The interactive toxic effect of homocysteine and copper on cardiac microvascular endothelial cells during ischemia-reperfusion injury

Hyperhomocysteinemia (HHcy) is associated with the development and progression of chronic cardiovascular diseases through the deleterious effects of high levels of homocysteine (Hcy) on the cardiovascular system. However, the exact mechanism of action of Hcy on the acute injury of the cardiovascular system following ischemia/reperfusion (I/R) remains unclear. The present study demonstrated that copper mobilization occurs during cardiac I/R, and the interactive toxic effect of Hcy and mobile Cu²⁺ during cardiac I/R induces necroptosis of cardiac microvascular endothelial cells (CMECs) and thus enhances cardiac dysfunction. In the present study, we utilized three cardiac I/R model: isolated rat heart, in vivo model as well as cell culture, and demonstrated that copper mobilization occurs during cardiac I/R, and the interactive toxic effect of Hcy and mobile Cu²⁺ during cardiac I/R induces necroptosis of cardiac microvascular endothelial cells (CMECs) and thus enhances cardiac dysfunction. Furthermore, we proved that the Cu²⁺ chelator TTM significantly mitigated the deleterious effects of Hcy and Cu²⁺ on CMECs and cardiac function both in vitro and in vivo. Mechanismly, the combinative effect of Hcy and Cu²⁺ are associated with the production of reactive oxygen species (ROS) and nitric oxide (NO) by NADPH oxidase (NOX) and endothelial nitric oxide synthase (eNOS), respectively. Subsequently, the overproduction of toxic peroxynitrite (ONOO^-) induces CMECs necroptosis. The application of ROS scavengers in CMECs resulted in a notable reduction in necroptosis mediated by Hcy and Cu²⁺ under hypoxia/reperfusion (H/R) condition. These findings indicate that the mechanism by which Hcy and Cu²⁺ enhances cardiac dysfunction under I/R condition may be attributed to the stimulation of both NOX and eNOS activity, resulting in the generation of excessive ONOO^- and subsequent necroptosis of CMECs.