Effect of fibroblasts small- conductance Ca2+ -activated potassium channel subtype 2 (SK2) on myocardial fibrosis in pressure overload mouse.

Studies have shown that Small conductance Ca2 + -activated K+ (SK) channel are expressed in fibroblasts. We aimed to determine the expression of SK2 channels in cardiac fibroblasts during myocardial hypertrophy and investigate its relationship with fibrotic remodeling. Myocardial hypertrophy and fibrotic remodeling induced by transverse aortic constriction (TAC) were assessed by echocardiography, Masson's trichrome staining and Western blot. Knockdown and overexpression of the SK2 protein were used to assess relationship between SK2 expression in fibroblasts and myocardial fibrosis. There is a positive correlation between myocardial fibrosis and SK2 channel protein expression during the development of myocardial hypertrophy. The differentiation and secretion of fibroblasts in mice with cardiac hypertrophy are enhanced, and the expression of SK2 channel protein is increased. Manipulating SK2 levels in fibroblasts can either promote or inhibit their differentiation and secretory function. Knocking down SK2 reduces the up-regulation of TGF ß1, p-Smad2/3/GAPDH, p-p38/GAPDH, p-ERK1/2/GAPDH, and p-JNK/GAPDH proteins induced by Ang II in cardiac fibroblasts without significantly affecting total protein levels. AAV9-SK2-RNAi injection in mice improves cardiac function. Collectively, our study suggests that the expression of the SK2 channel is significantly increased in fibroblasts of mice with myocardial hypertrophy, potentially impacting myocardial fibrosis remodeling via the TGF-ß signaling pathway.

Environment-compatible heavy metal risk prediction method created with multilevel ensemble learning.

Accurate health risk prediction (HRP) is an effective means of reducing the hazards of heavy metal (HM) exposure. It can address the drawbacks of lag and passivity faced by health risk assessment. This study innovatively proposed an HRP method, MEL-HR, based on multilevel ensemble learning (MEL) technology and environment compatibility. We conducted point and interval prediction experiments on health risks using 490 sets of data covering 17 environment factors. The point prediction results indicated that when the model predicts HI and TCR, the R2 values were 0.707 and 0.619, respectively. For P5, P50, and P95 in interval prediction, the R2 values of the model were 0.706, 0.703, and 0.672 for HI, and that for TCR were 0.620, 0.607, and 0.616, respectively. The analysis of feature importance indicated that, in addition to HM factors, longitude, mining area coefficient, and soil organic matter were key environmental factors affecting the MEL-HR model. Comparative experiments showed that compared to soil HMs-based MEL-HR, environment compatibility-based MEL-HR has improved the accuracy for HI and TCR by 19.83 % and 40.36 % for the point prediction and 22.06 % and 40.01 % for interval prediction. This study can provide technical support for targeted and resilient prevention and control of health risks.

Progress on role of ion channels of cardiac fibroblasts in fibrosis.

Cardiac fibrosis is defined as excessive deposition of extracellular matrix (ECM) in pathological conditions. Cardiac fibroblasts (CFs) activated by injury or inflammation differentiate into myofibroblasts (MFs) with secretory and contractile functions. In the fibrotic heart, MFs produce ECM which is composed mainly of collagen and is initially involved in maintaining tissue integrity. However, persistent fibrosis disrupts the coordination of excitatory contractile coupling, leading to systolic and diastolic dysfunction, and ultimately heart failure. Numerous studies have demonstrated that both voltage- and non-voltage-gated ion channels alter intracellular ion levels and cellular activity, contributing to myofibroblast proliferation, contraction, and secretory function. However, an effective treatment strategy for myocardial fibrosis has not been established. Therefore, this review describes the progress made in research related to transient receptor potential (TRP) channels, Piezo1, Ca2+ release-activated Ca2+ (CRAC) channels, voltage-gated Ca2+ channels (VGCCs), sodium channels, and potassium channels in myocardial fibroblasts with the aim of providing new ideas for treating myocardial fibrosis.

Comprehensive analysis of RNA m6A methylation in pressure overload-induced cardiac hypertrophy.

AIM: To analyze and compare the mRNA N6-methyladenosine modifications in transverse aortic constriction induced mice hearts and normal mice hearts. MATERIALS AND METHODS: Colorimetric quantification was used to probe the changes in m6A modifications in the total RNA. The expression of m6A-related enzymes was analyzed via qRT-PCR and western blotting. RNA-seq and MeRIP-seq were performed to identify genes with differences in m6A modifications or expression in the transcriptome profile. RESULTS: Compared with the control group, the TAC group exhibited higher m6A methylation levels. FTO and WTAP were downregulated after TAC, while METTL3 was significantly downregulated at the protein level. MeRIP-seq revealed that 1179 m6A peaks were upmethylated and 733 m6A peaks were downmethylated, and biological analysis of these genes exhibited a strong relationship with heart function. CONCLUSION: Our findings provide novel information regarding m6A modification and gene expression changes in cardiac hypertrophy, which may be fundamental for further research.

