Activated hedgehog gene pattern correlates with dismal clinical outcome and tumor microenvironment heterogeneity in hepatocellular carcinoma.

Background: Activation of the Hedgehog signaling pathway is linked to the initiation and development of human hepatocellular carcinoma (HCC). However, its impact on clinical outcomes and the HCC microenvironment remains unclear. Methods: We performed comprehensive analyses of Hedgehog pathway genes in a large cohort of HCC patients. Specifically, we utilized univariate Cox regression analysis to identify Hedgehog genes linked to overall survival, and the LASSO algorithm was used to construct a Hedgehog-related gene pattern. We subsequently examined the correlation between the Hedgehog pattern and the HCC microenvironment employing the CIBERSORT and ssGSEA algorithms. Furthermore, Tumor Immune Dysfunction and Exclusion (TIDE) algorithm and the anti-PD-L1 treatment dataset (IMvigor210) are used to evaluate the clinical response of the Hedgehog pattern in predicting immune checkpoint inhibitors. Results: We found that the Hedgehog activation score (HHAS), a prognostic score based on 11 Hedgehog genes, was significantly associated with HCC patient survival. Patients exhibiting high HHAS experienced markedly reduced survival rates compared to those with low HHAS, and HHAS emerged as an independent prognostic factor for HCC. Functional enrichment analysis unveiled the association of the HHAS phenotype with functions related to the immune system, and further investigation demonstrated that HCC patients exhibiting low HHAS displayed elevated levels of anti-tumor immune activation in CD8+ T cells, while high HHAS were linked to immune escape phenotypes and increased infiltration of immune suppressive cells. In addition, in the Immune Checkpoint Inhibitor (ICI) cohort of IMvigor210, patients with higher HHAS had worse ICI treatment outcomes and shortened survival time, indicating that the HHAS is a useful indicator for predicting patient response to immunotherapy. Conclusions: In summary, our study offers valuable insights for advancing research on Hedgehog and its impact on tumor immunity, which provides an opportunity to optimize prognosis and immune therapy for HCC.

Hepatitis E Virus Seroprevalence Indicated a Significantly Increased Risk Selectively in Patients with Gastric Cancer among 17 Common Malignancies.

Background: The impact of hepatitis E virus (HEV) infection on cancer development has been poorly investigated. This study aimed to explore the relationship between HEV seroprevalence and cancer risks and to identify high cancer risk subgroups in HEV-exposed populations. Methods: HEV seroprevalence status was determined in cancer and non-cancer subjects. Logistic regression and sensitivity analyses were used to assess the relationship between HEV antibody seropositivity and cancer risk for 17 cancer types. Additionally, interaction analyses were applied to interpret the association of HEV seroprevalence and other cancer risk factors. Results: Of the enrolled 4948 cancer and 4948 non-cancer subjects, cancer subjects had a higher anti-HEV seropositivity than non-cancer subjects (46.36% vs. 32.50%, p < 0.01). However, this divergency varied in degrees across different cancer types. Additionally, HEV seroprevalence was associated with cancer risk in young males (OR: 1.64, 95% CI: 1.19−2.27, p < 0.01). Remarkably, a significant association between HEV seroprevalence and cancer risk was observed only in gastric cancer patients (OR: 1.82, 95% CI: 1.07−3.09, p = 0.03). Conclusions: HEV seroprevalence was associated with cancer risk selectively in gastric cancer patients and young males, suggesting that cancer screening, particularly gastric cancer, should be regularly performed in young males with a history of HEV exposure.

Proteomic Analysis of Hypoxia-Induced Senescence of Human Bone Marrow Mesenchymal Stem Cells.

