Nuclear receptor Rev-erb-α mediates palmitic acid-induced lipid peroxidation in cardiomyocytes

Abnormal cardiac lipid metabolism is a major contributor to cardiovascular disease (CVD). The nuclear receptor Rev-erb-α is recognized as a regulator of lipid metabolism; however, its role in cardiomyocyte lipotoxicity remains undefined. This study aims to investigate the functional role of Rev-erb-α in palmitic acid (PA)-induced lipid accumulation and peroxidation in cardiomyocytes. H9c2 cardiomyocytes are treated with the canonical Rev-erb agonists SR9009 and GSK4112 in combination with PA. Intracellular lipid droplet accumulation is quantified using Oil Red O, Nile red, and BODIPY 493/503 staining. Cytoplasmic and mitochondrial reactive oxygen species (ROS) levels are measured using 2',7'-DCFDA, dihydroethidium, and Mito-SOX probes, respectively, while DNA damage is assessed by quantifying the markers 53BP1 and γ-H2AX. Additionally, siRNA-mediated knockdown and adenovirus-mediated overexpression of Rev-erb-α are employed to validate its role in H9c2 and/or neonatal rat ventricular cardiomyocytes. PA treatment downregulates Rev-erb-α protein expression. Surprisingly, both SR9009 and GSK4112 exacerbate lipid droplet production and ROS production while activating the nuclear factor erythroid 2-related factor 2 (Nrf2)/heme oxygenase-1 (HO-1) antioxidant pathway, independent of Rev-erb-α. However, direct adenovirus-mediated overexpression of Rev-erb-α significantly attenuates lipid droplet formation and mitochondrial ROS, which is reversed by the Nrf2 inhibitor brusatol. Mechanistically, Rev-erb-α functions as a transcriptional activator of Nrf2. Our results demonstrate a direct protective role for Rev-erb-α against lipotoxic stress and crucially reveal that its commonly used agonists have confounding, off-target pro-oxidant effects. These findings have critical implications for developing Rev-erb-α-targeted therapies for CVDs.