Mingda Han

Fetal alcohol syndrome: cardiac birth defects in mice and prevention with folate

OBJECTIVE Alcohol (ethanol) consumption during pregnancy is linked to congenital heart defects that are associated with fetal alcohol syndrome. Recent reports have associated ethanol exposure with the Wnt/beta-catenin pathway. Therefore, we defined whether ethanol affects Wnt/beta-catenin signaling during cardiac cell specification. STUDY DESIGN Pregnant mice on embryonic day 6.75 during gastrulation were exposed by an intraperitoneal injection to a binge-drinking dose of ethanol. Folic acid supplementation of mouse diet was tested for the prevention of ethanol-induced cardiac birth defects. RESULTS Acute ethanol exposure induced myocardial wall changes and atrioventricular and semilunar valve defects, which was determined by echocardiography on embryonic day 15.5. A high folate diet prevented the ethanol-induced cardiac defects. Ethanol exposure in avian embryos suppressed 2 key Wnt-modulated genes that are involved in cardiac induction; folic acid rescued normal gene expression. CONCLUSION Folic acid supplementation alone or with myoinositol prevented alcohol potentiation of Wnt/beta-catenin signaling that allowed normal gene activation and cardiogenesis.

Cardiac Morphogenesis: Matrix Metalloproteinase Coordination of Cellular Mechanisms Underlying Heart Tube Formation and Directionality of Looping

During heart organogenesis, the spatiotemporal organization of the extracellular matrix (ECM) undergoes significant remodeling. Because matrix metalloproteinases (MMPs) are known to be key regulators of cell–matrix interactions, we analyzed the role(s) of MMPs, and specifically MMP‐2, in early heart development. Both MMP‐2 neutralizing antibody and the broad‐spectrum MMP inhibitor Ilomastat in a temporal manner, when applied between chick embryonic stages 5 (primitive streak stage) to stage 12 (∼16‐somites), produced severe heart tube defects. Exposure to the MMP inhibitor at stage 5 produced various degrees of cardia bifida. At the seven‐somite stage, MMP‐2/Ilomastat inhibition caused a shift in normal left‐right patterning of cell proliferation within the dorsal mesocardium and mesoderm of the anterior heart field that correlated with a change in looping direction. MMP inhibition at the 10‐ to 12‐somite stage resulted in an arrest of heart tube bending by inhibiting the breakdown of the dorsal mesocardial ECM. The experimental observations suggest that MMP activity regulates the coordination of early heart organogenesis by affecting ventral closure of the heart and gut tubes, asymmetric cell proliferation in the dorsal mesocardium to drive looping direction, and ECM degradation within the dorsal mesocardium allowing looping to proceed toward completion. Developmental Dynamics 233:739–753, 2005.

Blood Flow Dynamics of One Cardiac Cycle and Relationship to Mechanotransduction and Trabeculation during Heart Looping

Analyses of form-function relationships during heart looping are directly related to technological advances. Recent advances in four-dimensional optical coherence tomography (OCT) permit observations of cardiac dynamics at high-speed acquisition rates and high resolution. Real-time observation of the avian stage 13 looping heart reveals that interactions between the endocardial and myocardial compartments are more complex than previously depicted. Here we applied four-dimensional OCT to elucidate the relationships of the endocardium, myocardium, and cardiac jelly compartments in a single cardiac cycle during looping. Six cardiac levels along the longitudinal heart tube were each analyzed at 15 time points from diastole to systole. Using image analyses, the organization of mechanotransducing molecules, fibronectin, tenascin C, α-tubulin, and nonmuscle myosin II was correlated with specific cardiac regions defined by OCT data. Optical coherence microscopy helped to visualize details of cardiac architectural development in the embryonic mouse heart. Throughout the cardiac cycle, the endocardium was consistently oriented between the midline of the ventral floor of the foregut and the outer curvature of the myocardial wall, with multiple endocardial folds allowing high-volume capacities during filling. The cardiac area fractional shortening is much higher than previously published. The in vivo profile captured by OCT revealed an interaction of the looping heart with the extra-embryonic splanchnopleural membrane providing outside-in information. In summary, the combined dynamic and imaging data show the developing structural capacity to accommodate increasing flow and the mechanotransducing networks that organize to effectively facilitate formation of the trabeculated four-chambered heart.

