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: 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

Journal: Science Advances

Article Title: Mapping cardiac remodeling in chronic kidney disease

doi: 10.1126/sciadv.adj4846

Figure Lengend Snippet: ( A ) Mice underwent sham surgery, STNx, or IRI and were sacrificed at 70 or 147 days after surgery. ( B ) Serum urea, serum creatinine (S-creatinine), heart weight, normalized to tibia length, and ejection fraction at the time point of sacrifice in sham (SH), STNx, and IRI mice. ( C ) Representative trichrome-stained images of kidney and heart. Scale bars, 50 μm. ( D ) UMAP embedding of nuclei isolated from left ventricle of all mice. n = 33,039 nuclei from n = 12 mice. ( E ) Expression of marker genes per cluster (cardiomyocytes: Ryr2 , Dmd , Myh6 , Fhl2 , and Ttn ; endothelial cells: Vwf , Cgnl1 , Smoc1 , Bmx , Pecam1 , Flt1 , Adgrl1 , and Prprb ; fibroblasts: Pdgra , Egfr , Dcn , Gsn , Col3a1 , and Col8a1 ; lymphatic endothelial cells: Flt4 , Prox1 , Lyve1 , and Pdpn ; lymphocytes: Btla , Cd2 , Bcl11b , and Sell ; macrophages: Mrc1 , Lyz2 , Cd86 , Cd68 , and Cd80 ; mesothelial cells: Muc16 , Msln , and Upk3b ; monocytes: Cd45 , Csfr1r; RBC: Lock2 , and Cd36 ; pericytes: Pdgfrb , Notch3 , Mcam , and Rgs5 ; and VSMC : Myh11 , Myl9 , and Lmod ). ( F ) PROGENGY pathway analysis in all cell types, STNx versus sham and IRI versus sham. ( G ) Predicted receptor-ligand interactions per group, sham, STNx, and IRI. * P < 0.05; ** P < 0.001. CM, cardiomyocytes; EC, endothelial cells; Fib, fibroblasts; Lym, lymphocytes; LEC, lymphatic endothelial cells; Mac, macrophages; Mes, mesothelial cells; Mon, monocytes; Per, pericytes; RBC, red blood cells; VSMC, vascular smooth muscle cells.

Article Snippet: Human AC16 cardiomyocytes (Merck Millipore, USA) were cultured in Dulbecco’s modified Eagle’s medium (DMEM)–F12 (D6434 Sigma-Aldrich, Germany) supplemented with 2 mM glutamine (TMS-002-C Millipore, USA), 12.5% fetal bovine serum (ES-009-B Millipore, USA), and 1% penicillin/streptomycin.

Techniques: Staining, Isolation, Expressing, Marker

Journal: Science Advances

Article Title: Mapping cardiac remodeling in chronic kidney disease

doi: 10.1126/sciadv.adj4846

Figure Lengend Snippet: ( A ) Representative confocal images of WGA-stained left ventricular myocardium from mice after sham surgery, STNx, or IRI. Scale bars, 20 μm. ( B ) Feret diameter of cardiomyocytes from sham, STNx, and IRI mice. ( C ) Hypertrophy gene set enrichment in cardiomyocytes 1 to 4 from sham, STNx, and IRI mice (GSEA). ( D ) Diffusion map (DM) embedding of cardiomyocytes 1 to 4 from sham, STNx, and IRI mice color-coded per condition, ( E ) See (D) but color-coded per cardiomyocyte cluster. ( F ) Differentiation trajectory toward hypertrophic cardiomyocytes within DM from (D) to (E). ( G ) Hypertrophy gene set enrichment in DM of cardiomyocytes from (D) to (E). ( H ) Genes sorted along the pseudotime trajectory of cardiomyocyte hypertrophy in (F); the color represents z score of the log-normalized gene expression across pseudotime (0 to 100) cells. ( I ) GO terms sorted along the pseudotime trajectory of cardiomyocyte hypertrophy in (F); the color represents the −log 10 transformed P value of GO terms derived from highly expressed genes across pseudotime (one to five) cells. ( J ) AC16 cardiomyocyte cell line, treated with 5 or 10% uremic serum, compared to healthy serum; planimetric analysis of cross-sectional area per cardiomyocyte from fluorescence microscopic images, TMRM staining. ( K ) Hypertrophic gene set, expressed in AC16 cells after treatment with 5% healthy or 5% uremic serum. ( L ) PROGENY pathway analysis in AC16 cells after treatment with 5% healthy or 5% uremic serum. ( M ) Planimetric analysis of cross-sectional area per cardiomyocyte from fluorescence microscopic images, TMRM staining, cells either treated with healthy serum, uremic serum + vehicle, uremic serum + ruxolitinib, or uremic serum + etanercept. * P < 0.05; # P < 0.001.

