  Citation: Rodríguez-Graciani, K.M.; Chapa-Dubocq, X.R.; Ayala-Arroyo, E.J.; Chaves-Negrón, I.; Jang, S.; Chorna, N.; S. Maskrey, T.; Wipf, P.; Javadov, S. Effects of Ferroptosis on the Metabolome in Cardiac Cells: The Role of Glutaminolysis. Antioxidants 2022, 11, 278. https://doi.org/10.3390/ antiox11020278 Academic Editors: Nicola King and M.-Saadeh Suleiman Received: 10 December 2021 Accepted: 27 January 2022 Published: 29 January 2022 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affil- iations. Copyright: © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). antioxidants Article Effects of Ferroptosis on the Metabolome in Cardiac Cells: The Role of Glutaminolysis Keishla M. Rodríguez-Graciani 1 , Xavier R. Chapa-Dubocq 1 , Esteban J. Ayala-Arroyo 1 , Ivana Chaves-Negrón 1 , Sehwan Jang 1 , Nataliya Chorna 2 , Taber S. Maskrey 3 , Peter Wipf 3 and Sabzali Javadov 1, * 1 Department of Physiology, School of Medicine, University of Puerto Rico, San Juan, PR 00936, USA; keishla.rodriguez20@upr.edu (K.M.R.-G.); xavier.chapa@upr.edu (X.R.C.-D.); esteban.ayala@upr.edu (E.J.A.-A.); ivana.chaves@upr.edu (I.C.-N.); sehwan.jang@upr.edu (S.J.) 2 Department of Biochemistry, School of Medicine, University of Puerto Rico, San Juan, PR 00936, USA; nataliya.chorna@upr.edu 3 Department of Chemistry, University of Pittsburgh, Pittsburgh, PA 15260, USA; taber.maskrey@pitt.edu (T.S.M.); pwipf@pitt.edu (P.W.) * Correspondence: sabzali.javadov@upr.edu; Tel.: +1-787-758-2525 (ext. 2909) Abstract: Ferroptosis is a novel iron-dependent regulated cell death mechanism that affects cell metabolism; however, a detailed metabolomic analysis of ferroptotic cells is not yet available. Here, we elucidated the metabolome of H9c2 cardioblasts by gas chromatography-mass spectrometry during ferroptosis induced by RSL3, a GPX4 inhibitor, in the presence of ferrostatin-1 (a ferroptosis inhibitor), XJB-5-131 (a mitochondrial-targeted ROS scavenger), or TSM-1005-44 (a newly developed cellular ROS scavenger). Results demonstrated that RSL3 decreased the levels of amino acids involved in glutathione synthesis more than two-fold. In contrast, saturated fatty acids levels were markedly increased in RSL3-challenged cells, with no effects on unsaturated fatty acids. RSL3 significantly altered the levels of mitochondrial tricarboxylic acid cycle intermediates; isocitrate and 2-oxoglutarate were found to increase, whereas succinate was significantly decreased in RSL3-challenged cells. Ferrostatin-1, XJB-5-131, and TSM-1005-44 prevented RSL3-induced cell death and conserved the metabolomic profile of the cells. Since 2-oxoglutarate is involved in the regulation of ferroptosis, particularly through glutamine metabolism, we further assessed the role of glutaminolysis in fer- roptosis in H9c2 cardioblasts. Genetic silencing of GLS1, which encodes the K-type mitochondrial glutaminase (glutaminase C), protected against ferroptosis in the early stage. In conclusion, our study demonstrates that RSL3-induced ferroptosis impairs the metabolome of H9c2 cardioblasts. Keywords: ferroptosis; cardiomyocytes; metabolome; mitochondria; glutaminolysis; anti-ferroptotic compounds; ferrostatin-1; XJB-5-131; TSM-1005-44 1. Introduction Ferroptosis is a newly discovered iron-dependent non-apoptotic programmed cell death pathway. It is characterized by the accumulation of oxidized phospholipids due to the excessive oxygenation of polyunsaturated fatty acid (FA) residues of phospholipids by lipoxygenases and the limited capability of glutathione peroxidase 4 (GPX4) to neutralize oxidized phospholipids [1–4]. Mitochondria are the main source of reactive oxygen species (ROS); electron transport chain (ETC) complexes, monoamine oxidase, α-ketoglutarate dehydrogenase, NADPH oxidase 4, among others, produce superoxide (•O 2 − ), which is reduced to hydrogen peroxide (H 2 O 2 ) upon interacting with superoxide dismutase [5]. Normally, catalase and H 2 O 2 -specific glutathione peroxidases convert H 2 O 2 into H 2 O; however, under pathological stimuli, free redox-active iron becomes available in the cytosol. Redox-active iron through the Fenton reaction further increases the accumulation of ROS and activates lipoxygenases, particularly 15-lipoxygenases. The latter stimulates oxidation Antioxidants 2022, 11, 278. https://doi.org/10.3390/antiox11020278 https://www.mdpi.com/journal/antioxidants