No description available.

Congenital heart defects due to the aberrant development of the atrioventricular (AV) valves and septum are among the most common developmental abnormality in newborns and cause significant neonatal morbidity and mortality. A key point in cardiac morphogenesis occurs when cells within the endocardial cushions (ECCs), the precursors for the AV valvoseptal complex, delaminate and undergo an epithelial-to-mesenchymal transformation (EMT). The mesenchymal cells then proliferate and the cushion area elongates to form the AV valves and portions of the AV septae. The signals that initiate region-specific EMT during heart development are unknown. Cilia, known for their role in establishing left-right (LR) asymmetry, function to receive and integrate extracellular signals, including fluid flow, in a range of other organ systems. We hypothesize that cilia could also have a direct role in heart development outside of their role in LR development. Using immunohistochemistry, we demonstrated the presence of cilia on the myocardium, epicardium, and ECCs of wild-type mouse hearts at embryonic day (e) 9.5 and e12.5. To characterize the potential role of these cilia, we compared mice with mutations affecting ciliary biogenesis, motility, and mechanosensation. Using bright field microscopy and in situ hybridization, we analyzed the embryonic heart structure and the expression pattern of Gata4, an EMT transcription factor. We showed that compared to mice with immotile but structurally normal cilia, the mice without cilia had hypocellular ECCs, a thinned compact myocardium (CM), and an up-regulated expression of Gata4. These observations suggest that a subset of cilia called cardiac cilia have a role in cardiogenesis outside of their role in LR development and affect Gata4 expression. One possible function of cardiac cilia is as mechanosensors, integrating fluid flow and influencing cardiac morphogenesis including EMT and development of the CM.

Congenital heart defects (CHDs) occur in 5% of births. While gene mutations have been identified in CHD patients, not much is known about coordinated signaling mechanisms during heart morphogenesis. Endocardial cushions of the atrioventricular canal and outflow tract contribute to the formation of valves and septa in the heart. Epithelial cell to mesenchymal cell transition (EMT) is a key process in cardiac cushions before this tissue undergoes remodeling into valves and septa. Defining complex signaling networks directing cardiac cushion epithelial to mesenchymal transition is essential for understanding the etiology of CHDs. We identified the MAP3Kinases, MEKK3 and MEKK4, as signaling components present during cardiovascular development. MEKK3 is detected in myocardium and endocardium surrounding the cardiac cushions of the atrioventricular canal during heart morphogenesis, while MEKK4 is found in the myocardium, endocardium, and cushion mesenchyme. Functional assays were employed to examine how MEKK3 and MEKK4 kinase activity contributes to endocardial EMT. Addition of dominant negative (dn)-MEKK3 or dn-MEKK4 to endocardial cushion explants, cultures that recapitulate in vivo EMT, causes a significant decrease in mesenchyme formation as compared to controls. Ventricular explant cultures, where the endocardial cells do not normally undergo EMT, provided with constitutively active (ca) MEKK3 activates mesenchyme production. ca-MEKK4 is not sufficient to cause EMT in ventricular endocardium. Furthermore, ca-MEKK3 expression in ventricular explants leads to increased secreted TGFβ2, which mediates mesenchyme formation. Blockade of TGFβ2 in ventricular explant cultures provided with ca-MEKK3 ablates the activation of EMT. In addition to in vitro studies, we show that mice expressing kinase inactive MEKK4 have myxomatous valves characterized by increased proliferation and changes in extracellular matrix molecules such as hyaluronan. We next investigated whether signal transduction is affected in cushions and valves of the MEKK4 kinase inactive mice. Abnormal TGFβ signaling is observed in MEKK4 mutant hearts, which is also seen with Marfan's sydrome. Remarkably, activated MEKK3 is maintained in cardiac cushions of these mice after EMT indicating compensation by MEKK3 for loss of MEKK4 catalytic activity. Our observations define MEKK3 and MEKK4 expression during cardiovascular development and suggest that MEKK3 and MEKK4 have diverse functions during development of heart valves.

