Klotho attenuates angiotensin II-induced cardiotoxicity through suppression of necroptosis and oxidative stress

Abstract

Hyperglycemia is known to lead to cardiac injury and inflammation through the reactive oxygen species (ROS)-Toll-like receptor 4 (TLR4)-necroptosis pathway. Similarly, angiotensin II (Ang II) activates the TLR4-nuclear factor κB (NF-κB) p65 pathway, while the protein Klotho is known to inhibit this pathway, protecting cardiac cells from Ang II-induced injury. However, there is currently a lack of data on whether necroptosis participates in Ang II-induced cardiac injury and whether the Klotho protein has an effect on this process. The present study aimed to explore whether inhibition of the TLR4/NF-κB p65 necroptosis pathway is involved in the Klotho protein-mediated protection against the Ang II-induced cardiac injury and inflammation. H9c2 cardiac cells were incubated with 0.01 mM Ang II. Western blotting was used to assess the expression of receptor-interacting protein kinase 3 (RIP3), mixed-lineage kinase domain-like protein (MLKL), TLR4 and NF-κB p65. The present study also assessed injury indexes: Inflammatory cytokine expression, mitochondrial membrane potential (ΔΨm), apoptosis, ROS production and cell viability. The expression of TLR4, phosphorylated (p)-NF-κB p65, RIP3 and MLKL were increased by incubation with Ang II in H9c2 cells. The pretreatment of H9c2 cells with necrostatin-1 (Nec-1, an inhibitor of necroptosis) or TAK-242 (a small molecule inhibitor of TLR4) attenuated the upregulation of RIP3 and MLKL caused by Ang II. Klotho protein cotreatment also reversed the Ang II-induced upregulation of TLR4, p-NF-κB p65, RIP3 and MLKL. Furthermore, Ang II decreased cell viability and upregulated the secretion of inflammatory cytokines, ΔΨm loss and ROS generation blocked by pretreatment with Nec-1 or Klotho protein. Thus, it was determined that Klotho can attenuate the Ang II-induced necroptosis of cardiomyocytes through the TLR4/NF-κB p65 pathway, which suggests that Klotho could be a potential therapeutic drug against Ang II-induced cardiotoxicity.