ABSTRACT: Aims and introduction: Cardiac fibrosis is the hallmark of cardiac disease, particularly myocardial infarction (MI), and is one of the most prevalent causes of heart failure. MI is intricately linked to inflammation, extracellular matrix (ECM) remodelling, and cardiac fibrosis, however the mechanisms underpinning these events remain poorly understood. We recently identified that ECM1 has potential to play a key role in cardiac wound healing but the cellular origins in the heart and specific mechanisms of ECM1 in fibrosis remain elusive. Therefore, we investigated the spatiotemporal, cell specific, expression profile of ECM1 in the healthy and diseased mouse and human heart and investigate ECM1 dependent human cardiac fibroblast (HuCFb) cell signalling mechanisms. Methods and results: Western blotting, immunohistochemistry (IHC) and mRNA in-situ hybridisation revealed that ECM1 protein expression was significantly upregulated in human ischaemic and dilated heart failure patients, and predominantly localised interstitially to fibrotic, inflammatory, and peri-vascular areas. ECM1 specific analysis of the Litviňuková et al. (2020) single-cell and -nuclei RNA sequencing (sc/snRNAseq) dataset of healthy human hearts, and the Farbehi et al. (2019) scRNAseq dataset of mouse hearts post-MI demonstrated that ECM1 expression originates from fibroblasts, macrophages, and pericytes/vascular mural cells in the heart. Further, we identify that post-MI ECM1 is expressed in a temporal dynamic flux from unactivated fibroblasts in sham-operated mice, to M1 macrophages (M1MΦ) at day-3, and myofibroblasts and M2MΦ at day-7. Phosphoproteomics of ECM1 treated human cardiac fibroblast cells (HuCFb) alongside ECM1 to HuCFb cell surface protein binding pull-down and mass spectrometry, revealed that ECM1 signalling goes via proteins associated with Rho protein signal transduction, cell-cell adhesion, microtubule dynamics, and cell chemotaxis pathways, potentially initiated by ECM1 to CTNND1 binding on the cell surface. Further mechanistic analyses identified that ECM1 inhibits HuCFb cell migration and stimulates inflammatory (IL-6 and IL-1β mRNA expression), fibrotic (TGF-β1, TGF-β2 and Col1a2 mRNA expression), and non-canonical Wnt signalling pathways (Wnt5a mRNA expression and JNK1/2/3 and MYPT1 phosphorylation). Conclusions: This study demonstrates that ECM1 expression originates from macrophages and fibroblasts in the mouse and human heart. ECM1 has significant effects on HuCFb migration, inflammatory, fibrotic, Rho protein, and Wnt5a/non-canonical Wnt signalling pathways. Together, ECM1 plays an important role in cardiac wound healing and may represent a novel mechanism of inflammation-fibrosis crosstalk and progression post-MI.
Barbara Darnhofer 
