Journal: Circulation Research

Article Title: Effects of Atrial Fibrillation on the Human Ventricle

doi: 10.1161/CIRCRESAHA.121.319718

Figure Lengend Snippet: Atrial fibrillation (AF)-simulation in induced pluripotent stem cell cardiomyocytes (iPSC-CMs). Human iPSC-CMs treated either with AF-simulation (arrhythmic pacing: Arr; 60 bpm, 40% beat-to-beat-variability) or rhythmic pacing (control [Ctrl]; 60 bpm) chronically for 7 d. A , Representative recordings of stimulated Ca 2+ transients (epifluorescence microscopy, Fura-2) and ( B ) mean values for Ca 2+ transient amplitude, ( C ) diastolic Ca 2+ levels, ( D ) time to peak 80%, ( E ) relaxation time 80% of human iPSC-CM upon chronic AF-simulation (n=69 cardiomyocytes/6 differentiations/4 donors) or rhythmic pacing (n=71/6/4). F , Original recordings of caffeine-induced Ca 2+ transients (10 mmol/l caffeine, epifluorescence microscopy, Fura-2), ( G ) mean caffeine-transient amplitude indicating the sarcoplasmic reticulum Ca 2+ load and ( H ) SERCA2a activity (K sys -K caff ) of iPSC-CM after chronic AF-simulation (n=13/6/4) vs control (n=12/6/4). I , Representative confocal line scans (Fluo-4) showing diastolic sarcoplasmic reticulum Ca 2+ sparks and ( J ) mean Ca 2+ spark frequency (CaSpF) after chronic AF-simulation (n=68/7/4) vs control (n=67/7/4). K , Original recordings of cytosolic Na + levels (epifluorescence microscopy, SBFI) and ( L ) mean values of cytosolic Na + concentration of human iPSC-CM after chronic AF-simulation (n=110/7/4) compared with control (98/7/4). Data are provided as scatter plot with mean±SD. Each data point is calculated as mean value per differentiation. P were calculated using Student t test ( A–C , E–L ) or Mann-Whitney U test ( D ).

Article Snippet: The expression of RYR2 (ryanodine-receptor type 2), NCX (Na + -Ca 2+ exchanger), SERCA2a, PLB (phospholamban), and CaMKII was studied using specific antibodies anti-RyR2 (mouse monoclonal antibody, dilution 1:1000, Santa Cruz Biotechnology), NCX (mouse monoclonal antibody, dilution 1:1000, Swant), SERCA2a (rabbit polyclonal antibody, dilution 1:1000, Alomone), PLB (mouse monoclonal antibody, dilution 1:1000, Thermo Fisher) and CaMKII-δ (rabbit polyclonal antibody, dilution 1:1000, Thermo Fisher).

Techniques: Epifluorescence Microscopy, Activity Assay, Concentration Assay, MANN-WHITNEY

Journal: Circulation Research

Article Title: Effects of Atrial Fibrillation on the Human Ventricle

doi: 10.1161/CIRCRESAHA.121.319718

Figure Lengend Snippet: Molecular remodeling in the atrial fibrillation (AF) ventricle. Original representative Western Blots of human left ventricular (LV) myocardium from aortic stenosis patients with preserved LV function with sinus rhythm (SR, n=6-7) or AF (n=7) and expression levels (normalized to SR) for ( A ) ryanodine receptor type 2 (RyR2), ( B ) RyR2 phosphorylation at Ser2814 (normalized to total RyR2 expression), ( C ) NCX (Na + /Ca 2+ exchanger), ( D ) SERCA (sarcoplasmic reticulum Ca 2+ ATPase 2a), and ( E ) PLB (phospholamban). Representative Western Blots for ( F ) CaMKII (Ca 2+ /calmodulin-dependent protein kinase IIδc), ( G ) CaMKII phosphorylation at Thr287 (CaMKII-P), and ( H ) CaMKII oxidation at Met281/282 (CaMKII-ox). GAPDH was used as loading control. I , CaMKII activity (CycLex CaMKII activity ELISA kit) and ( J ) H 2 O 2 levels (colorimetric peroxidase assay) in LV myocardium from patients with SR or AF (n=6–7 each). Data are provided as scatter plot with mean±SD. Groups were statistically analysed using Student t test or Mann-Whitney U test (for E and G ).