Article Snippet: Human AC16 cardiomyocytes (Merck Millipore, USA) were cultured in Dulbecco’s modified Eagle’s medium (DMEM)–F12 (D6434 Sigma-Aldrich, Germany) supplemented with 2 mM glutamine (TMS-002-C Millipore, USA), 12.5% fetal bovine serum (ES-009-B Millipore, USA), and 1% penicillin/streptomycin.

Techniques: Staining, Diffusion-based Assay, Gene Expression, Transformation Assay, Derivative Assay, Fluorescence

Journal: Science Advances

Article Title: Mapping cardiac remodeling in chronic kidney disease

doi: 10.1126/sciadv.adj4846

Figure Lengend Snippet: ( A ) Gene set enrichment of genes associated to UTs on all cell types; STNx versus sham (IRI versus sham is shown in fig. S13). ( B ) HUVEC tube formation assay, treatment with 5 or 10% uremic serum versus healthy control serum, quantification of isolated branches and bright-field microscopic images. ( C ) HUVEC tube formation assay, treatment with endothelin 1 (Edn1) or endothelin 1 neutralizing antibody, quantification of total mesh area and bright-field microscopic images. Scale bars, 500 μm. Sham: # P < 0.001. ( D ) Expression of endothelin converting enzyme 1 ( Ece1 ) in lymphatic endothelial cells and endothelial cells 1 to 3, per treatment group. ( E ) Size of AC16 cells after treatment with Edn1 or vehicle for 3 days. Scale bars, 25 μm.

Article Snippet: Human AC16 cardiomyocytes (Merck Millipore, USA) were cultured in Dulbecco’s modified Eagle’s medium (DMEM)–F12 (D6434 Sigma-Aldrich, Germany) supplemented with 2 mM glutamine (TMS-002-C Millipore, USA), 12.5% fetal bovine serum (ES-009-B Millipore, USA), and 1% penicillin/streptomycin.

Techniques: HUVEC Tube Formation Assay, Control, Isolation, Expressing

Journal: Journal of Translational Medicine

Article Title: CAV3 alleviates diabetic cardiomyopathy via inhibiting NDUFA10-mediated mitochondrial dysfunction

doi: 10.1186/s12967-024-05223-6

Figure Lengend Snippet: CAV3 was decreased in diabetic mice hearts and knock-down CAV3 induced mitochondria-dependent intrinsic apoptosis in cardiomyocytes. A Western blotting and associated quantitative analysis the CAV3 protein expression levels in the hearts of wt and db/db mice (n = 7 mice). B Representative immunohistochemical stains of CAV3 in the hearts of wt and db/db mice (n = 7 mice). C Representative Western blotting and quantitative analyses for CAV3, Bax, Cleaved caspase 3 and Bcl2 protein levels in cardiomyocytes treated with or without HGHF and with or without CAV3 downregulation (n = 4). D Representative Western blotting and quantitative analyses for the content of cytochrome C in cytoplasm treated with or without HGHF and with or without CAV3 downregulation (n = 3). E Flow cytometry and quantitative analysis of apoptosis cells by AnnexinV-FITC and propidium iodide (PI) staining in cardiomyocytes treated with or without HGHF and with or without CAV3 downregulation in vitro (n = 5). F Flow cytometry and quantification analysis of mitochondrial membrane potential by JC-1 in cardiomyocytes treated with or without HGHF and with or without CAV3 downregulation. High levels of green fluorescence (x-axis) represent reduced mitochondrial membrane potential (ΔΨm) and high levels of red fluorescence (y-axis) represents increased ΔΨm. A decrease in the red/green fluorescence is indicative of loss of ΔΨm (n = 6). G Normalized ATP production levels in cardiomyocytes treated with or without HGHF and with or without CAV3 downregulation in vitro (n = 6). H Representative confocal microscope of MitoSOX staining, immunofluorescence staining of 8-oxoG andquantitative analyses in cardiomyocytes treated with or without HGHF and with or without CAV3 downregulation (MitoSOX fluorescence, red; 8-oxoG fluorescence) (n = 5). I Immunofluorescence images of 8-oxoG and TOM20 in the AC16 cells treated with HGHF. AC16 cells were stained with 8-oxoG antibodies (Alexa Fluor 594), DAPI, and TOM20 (Alexa Fluor 488). Data are depicted as mean ± SD. *p < 0.05, **p < 0.01

Article Snippet: Human cardiomyocytes (AC16) and mouse cardiomyocytes (HL-1) were purchased from iCell Bioscience (Shanghai, China).

