Silencing MEKK3 attenuates cardiomyocyte injury caused by hypoxia/reoxygenation via the sonic hedgehog pathway.

Abstract

MEKK3 is a member of MAP3K, which plays a pivotal role in cardiac diseases. In this study, we aimed to investigate the effects and potential mechanisms of MEKK3 on hypoxia/reoxygenation (H/R) injury of cardiomyocytes. After exposing H9C2 cells to H/R insult, real-time polymerase chain reaction and western blot analysis showed that MEKK3 was highly expressed. Cell viability, cell apoptosis, caspase 3/7 activity, and cleaved-caspase 3 expression were tested using a CCK-8 assay, Cell Death Detection PLUS ELISA, Caspase-Glo 3/7 Assay Kit and western blot analysis, respectively. Mitochondrial membrane potential, cytochrome C expression, adenosine triphosphate (ATP), and reactive oxygen species also were measured using JC-1 staining, western blot analysis, an ATP Assay Kit, and DCFH 2 -DA staining, respectively. The messenger RNA (mRNA) levels and secretions of TNF-α, IL-6, and IL-1β were evaluated. The results revealed that MEKK3 silencing promoted cell survival and attenuated lactate dehydrogenase leakage, cell apoptosis, caspase 3/7 activity, and the protein level of cleaved-caspase 3. Moreover, knockdown of MEKK3 blocked mitochondrial impairment by inhibiting the loss of mitochondrial membrane potential and cytochrome C expression as well as promoting ATP synthesis. MEKK3 deficiency led to a decrease in reactive oxygen species and malondialdehyde (MDA) generation and an increase in superoxide dismutase (SOD) activity. Deletion of MEKK3 led to reduced inflammatory cytokines in mRNA level and secretion. MEKK3 suppression activated the sonic hedgehog (Shh) signaling pathway in H9C2 cells. After blocking the Shh signaling pathway with a specific inhibitor, cyclopamine, the cardioprotective functions of MEKK3 downregulation were partly abolished. In conclusion, downregulation of MEKK3 prevented apoptosis and inflammation in H9C2 cells via the Shh signaling pathway.