Junctophilin-2 allosterically interacts with ryanodine receptor type 2 to regulate calcium release units in mouse cardiomyocytes.

The interaction between junctophilin-2 (JPH2) and ryanodine receptor type 2 (RyR2) regulated Ca2+ signaling in mouse cardiomyocytes. However, their exact interaction remains unclear. This study elucidates the interaction between JPH2 with RyR2 using co-immunoprecipitation of cardiac sarcoplasmic reticulum vesicles. Additionally, a glutathione S-transferase (GST) pull-down analysis was performed to investigate the physical interaction between RyR2 and JPH2 fragments. JPH2 interacted with RyR2 and the C terminus of the JPH2 protein can pull-down RyR2 receptors. Confocal immunofluorescence imaging indicated that the majority of JPH2 and RyR2 proteins were colocalized near Z-lines in isolated mouse cardiomyocytes. Knockdown of JPH2 reduced the amplitude of Ca2+ transients and disrupted its interaction with RyR2. Therefore, the C-terminus domain of JPH2 is required for interactions with RyR2 in mouse cardiomyocytes, which provides a molecular mechanism for seeking Ca2+-related disease prevention strategies.

Targeting Glycogen Synthase Kinase 3 Beta Regulates CD47 Expression After Myocardial Infarction in Rats via the NF-κB Signaling Pathway.

The aim of this study was to investigate the effects of the GSK-3ß/NF-κB pathway on integrin-associated protein (CD47) expression after myocardial infarction (MI) in rats. An MI Sprague Dawley rat model was established by ligating the left anterior descending coronary artery. The rats were divided into three groups: Sham, MI, and SB + MI (SB216763) groups. Immunohistochemistry was used to observe the changes in cardiac morphology. A significant reduction in the sizes of fibrotic scars was observed in the SB + MI group compared to that in the MI group. SB216763 decreased the mRNA and protein expression of CD47 and NF-κB during MI. Primary rat cardiomyocytes (RCMs) and the H9c2 cell line were used to establish in vitro hypoxia models. Quantitative real-time PCR and western blotting analyses were conducted to detect mRNA and protein expression levels of CD47 and NF-κB and apoptosis-related proteins, respectively. Apoptosis of hypoxic cells was assessed using flow cytometry. SB216763 reduced the protein expression of CD47 and NF-κB in RCMs and H9c2 cells under hypoxic conditions for 12 h, and alleviated hypoxia-induced apoptosis. SN50 (an NF-κB inhibitor) also decreased CD47 protein expression in RCMs and H9c2 cells under hypoxic conditions for 12 h and protected cells from apoptosis. GSK-3ß upregulates CD47 expression in cardiac tissues after MI by activating NF-κB, which in turn leads to myocardial cell damage and apoptosis.

The MORN domain of Junctophilin2 regulates functional interactions with small-conductance Ca2+ -activated potassium channel subtype2 (SK2).

Small-conductance Ca2+ -activated K+ channel subtype2 (SK2) are stable macromolecular complexes that regulate myocardial excitability and Ca2+ homeostasis. Junctophilin-2 (JP2) is a membrane-binding protein, which provides functional crosstalk by physically linking with the cell-surface and intracellular ion channels. We previously demonstrated that the MORN domain of JP2 interacts with SK2 channels. However, the roles of the JP2 MORN domain in regulating the precise subcellular localization and molecular modulation of SK2 have not yet been incompletely understood. In the present study, in vitro and in vivo assays were used to confirm the physical interactions between the SK2 channel and JP2 in H9c2 and HEK293 cells, with a concentration on the association between the C-terminus of SK2 channels and the MORN domain of JP2. Furthermore, the membrane expression of SK2 were found to be significantly impaired by the mutation or knockdown of JP2. Using immunofluorescence staining along with Golgi/early endosome markers, we studied the mechanisms of JP2-regulated SK2 membrane trafficking, which indicates that the JP2 MORN domain is probably necessary for the retrograde trafficking of SK2 channels. The functional study demonstrates that whole cell SK2 current densities recorded from the HEK293 cells co-expressing the JP2-MORN domain with SK2 were significantly augmented, compared with cells expressing SK2 alone. Our findings suggest that the MORN domain of JP2 directly modulates SK2 channel current amplitude and trafficking, through its interaction with an overlapping region of the JP2 MORN domain on the SK2 C-terminus.

