Early Responses to Oxidative Stress In Heart Cells: Signals From The Cell Membrane To The Nucleus and Beyond

Abstract

Oxidative stress is known to contribute to many forms of heart disease. Oxidants such as H₂O₂ can cause hypertrophy of cardiomyocytes (CMCs). Heart fibroblasts (HFs) also contribute to oxidant-induced heart disease by disordering the extracellular matrix and causing fibrosis. Since both of these cells encounter the same stressors in vivo, we examined the signaling pathways involved in responding to oxidative stress in both cell types. We have established the EGF Receptor, Src and matrix metalloproteinases (MMPs) as key regulators of oxidant-mediated phosphorylation of the MAPKs ERK1/2 and JNKs but not p38 in CMCs and HFs. We used oligonucleotide microarrays to examine the differences in global gene expression after H₂O₂ treatment in CMCs and HFs. Twenty-four hours after treatment, significant numbers of upregulated genes could be classified as being related to antioxidant or detoxification responses in both cell types. This trend lead us to examine the role of activation of promoters containing the Antioxidant Response Element (ARE) in the reaction of CMCs to H₂O₂. We have shown that H₂O₂ activates the ARE in CMCs in a manner that is dependant on the transcription factor Nf-E2 related factor 2 (Nrf2). ARE activation by H₂O₂ seems to induce cytoprotection. CMCs pretreated with H₂O₂ showed significantly less activation of caspase-3 when exposed to another oxidant, Doxorubicin. Overexpression of Nrf2 mediates this cytoprotection, possibly by protecting the cells from caspase-independent cell death. Although ARE-dependant genes were upregulated in the presence of excess Nrf2, two contractile proteins were repressed, suggesting that Nrf2 overexpression may have unknown side-effects in CMCs. We also studied the activation mechanism of Nrf2 in CMCs. Nrf2 protein levels increased after 10 min of exposure to 100 μM H₂O₂ and peaked at about 1 hr. Pharmacological and genetic inhibition of the PI3-Kinase pathway blocked AREluciferase activity in these cells. The PI3-Kinase inhibitor LY294002 also blocked Nrf2 protein accumulation, but not nuclear translocation. Here I present evidence that Nrf2 accumulation after H₂O₂ exposure is due to PI3-Kinase-mediated translational regulation. Since phosphorylation of translation initiation factors eIF4E and eIF2alpha are both inhibited by LY294002, Nrf2 translation initiation may be through non-5’ cap-mediated means.