Article Snippet: The expression of RYR2 (ryanodine-receptor type 2), NCX (Na + -Ca 2+ exchanger), SERCA2a, PLB (phospholamban), and CaMKII was studied using specific antibodies anti-RyR2 (mouse monoclonal antibody, dilution 1:1000, Santa Cruz Biotechnology), NCX (mouse monoclonal antibody, dilution 1:1000, Swant), SERCA2a (rabbit polyclonal antibody, dilution 1:1000, Alomone), PLB (mouse monoclonal antibody, dilution 1:1000, Thermo Fisher) and CaMKII-δ (rabbit polyclonal antibody, dilution 1:1000, Thermo Fisher).

Techniques: Western Blot, Expressing, Activity Assay, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY

Journal: The Journal of investigative dermatology

Article Title: Impaired trafficking of the desmoplakins in cultured Darier's disease keratinocytes.

doi: 10.1046/j.1523-1747.2003.12557.x

Figure Lengend Snippet: Figure 6. Dp associates with SERCA2 during di¡erentiation. (A) NHK cells grown in low-calcium medium were pulse-labeled with [35S]methionine and cysteine and then chased in a normal-calcium med- ium for 4 h. Cells were lyzed and immunoprecipitated with the indicated antibody. (B) NHK cells were incubated in normal-calcium medium for 4 h to induce cell di¡erentiation. Cells were lyzed and immunoprecipitated with anti-SERCA2 antibody. SERCA2 immunoprecipitates were analyzed by SDS-PAGE and western blot using an anti-Dp antibody (lane 2). Lane 1, Western blot of total cell extract.

Article Snippet: Mouse monoclonal anti-PG antibody was purchased from Cymbus Bioscience (Southampton, UK), goat polyclonal anti-SERCA2 antibody was from Santa Cruz Biotechnology (Insight Biotechnology, Middlesex, UK), and rabbit polyclonal anti-calnexin antibody was obtained from Calbiochem (CN Biosciences, Nottingham, UK).

Techniques: Labeling, Immunoprecipitation, Incubation, SDS Page, Western Blot

Journal: Circulation Research

Article Title: Endothelial S100A1 Modulates Vascular Function via Nitric Oxide

doi: 10.1161/circresaha.108.172031

Figure Lengend Snippet: Figure 6. S100A1 colocalizes with both SERCA2 and the IP3R. A, C, D, and F, Representative immunolabeling of SERCA2 (red) (A), IP3R (red) (D), and S100A1 (green) (C and F). Overlays of A and C (B) and D and F (E) reveal similar intracellular localization of both SERCA2 and IP3R with S100A1 protein. Original magnifi- cation, 60.

Article Snippet: Co- immunofluorescence for S100A1 and sarcoplasmic reticulum (SR) Ca2+-ATPase (SERCA2) Online Supplement Pleger et al., Endothelial S100A1 Modulates Vascular... 2 as well as for S100A1 and the inositol 1,4,5- triphosphate receptor (IP3R) was performed in RCEC using the S100A1 AB as described above and, consecutively, a goat-anti SERCA2 (sc- 8095, 1:100) or a goat-anti IP3R AB (sc-7278, Santa Cruz, CA) followed by consecutive probing with a 488 donkey anti-rabbit and a 555 donkey anti-goat AB’s (Molecular Probes; 1:100).

