Fetal and Neonatal Circulatory Disorders in Twin to Twin Transfusion Syndrome (The Secondary Publication).

Twin to twin transfusion syndrome (TTTS) is a major complication of monochorionic diamniotic (MD) twins, and its onset is known to be associated with placental vascular anastomoses and blood flow imbalance. In a typical case of TTTS, the recipient develops polyhydramnios, weight gain, cardiomegaly and hydrops fetalis in the uterus. In contrast, the donor develops oligohydramnios and intrauterine growth restriction. Recently, the significance of the renin-angiotensin-aldosterone system (RAAS) that transfers from the donor to the recipient has attracted interest in the fetal circulation of TTTS. The donor has decreased renal blood flow due to decreased circulating blood volume. For this reason, the secretion of RAAS hormones is augmented in the fetal kidneys of the donor. In TTTS, these RAAS hormones from the donor transfer to the recipient through the anastomosed vessels. In addition to excess preload, the recipient heart is exposed to excess afterload due to systemic vasoconstriction through RAAS hormones. Commonly occurring complications in the recipient include myocardial hypertrophy, atrioventricular valve regurgitation, and pulmonary valve stenosis or pulmonary atresia. Fetoscopic laser photocoagulation (FLP) has been introduced recently because neither mortality nor neurological morbidity have been satisfactorily improved with conventional treatment. FLP is a curative method that may improve the prognosis of TTTS. In Japan, this procedure has been performed frequently, and positive neurological outcomes have been achieved.

Maternal high-fat diet alters angiotensin II receptors and causes changes in fetal and neonatal rats†.

Maternal high-fat diet (HFD) during pregnancy is linked to cardiovascular diseases in postnatal life. The current study tested the hypothesis that maternal HFD causes myocardial changes through angiotensin II receptor (AGTR) expression modulation in fetal and neonatal rat hearts. The control group of pregnant rats was fed a normal diet and the treatment group of pregnant rats was on a HFD (60% kcal fat). Hearts were isolated from embryonic day 21 fetuses (E21) and postnatal day 7 pups (PD7). Maternal HFD decreased the body weight of the offspring in both E21 and PD7. The ratio of heart weight to body weight was increased in E21, but not PD7, when compared to the control group. Transmission electron microscopy revealed disorganized myofibrils and effacement of mitochondria cristae in the treatment group. Maternal HFD decreased S-phase and increased G1-phase of the cellular cycle for fetal and neonatal cardiac cells. Molecular markers of cardiac hypertrophy, such as Nppa and Myh7, were found to be increased in the treatment group. There was an associated increase in Agtr2 mRNA and protein, whereas Agtr1a mRNA and AGTR1 protein were decreased in HFD fetal and neonatal hearts. Furthermore, maternal HFD decreased glucocorticoid receptors (GRs) binding to glucocorticoid response elements at the Agtr1a and Agtr2 promoter, which correlated with downregulation of GR in fetal and neonatal hearts. These findings suggest that maternal HFD may promote premature termination of fetal and neonatal cardiomyocyte proliferation and compensatory hypertrophy through intrauterine modulation of AGTR1 and AGTR2 expression via GR dependent mechanism.

Loss of embryonic neural crest derived cardiomyocytes causes adult onset hypertrophic cardiomyopathy in zebrafish.

Neural crest cells migrate to the embryonic heart and transform into a small number of cardiomyocytes, but their functions in the developing and adult heart are unknown. Here, we show that neural crest derived cardiomyocytes (NC-Cms) in the zebrafish ventricle express Notch ligand jag2b, are adjacent to Notch responding cells, and persist throughout life. Genetic ablation of NC-Cms during embryogenesis results in diminished jag2b, altered Notch signaling and aberrant trabeculation patterns, but is not detrimental to early heart function or survival to adulthood. However, embryonic NC-Cm ablation results in adult fish that show severe hypertrophic cardiomyopathy (HCM), altered cardiomyocyte size, diminished adult heart capacity and heart failure in cardiac stress tests. Adult jag2b mutants have similar cardiomyopathy. Thus, we identify a cardiomyocyte population and genetic pathway that are required to prevent adult onset HCM and provide a zebrafish model of adult-onset HCM and heart failure.

