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: Circulation Research

Article Title: Phosphodiesterase 4D Regulates Baseline Sarcoplasmic Reticulum Ca 2+ Release and Cardiac Contractility, Independently of L-Type Ca 2+ Current

doi: 10.1161/circresaha.111.250464

Figure Lengend Snippet: Figure 4. PDE4D interactions with SERCA2a in murine and human myocardium. A, Rep- resentative Western blots (repeated in 3 separate hearts) probing with PDE4D in heart lysates from a PDE4D/ and WT mouse heart homogenates that were immunoprecipitated (IP) using control IgG or using RyR2- or SERCA2a-specific antibodies. Results show that PDE4D antibodies recognized bands at molecular weights of 97 kDa (corresponding to PDE4D-3, -7, and -9 splice variants) in immunoprecipita- tion reactions with anti- SERCA2a antibodies, but not with anti-RYR2 antibodies or with IgG controls. A very weak nonspecific band having a molecular weight of 110 kDa was detected in all immuno- precipitation groups, as well as in homogenates from the PDE4D-null mice. B, Repre- sentative inputs for immuno- precipitation reactions shown in A. C, Results of SERCA2a immunoprecipitation in human hearts. D, Diagram summariz- ing implications of our results in WT (left) and PDE4D/

Article Snippet: Membranes were incubated with rabbit polyclonal SERCA2a (1:2000, Abcam, Cambridge, MA, USA) and RyR2 34C antibodies (1:5000).

Techniques: Western Blot, Immunoprecipitation, Control, Molecular Weight

Journal: Life Metabolism

Article Title: Impaired SERCA2a phosphorylation causes diabetic cardiomyopathy through impinging on cardiac contractility and precursor protein processing

doi: 10.1093/lifemeta/loac013

Figure Lengend Snippet: Effects of a WD on cardiac function and Ca 2+ homeostasis in primary cardiomyocytes. (a) Body weights of male C57/Bl6J mice fed with a CD or a WD. n = 9−12. (b) Glucose tolerance test of male C57/Bl6J mice at 24 weeks of age. The mice were fed with WD for 16 weeks. The values show the glucose area under the curve during the glucose tolerance test. AUC, area under the curve. n = 7−8. (c) Expression and phosphorylation of SERCA2a, SPEG, AS160, PKB, and IRβ in primary cardiomyocytes isolated from CD- or WD-fed male mice (10-month-old, fasted overnight). The mice were fed with WD for 32 weeks. Primary cardiomyocytes were stimulated with or without insulin (300 nM, 30 min) before lysed for immunoblotting analyses. (d and e) Ca 2+ transients elicited by electrical stimulation (0.5 Hz) in primary cardiomyocytes isolated from the CD- or WD-fed male mice (7-month-old, fed ad libitum). The mice were fed with WD for 20 weeks. (d) Representative Ca 2+ transient images and curves. (e) Amplitudes, FDHM and time constant Tau of Ca 2+ transients. Sixty cells from 2 CD-fed mice and 43 cells from 2 WD-fed mice were analyzed. (f and g) EF (f) and FS (g) measured via echocardiography in CD- or WD-fed C57/Bl6J male mice (WD feeding started at the age of 2-month). n = 6–12. The data are given as the mean ± SEM. Statistical analyses were carried out using Student’s t -test. * indicates P < 0.05, ** P < 0.01, and *** P < 0.001.

Article Snippet: The total SERCA2a antibody (Cat No. 13985-1-AP), total FURIN antibody (Cat No. 18413-AP), and GAPDH (Cat No. 60004-1-Ig) antibody were from Proteintech (Wuhan, China).

