Abstract Body (Do not enter title and authors here): Introduction: Prior studies support a potential role for the stress response protein regulated in development and DNA damage 1 (REDD1) in cardiac function deficits caused by diabetes. REDD1 targets protein phosphatase 2A to dephosphorylate Akt, resulting in reduced Akt-dependent suppression of glycogen synthase kinase 3β (GSK3β). REDD1-dependent GSK3β signaling promotes inflammation via activation of the transcription factor NF-κB. However, GSK3β has also been shown to act through a negative feedback loop to suppress REDD1 levels and thus prevent chronic activation of pro-inflammatory signaling. The studies herein were designed to investigate the interplay between GSK3β/NF-κB signaling and REDD1 levels in the context of diabetic cardiomyopathy.
Methods: Male B6:129 REDD1+/+ and REDD1-/- mice were administered low-dose streptozotocin (STZ) and/or fed a high-fat diet to induce diabetes. Echocardiography was performed. AC16 cardiomyocyte cultures were exposed to medium containing 30 mM glucose or an osmotic control. Immunofluorescence, western blotting, and RT-PCR were used to determine protein and mRNA levels. A constitutively active GSK3β S9A variant was expressed in cells and cycloheximide chase assays were performed. Chemical inhibitors and shRNAs were used to manipulate GSK3β and NF-κB signaling.
Results: REDD1 mRNA and protein levels were upregulated in the hearts of diabetic mice. Unlike diabetic REDD1+/+ mice, diabetic REDD1-/- mice did not exhibit enhanced GSK3β activity, pro-inflammatory cytokine levels, or reduced ejection fraction. In cell culture studies, GSK3β promoted REDD1 mRNA and protein levels and reduced the rate of REDD1 protein degradation. Inhibition of GSK3β or the NF-κB kinase IKK prevented increased REDD1 protein in cells exposed to hyperglycemic conditions. In diabetic mice treated with the GSK3 inhibitor VP3.15, cardiac REDD1 levels were normalized.
Conclusions: These studies support a new feed-forward loop that drives pro-inflammatory signaling in the context of diabetic cardiomyopathy. Enhanced REDD1 mRNA expression and reduced REDD1 protein degradation in response to activation of GSK3/NF-κB signaling support multiple nodes of regulatory action. Inhibition of this newly identified feed-forward loop may represent a therapeutic target for interventions to address heart disease caused by diabetes.