Figure 1.
Before ischemia, hearts were randomly allocated to 1 of the experimental groups. Sham groups hearts were buffer perfused for a total of 90 or 190-Post was infused during the initial 20 minutes of reperfusion only, inhibitors were infused during the final 5 minutes of stabilization, as indicated by the lines under the bars, and during the initial 20 minutes of reperfusion.
Figure 2.
I/R Injury (infarct size and apoptosis) after 30-min ischemia and 120-min reperfusion.
Infarct size (IS): the amount of necrotic tissue is expressed as percentage of the left ventricle (% IS/LV), which is considered the risk area. Panel A: effects of CST-Post in normotensive (WKY) or hypertensive (SHR) hearts. Panel B: effects of CST-Post in hypertensive (SHR) heart in the presence of antagonists. TUNEL analysis: the apoptotic index of the cardiac muscle is in panel C. Tunel positive cardiomyocyte nuclei are shown by red arrows in panel D (WKY_Sham), panel E (SHR_Sham), panel F (SHR_I/R), and panel G (SHR_CST-Post). Negative control (panel H) is obtained by using the same protocol without TdT enzyme. Immunohistochemical localization of connexin 43 (green arrows) in the ventricular sections of SHR_Sham (panel I), SHR_I/R (panel L), and SHR_CST-Post (panel M). **p<0.01, *p<0.05. Two way ANOVA, (n = 6 for each group).
Figure 3.
Apoptotic factors after 30-min ischemia and 120-min reperfusion.
Representative Western Blots and relative densitometry for ARC (panel A) and for cleaved caspase 3 (panel B). Individual values were compared to β-actin and the mean value of the Sham group was considered as the reference for all groups, including Sham. Immunohistochemical localization of the apoptosis repressor recruitment domain (ARC, white arrows) in the ventricular cardiomyocytes of SHR Sham (D), SHR_I/R (E), SHR_CST-post (F) rat. Immunohistochemical localization of caspase 3 (blue arrows) of WKY (G), SHR_Sham (H), SHR_I/R (I), SHR_CST-post (L) in the ventricular cells. (C) Negative control. *p<0.01. ANOVA followed by Bonferroni’s Multiple comparison Test, (n = 6 for each group).
Table 1.
Pre and post ischemic cardiac function.
Figure 4.
Phospho-eNOS localization after 30-min ischemia and 120-min reperfusion.
Immunolocalization of phospho-eNOS (B–G), in SHR_Sham (B, C), SHR_I/R (D, E), SHR_CST-post (F, G) rat ventricular sections. The enzyme is localized mainly in vascular (yellow arrows) and endocardial endothelium (red arrows). Negative control is shown in A. Nuclei are counterstained with Hoechst, (n = 3 for each group).
Figure 5.
HIF-α expression after 30-min ischemia and 120-min reperfusion.
Representative RT-PCR (panel A) and Western Blots (panel B) and relative densitometry for hypoxia-inducible factor-1α (HIF-1α). Individual values were compared to loading control and the mean value of the Sham group was considered as the reference for all groups, including Sham. Immunohistochemical localization of HIF-1α in the ventricular sections of SHR_Sham (D), SHR_I/R (E), SHR_CST-Post (F) rat. In E the inset shows a detail of HIF-1α labeled myocardiocytes. (C) Negative control. Blue arrows (cardiomyocytes), red arrows (vascular endothelium). *p<0.01. ANOVA followed by Bonferroni’s Multiple comparison test, (n = 6 for each group).
Figure 6.
RISK pathway activation after 30 min ischemia and 20 min reperfusion.
Western blot analysis for RISK pathway at 20-min of reperfusion. Representative Western blots and relative densitometry showing that CST-Post given in early reperfusion results in an increased phosphorylation of Akt, ERK1/2, PKCε and GSK3β with respect to I/R or Sham Group. Individual values were compared to β-actin and the mean value of the Sham group was considered as the reference for all groups, including Sham. **p<0.01 vs. I/R_short, #p<0.05 vs. Sham_short. ANOVA followed by Newman–Keuls multiple comparison test, (n = 6 for each group).
Figure 7.
S-nitrosylation of calcium channels after 30 min ischemia and 20 min reperfusion.
Western blot analysis of S-nitrosylated proteins in homogenized cardiac ventricles. S-nitrosylation of membrane protein fraction and stripped membrane incubated with an anti L-type calcium channel antibody showing S-nitrosylation at the migration position corresponding to the L-type calcium channel in WKY (panel A) and SHR (panel B) hearts. Individual values were compared to β-actin and the mean value of the Sham group was considered as the reference for all groups, including Sham. *p<0.05 ANOVA followed by Newman–Keuls multiple comparison test, (n = 6 for each group).