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 one-way ANOVA followed by Tukey’s multiple comparisons test was used to assess significance (*p = 0.0272 rWT vs rS663A, *p = 0.0136 rWT vs rS663E, ****p < 0.0001). F–I Evaluation of cardioprotective signaling pathways in lysates of cardiac area at risks. F Quantification of western blotting for Phospho-ERK over total ERK1-2 in rWT (N = 11), rS663A (N = 11) and rS663E (N = 11) SERCA2 rescued mice. Mean with a 95% confidence interval, from n = 3 independent experiments, is shown and one-way ANOVA followed by Tukey’s multiple comparisons test was used to assess significance (ns p > 0.05). G Quantification of western blotting for phospho-STAT3 over total STAT3 in rWT (N = 8), rS663A (N = 8) and rS663E (N = 8) SERCA2 rescued mice. Mean with a 95% confidence interval, from n = 3 independent experiments, is shown and one-way ANOVA followed by Tukey’s multiple comparisons test was used to assess significance (ns p > 0.05) H Quantification of western blotting for phospho S16-T17-PLN over total PLN, expressed as fold of rWT. Mean with a 95% confidence interval, from n = 3 independent experiments, is shown and one-way ANOVA followed by Tukey’s multiple comparisons test was used to assess significance (ns p = 0.8195, *p = 0.0102 rS663A vs rS663E, *p = 0.0424 rWT vs rS663E). I Quantification of the interaction of PLN with SERCA2 determined by co-immunoprecipitation on lysates from cardiac area at risks in rWT (N = 6), rS663A (N = 6) and rS663E (N = 6) SERCA2 rescued mice. Mean with a 95% confidence interval, from n = 3 independent experiments, is shown and one-way ANOVA followed by Tukey’s multiple comparisons test was used to assess significance (ns p > 0.05). J–L Evaluation of the functions of the mouse cardiomyocytes isolated from the AR after the in vivo ischemia-reperfusion insult. J Cell death evaluated by flow cytometry with propidium iodide (PI) in rWT (N = 7) and rS663A (N = 7) SERCA2 rescued mice. Median with interquartile interval is shown and two-tailed unpaired t test with Welch’s correction was used to assess significance (*p = 0.0104). K Mitochondrial membrane potential evaluated with TMRM in rWT (N = 7) and rS663A (N = 8) SERCA2 rescued mice. Median with interquartile interval is shown and two-tailed unpaired t test with Welch’s correction was used to assess significance (***p = 0.0002). L Resting cytosol Ca²⁺ evaluated with the ratiometric FuraRed sensor in rWT (N = 6) and rS663A (N = 8) SERCA2 rescued mice. Median with interquartile interval is shown and two-tailed unpaired t test with Welch’s correction was used to assess significance (*p = 0.0105). M–P Quantification of sarcomere shortening in mouse cardiomyocytes isolated from the AR after the in vivo ischemia-reperfusion insult measured with the software IonWizard; Ionoptix system. M Evaluation of peak h in µm in rWT (N = 135) and rS663A (N = 245) SERCA2 rescued cardiomyocytes. Median with interquartile interval is shown and two-tailed unpaired t test with Welch’s correction was used to assess significance (****p < 0.0001). N Evaluation of time to peak 50% in rWT (N = 135) and rS663A (N = 245) SERCA2 rescued cardiomyocytes. Median with interquartile interval is shown and two-tailed unpaired t test with Welch’s correction was used to assess significance (****p < 0.0001). O Evaluation of time to baseline 50% in rWT (N = 133) and rS663A (N = 244) SERCA2 rescued cardiomyocytes. Median with interquartile interval is shown and two-tailed unpaired t test with Welch’s correction was used to assess significance (***p = 0.0003). P Evaluation of area under curve in rWT (N = 135) and rS663A (N = 242) SERCA2 rescued cardiomyocytes. Median with interquartile interval is shown and two-tailed unpaired t test with Welch’s correction was used to assess significance (****p < 0.0001).