Abstract 295: Proteomic Profiling of Changes at Active Polysomes That Occur as a Response to Ischemic Injury and Reperfusion of the Heart

Abstract

Introduction: Heart ischemic injury and subsequent reperfusion activate dynamic changes in protein expression that, we hypothesize, are regulated by alternative mechanism at active polysomes. Since analysis of mRNA can correlate poorly with protein composition, we applied proteomic approach to characterize the changes related to ischemia and reperfusion in proteins that comprise the ribosomes, proteins associated with translation machinery and the newly synthesized proteins. Methods: Mouse heart tissue homogenates for conditions of sham, ischemia and ischemia/reperfusion (n=5) were subjected to separation in sucrose gradient and resulted fractions containing high-efficiency polysomes (heavy), low-efficiency polysomes (light) and non-translating fraction (NT) for each condition were subjected to proteomic analysis. Results: We identified more than 1300 proteins in total. We defined the core of active polysomes to be composed of 500 proteins which were always present in heavy fraction regardless of conditions. While 60S- and 80S ribosomal proteins were present in both heavy and light fractions, 66 proteins of mitochondrial ribosomal complex (28S and 39S) were captured exclusively in light fraction. Of interest were proteins that were increased or decreased in heavy fraction after ischemia and ischemia/reperfusion. In particular, mitochondrial elongation factor Tu (EFTU) was increased while nuclear transcriptional activator (PURA) was decreased after ischemia, and both returned to control levels upon reperfusion. Several 60S- and 40S ribosomal proteins remained unchanged after ischemia but their levels decreased after reperfusion (e.g. L37a, S11, S12 and S19). Transport proteins such as protein of transmembrane 9 superfamily (TM9S2) and monocarboxylate transporter 1 (MOT1) were decreased in ischemia and remained decreased after reperfusion. Conclusion: The proteins associated with polysomes and translation machinery of diverse activity were identified and their expression changes triggered by heart ischemic injury and reperfusion were characterized. To our knowledge, this is the first extensive proteomic characterization of polysome-related proteins revealing their dynamic profiles after ischemic stress in the heart.