KCNQ2 mutations cause unique neonatal behavior arrests without motor seizures: Functional characterization.

OBJECTIVE: KCNQ2 gene mutation usually manifests as neonatal seizures in the first week of life. Nonsense mutations cause a unique self-limited familial neonatal epilepsy (SLFNE), which is radically different from developmental epileptic encephalopathy (DEE). However, the exact underlying mechanisms remain unclear. METHODS: The proband, along with their mother and grandmother, carried the c.1342C > T (p.Arg448Ter) mutation in the KCNQ2 gene. The clinical phenotypes, electroencephalography (EEG) findings, and neurodevelopmental outcomes were comprehensively surveyed. The mutant variants were transfected into HEK293 cells to investigate functional changes. RESULTS: The proband exhibited behavior arrests, autonomic and non-motor neonatal seizures with changes in heart rate and respiration. EEG exhibited focal sharp waves. Seizures were remitted after three months of age. The neurodevelopmental outcomes at three years of age were unremarkable. A functional study demonstrated that the currents of p.Arg448Ter were non-functional in homomeric p.Arg448Ter compared with that of the KCNQ2 wild type. However, the current density and V1/2 exhibited significant improvement and close to that of the wild-type after transfection with heteromeric KCNQ2 + p.Arg448Ter and KCNQ2 + KCNQ3 + p.Arg448Ter respectively. Channel expression on the cell membrane was not visible after homomeric transfection, but not after heteromeric transfection. Retigabine did not affect homomeric p.Arg448Ter but improved heteromeric p. Arg448Ter + KCNQ2 and heteromeric KCNQ2 + Arg448Ter + KCNQ3. CONCLUSIONS: The newborn carrying the p. Arg448Ter mutation presented frequent behavioral arrests, autonomic, and non-motor neonatal seizures. This unique pattern differs from KCNQ2 seizures, which typically manifest as motor seizures. Although p.Arg448Ter is a non-sense decay, the functional study demonstrated an almost-full compensation mechanism after transfection of heteromeric KCNQ2 and KCNQ3.

Mechanistic study of the Aldo-keto reductase family 1 member A1 in regulating mesenchymal stem cell fate decision toward adipogenesis and osteogenesis.

AIMS: Akr1A1 is a glycolytic enzyme catalyzing the reduction of aldehyde to alcohol. This study aims to delineate the role of Akr1A1 in regulating the adipo-osteogenic lineage differentiation of mesenchymal stem cells (MSCs). MAIN METHODS: MSCs derived from human bone marrow and Wharton Jelly together with gain- and loss-of-function analysis as well as supplementation with the S-Nitrosoglutathione reductase (GSNOR) inhibitor N6022 were used to study the function of Akr1A1 in controlling MSC lineage differentiation into osteoblasts and adipocytes. KEY FINDINGS: Akr1A1 expression, PKM2 activity, and lactate production were found to be decreased in osteoblast-committed MSCs, but PGC-1α increased to induce mitochondrial oxidative phosphorylation. Increased Akr1A1 inhibited the SIRT1-dependent pathway for decreasing the expressions of PGC-1α and TAZ but increasing PPAR γ in adipocyte-committed MSCs, hence promoting glycolysis in adipogenesis. In contrast, Akr1A1 expression, PKM2 activity and lactate production were all increased in adipocyte-differentiated cells with decreased PGC-1α for switching energy utilization to glycolytic metabolism. Reduced Akr1A1 expression in osteoblast-committed cells relieves its inhibition of SIRT1-mediated activation of PGC-1α and TAZ for facilitating osteogenesis and mitochondrial metabolism. SIGNIFICANCE: Several metabolism-involved regulators including Akr1A1, SIRT1, PPARγ, PGC-1α and TAZ were differentially expressed in osteoblast- and adipocyte-committed MSCs. More importantly, Akr1A1 was identified as a new key regulator for controlling the MSC lineage commitment in favor of adipogenesis but detrimental to osteogenesis. Such information should be useful to develop perspective new therapeutic agents to reverse the adipo-osteogenic differentiation of BMSCs, in a way to increase in osteogenesis but decrease in adipogenesis.

Effects of aldo-keto reductase family 1 member A on osteoblast differentiation associated with lactate production in MC3T3-E1 preosteoblastic cells.

