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: American Journal of Physiology - Heart and Circulatory Physiology

Article Title: A new model of congestive heart failure in rats

doi: 10.1152/ajpheart.00245.2011

Figure Lengend Snippet: Atrial natriuretic factor (ANF), brain natriuretic peptide (BNP), and sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) 2a expression in remote myocardium. 1: ANF mRNA expression; 2: BNP mRNA expression; 3: SERCA2a protein expression, n = 3 rats in each group.

Article Snippet: Western blotting of myocardial sarco(endo)plasmic reticulum Ca 2+ -ATPase (SERCA) 2a expression levels was performed by using a primary anti-SERCA2a polyclonal antibody (1:3,000; 21st Century Biochemicals) and a secondary antibody conjugated with alkaline phosphatase (1:10,000; Sigma).

Techniques: Expressing

Journal: Cardiovascular research

Article Title: Electrical remodelling precedes heart failure in an endothelin-1-induced model of cardiomyopathy.

doi: 10.1093/cvr/cvq351

Figure Lengend Snippet: Figure 4 Reduced LV Cx43, Cx40, and Nav1.5 expression in mice as early as 4 weeks after ET-1 induction. mRNA and protein expression of SERCA2a (A and B), Cav1.2 (C and D), Cx45 (E and F), Cx40 (G and H ), Nav1.5 (I and J), and Cx43 (K and L); as well as hET-1 mRNA (M), p-Cx43/total Cx43 protein (N), IL-6 mRNA (O), and representative western blots of LV of BT and NBT mice within Groups I–III (P). n ¼ 6–8 for qRT–PCR, and n ¼ 3–6 for western blot, for each genotype per group. *P , 0.05, **P , 0.01. SERCA2a, sarcoplasmic reticulum Ca2+– ATPase; Cav1.2, a1C–subunit of the L-type Ca2+ channel; Cx, connexin; Nav1.5, a-subunit of the cardiac Na+ channel; hET-1, human–ET-1; IL-6, interleukin-6.

Article Snippet: Twenty micrograms of protein extract were run on 4–12% SDS–PAGE, transferred to PVDF membrane, blocked in 5% non-fat dry milk in TBS-T (Tris base, NaCl, 0.1% Tween 20, pH 7.6) for 1 h, and incubated overnight at 48C in TBS-T with 5% BSA containing primary rabbit Abs against Cx43 and phospho-Cx43 (p-Cx43) (Ser368; Cell Signaling, Danvers, MA, USA), Cx45 (Invitrogen), ovem ber 13, 2015 D ow nloaded from Cx40 (Millipore, Billerica, MA, USA), Nav1.5 (Sigma-Aldrich), and Cav1.2 (Alomone Laboratories, Jerusalem, Israel), or a primary mouse Ab against SERCA2a (Novus Biologicals, Littleton, CO, USA).

Techniques: Expressing, Western Blot, Quantitative RT-PCR

Journal: JCI insight

Article Title: Cardiac gene therapy treats diabetic cardiomyopathy and lowers blood glucose.

doi: 10.1172/jci.insight.166713

Figure Lengend Snippet: Figure 2. Exogenous cBIN1 normalizes membrane microdomains at t-tubules. (A) Representative transmission electron microscopy images of post- treatment hearts (scale bar: 1 μm) from each group (left). Quantification of the degree of contour of t-tubules (n = 100–101 t-tubules from 16–40 images of 2–3 myocardial sections and 3 hearts from each group). Data are presented as percentage of t-tubules. χ2 test was used to compare t-tubule contour between groups. *** indicates P < 0.001 for db/m + GFP versus db/db + GFP; ††† indicates P < 0.001 for db/db + GFP versus db/db + cBIN1. (B) Western blots of cBIN1, SERCA2a, CaV1.2, RyR2, GLUT4, and IRAP in total cardiac microsome and sucrose-gradient isolated TT/jSR fraction (F4) from each group (n = 5 hearts per group). (C) Representative spinning disc confocal images of posttreatment mouse myocardium with power spectrum analysis of boxed areas, and quantification of SERCA2a peak power density at t-tubules (n = 35–44 cells from 3 hearts per group). Nonparametric Kruskal-Wallis test fol- lowed by Dunn’s test was used for comparison between selected pairs. *, **, *** indicates P < 0.05, 0.01, and 0.001, respectively, for comparison versus db/m + GFP; †, ††, ††† indicates P < 0.05, 0.01, 0.001, respectively, for comparison between db/db + GFP and db/db + cBIN1.

Article Snippet: For V5, SERCA2a, GLUT4, and IRAP labeling, permeabilized and blocked tissue sections or fixed cardiomyocytes were incubated with primary antibodies against V5 (V8317-2MC, Sigma-Aldrich), SERCA2a (ab2861, Abcam), GLUT4 (4670-1725GA, Bio-Rad Laboratories), or IRAP (6918S, Cell Signaling Technology) overnight at 4°C.