METHODS: Hypoxia in hBMSCs was induced for 0, 4, and 12 hours, and cellular senescence was evaluated by senescence-associated ß-galactosidase (SA-ß-gal) staining. Tandem mass tag (TMT) labeling was combined with liquid chromatography-tandem mass spectrometry (LC-MS/MS) for differential proteomic analysis of hypoxia in hBMSCs. Parallel reaction monitoring (PRM) analysis was used to validate the candidate proteins. Verifications of signaling pathways were evaluated by western blotting. Cell apoptosis was evaluated using Annexin V/7-AAD staining by flow cytometry. The production of reactive oxygen species (ROS) was detected by the fluorescent probe 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA). RESULTS: Cell senescence detected by SA-ß-gal activity was higher in the 12-hour hypoxia-induced group. TMT analysis of 12-hour hypoxia-induced cells identified over 6000 proteins, including 686 differentially expressed proteins. Based on biological pathway analysis, we found that the senescence-associated proteins were predominantly enriched in the cancer pathways, PI3K-Akt pathway, and cellular senescence signaling pathways. CDK1, CDK2, and CCND1 were important nodes in PPI analyses. Moreover, the CCND1, UQCRH, and COX7C expressions were verified by PRM. Hypoxia induction for 12 hours in hBMSCs reduced CCND1 expression but promoted ROS production and cell apoptosis. Such effects were markedly reduced by the PI3K agonist, 740 Y-P, and attenuated by LY294002. CONCLUSIONS: Hypoxia of hBMSCs inhibited CCND1 expression but promoted ROS production and cell apoptosis through activating the PI3K-dependent signaling pathway. These findings provided a detailed characterization of the proteomic profiles related to hypoxia-induced senescence of hBMSCs and facilitated our understanding of the molecular mechanisms leading to stem cell senescence.

Association between hepatitis B and E virus infection and hepatocellular carcinoma risk.

The role of hepatitis E virus (HEV) in developing hepatocellular carcinoma (HCC) is unclear. Our study aimed to investigate the role of HE infection in HCC development and the effect of hepatitis B virus (HBV) and HEV coinfection on HCC risk. A hospital-based case-control study was conducted. A total of 474 eligible HCC cases and 586 control patients were successfully recruited. The fasting venous blood was collected from the patients at the first visited to hospital and HBV infection and HEV infection were examined within 5 days. Crude and adjusted odd ratios (ORs) with 95% confidence interval (95% CI) were estimated by using logistic regression model. HBV infection (OR: 63.10, 95% CI: 42.02-97.26) rather than HEV infection (OR: 1.08, 95% CI: 0.721-1.65) was associated with an increased risk of HCC after adjustment for confounders. The association between HBV infection and HCC risk was more remarkable in male (OR: 72.61, 95% CI: 45.10-121.38) than in female (OR: 61.89, 95% CI: 25.74-169.26). In comparison with patients who infected with neither HEV nor HBV, those who infected with only HBV (OR: 69.62, 95% CI: 40.90-123.52) and who coinfected with HEV and HBV (OR: 67.48, 95% CI:37.23-128.19) were significantly associated with an increased risk after adjustment for potential confounders. The results showed that HBV infection rather than HEV infection was associated with an increased risk of HCC, and the HEV infection may alleviate the promoting impact of HBV on HCC development.

Hepatitis E virus re-infection accelerates hepatocellular carcinoma development and relapse in a patient with liver cirrhosis: A case report and review of literature.

BACKGROUND: Hepatitis E virus (HEV) superinfection is a suspected promoting factor for hepatocellular carcinoma (HCC) in patients with chronic hepatitis and cirrhosis. However, to date, very few cases of HEV-related HCC have been reported. Nevertheless, the role of HEV re-infection in cirrhotic liver without other chronic hepatitis infections has rarely been explored. CASE SUMMARY: A 53-year-old male farmer was diagnosed with liver cirrhosis and splenomegaly in August 2016, accompanied with negative HEV-IgM and positive HEV-IgG. No evidence of hepatitis B virus or hepatitis C virus infection was found. Since then the patient was evaluated for liver function and viral parameters every 3 mo. In June 2017, the patient presented severe fatigue with whole body itching and was diagnosed with HCC. Afterwards this patient experienced quick HCC development, progression, relapse, and metastasis in the following 8 mo, and presented persistent dual positivity of HEV-IgM and HEV-IgG. This patient had a long history of smoking and alcohol consumption. CONCLUSION: This unique case invokes the importance of HEV surveillance and treatment among cirrhotic patients, HCC cases, and blood donors.

G protein-coupled receptor kinase-2: A potential biomarker for early diabetic cardiomyopathy.

