An Investigation of Fibulin-2 in Hypertrophic Cardiomyopathy

Abstract

Hypertrophic cardiomyopathy (HCM) is the most common inherited heart muscle disease, with a prevalence of at least 1 in 500 in the general population. The disease is pleiotropic and is characterized by an increased stiffness of the myocardium, partly due to changes in the extracellular matrix (ECM), with elevated levels of interstitial fibrosis. Myocardial fibrosis is linked to impaired diastolic function and possibly phenotypic heterogeneity of HCM. The ECM consists of a very large number of proteins, which actively interact with each other as well as with myocardial cells. The role of other multiple components of the ECM in HCM has not been defined. Fibulin-2 is a glycoprotein component of the ECM, which plays an important role during embryogenesis of the heart; however, its role in adult myocardium has not been adequately studied. We here describe, for the first time, abnormal expression of fibulin-2 in the myocardium in patients with HCM as compared to normal controls. This abnormal expression was localized in the cytoplasm of myocardial cells and in the interstitial fibroblasts. In addition, fibulin-2 levels, measured by ELISA, were significantly elevated in the serum of patients with HCM as compared to normal controls.

Keywords: cardiac remodeling; extracellular matrix; fibroblast; fibrosis; hypertrophy.

Figure 1

Histological characterization of HCM myectomy tissues (A) Histological characterization of HCM tissues compared to control tissues using hematoxylin and eosin (H&E) staining, WGA staining, Picro-sirius staining, and TGFβ1 expression, assessed by immunohistochemistry. Scale bars are 100 (overview) and 20 µm (magnified insets). (B) Bar plots show a significant increase of cardiomyocytes size (µm²) (6 HCM vs. 3 ctrls), interstitial fibrosis index (79 HCM vs. 9 ctrls), and TGFβ1 levels (79 HCM vs. 9 ctrls).

Figure 2

FBLN2 expression and localization in HCM myectomy tissues along with Col IV and TGFβ1. (A) A representative transmural section of a myectomy specimen stained with picro-Sirius red and shows fibrosis distribution in the myocardium and sub-endocardium. Scale bar is 1 mm. (B) Picro-sirius red and immunohistochemical analysis of HCM tissues at the myocardium and sub-endocardial (n = 79), compared to controls (n = 9), showing FBLN2, Col IV, and TGFβ1 expression. Scale bars are 100 (overview) and 20 µm (magnified insets). (C) Confocal microscopy imaging shows Vim+ and SMA+ cells (red) infiltrating the FBLN2 sheath (green) at the sub-endocardial region (representative of 3 HCMs vs. 3 controls). Scale bars are 20 µm.

Figure 3

Elevated expression of FBLN2 in HCM myectomy tissues and cultured fibroblasts. (A) A representative Immunoblot for HCM and ctrl tissues shows total FBLN2 protein expression normalized to Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) expression. (B) Bar plot shows a significant difference (P = 0.0009) between mean FBLN2 expression in HCM tissues (n = 44) compared to ctrl tissues (n = 7). (C) A representative Immunoblot for cultured HCM and ctrl fibroblasts shows total FBLN2 protein expression normalized to GAPDH expression. (D) Bar plot shows an elevated level of FBLN2 in cultured HCM fibroblasts (n = 34) compared to ctrl fibroblasts (n = 5). E and F: Confocal microscope imaging of cultured HCM (n = 3) and control fibroblasts (n = 3), shows FBLN2 expression pattern and localization. Cells were co-stained with FBLN2 and vimentin (mesenchymal fibroblasts marker) (E), and FBLN2 with smooth muscle actin (myofibroblasts marker) (F). Scale bars are 20 µm.

Figure 4

Increased FBLN2 expression in the serum HCM patients. A boxplot shows a significant increase in circulating FBLN2 (pg/mL) in HCM patients’ serum (n = 90) compared to healthy subjects (n = 37).