Abstract 15133: Matricellular Protein CCN1 Regulates Cardiac Fibroblast ECM Synthesis and Alignment

Abstract

Introduction: Cellular communication network (CCN) proteins constitute a 6-member family of extracellular matrix (ECM)-associated signaling proteins that have important roles in wound healing and tissue fibrosis. Though their function in the heart is not fully explicated, CCN1 is often indicated as an antifibrotic factor. Yet, CCN1 levels correlate with infarct size and ventricular dysfunction in myocardial infarct patients—signifying a pro-fibrotic role for this factor in the heart. Objective: To determine the contribution of endogenous CCN1 to fibroblast (FB) ECM synthesis and organization. Methods: In vitro murine cardiac FB ECM synthesis, activation, proliferation, and matrix organization was evaluated in cells treated with CCN1 or non-target (NT) siRNA, by qPCR/immunoblotting for collagens and synthetic enzymes, EdU incorporation, and type I collagen immunostaining of decellularized matrices, respectively. CCN1-dependent integrin pathways modulating FB collagen synthetic enzyme expression were identified and interrogated via in vitro immunoprecipitation and RNAi-mediated depletion. Results: CCN1-deficient FBs showed reduced expression of type I collagen and collagen processing enzymes ( P4ha1 , P4hb , Plod1 , Plod2 , Loxl2 ). siRNA-mediated knockdown of CCN1 did not impact FB activation (α-SMA), but significantly reduced the expression of collagen synthetic/processing enzymes ( P4ha1 , Plod1 , Plod2 , Loxl2 ). Deficiency of CCN1 in both unstimulated and TGFβ1-stimulated FBs resulted in reduced proliferative capacity (7.17%, p=0.0205 and 5.34%, p=0.0428, respectively, relative to NT, n=3). CCN1 depletion promoted the alignment of FB-derived collagen fibers (0.30 ± 0.03 vs. 0.58 ± 0.04; mean ± SEM, p<0.0001, n=6). An interaction between CCN1 and integrin αvβ3 was validated; moreover, knockdown of integrin β3 reduced the expression of enzymes associated with collagen cross-linking ( Plod1 , Plod2 , Loxl2 ), partly phenocopying CCN1 depletion. Conclusions: The data suggest that CCN1 drives pro-fibrotic signaling and limits ECM organization in cardiac FBs. This may have implications for CCN1 in the infarcted heart by promoting scar establishment; however, long-term expression could lead to progressive detrimental remodeling.