Elucidating the contribution of mitochondrial glutathione to ferroptosis in cardiomyocytes

Abstract

Ferroptosis is a programmed iron-dependent cell death associated with peroxidation of lipids particularly, phospholipids. Several studies suggested a possible contribution of mitochondria to ferroptosis although the mechanisms underlying mitochondria-mediated ferroptotic pathways remain elusive. Reduced glutathione (GSH) is a central player in ferroptosis that is required for glutathione peroxidase 4 to eliminate oxidized phospholipids. Mitochondria do not produce GSH, and although the transport of GSH to mitochondria is not fully understood, two carrier proteins, the dicarboxylate carrier (DIC, SLC25A10 ) and the oxoglutarate carrier (OGC, SLC25A11 ) have been suggested to participate in GSH transport. Here, we elucidated the role of DIC and OGC as well as mitochondrial bioenergetics in ferroptosis in H9c2 cardioblasts. Results showed that mitochondria are highly sensitive to ferroptotic stimuli displaying fragmentation, and lipid peroxidation shortly after the onset of ferroptotic stimulus. Inhibition of electron transport chain complexes and oxidative phosphorylation worsened RSL3-induced ferroptosis. LC-MS/MS analysis revealed a dramatic increase in the levels of pro-ferroptotic oxygenated phosphatidylethanolamine species in mitochondria in response to RSL3 (ferroptosis inducer) and cardiac ischemia-reperfusion. Inhibition of DIC and OGC aggravated ferroptosis and increased mitochondrial ROS, membrane depolarization, and GSH depletion. Dihydrolipoic acid, an essential cofactor for several mitochondrial multienzyme complexes, attenuated ferroptosis and induced direct reduction of pro-ferroptotic peroxidized phospholipids to hydroxy-phospholipids in vitro . In conclusion, we suggest that ferroptotic stimuli diminishes mitochondrial bioenergetics and stimulates GSH depletion and glutathione peroxidase 4 inactivation leading to ferroptosis.

Xanthohumol Protects the Rat Myocardium against Ischemia/Reperfusion Injury-Induced Ferroptosis

“…Recent studies have shown that using gas cluster ion beam secondary ion mass spectrometry imaging can map PEox at ferroptotic H9c2 cardiomyoblasts [ 48 ]. In addition, LC-MS/MS analysis revealed a dramatic increase in PEox species in mitochondria in response to RSL3 and cardiac I/R [ 9 ]. A previous study showed that XN markedly decreases the level of lipid peroxidation and ROS production in rat liver [ 49 ].…”

“…GPX4 not only catalyzes the peroxidation of hydrogen peroxide (H 2 O 2 ) but also detoxifies the lipid peroxides by reducing glutathione [ 8 ]. Jang et al reported that ferroptotic stimuli reduce mitochondrial bioenergetics, glutathione depletion, and GPX4 inactivation, leading to ferroptosis in cardiomyocytes [ 9 ]. Park et al indicated that downregulation of GPX4 during myocardial infarction contributes to ferroptotic cell death in cardiomyocytes upon metabolic stress [ 10 ].…”

Effects of Ferroptosis on the Metabolome in Cardiac Cells: The Role of Glutaminolysis

“…H9c2 cardioblasts were cultured according to the manufacturer’s recommendations (ATCC, Manassas, VA, USA) with minor modifications [ 12 ]. Briefly, the cells were incubated in DMEM based modified media containing 4 mM L-glutamine, 4.5 g/L glucose, 1 mM sodium pyruvate, and 1.5 g/L sodium bicarbonate, pH 7.4, and supplemented with 10% fetal bovine serum and 1% antibiotic solution (HyClone, Logan, UT, USA) in a CO 2 incubator containing 95% air and 5% CO 2 at 37 °C.…”

“…Recent studies suggested a direct link between ferroptosis and coronary heart diseases, particularly myocardial infarction and ischemia-reperfusion, in animal models and patients [ 8 , 9 , 10 , 11 , 12 ]. However, the mechanisms of ferroptosis signaling in the heart remain unknown, which represents a challenge for the development of new therapeutic strategies for coronary heart diseases.…”

“…However, the mechanisms of ferroptosis signaling in the heart remain unknown, which represents a challenge for the development of new therapeutic strategies for coronary heart diseases. Inhibition of ferroptosis by the synthetic antioxidant ferrostatin-1 (Fer-1) demonstrated strong protective effects against cell death in cultured cardiomyocytes and isolated perfused hearts [ 8 , 9 , 10 , 12 , 13 ]. Fer-1 inhibits lipid peroxidation more effectively than other phenolic antioxidants since it scavenges alkoxyl radicals without being consumed in the process and then is regenerated by ferrous iron, reducing back the Fer-1 radical [ 14 ].…”