GSK2795039 prevents Tumor necrosis factor-α-induced RIP1-RIP3-MLKL-mediated necroptosis in H9C2 cardiomyocytes through inhibition of NADPH oxidase

Inflammatory cytokines and oxidative stress contribute to heart failure. Tumor necrosis factor-α (TNF-α), a major inflammatory cytokine, induces reactive oxygen species (ROS) and necroptosis in cardiomyocytes. GSK2795039 is a novel NADPH oxidase 2 inhibitor. However, it is unknown whether GSK2795039 prevents TNF-α-induced cardiomyocyte necroptosis and the underlying mechanisms. In the present study, we tested the hypothesis that GSK2795039 prevents TNF-α-induced necroptosis in cardiomyocytes through inhibition of NADPH oxidase-mediated RIP1-RIP3-MLKL pathway. The H9C2 cardiomyocytes were divided into the following groups: control, necrostatin-1, TNF-α, and TNF-α plus necrostatin-1; control, GSK2795039, TNF-α, and TNF-α plus GSK2795039. ROS were assessed by dihydroethidium staining. Necroptotic and apoptotic proteins were measured by Western blot. The RIP1 inhibitor necrostatin-1 attenuated apoptosis, necrosis, phosphorylated RIP3 (p-RIP3) and p-MLKL protein and intracellular ROS in TNF-α-treated H9C2 cardiomyocytes. GSK2795039 inhibited TNF-α-induced Nox2 expression, intracellular ROS and cell death. GSK2795039 prevented the increases in phosphorylated RIP1 (p-RIP1), p-RIP3 and p-MLKL proteins, reflective of their respective kinase activities, markers of necroptosis, in TNF-α-treated H9C2 cardiomyocytes. GSK2795039 prevented the decrease in anti-apoptotic Bcl-2 protein and the increases in pro-apoptotic Bax protein, phosphorylated JNK protein and cleaved caspase 3 protein, indicative of caspase activity, a marker of apoptosis, in TNF-α-treated H9C2 cardiomyocytes. The results indicate that GSK2795039 prevented TNF-α-induced RIP1-RIP3-MLKL-mediated necroptosis in H9C2 cardiomyocytes through inhibition of NADPH oxidase-derived ROS. These findings advocate for further animal testing of the specific Nox2 inhibitor GSK2795039 in myocardial infarction and heart failure, which are characterized by elevated levels of inflammatory cytokines such as TNF-α and oxidative stress.