Effect of Neprilysin Inhibition for Ischemic Mitral Regurgitation after Myocardial Injury

Figures

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  Figure 1. (A) Two weeks after left circumflex coronary artery occlusion was performed to induce myocardial infarction and ischemic mitral regurgitation (IMR), left ventricle size and function were evaluated by magnetic resonance imaging (MRI). (B,C) Representative images of cardiac MRI and immunohistochemical staining of hearts from the IMR model group (left) and sham control group (right). Arrows indicate infarcted area and myocardial fibrosis. Scale bar = 3 mm. (D) Schematic illustration of animal experiment protocol. Rats were randomly assigned to LCZ696 treatment (sacubitril/valsartan, 60 mg/kg/day in corn oil, n = 10), valsartan treatment (30 mg/kg/d in corn oil, n = 10), or corn oil only (MR control group; n = 11) group. (E) Comparison of parameters at 2 weeks after myocardial infarction among the three groups. (F) Comparison of serial changes in body weight among the three groups. EDV indicates end-diastolic volume; ESV, end-systolic volume; EF, ejection fraction; MRI, magnetic resonance imaging; IMR, ischemic mitral regurgitation. * p < 0.05 for differences from the sham group.

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  Figure 2. (A) Comparison of serial changes in end-diastolic volume (EDV) of the left ventricle until 6 weeks after myocardial infarction (MI) among the three groups. (B) Quantitative graphs of cardiac magnetic resonance imaging (MRI) measurements, which showed that EDV was significantly lower in the LCZ696 group than in the control group. There were no significant differences between the valsartan group and the control group. (C) Comparison of representative M-mode images acquired by echocardiogram at 6 weeks after MI from the three groups. Scale bar = 5 mm. (D) Quantitative graphs of echocardiographic measurements, which showed that end-diastolic dimensions were also significantly lower in the LCZ696 group than in the control group. (E) Representative images of cardiac MRI at baseline and 6 weeks after MI and immunohistochemistry at 6 weeks from the three groups. Scale bar = 3 mm. (F) A quantitative graph, which shows that the extent of myocardial fibrosis was smaller in the LCZ696 group than in the control group. LV PSR means overall fibrotic area of LV myocardium regardless of infarct size. (G) The LCZ696 group also had significantly lower heart weight than the control group, while…

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  Figure 3. (A) Comparison of representative light microscopy image from immune-histochemical staining of the mitral leaflets among the three groups. Scale bar = 50 μm. PSR staining demonstrates marked fibrotic thickening of the mitral leaflets in the IMR control group. This thickening is associated with an increase in excessive endothelial-to-mesenchymal transition represented by α-SMA and TGF-β immunostains. (B) Quantification of the PSR collagen thickness (μm), the α-SMA- and the TGF-β-positive areas (% of the mitral leaflet). (C) Representative immunofluorescence staining images of CD31 and α-SMA in the mitral leaflets from the three groups. Scale bar = 50 μm. Nuclei were stained with DAPI (blue). Merged images are shown and quantitative graph indicates the proportion of α-SMA(+) CD31(+) cells of total CD31(+) endothelial cells. (D) Comparison of mitral leaflet area among the three groups of animal experiments. (E) Gene expression levels of neprilysin and natriuretic peptides from hMVECs. IMR indicates ischemic mitral regurgitation; PSR, picrosirius red stain; α-SMA, α-smooth muscle actin; TGF-β, transforming growth factor-β; CD31, cluster of differentiation 31; DAPI,…

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  Figure 4. (A) TGF-β protein levels in human mitral valve endothelial cells (hMVECs) treated with LCZ696 or valsartan as determined by Western blots. Values represent the fold changes relative to the mean value measured in the control group. (B) Immunoblot analysis of total ERK, phosphorylated ERK, and GAPDH from hMVECs treated with LCZ696 or valsartan after TGF-β stimulation. Quantification of ERK density is relative to GAPDH. Values represent fold changes of the mean values relative to the control group. (C) Immunoblot analysis of CD31, PECAM, and GAPDH from hMVECs treated with LCZ696 or valsartan after TGF-β stimulation. Quantification of CD31 and PECAM density is relative to GAPDH. Values represent fold changes of the mean values relative to the control group. (D) Immunoblot analysis of α-SMA and GAPDH from hMVECs treated with LCZ696 or valsartan after TGF-β stimulation. Quantification of α-SMA density is relative to GAPDH. Values represent fold changes of the mean values relative to the control group. hMVECs indicates human mitral valve endothelial cells; TGF-β, transforming growth factor-β; MV, mitral valve; hrs, hours; ERK, extracellular signal-regulated kinase; GAPDH,…

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  Figure 5. (A) Comparison of representative images from continuous-wave Doppler echocardiograms among the three groups at 6 weeks after the MI and a quantitative graph of MR VTI measurement. (B) Representative 3-chamber view images from cardiac MRI and a quantitative graph, which shows comparisons of MR jet area among the three groups. Scale bar = 3 mm. (C) Mitral annular diameter, left atrium (LA) area and regurgitant fraction were significantly lower in the LCZ696 group compared with the control group. The valsartan group was not significantly different from the control group. IMR indicates ischemic mitral regurgitation; VTI, time velocity integral; LA, left atrium. * p < 0.05 for differences from the control group. † p < 0.05 for differences from the valsartan group.

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  Figure 6. (A) Representative pressure-volume loops from the three groups. The LCZ696 and valsartan treatment groups had smaller left ventricular volume and higher end-systolic pressure-volume relation (red line) compared with the control group. (B) The NT-proBNP level after 6 weeks of LCZ696 treatment was significantly decreased compared with the baseline level immediately after MI and it was similar to that of the age-matched normal control group without MI. (C) Comparisons of treadmill exercise duration among the three groups at 6 weeks after MI. IMR indicates ischemic mitral regurgitation; NT-proBNP, N-terminal pro B -type natriuretic peptide; MI, myocardial infarction. * p < 0.05 for differences from the control group.