Techniques: Expressing, Phospho-proteomics, Isolation, Western Blot

Journal: Life Metabolism

Article Title: Impaired SERCA2a phosphorylation causes diabetic cardiomyopathy through impinging on cardiac contractility and precursor protein processing

doi: 10.1093/lifemeta/loac013

Figure Lengend Snippet: Ca 2+ homeostasis in cardiomyocytes of the SERCA2a T484A mice. (a) Diagrammatic illustration of the WT and T484A knockin allele of SERCA2a. The Thr 484 residue on SERCA2a was changed to alanine via CRISPR/Cas9-assisted knockin substitution to generate SERCA2a T484A knockin mice. (b) SERCA2a-Thr 484 phosphorylation in cardiomyocytes of the WT and SERCA2a T484A female mice (2-month-old, fed ad libitum). (c and d) Ca 2+ transients elicited by electrical stimulation in primary cardiomyocytes isolated from the WT and SERCA2a T484A mice (3-month-old, male and female, fed ad libitum). (c) Representative Ca 2+ transient images and curves. (d) Quantification of amplitudes, FDHM and Tau of Ca 2+ transients. Seventy cells from 3 WT mice and 57 cells from 3 SERCA2a T484A mice were analyzed. (e and f) Ca 2+ transients elicited by electrical stimulation in primary cardiomyocytes isolated from the WT and SERCA2a T484A mice (13-month-old, male, fed ad libitum). (e) Representative Ca 2+ transient images and curves. (f) Quantification of amplitudes, FDHM and Tau of Ca 2+ transients. Eighty-eight cells from 2 WT mice and 71 cells from 2 SERCA2a T484A mice were analyzed. (g) Expression of Rcan1.4 mRNA in primary cardiomyocytes from the WT and SERCA2a T484A mice (2-month-old, male). n = 6. (h and i) Ca 2+ sparks in primary cardiomyocytes isolated from the WT and SERCA2a T484A mice (17-month-old, male, fed ad libitum). (h) Quantification of Ca 2+ spark frequency. Eighteen cells from 1 WT mice and 15 cells from 1 knockin mice were analyzed. (i) Representative images of Ca 2+ sparks. (j) SR Ca 2+ load in primary cardiomyocytes isolated from the WT and SERCA2a T484A mice (4-month-old, female, fed ad libitum). Thirty cells from 5 WT mice and 35 cells from 6 knockin mice were analyzed. The data are given as the mean ± SEM. Statistical analyses were carried out using Student’s t -test. ** indicates P < 0.01 and *** P < 0.001.

Article Snippet: The total SERCA2a antibody (Cat No. 13985-1-AP), total FURIN antibody (Cat No. 18413-AP), and GAPDH (Cat No. 60004-1-Ig) antibody were from Proteintech (Wuhan, China).

Techniques: Knock-In, Residue, CRISPR, Phospho-proteomics, Isolation, Expressing

Journal: Life Metabolism

Article Title: Impaired SERCA2a phosphorylation causes diabetic cardiomyopathy through impinging on cardiac contractility and precursor protein processing

doi: 10.1093/lifemeta/loac013

Figure Lengend Snippet: Insulin signaling in primary cardiomyocytes upon SERCA2a inhibition. (a) Insulin signaling in primary cardiomyocytes isolated from the neonatal WT or SERCA2a T484A knockin mice (male and female). Cardiomyocytes were stimulated with or without insulin (300 nM, 30 min). Phosphorylation of PKB and GSK3, and PAS-reactive phosphorylation were detected via immunoblotting using GAPDH as a loading control. (b) Insulin signaling in primary cardiomyocytes isolated from the adult WT or SERCA2a T484A knockin mice (15-month-old, male, fasted overnight). Cardiomyocytes were stimulated with or without insulin (300 nM, 30 min). Phosphorylation of PKB and PAS-reactive phosphorylation were detected via immunoblotting using FLOT1 as a loading control. (c) Insulin signaling in primary cardiomyocytes isolated from the adult WT or SERCA2a +/− mice (3-month-old, male, fasted overnight). Cardiomyocytes were stimulated with or without insulin (300 nM, 30 min). Phosphorylation of PKB and GSK3, and PAS-reactive phosphorylation were detected via immunoblotting using FLOT1 as a loading control. (d) Insulin signaling in control or TG-pretreated NRVCs. NRVCs were pretreated with or without 2 μM TG for 24 h, and then stimulated with or without insulin (300 nM) for 30 min. Phosphorylation of PKB and GSK3, and PAS-reactive phosphorylation were detected via immunoblotting using GAPDH as a loading control.

