Journal: Frontiers in Genetics

Article Title: Circ_0124644 Serves as a ceRNA for miR-590-3p to Promote Hypoxia-Induced Cardiomyocytes Injury via Regulating SOX4

doi: 10.3389/fgene.2021.667724

Figure Lengend Snippet: The expression of circ_0124644 in AMI patients and hypoxia-induced AC16 cells. (A) The circ_0124644 expression in the serum of AMI patients and healthy control volunteers was measured using qRT-PCR. (B) The expression of circ_0124644 was detected using qRT-PCR in hypoxia-treated and normoxia-treated AC16 cells. RNase R assay (C) and Act D assay (D) were used to confirm the stability of circ_0124644. (E) Subcellular localization analysis was performed to assess the distribution of circ_0124644 in the cytoplasm and nuclear of AC16 cells. ** P < 0.01, *** P < 0.001.

Article Snippet: Human cardiomyocytes (AC16) (Biovector National Typical Culture Center, Beijing, China) were cultured in DMEM medium (Gibco, Grand Island, NY, United States) containing 10% fetal bovine serum (FBS; Gibco) and 1% penicillin/streptomycin (Invitrogen, Carlsbad, CA, United States).

Techniques: Expressing, Control, Quantitative RT-PCR

Journal: Frontiers in Genetics

Article Title: Circ_0124644 Serves as a ceRNA for miR-590-3p to Promote Hypoxia-Induced Cardiomyocytes Injury via Regulating SOX4

doi: 10.3389/fgene.2021.667724

Figure Lengend Snippet: The regulation of circ_0124644 silencing on hypoxia-induced cardiomyocytes injury. (A) AC16 cells were transfected with sh-NC or sh-circ_0124644. The expression of circ_0124644 was measured by qRT-PCR. (B–I) AC16 cells were transfected with sh-NC or sh-circ_0124644, followed by treated with hypoxia. AC16 cells treated with normoxia were used as control. (B) CCK8 assay was used to examine cell viability. (C,D) Flow cytometry was performed to detect cell cycle distribution and cell apoptosis. (E) The protein levels of CyclinD1 and Cleaved-casp3 were determined using WB analysis. (F–I) Corresponding Assay Kits were used to assess the levels of LDH, MDA, SOD, and CAT. ** P < 0.01, *** P < 0.001.

Article Snippet: Human cardiomyocytes (AC16) (Biovector National Typical Culture Center, Beijing, China) were cultured in DMEM medium (Gibco, Grand Island, NY, United States) containing 10% fetal bovine serum (FBS; Gibco) and 1% penicillin/streptomycin (Invitrogen, Carlsbad, CA, United States).

Techniques: Transfection, Expressing, Quantitative RT-PCR, Control, CCK-8 Assay, Flow Cytometry

Journal: Frontiers in Genetics

Article Title: Circ_0124644 Serves as a ceRNA for miR-590-3p to Promote Hypoxia-Induced Cardiomyocytes Injury via Regulating SOX4

doi: 10.3389/fgene.2021.667724

Figure Lengend Snippet: Circ_0124644 acted as a sponge of miR-590-3p. (A) The predicted and mutated binding sites between miR-590-3p and circ_0124644 were exhibited. (B) The transfection efficiency of miR-590-3p mimic was confirmed by detecting miR-590-3p expression in AC16 cells using qRT-PCR. Dual-luciferase reporter assay (C) , RIP assay (D) , and RNA pull-down assay (E) were employed to assess the interaction between miR-590-3p and circ_0124644. (F) AC16 cells were transfected with sh-NC or sh-circ_0124644, followed by treated with hypoxia. AC16 cells treated with normoxia were used as control. The expression of miR-590-3p was examined by qRT-PCR. (G) The expression of miR-590-3p in the serum of AMI patients and healthy control volunteers was determined by qRT-PCR. (H) Pearson correlation analysis was used to assess the correlation between miR-590-3p and circ_0124644. ** P < 0.01, *** P < 0.001.