Thesis (M.A.) -- University of Texas Southwestern Medical Center at Dallas, 2006. / Not embargoed. Vita. Bibliography: 101-102.

Determining how coordinated gene expression changes direct embryonic heart development is paramount to understanding the genetic causes and developmental origins of congenital cardiomyopathies. Towards this goal, I present an optimized protocol for mouse thiouracil tagging (TU-tagging), a novel transcriptomics methodology for defining dynamic and cell specific gene expression programs, and validate TU-tagging for cardiovascular research. I then apply related and additional high-resolution approaches to characterize how vascular endothelial growth factor (VEGF) signaling coordinates cell and gene expression dynamics underlying heart ventricle development. TU-tagging is a powerful approach to study dynamic gene expression programs of defined cell types while they are natively embedded within a complex organ. TU-tagging integrates genetic and chemical approaches to provide temporally controlled in vivo covalent labeling of cell type–specific RNA. Here, I describe two significant optimizations of the TU-tagging molecular biology and bioinformatics workflows that improve the method’s ability to identify differentially expressed genes and expand the technology’s utility. Next, using chemical inhibition of VEGF signaling in combination with high-resolution imaging and transcriptomic methods, I show that VEGF signaling directly promotes formation of the trabeculae that uniquely develop in the heart ventricle. By RNA-Seq, I identify VEGF-dependent target genes, including Gpr126 and Bmp10, which encode additional signaling proteins. I further show that myocardial Bmp10 expression and resulting endocardial Bmp10-driven pSMAD1/5/8 signaling is under sustained control by endocardial VEGF signaling. This continuous VEGF-BMP signaling interplay between endocardial and myocardial cells led me to examine the dynamic tissue arrangements between the two cell types during early stages of trabeculation. By extensive staining and high resolution imaging, I show that endocardial cells can be subdivided into three classes; 1) quiescent cavity cells that are well-separated from the myocardium, 2) proliferative and signal responsive transition zone/stalk cells that directly interact with myocardium to coordinate both cell types’ activities, and 3) CD34-expressing migratory tip-like cells uniquely found at the base of forming trabeculae. VEGF promotes trabeculation by 1) driving proliferation of endocardial transition zone/stalk cells and, secondarily, neighboring myocardium, and 2) directing the outward migration of endocardial tip cells that causes myocardial tissue to accumulate within individual and extensive ridge-like trabeculae. Defining these multiple roles of VEGF signaling during ventricle development reveals a novel conceptual framework for understanding trabeculation mechanisms and therefore processes likely to be disrupted in common congenital cardiomyopathies. This dissertation includes previously published and unpublished co-authored material. / 10000-01-01

No description available.

Isolated noncompaction of the left ventricular myocardium (INVM) is characterized by the presence of numerous prominent trabeculations and deep intertrabecular recesses within the left ventricle, sometimes also affecting the right ventricle and interventricular septum. Familial occurrence of this disorder was described previously. We present a family in which 6 affected individuals demonstrated X-linked recessive inheritance of this trait. Affected relatives presented postnatally with left ventricular failure and arrhythmias, associated with the pathognomonic echocardiographic findings of INVM. The usual findings of Barth syndrome (neutropenia, growth retardation, elevated urinary organic acids, low carnitine levels, and mitochondrial abnormalities) were either absent or found inconsistently. Fetal echocardiograms obtained between 24-30 weeks of gestation in 3 of the affected males showed a dilated left ventricle in one heart, but were not otherwise diagnostic of INVM in any of the cases. Four of the affected individuals died during infancy, one is in cardiac failure at age 8 months, and one is alive following cardiac transplant at age 9 months. The hearts from infants who died or underwent transplantation appeared, on gross examination, to be enlarged, with coarse, deep ventricular trabeculations and prominent endocardial fibroelastosis. Histologically, there were loosely organized fascicles of myocytes in subepicardial and midmyocardial zones of both ventricles, and the myocytes showed thin, often angulated fibers with prominent central clearing and reduced numbers of filaments. Markedly elongated mitochondria were present in some ventricular myocytes from one specimen, but this finding was not reproducible. Genetic linkage analysis has localized INVM to the Xq28 region, where other myopathies with cardiac involvement have been located.