Carbonic Anhydrase Inhibitors Induce Developmental Toxicity During Zebrafish Embryogenesis, Especially in the Inner Ear.

In vertebrates, carbonic anhydrases (CAs) play important roles in ion transport and pH regulation in many organs, including the eyes, kidneys, central nervous system, and inner ear. In aquatic organisms, the enzyme is inhibited by various chemicals present in the environment, such as heavy metals, pesticides, and pharmaceuticals. In this study, the effects of CA inhibitors, i.e., sulfonamides [ethoxyzolamide (EZA), acetazolamide (AZA), and dorzolamide (DZA)], on zebrafish embryogenesis were investigated. In embryos treated with the sulfonamides, abnormal development, such as smaller otoliths, an enlarged heart, an irregular pectoral fin, and aberrant swimming behavior, was observed. Especially, the development of otoliths and locomotor activity was severely affected by all the sulfonamides, and EZA was a consistently stronger inhibitor than AZA or DZA. In the embryos treated with EZA, inner ear hair cells containing several CA isoforms, which provide HCO3- to the endolymph for otolith calcification and maintain an appropriate pH there, were affected. Acridine orange/ethidium bromide staining indicated that the hair cell damage in the inner ear and pectral fin is due to apoptosis. Moreover, RNA measurement demonstrated that altered gene expression of cell cycle arrest- and apoptosis-related proteins p53, p21, p27, and Bcl-2 occurred even at 0.08 ppm with which normal development was observed. This finding suggests that a low concentration of EZA may affect embryogenesis via the apoptosis pathway. Thus, our findings demonstrated the importance of potential risk assessment of CA inhibition, especially regarding the formation of otoliths as a one of the most sensitive organs in embryogenesis.

Pregestational type 2 diabetes mellitus induces cardiac hypertrophy in the murine embryo through cardiac remodeling and fibrosis.

BACKGROUND: Cardiac hypertrophy is highly prevalent in patients with type 2 diabetes mellitus. Experimental evidence has implied that pregnant women with type 2 diabetes mellitus and their children are at an increased risk of cardiovascular diseases. Our previous mouse model study revealed that maternal type 2 diabetes mellitus induces structural heart defects in their offspring. OBJECTIVE: This study aims to determine whether maternal type 2 diabetes mellitus induces embryonic heart hypertrophy in a murine model of diabetic embryopathy. STUDY DESIGN: The type 2 diabetes mellitus embryopathy model was established by feeding 4-week-old female C57BL/6J mice with a high-fat diet for 15 weeks. Cardiac hypertrophy in embryos at embryonic day 17.5 was characterized by measuring heart size and thickness of the right and left ventricle walls and the interventricular septum, as well as the expression of ß-myosin heavy chain, atrial natriuretic peptide, insulin-like growth factor-1, desmin, and adrenomedullin. Cardiac remodeling was determined by collagen synthesis and fibronectin synthesis. Fibrosis was evaluated by Masson staining and determining the expression of connective tissue growth factor, osteopontin, and galectin-3 genes. Cell apoptosis also was measured in the developing heart. RESULTS: The thicknesses of the left ventricle walls and the interventricular septum of embryonic hearts exposed to maternal diabetes were significantly thicker than those in the nondiabetic group. Maternal diabetes significantly increased ß-myosin heavy chain, atrial natriuretic peptide, insulin-like growth factor-1, and desmin expression, but decreased expression of adrenomedullin. Moreover, collagen synthesis was significantly elevated, whereas fibronectin synthesis was suppressed, in embryonic hearts from diabetic dams, suggesting that cardiac remodeling is a contributing factor to cardiac hypertrophy. The cardiac fibrosis marker, galectin-3, was induced by maternal diabetes. Furthermore, maternal type 2 diabetes mellitus activated the proapoptotic c-Jun-N-terminal kinase 1/2 stress signaling and triggered cell apoptosis by increasing the number of terminal deoxynucleotidyl transferase 2'-deoxyuridine 5'-triphosphate nick end labeling-positive cells (10.4 ± 2.2% of the type 2 diabetes mellitus group vs 3.8 ± 0.7% of the nondiabetic group, P < .05). CONCLUSION: Maternal type 2 diabetes mellitus induces cardiac hypertrophy in embryonic hearts. Adverse cardiac remodeling, including elevated collagen synthesis, suppressed fibronectin synthesis, profibrosis, and apoptosis, is implicated as the etiology of cardiac hypertrophy.

