Hyaluronidase 2 Deficiency Causes Increased Mesenchymal Cells, Congenital Heart Defects, and Heart Failure

Abstract

Background: Hyaluronan (HA) is required for endothelial-to-mesenchymal transition and normal heart development in the mouse. Heart abnormalities in hyaluronidase 2 (HYAL2)-deficient (Hyal2^-/- ) mice and humans suggested removal of HA is also important for normal heart development. We have performed longitudinal studies of heart structure and function in Hyal2^-/- mice to determine when, and how, HYAL2 deficiency leads to these abnormalities.

Methods and results: Echocardiography revealed atrial enlargement, atrial tissue masses, and valvular thickening at 4 weeks of age, as well as diastolic dysfunction that progressed with age, in Hyal2^-/- mice. These abnormalities were associated with increased HA, vimentin-positive cells, and fibrosis in Hyal2^-/- compared with control mice. Based on the severity of heart dysfunction, acute and chronic groups of Hyal2^-/- mice that died at an average of 12 and 25 weeks respectively, were defined. Increased HA levels and mesenchymal cells, but not vascular endothelial growth factor in Hyal2^-/- embryonic hearts, suggest that HYAL2 is important to inhibit endothelial-to-mesenchymal transition. Consistent with this, in wild-type embryos, HYAL2 and HA were readily detected, and HA levels decreased with age.

Conclusions: These data demonstrate that disruption of normal HA catabolism in Hyal2^-/- mice causes increased HA, which may promote endothelial-to-mesenchymal transition and proliferation of mesenchymal cells. Excess endothelial-to-mesenchymal transition, resulting in increased mesenchymal cells, is the likely cause of morphological heart abnormalities in both humans and mice. In mice, these abnormalities result in progressive and severe diastolic dysfunction, culminating in heart failure.

Keywords: cor triatriatum; developmental biology; endocardium; extracellular matrix; live birth.

Figure 1.

Structural abnormalities in hyaluronidase 2 (HYAL2)–deficient (Hyal2^−/−) mouse hearts. A–C, High-resolution micro-computed tomographic images of Hyal2^−/− and control hearts. Micro-computed tomographic images were reconstructed in 3-dimensional and colorized to enhance structural visualization of the atria (dashed lines). Hyal2^−/− mice with a grossly enlarged atrium (A) were deemed acute, whereas those with a mildly enlarged atrium (B) were deemed as chronic. A heart from a control mouse is shown in (C). D–F, Ultrasound images of Hyal2^−/− and control hearts. Bright-mode images of the heart showed an enlarged and dense left atrium (dashed lines) in the acute Hyal2^−/− mice compared with chronic Hyal2^−/− and control mice. The increased density is indicated in the image by the stronger white signal. G, Atrium diameter in Hyal2^−/− and control mice. The diameter of the atrium was significantly larger in both acute and chronic Hyal2^−/− mice compared with controls. H, Atrial valve (AV) and (I) mitral valve (MV) thickness was significantly increased in acute and chronic Hyal2^−/− mice compared with controls. *, †, ‡P<0.05; **, ††P<0.001; ***, †††, ‡‡‡, §§§P<0.0001. The number of animals used were for atrial enlargement control and chronic (n=6); acute (at 1 mo n=7; 2 mo n=4; and 3 mo n=3), for AV thickness control and chronic (n=6); acute (at 1 mo n=7; 2 mo n=4, and 3 mo n=3), for MV thickness control and chronic (n=6), acute (at 1 mo n=6; 2 mo n=4, and 3 mo n=3).

Figure 2.

Cardiac function in hyaluronidase 2 (HYAL2)–deficient (Hyal2^−/−) and control mice. Pulse-wave Doppler images were used to measure early to late atrial filling (E/A; A) and isovolumic relaxation time (IVRT; B). A and B, Acute and chronic Hyal2^−/− mice showed significant impairment at all ages compared with controls. There was no significant difference between acute and chronic Hyal2^−/− mice except at 4 wk where the IVRT of the acute group was significantly prolonged compared with the chronic group. C and D, Motion-mode images were used to measure systolic parameters including ejection fraction (EF) and fractional shortening (FS). No significant difference in the EF or FS was found between Hyal2^−/− and control mice, although the function was trending downward in the acute Hyal2^−/− mice at the last measurement before a humane end point was reached. E, Left ventricular (LV) mass. LV mass increased progressively in both the acute and chronic groups of Hyal2^−/− mice compared with controls. F, The myocardial performance index (MPI)=(IVRT+IVCT)/AET was increased significantly at all ages in acute and chronic groups of Hyal2^−/− mice compared with controls (AET indicates aortic ejection time; and IVCT, isovolumic contraction time). G, Cardiac output (CO) was significantly impaired in the acute group of Hyal2^−/− mice compared with controls, although the chronic group of Hyal2^−/− mice also showed decreased CO at most ages. *, †, ‡P<0.05; **, ††P<0.001; ***, †††, ‡‡‡P<0.0001, n=3 to 7 per group at all ages.

Figure 3.

