The AP-1 transcription factor Fosl-2 drives cardiac fibrosis and arrhythmias under immunofibrotic conditions

Figures

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  Fig. 1. Lin-gp38+ cell phenotyping during SSc myocardial fibrogenesis. a Quantification of Sirius Red staining for collagen, IHC for periostin (Postn) and αSMA of myocardial sections from 16 to 22-week-old mice (n = 5–9) (Mann–Whitney test, mean ± SEM). b Representative WB and densitometric analysis of αSMA protein content in homogenized hearts isolated from 16 to 22-week-old mice (n = 14–15, unpaired t-test, mean ± SEM). c Heart weight (HW)/body weight (BW) and HW/tibial length (TL) ratios at week 16–22 (n = 24–27, unpaired t-test, mean ± SEM). d Quantification of myocardial sections of gp38 IHC staining from 16–22-week-old mice (n = 7, Mann–Whitney test, mean ± SEM). e Representative flow cytometry pictures and corresponding quantification of Lin-gp38+ fibroblast frequency from the myocardium of 16–22-week-old mice (n = 13–14, Mann–Whitney test, mean ± SEM). f Co-localization of collagen and gp38 expression in myocardial sections (n = 5, scale bars: 100 µm) and IF of heart sections from 16 to 22-week-old Fosl-2wt and Fosl-2tg mice (n = 5, scale bars: 50 µm). g Spearman correlation between collagen expression quantified by Sirius Red staining and gp38 expression (n = 7–9). h…

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  Fig. 2. Fosl-2tg mice show changes in ECG and in parasympathetic/sympathetic balance. a Representative pictures of radiotelemetry ECG parameters and the heart rate variability parameters recorded within 24 h in Fosl-2wt and Fosl-2tg mice at week 16–22 (n = 12; unpaired t-test, mean ± SEM). b Analyses of PVC and VT events and AV blocks second-degree assessed by radiotelemetry and calculated as the average of 24 h of continuous ECG recording in awake Fosl-2wt and Fosl-2tg mice (n = 10, unpaired t-test, mean ± SEM). c Representative radiotelemetry ECG pictures of PVCs and VTs in Fosl-2tg mouse. d AV block second-degree and representative pictures of AV blocks in Fosl-2tg mouse. Arrows indicate P waves. HR heart rate, BPM beatings per minute, PVCs premature ventricular contractions, VTs ventricular tachycardias, AV block atrio-ventricular block, SDNN standard deviation of all NN intervals, RMSSD the square root of the root mean square of the sum of all differences between successive NN intervals, NN intervals interval between two normal heartbeats.

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  Fig. 3. Altered response to stress between Fosl-2wt and Fosl-2tg mice. a Analyses of HR before isoproterenol (ISO) (PRE ISO, 24 h) and after ISO (POST ISO, 150 min) stimulation assessed by radiotelemetry and calculated as the average of continuous ECG recording in awake Fosl-2wt and Fosl-2tg mice (n = 6, unpaired t-test, mean ± SEM). b Analyses of time response assessed based of the evaluation of HR after ISO (POST ISO, 150 min) stimulation in awake Fosl-2wt and Fosl-2tg mice (n = 6, unpaired t-test, mean ± SEM). Mean of the HR measured before ISO (PRE ISO, 24 h) stimulation has been assessed as a time response threshold. c Analyses of HR fold changes before ISO (PRE ISO, 24 h) compared to after ISO (POST ISO, 150 min) stimulation in Fosl-2wt or Fosl-2tg mice (n = 6, unpaired t-test, mean ± SEM). d Analyses of HR fold changes before ISO (PRE ISO) and after ISO (POST ISO) stimulation of Fosl-2wt and Fosl-2tg hearts in ex-vivo Langendorff reperfusion system (n = 4, paired t-test). e Analyses of the gene regulatory elements in the cardiac conduction system analysed by qPCR in Fosl-2wt and Fosl-2tg hearts (n = 6–10, unpaired t-test, mean ± SEM).

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  Fig. 4. Alteration in the gap-junction and association of cardiac conduction system with fibrosis in Fosl-2tg mice. a Representative pictures of contactin-2 IHC and Sirius Red staining in the myocardial sections from 16 to 22-week-old Fosl-2wt and Fosl-2tg mice (n = 6). Scale bar: 500 µm, insert scale bar: 50 µm. b Representative pictures of contactin-2 (green) and gp38 (red) immunofluorescence counterstained for nuclei with DAPI (blue) in the myocardial sections from 16 to 22-week-old Fosl-2wt and Fosl-2tg mice (n = 6). Scale bars: 100 µm. c Representative pictures of connexin-43 (Cx43, green) or E-cadherin (red), gp38 (red) and sacromeric α-actinin (Sarc αAct, pink) immunofluorescence counterstained for nuclei with DAPI (blue) in the myocardial sections from 16–22-week-old Fosl-2wt and Fosl-2tg mice (n = 6). Scale bars: 50 µm. d Representative pictures of connexin-40 (Gja5, green) and gp38 (red) immunofluorescence counterstained for nuclei with DAPI (blue) in the myocardial sections from 16 to 22-week-old Fosl-2wt and Fosl-2tg mice (n = 6). Scale bars: 50 µm. e Representative pictures of Rgs4 (red) immunofluorescence counterstained for nuclei with DAPI (blue) in the myocardial sections…

