Altered Ca²⁺ concentration, permeability and buffering in the myofibre Ca²⁺ store of a mouse model of malignant hyperthermia

Abstract

Key points• Malignant Hyperthermia (MH) affects the Ca 2+ movements that control muscle contraction. We measured Ca 2+ movements in skeletal muscle of "Y522S" mice, with a tyrosine-to-serine mutation in the RyR channel that causes MH in mice and humans.• In YS cells, [Ca 2+ ] inside the Ca 2+ store (sarcoplasmic reticulum, SR) was 45% of that in the wild type (WT), but the SR membrane permeability increased 2-fold, resulting in Ca 2+ release of initially normal value.• During Ca 2+ release, cytosolic [Ca 2+ ] and SR Ca 2+ buffering power evolved differently in YS and WT. These variables became similar in WT exposed to BAPTA, an inhibitor of Ca 2+ -dependent inactivation (CDI) of the RyR, suggesting that tyrosine 522 is involved in CDI.• Similar paradoxical observations in YS and WT cells with reduced content of the SR protein calsequestrin, revealed the importance of balance between SR Ca permeability (increased in YS) and storage capability (decreased when calsequestrin is low).Abstract Malignant hyperthermia (MH) is linked to mutations in the type 1 ryanodine receptor, RyR1, the Ca 2+ channel of the sarcoplasmic reticulum (SR) of skeletal muscle. The Y522S MH mutation was studied for its complex presentation, which includes structurally and functionally altered cell 'cores' . Imaging cytosolic and intra-SR [Ca 2+ ] in muscle cells of heterozygous YS mice we determined Ca 2+ release flux activated by clamp depolarization, permeability (P) of the SR membrane (ratio of flux and [Ca 2+ ] gradient) and SR Ca 2+ buffering power (B). In YS cells resting [Ca 2+ ] SR was 45% of the value in normal littermates (WT). P was more than doubled, so that initial flux was normal. Measuring [Ca 2+ ] SR (t) revealed dynamic changes in B(t). The alterations were similar to those caused by cytosolic BAPTA, which promotes release by hampering Ca 2+ -dependent inactivation (CDI). The [Ca 2+ ] transients showed abnormal 'breaks' , decaying phases after an initial rise, traced to a collapse in flux and P. Similar breaks occurred in WT myofibres with calsequestrin reduced by siRNA; calsequestrin content, however, was normal in YS muscle. Thus, the Y522S mutation causes greater openness of the RyR1, lowers resting [Ca 2+ ] SR and alters SR Ca 2+ buffering in a way that copies the functional instability observed upon reduction of calsequestrin content. The similarities with the effects of BAPTA suggest that the mutation, occurring near the cytosolic vestibule of the channel, reduces CDI as one of its primary effects. The unstable SR buffering, mimicked by silencing of calsequestrin, may help precipitate the loss of Ca 2+ control that defines a fulminant MH event.

Ca2+ Influx via the Na+/Ca2+ Exchanger Is Enhanced in Malignant Hyperthermia Skeletal Muscle

“…Furthermore, it is well established that up to 1 M (dose used in the present study) does not affect the forward mode (37 The increased NCX3 reverse mode activity in MHS murine muscle cells suggest a compensatory mechanism to overcome the lower SR Ca 2ϩ loading described in MHS muscle cells (19,25,64) as a result of RyR1-MH leakiness (21). The reduced SR Ca 2ϩ levels are likely to promote an increased Ca 2ϩ influx through Orai and TRPC channels (19), and possibly other Ca 2ϩ channels (34).…”

Malignant hyperthermia: to buffer or not to buffer

“…Furthermore, the threshold for voltage-and heat stress-dependent activation of RYR1 is lowered. In the current study, Manno et al (2013) employ imaging and patch clamp techniques to demonstrate that the SR [Ca 2+ ], buffering and permeability are altered in YS mutant cells. BAPTA-treated WT cells show similar alteration in calcium dynamics to YS mutants.…”

“…The mice homozygous for the mutation fail to survive but the heterozygous mice exhibit elevated temperature and whole body contractures in response to isoflurane and heat. In their paper, Manno et al (2013) Manno et al (2013) ] also helps in determining the capacity of SR to bind Ca 2+ (buffering capacity). In the YS mutants there is a faster decay of buffering capacity during the release process, which is similarly observed in BAPTA-treated WT cells.…”

“…Even a century later, the mechanism behind muscle-based non-shivering heat production remains elusive. In a recent issue of Journal of Physiology, Manno et al (2013) studied the role of abnormal calcium dynamics in a mouse model of MH. We read this paper with great interest as it shed some light on the plausible mechanism behind muscle-based heat production.…”

“…Calsequestrin null mice are also known to be susceptible to heat-and anaesthesia-induced MH due to deregulated RYR1 function (Dainese et al 2009). Manno et al (2013) transfected the muscles of adult mice with calsequestrin small interfering RNA to obtain cells with reduced calsequestrin content. They demonstrate that these silenced cells exhibit similar alterations in calcium dynamics as seen in YS mutant cells.…”