Abstract 17228: Rassf1A Inhibits Akt Activation in Cardiac Fibroblasts and Protects Against Ischemia Reperfusion Injury in the Heart
Our previous work demonstrated Rassf1A to be a critical mediator of Mst1 activation, the chief component of the mammalian Hippo pathway, in heart failure. In the setting of ischemia/reperfusion (I/R) injury, Mst1 is robustly activated and promotes injury in the heart; however, its regulation remains unclear. Using genetically modified mice in which expression of Rassf1A is altered in a cell type-specific manner, we demonstrate that systemic deletion of rassf1a-/- (KO) does not alter infarct size (36±4% vs 33±3%) or cardiac myocyte apoptosis (1.7±0.2% vs 2.1±0.4%) following I/R versus WT. Conversely, mice harboring deletion of rassf1a in cardiac myocytes (rassf1aflox/floxαMHC-Cre, CKO) have smaller infarcts (23±2 vs 38±3%, p<0.05) and less apoptosis (0.9±0.2 vs 2.0±0.2%, p<0.05) after I/R. Importantly, attenuation of Mst1 activation in ventricular homogenates was observed in both deletion models, implicating Rassf1A as a positive regulator of Mst1 during I/R. Langendorff global I/R injury yielded similar results - no protection in KO, yet significant protection in CKO hearts versus control mice, suggesting that native cardiac cells are sufficient to mediate this response. Using isolated cardiac fibroblast cultures we determined that Rassf1A negatively regulates activation of Akt in response to oxidative stress. Further, knockdown of endogenous Rassf1A causes increased NF-κB activity, a response mediated by Akt. A candidate molecule screen found exaggerated TNF-α expression in non-myocytes of KO hearts compared to WT (4.8±0.7fold, p<0.05), whereas TNF-α expression was attenuated in CKO hearts (reduced 65±8% vs control, p<0.05). Finally, WT and KO mice were administered TNF-α neutralizing antibody or control IgG and subjected to I/R. KO hearts treated with TNF-α Ab, but not IgG, had reduced infarcts (19±4% vs 34±5%, p<0.05), but no significant reduction in infarct size was observed in WT mice given TNF-α Ab. Taken together these data suggest that TNF-α blockade prevents the deleterious consequences of Rassf1A deletion in non-myocytes while unmasking the protective effect of Rassf1A deletion in cardiac myocytes following I/R. Further, these results demonstrate the importance of non-myocytes in modulating cardiac myocyte survival and I/R injury
- © 2011 by American Heart Association, Inc.