TBX5: A Key Regulator of Heart Development.

TBX5 is a member of the T-box transcription factor family and is primarily known for its role in cardiac and forelimb development. Human patients with dominant mutations in TBX5 are characterized by Holt-Oram syndrome, and show defects of the cardiac septa, cardiac conduction system, and the anterior forelimb. The range of cardiac defects associated with TBX5 mutations in humans suggests multiple roles for the transcription factor in cardiac development and function. Animal models demonstrate similar defects and have provided a useful platform for investigating the roles of TBX5 during embryonic development. During early cardiac development, TBX5 appears to act primarily as a transcriptional activator of genes associated with cardiomyocyte maturation and upstream of morphological signals for septation. During later cardiac development, TBX5 is required for patterning of the cardiac conduction system and maintenance of mature cardiomyocyte function. A comprehensive understanding of the integral roles of TBX5 throughout cardiac development and adult life will be critical for understanding human cardiac morphology and function.

Characterization of sinoatrial node in four conduction system marker mice.

The specialized cardiac conduction system (CCS) consists of the sinoatrial node (SAN) and the atrioventricular (AV) conduction system (AVCS), which includes proximal (AV node, bundle of His and bundle branches) and distal (Purkinje fibers) components. In four CCS marker mice [two transgenic (cGATA6|lacZ, CCS|lacZ) and two targeted gene knock-in (minK|lacZ, Hop|lacZ)] the expression of the lacZ gene (beta-gal) has been reported to mark portions of the proximal and distal AVCS; the expression of this marker in the adult SAN is unknown. The primary objective of this study was to analyze the utility of these marker mice in the identification of the SAN. Intercaval and interventricular septal regions, containing all the components of the CCS, were freshly dissected from adult mice based on the anatomical landmarks and sectioned. Immunohistochemical characterization was performed with SAN markers (Cx45, HCN4), compared to the reporter expression (beta-gal) and markers of the working myocardium (Cx40 and Cx43). In all four of the CCS marker mice, we found that beta-gal expression is consistently observed in the proximal and distal AVCS. However, the presence of lacZ gene expression in the working myocardium outside the CCS and/or the absence of this reporter expression in the SAN prevent the effective use of these mice to identify the SAN, leading us to conclude that none of the four CCS marker mice we studied specifically mark the SAN.

Tetralogy of Fallot with congenital aortic valvar stenosis: the tetralogy-truncus interrelationship.

Two rare patients are reported with tetralogy of Fallot and congenital aortic valvar stenosis. The anatomic and developmental interrelationship between tetralogy of Fallot and truncus arteriosus is summarized. A study of 100 randomly selected postmortem cases of tetralogy revealed aortic valve pathology in 8%, myxomatous aortic valve leaflets without stenosis in 4%, bicuspid aortic valves without stenosis in 3%, and congenital aortic valvar stenosis in 1%. The frequency of systemic semilunar valve pathology in truncus was much higher (66%): moderate to marked myxomatous change in 44%, mild myxomatous change in 22%, truncal valvar stenosis in 11%, and truncal valvar regurgitation in 15%. Being aware of the tetralogy-truncus interrelationship and knowing that myxomatous aortic valves are prone to premature calcific aortic stenosis and/or regurgitation, physicians should follow the aortic valves of surgically repaired patients with tetralogy of Fallot and truncus arteriosus long term with great care. Timely aortic valvuloplasty or replacement may well prove life-saving in such patients.

Ventricular arrhythmia vulnerability in cardiomyopathic mice with homozygous mutant Myosin-binding protein C gene.