Techniques: Membrane, Transmission Assay, Electron Microscopy, Western Blot, Isolation, Comparison

Journal: JCI insight

Article Title: Cardiac gene therapy treats diabetic cardiomyopathy and lowers blood glucose.

doi: 10.1172/jci.insight.166713

Figure Lengend Snippet: Figure 5. AAV9-cBIN1 normalizes cardiac proteomics in diabetic mice. (A) Bar graphs of LFQ LC-MS/MS data (fold changes over control db/m mice) of SERCA2a, RyR2, GLUT4, and IRAP from the db/m mice treated with AAV9-GFP (n = 4 hearts) and db/db mice treated with AAV9-GFP (n = 4 hearts) or cBIN1 (n = 3 hearts). (B) PCA plot of all 3 groups (n = 2 repeats/heart × 3–4 hearts/group) generated based on LFQ LC-MS/MS proteomics. All data are presented as mean ± SEM. One-way ANOVA followed by Bonferroni’s test or Kruskal-Wallis test followed by Dunn’s test was used for comparison between the selected pairs. * indicates P < 0.05 for comparison versus db/m + GFP; † indicates P < 0.05 for comparison between db/db + GFP and db/db + cBIN1.

Article Snippet: For V5, SERCA2a, GLUT4, and IRAP labeling, permeabilized and blocked tissue sections or fixed cardiomyocytes were incubated with primary antibodies against V5 (V8317-2MC, Sigma-Aldrich), SERCA2a (ab2861, Abcam), GLUT4 (4670-1725GA, Bio-Rad Laboratories), or IRAP (6918S, Cell Signaling Technology) overnight at 4°C.

Techniques: Liquid Chromatography with Mass Spectroscopy, Control, Generated, Comparison

Journal: Anatolian Journal of Cardiology

Article Title: Hypoxia-Induced Sarcoplasmic Reticulum Ca 2+ Leak Is Reversed by Ryanodine Receptor Stabilizer JTV-519 in HL-1 Cardiomyocytes

doi: 10.5152/AnatolJCardiol.2022.1223

Figure Lengend Snippet: The effect of SERCA2a blockade with thapsigargin. Representative example of Ca +2 levels recording (fluorescence of Fluo-5N) after adding thapsigargin to the HL-1 cardiomyocytes.

Article Snippet: After blocking, the membranes were washed using Tris-buffered saline with Tween (TBS-T) and then incubated with primary antibody SERCA2a (A010-23S, Badrilla, Leeds, UK, dilution 1:1000), RyR2 (ARR-002, Alomone Labs, Jerusalem, Israel, dilution 1:1000) and β-tubulin (ab6046, Abcam, Cambridge, UK, dilution 1:1000) overnight.

Techniques: Fluorescence

Journal: Anatolian Journal of Cardiology

Article Title: Hypoxia-Induced Sarcoplasmic Reticulum Ca 2+ Leak Is Reversed by Ryanodine Receptor Stabilizer JTV-519 in HL-1 Cardiomyocytes

doi: 10.5152/AnatolJCardiol.2022.1223

Figure Lengend Snippet: The effect of hypoxia and Rycal treatment on RyR2 and SERCA2a gene expression. The effect of 7-day hypoxic and normoxic exposure with Rycals/Vehicle (DMSO) treatment on RyR2 and SERCA2a gene expression. n = 6, * P < .050 for comparison with 1% O 2 , # P < .050 for comparison with Vehicle, $ P < .050 for comparison with 0.3 mM (all 1-way ANOVA with Tukey post hoc test).

Article Snippet: After blocking, the membranes were washed using Tris-buffered saline with Tween (TBS-T) and then incubated with primary antibody SERCA2a (A010-23S, Badrilla, Leeds, UK, dilution 1:1000), RyR2 (ARR-002, Alomone Labs, Jerusalem, Israel, dilution 1:1000) and β-tubulin (ab6046, Abcam, Cambridge, UK, dilution 1:1000) overnight.

Techniques: Expressing

Journal: Anatolian Journal of Cardiology

Article Title: Hypoxia-Induced Sarcoplasmic Reticulum Ca 2+ Leak Is Reversed by Ryanodine Receptor Stabilizer JTV-519 in HL-1 Cardiomyocytes

doi: 10.5152/AnatolJCardiol.2022.1223

Figure Lengend Snippet: The effect of hypoxia and Rycal treatment on RyR2 and SERCA2a protein expression. The effect of 7-day hypoxic and normoxic exposure with Rycals/Vehicle (DMSO) treatment on RyR2 and SERCA2a protein expression. n = 6. No statistically significant comparison.

Article Snippet: After blocking, the membranes were washed using Tris-buffered saline with Tween (TBS-T) and then incubated with primary antibody SERCA2a (A010-23S, Badrilla, Leeds, UK, dilution 1:1000), RyR2 (ARR-002, Alomone Labs, Jerusalem, Israel, dilution 1:1000) and β-tubulin (ab6046, Abcam, Cambridge, UK, dilution 1:1000) overnight.

Techniques: Expressing