Human Lysyl Oxidase Over-Expression Enhances Baseline Cardiac Oxidative Stress but Does Not Aggravate ROS Generation or Infarct Size Following Myocardial Ischemia-Reperfusion

Figures

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  Figure 1. TgLOX mice show increased LOX expression in the myocardium and enhanced ROS production. (A) Human LOX (hLOX) mRNA levels were analyzed in hearts from wild-type (WT) and TgLOX mice by real-time PCR. Levels of human LOX mRNA were undetectable in WT mice, but an arbitrary value of 1 was attributed for comparative purposes only (WT, n = 6; TgLOX, n = 8). (B) Protein levels of LOX were assessed by Western blot in heart lysates from WT and TgLOX mice. GAPDH levels are shown as a loading control. Representative immunoblot images have been included. The histogram shows the result from the densitometric analysis (WT, n = 4; TgLOX, n = 6). Data are expressed as mean ± SEM. * p < 0.05 vs. WT. t-test (A) and Mann–Whitney test (B) were applied. (C) Immunohistochemical analysis of LOX in hematoxylin counterstained myocardial sections from these animals. A negative control, in which the primary antibody was excluded, was used to test for non-specific binding of the secondary antibody (bars: 100 µm). (D) Representative dihydroethidium (DHE) staining in WT and TgLOX mice hearts (bars: 100 µm).

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  Figure 2. Impact of LOX transgenesis on rigor onset, peak hypercontracture, and functional recovery in isolated mice hearts. Rigor onset during ischemia (A) and peak hypercontracture (B) during initial reperfusion in isolated wild-type (WT) and TgLOX mice hearts submitted to global ischemia-reperfusion (n = 5/group). No significant differences between both groups were observed (Student’s t-test). (C,D) show functional recovery during reperfusion (LVdevP, (n = 4/group)) (C) and changes in perfusion pressure (D) (n = 5/group) in isolated hearts from both groups. No significant differences between both groups were observed (repeated measures ANOVA and Dunnett’s tests). Data are expressed as mean ± SEM.

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  Figure 3. Ischemia-reperfusion injury in isolated mice hearts from wild-type (WT) and TgLOX animals (n = 5/group). No differences were observed between both groups in infarct size ((A); Student’s t-test), cumulative lactate dehydrogenase (LDH) release during the first 15 min of reperfusion ((B); Student’s t-test) or LDH release during the whole reperfusion period ((C); MANOVA). Data are expressed as mean ± SEM.

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  Figure 4. Influence of LOX transgenesis on cardiac reactive oxygen species (ROS) generation after ischemia-reperfusion injury. Superoxide anion levels were assessed by dihydroethidium (DHE) staining in isolated hearts from wild-type (WT) and TgLOX mice subjected to normoxic perfusion (control) or to global ischemia-reperfusion (I/R). DHE staining was quantified at 5 min of reperfusion. Representative images are shown (bars: 100 µm). At least 4 fields from n = 3 mice in each group were quantified. Data, are expressed as mean ± SEM. * p < 0.05 vs. WT normoxic hearts (Kruskal–Wallis test).

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  Figure 5. Changes in left ventricular end-diastolic internal diameter (LVEDD; (A)), left ventricular end-systolic internal diameter (LVESD; (B)), posterior wall thickness (LVPW; (C)), interventricular septum thickness (IVS; (D)), ejection fraction (E) and cardiomyocyte cross-sectional area assessed on WGA stained sections (F) in wild-type (WT; n = 6) and TgLOX (n = 7) mice subjected to a brief period of coronary artery occlusion (45 min) and 28 days of reflow. Representative images of hematoxylin-eosin (upper panels) and WGA (lower panels)-stained sections are shown in (F) (bars: 25 µm).

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  Figure 6. (A) Scar area, expressed as percentage of left ventricular (LV) area in the scanned images, in wild-type (WT; n = 6) and TgLOX (n = 7) mice subjected to a 45-min period of coronary artery occlusion and 28 days of reperfusion. Representative images of myocardial sections stained with picrosirius red are shown for each group (bars: 1000 µm). Squared areas magnified on the right evidence collagen content in scar area (bars: 100 µm). (B) Interstitial collagen deposition in remote myocardium. Representative images are shown (bars: 100 µm). No significant differences between both groups were observed (Student’s t-test).