Aldo-keto reductase family 1 member A (AKR1A) is an NADPH-dependent aldehyde reductase widely expressed in mammalian tissues. In this study, induced differentiation of MC3T3-E1 preosteoblasts was found to increase AKR1A gene expression concomitantly increased NOx- (nitrite + nitrate), increased glucose uptake, increased [NAD(P)+]/[NAD(P)H] and lactate production but decreased reactive oxygen species (ROS) without changes in endothelial nitric oxide synthase (eNOS) expression in differentiated osteoblasts (OBs). A study using gain- and loss-of-function MC3T3-E1 cells indicated that AKR1A is essential for modulating OB differentiation and gene expression of collagen 1 A1, receptor activator of nuclear factor kappa-B ligand, and osteoprotegerin in OBs. Immunofluorescence microscopy also revealed that changes in AKR1A expression altered extracellular collagen formation in differentiated OBs. Consistently, analyses of alkaline phosphatase activity and calcium deposits of matrix mineralization by Alizarin Red S staining verified that AKR1A is involved in the regulation of OB differentiation and bone matrix formation. In addition, AKR1A gene alterations affected the levels of NOx-, eNOS expression, glucose uptake, [NAD(P)+]/[NAD(P)H] dinucleotide redox couples, lactate production, and ROS in differentiated OBs. Herein, we report that AKR1A-mediated denitrosylation may play a role in the regulation of lactate metabolism as well as redox homeostasis in cells, providing an efficient way to quickly gain energy and to significantly reduce oxidative stress for OB differentiation.

ZAKß Alleviates Oxidized Low-density Lipoprotein (ox-LDL)-Induced Apoptosis and B-type Natriuretic Peptide (BNP) Upregulation in Cardiomyoblast.

Oxidized low-density lipoprotein (ox-LDL) is a type of modified cholesterol that promotes apoptosis and inflammation and advances the progression of heart failure. Leucine-zipper and sterile-α motif kinase (ZAK) is a kinase of the MAP3K family which is highly expressed in the heart and encodes two variants, ZAKα and ZAKß. Our previous study serendipitously found opposite effects of ZAKα and ZAKß in which ZAKß antagonizes ZAKα-induced apoptosis and hypertrophy of the heart. This study aims to test the hypothesis of whether ZAKα and ZAKß are involved in the damaging effects of ox-LDL in the cardiomyoblast. Cardiomyoblast cells H9c2 were treated with different concentrations of ox-LDL. Cell viability and apoptosis were measured by MTT and TUNEL assay, respectively. Western blot was used to detect apoptosis, hypertrophy, and pro-survival signaling proteins. Plasmid transfection, pharmacological inhibition with D2825, and siRNA transfection were utilized to upregulate or downregulate ZAKß, respectively. Ox-LDL concentration-dependently reduces the viability and expression of several pro-survival proteins, such as phospho-PI3K, phospho-Akt, and Bcl-xL. Furthermore, ox-LDL increases cleaved caspase-3, cleaved caspase-9 as indicators of apoptosis and increases B-type natriuretic peptide (BNP) as an indicator of hypertrophy. Overexpression of ZAKß by plasmid transfection attenuates apoptosis and prevents upregulation of BNP. Importantly, these effects were abolished by inhibiting ZAKß either by D2825 or siZAKß application. Our results suggest that ZAKß upregulation in response to ox-LDL treatment confers protective effects on cardiomyoblast.

KCNQ2 Selectivity Filter Mutations Cause Kv7.2 M-Current Dysfunction and Configuration Changes Manifesting as Epileptic Encephalopathies and Autistic Spectrum Disorders.