Techniques: Western Blot, Expressing, Immunohistochemical staining, Flow Cytometry, Staining, In Vitro, Membrane, Fluorescence, Microscopy, Immunofluorescence

Journal: Journal of Translational Medicine

Article Title: CAV3 alleviates diabetic cardiomyopathy via inhibiting NDUFA10-mediated mitochondrial dysfunction

doi: 10.1186/s12967-024-05223-6

Figure Lengend Snippet: Overexpression of CAV3 protected against cardiac dysfunction and cardiac remodeling in db/db mice. A Western blotting and associated quantitative analysis the CAV3 protein expression levels in the hearts of db/db mice with or without CAV3 overexpression in vivo (n = 7 mice). B – F The body weight, food intake and blood glucose levels of wt and db/db mice among each group were showed by a line chart (n = 7 mice). G Hemodynamic analysis (dp/dtmin) of wt mice and db/db mice with or without CAV3 overexpression. (n = 7 mice). H Representative images of echocardiograph (E/A) and relevant quantification analysis in wt mice and db/db mice with or without CAV3 overexpression (n = 7 mice). I The HW/TL ratio was quantitatively analyzed in wt mice and db/db mice with or without CAV3 overexpression (n = 7 mice). J Representative images of H&E staining and WGA staining to detect cardiomyocyte cross-sectional area, Sirius red staining to detect myocardial interstitial collagen and relevant quantification analysis in wt mice and db/db mice with or without CAV3 overexpression (n = 7 mice). Data are depicted as mean ± SD. *p < 0.05, **p < 0.01

Article Snippet: Human cardiomyocytes (AC16) and mouse cardiomyocytes (HL-1) were purchased from iCell Bioscience (Shanghai, China).

Techniques: Over Expression, Western Blot, Expressing, In Vivo, Staining

Journal: Journal of Translational Medicine

Article Title: CAV3 alleviates diabetic cardiomyopathy via inhibiting NDUFA10-mediated mitochondrial dysfunction

doi: 10.1186/s12967-024-05223-6

Figure Lengend Snippet: Overexpression of CAV3 prevented mitochondrial dysfunction in diabetic cardiomyocytes in vivo and in vitro models. A Representative transmission electron microscope images of cardiac mitochondria and quantification analysis of mitochondrial cristae number and the proportion of mitochondria with disorganized cristae in wt mice and db/db mice with or without CAV3 overexpression (n = 7 mice). B Normalized ATP production levels in cardiomyocytes of wt mice and db/db mice with or without CAV3 overexpression (n = 7 mice). C Flow cytometry analysis and quantification of mitochondrial membrane potential by JC-1 in cardiomyocytes treated with or without HGHF and with or without CAV3 overexpression. (n = 5). D Extracellular flux analysis of OCRs and respective quantitative analysis in cardiomyocytes treated with or without HGHF and with or without CAV3 overexpression (n = 3). E Representative images of DHE staining and quantitative analysis of ROS levels in the hearts of wt mice and db/db mice with or without CAV3 overexpression (n = 7 mice). F Representative images of immunofluorescence staining of 8-oxoG and quantitative analyses in cardiomyocytes treated with or without HGHF and with or without CAV3 overexpression (8-oxoG fluorescence, red) (n = 5). G Representative Western blotting and quantitative analyses for SOD2, Bcl2, Bax and Cleaved caspase 3 protein levels in cardiomyocytes of wt mice and db/db mice with or without CAV3 overexpression (n = 7 mice). H TUNEL assay by double staining with TUNEL (red) and DAPI (blue) detected apoptotic cells in mice hearts and quantitative analysis of wt mice and db/db mice with or without CAV3 overexpression (n = 7 mice). Data are depicted as mean ± SD. *p < 0.05, **p < 0.01

Article Snippet: Human cardiomyocytes (AC16) and mouse cardiomyocytes (HL-1) were purchased from iCell Bioscience (Shanghai, China).