Article Snippet: Human cardiomyocytes (AC16) (Biovector National Typical Culture Center, Beijing, China) were cultured in DMEM medium (Gibco, Grand Island, NY, United States) containing 10% fetal bovine serum (FBS; Gibco) and 1% penicillin/streptomycin (Invitrogen, Carlsbad, CA, United States).

Techniques: Binding Assay, Transfection, Expressing, Quantitative RT-PCR, Luciferase, Reporter Assay, Pull Down Assay, Control

Journal: Frontiers in Genetics

Article Title: Circ_0124644 Serves as a ceRNA for miR-590-3p to Promote Hypoxia-Induced Cardiomyocytes Injury via Regulating SOX4

doi: 10.3389/fgene.2021.667724

Figure Lengend Snippet: The regulation of miR-590-3p overexpression on cardiomyocytes injury induced by hypoxia. AC16 cells were transfected with miR-NC or miR-590-3p mimic, followed by treated with hypoxia. Normoxia-treated AC16 cells were used as control. (A) Cell viability was determined using CCK8 assay. (B,C) Cell cycle distribution and cell apoptosis were detected by flow cytometry. (D) WB analysis was performed to evaluate the protein levels of CyclinD1 and Cleaved-casp3. (E–H) The levels of LDH, MDA, SOD, and CAT were determined using corresponding Assay Kits. ** P < 0.01, *** P < 0.001.

Article Snippet: Human cardiomyocytes (AC16) (Biovector National Typical Culture Center, Beijing, China) were cultured in DMEM medium (Gibco, Grand Island, NY, United States) containing 10% fetal bovine serum (FBS; Gibco) and 1% penicillin/streptomycin (Invitrogen, Carlsbad, CA, United States).

Techniques: Over Expression, Transfection, Control, CCK-8 Assay, Flow Cytometry

Journal: Frontiers in Genetics

Article Title: Circ_0124644 Serves as a ceRNA for miR-590-3p to Promote Hypoxia-Induced Cardiomyocytes Injury via Regulating SOX4

doi: 10.3389/fgene.2021.667724

Figure Lengend Snippet: Effects of miR-590-3p inhibitor and circ_0124644 silencing on hypoxia-induced cardiomyocytes injury. (A) AC16 cells were transfected with anti-NC or anti-miR-590-3p. MiR-590-3p expression was detected by qRT-PCR. (B–I) AC16 cells were transfected with sh-NC + anti-NC, sh-circ_0124644 + anti-NC, sh-circ_0124644 + anti-miR-590-3p, followed by treated with hypoxia. Normoxia-treated AC16 cells transfected with sh-NC + anti-NC were used as control. (B) CCK8 assay was employed to measure cell viability. (C,D) Cell cycle distribution and cell apoptosis were evaluated using flow cytometry. (E) The protein levels of CyclinD1 and Cleaved-casp3 were examined by WB analysis. (F–I) The levels of LDH, MDA, SOD, and CAT were analyzed by corresponding Assay Kits. ** P < 0.01, *** P < 0.001.

Article Snippet: Human cardiomyocytes (AC16) (Biovector National Typical Culture Center, Beijing, China) were cultured in DMEM medium (Gibco, Grand Island, NY, United States) containing 10% fetal bovine serum (FBS; Gibco) and 1% penicillin/streptomycin (Invitrogen, Carlsbad, CA, United States).

Techniques: Transfection, Expressing, Quantitative RT-PCR, Control, CCK-8 Assay, Flow Cytometry

Journal: Frontiers in Genetics

Article Title: Circ_0124644 Serves as a ceRNA for miR-590-3p to Promote Hypoxia-Induced Cardiomyocytes Injury via Regulating SOX4

