Upregulated FoxO1 promotes arrhythmogenesis in mice with heart failure and preserved ejection fraction

Figures

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  Fig. 1. Impaired electrical impulse propagation underlies arrhythmia in HFpEF. a Experimental design. b Representative left ventricular M-mode echocardiographic images, pulse-wave Doppler showing E (early filling)– and A (atrial filling)–wave, and tissue Doppler showing E′. Quantification of ejection fraction (c, EF) and diastolic function (d, E/E’ ratio), n = 7 (Ctr) and n = 8 (HFpEF). Representative programmed electrical stimulation tracings and pacing protocol (e) and Incidence of pacing-induced ventricular tachycardia (VT, f). g Representative isochronal voltage maps. Action potential duration at 80% of repolarization (APD80, h) and conduction velocity (i) at different basic cycle lengths (BCL), n = 8 (Ctr) and n = 7 (HFpEF). j Representative isochronal voltage maps (*indicates the location of pacing electrode stimulating at 80-ms BCL). Conduction velocity (CV) in transverse (k) and longitudinal (l) directions, n = 5 (Ctr and HFpEF). m Representative picrosirius red images (scale bar = 100 µm) and fibrosis quantification (n), n = 7 (Ctr and HFpEF). o Quantification of hydroxyproline content, n = 6 (Ctr) and n = 7 (HFpEF). Data are expressed as mean ± SEM. Unpaired two-sided Student’s…

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  Fig. 2. Transcriptomics of HFpEF hearts revealed a high convergence of metabolic signals toward FoxO1 activation. a Heatmap of difference expressed genes in the heart. b Biologically relevant pathways based on Kyoto Encyclopedia of Genes and Genomes enrichment analysis. c Schematic illustration highlighting the multiple altered pathways in HFpEF hearts converge to Forkhead Box O signaling. d Representatives immunoblot images of major cardiac FoxO isoforms in the enriched nuclear fraction. e Protein quantification, n = 3 (Ctr) and n = 4 (HFpEF). f Relative expression of known downstream targets of FoxO1 and different fibroblast states in left ventricular HFpEF samples relative to controls based on RNA sequencing. g Experimental design of adult cardiac fibroblast isolation and mRNA quantification of fibrosis markers by qPCR (h), n = 10 (Ctr) and n = 9 (HFpEF). Data are expressed as mean ± SEM. Two-sided Fisher’s exact test (b). Unpaired two-sided Student’s t-test (e, h). Source data are provided as a Source Data file. Parts of Fig. 2g were created in BioRender. Mesquita, T. (2024) https://BioRender.com/e91m165. Released under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0…

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  Fig. 3. Downregulation of FoxO1 enhances the propagation of electrical impulses and alleviates the arrhythmogenesis in HFpEF. a Experimental design. b Cardiac transduction by AAV vectors showing paired brightfield (top) and epifluorescent (bottom) images of mCherry 4 weeks after AAV injection. Quantification of ejection fraction (c, EF) and diastolic function (d, E/E’ ratio), n = 10 (Ctr), n = 9 (HFpEF AAV9 scramble-sh) and n = 10 (HFpEF AAV9 FoxO1-sh). e Exercise capacity. Lung weight (f, LW) and heart weight (g, HW) normalized by the tibia length (TL). e–g n = 9 (Ctr), n = 9 (HFpEF AAV9 scramble-sh) and n = 10 (HFpEF AAV9 FoxO1-sh). Representative programmed electrical stimulation tracings and pacing protocol (h) and Incidence of pacing-induced ventricular tachycardia (VT, i). Action potential duration at 80% of repolarization (APD80, j) and conduction velocity (k) at different basic cycle lengths (BCL), n = 7 (HFpEF AAV9 scramble-sh) and n = 6 (HFpEF AAV9 FoxO1-sh). l Representative isochronal voltage maps (*indicates the location of pacing electrode stimulating at 80-ms BCL). Conduction velocity (CV) in transverse (m) and longitudinal (n) directions, n = 5 (HFpEF AAV9 scramble-sh and…