BACKGROUND: Homozygous mutant mice expressing a truncated form of myosin-binding protein C (MyBP-C(t/t)) develop severe dilated cardiomyopathy, whereas the heterozygous mutation (MyBP-C(t/+)) causes mild hypertrophic cardiomyopathy. Adult male MyBP-C(t/t) and MyBP-C(t/+) mice were evaluated for arrhythmia vulnerability with an in vivo electrophysiology study. METHODS AND RESULTS: Surface ECGs were obtained for heart rate, rhythm, and conduction intervals. Atrial, atrioventricular, and ventricular conduction parameters and refractoriness were assessed in 22 MyBP-C(t/t), 10 MyBP-C(t/+), and 17 wild-type MyBP-C(+/+) mice with endocardial pacing and intracardiac electrogram recording. Arrhythmia induction was attempted with standardized programmed stimulation at baseline and with isoproterenol. Heart rate variability and ambient arrhythmia activity were assessed with telemetric ECG monitors. Quantitative histological characterization was performed on serial sections of excised hearts. MyBP-C(t/t) and MyBP-C(t/+) mice have normal ECG intervals and sinus node, atrial, and ventricular conduction and refractoriness. Ventricular tachycardia was reproducibly inducible in 14 of 22 MyBP-C(t/t) mice (64%) during programmed stimulation, compared with 2 of 10 MyBP-C(t/+) mice (20%) and 0 of 17 wild-type controls (P<0.001). Ventricular ectopy was present only in MyBP-C(t/t) mice during ambulatory ECG recordings. There were no differences in heart rate variability parameters. Interstitial fibrosis correlated with genotype but did not predict arrhythmia susceptibility within the MyBP-C(t/t) group. CONCLUSIONS: MyBP-C(t/t) mice, despite prominent histopathology and ventricular dysfunction, exhibit normal conduction and refractoriness, yet are vulnerable to ventricular arrhythmias. Somatic influences between genetically identical mutant mice most likely account for variability in arrhythmia susceptibility. A sarcomeric protein gene mutation leads to a dilated cardiomyopathy and ventricular arrhythmia vulnerability phenotype.

Comparison of two murine models of familial hypertrophic cardiomyopathy.

Although sarcomere protein gene mutations cause familial hypertrophic cardiomyopathy (FHC), individuals bearing a mutant cardiac myosin binding protein C (MyBP-C) gene usually have a better prognosis than individuals bearing beta-cardiac myosin heavy chain (MHC) gene mutations. Heterozygous mice bearing a cardiac MHC missense mutation (alphaMHC(403/+) or a cardiac MyBP-C mutation (MyBP-C(t/+)) were constructed as murine FHC models using homologous recombination in embryonic stem cells. We have compared cardiac structure and function of these mouse strains by several methods to further define mechanisms that determine the severity of FHC. Both strains demonstrated progressive left ventricular (LV) hypertrophy; however, by age 30 weeks, alphaMHC(403/+) mice demonstrated considerably more LV hypertrophy than MyBP-C(t/+) mice. In older heterozygous mice, hypertrophy continued to be more severe in the alphaMHC(403/+) mice than in the MyBP-C(t/+) mice. Consistent with this finding, hearts from 50-week-old alphaMHC(403/+) mice demonstrated increased expression of molecular markers of cardiac hypertrophy, but MyBP-C(t/+) hearts did not demonstrate expression of these molecular markers until the mice were >125 weeks old. Electrophysiological evaluation indicated that MyBP-C(t/+) mice are not as likely to have inducible ventricular tachycardia as alphaMHC(403/+) mice. In addition, cardiac function of alphaMHC(403/+) mice is significantly impaired before the development of LV hypertrophy, whereas cardiac function of MyBP-C(t/+) mice is not impaired even after the development of cardiac hypertrophy. Because these murine FHC models mimic their human counterparts, we propose that similar murine models will be useful for predicting the clinical consequences of other FHC-causing mutations. These data suggest that both electrophysiological and cardiac function studies may enable more definitive risk stratification in FHC patients.

An abnormal Ca(2+) response in mutant sarcomere protein-mediated familial hypertrophic cardiomyopathy.