Sodium-calcium exchanger (NCX-1) and calcium modulation: NCX protein expression patterns and regulation of early heart development

Ouabain‐induced inhibition of early heart development indicated that Na/K‐ATPase plays an important role in maintaining normal ionic balances during differentiation of cardiomyocytes (Linask and Gui [1995] Dev Dyn 203:93–105). Inhibition of the sodium pump is generally accepted to affect the activity of the Na+‐Ca++ exchanger (NCX) to increase intracellular [Ca++]. These previous findings suggested that Ca++ signaling may be an important modulator during differentiation of cardiomyocytes. In order to identify a connection between heart development and NCX‐mediated Ca++ regulation, we determined the embryonic spatiotemporal protein expression pattern of NCX‐1 during early developmental stages. In both chick and mouse embryos, NCX‐1 (the cardiac NCX isoform) is asymmetrically expressed during gastrulation; in the right side of the Hensens node in the chick, in the right lateral mesoderm in the mouse. At slightly later stages, NCX‐1 is expressed in the heart fields at comparable stages of heart development, in the chick at stage 7 and in the mouse at embryonic day (ED) 7.5. By ED 8 in the mouse, the exchanger protein displays a rostrocaudal difference in cardiac expression and an outer curvature‐inner curvature ventricular difference. By ED 9.5, cardiac expression has increased from that seen at ED8 and NCX‐1 is distributed throughout the myocardium consistent with the possibility that it is important in regulating initial cardiac contractile function. Only a low level of expression is detected in inflow and outflow regions. To substantiate a role for the involvement of calcium‐mediated signaling, using pharmacologic approaches, ionomycin (a Ca++ ionophore) was shown to perturb cardiac cell differentiation in a manner similar to ouabain as assayed by cNkx2.5 and sarcomeric myosin heavy chain expression. In addition, we show that an inhibitor of NCX, KB‐R7943, can similarly and adversely affect early cardiac development at stage 4/5 and arrests cardiac cell contractility in 12‐somite embryos. Thus, based upon NCX‐1 protein expression patterns in the embryo, experimental Ca++ modulation, and inhibition of NCX activity by KB‐R7943, these results suggest an early and central role for calcium‐mediated signaling in cardiac cell differentiation and NCXs regulation of the initial heartbeats in the embryo.

Early temporal‐specific responses and differential sensitivity to lithium and Wnt‐3A exposure during heart development

Members of both Wnt and bone morphogenetic protein (BMP) families of signaling molecules are important in heart development. We previously demonstrated that β‐catenin, a key downstream intermediary of the canonical Wnt signaling pathway, delineates the dorsal boundary of the cardiac compartments in an anteroposterior progression. We hypothesized the progression involves canonical Wnt signaling and reflects development of the primary body axis of the embryo. A similar anteroposterior signaling wave leading to cardiac cell specification involves inductive signaling by BMP‐2 synthesized by the underlying endoderm in anterior bilateral regions. Any molecule that disrupts the normal balance of Wnt and BMP concentrations within the heart field may be expected to affect early heart development. The canonical Wnt signaling step mimicked by lithium involves inactivation of glycogen synthase kinase‐3β (GSK‐3β; Klein and Melton [ 1996 ] Proc. Natl. Acad. Sci. U. S. A. 93:8455–8459). We show that lithium, Wnt‐3A, and an inhibitor of GSK‐3β, SB415286, affect early heart development at the cardiac specification stages. We demonstrate that normal expression patterns of key signaling molecules as Notch‐1 and Dkk‐1 are altered in the anterior mesoderm within the heart fields by a one‐time exposure to lithium, or by noggin inhibition of BMP, at Hamburger and Hamilton (HH) stage 3 during chick embryonic development. The severity of developmental defects is greatest with exposure to lithium or Wnt‐3A at HH stage 3 and decreases at HH stage 4. Taken together, our results demonstrate that there are temporal‐specific responses and differential sensitivities to lithium/Wnt‐3A exposure during early heart development. Developmental Dynamics 235:2160–2174, 2006.

