SERCA2 phosphorylation at serine 663 is a key regulator of Ca2+ homeostasis in heart diseases

Figures

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  Fig. 1. GSK3β-dependent phosphorylation of SERCA2 at serine 663 in human and mouse hearts. A Representative curves of normalized [Ca2+]ER ratio signals over time measured with the genetically encoded Ca2+ probe D1ER in HEK293-T cells in Ca2+ free and 1 mM EGTA-containing buffer for three minutes before depleting the ER Ca2+ store by adding 100 µM cyclopiazonic acid (CPA) and 500 µM ATP-Na2+ for seven minutes and prior to ER Ca2+ refilling by switching for six minutes and a half to 2 mM Ca2+, 500 µM ATP-Mg2+ and 1 µM ionomycin-containing buffer (green curve), or supplemented with 10 µM TDZD8 (orange curve), or 1 µM thapsigargin (gray curve) to compare SERCA activity. Ratio signals were normalized as following: [1+ (R − Rt=600s)/(Rt=180s. − Rt=600s)]. B Violin plot of ER Ca2+ refilling slope over two minutes from time 630 s of control (n = 199, on 11 experimental days), TDZD8 (n = 210, on 7 experimental days), thapsigargin (n = 42, on 2 experimental days) and 1 µM SB216763 (N = 212 cells, on 6 experimental days). Median with a 95% confidence interval is shown and one-way ANOVA followed by Tukey’s multiple comparisons test was used to assess significance versus Control condition (****p <…

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  Fig. 2. Phosphorylation of SERCA2 at serine 663 regulates SERCA2 activity. A Representative curves of normalized [Ca2+]ER ratio signals over time measured with the genetically encoded Ca2+ probe D1ER in HEK293-T wild-type (green curve), Serca2[S663A] phosphoresistant mutant (red curve) and Serca2[S663A] phosphoresistant mutant supplemented with 10 µM TDZD8 for ER Ca2+ refilling (dashed orange curve). Ratio signals were normalized as following: [1 + (R − Rt=600s)/(Rt=180sec. − Rt=600s)]. B Violin plot of the ER Ca2+ refilling slope over two minutes from time 630 s of WT (N = 108 cells on 4 experimental days), Serca2[S663A] (N = 108 cells on 9 experimental days) and Serca2[S663A] + TDZD8 (N = 100 cells on 4 experimental days) respectively. Median with a 95% confidence interval is shown and one-way ANOVA followed by Tukey’s multiple comparisons test was used to assess significance (****p < 0.0001) (ns p = 0.3953, ****p < 0.0001). C Violin plot of basal [Ca2+]ER (endoplasmic reticulum) ratio measured with D1ER probe in HEK293-T wild-type (WT) (N = 117) and Serca2[S663A] phosphoresistant mutant cells (N = 108). Median with distribution is shown and two-tailed unpaired t test with Welch’s…

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  Fig. 3. Phosphoresistance of SERCA2 at S663 confers in vitro protection against H/R injury. A Cell death evaluated by flow cytometry with propidium iodide (PI) in normoxic and hypoxic WT (N = 11) and Serca2[S663A] (N = 12) groups. Cyclosporin A (CsA) (N = 11) was used as a positive control. Median with a 95% confidence interval from 4 independent days is shown and two-way ANOVA followed by Tukey’s multiple comparisons test versus WT condition was used to assess significance (ns p ≥ 0.05, *p = 0.0119, ***p = 0.004. B Kinetic curves of [Ca2+]ER (endoplasmic reticulum) ratio measured with D1ER probe at 0.5 h/1 h/2 h reoxygenation of HEK293-T wild-type (WT) (dotted green curve, N = 103/86/89 for respective reperfusion time, on 4 experimental days) and Serca2[S663A] phosphoresistant mutant (continuous red curve, N = 90/95/95 for respective reperfusion time, on 4 experimental days) cells exposed to 18 h hypoxia (1%). Mean with a 95% confidence interval is shown and two-tailed unpaired t test with Welch’s correction was used to assess significance (****p < 0.0001). C Kinetic curves of [Ca2+]CYTO (cytosol) ratio measured with D3cpV probe at 0.5 h/1 h/2 h reoxygenation of HEK293-T wild-type (WT)…

