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Mechanisms of Ca2+ release in human and toad skeletal muscle in response to halothane

D.P. Singh, L. Pearce, C.R. Lamboley and B.S. Launikonis, School of Biomedical Sciences, The University of Queensland, St Lucia, QLD 4072, Australia.

Ca2+ release through the ryanodine receptor (RyR) can be directly activated by agonists such as volatile anaesthetics (e.g. halothane). In humans with a RyR mutation, typically used concentrations of halothane can cause Ca2+ release, making them susceptible to malignant hyperthermia (MH). The mechanism of Ca2+ release through the RyR of people who are MH susceptible is not well understood. While it is known that direct activation of RyR opening is dependent on local Ca2+ levels, how Ca2+ is interacting with the RyR, and adjacent RyRs, to open these channels and cause propagating Ca2+ that probably underlie an MH episode require examination.

In skeletal muscle, RyRs can be opened by Ca2+ induced Ca2+ release (CICR) or activated by luminally high Ca2+. The latter mechanism has been referred to as store overload-induced Ca2+ release (SOICR). In amphibian skeletal muscle, two RyR isoforms exist and a prominent CICR mechanism is active when cytoplasmic Ca2+ is raised. In mammalian skeletal muscle CICR is either weak, not present at all or completely inhibited by an interaction with adjacent voltage sensors. We suspected that comparing how halothane induced Ca2+ release in toad and human muscle susceptible to MH would assist in distinguishing between the mechanisms that work during an MH episode. Therefore we aimed to compare halothane-induced Ca2+ release in toad and MH susceptible human muscle fibres under identical conditions.

All experiments performed were approved by The University of Queensland Human Ethics & Animal Ethics Committees. Human muscle biopsies were collected under local anaesthesia from the Vastus Lateralis (VL) muscle. Cane toads (Bufo Marinus) were euthanized by double pithing and the Iliofibularis (IL) muscle was extracted. Single fibres were isolated and mechanically skinned under paraffin oil.

We hypothesized that by using mechanically skinned fibres from toad and MHS humans in the same experimental chamber that any differences in Ca2+ release properties under 1 mM halothane would be observed by rapidly imaging cytoplasmic Ca2+ in a K+-based cytoplasmic solution containing rhod-2 and 0.1 mM EGTA (0.1 - 0.2 μM [Ca2+]) on a Zeiss LSM 5 live microscope (Cully et al., 2016). To do this we positioned two fibres perpendicularly to each other, that is, they crossed over to form a junction between to the two preparations. Ca2+ waves reaching the junction allowed the effect of locally increased cytoplasmic Ca2+ to be observed as a new wave was established on the adjacent fibre. The two fibres placed in the chamber were placed in the combinations: toad v toad; human v human; and toad v human.

In toad vs toad experiments, Ca2+ wave propagation into the quiescent fibre from the active fibre occurred rapidly (1.02 ± 0.08 s; n = 10). In human vs human (MHS muscle), a delay of 4.17 ± 0.51 s (n = 7) in the propagation of Ca2+ release from the active to quiescent fibres was observed. In toad vs human fibre experiments (n=11), the characteristics of wave initiation in the quiescent fibres were maintained in each taxa. These Ca2+ wave propagation rates differed significantly between toad (1.02 ± 0.08 secs) vs human MHS (4.17 ± 0.51 s, T-test, P < 0.05). The delay in initiation of Ca2+ waves in the quiescent fibre from the local Ca2+ rise in the active fibre indicate that the cytoplasmic Ca2+ immediately causes Ca2+ release in the toad (CICR) whereas the delay in human fibres indicates that the SR needs to load Ca2+ to reach the threshold for luminal activation of Ca2+ release.

Cully TR, Edwards JN, & Launikonis BS (2014). Activation and propagation of Ca2+ release from inside the sarcoplasmic reticulum network of mammalian skeletal muscle. J Physiol, 592, 3727-3746.