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

Abstract

Objectives: Coronary reperfusion procedure leads to ischemia/reperfusion injury of the heart (IRI). IRI arises from increased degradation of myosin light chains and increased activity of matrix metalloproteinase 2 (MMP-2). Increased production of toxic peroxynitrite (ONOO^-) during oxidative stress is a source of increased nitration/nitrosylation of contractile proteins, which enhance their degradation through MMP-2. Hence, an imbalance in nitric oxide (NO) metabolism along with oxidative stress is an important factor contributing to pathophysiology of cardiovascular disorders, including myocardial infarction. The aim of the current study was to provide an important insight into understanding the interaction of iNOS, eNOS, and ADMA during oxidative stress and to propose the beneficial therapy to modulate this interaction. Material and Methods. Pathogen-free Wistar rats were used in this study as a surrogate heart model ex vivo. Rat hearts perfused using the Langendorff method were subjected to global no-flow ischemia with or without administration of DOXY (1 µM), ML-7 (0.5 µM), and L-NAME (2 µM) mixture. Haemodynamic parameters of heart function, markers of I/R injury, tissue expression of iNOS, eNOS, and phospho-eNOS, asymmetric dimethylarginine, and NO production as well as MMP-2 activity were measured.

Results: Mechanical heart function and coronary flow (CF) were decreased in the hearts subjected to I/R. Treatment of the hearts with the tested mixture resulted in a recovery of mechanical function due to decreased activity of MMP-2. An infusion of Doxy, ML-7, and L-NAME mixture into I/R hearts decreased the expression of iNOS, eNOS, and phospho-eNOS and in consequence reduced ADMA expression. Decreased ADMA production led to enhanced NO synthesis and improvement of cardiac function at 85% of aerobic control.

Conclusions: Synergistic effect of the multidrug therapy with the subthreshold doses allows addressing a few pathways of I/R injury simultaneously to achieve protection of cardiac function during I/R.

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.