Effect of LYRM1 knockdown on proliferation, apoptosis, differentiation and mitochondrial function in the P19 cell model of cardiac differentiation in vitro.

To explore the effects of LYRM1 knockdown on proliferation, apoptosis, differentiation and mitochondrial function in the embryonic carcinoma (P19) cell model of cardiac differentiation. Knockdown of LYRM1 using small interfering RNA (siRNA) was confirmed by quantitative real-time PCR. Cell Counting Kit-8(CCK-8) proliferation assays and cell cycle analysis demonstrated that LYRM1 gene silencing significantly inhibited P19 cell proliferation. Flow cytometry and measurement of their caspase-3 activities revealed that knockdown of LYRM1 increased P19 cell apoptosis. Observation of morphological changes using an inverted microscope and expression analysis of specific differentiation marker genes using quantitative real-time PCR and Western blotting revealed that knockdown of LYRM1 significantly inhibited the differentiation of P19 cells into cardiomyocytes. Furthermore, real-time quantitative PCR applied to detect mitochondrial DNA (mtDNA) copy number implied that there was no significant difference in the LYRM1 knockdown group compared with the control group. Cellular ATP production investigated by luciferase-based luminescence assay was dramatically decreased in differentiated cells transfected with LYRM1 RNAi. Fluorescence microscopy and flow cytometery were used to detect the reactive oxygen species (ROS) and the mitochondrial membrane potential (MMP) showed that the level of ROS was dramatically increased and MMP was obviously decreased in differentiated cells transfected with LYRM1 RNAi. Collectively, knockdown of LYRM1 promoted apoptosis and suppressed proliferation and differentiation in P19 cells. In addition, knockdown of LYRM1 induced mitochondrial impairment in P19 cells during differentiation, which was reflected by decreased ATP synthesis, lower MMP and increased ROS levels.

Prediction of spontaneous closure of isolated ventricular septal defects in utero and postnatal life.

BACKGROUND: Ventricular septal defect (VSD) is a highly prevalent fetal congenital heart defect, which can become spontaneously closed during infancy. The current study aims to characterize fetal VSDs that were subsequently spontaneously closed in the first 2 years of life in eastern China. METHODS: Between January 2011 and December 2013, 257 fetal patients diagnosed with isolated VSD by fetal echocardiography at Nanjing Maternity and Child Health Care Hospital, China, were enrolled in the study. Subjects were divided into three groups: group 1 = persistent VSD; group 2 = closed after birth; group 3 = closed during gestation. Fetal echocardiography data, physical features at birth and follow-up outcomes for 2 years were compared to identify factors contributing to spontaneous closure (SC) of VSD. A predictive formula was applied to patients admitted to hospital in the first quarter of 2014 (n = 23) for validation. RESULTS: SC occurred in 42.8% patients. Birth weight (3.095 ± 0.774, 3.174 ± 0.535, 3.499 ± 0.532 kg in groups 1, 2 and 3, respectively) and defect diameter (3.422 ± 0.972, 2.426 ± 0.599, 2.292 ± 0.479 mm, in groups 1, 2 and 3, respectively) showed statistically significant differences between the three groups (P = 0.004 and P = 0.000, respectively). Receiver operating characteristic (ROC) curves identified cut-off value for the defect diameter as 2.55 mm, and logistic regression analysis identified the SC probability = (1 + exp -[-2.151 - 0.716*birth weight + 1.393*diameter])-1. Results indicated that male fetuses, full-term birth, muscular VSD, and defects without blood flow crossing the septum, have higher incidence of SC. CONCLUSIONS: The major determinants of SC of isolated VSD are birth weight and diameter of the defect. In addition, VSD location may also affect the SC incidence.

[Interaction of Cucurbit[8]uril with ß-Indoleacetic Acid and Methylviologen].

