T-Cell–Derived IFNγ Instructs ECM Crosslinking by Cardiac Fibroblasts Through LOXL3 in Experimental Cardiometabolic HFpEF

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  Figure 1:. Mechanical ECM remodeling correlates with diastolic dysfunction in cardiometabolic HFpEF. A: 8–12-week-old wild-type C57Bl/6J mice received standard chow and water (STD) or the combination of high fat diet (HFD, 60% fat) and drinking water containing 0.5 g/L of the hypertension-inducing agent L-nitro-arginine-methyl-ester (L-NAME) for 5 weeks. B+C: Diastolic function assessed by Doppler-mode echocardiography: representative recordings of mitral valve inflow velocity (B) and ratio of early/late mitral valve inflow velocity (E/A, N=11–12/group, C). D: Preparation of decellularized left ventricular (LV) ECM samples for uniaxial tensile test. E: Representative recordings of uniaxial tensile test on LV-ECM samples from STD vs H/L-treated mice. F: Elastic modulus of LV-ECM (N=12/group). G: Elastic modulus of LV-ECM from mice on STD diet, HFD, L-NAME in drinking water, or the combination of HFD and L-NAME (H/L) for 5 weeks. H: Simple linear regression between E/A and LV-ECM Elastic modulus after 5 weeks of STD- or H/L-treatment. I: Representative images of Picrosirius red staining of LV sections of STD- or H/L-treated mice with quantification of collagen area fraction (N=3/group). J:…

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  Figure 2:. Mechanical ECM remodeling in murine cardiometabolic HFpEF is T cell-dependent and human HFpEF transcriptome is characterized by a T cell signature. A: Number of CD4+ T cells identified by flow cytometry in mice fed a STD diet for 3 weeks or H/L for 3 or 5 weeks (N=12/group). B: Elastic modulus of LV-ECM of STD- (5 weeks) or H/L-treated mice (3 or 5 weeks, N=12/group). C+D: Diastolic function assessed by Doppler-mode echocardiography in wild-type or Tcra−/− C57Bl/6J mice after 5 weeks of STD or H/L treatment: representative recordings of mitral valve inflow velocity (C) and ratio of early/late mitral valve inflow velocity (E/A, D, N=6–8/group). E+F: Uniaxial tensile testing of decellularized LV-ECM derived from WT or Tcra−/− mice after 5 weeks of STD- or H/L-treatment: Representative recordings (E) and Elastic modulus (F, N=6–8/group). G+H: Bulk RNA sequencing of human LV tissue from control and HFpEF patients. GO analysis of significantly upregulated genes in HFpEF compared to control samples. Shown are the 10 most significantly enriched biological processes (G) and cellular compartments (H). I: Correlation analysis of collagen (green) and lysyl oxidase (blue) encoding genes with…

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  Figure 3:. Cardiac fibroblasts express higher levels of lysyl oxidases in cardiometabolic HFpEF. A: Lysyl oxidase expression score across all identified cell types including data from STD- and H/L-treated mice. B: UMAP of CFB isolated from the full data set reveals 6 distinct CFB subclusters. C: Relative abundance of each CFB subcluster in STD- compared to H/L-treated mice. D: Gene ontology (GO) analysis of CFB-cluster 0 marker genes. Shown are the most significantly enriched biological processes. E: Lysyl oxidase expression score across CFB in STD vs H/L-treated mice. F: Cardiac LOXL3 protein levels assessed by ELISA in wild-type C57Bl/6J mice after 3 or 5 weeks of STD- or H/L-treatment (N=12/group).

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  Figure 4:. CD4+ T cell-derived IFNγ induces LOXL3 expression in CFB through HIF1α which is sufficient for ECM stiffening in vitro. A: Cardiac fibroblasts (CFB) isolated from C57Bl/6J mice were treated with the secretome of activated CD4+ T cells. B: Loxl3 mRNA expression levels in CFB treated with the secretome of activated CD4+ T cells for the indicated durations (expressed as fold change compared to non-treated CFB (NC), N=6). C: Relative lysyl oxidase activity in culture media from CFB after treatment with CD4+ T cell secretome for 24 h (N=6). D: In vitro collagen gel contraction by CFB treated with control (NC), CD4+ T cell secretome or recombinant TGFβ (100 ng/mL) after 24 h (N=4). E+F: Uniaxial tensile testing of dECM after incubation with CFB secretome. Representative recordings (E) and fold change of Elastic modulus (F) of dECM preparations after incubation with CFB secretome after the indicated treatments in presence or absence of β-aminopropionitrile (BAPN, 500 uM, N=4). G+H: Relative Loxl3 mRNA levels in CFB after 24 h of treatment with the indicated recombinant cytokines (IL-2: 25 U/mL, IFNγ: 100 U/mL, IL-4: 500 ng/mL, N=5, G) or CD4+ T cell secretome in presence or absence of…

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  Figure 5:. In vivo, IFNγ and lysyl oxidation are required for ECM stiffening and diastolic dysfunction. A: Wild-type C57Bl/6J mice received 25 kU recombinant IFNγ or PBS for 5 consecutive days by i.p. injection (N=6). B-C: Relative Hif1α (B) and Loxl3 (C) mRNA as well as LOXL3 protein (D) levels in the LV of PBS or IFNγ treated mice. E: Wild-type or Ifng−/− C57Bl/6J mice were subjected to H/L treatment for 5 weeks. F-G: Elastic modulus of LV-ECM (F) and diastolic function expressed as E/A ratio (G) in wild-type and Ifng−/− mice after 5 weeks of HFD/L-NAME treatment (N=12/group). H: Wild-type C57Bl/6J were subjected to HFD/L-NAME treatment for 5 weeks with daily PBS or β-aminopropionitrile (BAPN, 100 mg/kg) injections from week 3 to 5. I-J: Elastic modulus of LV-ECM (I) and diastolic function expressed as E/A ratio (H) in wild-type mice receiving PBS or BAPN during weeks 3–5 of HFD/L-NAME treatment (N=12/group).