Techniques: Over Expression, In Vivo, In Vitro, Transmission Assay, Microscopy, Flow Cytometry, Membrane, Staining, Immunofluorescence, Fluorescence, Western Blot, TUNEL Assay, Double Staining

Journal: Journal of Translational Medicine

Article Title: CAV3 alleviates diabetic cardiomyopathy via inhibiting NDUFA10-mediated mitochondrial dysfunction

doi: 10.1186/s12967-024-05223-6

Figure Lengend Snippet: CAV3 interacted with mitochondrial complex I by anchoring NDUFA10 and maintaining its stability. A IP assay was carried out using CAV3 antibody and Coomassie Brilliant blue was stained to indicate CAV3 bands. B Through bioinformatics methods, NDUFA10 and TARDBP were overlapped from the LC–MS/MS analysis, DCM-related Genes (DisGeNet & Gencards) and Mitochondrial dysfunction-related genes (GeneCards). C , D IP assay was carried out using CAV3 or NDUFA10 antibody in cardiomyocytes. E Representative Western blotting assessing the purity of the mitochondria isolated. F – G IP assay was carried out using CAV3 or NDUFA10 antibody in mitochondria isolated from AC16 cells. H Immunofluorescence images of cardiomyocytes were stained with mitochondria-targeting dye (Mito-Tracker, Deep Red, Alexa Fluor 647), DAPI, NDUFA10 antibodies (Alexa Fluor 594), and CAV3 antibodies (Alexa Fluor 488). I Western blotting and quantitative analyses protein levels in cardiomyocytes of wt mice and db/db mice with or without CAV3 overexpression (n = 7 mice). J The levels of NDUFA10 mRNA in cardiomyocytes treated with or without HGHF and with or without CAV3 overexpression (n = 6). K CAV3 plasmid (pcDNA3.1-CAV3) was transfected into cardiomyocytes using Lipofectamine 2000 and treated with HGHF. The transfected cells were treated with 100 µmol/L cycloheximide for 0, 2, 4, 6, 8, 10 h. The protein levels of NDUFA10 were shown by a line chart (n = 3). L Cardiomyocytes were treated with or without HGHF, CHX (100 µmol/L), and MG132 (10 µmol/L) or CQ (50 µmol/L) for 24 h. Quantitative analyses of NDUFA10 protein levels among each group (n = 3). M Immunofluorescence images of NDUFA10 and lysosomes-targeting dye (Lamp1) in the cardiomyocytes treated with or without CAV3 overexpression. Cardiomyocytes were stained with NDUFA10 antibodies (Alexa Fluor 594), DAPI, and Lamp1 (Alexa Fluor 488). Data are depicted as mean ± SD. *p < 0.05, **p < 0.01

Article Snippet: Human cardiomyocytes (AC16) and mouse cardiomyocytes (HL-1) were purchased from iCell Bioscience (Shanghai, China).

Techniques: Staining, Liquid Chromatography with Mass Spectroscopy, Western Blot, Isolation, Immunofluorescence, Over Expression, Plasmid Preparation, Transfection

Journal: Journal of Translational Medicine

Article Title: CAV3 alleviates diabetic cardiomyopathy via inhibiting NDUFA10-mediated mitochondrial dysfunction

doi: 10.1186/s12967-024-05223-6

Figure Lengend Snippet: CAV3 overexpression restored mitochondrial function partially relied on NDUFA10 in vivo and in vitro models. A Representative transmission electron microscope images of cardiac mitochondria and quantification analysis of mitochondrial cristae number and the proportion of mitochondria with disorganized cristae in different db/db mice groups (n = 7 mice). B Normalized ATP production levels in cardiomyocytes of db/db mice transfected with or without AAV-CAV3 or AAV-shNDUFA10 (n = 7 mice). C Flow cytometry and quantitative analysis of mitochondrial membrane potential by JC-1 in cardiomyocytes treated with HGHF and transfected with or without pc3.1-CAV3 or siNDUFA10 (n = 5). D Extracellular flux analysis of OCRs and respective quantitative analysis in cardiomyocytes treated with HGHF and transfected with or without pc3.1-CAV3 or siNDUFA10 (n = 3). E The activity of mitochondrial complex I in cardiomyocytes of db/db mice transfected with or without AAV-CAV3 or AAV-shNDUFA10 (n = 7 mice). F Representative images of DHE staining and quantitative analysis of ROS levels in the hearts of db/db mice transfected with or without AAV-CAV3 or AAV-shNDUFA10 (n = 7 mice). G Representative images of immunofluorescence staining of 8-oxoG and quantitative analyses in cardiomyocytes treated with HGHF and transfected with or without pc3.1-CAV3 or siNDUFA10 (8-oxoG fluorescence, red) (n = 5). H Representative Western blotting and quantitative analyses for SOD2, Bcl2, Bax and Cleaved caspase 3 protein levels in cardiomyocytes of db/db mice transfected with or without AAV-CAV3 or AAV-shNDUFA10 (n = 7 mice). I TUNEL assay by double staining with TUNEL (red) and DAPI (blue) detected apoptotic cells in the hearts and quantitative analysis of different db/db mice groups (n = 7 mice). Data are depicted as mean ± SD. *p < 0.05, **p < 0.01

Article Snippet: Human cardiomyocytes (AC16) and mouse cardiomyocytes (HL-1) were purchased from iCell Bioscience (Shanghai, China).