The interaction between Q[8] with ß-indoleacetic acid and the methylviologen was studied in aqueous solution with electronic absorption spectroscopy (UV-Vis), fluorescence spectroscopy, 1H NMR spectroscopy and isothermal titration calorimetry (ITC) in details. The authors explored the mode of action, action site and thermodynamic properties of the host-guest system. The electronic absorption and fluorescence spectroscopy data showed that the Q[8]/IAA system and Q[8]/MV²âº system informed 1:1 inclusion complexes in aqueous solution. ITC results showed that the changes of Gibbs free energy and enthalpy are all negative, it suggested that complex formation was spontaneous and exothermic reaction. Moreover, ITC results for the Q [8] and IAA with MV²âº indicate that the association constants of the Q[8]-IAA and Q[8]-MV²âº complexes were (3.22 ± 0.96) x 105 L · mol⁻¹ and (3.90 ± 0.91) x 106 L · mol⁻¹, respectively. Therefore, the interaction between Q[8] and IAA with the MV²âº was a competitive process. This likely occurs because the MV²âº and IAA molecules attempt to occupy the Q[8] cavity, which reduces the fluorescence and absorption spectra intensity of Q[8]-IAA because of the formation of a new inclusion complex between Q[8] and IAA with MV²âº. In addition, with the addition of MV²âº to a Q[8]/IAA complex, 1H NMR results showed that the indole moieties of ß-indoleacetic acid and bipyridyl group of methylviologen can be incorporated into Q[8] cavities because of electronic transfer MV²âº with PQ in a Q[8] cavity with ternary complexes. These results provides the potential applications for the supramolecular self-assembly in cucurbit[n]urils field.

Long noncoding RNAs expression profile of the developing mouse heart.

Long noncoding RNAs (lncRNAs) represent a sub-group of noncoding RNAs that are longer than 200 nucleotides. The characterization of lncRNAs and their acceptance as crucial regulators of numerous developmental and biological pathways have suggested that the lncRNA study has gradually become one of the hot topics in the field of RNA biology. Many lncRNAs show spatially and temporally restricted expression patterns during embryogenesis and organogenesis. This study aimed to characterize the lncRNA profile of the fetal mouse heart at three key time points (embryonic day E11.5, E14.5, and E18.5) in its development, by performing a microarray lncRNAs screen. Gene Ontology analysis and ingenuity pathway analysis showed some significant gene functions and pathways were altered in heart development process. We compared lncRNAs profile between the three points (E14.5 vs. E11.5 [early development]; E18.5 vs. E14.5 [later development]). A total of 1,237 lncRNAs were found to have consistent fold changes (>2.0) between the three time points. Among them, 20 dysregulated lncRNAs were randomly selected and confirmed by real-time qRT-PCR. Additionally, bioinformatics analysis of AK011347 suggested it may be involved in heart development through the target gene Map3k7. In summary, this study identified differentially expressed lncRNAs in the three time points studied, and these lncRNAs may provide a new clue of mechanism of normal heart development.

Overexpression of FABP3 promotes apoptosis through inducing mitochondrial impairment in embryonic cancer cells.

Fatty acid-binding protein 3 (FABP3) is a low-molecular-weight protein with a distinct tissue distribution that may play an important role in fatty acid transport, cell growth, cellular signaling, and gene transcription. Previously, we have found that FABP3 was involved in apoptosis-associated congenital cardiac malformations, but the underlying mechanisms have not yet been described. In the present study, we investigated the characteristics of mitochondrial dysfunction in embryonic cancer cells (P19 cells) that overexpressed FABP3. We demonstrated that in FABP3-overexpressing P19 cells a lower cellular ATP production was accompanied by a dramatic decrease in mitochondrial membrane potential (MMP), despite the lack of a substantial decrease in the mtDNA copy number. In addition, FABP3 overexpression also led to an imbalance in mitochondrial dynamics and to excess intracellular reactive oxygen species production. Collectively, our results indicated that overexpression of FABP3 in P19 cells caused mitochondrion dysfunction that might be responsible for the development of FABP3-induced apoptosis.

Silencing of FABP3 promotes apoptosis and induces mitochondrion impairment in embryonic carcinoma cells.

Fatty acid binding protein 3 (FABP3) (also known as H-FABP) is a member of the intracellular lipid-binding protein family, and is mainly expressed in cardiac muscle tissue. The in vivo function of FABP3 is proposed to be in fatty acid metabolism, trafficking, and cell signaling. Our previous study found that FABP3 is highly regulated in patients with ventricular septal defect (VSD), and may play a significant role in the development of human VSD. In the present study, we aimed to investigate the impact of FABP3 knockdown by RNA interference (RNAi) on apoptosis and mitochondrial function of embryonic carcinoma (P19) cells. The results revealed that downregulated FABP3 expression promoted apoptosis, and resulted in mitochondrial deformation, increased mitochondrial membrane potential (MMP), and decreased intracellular ATP synthesis. In addition, the knockdown of FABP3 also led to excess intracellular ROS production. However, there was no obvious influence on the amount of mitochondrial DNA. Collectively, our results indicated that FABP3 knockdown promoted apoptosis and caused mitochondrial dysfunction in P19 cells, which might be responsible for the development of human VSD.

