Sickle cell disease (SCD) is characterized by increased hemolysis which results in plasma heme overload and ultimately cardiovascular complications. Here, we hypothesized that increased heme in SCD causes upregulation of heme oxygenase 1 (Hmox1) which consequently drives cardiomyopathy through ferroptosis, an iron-dependent non-apoptotic form of cell death. First, we demonstrated that the Townes SCD mice had higher levels of hemopexin-free heme in the serum and increased cardiomyopathy, which was corrected by hemopexin supplementation. Cardiomyopathy in SCD mice was associated with upregulation of cardiac Hmox1, and inhibition or induction of Hmox1 improved or worsened cardiac damage, respectively. Since free iron, a product of heme degradation through Hmox1, has been implicated in toxicities including ferroptosis, we evaluated the downstream effects of elevated heme in SCD. Consistent with Hmox1 upregulation and iron overload, levels of lipid peroxidation and ferroptotic markers increased in SCD mice, which were corrected by hemopexin administration. Moreover, ferroptosis inhibitors decreased cardiomyopathy, whereas a ferroptosis inducer erastin exacerbated cardiac damage in SCD and induced cardiac ferroptosis in non-sickling mice. Finally, inhibition or induction of Hmox1 decreased or increased cardiac ferroptosis in SCD mice, respectively. Together, our results identify ferroptosis as a key mechanism of cardiomyopathy in SCD.

Ferroptosis is a recently defined form of iron‐dependent, non‐apoptotic programmed cell death that is characterized by accumulation of lipid peroxides. Although ferroptosis was first identified in cancer cells, recent studies have demonstrated its implications in a wide range of physiological disorders such as ischemic and hemorrhagic stroke, Alzheimer’s disease, and epilepsy. Notably, there is a significant association between iron loading and heart failure. For example, cardiomyopathy is the leading cause of mortality in thalassemia and accounts for 30% of mortality in iron overload sickle cell disease (SCD). However, the underlying mechanisms of iron‐induced cardiotoxicity are not well understood. Although cell death mechanisms such as apoptosis and necrosis have been recognized in the context of heart disease, the role of ferroptosis in cardiac damage is largely unexplored. Here we hypothesized that cardiac iron overload promotes ferroptosis and cardiac dysfunction in the Townes mouse model of SCD. To test our hypothesis, we determined and compared iron levels, cardiotoxicity and ferroptotic markers between sickling HbSS (homozygous for the human βS globin gene) and their non‐sickling heterozygous HbAS littermates. First, we observed an increase in systemic and tissue iron levels in HbSS mice. We also found increased expression of the cardiotoxic markers ANP (atrial natriuretic peptide) and BNP (brain natriuretic peptide). These results were associated with an upregulation of the ferroptotic marker, Ptgs2, and increased levels of lipid peroxidation, a characteristic of ferroptosis, in the cardiac tissue of HbSS mice. However, no differences were found in caspase‐3 levels, suggesting that cardiotoxicity does not result from apoptotic mechanisms. To distinguish between the effects of the sickling gene and iron loading, we induced iron overload in non‐sickling mice by iron dextran treatment, an established model of secondary iron overload. Similar to our findings in sickling mice, we demonstrated that induced iron overload displayed increased lipid peroxidation and cardiac damage, consistent with cardiac ferroptosis. Finally, inhibition of ferroptosis, either by chelating excess iron (using an iron chelator deferoxamine) or by inhibiting lipid peroxidation (using ferrostatin‐1, a lipid radical scavenger), improved cardiac dysfunction in sickling mice. These findings suggest a novel mechanism of cardiac cell death in SCD and could provide potential protective targets against iron‐induced cardiotoxicity.

Decreased red blood cell (RBC) half‐life in sickle cell disease (SCD) results in increased hemolysis and is associated with stroke and other cardiovascular complications although the exact mechanisms remain unexplored. We hypothesized that increased circulating heme in SCD promotes cardiotoxicity through ferroptosis by upregulating heme oxygenase 1 (Hmox1). Our data demonstrated that sickling Townes mice had higher serum heme, lower serum levels of the heme scavenger protein hemopexin and increased cardiotoxicity and that supplementation with hemopexin decreased cardiotoxicity in sickling mice. We also demonstrated that cardiac expression of Hmox1 was increased in SCD mice; inhibition or induction of Hmox1 decreased or worsened cardiac damage, respectively. We also demonstrated that labile redox‐active iron, a product of heme degradation by Hmox1, was increased in SCD. Subsequently, we evaluated if ferroptosis, an iron‐dependent non‐apoptotic cell death mechanism, resulted from increased Hmox1 and drove cardiotoxicity in SCD. We found an increase in lipid peroxidation and ferroptotic markers in the cardiac tissue of SCD mice, which were corrected by hemopexin and Hmox1 inhibition whereas Hmox1 induction exacerbated lipid peroxidation and cardiac ferroptosis. Finally, inhibitors or inducers of ferroptosis decreased or exacerbated SCD cardiotoxicity respectively. Together, our results identify a novel mechanism for heme‐associated cardiac damage in SCD.

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