β3 Integrin in Cardiac Fibroblast Is Critical for Extracellular Matrix Accumulation during Pressure Overload Hypertrophy in Mouse

Sundaravadivel Balasubramanian,Michael R Zile,Harinath Kasiganesan,Amy D Bradshaw,Kamala P Sundararaj,Yuhua Zhang,Dhandapani Kuppuswamy,Dorea L Pleasant,Lakeya Quinones,Mirjam M Zegers

doi:10.1371/journal.pone.0045076

Sundaravadivel Balasubramanian, Michael R Zile + Show 8 more

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https://doi.org/10.1371/journal.pone.0045076

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Abstract

The adhesion receptor β3 integrin regulates diverse cellular functions in various tissues. As β3 integrin has been implicated in extracellular matrix (ECM) remodeling, we sought to explore the role of β3 integrin in cardiac fibrosis by using wild type (WT) and β3 integrin null (β3−/−) mice for in vivo pressure overload (PO) and in vitro primary cardiac fibroblast phenotypic studies. Compared to WT mice, β3−/− mice upon pressure overload hypertrophy for 4 wk by transverse aortic constriction (TAC) showed a substantially reduced accumulation of interstitial fibronectin and collagen. Moreover, pressure overloaded LV from β3−/− mice exhibited reduced levels of both fibroblast proliferation and fibroblast-specific protein-1 (FSP1) expression in early time points of PO. To test if the observed impairment of ECM accumulation in β3−/− mice was due to compromised cardiac fibroblast function, we analyzed primary cardiac fibroblasts from WT and β3−/− mice for adhesion to ECM proteins, cell spreading, proliferation, and migration in response to platelet derived growth factor-BB (PDGF, a growth factor known to promote fibrosis) stimulation. Our results showed that β3−/− cardiac fibroblasts exhibited a significant reduction in cell-matrix adhesion, cell spreading, proliferation and migration. In addition, the activation of PDGF receptor associated tyrosine kinase and non-receptor tyrosine kinase Pyk2, upon PDGF stimulation were impaired in β3−/− cells. Adenoviral expression of a dominant negative form of Pyk2 (Y402F) resulted in reduced accumulation of fibronectin. These results indicate that β3 integrin-mediated Pyk2 signaling in cardiac fibroblasts plays a critical role in PO-induced cardiac fibrosis.

Highlights

Integrins are cell surface glycoproteins involved in a variety of cellular functions including migration, adhesion, spreading, proliferation, transcription and translation
Upon 4 wk transverse aortic constriction (TAC) induced pressure overload, there is a marked increase in collagen accumulation in the case of wild type (WT) myocardium whereas the accumulation of collagen was significantly reduced in b32/2 myocardium
In the case of b32/2 mice, extending pressure overload (PO) beyond 4 wk might be useful to know if the loss of PO-induced cardiac fibrosis in b32/2 mice was due to a delay in the synthesis and/or deposition of extracellular matrix (ECM) proteins

Summary

Introduction

Integrins are cell surface glycoproteins involved in a variety of cellular functions including migration, adhesion, spreading, proliferation, transcription and translation. In PO myocardium, others and we have documented integrin-mediated survival signaling via NFkB activation [5], cytoskeletal rearrangement via focal adhesion complex formation [6,7] and cell growth via S6K activation [8,9,10,11]. While these studies point to a direct role of integrins on cardiomyocytes, their role in cardiac fibroblasts, the other major cell type in the myocardium, has not been extensively studied. Our subsequent analysis showed a high degree of myocardial calpain activation and apoptosis in the b32/2 PO mice [14]

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