Anti-apoptotic peptide for long term cardioprotection in a mouse model of myocardial ischemia–reperfusion injury

Figures

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  Figure 1. Study design and experimental protocol. (A): Study design. Mice (n = 66) were included into the SHAM (surgery placebo; n = 18) or the IR (ischemia–reperfusion, n = 48) sub-groups. In the IR group (with coronary artery ligation), injection of saline buffer (Ctrl) or TD peptide (TD) was blinded to the experimenter by assigning an anonymity number to each mouse. Anonymity was lifted after the completion of all steps of follow-up of the IR group (dotted frame) including data analysis, just before the statistical tests. At this step only, each mouse included in the study was assigned to a study group and the “n” number of mice in each group mentioned in blue was determined. Two mice had to be removed from the study (1 TD and 1 Ctrl) at this step since the quality control applied to the body temperature recordings revealed a slight decrease during myocardial infarction (MI). (B): Experimental and treatment protocols. MI was induced by transient coronary artery ligation and reperfusion. Peptide treatment (red arrow) was administered intravenously 5 min before the onset of reperfusion. Blood samples were collected at 24 h, 1, 2, 3 and 6 months post-surgery to assess cardiac Troponin I…

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  Figure 2. Decreased cTnI levels and evaluation of cohort survival. (A): Cardiac Troponin I (cTnI) levels were measured using the high sensitivity mouse cardiac Troponin-I Elisa on plasmatic fractions of blood samples from SHAM (n = 17), Ctrl (n = 20) and TD (n = 16) mice. Scatter dot blots and mean ± SEM were plotted for cTnI measured at 24 h (peak level). Statistical analysis was performed using ANOVA test with the Tukey’s post hoc test for multiple comparisons at 24 h. *** was noted for p = 0.0009 and **** for p < 0.0001 for TD versus Ctrl and # for p = 0.5527 for TD versus SHAM. (B): Mean values of cTnI levels evaluated at 1, 30, 60, 90 and 180 days during the clinical follow-up were plotted in SHAM (n = 13), Ctrl (n = 17) and TD (n = 13) mice. Statistical analysis was performed using Two-way ANOVA followed by the Tukey’s post hoc test. **** is given for pgroup < 0.0001. (C): Survival curves are presented for SHAM, Ctrl and TD mice. Numbers represent total number of animals that survived (left) relative to those that were included in the study (right). The comparison between the 3 survival curves was performed using the Gehan–Breslow–Wilcoxon test. ns was noted for p = 0.2452.

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  Figure 3. Decreased cardiac dilatation upon TD-treatment. Measurements performed by conventional echocardiography on bi-dimensional images of the parasternal long axis (B-mode) using Vevolab software for LV end-systolic (A,B) and end-diastolic (C,D) cavity dimensions in SHAM (n = 15), Ctrl (n = 17) and TD (n = 12) mice. Graphs showing longitudinal changes for (A): LV end-systolic area (LVES area, mm3) with p** = 0.0012 for SHAM versus Ctrl, p* = 0.0280 for TD versus Ctrl and p# = 0.6366 versus SHAM; (B): for LV end-systolic volume (LVESV, µL) with p** = 0.0012 for SHAM versus Ctrl, p* = 0.0337 for TD versus Ctrl and p# = 0.5868 versus SHAM; (C): for LV end-diastolic area (LVED area, mm3) with pns = 0.0874 for SHAM versus Ctrl, pns = 0.7571 for TD versus Ctrl and p# = 0.4055 versus SHAM; (D): for LV end-diastolic volume (LVEDV, µL) with p* = 0.0364 for SHAM versus Ctrl, pns = 0.5560 for TD versus Ctrl and p# = 0.3835 versus SHAM. Data are presented as mean ± SEM and connecting lines. Statistical analysis for (A,B,C) was performed using the Two-way ANOVA test followed by the Tukey’s post test for repeated measures. Statistical analysis for (D; LVEDV with pinteraction* = 0.0282) was obtained…

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  Figure 4. Decreased heart fibrosis in TD-treated mice. At the end of the study, hearts were harvested in SHAM (n = 17), Ctrl (n = 20) and TD (n = 16) mice and weighted. Scatter dot blots and mean ± SEM were plotted for LV and left atria (LA) weights reported to body weight in (A): LVW/BW with pns = 0.1707 and in (B): LAW/BW with pns = 0.5561. (C): Histology was performed on paraffin-embedded sections from LV harvested at 6 months post-MI. Representative pictures of microscopic observations for Picrosirius red stained sections from SHAM, Ctrl and TD hearts. (D): Fibrosis area (Scatter dot blots and mean ± SEM) was decreased by 54% in TD-treated versus Ctrl LV samples. Statistical analysis was performed using one-way ANOVA with p**** < 0.0001 for SHAM versus Ctrl, p** = 0.0079 for TD versus Ctrl and p# = 0.3795 versus SHAM.

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  Figure 5. Improved cardiac performance in TD-treated mice. Echocardiographic parameters were calculated from end-diastolic and end-systolic LV volumes using Vevolab software in SHAM (n = 15), Ctrl (n = 17) and TD (n = 12) mice. (A): ejection fraction (EF, %) with p**** < 0.0001 for SHAM versus Ctrl, p**** < 0.0001 for TD versus Ctrl and p# = 0.9621 versus SHAM and (B): fractional shortening (FS, %) with p*** = 0.0005 for SHAM versus Ctrl, p*** = 0.0004 for TD versus Ctrl and p# = 0.9466 versus SHAM. Speckle-tracking imaging using Vevostrain software allowed to evaluate (C): the global longitudinal strain (peak average, in %) from a parasternal long axis view in SHAM (n = 14), Ctrl (n = 16) and TD (n = 12) mice with p**** < 0.0001 for SHAM versus Ctrl, p*** = 0.0003 for TD versus Ctrl and p# = 0.9562 versus SHAM; (D): the global circumferential strain (%) from a parasternal short axis view in SHAM (n = 12), Ctrl (n = 16) and TD (n = 10) mice with p** = 0.0096 for SHAM versus Ctrl, pns = 0.2557 for TD versus Ctrl and p# = 0.4163 versus SHAM. STI allowed also to record an automated measurement of ejection fraction (STI-derived EF, %) obtained from endocardial detection in end-diastole and…