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, prior to development of valve fibrosis and calcification, aortic regurgitation, but not aortic stenosis, was common in Wave mice. Aortic valve fibrosis, pro-fibrotic 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.

Discovery of an Experimental Model of Unicuspid Aortic Valve

“…Those mice, which are known as waved‐2 mice, however, are born with 3 discrete aortic valve cusps and rarely develop AS; the incidence of aortic valve dysfunction does not increase with age 13. Thus, to our knowledge, aortic valves in Velvet mice are unique among experimental models, in their resemblance to unicuspid valves in humans.…”

“…Slides were stained with Alizarin Red or Picrosirius Red to quantitate the amount of calcium or collagen, respectively, as described previously 12. Movat's Pentachrome stain was used to visualize proteoglycans and collagen in the aortic valve, as described previously 13. Sources of primary antibodies for immunostaining were as follows: collagen‐1 (Abcam, Cambridge, MA), α‐smooth muscle actin (α‐SMA; Abcam), Runx2 (Santa Cruz Biotechnology, Dallas, TX), and CD31 (BD Biosciences, San Jose, CA).…”

“…The apical two thirds of the LV, which had been removed to facilitate sectioning of the aortic valve, was cut in 10‐μm transverse sections, and stained with Masson's Trichrome, as previously described 13. For studies of gene expression, myocardium was homogenized in Trizol, and RNA was prepared using RNeasy mini kit (Qiagen, Germantown, MD).…”

Making “Waves” With a Murine Model of Aortic Insufficiency

“…2 This was paralleled by left ventricular hypertrophy, increased LV end diastolic dimension, and total stroke volume. Valve proteoglycan content was increased at this young age, but not calcification, demonstrating that myxomatous semilunar valves are present before the development of calcific disease.…”

“…1, 2 Hajj et al revisit a previously reported mouse homozygous for a single nucleotide mutation in the gene encoding the epidermal growth factor receptor (EGFR), resulting in a 90% global reduction in EGFR-tyrosine kinase activity. 3 Egfr Wa2/Wa2 , or waved-2 (Wave) mice, had been previously reported to develop abnormalities of the semilunar valves.…”