Abstract Session

Fibrosing rheumatic diseases (scleroderma, MCTD, IgG4-related disease, scleroderma mimics)

Session: Abstracts: Systemic Sclerosis and Related Disorders – Basic Science (1621–1624)

1623: Combined Single-Cell, Bulk RNA Sequencing and Proteomics Analysis Reveals New Candidate Targets Involved in Myocardial Fibrogenesis

Sunday, November 13, 2022

5:30 PM – 5:40 PM Eastern Time

Location: Room 201

Presenting Author(s)

IK

Ievgeniia Kocherova, MSc

University of Zurich
Schlieren, Switzerland

Ievgeniia Kocherova¹, Elena Pachera², Daria Nurzynska³, Franca Di Meglio³, Oliver Distler⁴, Przemysław Błyszczuk² and Gabriela Kania², ¹Center of Experimental Rheumatology, Department of Rheumatology, University Hospital Zurich, University of Zurich, Schlieren, Switzerland, ²Center of Experimental Rheumatology, Department of Rheumatology, University Hospital Zurich, University of Zurich, Zürich, Switzerland, ³Department of Public Health, University of Naples "Federico II", Naples, Italy, ⁴Department of Rheumatology, University Hospital Zurich, University of Zurich, Zürich, Switzerland

Background/Purpose: Inflammatory dilated cardiomyopathy (iDCM) often leads to heart failure (HF), which is the main cause of cardiac mortality in patients with systemic autoimmune diseases. Fibroblast activation, driven by the activator protein 1 family member Fos-related antigen-2 (FOSL-2), represents a critical step in cardiac fibrogenesis. The existing antifibrotic therapies demonstrate limited efficacy against myocardial fibrosis. We aimed to identify new candidate targets implicated in cardiac fibrogenesis under immunofibrotic conditions.

Methods: Cardiac fibroblasts were isolated from the left atria of patients (n=5) undergoing heart transplantation due to HF associated with iDCM and from unaffected hearts of brain-dead donors (Ctrl, n=5). Protein identification and quantification was performed using liquid chromatography tandem-mass spectrometry (LC–MS/MS). The data were analysed with MaxQuant v1.6.2.3 software. Bulk RNA sequencing (RNA-seq) was conducted using the Illumina HiSeq platform. Differentially expressed genes were identified using DESeq2. Publicly available single-cell (sc) RNA sequencing datasets (GSE109816, GSE121893) on adult hearts from HF patients (n=6) and Ctrl (n=14) were analysed using Seurat package (V.2.3.4). Specific gene knockdown was achieved by siRNA transfection of human foetal cardiac fibroblasts (HCFs), untreated or stimulated with TGF-β for 48-72h. Profibrotic marker expression was assessed using RT-qPCR and Western Blot. Cell viability was measured using PrestoBlue HS reagent, and ATP production was quantified with CellTiter-Glo assay.

Results: Single-cell, bulk RNA sequencing and LC-MS/MS analysis identified matrix remodelling associated protein MXRA5, transcription factor FOXF1 and membrane protein dysferlin (DYSF) as candidate targets significantly upregulated in HF (Fig 1). Further in vitro studies on HCFs (n=4) showed that TGF-β upregulated MXRA5 (p< 0.01) and DYSF (p< 0.001) but downregulated FOXF1 (p< 0.05). STRING database indicated a putative interaction between the candidate targets and known profibrotic markers (Fig 2). In vitro, MXRA5 silencing (n=8) resulted in the upregulation of DYSF (p< 0.05), ACTA2 (p< 0.05) and COL1A1 (p< 0.001) in untreated HCFs, and also upregulated DYSF (p< 0.01) and COL1A1 (p< 0.05) after 48h of TGF-β stimulation. FOXF1 knockdown in HCFs (n=8) followed by 48h of TGF-β stimulation downregulated MXRA5 (p< 0.01) and ACTA2 (p=0.06), and upregulated DYSF (p< 0.001) and COL1A1 (p=0.05). DYSF silencing (n=4) upregulated MXRA5 after 48h of TGF-β stimulation (p< 0.05), downregulated ACTA2 (48h and 72h of TGF-β stimulation, p< 0.05), and upregulated FOSL-2 protein levels in untreated HCFs and 72h after TGF-β stimulation (n=3, p< 0.05). Candidate targets knockdown reduced cell viability in untreated (n=4, DYSF: p< 0.01, MXRA5: p< 0.001, FOXF1: p< 0.01) and TGF-β stimulated (n=4, DYSF: p< 0.05, MXRA5: p< 0.05, FOXF1: p< 0.01) HCFs. ATP levels were decreased in TGF-β-stimulated HCFs after DYSF silencing (n=6, p=0.05).

Conclusion: We identified DYSF, MXRA5 and FOXF1 in cardiac fibroblasts as potential therapeutic targets implicated in myocardial fibrogenesis, including the regulation of profibrotic transcription factor FOSL-2.

Figure 1. Multi-omics analysis of human cardiac fibroblasts in homeostasis and HF. (A) Bulk RNA-seq analysis identified a total of 67 significantly differentially expressed genes (absolute log2FC>1, adj. p < 0.05). (B) The comparative analysis of bulk RNA-seq results and publicly available scRNA-seq datasets revealed two commonly upregulated genes in HF fibroblasts or their subclusters, encoding FOXF1 (log2FC=3.51, adj.p < 0.05) and MXRA5 (log2FC=2.91, adj. p < 0.05). (C) Volcano plot and (D) heatmap illustrate 14 differentially expressed proteins (absolute log2FC>1, adj. p < 0.05) in the HF group compared to Ctrl. The most upregulated protein in HF fibroblasts is DYSF (log2FC=5.78, adj. p < 0.005).

Figure 2. STRING interaction network between the new candidate targets upregulated in HF and known profibrotic markers (COL1A1, FN1, ACTA2, FOSL2). Minimum required interaction score: (A) 0.400, (B) 0.190.
Disclosures: I. Kocherova, None; E. Pachera, None; D. Nurzynska, None; F. Di Meglio, None; O. Distler, AbbVie/Abbott, Amgen, GlaxoSmithKlein(GSK), Novartis, Roche, UCB, Kymera, Mitsubishi Tanabe, Boehringer Ingelheim, 4P-Pharma, Acceleron, Alcimed, Altavant Sciences, AnaMar, Arxx, AstraZeneca, Blade Therapeutics, Bayer, Corbus Pharmaceuticals, CSL Behring, Galapagos, Glenmark, Horizon, Inventiva, Lupin, Miltenyi Biotec, Merck/MSD, Prometheus Biosciences, Redx Pharma, Roivant, Sanofi, Topadur, Pfizer, Janssen, Medscape, Patent issued “mir-29 for the treatment of systemic sclerosis” (US8247389, EP2331143), FOREUM Foundation, ERS/EULAR Guidelines, EUSTAR, SCQM (Swiss Clinical Quality Management in Rheumatic Diseases), Swiss Academy of Medical Sciences (SAMW), Hartmann Müller Foundation; P. Błyszczuk, None; G. Kania, None.