SS-31@Fer-1 Alleviates ferroptosis in hypoxia/reoxygenation cardiomyocytes via mitochondrial targeting.

Purpose: Targeting mitochondrial ferroptosis presents a promising strategy for mitigating myocardial ischemia-reperfusion (I/R) injury. This study aims to evaluate the efficacy of the mitochondrial-targeted ferroptosis inhibitor SS-31@Fer-1 (elamipretide@ferrostatin1) in reducing myocardial I/R injury.

Methods: SS-31@Fer-1 was synthesized and applied to H9C2 cells subjected to hypoxia/reoxygenation (H/R) to assess its protective effects. Cytotoxicity was evaluated using a cell counting kit-8 (CCK-8) assay, with lactate dehydrogenase (LDH) and creatine kinase isoenzyme (CK-MB) levels measured. Mitochondrial reactive oxygen species (ROS) and mitochondrial membrane potential (MMP) were assessed using Mito-SOX and JC-1 fluorescent dyes, respectively. Lipid peroxidation products, malondialdehyde (MDA) and glutathione (GSH), were quantified. Mitochondrial structure, mt-cytochrome b (mt-Cytb), and mt-ATP synthase membrane subunit 6 (mt-ATP6) were analyzed. Additionally, iron homeostasis and ferroptosis markers were evaluated.

Results: SS-31@Fer-1 significantly improved H/R-induced cardiomyocyte viability and reduced LDH and CK-MB levels. Compared to the Fer-1 group, SS-31@Fer-1 reduced GSH and increased MDA levels, enhancing mitochondrial integrity and function. Notably, it increased mitochondrial ROS and decreased MMP, indicating a mitigation of H/R-induced cardiomyocyte cytotoxicity. Furthermore, SS-31@Fer-1 maintained cellular iron homeostasis, as evidenced by increased expression of FTH, FTMT, FPN, and ABCB8. Elevated levels of GPX4 and Nrf2 were observed, while ACSL4 and PTGS2 levels were reduced in the SS-31@Fer-1 group.

Conclusions: SS-31@Fer-1 effectively suppressed ferroptosis in H/R-induced cardiomyocytes by maintaining cellular iron homeostasis, improving mitochondrial function, and inhibiting oxidative stress. These findings provide novel insights and opportunities for alleviating myocardial I/R injury.