Effects of Remimazolam and Propofol on Ca2+ Regulation by Ryanodine Receptor 1 with Malignant Hyperthermia Mutation

Abstract

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.

Figure 1

Western blot image. Western blot image confirmed the presence of ryanodine receptor 1 (RYR1) protein in the human embryonic kidney- (HEK-) 293 cells transfected with wild-type or mutant RYR1, p.Thr84Met, p.Ser2345Arg, or p.Ala4894Thr.

Figure 2

Representative Ca²⁺ release response of wild-type ryanodine receptor 1- (RYR1-) transfected human embryonic kidney- (HEK-) 293 cells or non-RYR1-transfected HEK-293 cells. Representative traces of agonist-induced Ca²⁺ release by HEK-293 cells transfected with wild-type pcRYR1 cDNA or without any pcRYR1 cDNA. The vertical axis represents the Fura-2 ratio (340/380 nm), i.e., intracellular Ca²⁺ release, and the horizontal axis represents time (minutes). The response of wild-type RYR1 expressing HEK-293 cells to (a) caffeine, (b) caffeine with continuous reflux of remimazolam (546 μM, 100-fold higher than clinical dose), (c) caffeine with continuous reflux of propofol (100 μM, 100-fold higher than the clinical dose), (d) remimazolam (increasing concentrations up to 5020 μM), and (e) propofol (increasing concentrations up to 5000 μM). The response of HEK-293 cells without expressing RYR1 to caffeine (f). No increase in intracellular Ca²⁺ levels for caffeine was observed in HEK-293 cells without expressing RYR1.

Figure 3

Caffeine concentration-dependent Ca²⁺ release in human embryonic kidney- (HEK-) 293 cells transfected with wild-type or mutant ryanodine receptor 1 (RYR1). Caffeine dose-response of Ca²⁺ release in HEK-293 cells transfected with wild-type or mutant RYR1. Data were normalized to the maximal response of each cell type. The vertical axis represents the percentage of maximal Ca²⁺ response, and the horizontal axis represents caffeine concentration (mM). Data are means ± SD in mutants and wild-type. Circles and other symbols represent the wild-type and mutant RYR1-transfected groups. The response of (a) wild-type RYR1 and p.Thr84Met RYR1-transfected cells, (b) wild-type RYR1 and p.Ser2345Arg RYR1-transfected cells, and (c) wild-type RYR1 and p.Ala4894Thr RYR1-transfected cells to caffeine. Curves of all mutant RYR1-transfected cells were shifted leftward relative to those of wild-type RYR1-transfected cells.

Figure 4

Caffeine concentration-dependent Ca²⁺ release in human embryonic kidney (HEK-293) cells transfected with wild-type or mutant ryanodine receptor 1 (RYR1) perfused with remimazolam or propofol. Caffeine-induced Ca²⁺ release in HEK-293 cells transfected with wild-type or mutant RYR1 perfused with remimazolam or propofol. Data were normalized to the maximal response of each cell type. Vertical and horizontal axes represent percentage maximal Ca²⁺ response and caffeine concentration (mM). Data are means ± SD in cells perfused with or without remimazolam or propofol. Squares and circles represent remimazolam or propofol perfusion and nonperfused groups, respectively. (a–d) Remimazolam and (e–h) propofol perfused cells. Response of (a) wild-type, (b) p.Thr84Met, (c) p.Ser2345Arg, (d) p.Ala4894Thr, (e) wild-type, (f) p.Thr84Met RYR1, (g) p.Ser2345Arg, and (h) p.Ala4894Thr. Curves of all mutant and wild-type RYR1-transfected cells did not show a clear leftward shift under perfusion of remimazolam or propofol.

Figure 5

Remimazolam and propofol concentration-dependent Ca²⁺ release by human embryonic kidney- (HEK-) 293 cells transfected with or without wild-type ryanodine receptor 1 (RYR1). Remimazolam and propofol concentration-response curves of RYR1-untransfected cells and wild-type RYR1-transfected cells are shown. Data were normalized to the maximal response of each cell type. The vertical axis represents percentage maximal Ca²⁺ response, and the horizontal axis represents remimazolam or propofol concentration (mM). Data are means ± SD in nontransfected and wild-type RYR1. Circles and squares represent wild-type RYR1 and untransfected groups, respectively. Dose-response curves of (a) remimazolam and (b) propofol. Curves showed no significant leftward shift in wild-type RYR1-transfected cells compared to untransfected RYR1 cells.