After myocardial infarction (MI) cardiac repair starts right after reperfusion and involves cardiac fibroblasts (CF) as key players in cardiac remodeling. In the early phase, CF secrete proinflammatory cytokines and proteases which degrade the damaged tissue. Thereafter, cardiac fibroblasts are activated (aCF) and proliferate producing collagen and other extracellular matrix proteins that promote scar formation (Humeres and Frangogiannis, JAAC 4:449-467, 2019). In a recent single-cell mRNA sequencing study, our group has shown that activated cardiac fibroblasts (5 days post MI) express numerous paracrine factors which potentially may influence inflammation and myocardial contraction (Hesse et al., eLife 10:e65921, 2021). In the present study, we explored the secretome of cultured CF and aCF using metabolic labeling of proteins and subsequent mass spectrometric detection. We used a recently published technique that combines stable isotope (SILAC) labeling and Click-Chemistry (Eichelbaum and Krijgsveld, Mol Biol. 1174:101-14, 2014) which enables the quantitative secretome analysis of newly synthesized proteins of CF and aCF in-vitro.
Methods
Cardiac fibroblasts were isolated 5 days after MI (50 min ischemia followed by reperfusion) or 5 days after sham surgery (control). As SILAC uses the metabolic incorporation of “heavy” or “intermediate” 13C- or 15N-labeled amino acids into proteins followed by mass spectrometry (MS), we compared the secretome of CF and aCF obtained after MI to CF obtained after sham surgery. In brief: cultured CF and aCF were depleted of arginine (Arg), lysine (Lys) and methionine (Met) for 1h and thereafter labeled with heavy arginine and lysine isotopes or intermediate arginine and lysine isotopes respectively for 6h. In addition the cells were labeled with azidohomoalanine (AHA) which is analogue to methionine but carries an azide group. The azide group of AHA allows binding of these proteins to a biotin resin (CLICK reaction) so all other proteins and contaminants can be removed by washing.
Workflow
Results
We identified 84 proteins secreted from cardiac fibroblasts obtained 5 days post MI. Biologically interesting is the significant increase in Lysyl Oxidase Like 3 (Loxl3) compared to CF of sham operated hearts. Loxl3 oxidates lysyl and hydroxyl residues from collagen and plays a crucial role in epithelial-mesenchymal transition and matrix crosslinking after MI (Laurentino et al., Int. J. Mol. Sci. 20(14), 2019). In addition we found a higher abundance of proinflammatory proteins such as Chemokine (C-C motif) ligand 9 (CCL9). Furthermore we identified several proteins which play a role in cell signaling and migration e.g. Microfibril Associated Protein 5 (Mfap5), Sushi Repeat Containing Protein X-linked 2 (Srpx2) and Tyrosine-protein Kinase Receptor UFO (Axl) which regulates cell migration as well as cell proliferation (Zhu et al., Mol. Cancer 18(153), 2019).
Conclusion
Quantitative secretome analysis of CF and aCF formed after MI is possible using a combination of SILAC labeling and Click-Chemistry. Loxl3 was found to be significantly increased after MI other proteins such as CCL9, Mfap5, Srpx2 and Axl showed higher abundance. These proteins may be functionally relevant targets in future investigations.
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