[The changes of the expression of 2-type small conductance-Ca2+- activating-K+ (SK2) channel protein in hypertensive rat myocardium].

OBJECTIVE: To investigate the expression of 2-type small conductance-Ca2+-activating-K+ (SK2) channel protein in hypertensive rat myocardial cells. METHODS: Twelve healthy adult male SD rats were randomly divided into control group (n=5) and experimental group (n=7). The rats of experimental group were injected intraperitoneally with N'-nitro-L-arginine (L-NNA 15 mg/(kg·d))while the rats of control group were injected intraperitoneally with isometrical normal saline(15 ml/(kg·d )). The body weight, blood pressure and electrocardiogram of the rats were measured every week. After 4 weeks, the rats were sacrificed to obtain hearts, and the expression of SK2 channel protein in myocardium was detected by Western blot. RESULTS: After 4 weeks of administration, compared with the control group, the blood pressure in the experimental group was significantly elevated (P＜0.05), QRS duration and R-R interval were prolonged, and the expressions of SK2 channel in the atrial and ventricular tissue of the experimental group were significantly higher than those in the control group (1.12±0.18,1.64±0.26, P ＜ 0.05). CONCLUSION: The expressions of atrial and ventricular SK2 pathway are increased in hypertensive model rats. It may be one of the mechanism leading to arrhythmias in hypertensive model rats and can provide new ideas and strategies for the treatment and prognosis of hypertensive diseases.

Beclin 1 promotes apoptosis and decreases invasion by upregulating the expression of ECRG4 in A549 human lung adenocarcinoma cells.

Although Beclin 1 has been demonstrated to exert an important role in cell autophagy during carcinogenesis, its biological function in lung cancer has yet to be fully elucidated. A previous study by our laboratory identified that knockdown of Beclin 1 promoted cell growth and inhibited apoptosis in the A549 lung cancer cell line. In the present study, a Beclin 1 lentiviral expression vector was constructed, and an A549 cell line was established with a steady expression of Beclin 1. Furthermore, the effect of Beclin 1 overexpression on cell invasion and apoptosis, changes in the activities of the apoptosis­associated caspases­3 and ­9, and the overexpression of esophageal cancer­related gene 4 (ECRG4) were examined. The results demonstrated that the overexpression of Beclin 1 in A549 cells reduced cell invasion by Matrigel invasion assay and promoted apoptosis by flow cytometric analysis (P<0.01) compared with Lenex­packaged lentiviral particles and non­transfected control groups. Furthermore, the overexpression of Beclin 1 in A549 cells increased the activities of caspases­3 and ­9 and the expression of ECRG4 (P<0.01) compared with Lenex­packaged lentiviral particles and non­transfected control groups. In conclusion, the overexpression of Beclin 1 promoted apoptosis and decreased invasion by upregulating the expression of ECRG4 in A549 lung adenocarcinoma cells. Therefore, the selection of Beclin l as a target for gene therapy represents a more effective method for the treatment of lung cancer.

[Expression of choline acetyltransferase in the rat barrel cortex by electrical stimulation].

OBJECTIVE: To observe a turning performance in the rats excited by using a proper electrical stimuli of the barrel cortex region (BC), and the expression of choline acetyltransferase (ChAT) in the BC regions after electoral stimulation. METHODS: SD rats were divided into three groups. The stimulation electrodes were surgically implanted into the bilateral BC regions in the control group and the experimental group rats. The experiment group post surgery for seven days was given the electrical impulses via connection with the electrodes for three times each day through consecutive three days. Three groups of the rats were killed and the brains were quickly removed for frozen sections and then performed with conventional HE and immunohistochemistry staining. And protein samples were prepared from brain and the hippocampus tissues of the three groups to detect the level of the ChAT protein by Western blot. RESULTS: The experimental rats turn left or right when continuously stimulation in the bilateral BC regions with electric pulse. HE staining showed no significant damage around electrodes in the cerebral cortex. Compared with the control and blank groups, the ChAT positive rate in the brain section in the experimental rats was significantly high by immunohistochemistry assay; the level of the ChAT protein in the rats given the electrical stimulation increased. CONCLUSION: Turnings performance of the rat could be initiated hy electrical stimuli in the BC region. Expression of ChAT is significantly higher in the BC regions of rat under electrical stimulation, suggesting that acetylcholine might be associated with signal transmission between senses and movement behavior in the nervous central system.

[Construction and identification of lentiviral vector of siRNA specific for Beclin1 gene].

