Mural cell dysfunction contributes to diastolic heart failure by promoting endothelial dysfunction and vessel remodelling

Figures

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  Fig. 1. Db/db mice develop diastolic dysfunction after 8 weeks of 1% salt. (A) Body weight, (B) fasting glucose, (C) E/E’ ratio, and (D) ejection fraction (EF%) measured by echocardiography in db/+ and db/db + 1% salt mice. (E) Heart weight normalised to tibia length (HW/TL). (F, G) Cardiomyocyte cross-sectional area measured on laminin staining with representative images at 2 and 8 weeks (n = 5–8/group) (scalebar is 30 μm). (H, I) Total fibrosis measured on a Sirius red staining with representative images at 8 weeks (n = 5–8/group) (scalebar is 500 μm). Values are presented as mean ± SEM. Two-way ANOVA followed by Sidak’s multiple comparisons test with *p < 0.05, **p < 0.01, and ***p < 0.001. E, early mitral inflow peak velocity; E’, early diastolic annulus peak velocity; EF, ejection fraction; NS, non-significant. At 2 weeks: n = 8 F (1 db/+, 7 db/db) and 5 M (4 db/+, 1 db/db); at 4 weeks: n = 7 F (4 db/+, 3 db/db) and 8 M (4 db/+, 4db/db); at 6 weeks: n = 7 F (3 db/+, 4 db/db) and 6 M (3 db/+, 3 db/db), at 8 weeks: 9 F (6 db/+, 3 db/db) and 6 M (3 db/+ and 3 db/db)

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  Fig. 2. Db/db mice + 1% salt show microvascular dysfunction before the onset of diastolic dysfunction. (A, B) Cardiac capillary density measured as the number of IB4 + microvessels per area in db/+ and db/db + 1% salt mice at 2, 4, 6 and 8 weeks with representative images (n = 5–8/group). (C) Number of pericytes was determined by counting the cells that were double positive for NG2 and PDGFRb marker, and co-localised with IB4 + microvessels. (D) Pericyte coverage, assessed by the ratio of IB4 + microvessels covered by NG2/PDGFRb double positive cells over total IB4 + microvessels. (E, F) Active vessel regression, assessed by the ratio of Coll IV + IB4- microvessels over total IB4 + microvessels with representative images at 4 and 6 weeks (n = 5–8/group). White arrowheads indicate empty collagen sleeves. Scalebars are 6 μm. Values are presented as mean ± SEM. Two-way ANOVA followed by Sidak’s multiple comparisons test with *p < 0.05, **p < 0.01, and ***p < 0.001. IB4, isolectin B4; NG2, neural glial antigen 2; NS, non-significant; PDGFRb, platelet derived growth factor receptor beta; Coll IV, collagen IV. At 2 weeks: n = 8 F (1 db/+, 7 db/db) and 5 M (4db/+, 1 db/db); at 4 weeks: n = 7 F…

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  Fig. 3. Hearts of db/db + 1% salt mice show endothelial cell dysfunction and angiogenesis impairment. Single cell analysis on the hearts of PDGFRβ-CreERT2-EYFP-db/db mice given 1% salt for 4 weeks and PDGFRβ-CreERT2-EYFP-db/+ control mice (n = 4 male mice pooled per sample). UMAP showing clustering (A) identifying 17 clusters with 3 large cell types (ECs, fibroblasts, and mural cells) or split by genotype (B). (C) Distribution of cells in PDGFRβ-CreERT2tg/+-EYFPtg/+-db/+ (n = 3604 cells) and PDGFRβ-CreERT2tg/+-EYFPtg/+-db/db (n = 2741 cells) mice shown as percentage of cells. (D) GO term analysis (FC = 0.15) on top 500 DEG between PDGFRβ-CreERT2tg/+-EYFPtg/+-db/+ and PDGFRβ-CreERT2 tg/+-EYFPtg/+-db/db ECs. (E,F) Staining for Ki67 to assess EC proliferation with representative images at 4 weeks. White arrowheads indicate proliferating EC (scale bar: 10 μm). (G,H) Analysis of ‘jagged’ VE-Cadherin junctions with representative image at 4 weeks (scale bar: 6 μm). Junctions were scored jagged, mixed or straight. All data were analysed by a Student’s t-test with *p < 0.05, **p < 0.01, and ***p < 0.001 except for data presented in (G) which were analysed by a Pearson’s Chi-squared test. VE-Cad,…

