Early donepezil monotherapy or combination with metoprolol significantly prevents subsequent chronic heart failure in rats with reperfused myocardial infarction

Figures

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  Fig. 1. Experimental design and timeline. a Experimental design of UT, DT, MT, and DMT therapy in rats with chronic heart failure with reperfused myocardial infarction (RMI). b, c Experimental events and timelines. UT, untreated; DT, donepezil treatment; MT, metoprolol treatment; DMT, donepezil plus metoprolol treatment; BP, blood pressure; HR, heart rate

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  Fig. 2. Effects of treatment on heart rate (HR) and mean blood pressure (MBP) of rats with RMI-induced CHF, recorded using telemetry. a Weekly HR. Each point represents averaged 1-week data for all animals in each group. b Daily average HR during week 1. Each point represents the averaged 1-day data for all animals in each group. (UT, n = 14; DT, n = 16; MT, n = 18; DMT, n = 14). c Weekly MBP. Each point represents averaged 1-week data for all animals in each group. Values are expressed as means ± SEMs. a–d P < 0.05; aa, bb, cc and dd P < 0.01 vs. pretreatment value (week 0, day 0, at 1-h post-RMI) within each group (UT, DT, MT or DMT); *P < 0.05, **P < 0.05 vs. UT groups, using one-way ANOVA and post-hoc Dunnett’s test. CHF, chronic heart failure

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  Fig. 3. Masson’s trichrome staining of ventricles in rats with CHF 10 weeks after treatment. a Representative biventricular sections of RMI. The region surrounded by a green line showed area at risk. Scale bars, 2 mm. b Representative images of cardiac fibrosis in the noninfarcted area. c Representative myocyte cross sections (papillary cardiomyocytes). Scale bars, 50 μm. d Area at risk (UT, 38 ± 5%, n = 12; DT, 38 ± 6%, n = 15; MT, 38 ± 4%, n = 18; DMT, 39 ± 5%, n = 14). e Infarct size (infarcted area/area at risk) (UT, 51 ± 2%, n = 12; DT, 33 ± 3%, n = 15; MT, 56 ± 3%, n = 18; DMT, 35 ± 3%, n = 14). f Cardiac fibrosis index in the noninfarcted regions (UT, 5.0 ± 0.6%/field, n = 10, 60 fields of view; DT, 2.7 ± 0.2%/field, n = 14, 84 fields; MT, 4.9 ± 0.3%/field, n = 18, 108 fields; DMT, 2.4 ± 0.2%/field, n = 14, 84 fields). g Myocyte cross-sectional area in the noninfarcted region (UT, 1118 ± 98 μm2/cell, n = 7, 312 cells; DT, 683 ± 34 μm2/cell, n = 12, 668 cells; MT, 1218 ± 123 μm2/cell, n = 7, 264 cells; DMT, 465 ± 33 μm2/cell, n = 6, 869 cells). h Representative micrographs of blood microvessels (von Willebrand factor [vWF, red]) immunostaining in the peri-infarct and noninfarcted…

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  Fig. 4. Neutrophil infiltration of rat myocardium and histological analysis on day 8 after reperfused myocardial infarction (RMI). Representative photomicrographs of Masson’s trichrome staining (a), and hematoxylin and eosin staining of the heart (b) and the ischemia/reperfusion (I/R) region. The whole heart scale bars are 2 mm. Infiltrating neutrophils in the I/R region, scale bars are 50 μm. c Box-and-whisker plots of neutrophil counts in the I/R region (UT: 271 ± 15/field, n = 5, 30 fields; DT: 123 ± 7/field, n = 5, 30 fields; MT: 257 ± 10/field, n = 4, 24 fields; DMT: 136 ± 4/field, n = 4, 24 fields). Data are expressed as means ± SEMs. **P < 0.01 vs. UT; ††P < 0.01 vs. MT, using nonparametric Kruskal–Wallis tests, followed by Dunn’s test

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  Fig. 5. Immunohistological staining of macrophages on day 8 post-RMI. Representative micrographs of CD68 (a), and triple CD68, vWF, and DAPI staining of the I/R region (b). Representative macrographs of CD68 and CD80 cells (proinflammatory M1) (c), and CD68 and CD163 cells (anti-inflammatory M2) (d) in the I/R region. Scale bars, 50 μm. e Quantitative analysis of macrophage infiltration (UT:24 ± 3%/field, n = 4, 24 fields of view; DT: 11 ± 2%/field, n = 6, 36 fields; MT: 24 ± 2%/field, n = 5, 30 fields; DMT: 10 ± 3%/field, n = 4, 32 fields). f M1 infiltration (UT: 9.4 ± 0.8%/field, n = 4, 24 fields; DT: 1.6 ± 0.9%/field, n = 5, 30 fields; MT: 9.8 ± 0.5%/field, n = 5, 30 fields; DMT: 1.5 ± 0.2%/field, n = 4, 24 fields). g M2 infiltration (UT: 1.5 ± 0.1%/field, n = 4, 24 fields; DT: 3.1 ± 0.5%/field, n = 5, 30 fields; MT: 1.5 ± 0.2%/field, n = 5, 30 fields; DMT: 4.8 ± 0.6%/field, n = 4, 24 fields). h M1 to M2 ratio (UT: 6.9 ± 1.7, n = 4; DT: 1.1 ± 0.6, n = 5; MT: 6.0 ± 0.9, n = 5; DMT: 0.4 ± 0.1, n = 4). M1, macrophage type 1; M2, macrophage type 2. Data are expressed as means ± SEMs. e–h Box-and-whisker plots based on macrophage, M1, and M2 infiltration and the M1/M2 ratio in all fields.…

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  Fig. 6. Analysis of myocardial apoptosis on day 8 post-RMI. a Representative micrographs of TUNEL, Cox43 (connexin 43), and DAPI staining of the I/R region. Scale bars, 50 μm. b Quantitative analysis of apoptotic cells (UT: 213 ± 3/field, n = 4, 24 fields; DT: 99 ± 8/field, n = 6, 36 fields; MT: 236 ± 31/field, n = 5, 30 fields; DMT: 39 ± 2/field, n = 4, 24 fields). Data are expressed as means ± SEMs. B Box-and-whisker plots based on apoptotic cells in all fields of each group. **P < 0.01 vs. UT; ††P < 0.01 vs. MT, using nonparametric Kruskal–Wallis tests, followed by Dunn’s test