Cardiac leptin overexpression in the context of acute MI and reperfusion potentiates myocardial remodeling and left ventricular dysfunction

Figures

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  Fig 1. Study design. (A) Mice in both groups underwent ischemic injury by LAD ligation, which lasted 45 minutes, and received a single injection of either LepA 250μg in 200μl saline solution or 200μl saline alone at reperfusion. Echocardiography was performed at baseline, 24 hours and 30 days postoperatively; (B) Body weight (mean±SE) time course analysis.

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  Fig 2. Echocardiographic analysis of multiple morphological and functional parameters in IR/LepA and IR/saline treated mice. (A-E): Echocardiography was performed at baseline, 24 hours and 30 days postoperatively. A: Increase in left ventricle (LV) end-diastolic diameter (EDD) and end-systolic diameter (ESD); B. Increase in LV volume at diastole and systole; C. Increase in endocardial (Endo) area in diastole and systole; D. Endo area change, Endo fractional area change (FAC)%, and LV fractional shortening (FS)%; E. Ejection fraction (EF)% on day1 and day 30. Values show average percent of change ± SE from baseline measurements. In all figures orange bar depicts IR/LepA treated mice, and blue bars IR/Saline treated mice. * p<0.05, ** p<0.01 by two-tailed unpaired student’s t-test between the two groups. Error bars indicate standard error. (data collected from 14 IR/saline and 16 IR/LepA treated mice).

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  Fig 3. Kinetics of leptin and leptin receptor levels. (A-D): Gene expression time course analysis: leptin mRNA log2 fold change in expression in IR/saline and IR/LepA treated mice in the heart (A), liver (C), adipose tissue (D), Cardiac leptin receptor (lepr) mRNA log2 fold change in expression in IR/saline and IR/LepA treated mice (B). (E): Immunohistochemical analysis of leptin antigen in hearts from IR/saline and IR/LepA treated mice, at 48 hours (E-1,2) and 30 days (E-3,4) postoperatively. (F): Plasma leptin level determined by ELISA (ng/ml). In one separate group mice were treated by a single IP injection of LepA or saline without IR, while other mice were subjected to IR/LepA or IR/saline treatment. Note, peak of plasma leptin at 7.5 hours in both groups, subsiding to normal levels by 24 hours. (6 samples from each group were used for leptin IHC analysis).

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  Fig 4. LV hypertrophy and perivascular fibrosis. (A1-2,B) Hypertrophy assessment—cardiac muscle fiber cross section area (μm2) in IR/saline vs IR/LepA treated mice on POD-30, using Masson trichrome stained slides, scale bar 50μm, p<0.001, two tailed t-test; (C) Heart weight to body weight ratio (HW/BW) in IR/saline vs IR/LepA treated mice, p<0.05; (D) LV mass on POD-30 in IR/saline vs IR/LepA. (E) Gene expression of anp mRNA log2 fold change in expression in IR/saline and IR/LepA treated mice in the heart; (F) Perivascular fibrosis in hearts of IR/saline vs IR/LepA treated mice on POD 30, scale bar 100μm, p<0.001, two tailed t-test (F). (6 samples from each group were assessed).

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  Fig 5. Inflammatory infiltration and angiogenesis. (A) Representative macrophage infiltration and angiogenesis in POD 30 heart samples: Macrophage infiltration in IR/saline vs (B) IR/saline treated mice demonstrated by IHC for Mac2 antigen, scale bar 400μm and 200 μm for both samples. (C,D) Angiogenesis assessment by immunohistochemistry for VWF in the same heart samples. Scale bar 100μm. (5 samples from each group were assessed).

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  Fig 6. Analysis of cardiac signaling pathways. Gene expression time course analysis of tgfb1 (A) and smad2 (B) log2 fold change in expression in IR/saline vs IR/LepA treated mice in the heart. IHC analysis of phosphorylated Smad2 in heart samples from POD-30 collected from IR/saline (C1) and IR/LepA (C2) treated mice. Scale bar 50μm; p<0.001, two tailed t-test (D). Arrows point at positive nuclear signal, stained brown. Time course analysis for STAT3 mRNA, log2 fold change in expression in IR/saline and IR/LepA POD-30 cardiac samples (F). IHC analysis of phosphorylated STAT3 in heart samples from POD-30 collected from IR/saline (E1) and IR/LepA (E2) treated mice. Scale bar 50μm. Arrowheads point at positive nuclear signal, stained light brown. (5 samples from each group were used for IHC analysis).