Up-regulation of heme oxygenase-1 after infarct initiation reduces mortality, infarct size and left ventricular remodeling: experimental evidence and proof of concept

Abstract

Background: Up-regulation of HO-1 by genetic manipulation or pharmacological pre-treatment has been reported to provide benefits in several animal models of myocardial infarction (MI). However, its efficacy following MI initiation (as in clinical reality) remains to be tested. Therefore, this study investigated whether HO-1 over-expression, by cobalt protoporphyrin (CoPP) administered after LAD ligation, is still able to improve functional and structural changes in left ventricle (LV) in a rat model of 4-week MI.

Methods: A total of 144 adult male Wistar rats were subjected to either left anterior coronary artery ligation or sham-operation. The effect of CoPP treatment (5 mg/kg i.p. at the end of the surgical session and, then, once a week for 4 weeks) was evaluated on the basis of survival, electro- and echocardiography, plasma levels of B-type natriuretic peptide (BNP), endothelin-1 and prostaglandin E2, coronary microvascular reactivity, MI size, LV wall thickness and vascularity. Besides, the expression of HO-1 and connexin-43 in different LV territories was assessed by western blot analysis and immunohistochemistry, respectively.

Results: CoPP induced an increased expression of HO-1 protein with >16 h delay. CoPP treatment significantly reduced mortality, MI size, BNP concentration, ECG alterations, LV dysfunction, microvascular constriction, capillary rarefaction and restored connexin-43 expression as compared to untreated MI. These functional and structural changes were paralleled by increased HO-1 expression in all LV territories. HO activity inhibition by tin-mesoporphyrin abolished the differences between CoPP-treated and untreated MI animals.

Conclusions: This is the first report demonstrating the putative role of pharmacological induction of HO-1 following coronary occlusion to benefit infarcted and remote territories, leading to better cardiac function in a 4-week MI outcome.

Figure 1

Histograms of infarct size and HO-1 expression in hearts at 4 weeks after LAD ligation. From left to right: sham operated (n = 6), vehicle-treated MI (n = 16), CoPP-treated MI (n = 14) and CoPP + SnMP-treated MI rats (n = 6). A. Representative freshly-cut transverse sections used for determination of infarct size (upper) and quantitative results on infarct size (lower) expressed as mean ± SE percentage of left ventricle. RV: right ventricle; LVP: left ventricular posterior free wall; LVA: left ventricular anterior free wall. B. Representative western blots (upper) and quantitative results from densitometric analysis of HO-1 and β-actin expression (lower) of the corresponding regions shown in the areas outlined by the black boxes and labeled with letters in the representative freshly-cut transverse section: a) right ventricle wall; b) left ventricle posterior wall, opposite to LAD territory; c) border region to LAD area and d) central zone of the infarcted area. Histograms are expressed as ratio between HO-1 and the comparative protein β-actin.

Figure 2

Distribution of connexins 43 in the remote region at 4 weeks after LAD ligation. Upper panels: representative immunostaining for Cx43 from sham-operated (a), vehicle treated MI (b), CoPP-treated MI (c), and CoPP + SnMP-treated MI groups (d) at low magnification (10×, calibration bar: 40 μm) and at higher magnification (400×, insets). Lower panel: histograms of the proportion of the total cell area occupied by Cx43 immunoreactive signal in the remote region. Data are expressed as means ± SE. *p < 0.001 compared with both sham-operated and CoPP-treated MI groups; #p < 0.02 compared with sham-operated group.

Figure 3

Histomorphometric analysis of arteriolar and capillary density in the border and the remote region at 4 weeks after LAD ligation. Histograms of arteriolar density (A) and capillary density (B) in the border (BZ) and the remote (RZ) zone of sham operated animals (white bar, n = 6), MI group (black bar, n = 6) and CoPP-treated MI group (dashed bar, n = 6). Data are expressed as means ± SE. *p < 0.05 and **p < 0.001 compared with sham operated; #p < 0.05 compared with CoPP-treated MI groups.