Contrary microRNA Expression Pattern Between Fetal and Adult Cardiac Remodeling: Therapeutic Value for Heart Failure.

microRNAs (miRNAs) belong to a class of non-coding RNAs that regulate post-transcriptional gene expression during development and disease. Growing evidence indicates abundant miRNA expression changes and their important role in cardiac hypertrophy and failure. However, the role of miRNAs in fetal cardiac remodeling is little known. Here, we investigated the altered expression of fifteen miRNAs in rat fetal cardiac remodeling compared with adult cardiac remodeling. Among fifteen tested miRNAs, eleven and five miRNAs (miR-199a-5p, miR-214-3p, miR-155-3p, miR-155-5p and miR-499-5p) are significantly differentially expressed in fetal and adult cardiac remodeling, respectively. After comparison of miRNA expression in fetal and adult cardiac remodeling, we find that miRNA expression returns to the fetal level in adult cardiac failure and is activated in advance of the adult level in fetal failure. The current study highlights the contrary expression pattern between fetal and adult cardiac remodeling and that supports a novel potential therapeutic approach to treating heart failure.

Periods of cardiovascular susceptibility to hypoxia in embryonic american alligators (Alligator mississippiensis).

During embryonic development, environmental perturbations can affect organisms' developing phenotype, a process known as developmental plasticity. Resulting phenotypic changes can occur during discrete, critical windows of development. Critical windows are periods when developing embryos are most susceptible to these perturbations. We have previously documented that hypoxia reduces embryo size and increases relative heart mass in American alligator, and this study identified critical windows when hypoxia altered morphological, cardiovascular function and cardiac gene expression of alligator embryos. We hypothesized that incubation in hypoxia (10% O2) would increase relative cardiac size due to cardiac enlargement rather than suppression of somatic growth. We exposed alligator embryos to hypoxia during discrete incubation periods to target windows where the embryonic phenotype is altered. Hypoxia affected heart growth between 20 and 40% of embryonic incubation, whereas somatic growth was affected between 70 and 90% of incubation. Arterial pressure was depressed by hypoxic exposure during 50-70% of incubation, whereas heart rate was depressed in embryos exposed to hypoxia during a period spanning 70-90% of incubation. Expression of Vegf and PdgfB was increased in certain hypoxia-exposed embryo treatment groups, and hypoxia toward the end of incubation altered ß-adrenergic tone for arterial pressure and heart rate. It is well known that hypoxia exposure can alter embryonic development, and in the present study, we have identified brief, discrete windows that alter the morphology, cardiovascular physiology, and gene expression in embryonic American alligator.

The cerebral cavernous malformation proteins CCM2L and CCM2 prevent the activation of the MAP kinase MEKK3.