Techniques: Over Expression, In Vivo, In Vitro, Transmission Assay, Microscopy, Transfection, Flow Cytometry, Membrane, Activity Assay, Staining, Immunofluorescence, Fluorescence, Western Blot, TUNEL Assay, Double Staining

Journal: Journal of Translational Medicine

Article Title: CAV3 alleviates diabetic cardiomyopathy via inhibiting NDUFA10-mediated mitochondrial dysfunction

doi: 10.1186/s12967-024-05223-6

Figure Lengend Snippet: Cardiac-restricted NDUFA10 expression partially attenuated CAV3 benefits in diabetic cardiomyopathy. A Hemodynamic analysis (dp/dtmin) of db/db mice transfected with or without AAV-CAV3 or AAV-shNDUFA10 (n = 7 mice). B Representative images of echocardiograph (E/A) and relevant quantification analysis in db/db mice transfected with or without AAV-CAV3 or AAV-shNDUFA10 (n = 7 mice). C The HW/TL ratio was quantitatively analyzed in db/db mice transfected with or without AAV-CAV3 or AAV-shNDUFA10 (n = 7 mice). D Representative images of H&E staining and WGA staining to detect cardiomyocyte cross-sectional area, Sirius red staining to detect myocardial interstitial collagen and relevant quantification analysis in db/db mice transfected with or without AAV-CAV3 or AAV-shNDUFA10 (n = 7 mice). Data are depicted as mean ± SD. *p < 0.05, **p < 0.01

Article Snippet: Human cardiomyocytes (AC16) and mouse cardiomyocytes (HL-1) were purchased from iCell Bioscience (Shanghai, China).

Techniques: Expressing, Transfection, Staining

Journal: Journal of Translational Medicine

Article Title: CAV3 alleviates diabetic cardiomyopathy via inhibiting NDUFA10-mediated mitochondrial dysfunction

doi: 10.1186/s12967-024-05223-6

Figure Lengend Snippet: The schematic representation of the mechanisms by which CAV3 downregulation exacerbates diabetic cardiomyopathy via inducing NDUFA10-mediated mitochondrial dysfunction in diabetes. CAV3 expression is decreased in cardiomyocytes under diabetic conditions. Downregulation of CAV3 leads to the downregulation of NDUFA10, which is degraded via the lysosomal pathway. Consequently, the reduction of NDUA10 in mitochondria lead to a decrease in the mitochondrial complex I activity, resulting in mitochondrial dysfunction. As a result, ATP production decreases, mitochondrial ROS levels increase and cell apoptosis occurs, thereby leading to cardiac dysfunction

Article Snippet: Human cardiomyocytes (AC16) and mouse cardiomyocytes (HL-1) were purchased from iCell Bioscience (Shanghai, China).

Techniques: Expressing, Activity Assay

Journal: Frontiers in Endocrinology

Article Title: Weighted Gene Co-Expression Network Analysis Identifies ANGPTL4 as a Key Regulator in Diabetic Cardiomyopathy via FAK/SIRT3/ROS Pathway in Cardiomyocyte

doi: 10.3389/fendo.2021.705154

Figure Lengend Snippet: Screening of a novel therapeutic target for DbCM. (A) Relative mRNA expression of hub genes normalized to a GAPDH internal control (n=6 each). Values are expressed relative to control groups. (B, C) Representative Western blots and analysis of ANGPTL4 expression; p-FAK(Y397)/FAK ratio; SIRT3 expression; Ac-SOD2/SOD2 ratio; P67phox expression; Gp91phox expression; P47phox expression; Cleaved caspase-3 expression; Bcl-2 expression; Bax expression. Equal protein loading was confirmed using an anti-GAPDH antibody (n=6 each). (D) Representative images of ANGPTL4 fluorescence in heart sections. Scale bar, 50 μm. (E) Representative images of DHE fluorescence in heart sections. Scale bar, 50 μm. (F) Representative images of apoptotic cardiomyocytes (200×). The apoptotic cells were detected by TUNEL (green), and the nuclei were detected by DAPI (blue). Scale bar, 50 μm. (G) Representative immunohistochemical stainings of ANGPTL4, P47phox, and Cleaved caspase-3 (n=6 each). Scale bars, 50 μm. (H) Quantification of ANGPTL4 fluorescence intensity in heart sections (n=6 each). (I) Quantification of DHE fluorescence intensity in heart sections (n=6). (J) Percentage of TUNEL positive cells. (K–M) Representative immunohistochemical quantification of ANGPTL4, P47phox, and Cleaved caspase-3. Scale bars, 50 μm. Data are shown as mean ± SD (n=6 each); * p < 0.05, ** p < 0.01 vs CON group (student t -test). ns represents statically non-significant; IOD, the integral optical density.

