Corin Overexpression Reduces Myocardial Infarct Size and Modulates Cardiomyocyte Apoptotic Cell Death

Figures

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  Figure 1. Cardiac-specific overexpression of corin decreases infarct size 24 h post-MI. (A) Area at risk for ischemia (AAR; Evans blue staining) and infarct area [IFA; 1% 2,3,5-triphenyl-2H-tetrazolium chloride (TTC)] in representative heart sections from wild-type (WT) & corin-Tg mice 24 h post-MI. (B) AAR and IFA were measured in heart sections using Image-Pro Plus software and are shown as the ratio of IFA to AAR. Data represent means ± SE of n = 7 mice per group. (C) Comparison of the IFA and the area of low corin expression in coronal heart sections. Representative images with H&E staining (top panel) and corin (green) & WGA (red) double-immunofluorescence (IF) staining (DAPI, blue, bottom panel), bar = 100 µm. The IFA was differentiated from non-infarct area by the characteristic eosinophilic staining (highlighted with a dotted line) on H&E sections. In contrast to the WT post-MI heart, there was residual myocardial corin expression (indicated by yellow arrows) in the ischemic area of corin-Tg hearts post-MI (IF sections). (D) The IFA (eosinophilic area) and total left ventricular area (LVA) of myocardium were measured using Image-Pro Plus software in H&E stained sections of each…

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  Figure 2. Cardiac-specific overexpression of corin affects heart function in both early and late phase post-MI. (A) Dynamic changes in ejection fraction (EF%) (assessed by echocardiography), (B) lung weight to body weight ratio (LW/BW%), (C) systemic extracellular water (edema) assessed by quantitative magnetic resonance and (D) heart weight to body weight ratio (HW/BW%) in mouse groups of WT vs. corin-Tg post-MI. Portions of data from panels A, B, and D (WT group at non-MI, 3 h, 24 h, and 3 days) have been published previously [19] and are included here to reduce animal numbers. Differences between WT and corin-Tg at each study time point were analyzed by 2-way ANOVA with Sidak’s multiple comparisons. Data represent means ± SE of n = 3–34 mice per group at each time point. * p < 0.05, ** p < 0.01, **** p < 0.0001 and not significant.

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  Figure 3. Cardiac-specific overexpression of corin attenuates cardiomyocyte apoptosis 24 h post-MI. (A) Representative images of TUNEL staining (green) with troponin (red, cardiac marker) and DAPI-stained nuclei (blue) in the ischemic area of left ventricles (LV) from WT and corin-Tg mouse hearts post-MI (40× magnification). (B,C) Quantitative digital analysis of TUNEL staining. The total numbers TUNEL-stained cells (TUNEL+) and DAPI-stained nuclei (total cell) in the LV were counted; the total left ventricular area (LVA) was measured at 5× using Image-Pro Plus software. The ratios of TUNEL+ cells per myocardial area (mm2) and the percentages of TUNEL+ cells vs. total cells were calculated. Data represent means ± SE of n = 4 mice per group. (D,F,H) Western blot analysis of tissue lysates, prepared using the infarct core and border zone myocardium from corin-Tg-MI (n = 3) and WT-MI (n = 4) hearts, with antibodies for cleaved (c) caspase 3 (17 kD), caspase 3 (34 kD) c-caspase 9 (37, 39 kD), caspase 9 (37 kD), c-caspase 8 (18 kD), and caspase 8 (55 kD) under reducing conditions. Actin (43 kD) was used as loading control. (E,G,I) Bar graphs represent the densitometry analysis of c-caspase 3, 9…

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  Figure 4. Cardiac corin overexpression modulates activity and expression level of pro-apoptotic and anti-apoptotic proteins in the infarcted heart. (A) Expression and phosphorylation of Akt (Ser473) and Bad (Ser136) were assessed by Western-blotting under reduced conditions in post-MI tissues from corin-Tg and WT hearts. Actin was used as loading control. (B,C) Densitometry analysis of the pAkt (60 kD) and pBad (23 kD) normalized to Akt (60 kD) and Bad (25 kD), respectively. (D) Expression of Bcl-2 family proteins, Bcl2, Bax, Bcl-xl, and Bak in corin-Tg and WT hearts post-MI. Cardiac corin (206 kD) overexpression was also confirmed. (E) Densitometry analysis of Bcl2 (26 kD), Bax (23 kD), Bcl-xl (30 kD), and Bak (30 kD) expression normalized to actin (43 kD) and, of the Bax/Bcl2 ratio. Heart tissue extracts were prepared as described in Figure 3. Data represent means ± SE of n = 3-4 mice per group. * p < 0.05, ** p < 0.01, *** p < 0.001 and ns, not significant.

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  Figure 5. Corin overexpression regulates expression of the Bcl-2 family of proteins expression in non-MI hearts. (A) Expression of Akt and Bad, as well as Akt phosphorylation (Ser473) and Bad phosphorylation (Ser136) were assessed by Western-blot under reduced conditions in corin-Tg and WT hearts. Actin (43 kD) was used as a loading control. (B,C) Densitometry analysis of the pAkt (60 kD) and pBad (23 kD) normalized to Akt (60 kD) and Bad (25 kD), respectively. (D) Expression of Bcl-2 family proteins Bcl2, Bax, Bcl-xl, and Bak in corin-Tg and WT mouse hearts. Corin (206 kD) overexpression was also confirmed. (E) Densitometry analysis of Bcl2 (26 kD), Bax (23 kD), Bcl-xl (30 kD), and Bak (30 kD) expression normalized to actin (43 kD) and of the Bax/Bcl2 ratio. Heart tissue extracts were prepared as in Figure 3 from WT and corin-Tg mouse hearts; the same antibodies were used for detection as in Figure 4. Data represent means ± SE of n = 4 mice per group. * p < 0.05 and ns, not significant.