Fibulin-3 is necessary to prevent cardiac rupture following myocardial infarction

Figures

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  Figure 1. Expression of Efemp1 in cardiac fibroblasts before and after myocardial infarction. Analysis of previously published single cell/nuclei and bulk RNA-seq datasets. (a) UMAP plots showing detected lineages in scRNA-seq of cardiac interstitial cells from sham and MI hearts37 with Efemp1 expression indicated. (b) UMAP plot showing Efemp1 expression in cardiac snRNA-seq38 of young vs old mice. (c) UMAP plot showing aggregate of Pdgfra-GFP+ cells from sham and MI hearts. (d) UMAP plot of GFP+ cells separated according to experimental condition (sham, MI-day 3, MI-day 7). (e,f) Expression of select genes in GFP+ cells as visualised I on UMAP coordinates according to condition or (f) according to box plots. (g) Expression of Efemp1 in a bulk RNA-Seq MI time-course of Pdgfra-GFP+/Sca1+ or Pdgfra-GFP+/Sca1- cells39. *indicates whether Efemp1 is differentially expressed (DESeq219; Padj < 0.05) at an MI time-point relative to undamaged control for the indicated Sca1 fraction.

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  Figure 2. Fibulin-3 is elevated in left ventricular tissue and plasma of heart failure patients. (a,b) Fibulin-3 was significantly elevated vs controls in the ventricular tissue (LC–MS/MS analysis) and plasma of heart failure (HF) patients. (c) Circulating fibulin-3 in HF patients was significantly positively correlated with established biomarkers N-terminal pro b-type natriuretic peptide (NT-proBNP), high sensitivity C-reactive protein (hs-CRP), and significantly negatively correlated with LV ejection fraction (LVEF). Ventricular tissue, HF n = 15, control n = 10; Plasma n ≥ 55 including non-failing and HF patient plasma.

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  Figure 3. Fibulin-3 deletion results in cardiac rupture and ventricular remodelling post-infarct. (a) Efemp1−/− mice had significantly lower survival rates post-MI compared to WT mice, post-mortem revealed cardiac rupture to be the predominant cause of death (n = 24 Efemp1−/−, n = 30 WT, Kaplan–Meier analysis). (b) Representative images of cardiac rupture post-MI along the border zone (white circle), sutured LAD is depicted by black arrow. (c,d) In the mice that survived to 28 days, collagen deposition was not different, and heart weight to tibia length ratio (HW:TL) was significantly larger in the Efemp1−/− mice (n = 16 Efemp1−/−, n = 28 WT, p = 0.03). (e) Ejection fraction (LVEF) was not different between the groups, however ventricular remodelling was evident at 28 days post-MI—intraventricular septum thickness (IVS, p = 0.02), end diastolic volume (EDV, p = 0.001), and left ventricular internal diameter (LVID, p = 0.003), were significantly different between groups (n = 14 Efemp1−/−, n = 12 WT). Mean ± SD.

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  Figure 4. Fibulin-3 deletion resulted in less collagen content, upregulated Mmp9 expression, and highly variable collagen organisation in the infarct zone at day-3 post-MI. (a) Collagen deposition was significantly reduced in the Efemp1−/− mice at 3-days post-MI (n = 10 Efemp1−/−, n = 10 WT). (b) No significant differences were seen between Efemp1−/− and WT mice in collagen-I or collagen-III mRNA expression at 3 days post-infarct, Mmp2 showed a trend to decreased mRNA expression, and Mmp9 border zone mRNA expression was significantly increased in Efemp1−/− vs WT mice, fold change relative to WT control, dotted line; relative levels of genes normalised to a reference geomean and converted to fold change, bars represent the mean ± SD. Infarct Zone (IZ), Border Zone (IZ), and Remote Zone (RZ); all n = 5/group. (c) No significant difference in total group-wise collagen fibre length, nor in fibre-alignment was seen between Efemp1−/− and WT mice at 3 days post-MI (infarct-zone tissue), however a distinct dichotomy in the Efemp1−/− mice was seen with 2 mice displaying high fibre-alignment, and 4 with fibre disarray, n = 5–6/group. Representative images of second harmonic generation two-photon…

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  Figure 5. Bulk RNA-seq reveals differential expression of ECM and muscle fibre organisation genes, inflammatory genes and Mmp9 in the infarct zone of Efemp1−/− at day-3 post-MI. (a) Bulk RNA-seq was conducted on isolated infarct zone tissue from WT mice (n = 7) and Efemp1−/− (n = 5) at day-3 post-MI. A condensed list of genes of interest are presented ranked by fold-change; edgeR analysis, all presented genes FDR ≥ 0.05. (b) Gene lists derived from RNA-seq underwent GSEA pathway analysis to identify pathways enriched in the WT and Efemp1−/− mice infarct zones. Pathway enrichment results were visualized in Cytoscape using the EnrichmentMap, AutoAnnotate and ClusterMaker2 apps before being manually laid out and labels removed for visual purposes. Enriched pathways for WT are shown in blue and the pathways enriched in Efemp1−/− mice are in red. Each node (circle) represents a distinct pathway, and edges (lines) represent overlapping genes between pathways, determined using a similarity coefficient. All the genes involved in the enriched pathways were then used to produce a heatmap, shown on the right.