Mixture of Doxycycline, ML-7 and L-NAME Restores the Pro- and Antioxidant Balance during Myocardial Infarction-In Vivo Pig Model Study

Abstract

The restoration of blood flow to the ischemic myocardium inflicts ischemia/reperfusion (I/R) heart injury (IRI). The main contributors to IRI are increased oxidative stress and subsequent excessive production of ROS, increased expression of NOS and peroxinitate, activation of MMPs, and enhanced posttranslational modifications of contractile proteins, which make them more susceptible to proteolytic degradation. Since the pathophysiology of IRI is a complex issue, and thus, various therapeutic strategies are required to prevent or reduce IRI and microvascular dysfunction, in the current study we proposed an innovative multi-drug therapy using low concentrations of drugs applied intracoronary to reach microvessels in order to stabilize the pro- and antioxidant balance during a MI in an in vivo pig model. The ability of a mixture of doxycycline (1 μM), ML-7 (0.5 μM), and L-NAME (2 μM) to modulate the pro- and antioxidative balance was tested in the left ventricle tissue and blood samples. Data showed that infusion of a MIX reduced the total oxidative status (TOS), oxidative stress index (OSI), and malondialdehyde (MDA). It also increased the total antioxidant capacity, confirming its antioxidative properties. MIX administration also reduced the activity of MMP-2 and MMP-9, and then decreased the release of MLC1 and BNP-26 into plasma. This study demonstrated that intracoronary administration of low concentrations of doxycycline in combination with ML-7 and L-NAME is incredibly efficient in regulating pro- and antioxidant balance during MI.

Keywords: MI pig model; ischemia-reperfusion injury; oxidative stress; pro- and antioxidant balance.

Figure 1

Angiographic image of the left anterior descending artery before ischemia (A) and at the time of occlusion (B). Representative ECG recording confirming normal heart’s electrical activity before (C) and during (D) myocardial (MI) infarction.

Figure 2

Representative images of the heart subjected to myocardial infarction show a macroscopic evaluation of necrotic tissue in the MI area and healthy tissue (A). Representative microscopic images of HE stained tissue from infarcted and non-infarcted areas of the heart (B), magnification factor 200 µm. H—healthy myocardial tissue; N—necrotic changes in the area of infarction.

Figure 3

Changes in oxidative status in left ventricle heart tissue from infarct area and non-infarct area, independently of drug administration. Notes: TOS—total oxidative status; OSI—oxidative stress index; MDA—malondialdehyde; LVIA—left ventricle heart tissue from infarct area; LVCA—left ventricle heart tissue from non-infarct area (control); MI—myocardial infarction; MI-MIX—myocardial infarction with administration of MIX; MIX—mixture of doxycycline (1 μM), ML-7 (0.5 μM) and L-NAME (2 μM); ***—p < 0.001, **—p < 0.01.

Figure 4

Changes in antioxidative status in left ventricle heart tissue from an infarct area and a non-infarct area independently of drug administration. Notes: TAC—total antioxidant capacity; act_GPx—activity of glutathione peroxidase; act_GR—activity of glutathione reductase; act_GST—activity of glutathione S-transferase; LVIA—left ventricle heart tissue from infarct area; LVCA—left ventricle heart tissue from non-infarct area (control); MI—myocardial infarction; MI-MIX—myocardial infarction with administration of MIX; MIX—mixture of doxycycline (1 μM), ML-7 (0.5 μM) and L-NAME (2 μM); ***—p < 0.001.

Figure 5

The correlation between tissue antioxidative markers and the release of MLC1 or BNP-26 into the plasma: act_GPX vs. MLC1 (A), act_GR vs. MLC1 (B), TAC vs. BNP-26 (C), act_GPX vs. BNP-26 (D), and act_GR vs. BNP-26 (E). Notes: TAC—total antioxidant capacity; act_GPx—activity of glutathione peroxidase; act_GR—activity of glutathione reductase; act_GST—activity of glutathione S-transferase; MLC1—myosin light chains type 1; BNP-26—B-type natriuretic protein.

Figure 6

Percentage impact of intracoronary MIX treatment on changes in pro- and antioxidant status. Notes: TAC—total antioxidant capacity; TOS—total oxidative status; OSI—oxidative stress index; MDA—malondialdehyde; act_GPX—activity of glutathione peroxidase; act_GR—activity of glutathione reductase; act_GST—activity of glutathione S-transferase; MIX—mixture of doxycycline (1 μM), ML-7 (0.5 μM); baseline (green) refers to the tissue not treated with MIX.

Figure 7

Serum concentrations (A) and MIX treatment effect (B) on serum concentrations of pro- and antioxidant markers at different time points. Notes: TAC—total antioxidant capacity; TOS—total oxidative status; OSI—oxidative stress index; LF—lipofuscin; MI—myocardial infarction; MIX—mixture of doxycycline, ML-7 and L-NAME; T0—before ischemia; TR—during reperfusion; TE—before euthanasia; ***—p < 0.001; **—p < 0.01; *—p < 0.05.

Figure 8

Analysis of MMP-2 (A), MMP-9 (B), MLC1 (C), and BNP-26 (D) in porcine plasma at reperfusion. Notes: MMP-2/MMP-9—matrix metalloproteinases 2 and 9; MI—myocardial infarction; MI-MIX—myocardial infarction with administration of MIX; MIX—mixture of doxycycline (1 μM), ML-7 (0.5 μM), and L-NAME (2 μM); ***—p < 0.001; **—p < 0.01; *—p < 0.05.