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  Fig. 5. Inflammation in the heart of Fosl-2tg mice and myocardial phenotype in Rag2−/−Fosl-2tg mice. a Quantification of CD45 IHC staining on myocardial sections from 16 ti 22-week-old mice (n = 7–9, Mann–Whitney test, mean ± SEM). b Representative flow cytometry t-SNE2 blots and corresponding quantification of immune cell populations (Ly6G high (hi) neutrophils, Ly6G intermediate (int) monocytes, Ly6c int monocytes, Lin-CD11b+ monocytes, CD11b+MHCII hi dendritic cells (DCs), CD11b–MHCII hi DCs, Lin-CD45+ cells and T cells) from the myocardium of 16–22-week-old mice (n = 5–6, unpaired t-test). Gating strategy is presented in Supplementary Fig. 10. c Quantification of Sirius Red, periostin (Postn) and gp38 IHC staining of myocardial sections from 16 to 22-week-old mice (n = 6–8, Mann–Whitney test, mean ± SEM). d Representative flow cytometry pictures and corresponding quantification of Lin-gp38+ fibroblast frequency from the myocardium of 16–22-week-old mice (n = 5–8, Mann–Whitney test, mean ± SEM). e HR and QT intervals recorded in 16–22-week-old mice (n = 11, unpaired t-test, mean ± SEM). f Representative pictures of connexin-43 (Cx43, green), gp38 (red) or E-cadherin (red) and…

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  Fig. 6. Lin-gp38+Fosl-2tg fibroblasts are committed to myofibroblast phenotype. a Heatmap of significantly deregulated genes between Lin-gp38+Rag2−/−Fosl-2tg and Lin-gp38+Fosl-2tg expressed in Transcripts Per Million (TPM) for Rag2−/−Fosl-2wt, Rag2−/−Fosl-2tg and Fosl-2tg groups. Analysis was performed with the comprehensive gene set enrichment web server EnrichR for Transcription (database ChEA 2016) and Pathways (database BioPlanet 2016) areas of significantly deregulated genes uniquely upregulated in Lin-gp38+Fosl-2tg vs. Lin-gp38+Rag2−/−Fosl-2tg fibroblasts. Cardiac fibroblasts were isolated for the hearts of 16-20-week-old mice. b Heatmap of significantly deregulated genes between Lin-gp38+Rag2−/−Fosl-2wt and Lin-gp38+Rag2−/−Fosl-2tg expressed in TPM for Rag2−/−Fosl-2wt, Rag2−/−Fosl-2tg, and Fosl-2tg groups. Analysis as in a of significantly deregulated genes uniquely upregulated in Lin-gp38+Rag2−/−Fosl-2tg vs. Lin-gp38+Rag2−/−Fosl-2wt. Quantification of: c 5-bromo-2’-deoxyuridine (BrdU) proliferation assay, d Caspase 3/7 activity assay, e Quantification of Senescence Associated β-galactosidase (SA-β-gal) activity of cardiac Lin-gp38+ fibroblasts from 16 to 22-week-old Fosl-2wt and…

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  Fig. 7. GP38 and FOSL-2 expression in human specimens. Representative pictures and relative quantification of Sirius red (a), GP38 IHC (b), and FOSL-2 IHC (c) staining of EMBs from HF/SSc patients and donors with healed myocarditis (n = 10–24, unpaired t-test, mean ± SEM, scale bar: 20 µm). d Representative flow cytometry plot of GP38-expressing human foetal cardiac fibroblasts (fCFs). Panel e illustrates representative immunoblots of FOSL-2 in fCFs in response to treatment with TGF-β (10 ng/mL) for 24 h. f hFOSL-2 overexpression in fCFs assessed by qPCR and WB analysis (n = 3, mean ± SEM). Panels g and h show representative immunoblots and WB analysis of fCFs transfected with pCMV control and pCMV-FOSL-2 vectors at basal condition in 10% FBS DMEM (F) and after stimulation with TGF-β or LPS for 24 h (G) (all paired t-test, n = 5).