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  Fig. 4. scRNA-seq revealed transcriptome signatures of fibroblasts in HFpEF hearts. a Heatmap of difference expressed genes in the heart. b, c Gene set enrichment analysis. d Schematic depicting the experimental design of scRNA-seq. e Uniform manifold approximation and projection (UMAP) embedding of all cells and from all conditions. f Violin plots showing the expression of known markers of activated fibroblasts (FB) in all identified clusters. g Expression profiles and the number of cells expressing markers of FB activation within activated and quiescent FBs clusters. h Gene ontology terms in activated FBs clusters. i The relative number of cells within activated and quiescent FBs clusters. j Pseudo-bulk expression profiles and the number of cells expressing markers of FB activation in FBs cells. Kolmogorov-Smirnov test (b, c) and two-sided Fisher’s exact test (h). Source data are provided as a Source Data file. Parts of Fig. 4 d were created in BioRender. Mesquita, T. (2024) https://BioRender.com/x03s068. Released under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International license.

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  Fig. 5. Downregulation of FoxO1 alters intercellular communication between cardiomyocytes and fibroblasts in HFpEF hearts. a Gene set enrichment analysis plots within all FB clusters showing upregulation of the extracellular matrix organization term in scramble-treated HFpEF compared to control (top) and downregulation of this gene set term by the treatment with AAV9-FoxO1-sh (bottom); NES normalized enrichment score. b Representative immunofluorescence images for periostin and quantification (c), n = 4 (Ctr, HFpEF AAV9 scramble-sh and HFpEF AAV9 FoxO1-sh). d Representative immunofluorescence images for connexin 43 (Cx43), vimentin, and alpha sarcomeric actinin (αSA). e Relationship analysis between vimentin area and Cx43 fragmentation per field of view (scale bar = 25 µm), n = 4 (Ctr 11 random fields, HFpEF AAV9 scramble-sh 19 random fields and HFpEF AAV9 FoxO1-sh 15 random fields). Dashed line: one phase exponential fit. Data are expressed as mean ± SEM. Kolmogorov-Smirnov test (a) and one-way ANOVA followed by Bonferroni post hoc test (c). Source data are provided as a Source Data file.

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  Fig. 6. Nuclear translocation of FoxO1 is necessary to activate fibroblast reprogramming. a Experimental design of adenoviral overexpression of GFP and flag-fused mutant FoxO1 (FoxO1AAA) in fibroblasts from neonatal hearts. b Representative images showing the expression of GFP and Flag (fused with FoxO1, scale bar = 100 µm) and quantification of subcellular localization (c). d mRNA expression of FBs activation markers, n = 6 (Ad-GFP and Ad-FoxO1AAA). e mRNA expression at indicated times after actinomycin D treatment, presented as percent of transcript levels relative to time point zero, n = 3 (Ad-GFP and Ad-FoxO1AAA). Data are expressed as mean ± SEM. Unpaired two-sided Student’s t-test (d). Two-way repeated measures ANOVA followed by Bonferroni post hoc test (e). mRNA levels were fitted using one-phase exponential decay. a.u., arbitrary unit. Source data are provided as a Source Data file. Parts of (a) were created in BioRender. Mesquita, T. (2024) https://BioRender.com/a30n975. Released under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International license.

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  Fig. 7. Targeted downregulation of FoxO1 in activated fibroblasts alleviates fibrotic remodeling and arrhythmias in HFpEF. a Experimental design. b Representative left ventricular M-mode echocardiographic images, pulse-wave Doppler showing E (early filling)– and A (atrial filling)–wave, and tissue Doppler showing E′. Quantification of ejection fraction (c, EF) and diastolic function (d, E/E’ ratio). e Exercise capacity. Lung weight (f, LW) and heart weight (g, HW) normalized by the tibia length (TL). c–g, n = 8 (HFpEF AAV9-GFP and HFpEF AAV9-Cre). Representative programmed electrical stimulation tracings and pacing protocol (h) and incidence of pacing-induced ventricular tachycardia (VT, i). j Representative picrosirius red images (scale bar = 200 µm) and fibrosis quantification (k), n = 5 (HFpEF AAV9-GFP and HFpEF AAV9-Cre). l Schematic illustration of our working hypothesis. Data are expressed as mean ± SEM. Unpaired two-sided Student’s t-test (c–g, k). Two-sided Fisher’s exact test (i). Source data are provided as a Source Data file. Parts of (a and l) were created in BioRender. Mesquita, T. (2024) https://BioRender.com/p96k811 and https://BioRender.com/f20a900. Released under a…