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  Fig. 4. S663A phosphorylation modulates Ca2+ signaling and function in mouse cardiomyocytes. A Typical curves of [Ca2+]CYTO (cytosol) F/F0 ratio signals over time measured with Fluo-4, in rWT (green curve), rS663A (red curve) and rS663E (blue curve) rescued cardiomyocytes, in 2 mM Ca2+ electro-stimulation buffer for 30 s, followed by 20 s electro-stimulation at 1.0 Hz and 40 V, and prior to 5 mM caffeine stimulation at time 60 s. B Violin plots of the transient amplitude during electro-stimulation in rWT (N = 184 cells on 5 experimental days), rS663A (N = 253 cells on 5 experimental days) and rS663E (N = 260 cells on 6 experimental days). Median with interquartile range is shown and one-way ANOVA followed by Tukey’s multiple comparisons test was used to assess significance (ns p = 0.0666, **p = 0.0014, ****p < 0.0001). C Violin plots of the [Ca2+] maximal increase in rWT (N = 214 on 5 experimental days), rS663A (N = 164 cells on 5 experimental days) and rS663E (N = 257 cells on 6 experimental days). Median with interquartile range is shown and one-way ANOVA followed by Tukey’s multiple comparisons test was used to assess significance (ns p = 0.9759, *p = 0.0241, **p = 0.0063). D Violin…

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  Fig. 5. Phosphorylation state of SERCA2 at S663 regulates in vivo myocardial infarct size. A Experimental design for in vivo gene therapy experiments with heart transverse section after TTC staining. Scale bar represents 500 μm. B Quantification of the area at risk (AR) expressed as percentage of left ventricle (LV) of SERCA2-KD mice rescued with SERCA2 -WT (N = 13), -S663A (N = 11) or -S663E (N = 14). Mean with a 95% confidence interval is shown and one-way ANOVA followed by Tukey’s multiple comparisons test was used to assess significance (ns p ≥ 0.05). C Scatterplot of AN over the AR of mice rescued with SERCA2 -WT (N = 13), -S663A (N = 11) or -S663E (N = 14). D Quantification of infarct size (AN) expressed as percentage of AR of mice rescued with SERCA2 -WT (N = 13), -S663A (N = 11) or -S663E (N = 14). Mean with a 95% confidence interval is shown and one-way ANOVA followed by Tukey’s multiple comparisons test was used to assess significance (*p = 0.0292 rWT vs rS663A, *p = 0.0144 rWT vs rS663E, ****p < 0.0001). E Dot plot of fitted linear regression slope of AN/AR of rWT (N = 13), rS663A (N = 11) or rS663E (N = 14) SERCA2 rescued mice. Mean with a 95% confidence interval is shown and…

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  Fig. 6. Mechanistic role of SERCA2 phosphorylation at S663 in ischemic and healthy/protected hearts. In healthy heart, during excitation-contraction coupling (ECC) induced after depolarization of the sarcolemma, a small amount of Ca2+ enters to the sarcoplasm through the voltage sensitive L-type calcium dihydropyridine channels (LTCC) of the T-tubule, targeting a rapid and large amount of Ca2+ release (systolic Ca2+) from inside the sarcoplasmic reticulum (SR/ER) through the Ca2+ ryanodine channels (RyR), which subsequently activates myofilaments (MF) for muscle contraction. Relaxation occurs when SR Ca2+ ATPase (SERCA2) reuptakes Ca2+, which is regulated by the balance of the phosphorylation level of SERCA2 at serine 663 (S663) via the GSK3β, lowering cytosolic Ca2+ concentration in combination with Ca2+ extrusion via Na+–Ca2+ exchanger (NCX) working in reverse mode. Mitochondria (Mito) also participate taking and extruding Ca2+ from the cytosol during Ca2+ cycle. During ischemic heart disease, the level of SERCA2 phosphorylation at S663 is increased, reducing the reuptake of Ca2+ into SR/ER during the relaxation phase, which contributes to the increase of both intracellular (Ca2+ i)…