siRNA-mediated inhibition of NTT-MMP-2 reduces oxidative stress and apoptotic signaling in an ex vivo model of ischemia/reperfusion injury

Figures

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  Fig. 1. Scheme of the experimental protocol. Hearts underwent 25 min of aerobic stabilization (Aero 25’), 22 min of global no-flow ischemia (Ischemia 22’), and 30 min of reperfusion at 37 °C (Reperfusion 30’). In the I/R + siRNA group, and Aero siRNA groups, MMP-2 siRNA mixed with siPORT Amine in Krebs–Henseleit buffer and in group I/R + scrRNA scrambled RNA with siPORT in K-H buffer was infused for 10 min before ischemia and again during the first 10 min of reperfusion. Coronary effluents were collected at 47 min (onset of reperfusion). Aero group was permanently perfused aerobically during the whole experiment. Figure created with BioRender.com.

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  Fig. 2. Heart mechanical function recovery (A) tissue injury (B) and MMP2 (C) and MMP-9 (D) synthesis in hearts undergoing I/R injury. fractions. ScrRNA- scrambled RNA; siRNA- MMP-2 silencing RNA.

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  Fig. 3. MMP-2 concentration in mitochondrial (A) and cytoplasmic (E) fractions. Activity of NTT-MMP-2 (B,F), total MMP-2 (C,G) and pro-MMP-2 (D,H) in mitochondrial and cytoplasmic fractions. Representative zymogram of MMPs activities in heart homogenates (I). HT10-80 was used as standard for pro-/active MMP-2 and MMP-9.

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  Fig. 4. Changes of heart rate (A) and RPP (calculated as the product of the heart rate and left ventricular developed pressure (heart rate x left ventricular developed pressure/1000)) (B) during the experiment. Recovery of heart mechanical function (difference between RPP at 25th and 77th min of perfusion expressed as a percentage of RPP recovery) (C). LDH activity in coronary effluents (D). Correlation between NTT-MMP-2 activity in mitochondrial fraction and LDH activity (E), and HR (F).*p < 0.05 Aero vs I/R, # p < 0.05 I/R vs I/R + siRNA.

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  Fig. 5. ROS/RNS level in heart homogenates (A); Correlation of ROS level in hearts with NTT-MMP-2 (B), HR (C), recovery of RPP (D) and LDH activity (E).

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  Fig. 6. Correlation of mitochondrial NTT-MMP-2 and NF-κB concentration in cytoplasm (A); Cytoplasmic concentration of NF-κB (B); Cytochrome c level in mitochondrial (C) and cytoplasmic (D) fractions. Correlation of cytochorme c in mitochondrial fraction and NF-κB (E), LDH as a marker of injury (F), HR (G) and RPP (H).

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  Fig. 7. BAX (A) and BCL2 (C) gene expression in rat hearts. Correlation of BAX (B) and BCL2 (D) genes expression and NF-κB concentration in cytoplasm.

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  Fig. 8. Concentration of NFATc1 (A) and IL-6 (B) in the cytoplasmic fractions. Correlations of NFATc1 and IL-6 concentrations (C).