Article Snippet: The total SERCA2a antibody (Cat No. 13985-1-AP), total FURIN antibody (Cat No. 18413-AP), and GAPDH (Cat No. 60004-1-Ig) antibody were from Proteintech (Wuhan, China).

Techniques: Inhibition, Isolation, Knock-In, Phospho-proteomics, Western Blot, Control

Journal: Life Metabolism

Article Title: Impaired SERCA2a phosphorylation causes diabetic cardiomyopathy through impinging on cardiac contractility and precursor protein processing

doi: 10.1093/lifemeta/loac013

Figure Lengend Snippet: FURIN-dependent protein precursor processing in cells with impaired Ca 2+ homeostasis. (a, b) Expression of FURIN, IRβ, and IGF1Rβ in primary cardiomyocytes of the WT and SERCA2a T484A mice (2-month-old, male). GAPDH was used as a loading control. (a) Immunoblots. (b) Quantification of immunoblot signals. n = 6. (c and d) Expression of FURIN, IRβ, and IGF1Rβ in primary cardiomyocytes of the WT and SERCA2a +/− mice (3-month-old, male). GAPDH was used as a loading control. (c) Immunoblots. (d) Quantification of immunoblot signals. n = 6. (e and f) Expression of FURIN and IRβ in the heart of CD- or WD-fed mice (12-month-old, male). The mice were fed with WD for 40 weeks. (e) Representative immunoblots. (f) Quantification of immunoblot signals. n = 4. (g, h) Expression of FURIN, precursor IR, IRβ, precursor IGF1R, and IGF1Rβ in NRVCs stimulated with or without TG (2 μM, 24h). (g) Immunoblots. (h) Quantification of immunoblot signals. n = 6. (I, j) IR processing in cells upon treatment with TG. HEK293 cells transfected with Flag-IR were treated with Click-iT TM AHA for the indicated time in the presence or absence of TG (2 μM, 90 min). After immunoprecipitation using the Flag beads, nascent precursor IR, and mature IRβ were reacted with Click-iT TM Protein Reaction Buffer Kit, and detected using HRP-labeled Avidin antibody. (i) Representative blots. (j) Quantification of IR processing that was defined as mature IRβ relative to precursor IR. n = 5. (k, l) Effects of FURIN expression on IR and IGF1R processing in TG-treated HEK293 cells. FURIN-mCherry or free mCherry was expressed in HEK293 cells that were stimulated with or without TG (2 μM) for 24 h. FURIN, precursor IR, IRβ, precursor IGF1R, and IGF1Rβ proteins were detected in cell lysates via immunoblotting. (k) Representative blots. (l) Quantitative data. n = 6. † indicates P < 0.001 (TG vector vs Control vector), and ‡ indicates P < 0.001 (TG FURIN-mCherry vs Control FURIN-mCherry) except for precursor IGF1R where ‡ indicates P < 0.05 (TG FURIN-mCherry vs Control FURIN-mCherry). (m, n) FURIN protein levels in HEK293 cells that were treated with the proteosome inhibitor MG-132 (10 μM) or the lysosome inhibitor Bafilomycin A1 (400 nM) in the presence of TG (2 μM) for 4 h. (m) Representative blots. (n) Quantitative data. n = 3. The data are given as the mean ± SEM. Statistical analyses were carried out via Student’s t -test for (b), (d), (f), and (h), via one-way ANOVA for (n), and via two-way ANOVA for (l) and (j). * indicates P < 0.05, ** P < 0.01, and *** P < 0.001.

Article Snippet: The total SERCA2a antibody (Cat No. 13985-1-AP), total FURIN antibody (Cat No. 18413-AP), and GAPDH (Cat No. 60004-1-Ig) antibody were from Proteintech (Wuhan, China).

