Myosin VI and cardiomyopathy: Left ventricular hypertrophy, fibrosis, and both cardiac and pulmonary vascular endothelial cell defects in the Snell's waltzer mouse

Abstract

In mice and humans, loss of myosin VI (Myo6) function results in deafness, and certain Myo6 mutations also result in cardiomyopathies in humans. The current studies have utilized the Snell's waltzer (sv) mouse (a functional null mutation for Myo6) to determine if this mouse also exhibits cardiac defects and thus used to determine the cellular and molecular basis for Myo6-associated heart disease. Myo6 is expressed in mouse heart where it is predominantly expressed in vascular endothelial cells (VECs) based on co-localization with the VEC cell marker CD31. Sv/sv heart mass is significantly greater than that of sv/+ littermates, a result of left ventricle hypertrophy. The left ventricle of the sv/sv exhibits extensive fibrosis, both interstitial and perivascular, based on histologic staining, and immunolocalization of several markers for fibrosis including fibronectin, collagen IV, and the fibroblast marker vimentin. Myo6 is also expressed in lung VECs but not in VECs of intestine, kidney, or liver. Sv/sv lungs exhibit increased periaveolar fibrosis and enlarged air sacs. Electron microscopy of sv/sv cardiac and lung VECs revealed abnormal ultrastructure, including luminal protrusions and increased numbers of cytoplasmic vesicles. Previous studies have shown that loss of function of either Myo6 or its adaptor binding partner synectin/GIPC results in impaired arterial development due to defects in VEGF signaling. However, examination of synectin/GIPC-/- heart revealed no fibrosis or significantly altered VEC ultrastructure, suggesting that the cardiac and lung defects observed in the sv/sv mouse are not due to Myo6 function in arterial development.

Keywords: GIPC; Myo6; caveoli; fibrosis; vascular endothelial cell.

Fig. 1. Myo6 is expressed in the heart

Localization of Myo6 (A, C, E, F) and F-actin (B, C, E, F) in sv/+ (A–C) and sv/sv (D–F) heart. Merged images (C, F) also show Hoechst nuclear staining in blue. Myo6 is predominantly expressed in presumed VECs (see Fig. 2) although there is diffuse muscle staining above background levels observed in sv/sv heart. Inset Fig. 1A: Myo6 immunoblot of heart tissues homogenates from sv/+ and sv/sv mice. Bar: 35 μm.

Fig. 2. Myo6 is highly expressed in cardiac VECs

Localization of Myo6 (A, C, D, F) and the VEC marker CD31 (B, C, E, F) in sv/+ heart. Low (A–C) and higher magnification (D–F) images show colocalization of Myo6 and CD31 in both capillaries and larger vessels. Merged images also show Hoechst stained nuclei in blue. Bar, A–C: 35 μm; D–F: 17 μm.

Fig. 3. Myo6 is expressed in VECs of the lung

Localization of Myo6 (A, C) and CD31 (B, C) in sv/+ lung. Bar: 35 μm.

Fig. 4. Myo6 is not expressed in the VECs of intestine, kidney or liver

Localization of CD31 (A, C, D, F, G, I) and Myo6 (B, C, E, F, H, I) in sv/+ intestine (A–C), kidney (D–F), and liver (G–I). Merged images show nuclear staining in blue. Bar: 35 μm.

Fig. 5. Sv/sv mice have enlarged hearts in comparison to sv/+ littermates

(A) Images of dissected sv/+ and sv/sv hearts. B. The loss of myo6 causes a statistically significant increase in heart / body mass ratio. Sv/+ mean ratio = 4.8 ± 0.69, n = 12 mice vs. sv/sv mean ratio = 6.2 ± 0.80, n = 12 mice, *p < 0.00005. Mean body mass for sv/+ = 37.0g and mean heart weight = 171mg vs. sv/sv mean body weight = 28.4g and mean heart weight = 178mg. C. heart/body mass ratios in sh-1/+ and sh-1/sh-1 mice. Sh-1/+ mean ratio = 4.6 ± 0.50, n = 5 mice. sh-1/sh-1 mean ratio 5.4 ± 0.29, n = 5. P=0.05. Mean body mass for Sh-1/+ = 38.9g and mean heart mass = 179mg vs. sh-1/sh-1 mean body mass = 27.6 and mean heart mass = 160mg.

