Ferroptosis is a recently identified iron‐dependent form of nonapoptotic cell death implicated in brain, kidney, and heart pathology. However, the biological roles of iron and iron metabolism in ferroptosis remain poorly understood. Here, we studied the functional role of iron and iron metabolism in the pathogenesis of ferroptosis. We found that ferric citrate potently induces ferroptosis in murine primary hepatocytes and bone marrow–derived macrophages. Next, we screened for ferroptosis in mice fed a high‐iron diet and in mouse models of hereditary hemochromatosis with iron overload. We found that ferroptosis occurred in mice fed a high‐iron diet and in two knockout mouse lines that develop severe iron overload (Hjv–/– and Smad4Alb/Alb mice) but not in a third line that develops only mild iron overload (Hfe –/– mice). Moreover, we found that iron overload–induced liver damage was rescued by the ferroptosis inhibitor ferrostatin‐1. To identify the genes involved in iron‐induced ferroptosis, we performed microarray analyses of iron‐treated bone marrow–derived macrophages. Interestingly, solute carrier family 7, member 11 (Slc7a11), a known ferroptosis‐related gene, was significantly up‐regulated in iron‐treated cells compared with untreated cells. However, genetically deleting Slc7a11 expression was not sufficient to induce ferroptosis in mice. Next, we studied iron‐treated hepatocytes and bone marrow–derived macrophages isolated from Slc7a11–/– mice fed a high‐iron diet. Conclusion: We found that iron treatment induced ferroptosis in Slc7a11–/– cells, indicating that deleting Slc7a11 facilitates the onset of ferroptosis specifically under high‐iron conditions; these results provide compelling evidence that iron plays a key role in triggering Slc7a11‐mediated ferroptosis and suggest that ferroptosis may be a promising target for treating hemochromatosis‐related tissue damage. (Hepatology 2017;66:449–465).

Rationale: Maintaining iron homeostasis is essential for proper cardiac function. Both iron deficiency and iron overload are associated with cardiomyopathy and heart failure via complex mechanisms. Although ferritin plays a central role in iron metabolism by storing excess cellular iron, the molecular function of ferritin in cardiomyocytes remains unknown. Objective: To characterize the functional role of ferritin H (Fth) in mediating cardiac iron homeostasis and heart disease. Methods and Results: Mice expressing a conditional Fth knockout allele were crossed with two distinct Cre recombinase-expressing mouse lines, resulting in offspring that lack Fth expression specifically in myocytes (MCK-Cre) or cardiomyocytes (Myh6-Cre). Mice lacking Fth in cardiomyocytes had decreased cardiac iron levels and increased oxidative stress, resulting in mild cardiac injury upon aging. However, feeding these mice a high-iron diet caused severe cardiac injury and hypertrophic cardiomyopathy, with molecular features typical of ferroptosis, including reduced glutathione (GSH) levels and increased lipid peroxidation. Ferrostatin-1, a specific inhibitor of ferroptosis, rescued this phenotype, supporting the notion that ferroptosis plays a pathophysiological role in the heart. Finally, we found that Fth-deficient cardiomyocytes have reduced expression of the ferroptosis regulator Slc7a11, and overexpressing Slc7a11 selectively in cardiomyocytes increased GSH levels and prevented cardiac ferroptosis. Conclusions: Our findings provide compelling evidence that ferritin plays a major role in protecting against cardiac ferroptosis and subsequent heart failure, thereby providing a possible new therapeutic target for patients at risk of developing cardiomyopathy.

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Although the serum-abundant metal-binding protein transferrin (encoded by the Trf gene) is synthesized primarily in the liver, its function in the liver is largely unknown. Here, we generated hepatocyte-specific Trf knockout mice (Trf-LKO), which are viable and fertile but have impaired erythropoiesis and altered iron metabolism. Moreover, feeding Trf-LKO mice a high-iron diet increased their susceptibility to develop ferroptosis-induced liver fibrosis. Importantly, we found that treating Trf-LKO mice with the ferroptosis inhibitor ferrostatin-1 potently rescued liver fibrosis induced by either high dietary iron or carbon tetrachloride (CCl4) injections. In addition, deleting hepatic Slc39a14 expression in Trf-LKO mice significantly reduced hepatic iron accumulation, thereby reducing ferroptosis-mediated liver fibrosis induced by either high dietary iron diet or CCl4 injections. Finally, we found that patients with liver cirrhosis have significantly lower levels of serum transferrin and hepatic transferrin, as well as higher levels of hepatic iron and lipid peroxidation compared to healthy controls. Taken together, these data indicate that hepatic transferrin plays a protective role in maintaining liver function, providing a possible therapeutic target for preventing ferroptosis-induced liver fibrosis.