Abstract 5294: Sequestration of Prosurvival Akt Pathway by Reductive Stress Underlies hR120GCryAB Protein Aggregation Cardiomyopathy in Mice
Recent genetic studies of protein aggregation R120GCryAB cardiomyopathy in mice have implicated a novel ‘toxic’ gain-of-function mechanism linked to increased G6PD activity and levels of reduced glutathione (GSH), termed ‘reductive stress.’ Because the intercrosses between hR120GCryAB High Tg and G6PD-deficient (20% normal activity) rescues the phenotype, we hypothesized that the pathogenesis of hR120GCryAB cardiomyopathy are causally linked to redox sensitive signaling transduction pathways controlling cardiac hypertrophy and failure. We examined the temporo-spatial effects on activation of the major stress-dependent survival pathways. Human R120GCryAB expression in mice induces modest activation of total and phosphospecific p38 and ERK1/2 kinases but significantly increases phospho-specific JNK1/2 in vivo. Both total and phosphospecific AKT1/2 expression were similarly increased in hR120GCryAB and hR120GCryAB/G6PDmut in vivo. In contrast, immunohistochemical analysis indicated cytosolic Akt expression co-localized with in large protein aggregates, whereas G6PD deficiency completely prevented protein aggregation, indicating hR120GCryAB-induced reductive stress governs conformational states of PI3-kinase/AKT signaling in compartment-specific manner. Consistent with this notion, the cytosolic abundance of forkhead FOXO3a, a phosphorylation-dependent downstream target of nuclear AKT1/2 signaling, was decreased significantly in R120GCryAB High Tg heart homogenates and reciprocally increased in hR120GCryAB/G6PDmut in vivo. To investigate the critical roles of AktCys60 and AktCys77 residues, which form disulfide bond pairing under oxidized conditions, we next performed site-directed mutagenesis and demonstrated that redox-insensitive Akt mutants (AktC60A and AktC77A, respectively) were exclusively localized in the nucleus when expressed transiently in H9c2 myocytes. Redox-dependent mechanisms play a key role in the cardiotoxicity associated with hR120GCryAB-induced reductive stress. Furthermore, G6PD inhibition is a novel therapeutic strategy for mitigating cardiac hypertrophy and heart failure in hR120GCryAB cardiomyopathy.