Analysis of mesenchymal stem cell proteomes in situ in the ischemic heart

Abstract

Rationale: Cell therapy for myocardial infarction is promising but largely unsuccessful in part due to a lack of mechanistic understanding. Techniques enabling identification of stem cell-specific proteomes in situ in the injured heart may shed light on how the administered cells respond to the injured microenvironment and exert reparative effects. Objective: To identify the proteomes of the transplanted mesenchymal stem cells (MSCs) in the infarcted myocardium, we sought to target a mutant methionyl-tRNA synthetase (MetRS L274G ) in MSCs, which charges azidonorleucine (ANL), a methionine analogue and non-canonical amino acid, to tRNA and subsequently to nascent proteins, permitting isolation of ANL-labeled MSC proteomes from ischemic hearts by ANL-alkyne based click reaction. Methods and Results: Murine MSCs were transduced with lentivirus MetRS L274G and supplemented with ANL; the ANL-tagged nascent proteins were visualized by bio-orthogonal non-canonical amino-acid tagging, spanning all molecular weights and by fluorescent non-canonical amino-acid tagging, displaying strong fluorescent signal. Then, the MetRS L274G -transduced MSCs were administered to the infarcted or Sham heart in mice receiving ANL treatment. The MSC proteomes were isolated from the left ventricular protein lysates by click reaction at days 1, 3, and 7 after cell administration, identified by LC/MS. Among all identified proteins (in Sham and MI hearts, three time-points each), 648 were shared by all 6 groups, accounting for 82±5% of total proteins in each group, and enriched under mitochondrion, extracellular exosomes, oxidation-reduction process and poly(A) RNA binding. Notably, 26, 110 and 65 proteins were significantly up-regulated and 11, 28 and 19 proteins were down-regulated in the infarcted vs. Sham heart at the three time-points, respectively; these proteins are pronounced in the GO terms of extracellular matrix organization, response to stress and regulation of apoptotic process and in the KEGG pathways of complements and coagulation cascades, apoptosis, and regulators of actin cytoskeleton. Conclusions: MetRS L274G expression allows successful identification of MSC-specific nascent proteins in the infarcted hearts, which reflect the functional states, adaptive response, and reparative effects of MSCs that may be leveraged to improve cardiac repair.

Deciphering the in vivo Dynamic Proteomics of Mesenchymal Stem Cells in Critical Limb Ischemia

“…The change of ribosomal proteins to ischemic insults has been shown to persist for up to 14 days. We demonstrated that the increased expression of RPL28 and RPL26 at days 1 and 3 postsurgery, consistent with other reports (Lee et al, 2013;Ong et al, 2013;Han et al, 2020), and elevations declined to normal values by day 14. RPL28 belongs to the ribosomal proteins family.…”

“…Flow cytometry was performed as we previously described ( Han et al, 2020 ). Briefly, BMSCs cells were digested by trypsin and collected by centrifugation.…”

“…In this study, we utilized a metabolic labeling technique to tag and further enrich nascent proteins in the transplanted cells in situ ( Erdmann et al, 2015 ; Sadlowski et al, 2018 ; Alvarez-Castelao et al, 2019 ; Han et al, 2020 ). Mutation of MetRS from L to G at 274 site allowed the utilization of ANL instead Met during protein synthesis.…”

“…Mutation of MetRS from L to G at 274 site allowed the utilization of ANL instead Met during protein synthesis. In this way, nascent proteins are tagged with ANL in cells transduced with mutant MetRS ( Han et al, 2020 ). Further identification, enrichment and analysis of ANL tagged proteins will allow us to decipher the dynamic cell functions in vivo .…”

Visualization and Identification of Bioorthogonally Labeled Exosome Proteins Following Systemic Administration in Mice

“…Thus, when exosomes produced by the MetRS* MSCs were systemically administered to mice, the protein components of their cargo could be accurately and reliably tracked, visualized, identified, and quantified in tissues from a variety of organs, including both intact and infarcted hearts. Notably, as bioorthogonal chemistry can occur in living systems without disrupting native biochemical processes ( Prescher et al, 2004 ; Prescher and Bertozzi, 2005 ; Sletten and Bertozzi, 2011 ; Ngo et al, 2013 ; Han et al, 2020a ), this approach can be applied to other cell populations and even extended to wholly in vivo investigations by generating MetRS ∗ -transgenic mice ( Alvarez-Castelao et al, 2017 ; Han et al, 2020a ).…”