Dominant-negative sarcomere protein gene mutations cause familial hypertrophic cardiomyopathy (FHC), a disease characterized by left-ventricular hypertrophy, angina, and dyspnea that can result in sudden death. We report here that a murine model of FHC bearing a cardiac myosin heavy-chain gene missense mutation (alphaMHC(403/+)), when treated with calcineurin inhibitors or a K(+)-channel agonist, developed accentuated hypertrophy, worsened histopathology, and was at risk for early death. Despite distinct pharmacologic targets, each agent augmented diastolic Ca(2+) concentrations in wild-type cardiac myocytes; alphaMHC(403/+) myocytes failed to respond. Pretreatment with a Ca(2+)-channel antagonist abrogated diastolic Ca(2+) changes in wild-type myocytes and prevented the exaggerated hypertrophic response of treated alphaMHC(403/+) mice. We conclude that FHC-causing sarcomere protein gene mutations cause abnormal Ca(2+) responses that initiate a hypertrophic response. These data define an important Ca(2+)-dependent step in the pathway by which mutant sarcomere proteins trigger myocyte growth and remodel the heart, provide definitive evidence that environment influences progression of FHC, and suggest a rational therapeutic approach to this prevalent human disease.

Low-dose cyclosporin A therapy in children with refractory immune thrombocytopenic purpura.

Although splenectomy is the most effective treatment for chronic idiopathic thrombocytopenic purpura (ITP), many post-splenectomy patients have recurrent thrombocytopenia refractory to multiple medical therapies. Three consecutive patients with relapsed ITP after splenectomy and who were refractory to multiple medical therapies were treated with low dose cyclosporin A (CsA). In all 3 patients, the platelet count increased dramatically within 1 month from the onset of CsA therapy. The only detectable toxicity was hypomagnesemia and mild hypertension in 1 patient. CsA may be efficacious in treating patients with chronic ITP, which is refractory to all medical and surgical therapies currently being used.

lin-12 and glp-1 are required zygotically for early embryonic cellular interactions and are regulated by maternal GLP-1 signaling in Caenorhabditis elegans.

Cell-cell interactions mediated by LIN-12 and GLP-1, members of the LNG (LIN-12, Notch, GLP-1) family of receptors, are required to specify numerous cell fates during development of the nematode Caenorhabditis elegans. Maternally expressed GLP-1 participates in two of at least four sequential inductive interactions that specify the fates of early embryonic descendants of the AB founder cell. We report that GLP-1 and LIN-12, and apparently their ligand, LAG-2, as well as a downstream component, LAG-1, are required in the latter two inductions. We find that LAG-2 is expressed in the signaling cells and LIN-12 is expressed in cells receiving the inductions, consistent with their proposed roles as ligand and receptor, respectively. Furthermore, we report that maternal GLP-1 activity is required (1) to repress early zygotic lag-2 expression and (2) to activate zygotic lin-12 expression in the early embryo. The patterning of both receptor and ligand expression by maternal GLP-1 signaling establishes competence for the zygotic LNG-mediated cellular interactions and localizes these interactions to the appropriate cells. We propose that activation of maternal GLP-1 regulates zygotic lin-12 and lag-2 expression by a regulatory mechanism analogous to that described for the post-embryonic gonad.

The potential to differentiate epidermis is unequally distributed in the AB lineage during early embryonic development in C. elegans.

In the early Caenorhabditis elegans embryo most of the ectoderm arises from the AB blastomere, one of the six founder cells. We report that nonequivalent blastomeres are generated at the third division round in the AB lineage. Each AB granddaughter divides to produce one cell that has the potential to make abundant epidermis and one that instead produces primarily nervous system. This unequal distribution of the potential to make epidermis occurs in an AB granddaughter that is isolated by laser-ablation of all other cells or during the development of an isolated AB blastomere in culture. The fidelity of this event is normally masked by a signal from the MS founder cell, which induces mesoderm in particular AB descendants. When MS induction is prevented by laser cell-ablation or by a mutation in the glp-1 gene, the epidermal fate map of the AB great granddaughters becomes left-right symmetrical. Cell lineage analyses demonstrate that, in fact, the AB lineage becomes entirely left-right symmetrical in the absence of MS induction. This accounts for the extra epidermal cells previously observed in a glp-1 mutant. Our results suggest that epidermal differentiation in the nematode may be controlled by a cell-autonomous mechanism that differentially allocates epidermal potential during AB development and that MS induction generates the left-right asymmetry in the fates of AB descendants in part by overriding this potential.

Combinatorial specification of blastomere identity by glp-1-dependent cellular interactions in the nematode Caenorhabditis elegans.