BACKGROUND: G protein-coupled receptor kinase-2 (GRK2) has been shown as a key regulator of cardiac function, and the myocardial GRK2 levels are mirrored by the levels in peripheral blood mononuclear cells (PBMCs). In this study, we evaluated the myocardial and PBMCs GRK2 levels in early diabetic cardiomyopathy (DCM). METHODS: C57BL/KS-db/db male diabetic mice at 12 weeks of age, as the type 2 diabetes (T2DM) animal model of early DCM were evaluated. Forty-four T2DM patients with left ventricular diastolic dysfunction (LVDD), without evidence of hypertension, coronary artery diseases, congestive heart failure, and diabetic complications and without evidence of ischemia in a maximal treadmill exercise test, were recruited as the DM + LVDD group; 30 age-matched T2DM patients without LVDD were recruited as the DM control group. Left ventricular diastolic function was evaluated by cardiac tissue Doppler. The pseudonormal pattern of ventricular filling and E'/A' < 1 were regarded as LVDD. RESULTS: Compared to 8-week-old diabetic mice and 12-week-old control mice, GRK2-mRNA level and expression in myocardial tissues of 12-week-old diabetic mice were significantly increased, as well as the left ventricular wall thickness and systolic function. And the collagen volume fraction (CVF), collagen-3 expression, P53 expression, and cell apoptotic rate in the myocardium of 12-week-old diabetic mice elevated as well. The GRK2-mRNA level in PBMCs of DM with LVDD was significantly higher than in DM control without LVDD. CONCLUSIONS: GRK2 expression increased in the myocardial tissue and the PBMCs at the early stage of DCM. These data support further research on the role of GRK2 as the clinical biomarker for early DCM.

Comparison of Ca2+ Handling for the Regulation of Vasoconstriction between Rat Coronary and Renal Arteries.

BACKGROUND: Ca2+ plays an important role in the regulation of vasoconstriction. Ca2+ signaling is regulated by a number of Ca2+-handling proteins. However, whether differences in Ca2+ handling affect the regulation of vasoconstriction in different arteries remains elusive. OBJECTIVE: To determine whether differences in Ca2+ handling affect the response to vasoconstrictors in different arteries. METHODS: Arterial ring contraction was measured using a Multi Myograph System. Vascular smooth muscle cells (VSMCs) were digested with type 2 collagenase in DMEM, then intracellular calcium concentration was measured with the Ca2+ probe fluo-4/AM in the isolated cells. Calcium-related proteins were assayed by Western blotting. RESULTS: Phenylephrine did not induce -coronary arterial contraction. There were differences in -5-hydroxytryptamine, 9,11-dideoxy-11a,9a-epoxymethano-prostaglandin F2a, and endothelin 1-induced vasoconstriction in different solutions between coronary and renal arteries. Vasoconstrictions in the presence of Bay K8644 were stronger in coronary than in renal arteries. Store-operated calcium (SOC) channels could mediate Ca2+ influx in VSMCs of both groups. SOC channels did not participate in the contraction of coronary arteries. In addition, there were significant differences in the expressions of receptors and ion channels between the two groups. CONCLUSIONS: Ca2+ handling contributed to the different responses to vasoconstrictors between coronary and renal arteries.

Mechanisms of U46619-induced contraction in mouse intrarenal artery.

Thromboxane A2 (TXA2 ) has been implicated in the pathogenesis of vascular complications, but the underlying mechanism remains unclear. The contraction of renal arterial rings in mice was measured by a Multi Myograph System. The intracellular calcium concentration ([Ca2+ ]i ) in vascular smooth muscle cells (VSMCs) was obtained by using a fluo-4/AM dye and a confocal laser scanning microscopy. The results show that the U46619-induced vasoconstriction of renal artery was completely blocked by a TXA2 receptor antagonist GR32191, significantly inhibited by a selective phospholipase C (PI-PLC) inhibitor U73122 at 10 µmol/L and partially inhibited by a Phosphatidylcholine - specific phospholipase C (PC-PLC) inhibitor D609 at 50 µmol/L. Moreover, the U46619-induced vasoconstriction was inhibited by a general protein kinase C (PKC) inhibitor chelerythrine at 10 µmol/L, and a selective PKCδ inhibitor rottlerin at 10 µmol/L. In addition, the PKC-induced vasoconstriction was partially inhibited by a Rho-kinase inhibitor Y-27632 at 10 µmol/L and was further completely inhibited together with a putative IP3 receptor antagonist and store-operated Ca2+ (SOC) entry inhibitor 2-APB at 100 µmol/L. On the other hand, U46619-induced vasoconstriction was partially inhibited by L-type calcium channel (Cav1.2) inhibitor nifedipine at 1 µmol/L and 2-APB at 50 and 100 µmol/L. Last, U46619-induced vasoconstriction was partially inhibited by a cell membrane Ca2+ activated C1- channel blocker 5-Nitro-2-(3-phenylpropylamino) benzoic acid (NPPB) at 50 and 100 µmol/L. Our results suggest that the U46619-induced contraction of mouse intrarenal arteries is mediated by Cav1.2 and SOC channel, through the activation of thromboxane-prostanoid receptors and its downstream signaling pathway.