Indiana University-Purdue University Indianapolis (IUPUI) / Neural crest cells (NCC) are a multipotent population of cells that form at the dorsal region of neural tube, migrate and contribute to a vast array of embryonic structures, including the majority of the head, the septum of the cardiac outflow tract (OFT), smooth muscle subpopulations, sympathetic nervous system and many other organs. Anomalous NCC morphogenesis is responsible for a wide variety of congenital defects. Importantly, several individual members of the TGFβ superfamily have been shown to play essential roles in various aspects of normal NCC development. However, it remains unclear what role Smad7, a negative regulator of TGFβ superfamily signaling, plays during development and moreover what the spatiotemporal effects are of combined suppression of TGFβ superfamily signaling during NCC formation and colonization of the developing embryo. Using a cre/loxP three-component triple transgenic system, expression of Smad7 was induced via doxycycline in the majority of pre- and post-migratory NCC lineages (via Wnt1-Cre mice). Further, expression of Smad7 was induced via doxycycline in a subset of post-migratory NCC lineages (via Periostin-Cre mice, after the NCC had reached their target organs and undergone differentiation). Induction of Smad7 within NCC significantly suppressed TGFβ superfamily signaling, as revealed via diminished phosphorylation levels of both Smad1/5/8 and Smad2/3 in vivo. This resulted in subsequent loss of NCC-derived craniofacial, pharyngeal and cardiac OFT cushion tissues. ROSA26r NCC lineage mapping demonstrated that cardiac NCC emigration and initial migration were unaffected, but subsequent colonization of the OFT was significantly reduced. At the cellular level, increased cell death was observed, but cell proliferation and NCC-derived smooth muscle differentiation were unaltered. Molecular analysis demonstrated that Smad7 induction resulted in selective increased phospho-p38 levels, which in turn resulted in the observed initiation of apoptosis in trigenic mutant embryos. Taken together, these data demonstrate that tightly regulated TGFβ superfamily signaling is essential for normal craniofacial and cardiac NCC colonization and cell survival in vivo.

Heart disease is the main cause of death today, and studying causes and treatments are of great interest. Blood flow simulations using computational fluid dynamics shows promise in providing insight into this area. This study builds upon previous work by Larsson et al. and Kronborg et al. who have developed a program for simulating the blood flow through patient specific left ventricles. More specifically we aimed to improve the accuracy of their blood flow simulation by accounting for the protruding structure of the endocardial wall, previously disregarded in the model due to the limitations in spacial accuracy of echocardiography. These structures, consisting of trabeculae carneae and papillary muscles, have been shown to have a significant impact on the blood flow. In a recent study, Sacco et al. proposed a solution were a porous layer could mimic the effects on the blood flow from these structures in a rigid heart model. Our study aimed to apply this modification to the left ventricle of the dynamic model using the Navier-Stokes-Brinkman flow equation and a subdomain defining the porous region. This study has been working towards the end goal of fully implementing the porous layer into the heart simulation. The equations needed have been formulated and simulations have been run on flow in a more simple setting to verify the model. The simulations show promise in being able to recreate the results from Sacco et al. but further development is needed before the porous model can be tested in the dynamic left ventricle model, most notably defining the porous subdomain in the dynamic model. We conclude that the porous domain will affect the flow, possibly breaking up vortices and reducing the wall shear stress. Confirming this requires additional studies, but the implementation of a porous domain would likely result in a more accurate simulation.

No description available.