Donepezil, Anti-Alzheimer's Disease Drug, Prevents Cardiac Rupture during Acute Phase of Myocardial Infarction in Mice

Background: We have previously demonstrated that the chronic intervention in the cholinergic system by donepezil, an acetylcholinesterase inhibitor, plays a beneficial role in suppressing long-term cardiac remodeling after myocardial infarction (MI). In comparison with such a chronic effect, however, the acute effect of donepezil during an acute phase of MI remains unclear. Noticing recent findings of a cholinergic mechanism for anti-inflammatory actions, we tested the hypothesis that donepezil attenuates an acute inflammatory tissue injury following MI. Methods and Results: In isolated and activated macrophages, donepezil significantly reduced intra- and extracellular matrix metalloproteinase-9 (MMP-9). In mice with MI, despite the comparable values of heart rate and blood pressure, the donepezil-treated group showed a significantly lower incidence of cardiac rupture than the untreated group during the acute phase of MI. Immunohistochemistry revealed that MMP-9 was localized at the infarct area where a large number of inflammatory cells including macrophages infiltrated, and the expression and the enzymatic activity of MMP-9 at the left ventricular infarct area was significantly reduced in the donepezil-treated group. Conclusion: The present study suggests that donepezil inhibits the MMP-9-related acute inflammatory tissue injury in the infarcted myocardium, thereby reduces the risk of left ventricular free wall rupture during the acute phase of MI

Histological analysis in heart sections at 3 days after MI.

<p>Values are means ± SD. DPZ, donepezil-treated group. #, <i>P<0.01</i>. ##, <i>P<0.05</i>.</p

The involvement of ACh in the inhibitory effect of donepezil on macrophage MMP-9.

<p>(a) LPS treatment increased MMP-9 (LPS) in culture medium, which was inhibited by donepezil pretreatment (DPZ/LPS). However, ACh could not reproduce the inhibitory effect of donepezil (ACh/LPS). (b) Donepezil inhibited the LPS-induced MMP-9 even in the presence of a muscarinic ACh receptor blocker, atropine (Atr/DPZ/LPS), or a nicotinic ACh receptor blocker, mecamylamine (Mec/DPZ/LPS). Both blockers showed no effect on LPS-induced MMP-9 independently (Atr/LPS and Mec/LPS).</p

Western blot and zymographic analysis on macrophage MMP-9.

<p>(a) Western blot analysis of MMP-9 content in culture media and macrophage cells before and after LPS treatment with or without donepezil pretreatment. In control (control), MMP-9 was stored within cells and also secreted to culture medium. In LPS-treated cells (LPS), MMP-9 was significantly decreased within cells and increased in culture medium. In donepezil-pretreated and LPS-treated cells (DPZ/LPS), MMP-9 within both cells and medium were as low as undetectable level. The expression of α-tubulin was shown as an internal control. #, <i>P<0.01</i> versus control. ##, <i>P<0.01</i> versus LPS. *, <i>P<0.05</i> versus control. (b) Gelatin zymography for analysis of MMP-9 enzymatic activity. Compared to control (control), proteinase activity of MMP-9 secreted to culture medium was significantly high in LPS-treated cells (LPS) and low in donepezil-pretreated and LPS-treated cells (DPZ/LPS). #, <i>P<0.01</i>.</p

Effect of donepezil on MMP-9 in macrophages.

<p>MMP-9 in culture medium was evaluated by Western blot analysis before and after LPS (10 ng/ml) treatment with or without donepezil pretreatment (100 µM). Compared to control (control), LPS treatment significantly increased MMP-9 in culture medium (LPS), whereas donepezil pretreatment significantly decreased MMP-9 even in the presence of LPS (DPZ/LPS). #, <i>P<0.01</i>.</p

Immunocytochemistry with anti MMP-9 antibody in macrophages.

<p>DIC images showed that control macrophages had a spindle shape (a), whereas LPS-treated macrophages had a relatively round shape and numerous intracellular vacuoles regardless of donepezil pretreatment (c and e). Immunofluorescence micrographs showed that MMP-9 signals were observed around cytoplasm in the control (b) and the LPS-treated cells (d), which was lowered to undetectable levels by donepezil pretreatment (f).</p

Physiological characteristics in sham operated and infarcted mice at 3 days after MI.

<p>Values are means ± SD. HR, heart rate; SBP, systolic blood pressure; HW/BW, heart weight to body weight ratio; DPZ, donepezil-treated group. #, <i>P<0.01</i> versus sham group.</p

Cardiac rupture rate at 4 days after MI.

<p>DPZ, donepezil-treated group. #, <i>P<0.01</i> versus untreated group.</p

Immunohistochemistry of MMP-9 in infarcted myocardium.

<p>(a) A DIC image of the left ventricular infarct area. (b and c) The same fields stained with Hoechst 33258 to indicate the position of nuclei (b) and with a rabbit anti mouse MMP-9 antibody to indicate the localization of MMP-9 (c). (d) A schematic drawing of the field observed. IA and NIA represent infarct and non-infarct area, respectively.</p