Techniques: Immunolabeling

Journal: Journal of Molecular Histology

Article Title: LOC102549726/miR-760-3p network is involved in the progression of ISO-induced pathological cardiomyocyte hypertrophy via endoplasmic reticulum stress

doi: 10.1007/s10735-023-10166-1

Figure Lengend Snippet: LOC102549726 promotes CH via ER stress in ISO-treated NRCMs. ( a ) The interfering effect of the three synthesized siRNA-LOC102549726 was evaluated and compared with the NC group by real-time PCR, and Si-435 was selected for subsequent experiments. n = 3 in each group. Values are expressed as mean ± SD. * p < 0.05, vs. NC group, using an ANOVA test. ( b ) Knockdown of LOC102549726 by siRNA reversed the ISO-induced increase in mRNA levels of ANP, ATF 6 induced by ISO in NRCMs. n = 3 in each group. Values are mean ± SD. * p < 0.05, vs. control group; # p < 0.05, vs. ISO + NC group, respectively using Student’s t-test and one way ANOVA test. ( c,d ) Representative H&E staining images and quantitative analyses of myocardial hypertrophy based on cell surface area in the indicated groups. The hypertrophic response was reversed after transfection with si-LOC102549726 as compared to ISO-treated and negative control (NC) transfected groups. The datas are represented as mean ± SD, n = 3 for each group. Scale bar = 100 μm. * p < 0.05 vs. control group; # p < 0.05 vs. ISO + NC group, respectively using Student’s t-test and one way ANOVA test. ( e,f ) Representative immunoblots and quantitative analyses of ATF4, ATF6, eIF2α, and p-eIF2α in the indicated groups. si-LOC102549726 treatment significantly reduced ISO-induced overexpression of ATF6, eIF2α and p-eIF2α in NRCMs. Bars show the mean values ± SD. n = 3, * p < 0.05 vs. control group; # p < 0.05 vs. ISO + NC group, respectively using Student’s t-test and one way ANOVA test. ( g,h ) Representative immunoblots and quantitative analyses of p-IRE, XBP-1, HERG, SERCA2 and BNP in the indicated groups. si-LOC102549726 treatment enhanced the expression levels of HERG and SERCA2 than those in the control group, whereas the levels of p-IRE, XBP-1 and BNP reduced. Bars show the mean values ± SD. n = 3, * p < 0.05 vs. control group; # p < 0.05 vs. ISO + NC group, respectively using Student’s t-test and one way ANOVA test

Article Snippet: The membranes were blocked with 5% skimmed milk in Tris-buffered saline Tween (TBST) for 1 h at room temperature and then incubated with the following primary antibodies; Rabbit anti rat ATF6 (1:1000; cat. no. ab37149; Abcam), Rabbit Anti rat p-IRE1 (1:1000; cat. no.14C10; CST), Rabbit Anti rat p-eIF2α (1:1000; cat. no. #9721; CST), Rabbit Anti rat XBP-1 (1:1000; cat. no. abs115725; absin), Rabbit Anti rat ATF4 (1:1000; cat. no. abs135528; absin), Rabbit Anti rat HERG (1:1000; cat. no. APC-062; alomone labs), and Rabbit Anti rat SERCA2 (1:1000; cat. no. 4388; CST) overnight at 4 °C.

Techniques: Synthesized, Real-time Polymerase Chain Reaction, Knockdown, Control, Staining, Transfection, Negative Control, Western Blot, Over Expression, Expressing

Journal: Journal of Molecular Histology

Article Title: LOC102549726/miR-760-3p network is involved in the progression of ISO-induced pathological cardiomyocyte hypertrophy via endoplasmic reticulum stress

doi: 10.1007/s10735-023-10166-1

Figure Lengend Snippet: Roles of miR-760-3p in mediating the anti-hypertrophic effect in ISO-induced cardiomyocyte hypertrophy via ER stress. ( a ) The miRNA relative expression level of synthesized miR-760-3p mimic and miR-760-3p inhibitor were verified using qPCR. n = 3 in each group. Values are mean ± SD. * p < 0.05, vs. NC group, using an ANOVA test. ( b,c ) Representative H&E staining images of cardiomyocytes and quantitative analyses of myocardial hypertrophy based on cell surface area in the indicated groups. Transfection with miR-760-3p inhibitor resulted in larger cell size than NC transfection, whereas miR-760-3p mimic significantly decreased cell surface area in NRCMs. The datas are represented as mean ± SD, n = 3 for each group. Scale bar = 100 μm. * p < 0.05 vs. control group; # p < 0.05 vs. ISO + NC group, respectively using Student’s t-test and one way ANOVA test. ( d-f ) Representative immunoblots and quantitative analyses of ATF4, ATF6, eIF2α, p-eIF2α, p-IRE, XBP-1, HERG, SERCA2 and BNP in NRCMs transfected with miR-760-3p mimic and inhibitor upon ISO treatment in the indicated groups. MiR-760-3p inhibitor enhanced the expression of ATF4, ATF6, eIF2α, p-eIF2α, p-IRE, XBP-1 and BNP, while simultaneously reducing p-eIF2α, p-eIF2α/eIF2α ratio, HERG, and SERCA2. Conversely, transfection with miR-760-3p mimic showed the opposite effect. Bars show the mean values ± SD. n = 3, *p < 0.05 vs. control group; # p < 0.05 vs. ISO + NC group, respectively using Student’s t-test and one way ANOVA test

