Spontaneous Aortic Regurgitation and Valvular Cardiomyopathy in Mice

Abstract

Objective: We studied the mechanistic links between fibrocalcific changes in the aortic valve and aortic valve function in mice homozygous for a hypomorphic epidermal growth factor receptor mutation (Wave mice). We also studied myocardial responses to aortic valve dysfunction in Wave mice.

Approach and results: At 1.5 months of age, before development of valve fibrosis and calcification, aortic regurgitation, but not aortic stenosis, was common in Wave mice. Aortic valve fibrosis, profibrotic signaling, calcification, osteogenic markers, lipid deposition, and apoptosis increased dramatically by 6 and 12 months of age in Wave mice. Aortic regurgitation remained prevalent, however, and aortic stenosis was rare, at all ages. Proteoglycan content was abnormally increased in aortic valves of Wave mice at all ages. Treatment with pioglitazone prevented abnormal valve calcification, but did not protect valve function. There was significant left ventricular volume overload, hypertrophy, and fetal gene expression, at all ages in Wave mice with aortic regurgitation. Left ventricular systolic function was normal until 6 months of age in Wave mice, but became impaired by 12 months of age. Myocardial transverse tubules were normal in the presence of left ventricular hypertrophy at 1.5 and 3 months of age, but became disrupted by 12 months of age.

Conclusions: We present the first comprehensive phenotypic and molecular characterization of spontaneous aortic regurgitation and volume-overload cardiomyopathy in an experimental model. In Wave mice, fibrocalcific changes are not linked to valve dysfunction and are epiphenomena arising from structurally incompetent myxomatous valves.

Keywords: aortic regurgitation; aortic stenosis; aortic valve disease; cardiomyopathy.

Conflict of interest statement

Disclosures: The authors have no potential conflicts of interest.

© 2015 American Heart Association, Inc.

Figures

Figure 1. Histology of aortic valve

A – C: Aortic valve collagen detected by Masson’s Trichrome or Picrosirius Red staining (for pioglitazone-treated [Pio] mice). D – F: Aortic valve calcification, assessed using Alizarin Red staining. G – I: Aortic valve lipid content, detected using Oil Red-O staining. J – L: Proteoglycan, assessed using Movat’s Pentachrome staining (blue-green). % refers to the proportion of valve tissue exhibiting positive staining. Scale bar = 500 μm. *p < 0.05 vs. age-matched Control; **p < 0.025 vs. 6 mo. Wave.

Figure 2. Immunostaining for pro-fibrotic signaling molecules in the aortic valve

Examples are shown from mice at 12 months of age. A–C: transdifferentiation from quiescent valve interstitial cells to pro-fibrotic myofibroblasts, indicated by staining for α-smooth muscle actin (α-SMA). D–F: staining for transforming growth factor-β1 (TGF-β1), a pro-fibrotic signaling molecule. G–I: staining for p-Smad2, a mediator of pro-fibrotic TGF-β1 signaling. J–L: antibody-free negative controls for immunostaining. The image in Panel L is identical to the image in Panel K, except that Panel L was electronically brightened to show the location of valve tissue (V). % refers to the proportion of valve tissue exhibiting positive staining. Autofluorescence (AF) arises from coherently arranged fibers in valve annulus, but not in valve. Scale bar = 100 μm. *p < 0.05 vs. age-matched and treatment-matched Control.

Figure 3. Immunostaining for osteogenic differentiation in the aortic valve

Examples were obtained at 12 months of age. % refers to the proportion of valve tissue exhibiting positive staining. Scale bar = 100 μm. *p

Figure 4. Proteoglycan homeostasis in aortic valve

Histologic data were obtained at 6 months of age (N = 4). Red stain = intact versican or cleaved versican, respectively (arrows); to-Pro Blue stain = nuclei. PCR data were obtained at 12 months of age (N = 6). Scale bar = 100 μm. *p vs. untreated Control.

Figure 5. Aortic valve function

A–C: M-mode echocardiograms demonstrating aortic cusp separation (blue-green line). D–F: Color Doppler frames acquired in mid-diastole. Blue jet indicates regurgitant flow across the aortic valve (AoV, white arrow). G–I: Pressure measurements during catheter pullback from left ventricle to aorta, which indicate a significant pressure gradient in the Wave mouse with normal aortic cusp separation (B, E, H). AoW aortic root wall.

Figure 6. Aortic Valve Function

A–C: echocardiographic findings. D: MRI findings at 6 mo. E, F: invasive hemodynamic findings. G: Correlation between LV stroke volume and aortic valve peak gradient. H: Lack of correlation between aortic cusp separation and peak aortic valve gradient. ACS aortic cusp separation, AS aortic stenosis, AR aortic regurgitation, AoV aortic valve, Ao aorta, LV SV left ventricular stroke volume. *p < 0.05 vs. age-matched and treatment-matched Control.

Figure 6. Aortic Valve Function

A–C: echocardiographic findings. D: MRI findings at 6 mo. E, F: invasive hemodynamic findings. G: Correlation between LV stroke volume and aortic valve peak gradient. H: Lack of correlation between aortic cusp separation and peak aortic valve gradient. ACS aortic cusp separation, AS aortic stenosis, AR aortic regurgitation, AoV aortic valve, Ao aorta, LV SV left ventricular stroke volume. *p < 0.05 vs. age-matched and treatment-matched Control.

Figure 7. Ventricular structure and function

A–D: Echocardiography. For Wave mice, data are shown only for mice with moderate or severe aortic regurgitation. E: RVEF, assessed by MRI, at 6 months of age. LV left ventricular, EDV end-diastolic volume, SV stroke volume, EF ejection fraction, RV right ventricular. *p < 0.05 vs. age-matched and treatment-matched Control.

Figure 8. T-tubule organization in myocardium

At 1.5 months of age, and at 3 months of age, when left ventricular hypertrophy is already present in Wave mice, T-tubules are regularly spaced and oriented perpendicular to myocyte long-axes in Wave myocardium (arrow). By 12 months of age, when left ventricular systolic dysfunction occurs in Wave mice, T-tubules have become disorganized. TTpower is a statistical convention used to quantify the level of organization of T-tubules. (See Reference .) Sample sizes for WT and Wave mice at the 3 ages are 3,3; 2,3; and 4,8. Ten confocal micrographs were analyzed for each mouse. Scale bar = 50 μm. *p < 0.05 vs. Control.