Targeted Mybpc3 Knock-Out Mice with Cardiac Hypertrophy Exhibit Structural Mitral Valve Abnormalities

Abstract

MYBPC3 mutations cause hypertrophic cardiomyopathy, which is frequently associated with mitral valve (MV) pathology. We reasoned that increased MV size is caused by localized growth factors with paracrine effects. We used high-resolution echocardiography to compare Mybpc3-null, heterozygous, and wild-type mice (n = 84, aged 3-6 months) and micro-CT for MV volume (n = 6, age 6 months). Mybpc3-null mice showed left ventricular hypertrophy, dilation, and systolic dysfunction compared to heterozygous and wild-type mice, but no systolic anterior motion of the MV or left ventricular outflow obstruction. Compared to wild-type mice, echocardiographic anterior leaflet length (adjusted for left ventricular size) was greatest in Mybpc3-null mice (1.92 ± 0.08 vs. 1.72 ± 0.08 mm, p < 0.001), as was combined leaflet thickness (0.23 ± 0.04 vs. 0.15 ± 0.02 mm, p < 0.001). Micro-CT analyses of Mybpc3-null mice demonstrated increased MV volume (0.47 ± 0.06 vs. 0.15 ± 0.06 mm³, p = 0.018) and thickness (0.35 ± 0.04 vs. 0.12 ± 0.04 mm, p = 0.002), coincident with increased markers of TGFβ activity compared to heterozygous and wild-type littermates. Similarly, excised MV from a patient with MYBPC3 mutation showed increased TGFβ activity. We conclude that MYBPC3 deficiency causes hypertrophic cardiomyopathy with increased MV leaflet length and thickness despite the absence of left ventricular outflow-tract obstruction, in parallel with increased TGFβ activity. MV changes in hypertrophic cardiomyopathy may be due to paracrine effects, which represent targets for therapeutic studies.

Keywords: TGFβ; hypertrophic cardiomyopathy; mitral valve.

Figure 1

Imaging of the MV by high-resolution echocardiography. Representative echocardiographic images are shown from Mybpc3 (−/−) (A,C,D) and (+/+); (B) mice; arrows show AML; Systolic (C) and diastolic (D) images are shown with AML and PML highlighted.

Figure 2

Mybpc3 (−/−) MVs have increased nuclear pSmad2. Immunostaining for pSmad2 was performed using AEC as the chromogen (red) according to standard protocols (Vectorlabs). Representative 40× images are shown from (+/+) (A,C) and Mybpc3 (−/−) (B,D) mice. Nuclear pSmad2 staining (arrows) is a marker for TGFβ activity. Relative quantification of immunostaining (E) was performed by blinded reviewers, using a scale from 1 (faint staining) to 5 (strong staining). Error bars show SEM.

Figure 3

Mybpc3 (−/−) MVs have increased periostin. Immunostaining for periostin was performed using DAB as the chromogen (brown). Representative low power (40×, A,B) images are shown from (+/+) (A) and Mybpc3 (−/−) (B) mice. Increased periostin (brown) is evident in the Mybpc3 (−/−) MV leaflets; (C) Western blot for quantification of periostin in MV leaflets comparing 4 (+/+) and 4 (−/−) leaflets, with use of β-actin to normalize for protein loading. Relative quantification of immunostaining (D) was performed by blinded reviewers using a scale from 1 (faint staining) to 5 (strong staining). Quantification of Western blots was performed by NIH ImageJ software, demonstrating increased periostin in both MV leaflets (E) and adjacent myocardium (F) in Mybpc3 (−/−) mice compared to wild-type controls (+/+). Error bars show SEM.

Figure 4

Mybpc3 (−/−) MVs have more collagen. MVs were stained with modified Movat’s (A–D). The Mybpc3 (−/−) MV leaflets (C,D) show more collagen (yellow−brown in C and D) than the Wild-Type (+/+) MV leaflets (A,B). All size bars correspond to 100 μM. Relative quantification of collagen (E) on Movat’s staining was performed by blinded reviewers, using a scale from 1 (blue valves without brown) to 5 (brown valves without blue). Error bars show SEM.

Figure 5

Increased expression of Ctgf and Postn in Mybpc3 (−/−) hearts. The relative mRNA expression of Ctgf, encoding connective tissue growth factor, and Postn, encoding periostin, is shown with the wild-type (+/+) level set at 1.0. Bar graphs show the average relative gene quantification (N = 3 for each genotype and each tissue) with SEM.

Figure 6

Comparison of excised MVs in an HCM patient with a MYBPC3 mutation and in two normal hearts (controls, n = 2). Findings in the patient (A) and in a representative control (B) with histological (Movat’s) and immunohistochemical stainings are shown. Boxed areas represent regions where protein stainings were captured. (A) The MV from the HCM patient exhibits a disruption of the normal stratified layers of the mitral leaflets with an increase in TGFβ downstream extracellular matrix proteins (collagen I, periostin, and hyaluronan). Higher levels of TGFβ1 were observed in the leaflets of the HCM valve as well as the TGFβ1 signaling intermediate, pSmad3. Moreover, α-SMA, a TGFβ-regulated gene and a marker of myofibroblast phenotype, was also increased in these leaflets. All proteins are depicted in red except hyaluronan, which is in green, while nuclei are blue; (C) Western analysis of excised proteins collected from the valve revealed a significant increase in both collagen 1α1 and collagen 1α2 in the HCM patient with MYBPC3 mutation compared to the controls. Shown are each of the three Westerns performed on three different areas of the same human valve on the de-crosslinked proteins from human valve sections; they were compared against comparable areas in three separate controls (two controls are shown on the Westerns). Sample size was normalized based on similar tissue volumes used in the valve extraction procedure (see Experimental Section) (C = controls, P = patient); (D) Graphical depiction of fold-change in collagen expression (N = 3).