doi: 10.3389/fgene.2021.667724

Figure Lengend Snippet: SOX4 could be targeted by miR-590-3p. (A) The predicted and mutated binding sites between SOX4 3′UTR and miR-590-3p were shown. (B) Dual-luciferase reporter assay was used to confirm the interaction between SOX4 and miR-590-3p. (C) AC16 cells were transfected with miR-NC or miR-590-3p mimic, followed by treated with hypoxia. AC16 cells treated with normoxia were used as control. The protein expression of SOX4 was analyzed using WB analysis. (D) AC16 cells were transfected with sh-NC + anti-NC, sh-circ_0124644 + anti-NC, sh-circ_0124644 + anti-miR-590-3p, followed by treated with hypoxia. Normoxia-treated AC16 cells transfected with sh-NC + anti-NC were used as control. WB analysis was used to detect SOX4 protein expression. (E,F) The protein and mRNA expression levels of SOX4 in the serum of AMI patients and healthy control volunteers were evaluated using WB analysis and qRT-PCR. (G,H) The correlation between SOX4 and miR-590-3p or circ_0124644 was assessed by Pearson correlation analysis. ** P < 0.01, *** P < 0.001.

Article Snippet: Human cardiomyocytes (AC16) (Biovector National Typical Culture Center, Beijing, China) were cultured in DMEM medium (Gibco, Grand Island, NY, United States) containing 10% fetal bovine serum (FBS; Gibco) and 1% penicillin/streptomycin (Invitrogen, Carlsbad, CA, United States).

Techniques: Binding Assay, Luciferase, Reporter Assay, Transfection, Control, Expressing, Quantitative RT-PCR

Journal: Frontiers in Genetics

Article Title: Circ_0124644 Serves as a ceRNA for miR-590-3p to Promote Hypoxia-Induced Cardiomyocytes Injury via Regulating SOX4

doi: 10.3389/fgene.2021.667724

Figure Lengend Snippet: Effects of SOX4 and miR-590-3p overexpression on hypoxia-induced cardiomyocytes injury. (A) WB analysis was used to detect SOX4 protein expression in AC16 cells to evaluate the transfection efficiency of pcDNA SOX4 overexpression vector. (B–I) AC16 cells were transfected with miR-NC + pcDNA, miR-590-3p + pcDNA, or miR-590-3p + SOX4, followed by treated with hypoxia. Normoxia-treated AC16 cells transfected with miR-NC + pcDNA were used as control. (B) Cell viability was detected by CCK8 assay. (C,D) Flow cytometry was utilized for assessing cell cycle distribution and cell apoptosis. (E) WB analysis was used to test the protein levels of CyclinD1 and Cleaved-casp3. (F–I) Corresponding Assay Kits were employed to detect the levels of LDH, MDA, SOD, and CAT. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Human cardiomyocytes (AC16) (Biovector National Typical Culture Center, Beijing, China) were cultured in DMEM medium (Gibco, Grand Island, NY, United States) containing 10% fetal bovine serum (FBS; Gibco) and 1% penicillin/streptomycin (Invitrogen, Carlsbad, CA, United States).

Techniques: Over Expression, Expressing, Transfection, Plasmid Preparation, Control, CCK-8 Assay, Flow Cytometry

Journal: Experimental Animals

Article Title: Ectodysplasin-A2 receptor (EDA2R) knockdown alleviates myocardial ischemia/reperfusion injury through inhibiting the activation of the NF-κB signaling pathway

doi: 10.1538/expanim.24-0020

Figure Lengend Snippet: Dexmedetomidine (DEX) has a protective effect on hypoxia/reoxygenation (H/R)-induced cardiomyocyte injury. (A) Cell -Counting Kit 8 (CCK-8) was used to measure cell viability. (B, C) Apoptosis and apoptotic rate were determined using flow cytometry. (D) GSE126104 showed that DEX decreased ectodysplasin-A2 receptor (EDA2R) expression in the left ventricle of rats. ***, P <0.001 vs. con. (E) The expression of EDA2R in cardiomyocytes was detected by RT-qPCR and western blotting. ** P <0.01; *** P <0.001; **** P <0.0001 vs. con. ## P <0.01; #### P <0.0001 vs. H/R. n=3.

Article Snippet: AC16 human cardiomyocytes were purchased from iCell Bioscience Inc. (Shanghai, China).

