Melzer discusses a recent JGP study showing that alternative splicing of the skeletal muscle L-type calcium channel impacts on a modulatory effect of its γ subunit.

In this issue, Michelucci et al. report the existence of specific sites acting as Ca2+ entry units (CEUs) in fast skeletal muscle of mice lacking calsequestrin (CASQ1), the major Ca2+ binding protein of the SR. The CEU provides constitutive and store-operated Ca2+ entry (SOCE) and resistance to force decline resulting from SR Ca2+ depletion during repetitive muscle activity.

Calcium ions control multiple physiological functions by binding to extracellular and intracellular targets. One of the best-studied Ca2+-dependent functions is contraction of smooth and striated muscle tissue, which results from Ca2+ ligation to calmodulin and troponin C, respectively. Ca2+ signaling typically involves flux of the ion across membranes via specifically gated channel proteins. Because calcium ions are charged, they possess the ability to generate changes in the respective transmembrane voltage. Ca2+-dependent voltage alterations of the surface membrane are easily measured using microelectrodes. A well-known example is the characteristic plateau phase of the action potential in cardiac ventricular cells that results from the opening of voltage-gated L-type Ca2+ channels. Ca2+ ions are also released from intracellular storage compartments in many cells, but these membranes are not accessible to direct voltage recording with microelectrodes. In muscle, for example, release of Ca2+ from the sarcoplasmic reticulum (SR) to the myoplasm constitutes a flux that is considerably larger than the entry flux from the extracellular space. Whether this flux is accompanied by a voltage change across the SR membrane is an obvious question of mechanistic importance and has been the subject of many investigations. Because the tiny spaces enclosed by the SR membrane are inaccessible to microelectrodes, alternative methods have to be applied. In a study by Sanchez et al. (2018. J. Gen. Physiol. https://doi.org/10.1085/jgp.201812035) in this issue, modern confocal light microscopy and genetically encoded voltage probes targeted to the SR were applied in a new approach to search for changes in the membrane potential of the SR during Ca2+ release.

Calmodulin (CaM) and S100A1 fine-tune skeletal muscle Ca2+ release via opposite modulation of the ryanodine receptor type 1 (RyR1). Binding to and modulation of RyR1 by CaM and S100A1 occurs predominantly at the region ranging from amino acid residue 3614-3640 of RyR1 (here referred to as CaMBD2). Using synthetic peptides, it has been shown that CaM binds to two additional regions within the RyR1, specifically residues 1975-1999 and 4295-4325 (CaMBD1 and CaMBD3, respectively). Because S100A1 typically binds to similar motifs as CaM, we hypothesized that S100A1 could also bind to CaMBD1 and CaMBD3. Our goals were: (1) to establish whether S100A1 binds to synthetic peptides containing CaMBD1 and CaMBD3 using isothermal calorimetry (ITC), and (2) to identify whether S100A1 and CaM modulate RyR1 Ca2+ release activation via sites other than CaMBD2 in RyR1 in its native cellular context. We developed the mouse model (RyR1D-S100A1KO), which expresses point mutation RyR1-L3625D (RyR1D) that disrupts the modulation of RyR1 by CaM and S100A1 at CaMBD2 and also lacks S100A1 (S100A1KO). ITC assays revealed that S100A1 binds with different affinities to CaMBD1 and CaMBD3. Using high-speed Ca2+ imaging and a model for Ca2+ binding and transport, we show that the RyR1D-S100A1KO muscle fibers exhibit a modest but significant increase in myoplasmic Ca2+ transients and enhanced Ca2+ release flux following field stimulation when compared to fibers from RyR1D mice, which were used as controls to eliminate any effect of binding at CaMBD2, but with preserved S100A1 expression. Our results suggest that S100A1, similar to CaM, binds to CaMBD1 and CaMBD3 within the RyR1, but that CaMBD2 appears to be the primary site of RyR1 regulation by CaM and S100A1.

