Penehyclidine hydrochloride protects against anoxia/reoxygenation injury in cardiomyocytes through ATP-sensitive potassium channels, and the Akt/GSK-3β and Akt/mTOR signaling pathways

Abstract

Penehyclidine hydrochloride (PHC) can protect against myocardial ischemia/reperfusion (I/R) injury. However, the possible mechanisms of PHC in anoxia/reoxygenation (A/R)-induced injury in H9c2 cells remain unclear. In the present study, H9c2 cells were pretreated with PI3K/Akt inhibitor LY294002, ATP-sensitive K⁺ (KATP) channel blocker 5-hydroxydecanoate (5-HD), PHC, or KATP channel opener diazoxide (DZ) before subjecting to A/R injury. Cell viability and cell apoptosis were determined by cell counting kit-8 assay and annexin V/PI assay, respectively. Myocardial injury was evaluated by measuring creatine kinase (CK) and lactate dehydrogenase (LDH) activities. Intracellular Ca²⁺ levels, reactive oxygen species (ROS) generation, mitochondrial membrane potential (ΔΨ_m ), and mitochondrial permeability transition pore (mPTP) were measured. The levels of cytoplasmic/mitochondrial cytochrome c (Cyt-C), Bax, Bcl-2, cleaved caspase-3, KATP channel subunits (Kir6.2 and SUR2A), and the members of the Akt/GSK-3β and Akt/mTOR signaling pathways were determined by western blotting. We found that PHC preconditioning alleviated A/R-induced cell injury by increasing cell viability, reducing CK and LDH activities, and inhibiting cell apoptosis. In addition, PHC preconditioning ameliorated intracellular Ca²⁺ overload and ROS production, accompanied by inhibition of both mPTP opening and Cyt-C release into cytoplasm, and maintenance of ΔΨ_m . Moreover, PHC preconditioning activated mitochondrial KATP channels, and modulated the Akt/GSK-3β and Akt/mTOR signaling pathways. Similar effects were observed upon treatment with DZ. Pretreatment with LY294002 or 5-HD blocked the beneficial effects of PHC. These results suggest that the protective effects of PHC preconditioning on A/R injury may be related to mitochondrial KATP channels, as well as the Akt/GSK-3β and Akt/mTOR signaling pathways.

Keywords: ATP-sensitive K+channel; Akt/GSK-3β signaling; Akt/mTOR signaling; anoxia/reoxygenation injury; penehyclidine hydrochloride.