The lentiviral vector was used for construction of a recombinant mediating RNA interference (RNAi) against Beclin1 gene in this study. Recombinant vector plasmid was transfected into non small cell lung cancer (NSCLC) A549 cells by liposome. PCR results showed that three amplified positive fragments were inserted into pRNAT-U6. 2/Lenti vectors. DNA sequencing results showed that the three recombinant lentivirus plasmids, pRNAT-U6. 2/Lenti-si356, pRNAT-U6. 2/Lenti-si423 and pRNAT-U6. 2/ Lenti-si684 were constructed successfully. After transfection with liposome, RT-PCR and Western blot analysis confirmed that the expression of Beclin1 mRNA and protein was inhibited in the three recombinant lentivirus plasmids transfected groups, and gene silencing efficacy was 35.56%, 89.22% and 66.78%, respectively. The results demonstrated that the lentiviral vectors of RNAi targeting Beclin1 gene were successfully constructed, and NSCLC A549 stable cell line with Beclin1 gene knockdown was established. This study finally provided a new cell model to explore the biological behavior of the Beclin1 gene in NSCLC A549 cells.

Knockdown of autophagy-related gene BECLIN1 promotes cell growth and inhibits apoptosis in the A549 human lung cancer cell line.

The expression of BECLIN1 is significantly reduced in non­small cell lung cancer (NSCLC) compared with non­cancerous tissue. However, the role of BECLIN1 in lung cancer is unclear. Using the RNA interference (RNAi) technique the present study investigated the effect of the knockdown of BECLIN1 on the cell growth and proliferation of the A549 human lung cancer cell line. The target site for the RNAi technique was designed and the lentivirus vector for the small interfering (si)RNA expression was constructed according to the encoding sequence of the mRNA for BECLIN1. The A549 cells were transfected with the siRNA virus against BECLIN1. BECLIN1 expression was detected by reverse transcription (RT)­PCR and western blot analysis. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method was applied to detect cell growth. Flow cytometry was used to determine cell apoptosis. The activity of caspase­3 and caspase­9 was also detected in the A549 cells with BECLIN1 knockdown. The results showed that siRNA virus transfection significantly decreased the mRNA and protein expression of BECLIN1 in the transfected A549 cells. The knockdown of BECLIN1 promoted cell growth and decreased apoptosis. Caspase­3 and ­9 activity in the A549 cells with BECLIN1 knockdown was significantly reduced. In conclusion, the siRNA expression vector effectively inhibited the expression of BECLIN1 in the A549 human lung cancer cell line and promote the growth and proliferation of the A549 cells.

[Effects of CRF receptor antagonist on rem sleep in neonatal rat].

OBJECTIVE: To observe the role of NB127914, a CRF R1 receptor antagonist, in the regulation of neonatal sleep/wake cycle. METHODS: Rat pups were surgically implanted with electrodes at postnatal day(PN) 13. At PN 14, 6 hours polysomnographic recording data were continuously collected before and after administration of various doses of NBI 27914, atropine and the same amount of saline. RESULTS: Compared with baseline, rapid eye movement (REM) sleep was significantly reduced and was replaced primarily by non-REM (NREM) sleep in all groups treated with NBI, but not with dimethyl sulfoxide/saline. Atropine suppressed REM sleep significantly and increased wakefulness simultaneously. CONCLUSION: Blockage of corticotropin-releasing factor (CRF) R1 receptors deprives neonatal rat REM sleep.

Neonatal REM sleep is regulated by corticotropin releasing factor.

Sleep/wake regulation is quite different during the neonatal and adult periods. Although cholinergic neurons have been recognized to be the major source of rapid eye movement (REM) sleep regulation in adulthood, their effect on neonatal REM sleep remains to be discovered. Current evidence suggests that corticotropin-releasing factor (CRF) may play a role in REM promotion during the neonatal period. We conducted the following study to test our hypothesis that blocking CRF R1 receptor would reduce REM sleep in developing rat pups. First, rat pups were surgically implanted with electrodes on postnatal day (PN) 13. On PN 14, six hours of polysomnographic (PSG) data were collected before and after administration of three different doses of NBI 27914 (NBI), a CRF R1 receptor antagonist. Compared with baseline, REM sleep was significantly reduced in all groups treated with NBI but not with dimethyl sulfoxide/saline. The reduction of REM sleep was dose-related and was replaced primarily by non-REM (NREM) sleep. Second, two groups of rat pups were given a single dose of either NBI or vehicle on PN 14 for quantification of ACTH and acetylcholine without PSG recording. NBI induced no change of either ACTH or acetylcholine. Third, the effect of administering atropine (6 mg/kg) on sleep/wake in two-week-old rats was investigated. Atropine suppressed REM sleep significantly and increased wakefulness simultaneously. Our data revealed that blockage of CRF R1 receptors deprives neonatal REM sleep. The mechanism for CRF in enhancing REM sleep may be associated with but not be similar to the cholinergic mechanism.