We studied the effect of halothane and its interference with membrane voltage in malignant hyperthermia (MH) susceptible muscle by using enzymatically isolated single fibers (m. interosseus) of knock-in mice with the RyR1 mutation Y524S (human MH mutation Y522S). To investigate anesthetic- and voltage-triggered changes of Ca2+ concentration, we measured fluorescence from fibers loaded with Fura-2-AM. Similar as in the diagnostic in-vitro contracture test (IVCT) for MH susceptibility, cells were exposed to different halothane-containing solutions using a vaporizer system. The percentage of halothane in the gas phase was determined using infrared photometry. The temporal change of the halothane concentration in the recording chamber was checked using gas chromatography. In Krebs-Ringer's solution at room temperature, a step from 0 to 0.5% halothane in the gas phase caused a clear increase in resting Ca2+ concentration in most of the investigated fibers of mutant mice but very little response in fibers of WT littermates. The Ca2+ response was phasic: after reaching a peak the Ca2+ signal decreased at a rate of about 10%/min indicating either reduced sensitivity to halothane or inhibition of Ca2+ release after activation. A likely explanation is Ca2+-induced inactivation of RyR1. In further experiments, we applied halothane to single fibers which were voltage-clamped using two intracellular microelectrodes. Depolarizing pulses to different membrane potentials from a holding potential of −80 mV revealed a strong shift in the voltage threshold for activation of Ca2+ release to more negative potentials. Correspondingly, hyperpolarizing steps led to a rapid partial recovery from the halothane-induced increase in basal Ca2+ concentration. These results demonstrate reciprocal effects of a volatile anesthetic drug and membrane voltage on Ca2+ release in MH-susceptible skeletal muscle.

Malignant hyperthermia (MH) is a fatal hypermetabolic state that may occur during general anesthesia in susceptible individuals. It is often caused by mutations in the ryanodine receptor RyR1 that favor drug-induced release of Ca2+ from the sarcoplasmic reticulum. Here, knowing that membrane depolarization triggers Ca2+ release in normal muscle function, we study the cross-influence of membrane potential and anesthetic drugs on Ca2+ release. We used short single muscle fibers of knock-in mice heterozygous for the RyR1 mutation Y524S combined with microfluorimetry to measure intracellular Ca2+ signals. Halothane, a volatile anesthetic used in contracture testing for MH susceptibility, was equilibrated with the solution superfusing the cells by means of a vaporizer system. In the range 0.2 to 3%, the drug causes significantly larger elevations of free myoplasmic [Ca2+] in mutant (YS) compared with wild-type (WT) fibers. Action potential–induced Ca2+ signals exhibit a slowing of their time course of relaxation that can be attributed to a component of delayed Ca2+ release turnoff. In further experiments, we applied halothane to single fibers that were voltage-clamped using two intracellular microelectrodes and studied the effect of small (10-mV) deviations from the holding potential (−80 mV). Untreated WT fibers show essentially no changes in [Ca2+], whereas the Ca2+ level of YS fibers increases and decreases on depolarization and hyperpolarization, respectively. The drug causes a significant enhancement of this response. Depolarizing pulses reveal a substantial negative shift in the voltage dependence of activation of Ca2+ release. This behavior likely results from the allosteric coupling between RyR1 and its transverse tubular voltage sensor. We conclude that the binding of halothane to RyR1 alters the voltage dependence of Ca2+ release in MH-susceptible muscle fibers such that the resting membrane potential becomes a decisive factor for the efficiency of the drug to trigger Ca2+ release.

Skeletal muscle excitation–contraction (EC) coupling is initiated by sarcolemmal depolarization, which is translated into a conformational change of the dihydropyridine receptor (DHPR), which in turn activates sarcoplasmic reticulum (SR) Ca 2+ release to trigger muscle contraction. During EC coupling, the mammalian DHPR embraces functional duality, as voltage sensor and l -type Ca 2+ channel. Although its unique role as voltage sensor for conformational EC coupling is firmly established, the conventional function as Ca 2+ channel is still enigmatic. Here we show that Ca 2+ influx via DHPR is not necessary for muscle performance by generating a knock-in mouse where DHPR-mediated Ca 2+ influx is eliminated. Homozygous knock-in mice display SR Ca 2+ release, locomotor activity, motor coordination, muscle strength and susceptibility to fatigue comparable to wild-type controls, without any compensatory regulation of multiple key proteins of the EC coupling machinery and Ca 2+ homeostasis. These findings support the hypothesis that the DHPR-mediated Ca 2+ influx in mammalian skeletal muscle is an evolutionary remnant.