KCNQ2 mutations can cause benign familial neonatal convulsions (BFNCs), epileptic encephalopathy (EE), and mild-to-profound neurodevelopmental disabilities. Mutations in the KCNQ2 selectivity filter (SF) are critical to neurodevelopmental outcomes. Three patients with neonatal EE carry de novo heterozygous KCNQ2 p.Thr287Ile, p.Gly281Glu and p.Pro285Thr, and all are followed-up in our clinics. Whole-cell patch-clamp analysis with transfected mutations was performed. The Kv7.2 in three mutations demonstrated significant current changes in the homomeric-transfected cells. The conduction curves for V1/2, the K slope, and currents in 3 mutations were lower than those for the wild type (WT). The p.Gly281Glu had a worse conductance than the p.Thr287Ile and p.Pro285Thr, the patient compatible with p.Gly281Glu had a worse clinical outcome than patients with p.Thr287Ile and p.Pro285Thr. The p.Gly281Glu had more amino acid weight changes than the p.Gly281Glu and p.Pro285Thr. Among 5 BFNCs and 23 EE from mutations in the SF, the greater weight of the mutated protein compared with that of the WT was presumed to cause an obstacle to pore size, which is one of the most important factors in the phenotype and outcome. For the 35 mutations in the SF domain, using changes in amino acid weight between the WT and the KCNQ2 mutations to predict EE resulted in 80.0% sensitivity and 80% specificity, a positive prediction rate of 96.0%, and a negative prediction rate of 40.0% (p = 0.006, χ2 (1, n = 35) = 7.56; odds ratio 16.0, 95% confidence interval, 1.50 to 170.63). The findings suggest that p.Thr287Ile, p.Gly281Glu and p.Pro285Thr are pathogenic to KCNQ2 EE. In mutations in SF, a mutated protein heavier than the WT is a factor in the Kv7.2 current and outcome.

Effects of varying gelatin coating concentrations on RANKL induced osteoclastogenesis.

Here, we assessed the effects of varying concentrations of gelatin coating on Receptor Activator of Nuclear Factor κ-B Ligand (RANKL)-induced RAW264.7 murine macrophage differentiation into osteoclast (OC) via osteoclastogenesis. The microstructures of coating surfaces with different concentrations of gelatin were examined by scanning electron microscopy and atomic force microscopy. Increased gelatin coating concentrations led to decreased gel rigidity but increased surface adhesion force attenuated OC differentiation and the decreased actin ring formation in RANKL-induced osteoclastogenesis. The decreased actin ring formation is associated with decreased lysosomal-associated membrane protein 1 (LAMP1) activity and bone resorption in the differentiated OCs with different gelatin coating concentrations as compared to the cells differentiated without gelatin coatings. In addition, increasing concentrations of gelatin coating attenuated the medium TGF-ß1 protein levels and the expression levels of TGF-ß and type-I (R1) and type-II (R2) TGF-ß receptors in OCs, suggesting the gelatin-induced suppression of TGF-ß signaling for the regulation of RNAKL-induced OC differentiation. Taken together, these findings showed that changes in gelatin coating concentrations, which were associated with altered gel thickness and substrate rigidity, might attenuate TGF-ß signaling events to modulate OC differentiation and concomitant actin ring formation and bone matrix resorption in RANKL-induced osteoclastogenesis.

Early Blood Glucose Level Post-Admission Correlates with the Outcomes and Oxidative Stress in Neonatal Hypoxic-Ischemic Encephalopathy.

The antioxidant defense system is involved in the pathogenesis of neonatal hypoxic-ischemic encephalopathy (HIE). To analyze the relationship between first serum blood glucose levels and outcomes in neonatal HIE, seventy-four patients were divided, based on the first glucose level, into group 1 (>0 mg/dL and <60 mg/dL, n =11), group 2 (≥60 mg/dL and <150 mg/dL, n = 49), and group 3 (≥150 mg/dL, n = 14). Abnormal glucose levels had poor outcomes among three groups in terms of the clinical stage (p = 0.001), brain parenchymal lesion (p = 0.004), and neurodevelopmental outcomes (p = 0.029). Hearing impairment was more common in group 3 than in group 1 (p = 0.062) and group 2 (p = 0.010). The MRI findings of group 3 exhibited more thalamus and basal ganglion lesions than those of group 1 (p = 0.012). The glucose level was significantly correlated with clinical staging (p< 0.001), parenchymal brain lesions (p = 0.044), hearing impairment (p = 0.003), and neurodevelopmental outcomes (p = 0.005) by Pearson's test. The first blood glucose level in neonatal HIE is an important biomarker for clinical staging, MRI findings, as well as hearing and neurodevelopment outcomes. Hyperglycemic patients had a higher odds ratio for thalamus, basal ganglia, and brain stem lesions than hypoglycemic patients with white matter and focal ischemic injury. Hyperglycemia can be due to prolonged or intermittent hypoxia and can be associated with poor outcomes.