Involvement of Orai1 in tunicamycin-induced endothelial dysfunction.

Endoplasmic reticulum (ER) stress is mediated by disturbance of Ca2+ homeostasis. The store-operated calcium (SOC) channel is the primary Ca2+ channel in non-excitable cells, but its participation in agent-induced ER stress is not clear. In this study, the effects of tunicamycin on Ca2+ influx in human umbilical vein endothelial cells (HUVECs) were observed with the fluorescent probe Fluo-4 AM. The effect of tunicamycin on the expression of the unfolded protein response (UPR)-related proteins BiP and CHOP was assayed by western blotting with or without inhibition of Orai1. Tunicamycin induced endothelial dysfunction by activating ER stress. Orai1 expression and the influx of extracellular Ca2+ in HUVECs were both upregulated during ER stress. The SOC channel inhibitor SKF96365 reversed tunicamycin-induced endothelial cell dysfunction by inhibiting ER stress. Regulation of tunicamycin-induced ER stress by Orai1 indicates that modification of Orai1 activity may have therapeutic value for conditions with ER stress-induced endothelial dysfunction.

[MicroRNA-199a-3p enhances expressions of fibrosis-associated genes through targeting Smad1 in mouse cardiac fibroblasts].

OBJECTIVE: To investigate the role of miR-199a-3p in cardiac fibrosis and the potential target of miR-199a-3p. METHODS: Cardiac fibroblasts were isolated from C57BL/6 mice and cultured. The miR-199a-3p mimic and Smad1 siRNA were transiently transfected into the cardiac fibroblasts via liposome. Dual luciferase reporter assay was performed to confirm the interaction between miR-199a-3p and the 3'-UTR of Smad1. The expressions of Smad1 and fibrosis-related genes at the mRNA and protein levels in the cells after miR-199a-3p mimic transfection were determined using RT-qPCR and Western blotting, respectively. The expressions of Smad1, Smad3 and fibrosis-related genes at the protein level in cells transfected with miR-199a-3p mimic and Smad1 siRNA were detected using Western blotting. RESULTS: Over-expression of miR-199a-3p significantly increased the expression of cardiac fibrosis-related genes in cultured mouse cardiac fibroblasts. Dual luciferase reporter assay revealed the interaction of miR-199a-3p with the 3'-UTR of Smad1. The results of RT-qPCR and Western blotting confirmed that miR-199a-3p inhibited Smad1 expression at the post- transcriptional level. Transfection with miR-199a-3p mimic and siRNA-mediated Smad1 silencing consistently activated the Smad3 signaling pathway and enhanced the expressions of cardiac fibrosis-related genes in the cardiac fibroblasts. CONCLUSIONS: As the target gene of miR-199a-3p, Smad1 mediates the pro-fibrotic effect of miR-199a-3p by activating the Smad3 signaling in cultured mouse cardiac fibroblasts.

High Plasma Exposure of Statins Associated With Increased Risk of Contrast-Induced Acute Kidney Injury in Chinese Patients With Coronary Artery Disease.

