In Vivo Study on Doxycycline Protective Mechanisms during Myocardial Ischemia Injury in Rats

Abstract

Background: The fact that during myocardial ischemia/reperfusion (I/R) injury, myosin light chain 1 (MLC1) and troponin I (TnI) are degraded by matrix metalloproteases activity has already been well established in both in vitro and ex vivo studies. However, I/R injury is a complex issue based on several overlapping mechanisms. Increased activity of myosin light chain kinase and nitric oxide synthase due to oxidative stress leads to post-translational modifications of MLC1, thus leading to the increased degradation of these proteins.

Methods: Wistar rats were subjected to left anterior descending coronary artery occlusion. To measure the pharmacological effect of doxycycline, transthoracic echocardiography as well as biochemical tests, concentrations of TnI, LDH, MLC1, MMP-2 and MMP-9 were performed. Gelatinize activity and cytotoxicity level were also assessed; Results: I.p., administration of doxycycline before LAD occlusion surgery increased TnI and LDH content in the heart and decreased cytotoxicity. A reduction of MMP-2 and MMP-9 concentration and MMP-2 activity after administration of Doxy was also observed, as well as improvement in echocardiographic parameters just 7 days after surgery.

Conclusions: Inhibition of MMPs by doxycycline, in vivo, may serve as a protective agent in future therapy.

Keywords: LAD; MLC1; MMPs; doxycycline; ischemic heart disease; rat myocardial infraction.

Figure 1

(A) Evans Blue followed by 2,3,5-triphenyltetrazolium chloride (TTC) staining of rats’ hearts. Representative heart slices are shown for I/R hearts and IR hearts subjected to doxycycline treatment. White/yellow areas indicated infarct area. (B) Hematoxylin/eosin staining of hearts. Representative heart images are shown for I/R hearts and I/R hearts subjected to doxycycline treatment. Magnification 100×.

Figure 2

An influence of Doxy on the magnitude of heart ischemia injury: (A) Lower TnI concentration in rat hearts served as a marker of damage; (B) the number of dead cells in rat hearts based on the activity of dead cell protease, tested by cytotoxicity assay; (C) the activity of LDH in hearts as a marker of heart injury. (D) Correlation between TnI and other markers of injury: the activity of dead cell protease and LDH. All markers of injury were normalized to protein concentration. mU/mL: milliinternational enzyme units per milliliter; RLU: relative light units; Data are presented as mean ± SEM; n = 3–8; * p < 0.05 vs. Aero; # p < 0.05 vs. I/R; ANOVA with Tukey’s multiple comparisons tests were used.

Figure 3

Transthoracic echocardiography images of the rat heart on the 14th day of the experiment, showing representative pictures for both the IR and IR doxy groups.

Figure 4

Reduction of MMP-2 and MMP-9 concentration and MMP-2 activity after administration of Doxy 100 µM. MMP-2 (A) and MMP-9 (B) concentration was normalized to total protein concentration. MMP-2 activity (C) was normalized to total protein concentration and expressed in AU. (D) Correlation between concentration and activity of MMP-2. MMP, matrix metalloproteinase; * p < 0.05 vs. aerobic control; # p < 0.05 vs. I/R; n = 3–8.

Figure 5

Protective role of Doxy on ventricular MLC1 content in rat hearts (A). Correlation of MMP-2 activity and MLC1 tissue content in rat hearts (B). Correlation of fractional shortening after 14 days post-surgery (%) and MMP-2 activity and MLC1 tissue content in rat hearts (C). MLC1, myosin light chain type 1; * p < 0.05 vs. aerobic control; # p < 0.05 vs. I/R; n = 3–8.