Plant Natural Product Formononetin Protects Rat Cardiomyocyte H9c2 Cells against Oxygen Glucose Deprivation and Reoxygenation via Inhibiting ROS Formation and Promoting GSK‐3β Phosphorylation

Abstract

The opening of mitochondrial permeability transition pore (mPTP) is a major cause of cell death in ischemia reperfusion injury. Based on our pilot experiments, plant natural product formononetin enhanced the survival of rat cardiomyocyte H9c2 cells during oxygen glucose deprivation (OGD) and reoxygenation. For mechanistic studies, we focused on two major cellular factors, namely, reactive oxygen species (ROS) and glycogen synthase kinase 3β (GSK-3β), in the regulation of mPTP opening. We found that formononetin suppressed the formation of ROS and superoxide in a concentration-dependent manner. Formononetin also rescued OGD/reoxygenation-induced loss of mitochondrial membrane integrity. Further studies suggested that formononetin induced Akt activation and GSK-3β (Ser9) phosphorylation, thereby reducing GSK-3β activity towards mPTP opening. PI3K and PKC inhibitors abolished the effects of formononetin on mPTP opening and GSK-3β phosphorylation. Immunoprecipitation experiments further revealed that formononetin increased the binding of phosphor-GSK-3β to adenine nucleotide translocase (ANT) while it disrupted the complex of ANT with cyclophilin D. Moreover, immunofluorescence revealed that phospho-GSK-3β (Ser9) was mainly deposited in the space between mitochondria and cell nucleus. Collectively, these results indicated that formononetin protected cardiomyocytes from OGD/reoxygenation injury via inhibiting ROS formation and promoting GSK-3β phosphorylation.

Mitochondria and aging: A role for the mitochondrial transition pore?

“…An increased phospho‐GSK‐3β binding to ANT was suggested to be responsible for the inhibition of mPTP opening (Miura & Tanno, 2010; Nishihara et al., 2007). This mechanism would contribute to the cardioprotective effect of several drugs such as formononetin or resveratrol and of ischemic preconditioning (Cheng, Xia, Han, & Rong, 2016; Xi, Wang, Mueller, Norfleet, & Xu, 2009; Zhu, Rebecchi, Glass, Brink, & Liu, 2013; Zhu, Rebecchi, Wang, et al., 2013). In the aging heart, failure to reduce ANT/CypD interactions or decreased pGSK‐3β responsiveness of ANT could be responsible for the attenuation of cardioprotection afforded by ischemic preconditioning (Zhu, Rebecchi, Glass, et al., 2013; Zhu, Rebecchi, Wang, et al., 2013).…”

The Key Roles of GSK-3β in Regulating Mitochondrial Activity

“…Formononetin also promotes the binding of phosphor-Ser9-GSK-3β of ANT and disruption of the complex formation of CyP-D-ANT, leading to abolishment of mPTP opening and reduction of ROS generation [86]. …”

Pure Polyphenols Applications for Cardiac Health and Disease

“…Other isoflavones have been revealed as cardioprotective, for instance formononetin is the major isoflavone found in Radix Astragali, a Chinese medicinal plant widely used to treat cardiovascular diseases. It has been described that this compound reduces ROS formation, as well as mitochondrial damage, increasing cell viability of rat cardiomyocyte H9c2 cell line, after oxygen-glucose deprivation and reoxygenation [115]. Thus, by activating Akt and increasing glycogen synthase kinase-3β (GSK-3β) phosphorylation, formononetin enhances the interaction between phospho-GSK-3β and adenine nucleotide translocase, the mitochondrial permeability transition pore (mPTP) major component, which decreases mPTP opening and prevents apoptosis [115].…”