Fig. 6. Left ventricular hypertrophy and fibrosis in the sv/sv heart

Masson trichrome stained sections of sv/+ (A) and sv/sv (B–D) heart. Note the enlargement of the left ventricle (B) and trichrome (blue) stained interstitial and perivascular fibrotic regions in the sv/sv heart (C, D). (E, F)) H&E staining of non-fibrotic (E) and fibrotic regions (F) of sv/sv heart reveals the presence of non-cardiomyocte cells in fibrotic regions. Bar A, B: 1 mm; C, D: 100 μm; E, F: 25 μm.

Fig. 7. Elevated expression of markers for fibrosis in the sv/sv heart

Localization of F-actin (A–F; red), collagen (A, D; green), fibronectin (B, E; green), and vimentin (C, F) in sv/+ (A–C) and sv/sv (D–F) heart. Hoechst-stained nuclei are shown in blue. Bar: 35 μm.

Fig. 8. Young sv/sv mice exhibit perivascular fibrosis

Localization of the fibrosis marker, fibronectin (A, C) and F-actin (B, C) in 5 week old sv/sv heart. Merged image (C) also shows nuclei in blue. Note the prominent perivascular fibrosis. Bar: 50 μm.

Fig. 9. Increased fibrosis and enlarged air sacs in the sv/sv lung

Low (A, B) and higher magnification (C, D) images of Masson trichrome stained sections of sv/+ (A, C) and sv/sv (B, D) lung. Note the increased peri-aveolar air sac trichrome ECM staining and enlarged air sacs in the sv/sv lung. Bar A, B: 50 μm; C, D: 20 μm.

Fig. 10. Elevated expression of fibronectin in the sv/sv lung

Localization of CD31 (A, C, D, F) and the fibrosis marker fibronectin (B, C, E, F) in sv/+ (A–C) and sv/sv (D–F) lung. Merged images (C, F) show nuclei stained in blue. Bar: 35 μm.

Fig. 11. Increased apoptosis in sv/sv lung

(A, B) TUNEL staining of apoptotic nuclei (green) in sv/+ (A) and sv/sv (B) lung. F-actin staining is shown in red. (C) Quantification of TUNEL positive nuclei/ 450 μm × 450 μm image (10 images/genotype). There is a significant increase (p=0.0004) in TUNEL positive nuclei in sv/sv lung.

Fig. 12. Cardiac VEC ultrastructure is perturbed in the sv/sv mouse

TEM of capillaries in sv/+ (A) and sv/sv (B–F) heart. Note the luminal protrusions (Fig 12C–E) and increased numbers of cytoplasmic vesicles in the sv/sv (B–F). Bars: 500 nm

Fig. 13. Pulmonary VEC ultrastructure is perturbed in the sv/sv mouse

TEM of capillaries in sv/+ (A) and sv/sv (B–D) lung. Note the thickening and involution of the basement membrane (B), luminal protrusions (C) and increased numbers of VEC cytoplasmic vesicles (B–D) in the sv/sv. Bars: 500 nm.

Fig. 14. Loss of synectin/GIPC function does not result in ventricular fibrosis or altered VEC ultrastructural organization

(A, B) Localization of fibronectin (green) and F-actin (red) in WT (A) and synectin/GIPC −/− heart. (C, D). TEM of capillaries in WT (A) and synectin/GIPC −/− heart. Note the absence of fibrotic regions and normal VEC ultrastructure in the synectin/GIPC −/− although as shown here there are often increased numbers of surface caveolae and cytoplasmic vesicles. Bar A, B: 100 μm. Bar C, D: 500 nm