335 - Frataxin-Deficiency in the Heart Results in an Impaired Nrf2 Response: A Dual Mechanism Mediated via Up-Regulation of Keap1 and GSK3β Axis

Abstract

Friedreich's ataxia (FA) is a neuro- and cardio-degenerative disease due to frataxin-deficiency and leads to marked mitochondrial iron loading. The ensuing oxidative stress is likely a key contributor to the pathology. Recent studies in neuronal FA models have reported defective expression of the transcription factor, nuclear factor E2-related factor 2 (Nrf2), which regulates the cellular antioxidant response. However, the mechanism has not been elucidated and no molecular examination of the anti-oxidant response has been conducted in the FA heart where fatal cardiomyopathy develops. Using the MCK conditional frataxin knockout (KO) mouse, which exhibits a cardiomyopathy that mimics the human disease, we examined the Nrf2 antioxidant response pathway in the frataxin-deficient heart. Our studies demonstrated protein and GSH oxidation in the KO relative to wild-type (WT) littermates. Despite this, we found decreased total and nuclear Nrf2 protein levels and increased Keap1-mediated Nrf2 degradation in the heart. Moreover, we also demonstrated the involvement of Gsk3β-dependent export and degradation of nuclear Nrf2 in the heart, as evident by: (i) increased Gsk3β activation; (ii) increased Fyn phosphorylation, mediating nuclear export; and (iii) increased expression of β-TrCP, a substrate recognition subunit of the E3 ubiqitin ligase complex that is involved in Nrf2 degradation. Furthermore, electrophoretic mobility shift assays demonstrated decreased Nrf2-DNA-binding activity and a general decrease in target gene expression in frataxin KO hearts. These results indicate the blunted anti-oxidant response in the frataxin-deficient heart is, at least in part, due to reduced Nrf2 activity caused by its increased degradation via the Keap1 and Gsk3β pathways.