Transcriptomic and Lipidomic Mapping of Macrophages in the Hub of Chronic Beta-Adrenergic-Stimulation Unravels Hypertrophy-, Proliferation-, and Lipid Metabolism-Related Genes as Novel Potential Markers of Early Hypertrophy or Heart Failure

Figures

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  Figure 1. Schematic representation of iso-pump protocol with time-lapse of echocardiographic and morphologic measurements (A) and echocardiography and morphology parameters (B). HR, heart rate; IVSd, end-diastolic interventricular septum thickness; LVd, end-diastolic left ventricular diameter; PWd, end-diastolic posterior wall thickness; IVSs, end-systolic interventricular septum thickness; LVs, end-systolic left ventricular diameter; PWs, end-systolic posterior wall thickness; h/r, diastolic wall thickness to radius ratio; EF, ejection fraction; FS, fractional shortening; HW/TL, heart weight/tibia length. Kruskal–Wallis followed by Dunn post-hoc tests. * p < 0.05 ECH or HF vs. Ct; † p < 0.05 ECH vs. HF.

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  Figure 2. Transcriptomic characterization of cardiac Ct, ECH, and HF CD64+ macrophages. (A) Number of selectively upregulated and downregulated genes in Ct, ECH and HF macrophages. (B) Heat map showing the top genes selectively regulated in ECH (left) and HF (right) macrophages as compared to Ct and HF or Ct and ECH counterparts, respectively (with red and blue indicating increased and decreased expression, respectively). n = 4 mice/group. Normalization and differential statistical analysis were performed with the glm edgeR package. (C) RNAseq analysis. Ingenuity Pathway analysis showing specific canonical pathways and functions statistically enriched in ECH (left) and HF (right) macrophages. Part of the results concerning ECH macrophages specificities were published in Flamant et al.

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  Figure 3. Transcriptomic characterization of Ct, ECH, and HF cardiac CD64+ macrophages. Beta--adrenergic--, hypertrophy--, and proliferation--signaling related genes. Analysis of mRNA levels in isolated cardiac CD64+ macrophages by RNAseq (n = 4 mice/group), normalization and differential statistical analysis were performed with the glm edgeR package, with genes selectively and significantly regulated in the ECH group (written in red) as compared to Ct and HF and significantly regulated in HF group (written in blue) as compared to Ct and ECH. Genes written in black are compared to Ct and genes underlined display statistical different expression between ECH and HF groups (p < 0.05 Mann–Whitney test or addition of the symbol ≈ means p = 0.05 HF vs. ECH). (Left column) Indication of the mean value of gene expression in Ct macrophages (expressed in counts per million mapped reads (cpm) estimates). (Middle columns) Heat map representation of log2 fold change. (Right column) Significantly regulated genes.

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  Figure 4. Transcriptomic characterization of inflammation, fibrosis, phagocytosis, angiogenesis, and Ab presentation related genes in Ct, ECH, and HF cardiac CD64+ macrophages. Analysis of mRNA levels in isolated cardiac CD64+ cells by RNAseq (n = 4 mice/group, normalization and differential statistical analysis were performed with the glm edgeR package, with genes selectively and significantly regulated in the ECH group (written in red) as compared to Ct and HF and significantly regulated in HF group (written in blue) as compared to Ct and ECH. Genes written in black are compared to Ct and genes underlined display statistical different expression between ECH and HF groups (p < 0.05 Mann–Whitney test or addition of the symbol ≈ means p = 0.05 HF vs. ECH). (Left column). Indication of the mean value of gene expression in Ct macrophages (expressed in counts per million mapped reads (cpm) estimates). (Middle columns). Heat map representation of log2 fold change. (Right column). Significantly regulated genes.

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  Figure 5. Transcriptomic characterization of Ct, ECH, and HF cardiac CD64+ macrophages. Cell metabolism related genes. Analysis of mRNA levels in isolated cardiac CD64+ cells by RNAseq (n = 4 mice/group, normalization and differential statistical analysis were performed with the glm edgeR package, with genes selectively and significantly regulated in the ECH group (written in red) as compared to Ct and HF and significantly regulated in HF group (written in blue) as compared to Ct and ECH. Genes written in black are compared to Ct and genes underlined display statistical different expression between ECH and HF groups (p < 0.05 Mann–Whitney test or addition of the symbol ≈ means p = 0.05 HF vs. ECH). (Left column). Indication of the mean value of gene expression in Ct macrophages (expressed in counts per million mapped reads (cpm) estimates). (Middle columns). Heat map representation of log2 fold change. (Right column). Significantly regulated genes.

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  Figure 6. ECH macrophages are characterized by a net enrichment in lipid regulatory pathways related with eicosanoids, phospholipids, and sphingolipids metabolism. (A) RNAseq analysis of lipid regulatory pathway gene expression. n = 4 mice/group. Normalization and differential statistical analysis were performed with the glm edgeR package, with genes selectively and significantly regulated in the ECH group (written in red) as compared to Ct and HF. Genes underlined display statistical different expression between ECH and HF groups (p < 0.05 Mann–Whitney test or addition of the symbol ≈ means p = 0.05 HF vs. ECH). (Left column). Indication of the mean value of gene expression in Ct macrophages (expressed in counts per million mapped reads (cpm) estimates). (Middle columns). Heat map representation of log2 fold change. (Right column). Significantly regulated genes. (B) Graphical abstract of associated lipid pathways. (C) Lipidomic characterization of cardiac CD64+ macrophages from Iso-induced ECH versus control hearts (n = 5/group) sorted as previously described (Flamant et al., 2021, [5]). Kruskal–Wallis followed by Dunn’s post-hoc test. Cer, ceramide; LPC, lysophosphatidylcholine; LPE,…

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  Figure 7. Transcriptomic characterization of Ct, ECH, and HF cardiac CD64+ macrophages. Apoptosis and survival, cell–cell communication, growth hormone signaling, UPR signaling, and cell recruitment related genes. Analysis of mRNA levels in isolated cardiac CD64+ cells by RNAseq (n = 4 mice/group, normalization and differential statistical analysis were performed with the glm edgeR package, with genes selectively and significantly regulated in the ECH group (written in red) as compared to Ct and HF and significantly regulated in HF group (written in blue) as compared to Ct and ECH. Genes written in black are compared to Ct and genes underlined display statistical different expression between ECH and HF groups (p < 0.05 Mann–Whitney test or addition of the symbol ≈ means p = 0.05 HF vs. ECH). (Left column). Indication of the mean value of gene expression in Ct macrophages (expressed in counts per million mapped reads (cpm) estimates). (Middle columns). Heat map representation of log2 fold change. (Right column). Significantly regulated genes.

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  Figure 8. Specificities of ECH and HF cardiac CD64+ macrophages transcriptomic profiles.