Muscle weakness in Ryr1 I4895T/WT knock-in mice as a result of reduced ryanodine receptor Ca 2+ ion permeation and release from the sarcoplasmic reticulum

The type 1 isoform of the ryanodine receptor (RYR1) is the Ca 2+ release channel of the sarcoplasmic reticulum (SR) that is activated during skeletal muscle excitation–contraction (EC) coupling. Mutations in the RYR1 gene cause several rare inherited skeletal muscle disorders, including malignant hyperthermia and central core disease (CCD). The human RYR1 I4898T mutation is one of the most common CCD mutations. To elucidate the mechanism by which RYR1 function is altered by this mutation, we characterized in vivo muscle strength, EC coupling, SR Ca 2+ content, and RYR1 Ca 2+ release channel function using adult heterozygous Ryr1 I4895T/+ knock-in mice (IT/+). Compared with age-matched wild-type (WT) mice, IT/+ mice exhibited significantly reduced upper body and grip strength. In spite of normal total SR Ca 2+ content, both electrically evoked and 4-chloro- m -cresol–induced Ca 2+ release were significantly reduced and slowed in single intact flexor digitorum brevis fibers isolated from 4–6-mo-old IT/+ mice. The sensitivity of the SR Ca 2+ release mechanism to activation was not enhanced in fibers of IT/+ mice. Single-channel measurements of purified recombinant channels incorporated in planar lipid bilayers revealed that Ca 2+ permeation was abolished for homotetrameric IT channels and significantly reduced for heterotetrameric WT:IT channels. Collectively, these findings indicate that in vivo muscle weakness observed in IT/+ knock-in mice arises from a reduction in the magnitude and rate of RYR1 Ca 2+ release during EC coupling that results from the mutation producing a dominant-negative suppression of RYR1 channel Ca 2+ ion permeation

Voltage-gated calcium fluxes studied in skeletal muscle fibers of genetically engineered mice

Ca2+ channels play central roles in cellular signaling. In skeletal muscle, the dihydropyridine receptors (DHPRs), L-type Ca2+ channels, play a pivotal role as voltage sensors for the remote control of type 1 ryanodine receptors (RyR1). The general aim of this study was to characterize the alterations of voltage controlled fluxes of Ca2+ entry and Ca2+ release from internal stores in fully differentiated skeletal muscle fibers of normal and genetically modified mice. The first goal was to investigate the cross influence of the skeletal muscle-specific gamma1 subunit of the DHPR and of the PAA Ca2+ antagonist (-)D888. For this purpose, I studied voltage-dependent gating of both L-type Ca2+ current and Ca2+ release in muscle fibers of wildtype and gamma1 knock-out mice. The results indicate a common mechanism of modulation of voltage-dependent inactivation and suggest that the gamma1-subunit acts as an endogenous calcium antagonist. The second goal of the study was to assess the functional consequences of the Y522S MH mutation in RyR1 to EC coupling in adult skeletal muscle and to define the mechanisms that limit leakage of Ca2+ from the SR. For this purpose a recently developed transgenic mouse heterozygous for the RyR1 mutation was used. The investigations were focused on changes in the voltage window of Ca2+ flux generated by the overlap region of activation and inactivation curves, which likely contribute to the pathology. The results of this part of the study show evidence of a novel feedback mechanism that partially compensates for the tendency of the mutation to increase window Ca2+ flux. The findings provide useful information and are important for understanding the normal/dysfunctional Ca2+ handling in skeletal muscle of mammalian organism and can, therefore, be relevant to human muscle physiology

LIST OF ABBREVIATION

1.1 Excitation-contraction coupling machinery................................................................. 1 1.2 Signals in EC coupling under voltage-clamp condition..............................................

S100A1 promotes action potential-initiated calcium release flux and force production in skeletal muscle

The role of S100A1 in skeletal muscle is just beginning to be elucidated. We have previously shown that skeletal muscle fibers from S100A1 knockout (KO) mice exhibit decreased action potential (AP)-evoked Ca2+ transients, and that S100A1 binds competitively with calmodulin to a canonical S100 binding sequence within the calmodulin-binding domain of the skeletal muscle ryanodine receptor. Using voltage clamped fibers, we found that Ca2+ release was suppressed at all test membrane potentials in S100A1−/− fibers. Here we examine the role of S100A1 during physiological AP-induced muscle activity, using an integrative approach spanning AP propagation to muscle force production. With the voltage-sensitive indicator di-8-aminonaphthylethenylpyridinium, we first demonstrate that the AP waveform is not altered in flexor digitorum brevis muscle fibers isolated from S100A1 KO mice. We then use a model for myoplasmic Ca2+ binding and transport processes to calculate sarcoplasmic reticulum Ca2+ release flux initiated by APs and demonstrate decreased release flux and greater inactivation of flux in KO fibers. Using in vivo stimulation of tibialis anterior muscles in anesthetized mice, we show that the maximal isometric force response to twitch and tetanic stimulation is decreased in S100A1−/− muscles. KO muscles also fatigue more rapidly upon repetitive stimulation than those of wild-type counterparts. We additionally show that fiber diameter, type, and expression of key excitation-contraction coupling proteins are unchanged in S100A1 KO muscle. We conclude that the absence of S100A1 suppresses physiological AP-induced Ca2+ release flux, resulting in impaired contractile activation and force production in skeletal muscle

Muscle weakness in Ryr1I4895T/WT knock-in mice as a result of reduced ryanodine receptor Ca2+ ion permeation and release from the sarcoplasmic reticulum

The type 1 isoform of the ryanodine receptor (RYR1) is the Ca2+ release channel of the sarcoplasmic reticulum (SR) that is activated during skeletal muscle excitation–contraction (EC) coupling. Mutations in the RYR1 gene cause several rare inherited skeletal muscle disorders, including malignant hyperthermia and central core disease (CCD). The human RYR1I4898T mutation is one of the most common CCD mutations. To elucidate the mechanism by which RYR1 function is altered by this mutation, we characterized in vivo muscle strength, EC coupling, SR Ca2+ content, and RYR1 Ca2+ release channel function using adult heterozygous Ryr1I4895T/+ knock-in mice (IT/+). Compared with age-matched wild-type (WT) mice, IT/+ mice exhibited significantly reduced upper body and grip strength. In spite of normal total SR Ca2+ content, both electrically evoked and 4-chloro-m-cresol–induced Ca2+ release were significantly reduced and slowed in single intact flexor digitorum brevis fibers isolated from 4–6-mo-old IT/+ mice. The sensitivity of the SR Ca2+ release mechanism to activation was not enhanced in fibers of IT/+ mice. Single-channel measurements of purified recombinant channels incorporated in planar lipid bilayers revealed that Ca2+ permeation was abolished for homotetrameric IT channels and significantly reduced for heterotetrameric WT:IT channels. Collectively, these findings indicate that in vivo muscle weakness observed in IT/+ knock-in mice arises from a reduction in the magnitude and rate of RYR1 Ca2+ release during EC coupling that results from the mutation producing a dominant-negative suppression of RYR1 channel Ca2+ ion permeation