Article Snippet: Human cardiomyocytes (AC16) were purchased from Sciencell (Cat# 6200, San Diego, CA, USA).

Techniques: Expressing, Western Blot, Fluorescence, TUNEL Assay, Immunohistochemical staining

Journal: Frontiers in Endocrinology

Article Title: Weighted Gene Co-Expression Network Analysis Identifies ANGPTL4 as a Key Regulator in Diabetic Cardiomyopathy via FAK/SIRT3/ROS Pathway in Cardiomyocyte

doi: 10.3389/fendo.2021.705154

Figure Lengend Snippet: ANGPTL4 siRNA exhibited the anti-apoptotic and anti-oxidative effects in HG-treated AC16 cardiomyocytes. (A) Representative Western blots showing ANGPTL4, p-FAK(Y397), FAK, and SIRT3 levels in AC16 cardiomyocytes at indicated time points after treatment with HG (30 mM). (B) WB analysis of ANGPTL4 expression, p-FAK(Y397)/FAK ratio, and SIRT3 expression at indicated time points, respectively. (C, D) Representative Western blots and quantification of p-FAK(Y397), FAK, SIRT3, Ac-SOD2, SOD2, P67phox, P47phox, Cleaved caspase-3, Bcl-2, and Bax in cultured AC16 cardiomyocytes under the control or HG (30 mM) condition plus treatment with si-RNA targeting ANGPTL4 (si-ANGPTL4) or negative control siRNA (si-NC). Equal protein loading was confirmed using an anti-β-tubulin antibody. (E, G) Representative images and quantification of DHE fluorescence in cultured AC16 cardiomyocytes under the control or HG (30 mM) condition plus treatment with si-RNA targeting ANGPTL4 (si-ANGPTL4) or negative control siRNA (si-NC). Scale bars, 50 μm. (F, H) Representative images of apoptotic cardiomyocytes (200×) and percentage of TUNEL positive cells. The apoptotic cells were detected by TUNEL (green), and the nuclei were detected by DAPI (blue). Scale bar, 50 μm. Experiments were repeated at least three times independently with similar results. * p < 0.05, ** p < 0.01 vs CON group. # p < 0.05 vs the CON+SiNC group; & p < 0.05 vs the HG+SiNC group; $ p < 0.05 vs the HG+SiNC group (student t -test).

Article Snippet: Human cardiomyocytes (AC16) were purchased from Sciencell (Cat# 6200, San Diego, CA, USA).

Techniques: Western Blot, Expressing, Cell Culture, Negative Control, Fluorescence, TUNEL Assay

Journal: Frontiers in Endocrinology

Article Title: Weighted Gene Co-Expression Network Analysis Identifies ANGPTL4 as a Key Regulator in Diabetic Cardiomyopathy via FAK/SIRT3/ROS Pathway in Cardiomyocyte

doi: 10.3389/fendo.2021.705154

Figure Lengend Snippet: ANGPTL4 elevated SOD2 acetylation, oxidative stress, and apoptosis. (A, B) Representative immunoblots showing SIRT3, p-FAK(Y397), and FAK levels in AC16 cardiomyocytes at 48 h after different concentrations of ANGPTL4 treatment. (C, D) Representative Western blots and quantification of p-FAK(Y397), FAK, P67phox, SIRT3, SOD2,Ac-SOD2, Bax, Bcl-2, and Cleaved caspase-3 in AC16 cardiomyocytes after treatment with BSA or ANGPTL4 (200 ng/ml each) for 48 h. (E, G) Representative images of apoptotic cardiomyocytes (200×) after treatment with BSA or ANGPTL4 (200 ng/ml each) for 48 h and percentage of TUNEL positive cells. The apoptotic cells were detected by TUNEL (green), and the nuclei were detected by DAPI (blue). Scale bar, 50 μm. (F, H) Representative images and quantification of DHE fluorescence in AC16 cardiomyocytes after treatment with BSA or ANGPTL4 (200 ng/ml) for 48 h. Scale bar, 50 μm. (I) Schematic illustration shows ANGPTL4 interacting with integrin, inducing apoptosis in the Diabetic Cardiomyopathy via FAK/SIRT3/ROS pathway in cardiomyocyte. P represents phosphorylation, α and β represent subunit of the integrin. Data are shown as mean ± SD; * p < 0.05, ** p < 0.01 vs BSA group (student t test).

