文章标题：Multi-omics profifiling visualizes dynamics of cardiac development and functions
Published Journal: Cell Reports
Impact factor: 9.995
Author Unit: Nanjing Medical University
Baiqu provides services: discover metabolomics HD-MIX version
Baiqu Metabolomics Sharing-Research Background
Heart development in mammals is a multi-stage and strictly regulated complex process, which is precisely controlled by multiple signaling molecules and pathways in different time and space. If the gene regulatory network in this process is affected, it will lead to congenital The occurrence of heart disease. Therefore, it is particularly important to systematically elucidate the key molecules in cardiac development and maturation and analyze the molecular mechanisms of related diseases. Metabolomics sharing, however, most of the current omics studies on cardiac development are based on the transcriptional expression levels of genes, and no studies have explored the key factors of cardiac development and maturation through multi-omics studies throughout the life cycle, which hinders us from systematically Understand the overall process of cardiac development and the etiology of cardiac birth defects.
Baiqu Metabolomics Sharing - Research Methods
In order to draw a multi-omics map of the whole cycle of heart development, the research team of Nanjing Medical University based on various omics methods such as phosphoproteomics, proteomics, metabolomics, and single-cell transcriptomics, shared metabolomics, and described Molecular maps and switching patterns of key pathways and regulators in cardiac development and maturation.
Baiqu Metabolomics Sharing - Research Results 1 Multi-Omics Analysis of Mouse Heart
Figure 1. Temporal dynamics of the phosphoproteome, proteome, and transcriptome during heart development
Transcriptome- and proteome-based analyzes found that more than half of the genes showed relatively low correlations between mRNA and protein levels (Fig. 1A and 1B). But over time, the correlation gradually increased and eventually plateaued, suggesting that absolute mRNA levels may not predict protein abundance.
The 10 time points of mouse heart development were divided into 4 periods using K-means cluster analysis: Phase I of E10.5–14.5, Phase II–III of E16.5–2W, and Phase IV of 4W–8W ( Figure 1E). Metabolomics shared that the correlation between phase I gene expression and phosphorylation was significantly higher (Fig. 1F), which verifies that phosphorylation is strongly involved in signaling in early heart development.
2 Multiple sets of inferred and enriched pathways for cardiac development and maturation
Figure 2. Key pathways of tri-omics during cardiac development and maturation
The researchers performed GO analysis on the differentially expressed genes and differentially expressed proteins represented in each of the four periods in the transcriptome, proteome, and phosphoproteome (Fig. 2). The results showed that major pathways of cell proliferation and differentiation are enriched in phase I, including transcriptional regulation, RNA splicing, and mRNA processing. Stages II-III are stages in which the heart increases in shape and function. Pathways involved include mitochondrial translation, translation, cell adhesion, and cell matrix adhesion. Stage IV is the mature stage in which the heart acquires metabolic-related functions. Phase IV genes and proteins are involved in redox processes, metabolic processes (tricarboxylic acid [TCA] cycle and fatty acid metabolism), and transport.
The role of 3MAPK and AKT pathway balance in regulating phenotypic switching in cardiac development
Figure 3. Characterization of signaling dynamics and prediction of key kinase substrates during heart development
Protein phosphorylation is a key post-translational modification in signal transduction and cascades. Hypothesizing that phosphorylation sites with similar temporal kinetics are more likely to be substrates for the same kinase, the researchers grouped all phosphorylation sites into three major phosphopeptide clusters (Fig. 3A, 4B). The characteristics of different clusters were elucidated: cluster 1 was characterized by early significant activation followed by decreased phosphorylation levels, including MAPK, CDK, and CLK3 protein kinases (Figure 3B), and was significantly enriched in downstream pathways, such as protein translation, cellular adhesion and protein degradation (Fig. 3C). Metabolomics shared that cluster 2 showed delayed activation and decay in stages II-III (Fig. 3B). Significantly enriched pathways in cluster 2 were similar to those in cluster 1, except for further activation of cell-cell adhesion and switching to hormone signaling pathways (Fig. 3C). Cluster 3 showed a steady increase in phosphorylation levels, indicating activation later in cardiac development (Fig. 3B), and phosphoproteins at this cluster phosphorylation site were mainly enriched in physiological and pathological processes of the heart, including cardiac contraction (Fig. 3C).
In addition, the research team adopted the iGPS algorithm (GPS algorithm with interaction filter, or GPS in vivo), and determined that MAPK, CDK, and CLK play key roles in early embryonic development, and they were significantly enriched for cluster 1 phosphopeptides, Its role in initiating phosphorylation signaling is emphasized. A temporal concordance between kinase and substrate phosphorylation was also observed (Fig. 3D), suggesting a temporally specific function of the kinase. From E10.5 to E18.5 mouse hearts, the total protein level of p38 MAPK remained unchanged, while the protein level of phosphorylated (p-)p38 MAPK (180T) decreased significantly, further confirming that the phosphorylation of MAPK plays an important role in heart development. The early stages are important (Figure 3E). However, protein levels of phosphorylated AKT1 (S124) and phosphorylated AKT2 (S129) produced mutually exclusive activation of MAPKs in E14.5, suggesting their potentially opposing effects on heart development (Fig. 3D and 3E).
These results reflect the balanced effect of MAPK and AKT on cardiac development, in which the phosphorylation of MAPK mainly affects the differentiation of cardiomyocytes, while the phosphorylation of AKT mainly affects the proliferation of myocardium.