Lab-on-a-Molecule Probe: Multitarget Detection of Five Aromatic Pesticides Using a Supramolecular Probe under Single Wavelength Excitation.

In order to prevent and control the effects of pesticide residues on human health and the ecological environment, the rapid, highly sensitive, and selective detection of multiple pesticide residues has become an urgent problem to be solved. Herein, a lab-on-a-molecule probe based on a host-guest complex (ThT@Q[8] probe) has been developed to simultaneously analyze multiple aromatic pesticides under single wavelength excitation, such as fuberidazole, thiabendazole, carbendazim, thidiazuron, and tricyclazole. The fluorescence titration spectra of the ThT@Q[8] probe with the five pesticides mentioned above showed that the fluorescence intensity exhibited a good linear correlation with the pesticide concentration and the limit of detection was as low as 10-7 M. Because the ThT@Q[8] probe exhibits diverse fluorescence color changes to the five pesticides studied under a 365 nm ultraviolet lamp, we fabricated a single probe used to detect multiple analytes in the RGB triple channel by extracting the RGB variations. Principal component analysis and linear discriminant analysis proved that the ThT@Q[8] probe can recognize and distinguish five pesticides and can be applied at different concentrations. In real samples, the ThT@Q[8] probe recognized and distinguished five pesticides in tap water and Huaxi River water. The 1H NMR spectra results proved that a charge-transfer complex of ThT and pesticides in the Q[8] cavity may be formed. Moreover, we selected a test strip as a carrier to detect pesticides. The results indicate it can be used to quickly and conveniently detect different pesticides due to the rapid color change. Besides, the ThT@Q[8] probe has good cell permeability and can be used to detect pesticide residues in living cells. This work has laid the foundation for the qualitative and quantitative multitarget detection of pesticide residues.

miR-15a-5p regulates myocardial fibrosis in atrial fibrillation by targeting Smad7.

BACKGROUND: At present, there is no effective treatment for myocardial fibrosis in atrial fibrillation (AF). It is reported that miR-15a-5p is abnormally expressed in AF patients but its specific role remains unclear. This study aims to investigate the effect of miR-15a-5p in myocardial fibrosis. METHODS: Left atrial appendage (LAA) tissues were collected from AF and non-AF patients. In lipopolysaccharide (LPS) stimulated H9C2 cells, miR-15a-5p mimic, inhibitor, pcDNA3.1-Smad7 and small interfering RNA-Smad7 (siRNA-Smad7) were respectively transfected to up-regulate or down-regulate the intracellular expression levels of miR-15a-5p and Smad7. Quantitative real-time polymerase chain reaction (qRT-PCR) and western blot (WB) were used to determine the expression levels of miR-15a-5p, Smad7, transforming growth factor ß1 (TGF-ß1) and collagen I. Cell counting kit-8 (CCK-8) and ethylene deoxyuridine (EdU) were used to determine cell viability and proliferation capacity, respectively. Dual-luciferase was used to detect whether miR-15a-5p interacted with Smad7, hydroxyproline (HYP) and Hematoxylin-Eosin (HE) staining were used to detect tissue fibrosis. RESULTS: The expression levels of miR-15a-5p, TGF-ß1 and collagen I were up-regulated, while Smad7 was down-regulated in AF tissues and LPS-stimulated cells. MiR-15a-5p mimic can inhibit the expression of Smad7, and the dual-luciferase experiment confirmed their interaction. MiR-15a-5p inhibitor or pcDNA3.1-Smad7 can inhibit LPS-induced fibrosis and cell proliferation, while siRNA-Smad7 can reverse the changes caused by miR-15a-5p inhibitor. CONCLUSION: We combined clinical studies with LPS-stimulated H9C2 cell model to validate the role of miR-15a-5p in the regulation of Smad7 and fibrosis. Taken together, the miR-15a-5p/Smad7 pathway might be a potential target for AF therapy.

Identification of differentially expressed genes involved in transient regeneration of the neonatal C57BL/6J mouse heart by digital gene expression profiling.

