Excessive Accumulation of Ca2 + 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.

Parvalbumin affects skeletal muscle trophism through modulation of mitochondrial calcium uptake

“…The analysis of Ca 2+ dynamics of the three intracellular compartments ( Canato et al., 2019 ; Marcucci et al., 2018 ) confirmed the prolongation of the cytosolic transients in response to a single stimulus in PV −/− muscle fibers ( Schwaller et al., 1999 ). This is consistent with the view that PV facilitates the removal of Ca 2+ from the cytosol playing not only the role of buffer, which requires time for Mg 2+ replacement ( Hou et al., 1991 ), but also the role of shuttle between myofibrils and the SR ( Raymackers et al., 2000 ).…”

Altered Ca2+ Handling and Oxidative Stress Underlie Mitochondrial Damage and Skeletal Muscle Dysfunction in Aging and Disease

“…However, excessive RyR1 Ca 2+ leak can lead to local SR depletion that activates SOCE to further enhance resting Ca 2+ levels (see [ 126 ] for detailed discussion) ( Figure 1 ). In support of this idea, muscle fibers/myotubes from RYR1-Y522S were shown to exhibit reduced SR Ca 2+ content [ 118 , 127 ] and an increased rate of SOCE [ 128 ], suggesting increased STIM1 and ORAI1 expression/function. Consistent with this, increased SOCE activity was postulated to be an important mechanism for aberrant cytosolic Ca 2+ dynamics in muscle biopsies of MH patients [ 61 , 62 ].…”

“…Association of gain-of-function mutations in the RYR1 gene with MH (and some individuals with CCD) indicates that these disorders result, at least in part, from defective RyR1 function and Ca 2+ regulation in skeletal muscle. MH-related mutations in RyR1 destabilize the SR Ca 2+ release channel closed state, resulting in an increased susceptibility to opening in response to activators, SR Ca 2+ leak, and mitochondrial Ca 2+ uptake and subsequent ROS/RNS production [ 118 ]. In turn, RyR1 oxidative modifications (e.g., S-nitrosylation and S-gluthationylation) further destabilize the channel, and thus, further enhance RyR1 Ca 2+ leak.…”

A controversial issue: Can mitochondria modulate cytosolic calcium and contraction of skeletal muscle fibers?

“…A much faster calcium extrusion would lead to low levels of free Ca 2+ , even in the case that the total mitochondrial calcium required longer time to be fully extruded due to binding to matrix or IMM molecules, and this is not observed experimentally. This is even more remarkable considering that, after the end of the electrical stimulation, the cytosolic Ca 2+ decreases abruptly (see for example Canato et al, 2019), and therefore Ca 2+ entry through the MCU complex rapidly drops to basal levels, while the Ca 2+ extrusion should be kept high by the high intramitochondrial Ca 2+ . This supports the view that the calcium sequestration system inside the mitochondria acts as a buffer.…”