Abstract
Introduction
Hypertrophic cardiomyopathy (HCM) is an inherited cardiac disease caused by mutations in genes encoding cardiac sarcomere proteins. A keyhallmark of the disease is cardiac interstitial fibrosis, promoted by cardiac fibroblasts (CFs) [1]. Because CFs are mechanosensitive, extracellular matrix (ECM) stiffening may contribute to increase fibrosis and leading to disease progression. TGF-β is a knownprofibrotic regulator, able to activate fibroblasts into myofibroblasts causing a further ECM deposition and stiffening. Here, we report on the contribution of CFs in the composition and mechanics of cardiac microtissue.
Methods
Four different cell spheroid (CS) groups (CU: control untreated; CT: control+TGF-β; HCMU: diseased untreated; HCMT: diseased+TGF-β) were derived from primary human CFs. Samples were collected after informed consent and ethical approval. CSs were compressed (parallel-plate, MicroSquisher, CellScale) up to 50% apparent linear strain at 0.5µm/s displacement rate. The compression modulus of CSs was estimated by fitting force-displacement (F–δ) data with the extended Tatara model [2]. Viscoelasticity of CSs was studied by performing stress relaxation tests at 50% strain level with a ramp rate of 5µm/s and fitting stress-time data with the standard linear solid (SLS) model. Finally, the composition of CSs was assessed with immunofluorescent imaging (Vimentin for fibroblast identification) and Picrosirius red staining (for collagen) using the Zeiss LSM800 confocal microscope.
Results
HCMU spheroids exhibit a nearly three-fold higher stiffness compared to CU (i.e., diseased vs. healthy) both in untreated and treated conditions. Additionally, treatment with TGF-β resulted in approximately 2-fold stiffening of CSs in both healthy controls (CU vs. CT) and diseased samples (HCMU vs. HCMT) (Figure 1a). The viscosity was significantly higher in HCMT compared to CT, while no significant increase was observed in HCMU vs. CU samples (Figure 1b). Picrosirius red staining (Figure 1c) suggested increased amount of collagen in TGF-β – treated HCM CSs (Figure 1c), while vimentin signal increased significantly (p=0.0082) in HCMT vs. CT samples.
Discussion
CSs derived from HCM fibroblasts show an increased stiffness, in both treated and untreated samples. In particular stiffening in TGF-β – treated samples may be explained by the increased deposition of collagen, measured here as an increase in picrosirius red staining signal. Furthermore, the increased viscosity of HCMT vs. CT is accompanied by the increased vimentin signal intensity. This suggests that the increased number of fibroblasts may contribute further to the viscosity of CSs. HCM pathogenesis of cardiac fibrosis is yet to be fully understood, and multiple mechanisms could contribute to tissue stiffening and disease progression, which can be studied with the models presented in a patient-specific manner.