Heat-hypersensitive mutants of ryanodine receptor type 1 revealed by microscopic heating

Abstract

Thermoregulation is an important aspect of human homeostasis, and high temperatures pose serious stresses for the body. Malignant hyperthermia (MH) is a life-threatening disorder in which body temperature can rise to a lethal level. Here we employ an optically controlled local heat-pulse method to manipulate the temperature in cells with a precision of less than 1 °C and find that the mutants of ryanodine receptor type 1 (RyR1), a key Ca 2+ release channel underlying MH, are heat hypersensitive compared with the wild type (WT). We show that the local heat pulses induce an intracellular Ca 2+ burst in human embryonic kidney 293 cells overexpressing WT RyR1 and some RyR1 mutants related to MH. Fluorescence Ca 2+ imaging using the endoplasmic reticulum–targeted fluorescent probes demonstrates that the Ca 2+ burst originates from heat-induced Ca 2+ release (HICR) through RyR1-mutant channels because of the channels’ heat hypersensitivity. Furthermore, the variation in the heat hypersensitivity of four RyR1 mutants highlights the complexity of MH. HICR likewise occurs in skeletal muscles of MH model mice. We propose that HICR contributes an additional positive feedback to accelerate thermogenesis in patients with MH.

Rapid Dantrolene Administration with Body Temperature Monitoring Is Associated with Decreased Mortality in Japanese Malignant Hyperthermia Events

“…Recent studies have suggested that ryanodine receptor 1 (RYR1) plays an important role in heat-induced Ca 2+ release and the progression to MH [ 25 ]. Furthermore, these data suggest a positive feedback mechanism altering thermogenesis causing MH.…”

Mice with R2509C-RYR1 mutation exhibit dysfunctional Ca2+ dynamics in primary skeletal myocytes

“…The microscopy system used for optical heating has been described previously ( Oyama et al, 2022 ). Briefly, confocal images were obtained using an inverted microscope (IX73; Olympus) equipped with a Nipkow confocal scanner unit (CSU-X1; Yokogawa Electric), a dichroic mirror (Di01-T405/488/561; Semrock), a 20× objective lens (UPlanSApo, NA = 0.75; Olympus), and an EM-CCD camera (iXon Ultra; Andor Technology).…”

Thermal conductivity and conductance of protein in aqueous solution: Effects of geometrical shape

“…This remarkable observation has suggested a possibility that the intracellular temperature may play an important role as a biological signal, while a number of controversial issues such as “10 5 gap problem” 12–15 concerning the temperature relaxation remain. We then need further microscopic understanding of the biological roles of temperature and heat transfer at molecular level, whereas the experimental observations on the relationship between temperature and biomolecular behaviors are still difficult in spite of recent advances in experimental technologies 17–19 …”

“…Hence the use of molecular simulations may be promising to elucidate the microscopic mechanisms of thermal conduction at intracellular level. Substantial investigations have been carried out experimentally and computationally for both lipid membranes 24–29 and proteins 17–19,30–37 . Lipid membranes are usually in solid‐like state in the cell, thus being relatively easily investigated by conventional experimental and computational methods 20 .…”