The role of statins in reducing the incidence of contrast-induced acute kidney injury (CI-AKI) remains controversial. We sought to evaluate the association between CI-AKI and high plasma exposure of statins in coronary artery disease (CAD) patients undergoing coronary angiography (CAG). This association was first evaluated in 1,219 patients with CAD receiving atorvastatin (AT) therapy and validated in 635 patients receiving rosuvastatin (RST) therapy. The plasma concentrations of statins were quantified using validated UPLC-MS/MS methods and CI-AKI incidence was assessed during the first 48 h postoperatively. Among all participants (n = 1,854), AKI occurred in 57 of 1219 (4.7%) in the AT cohort and 30 of 635 (4.7%) in the RST cohort. High plasma AT-all exposure was associated with increased risk of CI-AKI (odds ratio [OR]: 2.265; 95% confidence interval [CI]: 1.609-3.187; p < 0.0001). Plasma AT-all concentration in the CI-AKI group (22.40 ± 24.63 ng/mL) was 2.6-fold higher than that in the control group (8.60 ± 9.65 ng/mL). High plasma RST exposure also significantly increased the risk of CI-AKI (OR: 2.281; 95% CI: 1.441-3.612; p = 0.0004). We further divided patients into two subgroups for each statin according to baseline renal function, and association between high plasma statin exposure and CI-AKI still remained highly significant in both subgroups. This study suggests for the first time that high plasma exposure of statins may significantly increase the risk of CI-AKI. Statins should be used with greater caution in CAD patients undergoing CAG to reduce the occurrence of CI-AKI.

Activation of miR-34a-5p/Sirt1/p66shc pathway contributes to doxorubicin-induced cardiotoxicity.

The molecular mechanisms underlying anthracyclines-induced cardiotoxicity have not been well elucidated. MiRNAs were revealed dysregulated in the myocardium and plasma of rats received Dox treatment. MicroRNA-34a-5p (miR-34a-5p) was verified increased in the myocardium and plasma of Dox-treated rats, but was reversed in rats received Dox plus DEX treatments. Human miR-34a-5p was also observed increased in the plasma of patients with diffuse large B-cell lymphoma after 9- and 16-week epirubicin therapy. Up-regulation of miR-34a-5p was observed in Dox-induced rat cardiomyocyte H9c2 cells. MiR-34a-5p could augment Bax expression, but inhibited Bcl-2 expression, along with the increases of the activated caspase-3 and mitochondrial potentials in H9C2 cells. MiR-34a-5p was verified to modulate Sirt1 expression post-transcriptionally. In parallel to Sirt1 siRNA, miR-34a-5p could enhance p66shc expression, accompanied by increases of Bax and the activated caspase-3 and a decrease of Bcl-2 in H9c2 cells. Moreover, enforced expression of Sirt1 alleviated Dox-induced apoptosis of H9c2 cells, with suppressing levels of p66shc, Bax, the activated caspase-3 and miR-34a-5p, and enhancing Bcl-2 expression. Therefore, miR-34a-5p enhances cardiomyocyte apoptosis by targeting Sirt1, activation of miR-34a-5p/Sirt1/p66shc pathway contributes to Dox-induced cardiotoxicity, and blockage of this pathway represents a potential cardioprotective effect against anthracyclines.

The enhancement of TXA2 receptors-mediated contractile response in intrarenal artery dysfunction in type 2 diabetic mice.

Thromboxane A2 (TXA2) has been implicated in the pathogenesis of diabetic vascular complications, although the underlying mechanism remains unclear. The present study investigated the alterations in TXA2 receptor signal transduction in type 2 diabetic renal arteries. The contraction of renal arterial rings in control (db/m+) mice and type 2 diabetic (db/db) mice was measured by a Multi Myograph System. Intracellular calcium concentration ([Ca2+]i) in vascular smooth muscle cells was measured by Fluo-4/AM dye and confocal laser scanning microscopy. Quantitative real-time PCR and Western blot analysis were used to determine gene and protein expression levels, respectively. A stable TXA2 mimic U46619 caused markedly stronger dose-dependent contractions in the renal arteries of db/db mice than in those of db/m+ mice. This response was completely blocked by a TXA2 receptor antagonist GR32191 and significantly inhibited by U73122. U46619-induced vasoconstriction was increased in the presence of nifedipine in db/db mice compared with that in db/m+ mice, whereas the response to U46619 did not differ between the two groups in the presence of SKF96365. Sarcoplasmic reticulum Ca2+ release-mediated and CaCl2-induced contractions did not differ between the two groups. In db/db mice, store-operated Ca2+(SOC) entry-mediated contraction in the renal arteries and SOC entry-mediated Ca2+ influx in smooth muscle cells were significantly increased. And the gene and protein expressions of TXA2 receptors, Orai1 and Stim1 were upregulated in the diabetic renal arteries. Therefore the enhancement of U46619-induced contraction was mediated by the upregulation of TXA2 receptors and downstream signaling in the diabetic renal arteries.

