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IntroductionHeat shock protein 70.3 (Hsp70.3) expression is increased in response to cellular stress and plays a cytoprotective role, including a cardioprotective role in the heart against ischemia/reperfusion (I/R) injury. Recent work from our lab shows that Hsp70.3 expression following I/R is controlled through alternative polyadenylation (APA). Truncation of the 3′UTR removes potential regulatory sequences and is observed concomitant with upregulation of Hsp70.3 expression. Herein, we investigated the hypothesis that APA truncation of the 3′UTR enhances polysome loading of the Hsp70.3 transcript.MethodsSucrose gradient ultracentrifugation was used to isolate polysome bound mRNA from HL‐1 cardiac myocyte cells subjected to control (long 3′UTR) or a 1 hr heat shock (HS; short 3′UTR).ResultsCells subjected to HS for one hour showed a decrease in the total amount of polysome loaded mRNA. The Hsp70.3 transcript found in the polysome dense fractions existed as a higher ratio of the APA truncated short 3′UTR, thus supporting our hypothesis that APA truncation of the Hsp70.3 3′UTR increases polysome loading.Our results provide an enhanced understanding of APA on the regulation of Hsp70.3 and will have a significant impact in understating the regulation of cardiac gene regulation after I/R injury.This work was partially funded by a University of Cincinnati Provost Pilot Research Project grant (MT).

IntroductionCardiovascular disease is the leading cause of death in the US with coronary heart disease (ischemia/reperfusion; I/R injury) accounting for approximately half of these deaths. Recent data from our lab suggests that alternative polyadenylation (APA) plays a role in cardiac gene expression following I/R via modulation of mRNA 3′UTR length, leading to the inclusion or exclusion of regulatory sequence elements. The goal of this work is to identify specific RNA binding proteins (RBPs) that are likely to mediate APA in the myocardium.MethodsThe 3′UTR of the heat shock protein 70.3 (Hsp70.3) mRNA was randomly biotinylated throughout and incubated with cardiac protein extract associated with differential APA of the Hsp70.3 3′UTR. Biotinylated RNA and RBPs were co‐precipitated and bound RBPs were identified using mass spectrometry.ResultsWe identified a total of 45 known RBPs interacting with the Hsp70.3 3′UTR. Of these, 9 unique RBPs were found to bind only under conditions associated with a long 3′UTR, whereas 10 unique RBPs were found to bind only in association with APA truncation of the 3′UTR. Our results identified RBPs previously indicated to play a role in APA and/or mRNA degradation as well as RBPs whose function remains unknown. Work is ongoing to determine the functional role of these RBPs in alternative polyadenylation and cardiac I/R injury.This work was partially funded by a UC CCTST Grant (MT).