Techniques: Cell Counting, CCK-8 Assay, Flow Cytometry, Expressing, Quantitative RT-PCR, Western Blot

Journal: Experimental Animals

Article Title: Ectodysplasin-A2 receptor (EDA2R) knockdown alleviates myocardial ischemia/reperfusion injury through inhibiting the activation of the NF-κB signaling pathway

doi: 10.1538/expanim.24-0020

Figure Lengend Snippet: Ectodysplasin-A2 receptor (EDA2R) knockdown suppresses hypoxia/reoxygenation (H/R)-induced cardiomyocyte apoptosis. (A) The knockdown efficiency of EDA2R in cardiomyocytes was detected by RT-qPCR and western blotting. (B) Cell -Counting Kit 8 (CCK-8) was applied to detect cell viability. (C, D) Apoptosis was determined using flow cytometry, and apoptotic rate was calculated. *** P <0.001; **** P <0.0001 vs. con. ## P <0.01; ### P <0.001; #### P <0.0001 vs. H/R+shNC. n=3.

Article Snippet: AC16 human cardiomyocytes were purchased from iCell Bioscience Inc. (Shanghai, China).

Techniques: Knockdown, Quantitative RT-PCR, Western Blot, Cell Counting, CCK-8 Assay, Flow Cytometry

Journal: Experimental Animals

Article Title: Ectodysplasin-A2 receptor (EDA2R) knockdown alleviates myocardial ischemia/reperfusion injury through inhibiting the activation of the NF-κB signaling pathway

doi: 10.1538/expanim.24-0020

Figure Lengend Snippet: Ectodysplasin-A2 receptor (EDA2R) knockdown represses mitochondria-mediated apoptosis. (A) Mitochondrial morphology of cardiomyocytes was observed by transmission electron microscopy (the arrows represented mitochondrial morphology, scale bar=500 nm). (B) Mitochondrial membrane potential (MMP) was detected by JC-1 (Scale bar=100 µ m, Red: **** P <0.0001 vs. con. or hypoxia/reoxygenation (H/R)+shNC; Green: #### P <0.0001 vs. con. H/R+shNC). (C, D) The levels of cytochrome C in mitochondria and cytoplasm of cardiomyocytes were determined by western blotting. (E) The expressions of Bax and Bcl-2 were determined in cardiomyocytes by western blotting. (F, G) Caspase-3 and Caspase-9 activity in cardiomyocytes was detected. *** P <0.001; **** P <0.0001 vs. con. # P <0.05; ## P <0.01; ### P <0.001; #### P <0.0001vs. H/R+shNC. n=3.

Article Snippet: AC16 human cardiomyocytes were purchased from iCell Bioscience Inc. (Shanghai, China).

Techniques: Knockdown, Transmission Assay, Electron Microscopy, Membrane, Western Blot, Activity Assay

Journal: Experimental Animals

Article Title: Ectodysplasin-A2 receptor (EDA2R) knockdown alleviates myocardial ischemia/reperfusion injury through inhibiting the activation of the NF-κB signaling pathway

doi: 10.1538/expanim.24-0020

Figure Lengend Snippet: Ectodysplasin-A2 receptor (EDA2R) knockdown inhibits the activation of the NF-κB signaling pathway. (A) The levels of IκBα, p-IκBα (Ser32), NF-κB p65 and p-NF-κB p65 (Ser536) were detected in cardiomyocytes by western blotting. (B) The expression distribution of NF-κB p65 in cardiomyocytes was determined by immunofluorescence, the fluorescence intensity of p65 in the nucleus was quantified in three fields of three sections (the arrows represented the distribution of p65 in the nucleus, scale bar=50 µ m). (C) The transcriptional activity of NF-κB in cardiomyocytes was measured by the electrophoretic mobility shift assay (EMSA). **** P <0.0001 vs. con. #### P <0.0001. n=3.

Article Snippet: AC16 human cardiomyocytes were purchased from iCell Bioscience Inc. (Shanghai, China).

Techniques: Knockdown, Activation Assay, Western Blot, Expressing, Immunofluorescence, Fluorescence, Activity Assay, Electrophoretic Mobility Shift Assay

Journal: Physiological Reports

Article Title: Dapagliflozin prevents ERK activation and SGLT2‐dependent endoglin upregulation in a mechanically provoked cardiac injury model

doi: 10.14814/phy2.15990

Figure Lengend Snippet: Troponin I and CD105 increased in cardiomyocytes after mechanical stretch. (a, b) Representative images showing troponin I and CD105‐positive cells in human cardiomyocytes after 24 and 48 h cyclic stretch. Cells were counterstained in 4,6‐diamidino‐2‐phenylindole. Scale bar = 50 μm. (c, d) Bar graphs representing troponin I and CD105 fluorescence intensity. Data represented at least three independent experiments in median (interquartile range). Data were analyzed with the Mann–Whitney U test.