Heteromeric Kv7.2 current changes caused by loss-of-function of KCNQ2 mutations are correlated with long-term neurodevelopmental outcomes.

Pediatric epilepsy caused by KCNQ2 mutations can manifest benign familial neonatal convulsions (BFNC) to neonatal-onset epileptic encephalopathy (EE). Patients might manifest mild to profound neurodevelopmental disabilities. We analysed c.853C > A (P285T) and three mutations that cause KCNQ2 protein changes in the 247 position: c.740C > T (S247L), c.740C > A (S247X), and c.740C > G (S247W). S247L, S247W, and P285T cause neonatal-onset EE and poor neurodevelopmental outcomes; S247X cause BFNC and normal outcome. We investigated the phenotypes correlated with human embryonic kidney 293 (HEK293) cell functional current changes. More cell-current changes and a worse conductance curve were present in the homomeric transfected S247X than in S247L, S247W, and P285T. But in the heteromeric channel, S247L, S247W and P285T had more current impairments than did S247X. The protein expressions of S247X were nonfunctional. The outcomes were most severe in S247L and S247W, and severity was correlated with heteromeric current. Current changes were more significant in cells with homomeric S247X, but currents were "rescued" after heteromeric transfection of KCNQ2 and KCNQ3. This was not the case in cells with S247L, S247W. Our findings support that homomeric current changes are common in KCNQ2 neonatal-onset EE and KCNQ2 BFNC; however, heteromeric functional current changes are correlated with long-term neurodevelopmental outcomes.

Matrix metalloproteinase 1 1 G/2 G gene polymorphism is associated with acquired atrioventricular block via linking a higher serum protein level.

Limited studies are available regarding the pathophysiological mechanism of acquired atrioventricular block (AVB). Matrix metalloproteinases (MMPs) and angiotensin-converting enzyme (ACE) have been implicated in the pathogenesis of arrhythmia. However, the relationship between these molecules and acquired AVB is still unclear. One hundred and two patients with documented acquired AVB and 100 controls were studied. Gene polymorphisms of the MMP1 and ACE encoding genes were screened by the gene sequencing method or polymerase chain reaction-fragment length polymorphism assay, followed by an association study. The frequencies of the MMP1 -1607 2G2G genotype and MMP1 -1607 2 G allele were significantly higher in the AVB group than that in the controls (OR = 1.933, P = 0.027 and OR = 1.684, P = 0.012, respectively). Consistently, the level of serum MMP1 was significantly greater in acquired AVB patients than that in controls (6568.9 ± 5748.6 pg/ml vs. 4730.5 ± 3377.1 pg/ml, P = 0.019). In addition, the MMP1 2G2G genotype showed a higher MMP-1 serum level than the other genotypes (1G1G/1G2G) (7048.1 ± 5683.0 pg/ml vs. 5072.4 ± 4267.6 pg/ml, P = 0.042). MMP1 1 G/2 G gene polymorphism may contribute to determining the disease susceptibility of acquired AVB by linking the MMP serum protein level.

L-caldesmon alters cell spreading and adhesion force in RANKL-induced osteoclasts.

BACKGROUND: Osteoclasts (OCs) are motile multinucleated cells derived from differentiation and fusion of hematopoietic progenitors of the monocyte-macrophage lineage that undergo a multistep process called osteoclastogenesis. The biological function of OCs is to resorb bone matrix for controlling bone strength and integrity, which is essential for bone development. The bone resorption function is based on the remodelling of the actin cytoskeleton into an F-actin-rich structure known as the sealing zone for bone anchoring and matrix degradation. Non-muscle caldesmon (l-CaD) is known to participate in the regulation of actin cytoskeletal remodeling, but its function in osteoclastogenesis remains unclear. METHODS/RESULTS: In this study, gain and loss of the l-CaD level in RAW264.7 murine macrophages followed by RANKL induction was used as an experimental approach to examine the involvement of l-CaD in the control of cell fusion into multinucleated OCs in osteoclastogenesis. In comparison with controls, l-CaD overexpression significantly increased TRAP activity, actin ring structure and mineral substrate resorption in RANKL-induced cells. In contrast, gene silencing against l-CaD decreased the potential for RANKL-induced osteoclastogenesis and mineral substrate resorption. In addition, OC precursor cells with l-CaD overexpression and gene silencing followed by RANKL induction caused 13% increase and 24% decrease, respectively, in cell fusion index. To further understand the mechanistic action of l-CaD in the modulation of OC fusion, atomic force microscopy was used to resolve the mechanical changes of cell spreading and adhesion force in RANKL-induced cells with and without l-CaD overexpression or gene silencing. CONCLUSIONS: l-CaD plays a key role in the regulation of actin cytoskeletal remodeling for the formation of actin ring structure at the cell periphery, which may in turn alter the mechanical property of cell-spreading and cell surface adhesion force, thereby facilitating cell-cell fusion into multinucleated OCs during osteoclastogenesis.