CircRNA_000203 enhances the expression of fibrosis-associated genes by derepressing targets of miR-26b-5p, Col1a2 and CTGF, in cardiac fibroblasts.

Circular RNAs (circRNAs) participate in regulating gene expression in diverse biological and pathological processes. The present study aimed to investigate the mechanism underlying the modulation of circRNA_000203 on expressions of fibrosis-associated genes in cardiac fibroblasts. CircRNA_000203 was shown upregulated in the diabetic mouse myocardium and in Ang-II-induced mouse cardiac fibroblasts. Enforced-expression of circRNA_000203 could increase expressions of Col1a2, Col3a1 and α-SMA in mouse cardiac fibroblasts. RNA pull-down and RT-qPCR assay indicated that circRNA_000203 could specifically sponge miR-26b-5p. Dual luciferase reporter assay revealed that miR-26b-5p interacted with 3'UTRs of Col1a2 and CTGF, and circ_000203 could block the interactions of miR-26b-5p and 3'UTRs of Col1a2 and CTGF. Transfection of miR-26b-5p could post-transcriptionaly inhibit expressions of Col1a2 and CTGF, accompanied with the suppressions of Col3a1 and α-SMA in cardiac fibroblasts. Additionally, over-expression of circRNA_000203 could eliminate the anti-fibrosis effect of miR-26b-5p in cardiac fibroblasts. Together, our results reveal that suppressing the function of miR-26b-5p contributes to the pro-fibrosis effect of circRNA_000203 in cardiac fibroblasts.

Targeting EZH1 and EZH2 contributes to the suppression of fibrosis-associated genes by miR-214-3p in cardiac myofibroblasts.

The role of microRNA-214-3p (miR-214-3p) in cardiac fibrosis was not well illustrated. The present study aimed to investigate the expression and potential target of miR-214-3p in angiotensin II (Ang-II)-induced cardiac fibrosis. MiR-214-3p was markedly decreased in the fibrotic myocardium of a mouse Ang-II infusion model, but was upregulated in Ang-II-treated mouse myofibroblasts. Cardiac fibrosis was shown attenuated in Ang-II-infused mice received tail vein injection of miR-214-3p agomir. Consistently, miR-214-3p inhibited the expression of Col1a1 and Col3a1 in mouse myofibroblasts in vitro. MiR-214-3p could bind the 3'-UTRs of enhancer of zeste homolog 1 (EZH1) and -2, and suppressed EZH1 and -2 expressions at the transcriptional level. Functionally, miR-214-3p mimic, in parallel to EZH1 siRNA and EZH2 siRNA, could enhance peroxisome proliferator-activated receptor-γ (PPAR-γ) expression and inhibited the expression of Col1a1 and Col3a1 in myofibroblasts. In addition, enforced expression of EZH1 and -2, and knockdown of PPAR-γ resulted in the increase of Col1a1 and Col3a1 in myofibroblasts. Moreover, the NF-κB signal pathway was verified to mediate Ang-II-induced miR-214-3p expression in myofibroblasts. Taken together, our results revealed that EZH1 and -2 were novel targets of miR-214-3p, and miR-214-3p might be one potential miRNA for the prevention of cardiac fibrosis.

Myocyte-specific enhancer factor 2C: a novel target gene of miR-214-3p in suppressing angiotensin II-induced cardiomyocyte hypertrophy.

