Lina Guan

CITED2 mutation links congenital heart defects to dysregulation of the cardiac gene VEGF and PITX2C expression.

CITED2, a cardiac transcription factor, plays an important role in cardiac development. CITED2 mutations lead to a constellation of cardiac defects, which include tetralogy of Fallot and outflow tract malformations. However, the mechanisms underlying these mutations are poorly understood. We investigated the function and mechanism of two missense mutations, G184S and S192G, responsible for tetralogy of Fallot and aortic stenosis, respectively. We found that CITED2 variants decreased its ability to mediate the expression of vascular endothelial growth factor (VEGF) and the expression of the paired-like homeodomain transcription factor 2-gamma (PITX2C), both of which are closely related to cardiac development. Luciferase reporter and mammalian two-hybrid assays showed that G184S and S192G in CITED2 restored the expression of VEGF, which was due to a reduction in its competitiveness with hypoxia inducible factor 1-alpha (HIF1-α) for binding to CBP/p300. In addition, we found that the G184S and S192G mutant decreased cooperation between CITED2 and transcription factor AP2-gamma (TFAP2C) in the transactivation of the PITX2C gene. These results provide important evidence that the mutation of CITED2 may play a role in the development of congenital heart disease (CHD) in humans.

Identification and Functional Analysis of GJA8 Mutation in a Chinese Family with Autosomal Dominant Perinuclear Cataracts

Congenital cataract is a clinically and genetically heterogeneous group of eye disorders that causes visual impairment and childhood blindness. The purpose of this study was to identify the genetic defect associated with autosomal dominant congenital perinuclear cataract in a Chinese family. A detailed family history and clinical data of the family were recorded, and candidate gene sequencing was performed to screen for mutation-causing disease in our study. Direct sequencing revealed a c.601G>A (p.E201K) transversion in exon 2 of GJA8. This mutation co-segregated with all affected individuals in the family and was not found in unaffected family members or 100 unrelated controls. The function and mechanism of novel GJA8 point mutation E201K in Chinese patients were then investigated in this study. We found E201K aberrantly located in cytoplasm and prevented its location in the plasma membrane. Induction of E201K expression led to a decrease in cell growth and viability by MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. Our study provides important evidence that GJA8 is a disease-causing gene for congenital cataract and that mutation of GJA8 has a potential causative effect.

Study on the mechanism of HIF1a-SOX9 in glucose-induced cardiomyocyte hypertrophy

A major cause of morbidity and mortality in cardiovascular disease is pathological cardiac hypertrophy. With an increase in the cellular surface area and upregulation of the atrial natriuretic peptide (ANP) gene, cardiac hypertrophy is a prominent feature of diabetic cardiomyopathy. ANP is a hypertrophic marker. Many works have been done to explore how the glucose induces the cardiac hypertrophy. However, it is not enough for us to figure it out. In this study, the influences of different glucose concentrations on cardiomyocytes were examined in vitro. The results showed that cardiomyocytes cultured with 25mM glucose tended to show a hypertrophic phenotype, while cardiomyocytes cultured with 35mM glucose tended to undergo apoptosis. An increased expression of SOX9 was observed when cardiomyocytes were cultured with 25mM glucose, but when the concentration of glucose was increased to 35mM, the expression of SOX9 decreased. We used the RNAi approach to knockdown SOX9 expression, to assess its effects on cardiomyocyte hypertrophy. The results showed that knockdown of the SOX9 gene suppressed the 25mM glucose-induced cardiomyocyte hypertrophy. The upregulation of the ANP gene was associated with overexpression of SOX9. Additionally, the results showed that high glucose (HG, 25mM) treatment increased the expression of hypoxia-inducible factor (HIF)1a. Further study showed that HIF1a participated in regulating SOX9 expression in response to HG. This study revealed a novel regulatory mechanism of HIF1a-SOX9 in high glucose-induced cardiomyocyte hypertrophy, as well as the related molecular mechanisms.

Down-regulation of EBAF in the heart with ventricular septal defects and its regulation by histone acetyltransferase p300 and transcription factors smad2 and cited2.

As a NODAL pathway inhibitor, EBAF plays a critical role during mammalian cardiac development. As recent tests that have been conducted on gene-targeted mice indicate, its expression is frequently altered where cardiac defects are present. We aimed to explore the EBAF expression pattern and molecular mechanism of EBAF gene for VSD genesis. In this report, we show that the average expression of EBAF in the disease tissues of VSD patients was lower than the expression in normal fetuses without VSD. Further study showed that the expression pattern of EBAF was potentially involved in cardiomyocyte apoptosis by Annexin-V and RT-PCR assays. We also found that abnormal activation of NODAL-PITX2C pathway was associated with down-regulation of EBAF. By luciferase reporter assays, we find that EBAF expression is mediated by transcriptional factors smad2 and cited2. In addition, ChIP assays showed that histone acetyltransferase p300 is involved in the activation of EBAF through inducing hyperacetylation of histone H4 at the EBAF promoter. Co-immunoprecipitation also indicates that the expression of EBAF is regulated by a transcriptional complex including p300, smad2, and cited2. This study revealed a novel regulator mechanism of EBAF, which may be a potential molecular target for halting the onset of VSDs. They also indicate that smad2, cited2, and p300 may play important roles in modulating the confirmation of ventricular septal defects.