Article Snippet: Human cardiomyocytes (AC16) were purchased from Sciencell (Cat# 6200, San Diego, CA, USA).

Techniques: Western Blot, TUNEL Assay, Fluorescence

Journal: Journal of Inflammation Research

Article Title: miR-16-5p Regulates Ferroptosis by Targeting SLC7A11 in Adriamycin-Induced Ferroptosis in Cardiomyocytes

doi: 10.2147/JIR.S393646

Figure Lengend Snippet: Ferroptosis involved in ADR-induced myocardial injury both in vivo and in vitro. ( A and B ) The responses of +dP/dt maximum and -dP/dt maximum in the study groups. ( C and D ) ADR-induced myocardial damage were assessed using biochemical cardiotoxicity markers LDH and CK-MB. ( E ) ADR-induced the lipid peroxidation in myocardial tissue. ( F ) We obtained the Fe 2+ data using iron colorimetric assay. ( G ) The immunofluorescence examination was performed to examine the distribution of 4-HNE. ( H ) Cell viability assays were conducted on H9c2 and AC16 cardiomyocytes in order to evaluate the cytotoxic effects of ADR. ( I ) H9c2 and AC16 cardiomyocytes were measured for lipid peroxidation. ( J ) Levels of Fe 2+ in the H9c2 and AC16 cells treated with ADR. ( K ) Levels of 4-HNE in the H9c2 and AC16 cells treated with ADR. Data are reported from at least three independent experiments and analysed by independent t -test, ***P < 0.001.

Article Snippet: Cell lines of human cardiomyocytes AC16 and rat cardiomyocytes H9c2 were acquired commercially (Biovector NTCC Inc., Beijing, China) and cultivated in DMEM with 10% fetal bovine serum (FBS) (Gibco, USA) and 1% penicillin-streptomycin in a humid atmosphere at 37 °C and CO2 (5%).

Techniques: In Vivo, In Vitro, Colorimetric Assay, Immunofluorescence

Journal: Journal of Inflammation Research

Article Title: miR-16-5p Regulates Ferroptosis by Targeting SLC7A11 in Adriamycin-Induced Ferroptosis in Cardiomyocytes

doi: 10.2147/JIR.S393646

Figure Lengend Snippet: Ferroptosis contribute to ADR-induced myocardial injury mechanism. ( A and B ) Co-administration of Fer-1/DFP alleviated ADR-induced myocardial damage assessed using biochemical cardiotoxicity markers LDH and CK-MB in mice. ( C ) Co-administration of Fer-1/DFP decreased ADR-induced the lipid peroxidation in myocardial tissue. ( D ) Co-administration of Fer-1/DFP decreased the expression of 4-HNE in myocardial tissue. ( E ) Cell viability assays were conducted on H9c2 and AC16 cardiomyocytes when co-administration with Erastin/Fer-1/DFP to evaluate the cytotoxic effects of ADR. ( F ) H9c2 and AC16 cardiomyocytes were measured for lipid peroxidation. ( G ) Levels of 4-HNE in the H9c2 and AC16 cells were examined when co-administration with Erastin/Fer-1/DFP under ADR. Data are reported from at least three independent experiments and analysed by one way ANOVA followed by Dunnett's post hoc test. ***P < 0.001 compared with Control; # P < 0.05, ## P < 0.01, ### P < 0.001 compared with ADR.

Article Snippet: Cell lines of human cardiomyocytes AC16 and rat cardiomyocytes H9c2 were acquired commercially (Biovector NTCC Inc., Beijing, China) and cultivated in DMEM with 10% fetal bovine serum (FBS) (Gibco, USA) and 1% penicillin-streptomycin in a humid atmosphere at 37 °C and CO2 (5%).