Accumulating evidence has revealed that the mammalian heart possesses a measurable capacity for renewal. Neonatal mice retain a regenerative capacity over a short time-frame (≤6 days), but this capacity is lost by 7 days of age. In the present study, differential gene expression profiling of mouse cardiac tissue was performed to further elucidate the mechanisms underlying this process. The global gene expression patterns of the neonatal C57BL/6J mouse heart were examined at three key time-points (1, 6 and 7 days old) using digital gene expression analysis. In the distribution of total clean tags, high-expression tags (>100 copies) were found to be predominant, whereas low expression tags (<5 copies) occupied the majority of distinct tag distributions. In total, 306 differentially expressed genes (DEGs) were detected in cardiac tissue, with the expression levels of 115 genes upregulated and those of 191 genes downregulated in 7-day-old mice compared with expression levels in 1- and 6-day-old mice, respectively. The expression levels of five DEGs were confirmed using quantitative polymerase chain reaction. Gene ontology analysis revealed a large proportion of DEGs distributed throughout the cell, and these DEGs were associated with binding as well as catalytic, hydrolase, transferase and molecular transducer activities. Furthermore, these genes were involved in cellular, metabolic and developmental processes, as well as biological regulation and signaling pathways. Pathway analysis identified the oxidative phosphorylation pathway to be the process most significantly putatively affected by the differential expression of these genes. These data provide the basis for future analysis of the gene expression patterns that regulate the molecular mechanism of cardiac regeneration.

Dynamic mitochondrial changes during differentiation of P19 embryonic carcinoma cells into cardiomyocytes.

Murine P19 embryonal carcinoma cells are multipotent cells that can differentiate into cardiomyocytes when treated with dimethyl sulfoxide. This experimental model provides an invaluable tool to study different aspects of cardiac differentiation, such as the function of cardiac­specific transcription factors and signaling pathways, and the regulation of contractile protein expression. The role of mitochondria during cardiac differentiation is unclear. In this context, we have examined the mitochondrial-related changes in undifferentiated and differentiated P19 cells. We observed that mitochondrial DNA content sharply decreased in P19 cell aggregates compared to undifferentiated cells, accompanied by decreased levels of adenosine triphosphate (ATP) and reactive oxygen species (ROS). Following the aggregation stage, the mitochondrial DNA content reached its highest level on day 7 of the differentiation process, with the intracellular ROS level showing a trend to increase, similar to cellular ATP production. In conclusion, our study on differentiating P19 embryonal carcinoma cells provides new insights into the role of mitochondria in the differentiation of P19 stem cells into beating cardiomyocytes.

Silencing of FABP3 leads to apoptosis-induced mitochondrial dysfunction and stimulates Wnt signaling in zebrafish.

Fatty acid binding protein 3 (FABP3, also termed heart-type fatty acid binding protein) is a member of the intracellular lipid-binding protein family that may be essential in fatty acid transport, cell growth, cellular signaling and gene transcription. Previously, we demonstrated that FABP3 was involved in apoptosis-associated congenital cardiac malformations; however, its mechanism of regulation remains unclear. Apoptosis has increasingly been considered to be important in cardiac development. In the present study, a zebrafish model was used to investigate the involvement of FABP3­morpholino (MO)-induced apoptosis and mitochondrial dysfunction in cardiac development. During the early stages of cardiac development, injection of FABP3­MO into zebrafish resulted in significant impairment in cardiac development and promoted the rate of apoptosis which was correlated with significant dysfunction of the mitochondria. For example, the ATP content was markedly decreased at 24 and 48 h post-fertilization (pf), reactive oxygen species production was significantly enhanced at 24 and 48 h pf and the mitochondrial DNA copy number was reduced at 24, 48 and 72 h pf. Additionally, Nkx2.5 expression was upregulated in FABP3-MO zebrafish, and Wnt signaling molecules (Wnt1, Wnt5 and Wnt11) were also highly expressed in FABP3-MO zebrafish at 24, 48 and 72 h pf. In conclusion, the results indicated that FABP3 knockdown exhibited significant toxic effects on cardiac development and mitochondrial function, which may be responsible for the knockdown of FABP3-induced apoptosis. Apoptosis was one of the mechanisms underlying this effect, and was correlated with the activation of Wnt signaling. These studies identified FABP3 as a candidate gene underlying the etiology of congenital heart defects.