Article Snippet: The membranes were blocked with 5% skimmed milk in Tris-buffered saline Tween (TBST) for 1 h at room temperature and then incubated with the following primary antibodies; Rabbit anti rat ATF6 (1:1000; cat. no. ab37149; Abcam), Rabbit Anti rat p-IRE1 (1:1000; cat. no.14C10; CST), Rabbit Anti rat p-eIF2α (1:1000; cat. no. #9721; CST), Rabbit Anti rat XBP-1 (1:1000; cat. no. abs115725; absin), Rabbit Anti rat ATF4 (1:1000; cat. no. abs135528; absin), Rabbit Anti rat HERG (1:1000; cat. no. APC-062; alomone labs), and Rabbit Anti rat SERCA2 (1:1000; cat. no. 4388; CST) overnight at 4 °C.

Techniques: Expressing, Synthesized, Staining, Transfection, Control, Western Blot

Journal: PLoS ONE

Article Title: Low Density Lipoproteins Promote Unstable Calcium Handling Accompanied by Reduced SERCA2 and Connexin-40 Expression in Cardiomyocytes

doi: 10.1371/journal.pone.0058128

Figure Lengend Snippet: Real-time PCR analysis showing SERCA2 (A), RyR2 (B), IP3-RI (C) and IP3-RIII mRNA expression levels in HL-1 cardiomyocytes exposed to increasing doses of LDL. Data were processed with a specially designed software programme based on Ct value of each sample and normalized to ARBP mRNA. Values significantly different from the level with 0 µg/ml LDL are indicated with asterisks.

Article Snippet: Blots were incubated with monoclonal antibodies against SERCA2 (Novus Biologicals NB100–237), Cx40 (Invitrogen, 36–4900) and Cx43 (Sigma-Aldrich C6219).

Techniques: Real-time Polymerase Chain Reaction, Expressing, Software

Journal: PLoS ONE

Article Title: Low Density Lipoproteins Promote Unstable Calcium Handling Accompanied by Reduced SERCA2 and Connexin-40 Expression in Cardiomyocytes

doi: 10.1371/journal.pone.0058128

Figure Lengend Snippet: Representative Western blot analysis showing SERCA2 bands in HL-1 cells exposed to increasing LDL concentrations (0, 50, 100 and 200 µg/mL) ( A ) or to similar dose (500 µg/ml) of LDL or HDL ( B ) for 24 hours. The bar graph below shows SERCA2 band quantification. Unchanged levels of β-tubulin are shown as loading control. Results are mean±SEM of three independent experiments performed in duplicate. *P<0.05 versus. HL-1 cells incubated in absence of LDL. C Calcium images acquired at rest before (CON) and during a rapid caffeine application. D Representative calcium transients recorded in cultures incubated without lipoproteins (CON), with 500 µg/ml LDL or with 500 µg/ml HDL. The lower panel shows the corresponding time integrals of the calcium transients. E summary of the effect of LDL and HDL on the time integral of the caffeine induced calcium transient. *P<0.05 vs. cells incubated with LDL

Article Snippet: Blots were incubated with monoclonal antibodies against SERCA2 (Novus Biologicals NB100–237), Cx40 (Invitrogen, 36–4900) and Cx43 (Sigma-Aldrich C6219).

Techniques: Western Blot, Control, Incubation