Effect of l-caldesmon on osteoclastogenesis in RANKL-induced RAW264.7 cells.

Non-muscle caldesmon (l-CaD) is involved in the regulation of actin cytoskeletal remodeling in the podosome formation, but its function in osteoclastogenesis remains to be determined. In this study, RANKL-induced differentiation of RAW264.7 murine macrophages to osteoclast-like cells (OCs) was used as a model to determine the physiological role of l-CaD and its phosphorylation in osteoclastogenesis. Upon RANKL treatment, RAW264.7 cells undergo cell-cell fusion into multinucleate, and TRAP-positive large OCs with a concomitant increase of l-CaD expression. Using gain- and loss-of-function in OC precursor cells followed by RANKL induction, we showed that the expression of l-CaD in response to RANKL activation is an important event for osteoclastogenesis, and bone resorption. To determine the effect of l-CaD phosphorylation in osteoclastogenesis, three decoy peptides of l-CaD were used with, respectively, Ser-to-Ala mutations at the Erk- and Pak1-mediated phosphorylation sites, and Ser-to-Asp mutation at the Erk-mediated phosphorylation sites. Both the former two peptides competed with the C-terminal segment of l-CaD for F-actin binding and accelerated formation of podosome-like structures in RANKL-induced OCs, while the third peptide did not significantly affect the F-actin binding of l-CaD, and decreased the formation of podosome-like structures in OCs. With the experiments using dephosphorylated and phosphorylated l-CaD mutants, we further showed that dephosphorylated l-CaD mutant facilitated RANKL-induced TRAP activity with an increased cell fusion index, whereas phosphorylated l-CaD decreased the TRAP activity and cell fusion. Our findings suggested that both the level of l-CaD expression and the extent of l-CaD phosphorylation play a role in RANKL-induced osteoclast differentiation.

Transforming Growth Factor-ß1 T869C Gene Polymorphism Is Associated with Acquired Sick Sinus Syndrome via Linking a Higher Serum Protein Level.

BACKGROUND: Familial sick sinus syndrome is associated with gene mutations and dysfunction of ion channels. In contrast, degenerative fibrosis of the sinus node tissue plays an important role in the pathogenesis of acquired sick sinus syndrome. There is a close relationship between transforming growth factor-ß1 mediated cardiac fibrosis and acquired arrhythmia. It is of interest to examine whether transforming growth factor-ß1 is involved in the pathogenesis of acquired sick sinus syndrome. METHODS: Overall, 110 patients with acquired SSS and 137 age/gender-matched controls were screened for transforming growth factor-ß1 and cardiac sodium channel gene polymorphisms using gene sequencing or restriction fragment length polymorphism methods. An enzyme-linked immunosorbent assay was used to determine the serum level of transforming growth factor-ß1. RESULTS: Two transforming growth factor-ß1 gene polymorphisms (C-509T and T+869C) and one cardiac sodium channel gene polymorphism (H588R) have been identified. The C-dominant CC/CT genotype frequency of T869C was significantly higher in acquired sick sinus syndrome patients than in controls (OR 2.09, 95% CI 1.16-3.75, P = 0.01). Consistently, the level of serum transforming growth factor-ß1 was also significantly greater in acquired sick sinus syndrome group than in controls (5.3±3.4 ng/ml vs. 3.7±2.4 ng/ml, P = 0.01). In addition, the CC/CT genotypes showed a higher transforming growth factor-ß1 serum level than the TT genotype (4.25 ± 2.50 ng/ml vs. 2.71± 1.76 ng/ml, P = 0.028) in controls. CONCLUSION: Transforming growth factor-ß1 T869C polymorphism, correlated with high serum transforming growth factor-ß1 levels, is associated with susceptibility to acquired sick sinus syndrome.

