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Many anesthetics, including Propofol, have been reported to induce elevation of intracellular calcium, and we were interested to investigate the possible contribution of calcium elevation to the mechanism of the newly approved remimazolam actions. Remimazolam is an intravenous anesthetic first approved in Japan in July 2020, and is thought to exert its anesthetic actions via γ-aminobutyric acid A (GABAA) receptors; however, the precise mechanisms of how remimazolam elevates intracellular calcium levels remains unclear. We examined the remimazolam-induced elevation of intracellular calcium using SHSY-5Y neuroblastoma cells, COS-7 cells, HEK293 cells, HeLa cells, and human umbilical vein endothelial cells (HUVECs) loaded with fluorescent dyes for live imaging. We confirmed that high concentrations of remimazolam (greater than 300 μM) elevated intracellular calcium in a dose-dependent manner in these cells tested. This phenomenon was not influenced by elimination of extracellular calcium. The calcium elevation was abolished when intracellular or intraendoplasmic reticulum (ER) calcium was depleted by BAPTA-AM or thapsigargin, respectively, suggesting that calcium was mobilized from the ER. Inhibitors of G-protein coupled receptors (GPCRs)-mediated signals, including U-73122, a phospholipase C (PLC) inhibitor and xestospongin C, an inositol 1,4,5-triphosphate receptors (IP3R) antagonist, significantly suppressed remimazolam-induced calcium elevation, whereas dantrolene, a ryanodine receptor antagonist, did not influence remimazolam-induced calcium elevation. Meanwhile, live imaging of ER during remimazolam stimulation using ER-tracker showed no morphological changes. These results suggest that high doses of remimazolam increased intracellular calcium concentration in a dose-dependent manner in each cell tested, which was predicted to be caused by calcium mobilization from the ER. In addition, our studies using various inhibitors revealed that this calcium elevation might be mediated by the GPCRs-IP3 pathway. However, further studies are required to identify which type of GPCRs is involved.

Background. We investigated the potential safety of remimazolam and propofol in malignant hyperthermia- (HM-) susceptible patients using ryanodine receptor 1- (RYR1-) expressing human embryonic kidney- (HEK-) 293 cells. Methods. We compared the enhanced responsiveness of HEK-293 cells expressing wild-type RYR1 with that of mutant RYR1 to caffeine following perfusion with remimazolam or propofol. Furthermore, we investigated whether RYR1 enhanced the responsiveness of cells to remimazolam or propofol and compared the median effective concentration (EC50; i.e., the concentration required to reach half-maximal activation) using an unpaired two-tailed t -test while a P < 0.05 was considered significant. Results. Remimazolam and propofol did not promote the caffeine-induced increase in intracellular Ca2+ levels in HEK-293 cells expressing mutant RYR1 even with exposure to approximately 100-fold the clinically used concentration. In wild-type RYR1, EC50 values of remimazolam following refusion vs. nonperfusion were 2.86 mM vs. 2.75 mM ( P = 0.76 ) while for propofol perfusion vs. nonperfusion, they were 2.76 mM vs. 2.75 mM, respectively ( P = 0.83 ). In mutant RYR1, EC50 values of remimazolam refusion vs. nonperfusion were 1.58 mM vs. 1.71 mM, respectively ( P = 0.63 ) while for propofol perfusion vs. nonperfusion, they were 1.65 mM vs. 1.71 mM, respectively ( P = 0.73 ). Remimazolam and propofol increased intracellular Ca2+ levels in a concentration-dependent manner, but the effect was not enhanced by RYR1. EC50 values of remimazolam with non-RYR1 vs. wild-type RYR1 were 1.00 mM vs. 0.92 mM, respectively ( P = 0.91 ) while those of propofol were 1.09 mM vs. 1.05 mM, respectively ( P = 0.84 ). Conclusions. The increase in intracellular Ca2+ concentration caused by remimazolam or propofol was not considered an RYR1-mediated reaction. We conclude that remimazolam and propofol can be safely used as an anesthetic in MH-susceptible patients with RYR1-mutation without causing MH and may be safely substituted for an MH-triggering anesthetic when RYR1-mediated MH occurs.