Folate rescues lithium-, homocysteine- and Wnt3A-induced vertebrate cardiac anomalies.

SUMMARY Elevated plasma homocysteine (HCy), which results from folate (folic acid, FA) deficiency, and the mood-stabilizing drug lithium (Li) are both linked to the induction of human congenital heart and neural tube defects. We demonstrated previously that acute administration of Li to pregnant mice on embryonic day (E)6.75 induced cardiac valve defects by potentiating Wnt–β-catenin signaling. We hypothesized that HCy may similarly induce cardiac defects during gastrulation by targeting the Wnt–β-catenin pathway. Because dietary FA supplementation protects from neural tube defects, we sought to determine whether FA also protects the embryonic heart from Li- or HCy-induced birth defects and whether the protection occurs by impacting Wnt signaling. Maternal elevation of HCy or Li on E6.75 induced defective heart and placental function on E15.5, as identified non-invasively using echocardiography. This functional analysis of HCy-exposed mouse hearts revealed defects in tricuspid and semilunar valves, together with altered myocardial thickness. A smaller embryo and placental size was observed in the treated groups. FA supplementation ameliorates the observed developmental errors in the Li- or HCy-exposed mouse embryos and normalized heart function. Molecular analysis of gene expression within the avian cardiogenic crescent determined that Li, HCy or Wnt3A suppress Wnt-modulated Hex (also known as Hhex) and Islet-1 (also known as Isl1) expression, and that FA protects from the gene misexpression that is induced by all three factors. Furthermore, myoinositol with FA synergistically enhances the protective effect. Although the specific molecular epigenetic control mechanisms remain to be defined, it appears that Li or HCy induction and FA protection of cardiac defects involve intimate control of the canonical Wnt pathway at a crucial time preceding, and during, early heart organogenesis.

Cross talk between cell-cell and cell-matrix adhesion signaling pathways during heart organogenesis: implications for cardiac birth defects.

The anterior-posterior and dorsal-ventral progression of heart organogenesis is well illustrated by the patterning and activity of two members of different families of cell adhesion molecules: the calcium-dependent cadherins, specifically N-cadherin, and the extracellular matrix glycoproteins, fibronectin. N-cadherin by its binding to the intracellular molecule beta-catenin and fibronectin by its binding to integrins at focal adhesion sites, are involved in regulation of gene expression by their association with the cytoskeleton and through signal transduction pathways. The ventral precardiac mesoderm cells epithelialize and become stably committed by the activation of these cell-matrix and intracellular signaling transduction pathways. Cross talk between the adhesion signaling pathways initiates the characteristic phenotypic changes associated with cardiomyocyte differentiation: electrical activity and organization of myofibrils. The development of both organ form and function occurs within a short interval thereafter. Mutations in any of the interacting molecules, or environmental insults affecting either of these signaling pathways, can result in embryonic lethality or fetuses born with severe heart defects. As an example, we have defined that exposure of the embryo temporally to lithium during an early sensitive developmental period affects a canonical Wnt pathway leading to beta-catenin stabilization. Lithium exposure results in an anterior-posterior progression of severe cardiac defects.