Three genes, CCM1, CCM2, and CCM3, interact genetically and biochemically and are mutated in cerebral cavernous malformations (CCM). A recently described member of this CCM family of proteins, CCM2-like (CCM2L), has high homology to CCM2. Here we show that its relative expression in different tissues differs from that of CCM2 and, unlike CCM2, the expression of CCM2L in endothelial cells is regulated by density, flow, and statins. In vitro, both CCM2L and CCM2 bind MEKK3 in a complex with CCM1. Both CCM2L and CCM2 interfere with MEKK3 activation and its ability to phosphorylate MEK5, a downstream target. The in vivo relevance of this regulation was investigated in zebrafish. A knockdown of ccm2l and ccm2 in zebrafish leads to a more severe "big heart" and circulation defects compared with loss of function of ccm2 alone, and also leads to substantial body axis abnormalities. Silencing of mekk3 rescues the big heart and body axis phenotype, suggesting cross-talk between the CCM proteins and MEKK3 in vivo. In endothelial cells, CCM2 deletion leads to activation of ERK5 and a transcriptional program that are downstream of MEKK3. These findings suggest that CCM2L and CCM2 cooperate to regulate the activity of MEKK3.

Alcohol-induced histone H3K9 hyperacetylation and cardiac hypertrophy are reversed by a histone acetylases inhibitor anacardic acid in developing murine hearts.

BACKGROUND: The expression of cardiac genes is precisely regulated, and any perturbation may cause developmental defects. In a previous study, we demonstrated that alcohol consumption during pregnancy could lead to uncontrolled expressions of cardiac genes and eventually result in cardiac dysplasia. However, the underlying mechanisms remain unclear. In the present study, we have investigated the alcohol-induced cardiac hypertrophy and its potential mechanisms. Furthermore, the protective effect of anacardic acid against the alcohol-induced cardiac hypertrophy has been explored in experimental mice. METHODS AND RESULTS: C57BL/6 pregnant mice were gavaged with 56% ethanol or saline and the hearts of their fetus were collected for analysis. Binding of p300, CBP, PCAF, SRC1, except GCN5, were increased to the NKX2.5 promoter in fetal mouse hearts exposed to alcohol. Increased acetylation of H3K9 and increased mRNA expression of NKX2.5, ß-MHC and Cx43 were observed in the same samples. Treatment with a pan-acetylase inhibitor, anacardic acid, reduced the binding affinity of p300 and PCAF to the NKX2.5, ß-MHC, Cx43 promoters and attenuated H3K9 hyperacetylation. Interestingly, anacardic acid down-regulated over-expression of these cardiac genes induced by alcohol and ultimately attenuated ethanol-induced cardiac hypertrophy in fetal mice. CONCLUSIONS: Our results indicate that alcohol exposure during pregnancy could lead to fetal cardiac hypertrophy. The over-expression of NKX2.5, ß-MHC, Cx43 mediated by p300 and PCAF may be critical mechanisms of alcohol-induced cardiac hypertrophy. Anacardic acid can down-regulate the over-expression of cardiac genes and reverse cardiac hypertrophy caused by alcohol treatment in pregnant mice, suggesting it could be a potential therapeutic agent for the treatment of cardiac hypertrophy.

Epigenetics in cardiac development, function, and disease.

Substantial new knowledge has accrued, over the past few years, concerning the epigenetic regulation of heart development and disease. Epigenetic mechanisms comprise DNA methylation, ATP-dependent chromatin remodeling, histone modifications, and non-coding RNAs. Many of these processes have been ascertained to influence the tight spatiotemporal control of gene expression during cardiac development. Nevertheless, the relative contribution of each mechanism and their potentially complex interplay remain largely unexplored. Cardiac development and disease are linked through the reactivation of fetal genes upon cardiac hypertrophy and failure. In cardiac disease, changes in gene expression are accompanied and influenced by distinct changes in histone modifications. Detailed knowledge about the epigenetic pathways of cardiac development and function is expected ultimately to lead to novel therapeutic strategies for heart disease and regenerative medicine.

Assessment of fetal cardiomyopathy in early-stage twin-twin transfusion syndrome: comparison between commonly reported cardiovascular assessment scores.

