In situ analysis of the proteome reveals how MSC is involved in repair after myocardial infarction

In order to understand the mechanism of stem cell treatment of ischemic heart disease, a research team led by the University of Alabama at Birmingham recently identified the proteome of ischemic heart mesenchymal stem cells in situ, which may help heart repair in the future
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The results were published in the "Theranostics" magazine

Ischemic heart disease and the heart failure caused by it is still the world's number one killer
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Regenerative medicine methods show good prospects

It is believed that MSCs mainly secrete biologically active molecules and microbubbles to promote tissue repair, but which factors they produce locally are still largely unknown
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In the past, most MSC secretome studies were conducted in cell culture, which may not necessarily represent their true condition in vivo

In recent years, scientists have successfully achieved cell type-specific labeling and separation of the nascent proteome
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They used mutant methionyl tRNA synthetase (MetRS) to target specific cells, incorporated the methionine substitute azidoleucine (ANL) into the newly synthesized peptide, and then used the click reaction ( click reaction) for visualization or subsequent separation and identification

To this end, the research team at the University of Alabama introduced MetRSL274G into MSCs, and then injected the transduced cells into the ischemic myocardium of mice with myocardial infarction (MI), and then passed liquid chromatography/mass spectrometry (LC/MS) and biological Informatics analysis is used to quantify the freshman MSC proteome (Figure 1)
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Figure 1.

Experimental process

Label the newly synthesized protein in MSC

The researchers purchased mouse-derived MSCs from Saiye Biology
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In order to achieve non-classical amino acid labeling in MSCs, they generated MetRSL274G-mCherry lentiviral vector.

Under the catalysis of copper ions, ANL-labeled proteins can react with dyes containing alkyne groups, thereby visualizing them through bioorthogonal non-classical amino acid markers (BONCAT) or fluorescent non-classical amino acid markers (FUNCAT), and can also be combined with alkyne-containing dyes.
The base resin reacts to achieve separation/enrichment and mass spectrometry identification
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They found that through this method, ANL-labeled proteins can be detected sensitively and specifically

Figure 2.

MSC's proteomic labeling strategy

In order to study the dynamic expression of MSC proteome in ischemic and normal hearts, they injected MetRSL274G-transduced MSCs into the infarct marginal zone of myocardial infarction mice or related areas of sham-operated mice, and then injected ANL into the intraperitoneal cavity at different time points.

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In the cells implanted in MSC, the expression of MetRSL274G produced a strong FUNCAT signal, indicating the presence of new ANL marker proteins in the cells

Proteome analysis at different time points

On the first 1, 3, and 7 days after the injection of the cells, the researchers isolated the MSC proteome from the left ventricular cell lysate through the click reaction, and identified it by LC/MS
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In a total of 6 groups (myocardial infarction group and sham operation group, three time points each), 648 kinds of proteins are shared, accounting for 82±5% of the total protein in each group
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Although there are a large number of common expression patterns, they found that the proteome of the myocardial infarction group and the sham operation group can still be distinguished well
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In order to identify the differentially expressed MSC proteins in ischemic and normal hearts, they compared the differences between the two using volcano plots
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Interestingly, on days 1, 3, and 7, compared with the control, the ischemic heart had 26, 110, and 65 proteins that were significantly up-regulated, and 11, 28, and 19 proteins were significantly down-regulated
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Through GO enrichment analysis, they found that the differences are mainly concentrated in the extracellular matrix structure, stress response and regulation of apoptosis process, which may reflect how MSC responds to the ischemic and hypoxic myocardial environment and exerts its ability to repair the heart The role of (Figure 3)
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Figure 3.
Identification of differentially expressed proteins in MSCs in ischemic and control hearts

Specifically, from day 1 to day 3, filaggrin A in the ischemic heart increased 12-fold, which was significantly higher than the control
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The detection levels of cofilin and fibronectin in the myocardial infarction group were also higher than those in the sham operation group
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These data indicate that the effect of MSCs on the ischemic heart may be mainly due to their production of extracellular matrix
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In addition, many damage-related molecular patterns (DAMP) have also been up-regulated, including heat shock protein, S100A9 protein, HMGB2 and complement factor H, indicating that MSC is involved in inflammation regulation
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In order to better understand the functional status of MSCs in ischemic myocardium, the researchers carried out KEGG pathway analysis
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They found that, compared with the control, pathways and proteins related to complement and coagulation cascade, actin cytoskeleton regulation, and apoptosis are highly enriched in ischemic hearts, indicating that the MSC proteome and secretome may It is a valuable resource for the treatment of cardiac ischemia
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Concluding remarks

Researchers believe that bio-orthogonal non-classical amino acid markers are a powerful tool for the overall analysis of the dynamic proteome in the body
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This method helps people understand the adaptability and repair activities of MSC in real time
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This result also indicates that the damage-related molecular patterns and complement brought by MSC may be involved in heart tissue repair
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Original Search

Han D, Yang J, Zhang E, Liu Y, Boriboun C, Qiao A, Yu Y, Sun J, Xu S, Yang L, Yan W, Luo B, Lu D, Zhang C, Jie C, Mobley J, Zhang J , Qin G.
Analysis of mesenchymal stem cell proteomes in situ in the ischemic heart.
Theranostics 2020; 10(24):11324-11338.
doi:10.
7150/thno.
47893.