Histological analysis of hyaluronidase 2 (HYAL2)–deficient (Hyal2^−/−) and control hearts. Transverse sections of hearts from Hyal2^−/− (acute and chronic) and control mice were compared for differences in morphology and structure. A–C, Images of hematoxylin and eosin–stained sections revealed an enlarged atrium (arrow) in both the acute (A) and chronic (B) groups of Hyal2^−/− mice compared with control mice (C). *A tissue mass in the atrium of the acute Hyal2^−/− mouse. D–F, Images of hyaluronan (HA) distribution in the Hyal2^−/− and control hearts. HA was detected as a brown precipitate using the HABP (HA-binding protein). There is intense brown staining in several regions of the Hyal2^−/− hearts (open arrows in D and E), whereas the intense brown staining is limited to the valves in the control hearts (open arrow in F). G–I, Masson trichrome staining of Hyal2^−/− and control hearts. Masson trichrome stains the extracellular matrix (ECM) components collagen and elastin as blue, and glycosaminoglycans remain unstained. Excess ECM indicating fibrosis (green arrows) is widespread in the Hyal2^−/− hearts compared with the control heart (I). G’–I’, Enlarged view of the area in the box in (G)–(I). J–L, Detection of mesenchymal cells in Hyal2^−/− and control hearts. Anti-vimentin (brown) indicates the presence of mesenchymal cells. There are increased numbers of vimentin-positive cells (arrow) in both the acute and chronic Hyal2^−/− atria (J and K) compared with the control atrium (L). M, Semi-quantitative analysis of vimentin-positive cells in Hyal2^−/− and control atria. Significantly increased numbers of vimentin-positive cells are present in Hyal2^−/− atria compared with control atria and significantly more in the acute Hyal2^−/− than in control Hyal2^−/− atria. N and O, Vimentin protein levels in Hyal2^−/− and controls hearts (atrium and ventricle). N, Western blot analysis showed increased expression of vimentin in the Hyal2^−/− heart compared with controls. GAPDH was used as protein-loading control. O, Quantification of vimentin levels in (N). The chemiluminescent images from Western blot analysis of vimentin from Hyal2^−/− and control hearts (n=4) were quantified using a BioRad ChemiDoc. The columns represent the average level of vimentin±SEM (n=4). Significance was determined using the Student t test. Bar=50 μm. The images in this figure are representative of those from 7 pairs of Hyal2^−/− and control mice.

Figure 4.

Histological analysis of Hyal2^−/− and control hearts at embryonic day (E) 18.5. Paraffin sections from E18.5 embryos were stained or used for immunohistochemistry. A and B, Images of hematoxylin and eosin–stained embryo sections revealed an enlarged atrium (open arrow) and the presence of fibrosis (arrow) in the Hyal2^−/− heart (A) compared with the control (B) heart. (Hyal2^−/−, n=8; control, n=6). C–F, Enlarged view of the area in the box in (A) and (B) stained for hyaluronan (HA). C and D, Images showing increased HA (brown) in the Hyal2^−/− atrium (C) compared with the control atrium (D; n=3). E and F, Detection of vimentin-positive cells (brown) revealed an excess in the Hyal2^−/− atrium (E) compared with the control (F; n=3). G–H, Semi-quantitative analysis of vimentin-positive cells in the atrium (G; n=3) and ventricle (H; n=4) of Hyal2^−/− and control hearts. There were significantly increased numbers of mesenchymal cells in the Hyal2^−/− hearts compared with controls. Bar=50 μm. LA, left atrium; LV, left ventricle; and RV; right ventricle. *P<0.05; **P<0.001.

Figure 5.

Hyaluronidase 2 (HYAL2) distribution in embryonic hearts. Sections of the embryonic heart at embryonic day (E) 8.5, from a previous study, were used for the detection of HYAL2 using immunofluorescent and immunohistochemical approaches. A–C, Detection of HYAL2 (red) in the endocardial lining of the blood vessels of the E8.5 heart. Nuclei are stained blue with Hoechst 33342. D–F, Enlarged view of the image in (A)–(C). G–J, HYAL2 distribution in E14.5 and E18.5 embryos. The brown staining indicates the presence of HYAL2 in the endothelial cells of blood vessels and heart valves of wild-type heart at E14.5 (H; n=3) and E18.5 (J; n=3), respectively. As expected, this signal is absent in the Hyal2^−/− hearts (G and I; n=3). Bar=50 μm.

Figure 6.

Histological analysis of Hyal2^−/− and control hearts at embryonic day (E) 14.5. A–B, Images of hematoxylin and eosin–stained sections show excess tissue in the ventricle in hyaluronidase 2 (HYAL2)–deficient (Hyal2^−/−; A) compared with control (B) hearts. C and D, Detection of hyaluronan (HA; brown) showed increased HA in the Hyal2^−/− ventricle (C) compared with the control (D). E and F, Vimentin-positive cells were found to be more abundant in the Hyal2^−/− heart (E) compared with the control (F). G and H, VEGF (brown) seemed to be more abundant in the Hyal2^−/− (G) than in control (H) heart. I and J, Semi-quantitative analysis of vimentin-positive cells in the atrium (I) and ventricle (J) of Hyal2^−/− and control hearts at E14.5. The number of mesenchymal cells in the Hyal2^−/− hearts was significantly increased compared with controls. Bar=50 μm. **P<0.001, n=3; LA indicates left atrium; and V, ventricle.