Huntington´s disease (HD) is a hereditary neurodegenerative disease resulting from an expanded polyglutamine sequence (poly-Q) in the protein huntingtin (HTT). Various studies report atrophy and metabolic pathology of skeletal muscle in HD and suggest as part of the process a fast-to-slow fiber type transition that may be caused by the pathological changes in central motor control or/and by mutant HTT in the muscle tissue itself. To investigate muscle pathology in HD, we used R6/2 mice, a common animal model for a rapidly progressing variant of the disease expressing exon 1 of the mutant human gene. We investigated alterations in the extensor digitorum longus (EDL), a typical fast-twitch muscle, and the soleus (SOL), a slow-twitch muscle. We focussed on mechanographic measurements of excised muscles using single and repetitive electrical stimulation and on the expression of the various myosin isoforms (heavy and light chains) using dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of whole muscle and single fiber preparations. In EDL of R6/2, the functional tests showed a left shift of the force-frequency relation and decrease in specific force. Moreover, the estimated relative contribution of the fastest myosin isoform MyHC IIb decreased, whereas the contribution of the slower MyHC IIx isoform increased. An additional change occurred in the alkali MyLC forms showing a decrease in 3f and an increase in 1f level. In SOL, a shift from fast MyHC IIa to the slow isoform I was detectable in male R6/2 mice only, and there was no evidence of isoform interconversion in the MyLC pattern. These alterations point to a partial remodeling of the contractile apparatus of R6/2 mice towards a slower contractile phenotype, predominantly in fast glycolytic fibers.

Malignant hyperthermia is a potentially fatal hypermetabolic state originating from excessive release of calcium stored in the sarcoplasmic reticulum (SR) of skeletal muscle. In most cases, MH susceptibility results from mutations in the type 1 ryanodine receptor (RyR1). In previous work (Andronache et al., PNAS 2009) we reported that heterozygous murine carriers of MH mutation Y524S (human Y522S) exhibit changes in steady state inactivation of the dihydropyridine receptor (DHPR), the sensor of the transverse tubular (TT) membrane potential. Availability curves were left shifted along the voltage axis suggesting that a feedback signal from RyR1 modulates DHPR inactivation. In the present study we investigated the hypothesis that junctional fluctuations of free Ca2+ concentration are involved in the feedback mechanism. We performed two-electrode voltage clamp experiments on enzymatically isolated toe muscle fibers of both WT and mutant mice (Y524S+/−) and measured L-type Ca2+ current and optical signals from fluorescent Ca2+ indicators. To test the hypothesis we applied conditions that would modify junctional Ca2+ levels. Millimolar concentrations of caffeine led to a left shift in the availability curve for L-type current indicating that drug-induced RyR1 hyperactivity can mimic the effect of the mutation. On the other hand, internal dialysis with an artificial solution containing 10 mM of BAPTA to effectively reduce the local Ca2+ transients near open RyR1 channels had little effect on the difference in steady state inactivation between WT and mutant fibers. We conclude that the altered inactivation depends on RyR1 hyperactivity but does not require the continuous presence of local Ca2+ fluctuations within the junctional gap separating TT and SR.

In contrast to cardiac excitation-contraction coupling (ECC), skeletal-muscle ECC is based on Ca2+-influx-independent inter-channel communications between the dihydropyridine receptor (DHPR) and the ryanodine receptor (RyR1). The role of the small Ca2+ influx through the DHPR in mammalian skeletal muscle, which is not (immediately) required for ECC, is still enigmatic. Previously, we discovered that zebrafish, as well as all higher teleost fish, lack DHPR Ca2+ conductivity in skeletal muscle (Schredelseker et al., PNAS, 2010). Point mutation N617D in pore loop II of zebrafish DHPRα1S-b explained non-conductivity in fast muscle. To investigate the fascinating skeletal muscle DHPR Ca2+-conductivity / non-conductivity phenomenon we generated a non-conducting-DHPR knock-in mouse (n.c.DHPR) by introducing the N→D mutation into gene CACNA1S. Interestingly, homozygous n.c.DHPR mice are viable, fertile, visually indistinguishable, and identical in body-weight development to wild-type (WT) siblings. Myotubes isolated from newborn n.c.DHPR mice display complete lack of DHPR Ca2+ influx without altered ECC. No difference in locomotor activity (home cage activity), motor coordination (rotarod, beam walking), and muscle strength (endurance test, wire hang test) is observed in 3-7 months-old homozygous n.c.DHPR mice compared to WT. Identical results were obtained from forced frequency and fatigue tests on isolated EDL (fast twitch) and soleus (slow twitch) muscle fibers. As soon feasible, tests will be repeated on aged (18 months-old) mice to test for putative age-related accumulative effects on muscle performance, in order to understand if DHPR Ca2+ influx in mammalian skeletal muscle is a physiological necessity or just a tolerated evolutionary remnant of the ancestral pure Ca2+-influx dependent ECC of early chordates and phylogenetic branches below. Supported: FWF P-23229-B09; DK-W1101-B12.

