Cardiac Ferroptosis Induced Through Novel Signaling Pathways

Background: Ferroptosis is a newly described form of non-apoptotic cell death caused by iron (Fe)-dependent lipid peroxidation. It remains unclear how different signaling pathways/molecules are involved in ferroptosis induction and prevention in cardiac myocytes. In the present study, we aimed to determine the presence of ferroptosis induction via different pathways and the effects of potential anti-ferroptotic strategies in mouse ventricular myocytes. Methods: Ferroptosis was analyzed using the Live/Dead cell viability assay in isolated mouse ventricular myocytes after primary culture and drug treatment. Lipid peroxidation was evaluated by using Liperfluo. Cardiac-specific mitochondrial calcium uniporter (mCU) knockout (KO) mice were used to determine the involvement of mCU. Results: Ferroptosis was induced and the level of lipid peroxidation was increased by the treatment with: 1) iron (Fe; ferric ammonium citrate, 0.1-10 mM), 2) RSL-3 (0.4 μM), a direct inhibitor of the glutathione peroxidase 4 (GPX4), 3) erastin (50 μM), an inhibitor of cystine/glutamate antiporter xCT, 4) the ferroptosis suppressor protein 1 inhibitor (iFSP1, 3 μM). Furthermore, the relationship between the induction of ferroptosis and the cardiotoxicity of doxorubicin (an anthracycline anti-cancer drug) was also evaluated. Ferroptosis induced by above-mentioned inducers was prevented by ferrostatin-1 (a lipid radical scavenger) alone or in combination with Fe chelators (deferoxamine or 2, 2’-bipyridyl) or antioxidants (Trolox or mitoTEMPO) consistent with the dependency of ferroptosis on Fe, oxidative stress and lipid oxidation. Importantly, Fe- and iFSP1- induced ferroptosis was abrogated by mCU KO suggesting mCU-mediated mitochondrial Fe uptake plays an essential role in the occurrence of ferroptosis. However, mCU KO did not prevent the occurrence of ferroptosis induced by RSL-3 or erastin. Conclusions: Ferroptosis induced via different signaling pathways exhibited differential sensitivities to the currently known anti-ferroptosis approaches reported in other cell types. Combination of various suppressers of ferroptotic pathways to alleviate the progression of ferroptosis may provide a promising therapeutic strategy for heart disease.