The role of microRNA-214-3p (miR-214-3p) in cardiac hypertrophy was not well illustrated. The present study aimed to investigate the expression and potential target of miR-214-3p in angiotensin II (Ang-II)-induced mouse cardiac hypertrophy. In mice with either Ang-II infusion or transverse aortic constriction (TAC) model, miR-214-3p expression was markedly decreased in the hypertrophic myocardium. Down-regulation of miR-214-3p was observed in the myocardium of patients with cardiac hypertrophy. Expression of miR-214-3p was upregulated in Ang-II-induced hypertrophic neonatal mouse ventricular cardiomyocytes. Cardiac hypertrophy was attenuated in Ang-II-infused mice by tail vein injection of miR-214-3p. Moreover, miR-214-3p inhibited the expression of atrial natriuretic peptide (ANP) and ß-myosin heavy chain (MHC) in Ang-II-treated mouse cardiomyocytes in vitro. Myocyte-specific enhancer factor 2C (MEF2C), which was increased in Ang-II-induced hypertrophic mouse myocardium and cardiomyocytes, was identified as a target gene of miR-214-3p. Functionally, miR-214-3p mimic, consistent with MEF2C siRNA, inhibited cell size increase and protein expression of ANP and ß-MHC in Ang-II-treated mouse cardiomyocytes. The NF-κB signal pathway was verified to mediate Ang-II-induced miR-214-3p expression in cardiomyocytes. Taken together, our results revealed that MEF2C is a novel target of miR-214-3p, and attenuation of miR-214-3p expression may contribute to MEF2Cexpressionin cardiac hypertrophy.

Left ventricular deformation associated with cardiomyocyte Ca(2+) transients delay in early stage of low-dose of STZ and high-fat diet induced type 2 diabetic rats.

BACKGROUND: In the early stage of diabetes, the cardiac ejection fraction is preserved, despite the existence of the subclinical cardiac dysfunction to some extent. However, the detailed phenotype of this dysfunction and the underlying mechanism remain unclear. To improve our understanding of this issue, we used low-dose STZ and high-fat diet to induce type 2 diabetic models in rats. The effects and the mechanism associated with the early stages of the disease were analyzed. METHODS: The type 2 diabetic mellitus (T2DM) in SD rats were induced through 30 mg/kg STZ and high-fat diet. Two-dimensional spackle-tracking echocardiography (STE) and the dobutamine test were performed to examine the cardiac function. Calcium transients of left ventricular myocytes were detected and the related intracellular signalling factors were analyzed by western blotting. RESULTS: After 6-weeks, T2DM rats in left ventricular (LV) diastole showed decreased global and segment strain(S) levels (P < 0.05), both in the radial and circumferential directions. Strain rate (Sr) abatement occurred in three segments in the radial and circumferential directions (P < 0.05), and the radial global Sr also decreased (P < 0.05). In the systolic LV, radial Sr was reduced, except the segment of the anterior septum, and the Sr of the lateral wall and post septum decreased in the circumferential direction (P < 0.05). Conventional M-mode echocardiography failed to detect significant alterations of cardiac performance between the two groups even after 12 weeks, and the decreased ejection fraction (EF%), fractional shortening (FS%) and end-systolic diameters (ESD) could be detected only under stress conditions induced by dobutamine (P < 0.05). In terms of calcium transients in cardiac myocytes, the Tpeak in model rats at 6 weeks was not affected, while the Tdecay1/2 was higher than that of the controls (P < 0.05), and both showed a dose-dependent delay after isoproterenol treatment (P < 0.05). Western blot analysis showed that in 6-week T2DM rats, myocardial p-PLB expression was elevated, whereas p-CaMKII, p-AMPK and Sirt1 were significantly down-regulated (P < 0.05). CONCLUSION: A rat model of T2DM was established by low dose STZ and a high-fat diet. LV deformation was observed in the early stages of T2DM in association with the delay of Ca(2+) transients in cardiomyocytes due to the decreased phosphorylation of CaMKII. Myocardial metabolism remodeling might contribute to the early LV function and calcium transportation abnormalities.

Species-specific differences in the role of L-type Ca²âº channels in the regulation of coronary arterial smooth muscle contraction.

The L-type calcium channel (LCC) plays a regulatory role in various physical and pathological processes. In the vasculature, LCCs mediate agonist-induced vascular smooth muscle contraction. However, whether LCC-mediated vessel responses to certain vasoconstrictors vary among species remains unclear. The coronary arteries were dissected from the hearts of rats and mice. Coronary arterial ring contraction was measured using the Multi Myograph system. High K+ (60 mM)-induced coronary artery contractions were stronger in rats than in mice, whereas CaCl2-induced contraction curves did not differ significantly between the two groups. Endothelin-1, U46619 (thromboxane A2 receptor agonist), and 5-hydroxytryptamine (5-HT) induced concentration-dependent vasoconstriction of coronary arterial rings in rats and mice. The vessel rings of mice were more sensitive to ET-1 and U46619 and less sensitive to 5-HT than those of rats. The LCC blocker nifedipine significantly inhibited coronary artery contractions induced by ET-1, U46619, and 5-HT. The inhibitory effect of 1 µM nifedipine on ET-1- and 5-HT-induced coronary artery contractions was stronger in mice than in rats, whereas its effect on U46619-induced vessel contractions was weaker in mice than in rats. The 5-HT2A receptor and LCC mRNA levels were higher in the coronary arteries of rats than in those of mice, whereas the expressions of the ETA and TXA2 receptors and Orai1 mRNA levels were comparable between the two groups. LCC plays an important role in coronary arterial contraction. Rats and mice show different responses to vasoconstrictors and LCC blockers, suggesting that the coronary arteries of rats and mice have different biological characteristics.