Techniques: Expressing, Control, Western Blot, Transfection, Immunoprecipitation, Labeling, Avidin-Biotin Assay, Plasmid Preparation

Journal: Life Metabolism

Article Title: Impaired SERCA2a phosphorylation causes diabetic cardiomyopathy through impinging on cardiac contractility and precursor protein processing

doi: 10.1093/lifemeta/loac013

Figure Lengend Snippet: Cardiac function and remodeling of the SERCA2a T484A knockin mice. (a−i) Echocardiography performed on the anaesthetized male SERCA2a T484A knockin mice and WT littermates at the indicated ages. (a) EF. (b) FS. (c) Systolic LV Vol. (d) Diastolic LV Vol. (e) Systolic LVAW. (f) Diastolic LVAW. (g) Systolic LVPW. (h) Diastolic LVPW. (i) Representative images of echocardiography. n = 18–24. (j) Expression of Anp and Bnp mRNA in the heart of WT and SERCA2a T484A knockin mice (4-month-old, male and female). n = 9–13. The data are given as the mean ± SEM. Statistical analyses were carried out using Student’s t -test. * indicates P < 0.05, ** P < 0.01, and *** P < 0.001.

Article Snippet: The total SERCA2a antibody (Cat No. 13985-1-AP), total FURIN antibody (Cat No. 18413-AP), and GAPDH (Cat No. 60004-1-Ig) antibody were from Proteintech (Wuhan, China).

Techniques: Knock-In, Expressing

Journal: Life Metabolism

Article Title: Impaired SERCA2a phosphorylation causes diabetic cardiomyopathy through impinging on cardiac contractility and precursor protein processing

doi: 10.1093/lifemeta/loac013

Figure Lengend Snippet: SERCA2a phosphorylation in the development of WD-induced DCM. (a and b) EF (a) and FS (b) measured via echocardiography in the male WT and SERCA2a T484A mice fed with a WD for periods indicated in the figure. Statistical analyses were performed via Student’s t -test for each time point between the WT and SERCA2a T484A mice. EF: * indicates P < 0.05 (WT/0 M vs SERCA2a T484A /0 M). FS: ** indicates P < 0.01 (WT/0 M vs SERCA2a T484A /0 M). n = 6−9. (c) Ca 2+ transients elicited by electrical stimulation in primary cardiomyocytes isolated from the WT and SERCA2a T484A male mice (ad libitum) fed with WD for 7 months (WD feeding started at the age of 4-month). Amplitudes, FDHM, and Tau of Ca 2+ transients from 59 WT cells (2 mice) and 83 SERCA2a T484A cells (2 mice), respectively. (d) Ca 2+ transients elicited by electrical stimulation in primary cardiomyocytes isolated from the WT and SERCA2a T484A mice (15-month-old, male, ad libitum) fed with CD. Amplitudes, FDHM, and Tau of Ca 2+ transients from 78 WT cells (2 mice) and 70 SERCA2a T484A cells (2 mice), respectively. (e) Phosphorylation and expression of SPEG, AS160, and PKB in primary cardiomyocytes isolated from the WT or SERCA2a T484A mice (15-month-old, male, fasted overnight) fed with a CD or a WD in response to insulin (300 nM, 30 min). (f) A diagram representing the proposed model in which SERCA2a is a critical mediator of insulin action in the heart. Insulin-stimulated SERCA2a-Thr 484 phosphorylation couples FURIN-dependent precursor protein processing with cardiac contraction through regulating SR Ca 2+ re-uptake to maintain cardiac function. Impaired phosphorylation of SERCA2a-Thr 484 inhibits SR Ca 2+ re-uptake and promotes FURIN degradation through lysosomes, which results in impaired processing of IR precursor. Therefore, impaired phosphorylation of SERCA2a-Thr 484 is both a cause and a consequence of cardiac insulin resistance, and underlies the early pathogenesis of DCM induced by WD. The data are given as the mean ± SEM. Statistical analyses were carried out using Student’s t -test. * indicates P < 0.05, and ** P < 0.01.

Article Snippet: The total SERCA2a antibody (Cat No. 13985-1-AP), total FURIN antibody (Cat No. 18413-AP), and GAPDH (Cat No. 60004-1-Ig) antibody were from Proteintech (Wuhan, China).

Techniques: Phospho-proteomics, Isolation, Expressing