Excessive Accumulation of Ca2 + in Mitochondria of Y522S-RYR1 Knock-in Mice: A Link Between Leak From the Sarcoplasmic Reticulum and Altered Redox State.

Marta Canato,Anna Raffaello,Lorenzo Marcucci,Luigi Leanza,Carlo Reggiani,Antonio Michelucci,Denis Vecellio Reane,Feliciano Protasi,Lina Cancellara,Paola Capitanio

doi:10.3389/fphys.2019.01142

Marta Canato, Anna Raffaello + Show 8 more

Open Access

https://doi.org/10.3389/fphys.2019.01142

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Abstract

Mice (Y522S or YS), carrying a mutation of the sarcoplasmic reticulum (SR) Ca2+ release channel of skeletal muscle fibers (ryanodine receptor type-1, RyR1) which causes Ca2+ leak, are a widely accepted and intensively studied model for human malignant hyperthermia (MH) susceptibility. Since the involvement of reactive oxygen species (ROS) and of mitochondria in MH crisis has been previously debated, here we sought to determine Ca2+ uptake in mitochondria and its possible link with ROS production in single fibers isolated from flexor digitorum brevis (FDB) of YS mice. We found that Ca2+ concentration in the mitochondrial matrix, as detected with the ratiometric FRET-based 4mtD3cpv probe, was higher in YS than in wild-type (WT) fibers at rest and after Ca2+ release from SR during repetitive electrical stimulation or caffeine administration. Also mitochondrial ROS production associated with contractile activity (detected with Mitosox probe) was much higher in YS fibers than in WT. Importantly, the inhibition of mitochondrial Ca2+ uptake achieved by silencing MCU reduced ROS accumulation in the matrix and Ca2+ release from SR. Finally, inhibition of mitochondrial ROS accumulation using Mitotempo reduced SR Ca2+ release in YS fibers exposed to caffeine. The present results support the view that mitochondria take up larger amounts of Ca2+ in YS than in WT fibers and that mitochondrial ROS production substantially contributes to the increased caffeine-sensitivity and to the enhanced Ca2+ release from SR in YS fibers.

Highlights

Malignant hyperthermia is a rare life-threatening response triggered by exposure to halogenated/volatile anesthetics commonly used during surgery interventions
The results obtained in this study show an increased Ca2+ entry in the mitochondria of YS fibers and support the view that Ca2+ accumulation in the mitochondrial matrix and mitochondrial ROS production are relevant to the feed forward mechanism which enhances sensitivity to caffeine and Ca2+ release through the mutated ryanodine receptor type-1 (RyR1) channel
Mice carrying a YS mutation of the amino acid 524 (522 in humans) of the Ca2+ channel RyR1 have provided for more than a decade an accepted model to study a group of human diseases: malignant hyperthermia (MH), environmental-exertional heat stroke (EHS), and central core disease (CCD) (Chelu et al, 2006; Durham et al, 2008; Boncompagni et al, 2009a; Michelucci et al, 2017a)

Summary

INTRODUCTION

Malignant hyperthermia is a rare life-threatening response triggered by exposure to halogenated/volatile anesthetics commonly used during surgery interventions (halothane, isofluorane, etc.). The relevance of the mitochondrial contribution to the free radicals overproduction during MH crises is still debated (Lanner et al, 2012), because several other possible sources of ROS/RNS are available in muscle fibers as NOX, NOS, PLA2, and xanthine oxidase (Powers and Jackson, 2008; Lanner et al, 2012) It is not known yet if Ca2+ uptake and ROS generation in the mitochondria of YS muscle fibers is abnormal during regular contractile activity and/or during hyperthermic crisis. The results obtained in this study show an increased Ca2+ entry in the mitochondria of YS fibers and support the view that Ca2+ accumulation in the mitochondrial matrix and mitochondrial ROS production are relevant to the feed forward mechanism which enhances sensitivity to caffeine and Ca2+ release through the mutated RyR1 channel

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CONCLUSIVE REMARKS