Involvement of gelsolin in TGF-beta 1 induced epithelial to mesenchymal transition in breast cancer cells.

BACKGROUND: Increasing evidence suggests that transforming growth factor-beta 1 (TGF-ß1) triggers epithelial to mesenchymal transition (EMT) and facilitates breast cancer stem cell differentiation. Gelsolin (GSN) is a ubiquitous actin filament-severing protein. However, the relationship between the expression level of GSN and the TGF-ß signaling for EMT progression in breast cancer cells is not clear. RESULTS: TGF-ß1 acted on MDA-MB231 breast cancer cells by decreasing cell proliferation, changing cell morphology to a fibroblast-like shape, increasing expressions for CD44 and GSN, and increasing EMT expression and cell migration/invasion. Study with GSN overexpression (GSN op) in both MDA-MB231 and MCF-7 cells demonstrated that increased GSN expression resulted in alterations of cell proliferation and cell cycle progression, modification of the actin filament assembly associated with altering cell surface elasticity and cell detachment in these breast cancer cells. In addition, increased cell migration was found in GSN op MDA-MB231 cells. Studies with GSN op and silencing by small interfering RNA verified that GSN could modulate the expression of vimentin. Sorted by flow cytometry, TGF-ß1 increased subpopulation of CD44+/CD22- cells increasing their expressions for GSN, Nanog, Sox2, Oct4, N-cadherin, and vimentin but decreasing the E-cadherin expression. Methylation specific PCR analysis revealed that TGF-ß1 decreased 50 % methylation but increased 3-fold unmethylation on the GSN promoter in CD44+/CD22- cells. Two DNA methyltransferases, DNMT1 and DNMT3B were also inhibited by TGF-ß1. CONCLUSIONS: TGF-ß1 induced epigenetic modification of GSN could alter the EMT process in breast cancer cells.

Molecular targets for anti-oxidative protection of green tea polyphenols against myocardial ischemic injury.

Ischemic heart disease is the leading cause of death worldwide. An improved understanding of the mechanisms involved in myocardial injury would allow intervention downstream in the pathway where certain drugs including natural products could be efficiently applied to target the end effectors of the cell death pathway. Green tea polyphenols (GTPs) have potent anti-oxidative capabilities, which may account for their beneficial effects in preventing oxidative stress associated with ischemia injury. Although studies have provided convincing evidence to support the protective effects of GTPs in cardiovascular system, the potential end effectors that mediate cardiac protection are only beginning to be addressed. Proteomics analyses widely used to identify the protein targets for many cardiovascular diseases have advanced the discovery of the signaling mechanism for GTPs-mediated cardio-protection. This review focuses on putative triggers, mediators, and end effectors for the GTPs-mediated cardio-protection signaling pathways engaged in myocardial ischemia crisis, allowing a promising natural product to be used for ameliorating oxidative stress associated with ischemic heart diseases.

Molecular identification for epigallocatechin-3-gallate-mediated antioxidant intervention on the H2O2-induced oxidative stress in H9c2 rat cardiomyoblasts.

