Mixture of MMP-2, MLC, and NOS Inhibitors Affects NO Metabolism and Protects Heart from Cardiac I/R Injury

Figure 1. Experimental protocol for ischemia/reperfusion (I/R) and aerobic control with or without administration of the mixture of doxycycline (MMP-2 inhibitor; 1.0 µM), L-NAME (NOS inhibitor; 2 µM), and ML-7 (inhibitor of MLC phosphorylation; 0.5 µM).

Figure 2. An effect of coadministration of doxycycline (MMP-2 inhibitor; 1.0 µM), L-NAME (NOS inhibitor; 2 µM), and ML-7 (inhibitor of MLC phosphorylation; 0.5 µM) on recovery of mechanical function of I/R hearts (a). An effect of I/R on the LDH level in coronary effluents (b). Doxy, doxycycline; MMP-2, matrix metalloproteinase-2; NOS, nitric oxide synthase; MLC, myosin light chain; I/R, ischemia/reperfusion; LDH, lactate dehydrogenase; ∗p < 0.05 vs. aerobic control; #p < 0.05 vs. I/R; mean ± SEM.

Figure 3. An effect of coadministration of doxycycline (1.0 µM), L-NAME (2 µM), and ML-7 (0.5 µM) on expression of iNOS (a), ADMA (b) and NO production (as total nitrite/nitrate) (c) in cardiac tissue. iNOS, inducible nitric oxide synthase; ADMA, asymmetric dimethylarginine; NO, nitric oxide; I/R, ischemia/reperfusion; ∗p < 0.05 vs. aerobic control; #p < 0.05 vs. I/R; mean ± SEM.

Figure 4. Correlations between iNOS, ADMA, and NO (a–c). iNOS, inducible nitric oxide synthase; ADMA, asymmetric dimethylarginine; NO, nitric oxide (measured indirectly as nitrite/nitrate).

Figure 5. Correlation between coronary flow and ADMA (a). An effect of I/R on coronary flow (b). ADMA, asymmetric dimethylarginine; I/R, ischemia/reperfusion; ∗p < 0.05 vs. aerobic control.

Figure 6. An effect of coadministration of doxycycline (1.0 µM), L-NAME (2 µM), and ML-7 (0.5 µM) on expression of eNOS (a) and phospho-eNOS (b) in cardiac tissue. eNOS, endothelial nitric oxide synthase; phospho-eNOS, phosphorylated (S1177) eNOS; I/R, ischemia/reperfusion; ∗p < 0.05 vs. aerobic control; #p < 0.05 vs. I/R; mean ± SEM.

Figure 7. An effect of coadministration of doxycycline (1.0 µM), L-NAME (2 µM), and ML-7 (0.5 µM) on activity of MMP-2 in cardiac tissue (a). Correlation between MMP-2 and iNOS (b) and MMP-2 and ADMA (c). MMP-2, matrix metalloproteinase-2; iNOS, inducible nitric oxide synthase; ADMA, asymmetric dimethylarginine; I/R, ischemia/reperfusion; ∗p < 0.05 vs. aerobic control; #p < 0.05 vs. I/R; mean ± SEM.

Figure 8. The elements of the cardiac ischemia/reperfusion injury pathway targeted by multidrug therapy with Doxy, L-NAME, and ML-7. Oxidative stress resulting from I/R leads to upregulation of iNOS, eNOS, phospho-eNOS (targeted by L-NAME), and ADMA. ADMA promotes uncoupling of NOS and production of superoxide (O2•−). Increased level of NOS-derived NO reacts with O2•− forming highly reactive peroxynitrite (ONOO−), which limits NO bioavailability. ONOO− activates MMP-2 (activity of MMP-2 was targeted by Doxy) which degrades MLC. Increased phosphorylation of MLC by MLCK (targeted by ML-7) increases its susceptibility to MMP-2 degradation. ADMA, asymmetric dimethylarginine; Doxy, doxycycline; eNOS, endothelial nitric oxide synthase; iNOS, inducible nitric oxide synthase; L-NAME, inhibitor of NOS; ML-7, inhibitor of MLC phosphorylation; MLC, myosin light chain; MLC-phos, phosphorylated myosin light chain; MLCK, myosin light chain kinase; MMP-2, matrix metalloproteinase-2; NO, nitric oxide; phopho-eNOS, phosphorylated (S1177) eNOS.