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

Figures

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  Figure 1. Effects of a WD on cardiac function and Ca2+ 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) Ca2+ 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 Ca2+ transient images and curves. (e) Amplitudes, FDHM and time constant Tau of Ca2+ 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…

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  Figure 2. Ca2+ homeostasis in cardiomyocytes of the SERCA2aT484A mice. (a) Diagrammatic illustration of the WT and T484A knockin allele of SERCA2a. The Thr484 residue on SERCA2a was changed to alanine via CRISPR/Cas9-assisted knockin substitution to generate SERCA2aT484A knockin mice. (b) SERCA2a-Thr484 phosphorylation in cardiomyocytes of the WT and SERCA2aT484A female mice (2-month-old, fed ad libitum). (c and d) Ca2+ transients elicited by electrical stimulation in primary cardiomyocytes isolated from the WT and SERCA2aT484A mice (3-month-old, male and female, fed ad libitum). (c) Representative Ca2+ transient images and curves. (d) Quantification of amplitudes, FDHM and Tau of Ca2+ transients. Seventy cells from 3 WT mice and 57 cells from 3 SERCA2aT484A mice were analyzed. (e and f) Ca2+ transients elicited by electrical stimulation in primary cardiomyocytes isolated from the WT and SERCA2aT484A mice (13-month-old, male, fed ad libitum). (e) Representative Ca2+ transient images and curves. (f) Quantification of amplitudes, FDHM and Tau of Ca2+ transients. Eighty-eight cells from 2 WT mice and 71 cells from 2 SERCA2aT484A mice were analyzed. (g) Expression of Rcan1.4 mRNA in primary…

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  Figure 3. Insulin signaling in primary cardiomyocytes upon SERCA2a inhibition. (a) Insulin signaling in primary cardiomyocytes isolated from the neonatal WT or SERCA2aT484A 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 SERCA2aT484A 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…

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  Figure 4. FURIN-dependent protein precursor processing in cells with impaired Ca2+ homeostasis. (a, b) Expression of FURIN, IRβ, and IGF1Rβ in primary cardiomyocytes of the WT and SERCA2aT484A 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-iTTM AHA for the indicated time in the presence or absence of TG (2 μM, 90 min). After immunoprecipitation using the Flag beads,…

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  Figure 5. Cardiac function and remodeling of the SERCA2aT484A knockin mice. (a−i) Echocardiography performed on the anaesthetized male SERCA2aT484A 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 SERCA2aT484A 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.

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  Figure 6. 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 SERCA2aT484A 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 SERCA2aT484A mice. EF: * indicates P < 0.05 (WT/0 M vs SERCA2aT484A/0 M). FS: ** indicates P < 0.01 (WT/0 M vs SERCA2aT484A/0 M). n = 6−9. (c) Ca2+ transients elicited by electrical stimulation in primary cardiomyocytes isolated from the WT and SERCA2aT484A male mice (ad libitum) fed with WD for 7 months (WD feeding started at the age of 4-month). Amplitudes, FDHM, and Tau of Ca2+ transients from 59 WT cells (2 mice) and 83 SERCA2aT484A cells (2 mice), respectively. (d) Ca2+ transients elicited by electrical stimulation in primary cardiomyocytes isolated from the WT and SERCA2aT484A mice (15-month-old, male, ad libitum) fed with CD. Amplitudes, FDHM, and Tau of Ca2+ transients from 78 WT cells (2 mice) and 70 SERCA2aT484A cells (2 mice), respectively. (e) Phosphorylation and expression of SPEG, AS160, and PKB in primary cardiomyocytes isolated from the WT or SERCA2aT484A mice…