Huntington's disease (HD) is caused by an expanded CAG trinucleotide repeat within the gene encoding the protein huntingtin. The resulting elongated glutamine (poly-Q) sequence of mutant huntingtin (mhtt) affects both central neurons and skeletal muscle. Recent reports suggest that ryanodine receptor-based Ca2+ signaling, which is crucial for skeletal muscle excitation-contraction coupling (ECC), is changed by mhtt in HD neurons. Consequently, we searched for alterations of ECC in muscle fibers of the R6/2 mouse, a mouse model of HD. We performed fluorometric recordings of action potentials (APs) and cellular Ca2+ transients on intact isolated toe muscle fibers (musculi interossei), and measured L-type Ca2+ inward currents on internally dialyzed fibers under voltage-clamp conditions. Both APs and AP-triggered Ca2+ transients showed slower kinetics in R6/2 fibers than in fibers from wild-type mice. Ca2+ removal from the myoplasm and Ca2+ release flux from the sarcoplasmic reticulum were characterized using a Ca2+ binding and transport model, which indicated a significant reduction in slow Ca2+ removal activity and Ca2+ release flux both after APs and under voltage-clamp conditions. In addition, the voltage-clamp experiments showed a highly significant decrease in L-type Ca2+ channel conductance. These results indicate profound changes of Ca2+ turnover in skeletal muscle of R6/2 mice and suggest that these changes may be associated with muscle pathology in HD.

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.

In vitro, calmodulin (CaM) and S100A1 activate the skeletal muscle ryanodine receptor ion channel (RyR1) at submicromolar Ca2+concentrations, whereas at micromolar Ca2+concentrations, CaM inhibits RyR1. One amino acid substitution (RyR1-L3625D) has previously been demonstrated to impair CaM binding and regulation of RyR1. Here we show that the RyR1-L3625D substitution also abolishes S100A1 binding. To determine the physiological relevance of these findings, mutant mice were generated with the RyR1-L3625D substitution in exon 74, which encodes the CaM and S100A1 binding domain of RyR1. Homozygous mutant mice ( Ryr1D/D) were viable and appeared normal. However, single RyR1 channel recordings from Ryr1D/Dmice exhibited impaired activation by CaM and S100A1 and impaired CaCaM inhibition. Isolated flexor digitorum brevis muscle fibers from Ryr1D/Dmice had depressed Ca2+transients when stimulated by a single action potential. However, during repetitive stimulation, the mutant fibers demonstrated greater relative summation of the Ca2+transients. Consistently, in vivo stimulation of tibialis anterior muscles in Ryr1D/Dmice demonstrated reduced twitch force in response to a single action potential, but greater summation of force during high-frequency stimulation. During repetitive stimulation, Ryr1D/Dfibers exhibited slowed inactivation of sarcoplasmic reticulum Ca2+release flux, consistent with increased summation of the Ca2+transient and contractile force. Peak Ca2+release flux was suppressed at all voltages in voltage-clamped Ryr1D/Dfibers. The results suggest that the RyR1-L3625D mutation removes both an early activating effect of S100A1 and CaM and delayed suppressing effect of CaCaM on RyR1 Ca2+release, providing new insights into CaM and S100A1 regulation of skeletal muscle excitation-contraction coupling.

In vitro, calmodulin (CaM) and S100A1 activate the skeletal muscle ryanodine receptor ion channel (RyR1) at submicromolar Ca(2+) concentrations, whereas at micromolar Ca(2+) concentrations, CaM inhibits RyR1. One amino acid substitution (RyR1-L3625D) has previously been demonstrated to impair CaM binding and regulation of RyR1. Here we show that the RyR1-L3625D substitution also abolishes S100A1 binding. To determine the physiological relevance of these findings, mutant mice were generated with the RyR1-L3625D substitution in exon 74, which encodes the CaM and S100A1 binding domain of RyR1. Homozygous mutant mice (Ryr1(D/D)) were viable and appeared normal. However, single RyR1 channel recordings from Ryr1(D/D) mice exhibited impaired activation by CaM and S100A1 and impaired CaCaM inhibition. Isolated flexor digitorum brevis muscle fibers from Ryr1(D/D) mice had depressed Ca(2+) transients when stimulated by a single action potential. However, during repetitive stimulation, the mutant fibers demonstrated greater relative summation of the Ca(2+) transients. Consistently, in vivo stimulation of tibialis anterior muscles in Ryr1(D/D) mice demonstrated reduced twitch force in response to a single action potential, but greater summation of force during high-frequency stimulation. During repetitive stimulation, Ryr1(D/D) fibers exhibited slowed inactivation of sarcoplasmic reticulum Ca(2+) release flux, consistent with increased summation of the Ca(2+) transient and contractile force. Peak Ca(2+) release flux was suppressed at all voltages in voltage-clamped Ryr1(D/D) fibers. The results suggest that the RyR1-L3625D mutation removes both an early activating effect of S100A1 and CaM and delayed suppressing effect of CaCaM on RyR1 Ca(2+) release, providing new insights into CaM and S100A1 regulation of skeletal muscle excitation-contraction coupling.

