Abstract 523: Analysis of Mesenchymal Stem Cell Proteomes In Situ in the Ischemic Heart

Abstract

Cell therapy for myocardial infarction is promising but largely unsuccessful partially 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. To identify the proteomes of the transplanted mesenchymal stem cells (MSCs) in the infarcted myocardium, we sought to engineer a mutant methionyl-tRNA synthetase (MetRS L274G ) in MSC, which charges the methionine analogue azidonorleucine (ANL) to tRNA and subsequently to nascent proteins, permitting isolation of ANL-labeled MSC proteomes from ischemic hearts by ANL-alkyne based click reaction. 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 ventricle 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 their GO terms are enriched under mitochondrion, extracellular exosomes, oxidation-reduction process and poly(A) RNA binding. Notably, 35, 131 and 84 proteins were significantly up-regulated and 12, 38 and 27 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 microvesicles, inflammatory response and apoptosis and in the KEGG pathways of complements and coagulation cascades, lysosomes, and regulators of actin cytoskeleton. 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.