OBJECTIVES: To assess the relationship between commonly reported fetal cardiomyopathy scoring systems in early-stage twin-twin transfusion syndrome (TTTS). METHODS: We reviewed retrospectively 100 cases of Quintero Stages I and II TTTS referred to our center for evaluation from 2008 to 2010. The cases were divided into groups of 25, representing each of four grades of TTTS cardiomyopathy as assessed by Cincinnati stage: no cardiomyopathy, Stage IIIa, Stage IIIb and Stage IIIc. Spearman correlation (rs ) was calculated between the Children's Hospital of Philadelphia (CHOP) score, cardiovascular profile score (CVPS), Cincinnati stage and myocardial performance index (MPI). RESULTS: There was a weak correlation between the Cincinnati stage and the CHOP score (rs = 0.36) and CVPS (rs = -0.39), while correlation was strong between the CHOP score and CVPS (rs = -0.72). MPI elevation was concordant with Cincinnati stage more frequently (82% of cases) than were ventricular hypertrophy (43%) or atrioventricular valve regurgitation (28%). 51% of fetuses with minimally elevated CHOP score (0-1) and 48% of fetuses with minimally depressed CVPS (9-10) had significant elevation (Z-score ≥ +3) in right ventricular or left ventricular MPI. CONCLUSIONS: MPI has a strong influence on grading the severity of fetal cardiomyopathy using the Cincinnati stage among fetuses with mild TTTS. Furthermore, significant elevation of the MPI is common among fetuses with mild disease as assessed by the CHOP score and CVPS. These differences should be understood when assessing and grading cardiomyopathy in TTTS, particularly in early (Quintero Stages I and II) disease.

Endothelial deletion of ADAM17 in mice results in defective remodeling of the semilunar valves and cardiac dysfunction in adults.

Global inactivation of the metalloproteinase ADAM17 during mouse development results in perinatal lethality and abnormalities of the heart, including late embryonic cardiomegaly and thickened semilunar and atrioventricular valves. These defects have been attributed in part to a lack of ADAM17-mediated processing of HB-EGF, as absence of soluble HB-EGF results in similar phenotypes. Because valvular mesenchymal cells are largely derived from cardiac endothelial cells, we generated mice with a floxed Adam17 allele and crossed these animals with Tie2-Cre transgenics to focus on the role of endothelial ADAM17 in valvulogenesis. We find that although hearts from late-stage embryos with ablation of endothelial ADAM17 appear normal, an increase in valve size and cell number is evident, but only in the semilunar cusps. Unlike Hbegf(-/-) valves, ADAM17-null semilunar valves do not differ from controls in acute cell proliferation at embryonic day 14.5 (E14.5), suggesting compensatory processing of HB-EGF. However, levels of the proteoglycan versican are significantly reduced in mutant hearts early in valve remodeling (E12.5). After birth, aortic valve cusps from mutants are not only hyperplastic but also show expansion of the glycosaminoglycan-rich component, with the majority of adults exhibiting aberrant compartmentalization of versican and increased deposition of collagen. The inability of mutant outflow valve precursors to transition into fully mature cusps is associated with decreased postnatal viability, progressive cardiomegaly, and systolic dysfunction. Together, our data indicate that ADAM17 is required in valvular endothelial cells for regulating cell content as well as extracellular matrix composition and organization in semilunar valve remodeling and homeostasis.

Developmental cardiac hypertrophy in a mouse model of prolidase deficiency.