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 Ca(2+) 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 Ca(2+) 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 Ca(2+) binding and transport processes to calculate sarcoplasmic reticulum Ca(2+) 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 Ca(2+) release flux, resulting in impaired contractile activation and force production in skeletal muscle.

Background and aims Some of the most obvious peripheral tissue changes in Huntington9s disease (HD) are found in skeletal muscle. Alterations in morphology, gene expression pattern, energy metabolism and differentiation have been described. However, it is still an open question to what extent these changes reflect cell autonomous effect of mutant huntingtin. As yet, surprisingly little information is available about contractile performance and excitation–contraction coupling in HD muscle. Recently we found that isometric contraction of fast twitch muscle in the R6/2 mouse model of HD exhibits significantly slower kinetics than WT muscle. To investigate the potential causes of the changes in muscle contraction we studied the kinetics of action potential triggered intracellular Ca2+ transients. Methods Enzymatically dissociated interosseus muscle fibres of male R6/2 (11–13 weeks old) exhibiting disease symptoms and of age matched WT mice were primary cultured up to 2 days. Ca2+-transients were elicited by extracellular electrical stimulation. Ca2+ dependent fluorescence signals of Fura2-AM loaded cells exhibiting all or none responses were analysed. A kinetic model was applied to estimate Ca2+ removal and Ca2+ release. Results In R6/2 fibres we observed significantly slower relaxation kinetics of Ca2+ transients elicited by single stimuli compared with WT. The mean time constant of relaxation was 31 ms±1 ms (SEM) for WT (n=25) and 53 ms±4 ms for R6/2 (n=73, p<0.01) at 25°C. Model analysis disclosed that these findings are compatible with a reduction by half of parvalbumin concentration and uptake rate constant in R6/2. Peak Ca2+ release flux was reduced to 45% of WT. Conclusion In our experiments we demonstrate that R6/2 animals transgenic for mutant huntingtin show slowed Ca2+ kinetics in fast twitch muscle. The analysis identifies a reduction of sarcoreticular Ca2+ release and reuptake as well as a decrease in myoplasmic Ca2+ binding as likely sources for the changes observed in muscle contraction.

Malignant hyperthermia (MH) is a life-threatening hypermetabolic condition caused by dysfunctional Ca 2+ homeostasis in skeletal muscle, which primarily originates from genetic alterations in the Ca 2+ release channel (ryanodine receptor, RyR1) of the sarcoplasmic reticulum (SR). Owing to its physical interaction with the dihydropyridine receptor (DHPR), RyR1 is controlled by the electrical potential across the transverse tubular (TT) membrane. The DHPR exhibits both voltage-dependent activation and inactivation. Here we determined the impact of an MH mutation in RyR1 (Y522S) on these processes in adult muscle fibers isolated from heterozygous RyR1 Y522S -knock-in mice. The voltage dependence of DHPR-triggered Ca 2+ release flux was left-shifted by ≈8 mV. As a consequence, the voltage window for steady-state Ca 2+ release extended to more negative holding potentials in muscle fibers of the RyR1 Y522S -mice. A rise in temperature from 20° to 30 °C caused a further shift to more negative potentials of this window (by ≈20 mV). The activation of the DHPR-mediated Ca 2+ current was minimally changed by the mutation. However, surprisingly, the voltage dependence of steady-state inactivation of DHPR-mediated calcium conductance and release were also shifted by ≈10 mV to more negative potentials, indicating a retrograde action of the RyR1 mutation on DHPR inactivation that limits window Ca 2+ release. This effect serves as a compensatory response to the lowered voltage threshold for Ca 2+ release caused by the Y522S mutation and represents a novel mechanism to counteract excessive Ca 2+ leak and store depletion in MH-susceptible muscle.