Cardioprotective effect of MMP‐2‐inhibitor‐NO‐donor hybrid against ischaemia/reperfusion injury

Figures

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  Figure 1. Constitutional formula of 5‐phenyloxyphenyl‐5‐aminoalkyl nitrate barbiturate

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  Figure 2. Experimental protocol for I/R and aerobic control with or without hybrid treatment. The hearts were perfused with 5‐phenyloxyphenyl‐5‐aminoalkyl nitrate barbiturate (0.1‐10.0 µmol/L) for the last 10 minutes of aerobic perfusion and the first 10 minutes of reperfusion. Arrows indicate time of start and the end of infusion of the compound. I/R, ischaemia/reperfusion

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  Figure 3. An effect of 5‐phenyloxyphenyl‐5‐aminoalkyl nitrate barbiturate on: (A) recovery of mechanical function of I/R hearts, (B) contractility of I/R cardiomyocytes and (C) LDH level in I/R hearts. *P < 0.05 vs aerobic control; # P < 0.05 vs I/R; mean ± SEM; n = 5‐10

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  Figure 4. An influence of 5‐phenyloxyphenyl‐5‐aminoalkyl nitrate barbiturate on expression of MMP‐2 (A), MMP‐9 (B) and iNOS (C) genes is human cardiomyocytes in a ratio to GAPDH. iNOS, inducible nitric oxide synthase; GAPDH, glyceraldehyde 3‐phosphate dehydrogenase; I/R, ischaemia/reperfusion; MMP, matrix metalloproteinase; *P < 0.05 vs aerobic control; # P < 0.05 vs I/R; mean ± SEM; n = 5‐10

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  Figure 5. Reduction of MMP‐2 synthesis and activity during infusion of 5‐phenyloxyphenyl‐5‐aminoalkyl nitrate barbiturate into rat heart (A‐B). Protective role of tested drug on MLC1 content in rat hearts (C). Correlation of MMP‐2 activity and MYL tissue content in rat hearts (D). MMP‐2 activity was normalized to protein concentration and expressed in AU. I/R, ischaemia/reperfusion; MMP, matrix metalloproteinase; MLC1, myosin light chain type 1 (ventricular); *P < 0.05 vs aerobic control; # P < 0.05 vs I/R; n = 5‐10

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  Figure 6. An influence of 5‐phenyloxyphenyl‐5‐aminoalkyl nitrate barbiturate on NO production (A). Correlation between iNOS and NO (B). iNOS, inducible nitric oxide synthase; I/R, ischaemia/reperfusion; *P < 0.05 vs aerobic control; # P < 0.05 vs I/R; n = 5‐10

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  Figure 7. An influence of 5‐phenyloxyphenyl‐5‐aminoalkyl nitrate barbiturate on iNOS (A), ADMA (B) tissue expression and synthesis as well as their correlation (C). ADMA‐asymmetric dimethylarginine; iNOS‐inducible nitric oxide synthase; I/R‐ischaemia/reperfusion; *P < 0.05 vs aerobic control; # P < 0.05 vs I/R; n = 5‐10

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  Figure 8. Potential regulation of MMP‐2 and iNOS‐ADMA pathway during oxidative stress in the absence (A) and the presence (B) of 5‐phenyloxyphenyl‐5‐aminoalkyl nitrate barbiturate‐ the scheme. NO is produced by inducible form of nitric oxide synthase (iNOS) from l‐arginine in the presence of oxygen, NADPH (nicotinamide adenine dinucleotide phosphate), BH4 (tetrahydrobiopterin) and FMN (flavin mononucleotide) and plays a crucial role in vasodilatation (increasing coronary flow‐CF), leukocytes adhesion, platelets aggregation and MMP‐2 expression. Oxidative stress leads to generation of ROS induced iNOS expression and production of endogenous asymmetric dimethylarginine (ADMA). ADMA in turn enhanced ‘iNOS uncoupling’ generating large amounts of O2-·, which in turn rapidly reacts with NO leading to peroxynitrite production (ONOO−). ONOO− plays multiple roles such as: inhibition of ADMA metabolism by DDAH, activates MMP‐2 and leads to nitration/nitrosylation of myosin light chains (MLC1s). Arrows mark the stimulating effect (→), lines terminated with a circle means an inhibition (‐o)