Ivabradine Induces Cardiac Protection against Myocardial Infarction by Preventing Cyclophilin-A Secretion in Pigs under Coronary Ischemia/Reperfusion

Figures

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  Figure 1. Ivabradine induces cardiac protection in pigs subjected to ischemia/reperfusion (IR). (A) Left ventricle ejection fraction values obtained at time points 0 and 7 days post-IR (n = 9 Ivabradine/8 placebo/3 sham. Mean ± SD. # p < 0.001 day 0 placebo vs. sham. $ p < 0.001 day 0 Ivabradine vs. sham. * p < 0.05 day 7 placebo vs. Ivabradine. ¶ p < 0.001 day 7 placebo vs. sham. ‡ p < 0.001 day 7 Ivabradine vs. sham. (B) Evans blue/TTC double staining performed in 0.5 cm heart sections isolated 7 days after IR from pigs treated with 0.3 mg/kg Ivabradine or placebo.

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  Figure 2. Ivabradine increases de-expression of cyclophilin-A in the hearts of pigs subjected to IR. (A) Immunoblot detection of cyclophilin-A (CypA) and Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as loading control in heart lysates from placebo and Ivabradine pigs subjected to IR. n = 9 Ivabradine/8 placebo. Mean ± SD; * p < 0.05 necrotic Ivabradine vs. placebo. (B) Confocal microscopy detection of cyclophilin-A (red) in heart sections of pigs treated with Ivabradine or placebo. n = 9 Ivabradine/8 placebo. Mean ± SD; * p < 0.05 necrotic Ivabradine vs. placebo.

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  Figure 3. Ivabradine reduces plasma CyPA after IR. (A) Immunoblot detection of CyPA and albumin as loading control in plasma samples from placebo- and Ivabradinetreated pigs subjected to IR (n = 9 Ivabradine/8 placebo. Mean ± SD; * p < 0.05 d7 placebo vs. Ivabradine). (B) mRNA expression from healthy and necrotic sections of hearts subjected to IR in response to Ivabradine or placebo (n = 9 Ivabradine/8 placebo).

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  Figure 4. Ivabradine increases the binding between CyPA and low-glycosylated extracellular matrix metalloproteinase inducer (EMMPRIN). (A) Immunoprecipitation of CyPA and EMMPRIN in heart extracts (n = 9 Ivabradine/8 placebo, mean ± SD, * p < 0.05 necrotic placebo vs. Ivabradine). (B) Immunoblot detection of MMP-9 and GAPDH as a loading control in heart lysates from placebo- and Ivabradine-treated pigs subjected to IR (n = 9 Ivabradine/8 placebo. Mean ± SD, * p < 0.05 necrotic placebo vs. Ivabradine).

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  Figure 5. Ivabradine reduces the secretion of CyPA in cardiac cells. H9c2 cells were treated with 5 mM Ivabradine for 0,1,2,4, and 8 h. CyPA (red) and VAMP (green) were visualized with specific antibodies by confocal microscopy. Merged panels show co-localization in yellow. Co-localized panels were generated through analyzing co-localizing pixels with the ImageJ software package. Nuclei were stained with dye Hoechst (n = 3).

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  Figure 6. Ivabradine has no effect on CyPA degradation. (A) Confocal microscopic detection of EEA-1 (red) and CyPA (green) in H9c2 cells incubated with Ivabradine, chloroquine, and a combination of both. Merged panels show co-localization in yellow (n = 3). (B) Immunoblot detection of CyPA and GAPDH as a loading control in protein lysates from cells incubated with Ivabradine, chloroquine, and a combination of both (n = 3, mean ± SD, * p < 0.03 control vs. Ivabradine. # p < 0.01 control vs. chloroquine (CQ), $ p < 0.006 control vs. CQ/iva). (C) Immunoblot detection of CyPA and GAPDH in protein lysates from cells incubated with Ivabradine, MG-132, and a combination of both (n = 3. Mean ± SD; * p < 0.003 control vs. Ivabradine. # p < 0.005 control vs. MG. $ p < 0.005 control vs. MG/iva).