Article Snippet: Human cardiomyocytes AC16 were purchased from MERCK (Catalogue #SCC109, Temecula, CA, USA).

Techniques: Fluorescence, MANN-WHITNEY

Journal: Physiological Reports

Article Title: Dapagliflozin prevents ERK activation and SGLT2‐dependent endoglin upregulation in a mechanically provoked cardiac injury model

doi: 10.14814/phy2.15990

Figure Lengend Snippet: Mechanical stretch upregulated SGLT1/2 protein levels, promoted nuclear translocation of p‐ERK, and induced p‐eNOS expression. (a–d) Green fluorescence indicating SGLT1, SGLT2, p‐ERK, and p‐eNOS‐positive cells in cardiomyocytes after 24 h of 5%, 25% cyclic stretch. Cells were counterstained in 4,6‐diamidino‐2‐phenylindole to exhibit blue fluorescence. Scale bar = 50 μm. Bar graphs representing SGLT1, SGLT2, p‐ERK, and p‐eNOS fluorescence intensity. Data represented at least three independent experiments in median (interquartile range). Data were analyzed with the Mann–Whitney U test.

Article Snippet: Human cardiomyocytes AC16 were purchased from MERCK (Catalogue #SCC109, Temecula, CA, USA).

Techniques: Translocation Assay, Expressing, Fluorescence, MANN-WHITNEY

Journal: Physiological Reports

Article Title: Dapagliflozin prevents ERK activation and SGLT2‐dependent endoglin upregulation in a mechanically provoked cardiac injury model

doi: 10.14814/phy2.15990

Figure Lengend Snippet: SGLT2 inhibitor decreased troponin I and CD105 protein expressions. (a) Representative images showing green fluorescence‐labeled troponin I and CD105‐positive cells in cardiomyocytes. Cardiomyocytes were treated with DAPA (SGLT2 inhibitor) or PD98059 (ERK inhibitor), followed by 25%, 24 h cyclic stretch. Scale bar = 50 μm. (b, c) Bar graphs showing troponin I and CD105 fluorescence intensity in the cardiomyocytes. (d) Correlation of troponin I and CD105 with cyclic‐stretch‐induced mechanical overloading was assessed. Data represented at least three independent experiments in median (interquartile range). Data were analyzed with the Mann–Whitney U test. Correlations were assessed using Spearman's rank correlation moment correlation coefficient.

Article Snippet: Human cardiomyocytes AC16 were purchased from MERCK (Catalogue #SCC109, Temecula, CA, USA).

Techniques: Fluorescence, Labeling, MANN-WHITNEY

Journal: Physiological Reports

Article Title: Dapagliflozin prevents ERK activation and SGLT2‐dependent endoglin upregulation in a mechanically provoked cardiac injury model

doi: 10.14814/phy2.15990

Figure Lengend Snippet: Mechanical stretch induced SGLT1/2 expression, upregulated ERK and eNOS activation. (a) Fluorescence images showing cardiomyocytes positive for SGLT1/2, p‐ERK, and p‐eNOS. DAPA (SGLT2 inhibitor) or PD98059 (ERK inhibitor) were added to cells, and stretched at 5%, 25% for 24 h. Scale bar = 50 μm. (b–d) Quantifications of SGLT1/2 and p‐ERK. (e) p‐eNOS fluorescence intensity in the stretched cells. Data represented at least three independent experiments in median (interquartile range). Data were analyzed with the Mann–Whitney U test.

Article Snippet: Human cardiomyocytes AC16 were purchased from MERCK (Catalogue #SCC109, Temecula, CA, USA).

