Abstract 18985: K-Ras Activates Mammalian Hippo Signaling to Promote Ischemia/Reperfusion Injury in the Heart

Abstract

Acute myocardial ischemia and reperfusion (I/R) cause damage to the heart, thereby impairing its ability to function effectively. Therapies aimed at preventing I/R injury are limited and a better understanding of the mechanisms involved remains critical. In hearts of mice subjected to I/R we found differing kinetics of H- and K-Ras activation, and only K-Ras modification following oxidative stress was observed. Expression of active H-Ras12V caused increased phosphorylation of AKT (1.8±0.3-fold vs LacZ) and ERK1/2 (3.3±0.3-fold vs LacZ) and protected cardiomyocytes against apoptosis, whereas K-Ras12V activated mammalian sterile 20-like kinase 1 (Mst1)(2.1±0.1-fold vs LacZ), which led to myocyte death. Endogenous K-Ras, but not H-Ras, was detected at mitochondria and associated with Ras association domain family 1 isoform A (RASSF1A) in both cardiomyocytes and mouse hearts. Genetic disruption of RASSF1A prevented K-Ras-induced Mst1 activation and inhibited Mst1 translocation to mitochondria in response to oxidative stress. Mass spectrometry analysis revealed that active Mst1 phosphorylates Bcl-xL at Ser14, a previously unreported residue, in cardiomyocytes and mouse heart, and mediates Bcl-xL phosphorylation in response to I/R in vivo. Ser14 phosphorylation attenuated Bcl-xL-Bax binding and the phosphomimetic Bcl-xL S14D mutant showed a weakened protective capacity against apoptotic insult (cell viability; LacZ + H2O2 45±5%, XL WT + H2O2 76±4%, XL SD + H2O2 51±5%, p<0.05). Following in vivo I/R, Kras+/- mice had significantly smaller myocardial infarct size vs WT mice (22±4% vs 39±2%, p<0.05), while no significant difference in infarct size was observed in Hras+/- mice (37±3% vs 38±3%). Conversely, cardiac-specific K-Ras12V TG mice had increased Mst1 activation and significantly larger infarcts vs NTG mice (67±10% vs 36±2%, p<0.05). This myocardial damage was rescued in bigenic mice expressing both K-Ras12V and Dn-Mst1 (41±7%, p<0.05), implicating Mst1 as a critical mediator of K-Ras in vivo. Our work demonstrates a distinct role for K-Ras as an endogenous promoter of mammalian Hippo signaling, and identifies a novel mechanism involving phosphorylation of Bcl-xL by which Mst1 mediates cardiomyocyte apoptosis and I/R injury in the heart.