Abstract 13051: A Lin28-let-7 Axis Modulates Adaptation of the Myocardium to Ischemia

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Background: The RNA binding protein Lin28 and microRNA-let-7 each regulate glucose metabolism, tissue repair and pluripotency; however, their roles in cardiac adaptation to ischemia are unknown. Methods and Results: miR-let-7c was significantly downregulated in myocardium of mice following a 2 hour coronary occlusion, compared to sham-operated controls (0.69 fold, p<0.05). In parallel, Lin28 protein was induced in the ischemic myocardium compared to adjacent non-ischemic tissue (~2.0-fold, p<0.05). Consistent with this, mature but not precursor forms of miR-let-7c were downregulated (0.7 fold, p<0.05) in neonatal rat ventricular myocytes (NRVMs) during both hypoxia and hypoxia with glucose depletion, but not in similarly treated fibroblasts, together with a time-dependent 6-fold increase in Lin28 protein compared to normoxic controls. mRNA levels of Lin28 were also increased, but less strikingly (2.2 fold vs normoxia control, p<0.05). Ischemia induced acetylation of Lin28, both in vivo and in NRVMs that was potentiated by the non-selective histone deacetylase (HDAC) inhibitor, trichostatin A, suggesting that Lin28 is post-translationally stabilized. Mechanistically, transduction of NRVM with a let-7g lentiviral expression vector rescued let-7 levels, prevented ischemia-induced myocyte death and caspase activation, and promoted phosphorylation and activation of Stat3. Knockdown of the let-7 family in hypoxic NRVMs using a sponge vector containing 6 let-7 binding sites derepressed predicted target genes Bcl2l1, DUSP1, INSR, Ras at both mRNA and protein levels and depressed STAT3 phosphorylation. Using PAR-CLIP, in which the RISC complex is crosslinked to its target transcripts, we observed direct let-7 targeting of Lin28A in cardiac myocytes. Conclusion: During ischemic stress, let-7 is post-transcriptionally downregulated in a myocyte-specific manner through the ischemia-mediated induction of its repressor, Lin28. Induction of Lin28 occurs through transcriptional induction as well as post-translational acetylation and stabilization. This Lin28-let-7 axis constitutes a bidirectional feedback loop that directly and indirectly regulates signaling cascades of known importance to myocardial adaption to ischemic stress.