Hyperglycemia-induced DNA damage response activates DNA-PK complex to promote endothelial ferroptosis in type 2 diabetic cardiomyopathy

<b>Rationale:</b> Hyperglycemia-induced endothelial dysfunction is a hallmark of diabetic cardiomyopathy, yet the underlying molecular mechanisms remain incompletely understood. This study aimed to investigate how the DNA damage response (DDR) pathway regulates endothelial cell ferroptosis under hyperglycemic conditions, potentially revealing new therapeutic targets for mitigating cardiac damage in type 2 diabetes mellitus (T2DM). <b>Methods:</b> We performed an integrated analysis of publicly available RNA sequencing datasets (GSE280770, GSE89475, GSE161931, CRA007245) to evaluate the role of DDR in hyperglycemia-induced endothelial dysfunction <i>in vitro</i> and <i>in vivo</i>, including in a T2DM mouse model. Key DDR and ferroptosis markers were validated in cardiac microvascular endothelial cells (CMECs) isolated from mice with streptozotocin (STZ)/high-fat diet (HFD)-induced T2DM, with and without treatment with the DNA-PK inhibitors NU7441 or M9831. <b>Results:</b> Hyperglycemia induced a robust DDR in endothelial cells, characterized by the upregulation of DNA-PK complex genes (PRKDC, XRCC5, XRCC6) and increased markers of DNA damage (γH2AX, 8-oxo-dG). This was accompanied by increased expression of pro-ferroptotic genes (Tfrc, Acsl4, Ptgs2), decreased expression of anti-ferroptotic genes (Gpx4, Slc7a11), and elevated lipid peroxidation (MDA, 4-HNE). Pharmacological inhibition of DNA-PK mitigated these effects, reducing oxidative stress, lipid peroxidation, and endothelial permeability, while improving cardiac contractile and relaxation parameters. <b>Conclusions:</b> Our findings implicate the DNA-PK complex as a key regulator of hyperglycemia-induced endothelial ferroptosis in T2DM cardiomyopathy. Targeting DNA-PK complex may represent a novel therapeutic strategy for mitigating microvascular dysfunction and cardiac decline in T2DM.