BACKGROUND: Epigallocatechin-3-gallate (EGCG) has been documented for its beneficial effects protecting oxidative stress to cardiac cells. Previously, we have shown the EGCG-mediated cardiac protection by attenuating reactive oxygen species and cytosolic Ca2+ in cardiac cells during oxidative stress and myocardial ischemia. Here, we aimed to seek a deeper elucidation of the molecular anti-oxidative capabilities of EGCG in an H2O2-induced oxidative stress model of myocardial ischemia injury using H9c2 rat cardiomyoblasts. RESULTS: Proteomics analysis was used to determine the differential expression of proteins in H9c2 cells cultured in the conditions of control, 400 µM H2O2 exposure for 30 min with and/or without 10 to 20 µM EGCG pre-treatment. In this model, eight proteins associated with energy metabolism, mitochondrial electron transfer, redox regulation, signal transduction, and RNA binding were identified to take part in EGCG-ameliorating H2O2-induced injury in H9c2 cells. H2O2 exposure increased oxidative stress evidenced by increases in reactive oxygen species and cytosolic Ca2+ overload, increases in glycolytic protein, α-enolase, decreases in antioxidant protein, peroxiredoxin-4, as well as decreases in mitochondrial proteins, including aldehyde dehydrogenase-2, ornithine aminotransferase, and succinate dehydrogenase ubiquinone flavoprotein subunit. All of these effects were reversed by EGCG pre-treatment. In addition, EGCG attenuated the H2O2-induced increases of Type II inositol 3, 4-bisphosphate 4-phosphatase and relieved its subsequent inhibition of the downstream signalling for Akt and glycogen synthase kinase-3ß (GSK-3ß)/cyclin D1 in H9c2 cells. Pre-treatment with EGCG or GSK-3ß inhibitor (SB 216763) significantly improved the H2O2-induced suppression on cell viability, phosphorylation of pAkt (S473) and pGSK-3ß (S9), and level of cyclin D1 in cells. CONCLUSIONS: Collectively, these findings suggest that EGCG blunts the H2O2-induced oxidative effect on the Akt activity through the modulation of PIP3 synthesis leading to the subsequent inactivation of GSK-3ß mediated cardiac cell injury.

Gelsolin (GSN) induces cardiomyocyte hypertrophy and BNP expression via p38 signaling and GATA-4 transcriptional factor activation.

Cardiomyocyte hypertrophy is an adaptive response of the heart to various types of stress. During the period of stress accumulation, the transition from physiological hypertrophy to pathological hypertrophy results in the promotion of heart failure. Gelsolin (GSN) is a member of the actin-binding proteins, which regulate dynamic actin filament organization by severing and capping. Moreover, GSN also regulates cell morphology, differentiation, movement, and apoptosis. In this study, we used H9c2 and H9c2-GSN stable clones in an attempt to understand the mechanisms of GSN overexpression in cardiomyocytes. These data showed that the overexpression of GSN in H9c2-induced cardiac hypertrophy and increased the pathological hypertrophy markers atrial natriuretic peptide brain natriuretic peptide. Furthermore, we found that E-cadherin expression decreased with the overexpression of GSN in H9c2, but ß-catenin expression increased. These data presume that the cytoskeleton is loose. Further, previous studies show that the mitogen-activated protein kinase pathway can induce cardiac hypertrophy. Our data showed that p-p38 expression increased with the overexpression of GSN in H9c2, and the transcription factor p-GATA4 expression also increased, suggesting that the overexpression of GSN in H9c2-induced cardiac hypertrophy seemed to be regulated by the p38/GATA4 pathway. Moreover, we used both the p38 inhibitor (SB203580) and GSN siRNA to confirm our conjecture. We found that both of these factors significantly suppressed gelsolin-induced cardiac hypertrophy through p38/GATA4 signaling pathway. Therefore, we predict that the gene silencing of GSN and/or the downstream blocking of GSN along the p38 pathway could be applied to ameliorate pathological cardiac hypertrophy in the future.

Epigallocatechin-3-gallate-mediated cardioprotection by Akt/GSK-3ß/caveolin signalling in H9c2 rat cardiomyoblasts.

BACKGROUND: Epigallocatechin-3-gallate (EGCg) with its potent anti-oxidative capabilities is known for its beneficial effects ameliorating oxidative injury to cardiac cells. Although studies have provided convincing evidence to support the cardioprotective effects of EGCg, it remains unclear whether EGCg affect trans-membrane signalling in cardiac cells. Here, we have demonstrated the potential mechanism for cardioprotection of EGCg against H2O2-induced oxidative stress in H9c2 cardiomyoblasts. RESULTS: Exposing H9c2 cells to H2O2 suppressed cell viability and altered the expression of adherens and gap junction proteins with increased levels of intracellular reactive oxygen species and cytosolic Ca2+. These detrimental effects were attenuated by pre-treating cells with EGCg for 30 min. EGCg also attenuated H2O2-mediated cell cycle arrest at the G1-S phase through the glycogen synthase kinase-3ß (GSK-3ß)/ß-catenin/cyclin D1 signalling pathway. To determine how EGCg targets H9c2 cells, enhanced green fluorescence protein (EGFP) was ectopically expressed in these cells. EGFP-emission fluorescence spectroscopy revealed that EGCg induced dose-dependent fluorescence changes in EGFP expressing cells, suggesting that EGCg signalling events might trigger proximity changes of EGFP expressed in these cells. Proteomics studies showed that EGFP formed complexes with the 67 kD laminin receptor, caveolin-1 and -3, ß-actin, myosin 9, vimentin in EGFP expressing cells. Using in vitro oxidative stress and in vivo myocardial ischemia models, we also demonstrated the involvement of caveolin in EGCg-mediated cardioprotection. In addition, EGCg-mediated caveolin-1 activation was found to be modulated by Akt/GSK-3ß signalling in H2O2-induced H9c2 cell injury. CONCLUSIONS: Our data suggest that caveolin serves as a membrane raft that may help mediate cardioprotective EGCg transmembrane signalling.