4 Identification of core TFs involved in early heart development
Figure 4. Epigenomic Approach to Identify Master Transcription Factors Regulating Heart Development
To further explore the importance of transcription factors (TFs) in mammalian heart development, chromatin transposase accessibility sequencing was performed on E10.5 prenatal mouse heart samples using the epiomics ATAC-seq technique analyze. The TF-TF regulatory network was constructed by predicting transcription factor binding sites, and footprint analysis was used to illustrate the regulatory lexicon at this stage (Fig. 4A). For metabolomics sharing, all TFs were divided into three groups: class I in the top 200, class II in the top 201–400, and class III in the rest (Fig. 4B). Among the top 10 TFs identified, multiple TFs are critical for heart development in mouse or embryonic stem cell models.
Furthermore, as higher temporal specificity and intolerance to functional mutations also suggest a potentially important role, we assessed mRNA expression in phase I (E10.5–E14.5), as well as all detected The probability of gene intolerance (pLI score) of TFs to loss-of-function (LoF) mutations was consistent with the above conclusion (Fig. 4C). Enrichment of class I TFs was observed among TFs highly expressed early in cardiac development and intolerant to LoF mutations (Fig. 4D). Metabolomics shared, in addition, experiments with TFRE18 demonstrated the expression and activity of these TFs, and the proportion of TFs detected in the class I group was significantly higher than that of class II and class III (Figure 4E and 4F), indicating that class I TFs Important role in heart development.
In addition, the LoF and deleterious missense mutations of class I TFs were significantly higher in patients with tetralogy of Fallot (TOF), the most common congenital heart disease, than in normal controls (Fig. 4G), while class II and III TFs do not. The key TFs identified by footprint analysis are very important for heart development and may be potential novel TOF causative gene candidates. These analyzes reveal a tight temporal regulation of gene expression programs during early cardiogenesis, with core TFs essential for proper cardiac development.
Meanwhile, to elucidate the transcriptional and chromatin regulators that mediate the signaling cascade, the team investigated protein-protein interactions (PPIs) between co-regulators of MAPK substrates. Metabolomics shared, results showed that based on experimentally validated STRING data, PPIs enriched in transcriptional regulatory pathways and class I TFs are thought to play key roles in early heart development.
5 Mature mouse heart TCA cycle and lipid metabolism pathway
Figure 5. TCA cycle and lipid metabolic pathways during heart development
The temporal metabolome profile based on KEGG enrichment analysis clearly showed that the number of metabolites changed significantly during heart development, mainly enriched in the TCA cycle and lipid metabolism pathways in the heart (Fig. 5A and 5B). In the mass spectrum of Figure 5C, it can be seen that metabolic substrates in fatty acid b oxidation (FAO) and TCA cycle are consumed, while key rate-limiting enzymes are highly expressed. Metabolomics shared, moreover, single-cell transcriptional profiling revealed that mature CMs were the major cell subpopulations involved in TCA and FAO metabolism, suggesting that these two metabolic pathways are the main energy source for myocardial contraction (Figure 5D and 5E).
Arachidonic acid metabolism in 6MHC-II+ resident macrophages promotes phagocytosis of apoptotic cells induced by efferent cell proliferation
Figure 6. Specific metabolism of arachidonic acid in adult mouse cardiac MHC-II+ native macrophages
Arachidonic acid (AA) also exhibited increased metabolic levels in the heart (Figures 5A, 5B, and 6B). High expression of AA biosynthetic genes in early stages and high expression of AA biosynthetic genes in adult mouse hearts indicated high biological activity of AA genes at later stages (Fig. 6A–6C).
Single-cell sequencing results revealed that prostaglandin endoperoxide synthase 1 (Ptgs1), a rate-limiting step in the conversion of AA to prostaglandin E2 (PGE2), was specifically expressed in macrophages of adult mouse hearts (Fig. 6D) . Metabolomics shared that macrophages were divided into three distinct clusters based on CCR2 expression and dependence, and Ptgs1 was mainly expressed in certain MHC-II+ native macrophages (Figures 6E and 6F). The proportion of Ptgs1+ cells of MHC-II+ native macrophages was significantly increased after transverse aortic constriction (TAC) (Fig. 6G). This suggests that Ptgs1 exerts a cardioprotective role by promoting the maturation of MHC-II+ native macrophages. Interestingly, further pathway analysis of upregulated genes in Ptgs1-high expressing cells revealed expression of key efferent cell receptors/enzymes, CX3C chemokine receptor 1 (Cx3cr1) and growth inhibitory specificity 6 (Gas6) Significantly higher than MHC-II+/Ptgs1– resident macrophages (Fig. 6I). Metabolomics sharing, the above data suggest that AA and its metabolites may play a potential role in tissue repair through efferent cell proliferation of apoptotic cells.
Baiqu Metabolomics Sharing - Research Conclusions
The team combined phosphoproteomics and machine learning methods to build an interaction network between phosphokinases and substrates, and found that the balanced activation of MAPKs and AKTs kinases is the key switch for the gradient conversion of cardiomyocyte differentiation and proliferation capabilities; also used epigenomics ATAC-seq and functional omics TFRE technology systematically identified the core transcription factors in the early heart development, organoids and genetic analysis found that the core transcription factors and their genetic mutations play an important role in the occurrence of congenital heart disease; finally, the author Combining metabolomics and single-cell transcriptomics data revealed that arachidonic acid metabolism is involved in the encapsulation of cardiac MHC-II+ native macrophages on apoptotic cardiomyocytes.
This study provides a comprehensive molecular description of cardiac development and maturation, significantly enriching existing datasets, which together reveal the complex but coordinated molecular mechanisms underpinning cardiac development and maturation. Shared by metabolomics, this approach provides an example of a general framework that enables a new understanding of the multilevel control of the development of different organs or tissues and provides functional annotations for validating pathogenic mutations and informing disease mechanisms.