Dataset Information

Excessive Accumulation of Ca^{2 +} in Mitochondria of Y522S-RYR1 Knock-in Mice: A Link Between Leak From the Sarcoplasmic Reticulum and Altered Redox State.

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.

SUBMITTER: Canato M

PROVIDER: S-EPMC6755340 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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Publications

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

Canato Marta M Capitanio Paola P Cancellara Lina L Leanza Luigi L Raffaello Anna A Reane Denis Vecellio DV Marcucci Lorenzo L Michelucci Antonio A Protasi Feliciano F Reggiani Carlo C

Frontiers in physiology 20190913

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 ...[more]

PMID: 31607937

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Project description:Ventricular arrhythmias can cause death in heart failure (HF). A trigger is the occurrence of Ca2+ waves which activate a Na+ -Ca2+ exchange (NCX) current, leading to delayed after-depolarisations and triggered action potentials. Waves arise when sarcoplasmic reticulum (SR) Ca2+ content reaches a threshold and are commonly induced experimentally by raising external Ca2+ , although the mechanism by which this causes waves is unclear and was the focus of this study. Intracellular Ca2+ was measured in voltage-clamped ventricular myocytes from both control sheep and those subjected to rapid pacing to produce HF. Threshold SR Ca2+ content was determined by applying caffeine (10 mM) following a wave and integrating wave and caffeine-induced NCX currents. Raising external Ca2+ induced waves in a greater proportion of HF cells than control. The associated increase of SR Ca2+ content was smaller in HF due to a lower threshold. Raising external Ca2+ had no effect on total influx via the L-type Ca2+ current, ICa-L , and increased efflux on NCX. Analysis of sarcolemmal fluxes revealed substantial background Ca2+ entry which sustains Ca2+ efflux during waves in the steady state. Wave frequency and background Ca2+ entry were decreased by Gd3+ or the TRPC6 inhibitor BI 749327. These agents also blocked Mn2+ entry. Inhibiting connexin hemi-channels, TRPC1/4/5, L-type channels or NCX had no effect on background entry. In conclusion, raising external Ca2+ induces waves via a background Ca2+ influx through TRPC6 channels. The greater propensity to waves in HF results from increased background entry and decreased threshold SR content. KEY POINTS: Heart failure is a pro-arrhythmic state and arrhythmias are a major cause of death. At the cellular level, Ca2+ waves resulting in delayed after-depolarisations are a key trigger of arrhythmias. Ca2+ waves arise when the sarcoplasmic reticulum (SR) becomes overloaded with Ca2+ . We investigate the mechanism by which raising external Ca2+ causes waves, and how this is modified in heart failure. We demonstrate that a novel sarcolemmal background Ca2+ influx via the TRPC6 channel is responsible for SR Ca2+ overload and Ca2+ waves. The increased propensity for Ca2+ waves in heart failure results from an increase of background influx, and a lower threshold SR content. The results of the present study highlight a novel mechanism by which Ca2+ waves may arise in heart failure, providing a basis for future work and novel therapeutic targets.

Dataset Information

Excessive Accumulation of Ca^{2 +} in Mitochondria of Y522S-RYR1 Knock-in Mice: A Link Between Leak From the Sarcoplasmic Reticulum and Altered Redox State.

Publications

Excessive Accumulation of Ca<sup>2 +</sup> in Mitochondria of Y522S-RYR1 Knock-in Mice: A Link Between Leak From the Sarcoplasmic Reticulum and Altered Redox State.

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