Cellular Nonmuscle Myosins NMHC-IIA and NMHC-IIB and Vertebrate Heart Looping

Flectin, a protein previously described to be expressed in a left‐dominant manner in the embryonic chick heart during looping, is a member of the nonmuscle myosin II (NMHC‐II) protein class. During looping, both NMHC‐IIA and NMHC‐IIB are expressed in the mouse heart on embryonic day 9.5. The patterns of localization of NMHC‐IIB, rather than NMHC‐IIA in the mouse looping heart and in neural crest cells, are equivalent to what we reported previously for flectin. Expression of full‐length human NMHC‐IIA and ‐IIB in 10 T1/2 cells demonstrated that flectin antibody recognizes both isoforms. Electron microscopy revealed that flectin antibody localizes in short cardiomyocyte cell processes extending from the basal layer of the cardiomyocytes into the cardiac jelly. Flectin antibody also recognizes stress fibrils in the cardiac jelly in the mouse and chick heart; while NMHC‐IIB antibody does not. Abnormally looping hearts of the NodalΔ 600 homozygous mouse embryos show decreased NMHC‐IIB expression on both the mRNA and protein levels. These results document the characterization of flectin and extend the importance of NMHC‐II and the cytoskeletal actomyosin complex to the mammalian heart and cardiac looping. Developmental Dynamics 237:3577–3590, 2008.

Effects of antisense misexpression of CFC on downstream flectin protein expression during heart looping

Dextral looping of the heart is regulated on multiple levels. In humans, mutations of the genes CFC and Pitx2/RIEG result in laterality‐associated cardiac anomalies. In animal models, a common read‐out after the misexpression of laterality genes is heart looping direction. Missing in these studies is how laterality genes impact on downstream morphogenetic processes to coordinate heart looping. Previously, we showed that Pitx2 indirectly regulates flectin protein by regulating the timing of flectin expression in one heart field versus the other (Linask et al. [ 2002 ] Dev. Biol. 246:407–417). To address this question further we used a reported loss‐of‐function approach to interfere with chick CFC expression (Schlange et al. [ 2001 ] Dev. Biol. 234:376–389) and assaying for flectin expression during looping. Antisense CFC treatment results in abnormal heart looping or no looping. Our results show that regardless of the sidedness of downstream Pitx2 expression, it is the sidedness of predominant flectin protein expression in the extracellular matrix of the dorsal mesocardial folds and splanchnic mesoderm apposed to the foregut wall that is associated directly with looping direction. Thus, Pitx2 can be experimentally uncoupled from heart looping. The flectin asymmetry continues to be maintained in the secondary heart field during looping. Developmental Dynamics 228:217–230, 2003.

Tropomyosin expression and dynamics in developing avian embryonic muscles

The expression of striated muscle proteins occurs early in the developing embryo in the somites and forming heart. A major component of the assembling myofibrils is the actin-binding protein tropomyosin. In vertebrates, there are four genes for tropomyosin (TM), each of which can be alternatively spliced. TPM1 can generate at least 10 different isoforms including the striated muscle-specific TPM1alpha and TPM1kappa. We have undertaken a detailed study of the expression of various TM isoforms in 2-day-old (stage HH 10-12; 33 h) heart and somites, the progenitor of future skeletal muscles. Both TPM1alpha and TPM1kappa are expressed transiently in embryonic heart while TPM1alpha is expressed in somites. Both RT-PCR and in situ hybridization data suggest that TPM1kappa is expressed in embryonic heart whereas TPM1alpha is expressed in embryonic heart, and also in the branchial arch region of somites, and in the somites. Photobleaching studies of Yellow Fluorescent Protein-TPM1alpha and -TPM1kappa expressed in cultured avian cardiomyocytes revealed that the dynamics of the two probes was the same in both premyofibrils and in mature myofibrils. This was in sharp contrast to skeletal muscle cells in which the fluorescent proteins were more dynamic in premyofibrils. We speculate that the differences in the two muscles is due to the appearance of nebulin in the skeletal myocytes premyofibrils transform into mature myofibrils.