Ablation of Skeletal Muscle Triadin Impairs FKBP12/RyR1 Channel Interactions Essential for Maintaining Resting Cytoplasmic Ca2+

Abstract

] rest in triadin-null myotubes is primarily driven by dysregulated RyR1 channel activity that results in part from impaired FKBP12/RyR1 functional interactions and a secondary increased Ca 2؉ entry at rest.In skeletal muscle, where control of cytosolic Ca 2ϩ concentration is key to muscle contraction, the overall Ca 2ϩ homeostasis is preserved by a concerted action of several ionic channels and transporters within the plasma membrane and the sarcoplasmic reticulum (SR).3 These proteins, including plasma membrane Ca 2ϩ ATPase, Na ϩ /Ca 2ϩ exchanger, dihydropyridine receptor, ryanodine receptor (RyRs), and the sarcoendoplasmic reticulum Ca 2ϩ ATPase, among others, interact functionally and physically with each other and with a plethora of regulatory proteins that ultimately modulate their activity, hence, the resting intracellular Ca 2ϩ levels. In muscle cells where SR Ca 2ϩ stores represent the main intracellular sources of Ca 2ϩ release, RyR1 seems positioned to play a key role in regulating myoplasmic [Ca 2ϩ ] rest . An effective example of this can be found in dyspedic cells that lack expression of all isoforms of RyRs (1), where we have recently demonstrated that RyR1 expression was sufficient to remodel the Ca 2ϩ regulatory machinery in a way that ultimately resulted in a significant shift in steady-state Ca 2ϩ fluxes contributing to [Ca 2ϩ ] rest (2). Alterations of RyR1 activity, like those conferred by malignant hyperthermia mutations, also resulted in long term changes in [Ca 2ϩ ] rest (3, 4), raising the possibility that indirect modulation of RyR1 by its protein binding partners may be important for homeostatic regulation of [Ca 2ϩ ] rest . Therefore, finding and understanding the role of RyR1 regulatory proteins could be instrumental in understanding the mechanisms that control long term Ca 2ϩ homeostasis in muscle.Among the endogenous regulators, the RyR1/triadin interactions are particularly intriguing, as its role in skeletal muscle function remains elusive (5). Although originally thought to be the link between RyR1 and DHPR (6 -8) and a key modulator of EC coupling (9 -11), it appears evident now that triadin acts primarily as a negative regulator of RyR1 activity (12-15). Although the mechanism by which triadin regulate RyRs remains unclear, there is support for the hypothesis that triadin is involved in facilitating the cross-communication be-* This work was supported, in whole or in part, by National Institute of Health Grants 5K01AR054818 (to C. F. P.), P01AR47605 (to P. D. A. and I. N. P.), and R01HL076433 (to B. R. F.

Reduced gain of excitation–contraction coupling in triadin-null myotubes is mediated by the disruption of FKBP12/RyR1 interaction

“…In an apparent disagreement with our results Oddoux et al [12] reported no statistical differences in RyR1 expression between Wt and triadin KO muscles, a difference that could be related to a higher dispersion in densitometric data in Oddoux’s report. As reported previously [13], expression of FKBP12 was also up-regulated in Tdn-null myotubes to 216 ± 21% of the Wt expression levels.…”

“…Instead, an impairment of either the allosteric transmission of the orthograde signal to RyR1 or RyR1-mediated Ca 2+ release itself seem more feasible. In this regard, our previous studies have shown that both a significant disruption FKBP12/RyR1 interaction and a sizable reduction in SR Ca 2+ content are associated with triadin ablation [13] and either of these effects by itself could account for the reduction in amplitude of the Ca 2+ release signal seen in triadin-null myotubes.…”

“…However, ablation of triadin did result in moderate alterations of DHPR Ca 2+ current kinetics and the amplitude of the voltage-dependent Ca 2+ release. Our data suggest that these changes are secondary to the disruption of the FKBP12/RyR1 complex induced by ablation of triadin [13] and can be overcome by over-expression of FKBP12.6. These results support the idea that triadins are not mandatory components of skeletal EC-coupling, but suggest that they may play an indirect role supporting the functional interaction between RyR1 and DHPR and other accessory proteins (i.e.…”

Review of RyR1 pathway and associated pathomechanisms

“…Similar findings were shown in isolated myotubes from the same mice as well as a significant increase in resting myoplasmic Ca ++ [131]. In support, Eltit et al (2010) demonstrated chronically elevated resting myoplasmic Ca ++ due to FKBP12-RyR1 dysfunction and SR store-operated Ca ++ entry [52, 53], suggesting increased basal RyR1 activity in triadin null myotubes isolated from skeletal muscle of mice. Additionally, Oddoux et al (2009) revealed a reduction of SR Ca ++ .…”

“…A number of studies have identified that triadin is “primarily a negative regulator of RyR1 [52]” [68, 70, 103, 131]. One study showed that amino acids 664 to 799 of DHPR alpha 1 subunit bind to triadin primarily at amino acids 68–278 [58].…”