Role of Nodal-PITX2C signaling pathway in glucose-induced cardiomyocyte hypertrophy

Pathological cardiac hypertrophy is a major cause of morbidity and mortality in cardiovascular disease. Recent studies have shown that cardiomyocytes, in response to high glucose (HG) stimuli, undergo hypertrophic growth. While much work still needs to be done to elucidate this important mechanism of hypertrophy, previous works have showed that some pathways or genes play important roles in hypertrophy. In this study, we showed that sublethal concentrations of glucose (25 mmol/L) could induce cardiomyocyte hypertrophy with an increase in the cellular surface area and the upregulation of the atrial natriuretic peptide (ANP) gene, a hypertrophic marker. High glucose (HG) treatments resulted in the upregulation of the Nodal gene, which is under-expressed in cardiomyocytes. We also determined that the knockdown of the Nodal gene resisted HG-induced cardiomyocyte hypertrophy. The overexpression of Nodal was able to induce hypertrophy in cardiomyocytes, which was associated with the upregulation of the PITX2C gene. We also showed that increases in the PITX2C expression, in response to Nodal, were mediated by the Smad4 signaling pathway. This study is highly relevant to the understanding of the effects of the Nodal-PITX2C pathway on HG-induced cardiomyocyte hypertrophy, as well as the related molecular mechanisms.

ROCK1/p53/NOXA signaling mediates cardiomyocyte apoptosis in response to high glucose in vitro and vivo

Gestational diabetes mellitus is a risk factor for congenital heart defects; however, the molecular basis of the congenital heart anomalies remains obscure. Previous reports showed a positive correlation between abnormal cardiomyocyte apoptosis and ventricular wall thinness, one type of congenital heart anomaly. This work explored the expression pattern and molecular mechanism of the Rho-associated coiled-coil containing protein kinase 1 (ROCK1) gene in cardiomyocyte apoptosis and genesis of ventricular wall thinness. In this report, we found a marked increase in the number of apoptotic cardiomyocytes in response to high glucose (HG) treatment. Moreover, up-regulation of ROCK1 expression observed in diabetic offspring compared with controls was potentially associated with cardiomyocyte apoptosis and the ventricular wall thinness. Further investigation showed that p53 and NOXA protein levels increased during ROCK1-meidated apoptosis in response to HG. In response to HG, whereby ROCK1 phosphorylated p53 at Ser15 to up-regulate its protein level. Furthermore, we found that p53 mediated the expression of NOXA during HG-induced apoptosis, and histone acetyltransferase p300 participated in this process. These findings reveal a novel regulatory mechanism of ROCK1/p53/NOXA signaling in modulating cardiomyocyte apoptosis in vitro and maternal diabetes-induced congenital heart defects in vivo.

Role of GAB1/PI3K/AKT signaling high glucose-induced cardiomyocyte apoptosis

Gestational diabetes mellitus (GDM) is a risk factor for abnormal heart development. Previous work showed that a decrease of myocardial cells and an increase of apoptotic cells leading to heart defects under hyperglycemia, and many genes and protein have been found to play important roles in cardiomyocyte apoptosis. However, there are still many blind nodes in HG-induced cardiac apoptosis. Our study showed that down-regulation of GAB1 occurred concurrently with HG-induced cardiomyocytes apoptosis and in the heart tissues of offspring of diabetic rats in vitro and in vivo. MTT and apoptosis assay showed GAB1 played a key role in mediating HG-induced apoptosis of cardiomyocytes. Down-regulation of XIAP and increased activities of Caspase3/7 was associated with GAB1-mediated cardiomyocyte apoptosis in response to HG treatment. Further study showed that the phosphorylation levels of AKT (Ser473) decreased after HG treatment. Over-expression of GAB1 resisted the reduction in AKT phosphorylation in response to HG. LY294002, which is an effective inhibitor of the PI3K/AKT signaling pathway, partly inhibited GAB1 to suppress apoptosis induced by HG in cardiomyocytes, and partly suppressed GAB1 to resist the decrease of XIAP in response to HG, indicating AKT signaling, XIAP, and Caspase3/7 participated in GAB1-mediated cardiomyocyte apoptosis in response to HG. Generally, we demonstrate a novel role of GAB1 and its down-stream signaling PI3K/AKT for modulating cardiomyocyte apoptosis in response to high glucose in vitro and vivo.

The connexin 46 mutant (V44M) impairs gap junction function causing congenital cataract

Connexin 46 (Cx46) is important for gap junction channels formation which plays crucial role in the preservation of lens homeostasis and transparency. Previously, we have identified a missense mutation (p.V44M) of Cx46 in a congenital cataract family. This study aims at dissecting the potential pathogenesis of the causative mutant of cataract. Plasmids carrying wild-type (wt) and mutant (V44M) of Cx46 were constructed and expressed in Hela cells respectively. Western blotting and fluorescence microscopy were applied to analyse the expression and subcellular localization of recombinant proteins, respectively. Scrape loading dye transfer experiment was performed to detect the transfer capability of gap junction channels among cells expressed V44M mutant. The results demonstrated that in transfected Hela cells, both wt-Cx46 and Cx46 V44M were localized abundantly in the plasma membrane. No significant difference was found between the protein expressions of the two types of Cx46. The fluorescent localization assay revealed the plaque formation, significantly reduced in the cells expressing Cx46 V44M. Immunoblotting analysis demonstrated that formation of Triton X-100 insoluble complex decreased obviously in mutant Cx46. Additionally, the scrape-loading dye-transfer experiment showed a lower dye diffusion distance of Cx46 V44M cells, which indicates that the gap junction intercellular communication activity was aberrant. Human Cx46 V44M mutant causing cataracts result in abnormally decreased formation of gap junction plaques and impaired gap junction channel function.