Techniques: Expressing, Activation Assay, Fluorescence, MANN-WHITNEY

Journal: Physiological Reports

Article Title: Dapagliflozin prevents ERK activation and SGLT2‐dependent endoglin upregulation in a mechanically provoked cardiac injury model

doi: 10.14814/phy2.15990

Figure Lengend Snippet: Proposed mechanism for Dapagliflozin on troponin I, endoglin (CD105) and ERK activation in the mechanically injured cardiac cell model. Our model depicts the upregulation of troponin I and endoglin (CD105) after mechanically triggered cardiac injury. (a) Cardiomyocytes were seeded on stretchable PDMS membrane, subjected to cyclic stretch at 5% and 25% elongation. After cyclic stretch, troponin I and CD105 were increased, triggering downstream p‐ERK to enter cell nucleus, potentially altered gene transcription that promoted upregulation of p‐eNOS. The accumulated p‐ERK nuclear translocation and p‐eNOS activation led to an imbalanced regulation of sodium/calcium ions. Finally, the cardiomyocytes were overwhelmed by loss of sodium/calcium ions homeostasis, renting the cells susceptible to CVD‐related risk factor. (b) Dapagliflozin (DAPA), the inhibitor having higher affinity for SGLT2, is able to prevent the characteristics of the mechanically provoked biomarkers for cardiac injury. As a result, SGLT2 and/or SGLT1 is crucial in maintaining the normal function of cardiomyocytes.

Article Snippet: Human cardiomyocytes AC16 were purchased from MERCK (Catalogue #SCC109, Temecula, CA, USA).

Techniques: Activation Assay, Membrane, Translocation Assay

Journal: Aging (Albany NY)

Article Title: Systems biology approach to exploring the effect of cyclic stretching on cardiac cell physiology

doi: 10.18632/aging.103465

Figure Lengend Snippet: Differentially expressed genes in human cardiomyocytes. Numbers of differentially expressed genes after cyclic stretching: ( A ) Genes showing an at least 2-fold difference in expression; ( B ) genes showing an at least 1.5-fold difference in expression. Venn diagrams show the overlapping genes with at least 2-fold and 1.5-fold differences in expression.

Article Snippet: Human ventricular cardiomyocyte cells (AC16) were maintained in DMEM/F12 (GeneDireX Incorporation, Taiwan) supplemented with 10% fetal bovine serum (Fisher Scientific, Pittsburgh, PA, USA), 100 IU/mL penicillin (Sigma-Aldrich) and 100 μg/mL streptomycin (Sigma-Aldrich).

Techniques: Expressing

Journal: Aging (Albany NY)

Article Title: Systems biology approach to exploring the effect of cyclic stretching on cardiac cell physiology

doi: 10.18632/aging.103465

Figure Lengend Snippet: Functional analysis of differentially expressed genes in human cardiomyocytes after 1, 4, 12, 24, and 48 hours of cyclic stretching.

Article Snippet: Human ventricular cardiomyocyte cells (AC16) were maintained in DMEM/F12 (GeneDireX Incorporation, Taiwan) supplemented with 10% fetal bovine serum (Fisher Scientific, Pittsburgh, PA, USA), 100 IU/mL penicillin (Sigma-Aldrich) and 100 μg/mL streptomycin (Sigma-Aldrich).

Techniques: Functional Assay

Journal: Aging (Albany NY)

Article Title: Systems biology approach to exploring the effect of cyclic stretching on cardiac cell physiology

doi: 10.18632/aging.103465

Figure Lengend Snippet: Differential expression profile of 29 genes after 1, 4, 12, 24, and 48 hours of cyclical stretching in human cardiomyocytes. The heat map diagram shows expression changes for 29 genes that were altered in cardiac myocytes in response to at least three time points of mechanical stretching.

Article Snippet: Human ventricular cardiomyocyte cells (AC16) were maintained in DMEM/F12 (GeneDireX Incorporation, Taiwan) supplemented with 10% fetal bovine serum (Fisher Scientific, Pittsburgh, PA, USA), 100 IU/mL penicillin (Sigma-Aldrich) and 100 μg/mL streptomycin (Sigma-Aldrich).

Techniques: Quantitative Proteomics, Expressing