Recent investigations have proposed a potential causal association between the occurrence of ferroptosis, nuclear factor kappa B (NF-κB) and ubiquitin-specific protease 24 (USP24). Nevertheless, the mechanism of USP24 and NF-κB regulation of ferroptosis in the context of diabetic cardiomyopathy (DCM) remain unclear.

In this study, a high-fat diet and a streptozotocin-induced mouse DCM model were established, and high glucose and palmitic acid treatment of H9c2 cells and neonatal mouse primary cardiomyocytes (NMPCs) was used as an in vitro DCM models. Utilizing both the in vivo and in vitro DCM models, we assessed of USP24, NF-κB, and ferroptosis levels, and explored the relationship among them.

In in vivo and in vitro DCM models, increased expression of USP24, NF-κB, phosphorylated NF-κB (p–NF–κB) and fatty acid-CoA ligase 4 (FACL4) were detected, along with accumulated iron, as well as reduced ferritin heavy chain 1 (FTH1), solute carrier family 7 member 11 (SLC7A11) and antioxidant capacity. Knockdown of USP24 resulted in a reduction of NF-κB levels, while knockdown of NF-κB did not lead to a decrease in USP24 expression. Moreover, in H9c2 cells, knockdown of USP24 and NF-κB separately resulted in reduced levels of FACL4, increased levels of SLC7A11 and FTH1, as well as improved antioxidant capacity and cell viability. In shUSP24 knockdown H9c2 cells, administration of phorbol 12-myristate 13-acetate (PMA) activated NF-κB, subsequently reversing the previously observed effect caused by USP24 knockdown.

These findings show that USP24 upregulates NF-κB to promote ferroptosis in DCM.

Schematic image of the study conclusions. Stimulation of myocardial cells by hyperglycemia and hyperlipidemia leads to an increase in USP24 expression. Subsequently, this triggers activation of NF-κB and initiates a cascade of molecular events. First and foremost, NF-κB downregulates the expression of SLC7A11, which restricts the exchange of cysteine and glutamate, leading to a reduction in intracellular cysteine levels and a consequent decline in GSH synthesis. In parallel, NF-κB upregulates the expression of FACL4, which catalyzes the conversion of arachidonic acid (AA) to AA-CoA by forming a reaction with acyl-coenzyme A (CoA). This AA-CoA species then undergoes a reaction with membrane phosphatidyl ethanolamine (PE) under the catalytic influence of lysophosphatidylcholine acyltransferase 3 (LPCAT3), resulting in the generation of PE-AA. Moreover, NF-κB promotes the degradation of FTH1, thereby releasing ferrous ions. These aforementioned biochemical reactions collectively culminate in the formation of PE-AA-OOH, ultimately culminating in ferroptosis.Download : Download high-res image (315KB)Download : Download full-size image