Short term doxycycline treatment induces sustained improvement in myocardial infarction border zone contractility

Abstract

Decreased contractility in the non-ischemic border zone surrounding a MI is in part due to degradation of cardiomyocyte sarcomeric components by intracellular matrix metalloproteinase-2 (MMP-2). We recently reported that MMP-2 levels were increased in the border zone after a MI and that treatment with doxycycline for two weeks after MI was associated with normalization of MMP-2 levels and improvement in ex-vivo contractile protein developed force in the myocardial border zone. The purpose of the current study was to determine if there is a sustained effect of short term treatment with doxycycline (Dox) on border zone function in a large animal model of antero-apical myocardial infarction (MI). Antero-apical MI was created in 14 sheep. Seven sheep received doxycycline 0.8 mg/kg/hr IV for two weeks. Cardiac MRI was performed two weeks before, and then two and six weeks after MI. Two sheep died prior to MRI at six weeks from surgical/anesthesia-related causes. The remaining 12 sheep completed the protocol. Doxycycline induced a sustained reduction in intracellular MMP-2 by Western blot (3649±643 MI+Dox vs 9236±114 MI relative intensity; p = 0.0009), an improvement in ex-vivo contractility (65.3±2.0 MI+Dox vs 39.7±0.8 MI mN/mm2; p<0.0001) and an increase in ventricular wall thickness at end-systole 1.0 cm from the infarct edge (12.4±0.6 MI+Dox vs 10.0±0.5 MI mm; p = 0.0095). Administration of doxycycline for a limited two week period is associated with a sustained improvement in ex-vivo contractility and an increase in wall thickness at end-systole in the border zone six weeks after MI. These findings were associated with a reduction in intracellular MMP-2 activity.

Fig 1. Experimental timeline.

Fig 2. Representative MRI imaging data.

Panel A is from control sheep two weeks prior to MI, panel B is from control animal six weeks after MI and panel C is from doxycycline animal six weeks after MI. All images are of the three-chamber long axis obtained at end systole. Epicardial and endocardial contour lines are in yellow. The red line is the manually determined MI perimeter. The red arrow marks the edge of the MI and white arrows mark the points of 1, 2, and 3 cm from the infarct edge.

Fig 3. LV wall thickness at end-systole six weeks after MI.

Panels A and B are color maps of LV wall thickness at end-systole six weeks after MI from representative MI control (A) and MI + Dox (B) animals. Maps are oriented with the anterior wall toward the viewer and colors range from black (< 2 mm) to brown (>14 mm). Panel C shows the effect of Doxycycline on wall thickness at end-systole in 1 cm increments from the MI edge. Note that data is from the entire LV. *p<0.05.

Fig 4. Effect of doxycycline on maximum myofilament developed force six weeks after MI.

The statistical comparison is with MI border zone control. * = p < 0.0001 with respect to MI border zone.

Fig 5. Effect of doxycycline on MMP-2 in the infarct border zone six weeks after MI.

Panel A shows the effect on MMP-2 using immunohistochemistry and panels B and C show western blot results. Panel A subpanels C and D were done using Nomarski optics. FL-MMP-2 staining is sarcomeric while the NTT-MMP-2 staining shows punctate, linear arrays of antibody staining between sarcomeres (black arrows). *p< 0.05. The effect of doxycycline on full length (D) and truncated (E) MMP-2 RNA. *p<0.05 for combined FL-MMP-2 and NTT-MMP-2 RNA effect. n = 4 for MI control and MI + Dox groups, n = 2 for non-operated control.

Fig 6. Effect of doxycycline on reactive oxygen species in the infarct border zone six weeks after MI.

Panel A shows the effect on superoxide and panel B on hydroxide. Superoxide was measured dihydroethidium (DHE) and hydroxide was measured with dichlorfluorescein (DCF). DAPI = 4',6-diamidino-2-phenylindole. Panels C and D show the % area of superoxide (DHE) and hydrogen peroxide (DCF) staining respectively. n = 3 for MI control and MI + Dox groups, n = 2 for non-operated control.