The effects of glutaminolysis inhibition on ferroptotic cell death signaling in cardiomyocytes

Abstract

Mitochondria play a central role in the regulation of cellular redox status. Reduced glutathione (GSH), a highly potent essential antioxidant which directly scavenges various oxidants such as superoxide anion, hydroxyl radical, nitric oxide, and carbon radicals in the cell. Moreover, GSH, through the glutathione peroxidase (GPX) family, is responsible for detoxifying H2O2, peroxynitrites, and lipid peroxides. Conversely, GSH depletion has been shown to trigger programed cell death pathways such as apoptosis and ferroptosis, however the molecular mechanisms underlying these death pathways remain unknown. The amino acids cysteine and glutamine are detrimental for sustaining cellular GSH levels, although during glucose deprivation intracellular glutamine can be transported to the mitochondria and catabolized through the TCA cycle to produce ATP through the process known as glutaminolysis. Glutamine is converted to glutamate through glutaminolysis catalyzed by glutaminase C (GAC), a mitochondrial localized K-type glutaminase, thereby, leading to the formation of 2-oxoglutarate, a TCA cycle intermediate. Excessive metabolism of glutamate by the TCA cycle can lead to GSH depletion and thus, stimulate ferroptosis. In this study, we evaluated if inhibition of glutaminolysis through genetic silencing of mitochondrial GAC can affect ferroptotic cell death and mitochondrial GSH levels/redox status in H9c2 cardiomyocytes. GAC expression was silenced in the cells with a GAC siRNA (GAC-KD cells) in the absence or presence of ferroptosis inducing agent, RSL3 (0.5 µM). Ferrostatin-1 (Fer-1, 2µM), a well-known anti-ferroptotic agent, was used to demonstrate the specificity of ferroptosis. In control group, the cells were treated with a non-coding siRNA (NC) under the same conditions. Cell death (LDH release to culture medium), mtROS production, GSH levels, and mitochondrial membrane potential were analyzed. We found that the cells treated with GAC siRNA showed a 51% reduction in GAC levels compared to NC cells. Analysis of LDH release in NC and GAC-KD cells incubated in the presence of RSL3 and/or Fer-1 for 1 h, 3 h, and 6 h demonstrated that LDH levels were significantly reduced in control, RSL3, and both treatment with GAC-KD compared to NC cells after 1 h incubation however, no differences were found in 3h and 6h of incubation. The GSH level was found a 30% higher in GAC-KD cells compared to NC cells. RSL3 induced a further increase (50%) of the GSH level in NC cells. In conclusion, our data demonstrate inhibition of glutaminolysis provides an early protection against RSL3-induced ferroptosis in H9c2 cardiomyocytes due to a surge in GSH levels suggesting a crucial role of glutaminolysis in early stages of ferroptosis.