The Stiffness of Cardiac Fibroblast Substrates Exerts a Regulatory Influence on Collagen Metabolism via α2β1 Integrin, FAK and Src Kinases.

Małgorzata Gałdyszyńska,Paulina Radwańska,Jacek Szymański,Jacek Drobnik

doi:10.3390/cells10123506

Abstract

Information about mechanical strain in the extracellular space is conducted along collagen fibers connected with integrins and then transmitted within cells. An aim of the study is to verify the hypothesis that the stiffness of cardiac human fibroblast substrates exerts a regulatory effect on collagen metabolism via integrin α2β1 and downstream signaling. The experiments were performed on human cardiac fibroblasts cultured on stiff or soft polyacrylamide gels. Extracellular and intracellular collagen content, metalloproteinase-1 (MMP-1), metalloproteinase-9 (MMP-9) and expression of the α1 chain of the procollagen type I gene (Col1A1) were elevated in cultures settled on soft substrate. The substrate stiffness did not modify tissue inhibitors of matrix metalloproteinase capacity (TIMPs 1–4). Integrin α2β1 inhibition (TC-I 15) or α2 subunit silencing resulted in augmentation of collagen content within the culture. Expression of Col1A1 and Col3A1 genes was increased in TC-I 15-treated fibroblasts. Total and phosphorylated levels of both FAK and Src kinases were elevated in fibroblasts cultured on stiff substrate. Inhibition of FAK (FAK kinase inhibitor 14) or Src kinase (AZM 47527) increased collagen content within the culture. The substrate stiffness exerted a regulatory influence on collagen metabolism via integrin α2β1 and its downstream signaling (FAK and Src kinases) in cardiac fibroblasts.

Highlights

The heart comprises several types of cells, including cardiomyocytes, cardiac fibroblasts and smooth muscle cells [1,2]
Cardiac fibroblasts account for 70% of heart cells; they are involved in the regulation of extracellular matrix metabolism (ECM) and release cytokines that may induce cardiomyocyte hypertrophy [4]
Our findings indicate that α2 integrin subunit expression occurs in cardiac fibroblasts settled on both soft and stiff gels (Figure 1A,B)

Summary

Introduction

The heart comprises several types of cells, including cardiomyocytes, cardiac fibroblasts and smooth muscle cells [1,2]. To function correctly as a pump, the cardiomyocytes provide contractility; these cells are knitted together by connective tissue scaffolding [1,3]. Cardiac fibroblasts account for 70% of heart cells; they are involved in the regulation of extracellular matrix metabolism (ECM) and release cytokines that may induce cardiomyocyte hypertrophy [4]. Several myocardial diseases are characterized by fibrosis, an excessive accumulation of collagen that leads to changes in the composition and structure of heart connective tissue. Excessive extracellular matrix accumulation modifies the mechanical properties of the heart, leading to an abnormal distribution of mechanical forces [3]. The synthesis of collagen is accompanied by secretion of matrix metalloproteinases (MMPs) [1,2]

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