CDK6 mediates the effect of attenuation of miR-1 on provoking cardiomyocyte hypertrophy.

MicroRNA-1 (miR-1) is approved involved in cardiac hypertrophy, but the underlying molecular mechanisms of miR-1 in cardiac hypertrophy are not well elucidated. The present study aimed to investigate the potential role of miR-1 in modulating CDKs-Rb pathway during cardiomyocyte hypertrophy. A rat model of hypertrophy was established with abdominal aortic constriction, and a cell model of hypertrophy was also achieved based on PE-promoted neonatal rat ventricular cardiomyocytes (NRVCs). We demonstrated that miR-1 expression was markedly decreased in hypertrophic myocardium and hypertrophic cardiomyocytes. Dual luciferase reporter assays revealed that miR-1 interacted with the 3'UTR of CDK6, and miR-1 was verified to inhibit CDK6 expression at the posttranscriptional level. CDK6 protein expression was observed increased in hypertrophic myocardium and hypertrophic cardiomyocytes. Morover, miR-1 mimic, in parallel to CDK6 siRNA, could inhibit PE-induced hypertrophy of NRVCs, with decreases in cell size, newly transcribed RNA, expressions of ANF and ß-MHC, and the phosphorylated pRb. Taken together, our results reveal that derepression of CDK6 and activation of Rb pathway contributes to the effect of attenuation of miR-1 on provoking cardiomyocyte hypertrophy.

Macrophage migration inhibitory factor promotes expression of GLUT4 glucose transporter through MEF2 and Zac1 in cardiomyocytes.

OBJECTIVE: Evidence shows that both macrophage migration inhibitory factor (MIF) and GLUT4 glucose transporter are involved in diabetic cardiomyopathy (DCM), but it remains largely unknown whether and how MIF regulates GLUT4 expression in cardiomyocytes. The present study aims to investigate the mechanism underlying the modulation of GLUT4 by MIF in cardiomyocytes. MATERIAL AND METHODS: Activations of AKT and AMPK signaling, and expressions of MIF, GLUT4 and the candidate GLUT4 regulation associated transcription factors in the diabetic mouse myocardium were determined. The screened transcription factors mediating MIF-promoted GLUT4 expression were verified by RNA interference (RNAi) and electrophoretic mobility shift assay (EMSA), respectively. RESULTS: MIF was increased, but GLUT4 was decreased in the diabetic mouse myocardium. MIF could enhance glucose uptake and up-regulate GLUT4 expression in NMVCs. Expressions of transcription factor MEF2A, -2C, -2D and Zac1 were significantly up-regulated in MIF-treated neonatal mouse ventricular cardiomyocytes (NMVCs), and markedly reduced in the diabetic myocardium. Knockdown of MEF2A, -2C, -2D and Zac1 could significantly inhibit glucose uptake and GLUT4 expression in cardiomyocytes. Moreover, EMSA results revealed that transcriptional activities of MEF2 and Zac1 were significantly increased in MIF-treated NMVCs. AMPK signaling was activated in MIF-stimulated NMVCs, and AMPK activator AICAR could enhance MEF2A, -2C, -2D, Zac1 and GLUT4 expression. Additionally, MIF effects were inhibited by an AMPK inhibitor compound C and siRNA targeting MIF receptor CD74, suggesting the involvement of CD74-dependent AMPK activation. CONCLUSIONS: Transcription factor MEF2 and Zac1 mediate MIF-induced GLUT4 expression through CD74-dependent AMPK activation in cardiomyocytes.