis. Further investigation will elucidate the cell-specific effects of PRKAR1alpha haploinsufficiency on PKA activity and the role of PKA in cardiac growth and differentiation.

Identification and localization of TBX5 transcription factor during human cardiac morphogenesis

Mutations in the TBX5 transcription factor gene cause human cardiac malformation in Holt‐Oram syndrome. To identify and localize TBX5 during cardiac morphogenesis, we performed immunohistochemical studies of TBX5 protein cardiac expression during human embryogenesis. Specific antibody to human TBX5 was generated in rabbits with a TBX5 synthetic peptide and affinity purification of antiserum. Anti‐TBX5 was used in immunohistochemical analyses of human cardiac tissue. In embryonic and adult heart, TBX5 is expressed throughout the epicardium and in cardiomyocyte nuclei in myocardium of all four cardiac chambers. Endocardial expression of TBX5 is only present in left ventricle. Asymmetric left‐sided transmyocardial gradients of TBX5 protein expression were observed in embryonic but not adult hearts. Human cardiac expression of TBX5 protein correlates with the cardiac manifestations of Holt‐Oram syndrome. TBX5 transmyocardial protein gradients may contribute to normal patterning of the human heart during embryogenesis.

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.

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.

Getting the T-box dose right

Holt–Oram syndrome has been associated with mutations in the T-box transcription factor TBX5, but little is known about the function of this protein or how mutations in it cause disease. A new mouse model of this syndrome will help to answer some of these questions.

Atrial Fibrillation and Other Clinical Manifestations of Altered TBX5 Dosage in Typical Holt–Oram Syndrome

To the editor: We were pleased to read the recent study in Circulation Research by Postma et al1 that describes an activation mutation in TBX5 that causes Holt–Oram syndrome. These exciting findings validate prior studies (reviewed elsewhere2) showing that cytogenetic abnormalities that produce TBX5 duplication (and presumed TBX5 overexpression) result in phenotypes that include Holt–Oram syndrome associated abnormalities. Moreover, we3,4 and others5 have previously demonstrated in experimental models that cell biological consequences of diminished and augmented Tbx5 expression are similar. In aggregate, these prior findings and the current data support a model6 in which Tbx5 dosage must …

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 (Tbx5epi-/) 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, Tbx5epi-/- hearts displayed a marked decrease in vascular density that was associated with myocardial hypoxia as exemplified by hypoxia inducible factor-1&agr; upregulation and increased binding of hypoxyprobe-1. Tbx5epi-/- 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.

Atrial form and function: Lessons from human molecular genetics

Molecular genetic analyses of human hereditary disorders that affect cardiac atrial structure and function have recently identified several genes that regulate atrial morphogenesis. Mutations of the TBX5, NKX2.5, EVC, and PRKAR1 alpha genes all result in abnormalities of human atrial growth and development, and mutations in at least one gene results in familial atrial fibrillation and is as yet unidentified. Ongoing studies to find interactions between these transcription factors and intracellular signaling molecules and other as yet unknown genes are establishing critical pathways in human cardiogenesis. Human investigation and experimental animal models of heart development synergize to elucidate etiologies of common congenital heart disease.

TGFβRIIb Mutations Trigger Aortic Aneurysm Pathogenesis by Altering Transforming Growth Factor β2 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), &agr;-smooth muscle actin (ACTA2), and transforming growth factor &bgr; receptors I and II (TGF&bgr;RI and TGF&bgr;RII). Methods and Results—We evaluated 100 probands to determine the mutation frequency in MYH11, ACTA2, TGF&bgr;RI, and TGF&bgr;RII 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 TGF&bgr;RII, and no mutations were found in TGF&bgr;RI. Additionally, we identified mutations in a 75 base pair alternatively spliced TGF&bgr;RII exon, exon 1a that produces the TGF&bgr;RIIb isoform and accounted for 2% of patients with mutations. Our in vitro analyses indicate that the TGF&bgr;RIIb activating mutations alter receptor function on TGF&bgr;2 signaling. Conclusions—We propose that TGF&bgr;RIIb expression is a regulatory mechanism for TGF&bgr;2 signal transduction. Dysregulation of the TGF&bgr;2 signaling pathway, as a consequence of TGF&bgr;RIIb mutations, results in aortic aneurysm pathogenesis.