Most somatic cells in the nematode Caenorhabditis elegans arise from AB, the anterior blastomere of the 2-cell embryo. While the daughters of AB, ABa and ABp, are equivalent in potential at birth, they adopt different fates as a result of their unique positions. One such difference is that the distribution of epidermal precursors arising from ABp is reversed along the anterior-posterior axis relative to those arising from ABa. We have found that a strong mutation in the glp-1 gene eliminates this ABa/ABp difference. Furthermore, extensive cell lineage analyses showed that ABp adopts an ABa-like fate in this mutant. This suggests that glp-1 acts in a cellular interaction that makes ABp distinct from ABa. One ABp-specific cell type was previously shown to be induced by an interaction with a neighboring cell, P2. By removing P2 from early embryos, we have found that the widespread differences between ABa and ABp arise from induction of the entire ABp fate by P2. Lineage analyses of genetically and physically manipulated embryos further suggest that the identifies of the AB great-granddaughters (AB8 cells) are controlled by three regulatory inputs that act in various combinations. These inputs are: (1) induction of the ABp-specific fate by P2, (2) a previously described induction of particular AB8 cells by a cell called MS, and (3) a process that controls whether an AB8 cell is an epidermal precursor in the absence of either induction. When an AB8 cell is caused to receive a new combination of these regulatory inputs, its lineage pattern is transformed to resemble the lineage of the wild-type AB8 cell normally receiving that combination of inputs. These lineage patterns are faithfully reproduced irrespective of position in the embryo, suggesting that each combination of regulatory inputs directs a unique lineage program that is intrinsic to each AB8 cell.

Configuration of DNA strands and mechanism of strand exchange in the Hin invertasome as revealed by analysis of recombinant knots.

The Hin recombinase of Salmonella normally catalyzes a site-specific DNA inversion reaction that is very efficient when the Fis protein and a recombinational enhancer sequence are present. The mechanism of this recombination reaction has been investigated by analyzing the formation and structure of knots generated in different plasmid substrates in vitro. Hin seldom knots the wild-type substrate under standard recombination conditions. However, we show that increasing the length of DNA between the recombination sites and the enhancer and changing the sequence of the core nucleotides where strand exchange occurs increases the efficiency of the knotting reaction. The structure of the knots generated by different mutant substrates strongly supports a model involving a unique configuration of DNA strands at synapsis and DNA strand exchange mediated by rotation of one set of Hin subunits after DNA cleavage. Analysis of the stereostructure of the knots by electron microscopy of RecA-coated DNA molecules demonstrates that the direction of subunit rotation is exclusively clockwise. Because multiple subunit rotations generating knotted molecules do not occur efficiently when the enhancer is located in its native position, we suggest that the enhancer normally remains associated with the two recombination sites in the invertasome structure during strand exchange to limit strand rotation once it has been initiated. Under certain conditions, however, complex knots are formed that are probably the result of the premature release of the enhancer and multiple, unrestrained subunit exchanges.

Alignment of recombination sites in Hin-mediated site-specific DNA recombination.

The Hin site-specific recombination system normally promotes inversion of DNA between two recombination sites in inverted orientation. We show that the rate of deletion of DNA between two directly repeated recombination sites is 10-300 times slower than inversion between sites in their native configuration as measured in vivo and in vitro, respectively. In vitro studies have shown that the deletion reaction has the same requirement for Fis, a recombinational enhancer, and DNA supercoiling as the inversion reaction. These requirements, together with the finding that the deletion products are interlinked once suggest that the deletion synaptic complex is similar to the invertasome intermediate that generates inversion. The inefficiency of the deletion reaction is not a function of a reduced ability to recognize or synapse recombination sites in direct orientation. Not only do these substrates support an efficient knotting reaction, but directly repeated recombination sites with symmetric core sequences also invert efficiently. These findings demonstrate that the recombination sites are preferentially assembled into the invertasome structure with the sites aligned in the configuration for inversion regardless of their starting orientation. We propose that the dynamics of a supercoiled DNA molecule biases the geometric assembly of specific intermediates. In the case of Hin-mediated recombination, inversion is overwhelmingly preferred over deletion because DNA supercoiling favors a specific alignment of DNA strands in the synaptic complex.