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. 2023 Oct 27;14(11):2009.
doi: 10.3390/genes14112009.

Effects of Remimazolam on Intracellular Calcium Dynamics in Myotubes Derived from Patients with Malignant Hyperthermia and Functional Analysis of Type 1 Ryanodine Receptor Gene Variants

Hirotsugu Miyoshi  1 , Sachiko Otsuki  1 , Keiko Mukaida  1 , Kenshiro Kido  1 , Ayako Sumii  1 , Tsuyoshi Ikeda  1 , Yuko Noda  1 , Toshimichi Yasuda  2 , Soshi Narasaki  1 , Takahiro Kato  1 , Satoshi Kamiya  1 , Yousuke T Horikawa  3 , Yasuo M Tsutsumi  1
Affiliations

Effects of Remimazolam on Intracellular Calcium Dynamics in Myotubes Derived from Patients with Malignant Hyperthermia and Functional Analysis of Type 1 Ryanodine Receptor Gene Variants

Hirotsugu Miyoshi et al. Genes (Basel). .

Abstract

Remimazolam is a novel general anesthetic and its safety in patients with malignant hyperthermia (MH) is unknown. We used myotubes derived from the skeletal muscle of patients with MH to examine the response to ryanodine receptor 1 (RYR1) agonist and remimazolam in MH-susceptible patients. Patients underwent muscle biopsy for the Ca2+-induced Ca2+ release (CICR) rate test, a diagnostic tool for MH in Japan. Ten patients had myotubes obtained from skeletal muscle cultures, and the genes associated with malignant hyperthermia in these patients were analyzed. The EC50 of caffeine, cresol, and remimazolam to induce intracellular calcium concentration change were compared between myotubes from CICR-negative genetic test patients and myotubes from other patients. Eight of the ten were CICR-positive, five of whom had RYR1 causative gene mutations or variants. Two patients had CICR-negative genetic tests, and as expected had the highest EC50 (the concentration of a drug that gives a half-maximal response) in response to caffeine, 4CmC and remimazolam. Three patients had a positive CICR but no known variants in RYR1 or CACNA1S (voltage-gated calcium channel subunit alpha1S). Myotubes in these patients had significantly lower EC50s for all agents than myotubes in CICR-negative patients. When myotubes from a patient who was CICR-negative and had no gene variant were used as a control, myotubes from CICR-positive patients were more hyper-responsive than controls to all stimulants used. The EC50 for remimazolam was lowest for myotubes from CICR-positive, RYR1-mutant patients, at 206 µM (corresponding to 123 µg/mL). The concentration was more than 80-times higher than the clinical concentration. RYR1 gene variants in R4645Q and W5020G were shown to be causative gene mutations for MH. Intracellular calcium in myotubes from MH patients are elevated at high concentrations of remimazolam but not at clinically used concentrations of remimazolam. Remimazolam appears to be safe to use in patients with MH.

Keywords: intracellular calcium dynamics; malignant hyperthermia; myotube; remimazolam; ryanodine receptor 1.

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Conflict of interest statement

The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

Figures

Figure 1
Figure 1
Myotubes were successfully isolated from patients at risk for MH. Myotubes with a negative CICR and no RYR1 or CACNA1S variant formed similar myotubes to CICR-positive with a known RYR1 mutation (A) Patient 1; (B) Patient 6).
Figure 2
Figure 2
Dosing protocol for remimazolam. (A) Typical intracellular calcium concentration fluctuations during remimazolam dosing protocols (presenting data from Patient 6). The X-axis shows the time-course, and the Y-axis shows the 340/380-nm wavelength ratio. After confirming the response to caffeine, the cells were exposed to a stepwise increase in remimazolam concentration. (B–K) Concentration–response curves for remimazolam and intracellular calcium. (B) shows representative dose–response curves for three patient groups divided by CICR test results and the presence or absence of RYR1 variants. The myotube concentration–response curve for Patients 3–10 is more left-shifted than the myotube concentration–response curve for Patient 1 (Circle mark), indicating that lower concentrations of remimazolam increase intracellular calcium in CICR-positive myotubes.
Figure 2
Figure 2
Dosing protocol for remimazolam. (A) Typical intracellular calcium concentration fluctuations during remimazolam dosing protocols (presenting data from Patient 6). The X-axis shows the time-course, and the Y-axis shows the 340/380-nm wavelength ratio. After confirming the response to caffeine, the cells were exposed to a stepwise increase in remimazolam concentration. (B–K) Concentration–response curves for remimazolam and intracellular calcium. (B) shows representative dose–response curves for three patient groups divided by CICR test results and the presence or absence of RYR1 variants. The myotube concentration–response curve for Patients 3–10 is more left-shifted than the myotube concentration–response curve for Patient 1 (Circle mark), indicating that lower concentrations of remimazolam increase intracellular calcium in CICR-positive myotubes.
Figure 2
Figure 2
Dosing protocol for remimazolam. (A) Typical intracellular calcium concentration fluctuations during remimazolam dosing protocols (presenting data from Patient 6). The X-axis shows the time-course, and the Y-axis shows the 340/380-nm wavelength ratio. After confirming the response to caffeine, the cells were exposed to a stepwise increase in remimazolam concentration. (B–K) Concentration–response curves for remimazolam and intracellular calcium. (B) shows representative dose–response curves for three patient groups divided by CICR test results and the presence or absence of RYR1 variants. The myotube concentration–response curve for Patients 3–10 is more left-shifted than the myotube concentration–response curve for Patient 1 (Circle mark), indicating that lower concentrations of remimazolam increase intracellular calcium in CICR-positive myotubes.
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