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  Fig. 4. Hearts of db/db mice show arteriole thickening and vessel enlargement after 4 weeks of salt treatment. (A) Feature plots for Pdgfrb, Kcnj8 (pericyte marker), Acta2 and Myh11 (VSMC markers) on single cell data of hearts of PDGFRβ-CreERT2-EYFP-db/db + salt and PDGFRβ-CreERT2-EYFP-db/+ mice. (B) Violin plot of Acta2 and Myh11 expression in the mural cell population of db/db + salt vs. db/+ mice. (C) GO term analysis on the top 500 DEG of the mural cells (FC = 0.25). (D) Percentage of ACTA2 positive area in the hearts of db/+ and db/db + 4-week salt mice. Percentage of ACTA2 positive area in (E) microvessels (< 10 μm) and (F) cardiac pericytes (identified as double positive for NG2 and PDGFRβ) of db/+ and db/db + 4-week salt mice. (G) ACTA2 + vessel density measured as the number of ACTA2 + blood vessels per total heart area. (H) Distribution of ACTA2 + blood vessels according to vessel size (µm). (I) Representative images of ACTA2 + blood vessels in the hearts of db/+ and db/db + 4-week salt mice. Scale bars: 500 μm. (J) Measurement of medial area and medial thickness to assess arteriole thickening. All data were analysed by a Student’s t-test with *p < 0.05 and **p < 0.01 except…

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  Fig. 5. Hearts of db/db mice show capillary dilatation and increased pericyte-EC intercellular space after 4 weeks of salt treatment. (A) Analysis of the capillary luminal diameter in db/db + 4 weeks 1% salt vs. db/+ mice with representative images (B). Scale bar: 10 μm. (C) Assessment of proliferation of cardiac pericytes (identified by PDGFRβ) by a Ki67 staining (n = 7–8/group). White arrowheads indicate proliferating pericytes (D). Scale bar: 25 μm. (E, F) TEM analysis (n = 3/group) to determine the intercellular area (E) between EC and pericytes with representative images (F). The red dotted line circles the intercellular area. Scale bar: 1 μm

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  Fig. 6. Pericyte dysfunction promotes EC inflammation and an EC proliferation arrest through TNFα-dependent paracrine signalling. Predictions of putative ligand-receptor interactions based on NicheNet analysis on the single cell RNA sequencing data between (A) the EC (sending) and VSMC_PC (receiver) population and (B) the VSMC_PC (sending) population and EC (receiver) population. Human pericytes were exposed to oxidative stress (OX, 100 µM H2O2) for 24 h or left untreated (ctrl) and then cocultured for 7 days with ECs. qPCR for (C) VCAM1, IL1A, IL8 and IL6 and (D)p21 on EC cocultured with healthy or OX-treated pericytes for 7 days. (E) Analysis of proliferation by EDU staining. (F) Western blot for cyclin D1, P-STAT1 and total STAT1 on EC cocultured with healthy or OX-treated pericytes for 7 days, with quantification. GAPDH was used as loading control. (G) qPCR for TNFA on pericytes exposed to oxidative stress (OX) for 24 h or left untreated (ctrl). (H, I) Silencing of TNFA in pericytes, followed by OX treatment (or left untreated), and cocultured with ECs for 7 days. qPCR for VCAM1 (H) and analysis of proliferation (I) on ECs 7 day cocultured with healthy or OX-treated siTNFA or siCtrl…