Matrix Metalloproteinase-2 Inhibition in Acute Ischemia-Reperfusion Heart Injury—Cardioprotective Properties of Carvedilol

Figures

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  Figure 1. (a–c). Effect of carvedilol, metoprolol and nebivolol on cardiac mechanical function after 20 min of ischemia (a) Effect of carvedilol on cardiac mechanical function after 20 min of ischemia. (b) Effect of metoprolol on cardiac mechanical function after 20 min of ischemia. (c) Effect of carvedilol on cardiac mechanical function after 20 min of ischemia. The result presented as the rate-pressure product (heart rate × left ventricular developed pressure) normalized to 15 min aerobic value. C—control group, aerobically perfused hearts, IR injury—ischemia-reperfusion injury, IR-CAR 0.1– hearts from IR injury model treated with 0.1 µM carvedilol, IR-CAR 1– hearts from IR injury model treated with 1 µM carvedilol, IR-CAR 10– hearts from IR injury model treated with 10 µM carvedilol, IR-MET 0.01—hearts from IR injury model treated with 0.01 µM metoprolol, IR-MET 0.1—hearts from IR injury model treated with 0.1 µM metoprolol, IR-MET 1—hearts from IR injury model treated with 1 µM metoprolol, IR-NEB 0.005—hearts from IR injury model treated with 0.005 µM nebivolol, IR-NEB 0.05—hearts from IR injury model treated with 0.05 µM nebivolol, IR-NEB 0.5—hearts from IR injury model treated with…

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  Figure 2. Influence of carvedilol, metoprolol and nebivolol on MMP-2 activity in coronary effluent. (a) Densitometric analysis of gelatinolytic MMP-2 activities in coronary effluent in samples from perfused hearts collected at different time points. (b) Representative zymogram showing gelatinolytic activities in coronary effluent samples from perfused hearts collected at different time points. The 72 kDa MMP-2 specific activity in coronary effluent samples was assessed by densitometric analysis (c) Log ratio of 45 vs. 25 min perfusion MMP-2 activities in coronary effluent in carvedilol treated groups. (d) Log ratio of 45 vs. 25 min perfusion MMP-2 activities in coronary effluent in metoprolol treated groups. (e) Log ratio of 45 vs. 25 min perfusion MMP-2 activities in coronary effluent in nebivolol treated groups. Only in the IR CAR 0.01 group there was no significant increase in MMP-2 activity. C—control group, aerobically perfused hearts, IR injury—ischemia-reperfusion injury, IR-CAR 0.1—hearts from IR injury model treated with 0.1 µM carvedilol, IR-CAR 1—hearts from IR injury model treated with 1 µM carvedilol, IR-CAR 10—hearts from IR injury model treated with 10 µM carvedilol, IR-MET…

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  Figure 3. Effect of CAR on MMP-2 activity in vitro. (a) Densitometric analysis of gelatinolytic MMP-2 activities in control gel and gel incubated with addition of 0.1 μM CAR. (b) Representative zymograms of MMP-2 activity in control gel (A) and gel incubated with addition of 0.1 μM CAR (B). C—control group, C+ CAR 0.1—in control gel and gel incubated with addition of 0.1 μM CAR. p > 0.05, n = 8, T-test.

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  Figure 4. Effect of carvedilol on MMP-2 mRNA expression in hearts subjected to ischemia-reperfusion. Fold of change was calculated by delta-delta Ct formula. Glyceraldehyde 3-phosphate dehydrogenase served as a normalizing gene. C—control group, aerobically perfused hearts, IR injury—ischemia-reperfusion injury, CAR 0.1—aerobically perfused hearts treated with 0.1 µM carvedilol, IR-CAR 0.1—hearts from IR injury model treated with 0.1 µM carvedilol. p > 0.05, n = 5, ANOVA.

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  Figure 5. MMP-2 content in coronary effluents and heart tissue. (a) Densitometric analysis of MMP-2 content in coronary effluent collected at 2 min of reperfusion after 20 min ischemia in IR CAR 0.1 group as determined by Western blot. (b) Representative western blot of MMP-2 content in coronary effluents collected at 2 min of reperfusion after 20 min ischemia in IR CAR 0.1 group. C—control group, aerobically perfused hearts, IR injury—ischemia-reperfusion injury, CAR 0.1, aerobically perfused hearts treated with 0.1 µM carvedilol, IR-CAR 0.1—hearts from IR injury model treated with 0.1 µM carvedilol. * C vs IR, C vs IR-CAR 0.1, CAR 0.1 vs IR, CAR 0.1 vs IR-CAR 0.1, p < 0.05, n = 5, ANOVA.

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  Figure 6. (a–d) Effect of carvedilol, metoprolol and nebivolol on troponin I content in heart tissue. (a) Densitometric analysis of TnI content in heart homogenates prepared at 30 min of reperfusion after 20 min ischemia in IR CAR 0.1 group as determined by Western blot. (b) Representative troponin I (TnI) protein level in heart homogenates prepared at the end of perfusion in IR CAR 0.1 group as determined by Western blot. (c) Densitometric analysis of TnI content in heart homogenates prepared at 30 min of reperfusion after 20 min ischemia in metoprolol treated hearts as determined by Western blot. (d) Densitometric analysis of TnI content in heart homogenates prepared at 30 min of reperfusion after 20 min ischemia in nebivolol treated hearts as determined by Western blot. Decrease of the level of 31 kDa troponin I was abolished only in the IR-CAR 0.1 μM group. C—control group, aerobically perfused hearts, IR injury—ischemia-reperfusion injury, IR-CAR 0.1—hearts from IR injury model treated with 0.1 µM carvedilol, IR-CAR 1—hearts from IR injury model treated with 1 µM carvedilol, IR-CAR 10– hearts from IR injury model treated with 10 µM carvedilol, IR-MET 0.01—hearts from IR injury model…

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  Figure 7. Experimental protocol for isolated heart perfusions. Detailed description in Material and methods part, “heart perfusion protocol” section. * show times when coronary effluent samples were collected during aerobic perfusion (23–25 min) and in the first 2 min of reperfusion (45–47 min).