Techniques: Expressing, Control

Journal: Journal of Inflammation Research

Article Title: miR-16-5p Regulates Ferroptosis by Targeting SLC7A11 in Adriamycin-Induced Ferroptosis in Cardiomyocytes

doi: 10.2147/JIR.S393646

Figure Lengend Snippet: SLC7A11-mediated ferroptosis involved in ADR-induced cardiac injury. ( A ) The ADR induces down-regulation of SLC7A11 and GPX4 expression both in vitro and in vivo. ( B ) The intervention of SLC7A11 mediated ADR-induced cardiac injury assessed using biochemical cardiotoxicity markers CK-MB in mice. ( C ) Cell viability assays were conducted on H9c2 and AC16 cardiomyocytes when SLC7A11 was interfered to mediated ADR-induced cardiac injury. ( D ) H9c2 and AC16 cardiomyocytes were measured for lipid peroxidation. ( E ) Levels of 4-HNE in the H9c2 and AC16 cells were examined when SLC7A11 was interfered under ADR. Data are reported from at least three independent experiments and analysed by one way ANOVA followed by Dunnett’s post hoc test. **P < 0.01, ***P < 0.001 compared with Control; ## P < 0.01, ### P < 0.001 compared with ADR+SC.

Article Snippet: Cell lines of human cardiomyocytes AC16 and rat cardiomyocytes H9c2 were acquired commercially (Biovector NTCC Inc., Beijing, China) and cultivated in DMEM with 10% fetal bovine serum (FBS) (Gibco, USA) and 1% penicillin-streptomycin in a humid atmosphere at 37 °C and CO2 (5%).

Techniques: Expressing, In Vitro, In Vivo, Control

Journal: Journal of Inflammation Research

Article Title: miR-16-5p Regulates Ferroptosis by Targeting SLC7A11 in Adriamycin-Induced Ferroptosis in Cardiomyocytes

doi: 10.2147/JIR.S393646

Figure Lengend Snippet: SLC7A11 interacted with miR-16-5p in cardiomyocyte. ( A ) Target prediction programs including miRNA.org and TargetScan were used to identify potential SLC7A11 miRNA targets. ( B ) The expression profiles of SLC7A11’s potential target miRNAs. ( C ) Spearman correlation analysis was used to analyze the relationship between the expression of SLC7A11 and miR-16-5p. ( D ) The binding sites between SLC7A11 and miR-16-5p sequences were listed using target prediction tools. ( E ) Following co-transfection of miR-16-5p mimics and wild-type luciferase reporters, the reporter intensity of the mutant reporter was not affected. ( F ) Western blot was used to detect levels of SLC7A11 in H9c2 and AC16 transfected with mimic or inhibitor of miR-16-5p. Data are reported from at least three independent experiments and analysed by independent t -test or one way ANOVA followed by Dunnett’s post hoc test. *P< 0.05, **P< 0.01, ***P < 0.001 compared with Control; ***P < 0.001 compared with miR-NC.

Article Snippet: Cell lines of human cardiomyocytes AC16 and rat cardiomyocytes H9c2 were acquired commercially (Biovector NTCC Inc., Beijing, China) and cultivated in DMEM with 10% fetal bovine serum (FBS) (Gibco, USA) and 1% penicillin-streptomycin in a humid atmosphere at 37 °C and CO2 (5%).

Techniques: Expressing, Binding Assay, Cotransfection, Luciferase, Mutagenesis, Western Blot, Transfection, Control

Journal: Journal of Inflammation Research

Article Title: miR-16-5p Regulates Ferroptosis by Targeting SLC7A11 in Adriamycin-Induced Ferroptosis in Cardiomyocytes

doi: 10.2147/JIR.S393646

Figure Lengend Snippet: MiR-16-5p functionally interacted with SLC7A11 to reversed the ADR-induced cardiac injury in vitro. ( A ) Analyzing the effect of miR-16-5p/SLC7A11 intervention on cell viability in H9c2 and AC16 cells. ( B ) Analyzing the effect of miR-16-5p/SLC7A11 intervention on lipid peroxidation levels in H9c2 and AC16 cells. ( C ) Analyzing the effect of miR-16-5p/SLC7A11 intervention on lipid peroxidation levels in H9c2 and AC16 cells. ( D ) Analyzing the effect of miR-16-5p/SLC7A11 intervention on Fe 2+ accumulation in AC16 cells. Data are reported from at least three independent experiments and analysed by independent one way ANOVA followed by Dunnett's post hoc test. ***P < 0.001 compared with miR-NC+shRNA-Con; # P < 0.05, ## P < 0.01, ### P < 0.001 compared with ADR+miR-NC+shRNA-Con; $$ P< 0.01, $$$ P < 0.001 compared with Control.

Article Snippet: Cell lines of human cardiomyocytes AC16 and rat cardiomyocytes H9c2 were acquired commercially (Biovector NTCC Inc., Beijing, China) and cultivated in DMEM with 10% fetal bovine serum (FBS) (Gibco, USA) and 1% penicillin-streptomycin in a humid atmosphere at 37 °C and CO2 (5%).

Techniques: In Vitro, shRNA, Control