BACKGROUND: Hypertrophic cardiomyopathy, characterized by thickened ventricular walls and reduced ventricular chamber volume, is a common cause of sudden cardiac death in young people. Most inherited forms result from mutations in genes encoding sarcomeric proteins. METHODS: Histologic analysis identified embryonic cardiac hypertrophy in dark-like mutant mice. BrdU analysis was performed to measure proliferation and cardiomyocytes were isolated to measure cell size. The dark-like mutation was identified by positional cloning. RESULTS: The dark-like mutation causes cardiomyocyte hypertrophy due to loss-of-function of peptidase d (Pepd), which encodes prolidase, a cytosolic enzyme that recycles proline for collagen re-synthesis. Prolidase deficiency is a rare autosomal recessive disease in humans with a broad phenotypic spectrum not reported to include heart defects, but a conserved role for prolidase in heart development was confirmed by morpholino knockdown in zebrafish. We tested the hypothesis that loss of prolidase function disrupts collagen-mediated integrin signaling and determined that the levels of several key integrin transducers were reduced in the hearts of dark-like mutant embryos. CONCLUSIONS: This work identifies dark-like mice as a model of prolidase deficiency that will be valuable for studying the role of proline metabolism in normal physiology and disease processes, and suggests that integrin signaling may regulate the onset of hypertrophic cardiac growth.

Gene coexpression network topology of cardiac development, hypertrophy, and failure.

BACKGROUND: Network analysis techniques allow a more accurate reflection of underlying systems biology to be realized than traditional unidimensional molecular biology approaches. Using gene coexpression network analysis, we define the gene expression network topology of cardiac hypertrophy and failure and the extent of recapitulation of fetal gene expression programs in failing and hypertrophied adult myocardium. METHODS AND RESULTS: We assembled all myocardial transcript data in the Gene Expression Omnibus (n=1617). Because hierarchical analysis revealed species had primacy over disease clustering, we focused this analysis on the most complete (murine) dataset (n=478). Using gene coexpression network analysis, we derived functional modules, regulatory mediators, and higher-order topological relationships between genes and identified 50 gene coexpression modules in developing myocardium that were not present in normal adult tissue. We found that known gene expression markers of myocardial adaptation were members of upregulated modules but not hub genes. We identified ZIC2 as a novel transcription factor associated with coexpression modules common to developing and failing myocardium. Of 50 fetal gene coexpression modules, 3 (6%) were reproduced in hypertrophied myocardium and 7 (14%) were reproduced in failing myocardium. One fetal module was common to both failing and hypertrophied myocardium. CONCLUSIONS: Network modeling allows systems analysis of cardiovascular development and disease. Although we did not find evidence for a global coordinated program of fetal gene expression in adult myocardial adaptation, our analysis revealed specific gene expression modules active during both development and disease and specific candidates for their regulation.

MicroRNAs in cardiac development and remodeling.

MicroRNAs (miRNAs) are a class of highly conserved, small, noncoding RNAs that play roles in a wide range of biologic processes. Dysregulated miRNA expression has been associated with human cardiovascular disease, and studies using animal models have shown that miRNAs are essential for cardiac development and remodeling. These previously unrecognized small molecules shed new light on the regulatory mechanisms underlying cardiac development and pathology, suggesting the potential importance of miRNAs as diagnostic markers and therapeutic targets for cardiovascular disease.

Tricuspid valve dysplasia with severe tricuspid regurgitation: fetal pulmonary artery size predicts lung viability in the presence of small lung volumes.

Congenital tricuspid valve disease (Ebstein's anomaly, tricuspid valve dysplasia) with severe tricuspid regurgitation and cardiomegaly is associated with poor prognosis. Fetal echocardiography can accurately measure right atrial enlargement, which is associated with a poor prognosis in the fetus with tricuspid valve disease. Fetal lung volumetric assessments have been used in an attempt to predict viability of fetuses using ultrasonogram and prenatal MRI. We describe a fetus with tricuspid dysplasia, severe tricuspid regurgitation, right atrial enlargement and markedly reduced lung volumes. The early gestational onset of cardiomegaly with bilateral lung compression raised the possibility of severe lung hypoplasia with decreased broncho-alveolar development. Use of fetal echocardiography with measurement of pulmonary artery size combined with prenatal MRI scanning of lung volumes resulted in an improved understanding of this anomaly and directed the management strategy towards a successful Fontan circulation.