Differential role of actin-binding proteins in controlling the adipogenic differentiation of human CD105-positive Wharton's Jelly cells.

BACKGROUND: Wharton's Jelly cells (WJCs) can be differentiated into adipocytes by cytoskeletal reorganisation in association with changes in the mechanical properties of cells. METHODS: WJCs subjected to adipocyte induction were observed changes in the cell morphology and alterations in actin filament formation. Transfection with either small interfering RNAs (siRNAs) against formin-2 (FMN-2), tropomyosin-1 (Tm-1), caldesmon (CaD), and profilin (Pro) or a pcDNA6-gelsolin (GSN)-constructed vector in WJCs was used to establish their regulatory roles in controlling adipogenesis. Phenotypic transformation of the cell shape and changes in cell surface adhesion force were determined in WJCs after transformation. RESULTS: The levels of protein and mRNA expression of ß-actin and several key actin binding ptoteins (ABPs) were decreased during the early stage of adipogenic induction but were recovered in the later induction. The siFMN-2, siTm-1, siCaD, and siPro gene knockdown in WJCs caused a widening of the cell shape, while WJCs overexpressing GSN retained a fibroblast cell shape. For both transformations, atomic force microscopy revealed alterations in the biomechanical signals on the cell surface. However, the adipogenic potency was increased after siFMN-2, siTm-1, siCaD, and siPro gene knockdown and decreased during GSN overexpression. CONCLUSIONS: siRNA gene knockdown of siFMN-2, siTm-1, siCaD, and siPro enhances the potency for WJCs commitment to adipocyte, while GSN overexpression modulates the PPAR-γ-independent pathway for the adipogenesis of WJCs. GENERAL SIGNIFICANCE: The phenotypic changes associated with decreased ABP gene expression are critical for regulating the adipogenic differentiation of WJCs through the temporal control of actin filament organisation.

Promoter polymorphism G-6A, which modulates angiotensinogen gene expression, is associated with non-familial sick sinus syndrome.

BACKGROUND: It is well known that familial sick sinus syndrome (SSS) is caused by functional alterations of ion channels and gap junction. Limited information is available on the mechanism of age-related non-familial SSS. Although evidence shows a close link between arrhythmia and the renin-angiotensin system (RAS), it remains to be determined whether the RAS is involved in the pathogenesis of non-familial SSS. METHODS: In this study, 113 patients with documented non-familial SSS and 125 controls were screened for angiotensinogen (AGT) and gap junction protein-connexin 40 (Cx40) promoter polymorphisms by gene sequencing, followed by an association study. A luciferase assay was used to determine the transcriptional activity of the promoter polymorphism. The interaction between nuclear factors and the promoter polymorphism was characterized by an electrophoretic mobility shift assay (EMSA). RESULTS: Association study showed the Cx40 -44/+71 polymorphisms are not associated with non-familial SSS; however, it indicated that four polymorphic sites at positions -6, -20, -152, and -217 in the AGT promoter are linked to non-familial SSS. Compared to controls, SSS patients had a lower frequency of the G-6A AA genotype (OR 2.88, 95% CI 1.58-5.22, P = 0.001) and a higher frequency of the G allele at -6 position (OR 2.65, 95% CI 1.54-4.57, P = 0.0003). EMSA and luciferase assays confirmed that nucleotide G at position -6 modulates the binding affinity with nuclear factors and yields a lower transcriptional activity than nucleotide A (P<0.01). CONCLUSION: G-6A polymorphism, which modulates the transcriptional activity of the AGT promoter, may contribute to non-familial SSS susceptibility.