The contribution of SLC7A11-mediated ferroptosis to cardiac injury in iron overload cardiomyopathy: an in vitro study

Abstract Background Recent studies have indicated that iron overload can induce myocardial ferroptosis in iron overload cardiomyopathy (IOC), but the specific mechanisms, including the role of SLC7A11, remain unclear. Purpose By delving into the role of SLC7A11-mediated ferroptosis in IOC, this research endeavors to provide novel insights and therapeutic strategies for understanding and treating IOC. Methods To induce an in vitro model of IOC, wild-type rat cardiomyocytes (H9C2) were cultured in a ferric citrate (FAC) medium. Cardiomyocytes were exposed to various concentrations of iron overload (ranging from 0 mM to 2 mM) for durations of 4, 8, 12, and 24 hours. Cell viability at different FAC concentrations was assessed by the cell counting kit-8 (CCK8) assay and the level of the ferroptosis indicator malondialdehyde (MDA) was measured to determine the optimal concentration and duration of FAC treatment which was required to establish the IOC cell model. Next, ferroptosis indicators, including MDA, prostaglandin peroxide synthase 2 (PTGS2), and glutathione peroxidase 4 (GPX4), and the pivotal factor, SLC7A11 of IOC cardiomyocytes were evaluated. The protein and mRNA levels of SLC7A11 were assessed by molecular biology techniques. Additionally, the mitochondrial morphology of the IOC cardiomyocytes was meticulously examined by transmission electron microscopy (TEM). Flow cytometry was employed for detecting reactive oxygen species (ROS) levels. Furthermore, we elucidated the underlying mechanism of myocardial injury in IOC by overexpressing SLC7A11 and subsequently detecting changes in ferroptosis markers. Results CCK-8 results showed that the viability of cardiomyocytes exhibited dose-dependently with increasing FAC concentration (R2=0.5483, p<0.0001). Furthermore, the viability of cardiomyocytes remained relatively stable, with survival rates of approximately 50% and not decreasing below 50%. Additionally, the MDA content in IOC cardiomyocytes increased dose-dependently with the elevation of FAC concentration (R2=0.75, p<0.05), with a significant increase observed at 0.75mM. Expression levels of ferroptosis marker genes, GPX4 and PTGS2, both at the mRNA and protein levels, are indicative of diminished antioxidant capacity. Meanwhile, the level of myocardial MDA and ROS were markedly elevated, indicating underscoring increased oxidative stress within the IOC cardiomyocytes. Moreover, there was a downregulation in the expression of SLC7A11 mRNA and protein in IOC cardiomyocytes, suggesting a potential compensatory mechanism to counteract oxidative damage. TEM further elucidated cardiac mitochondrial injuries in IOC rats, including focal vacuolization, swelling, crest disruption, and even disappearance. Overexpression of SLC7A11 resulted in a significant augmentation of GPX4 and a reduction of PTGS2mRNA and protein expression levels. Conclusions Ferroptosis, mediated by SLC7A11, may represent a pivotal mechanism underlying myocardial injury in IOC.