Cell Calcium 37 (2005) 121–127

Intracellular Ca2+ dynamics in malignant hyperthermia andcentral core disease: established concepts, new cellular

mechanisms involved

Guillermo Avila∗

Department of Biochemistry, Cinvestav-IPN, AP 14-740, Mexico City DF 07000, Mexico

Received 16 June 2004; received in revised form 2 August 2004; accepted 2 August 2004

Abstract

Malignant hyperthermia (MH) and central core disease (CCD) are inherited human disorders of skeletal muscle Ca2+ homeostasis. Both MHand CCD are linked to mutations and/or deletions in the gene encoding the skeletal muscle ryanodine receptor (RyR1), the intracellular Ca2+

release channel, which is essential to excitation–contraction (EC) coupling. Our knowledge on how mutations in RyR1 disrupt intracellularC ry studiesr historicalp ovel resultsi nt cellularm©

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a2+ homeostasis and EC coupling, eventually leading to MH and CCD has been recently improved, thanks to multidisciplinaanging from clinical, single channel recordings, patch-clamp experiments, and molecular biology. This review presents a brieferspective, on how pioneer studies resulted in associating MH and CCD to RyR1. The review is also focused on discussing n

n regard to pathophysiological consequences of specific MH/CCD RyR1 mutant proteins, which are representative of the differeechanisms that are linked to either phenotype.2004 Elsevier Ltd. All rights reserved.

eywords:Muscle disease; Ryanodine receptor; Excitation–contraction coupling

. Overview

Central core disease (CCD) is a human congenital my-pathy in which type 1 skeletal muscle fibers from affected

ndividuals exhibit amorphous areas (cores) that lack mito-hondria and oxidative enzyme activity. The most commonymptoms are lower limb skeletal muscle weakness and de-ormities. Malignant hyperthermia (MH) on the other hand,s a pharmacogenetic syndrome in which susceptible indi-iduals (MHS) exhibit attacks of tachycardia, hypoxia, highemperature, hypermetabolism, lactic acidosis, and skeletaluscle rigidity, in response to inhalation of volatile anes-

hetics (e.g. halothane), and administration of skeletal mus-le depolarizing agents (e.g. succinylcholine). MH episodesre death-threatening if not properly handled. A mutation in

he RyR1 gene that can be linked to MH has been identi-

∗ Tel.: +52 55 5061 3952; fax: +52 55 5747 7083.E-mail address:gavial@mail.cinvestav.mx.

fied in only about 50% of MH families but this numberlikely to increase as investigators move to analysis ofcomplete gene. A very few mutations in the CACN1AS gencoding the skeletal muscle dihydropyridine receptorthe SCN4A gene encoding the skeletal muscle Na+ channegene have also been linked to MH[1,2]. To date, only mutations in the RyR1 gene have been linked to CCD. CeRyR1 mutants produce both MH and CCD (MH + CCwhereas others result in either an MH-selective pheno(MH-only) or in apparently an exclusively form of CC(CCD-only). The review is focused on discussing howtered intracellular Ca2+ dynamics resulting from RyR1 mtant proteins might contribute to explain the pathophysioof MH and CCD, considering both established conceptsnovel theories/mechanisms involved. The following revion related aspects should be also consulted: moleculaphysiological basis[3–7], functional defects in RyR1 mtant proteins[8–10], congenital myopathies[11,12], ryan-odinopathies[13], and channelopathies[14–16].

143-4160/$ – see front matter © 2004 Elsevier Ltd. All rights reserved.

oi:10.1016/j.ceca.2004.08.001

122 G. Avila / Cell Calcium 37 (2005) 121–127

2. Earlier functional studies provide the key inassociating MH and CCD to RyR1

In a very recent and comprehensive review, Wappler[7]state that the first documented cases of MH appeared nearlya century ago (i.e. in 1900). Approximately 60 years there-after, Shy and Magee[17] reported what seems to be the firstdocumented pathologic case of CCD, and Denborough andLovell established[18] the first link between anesthesia asetiologic agent for malignant hyperthermia and the familialnature of the disease. The fact that certain MH patients exhibitalso muscle rigidity[19], strongly suggested that MH mightinvolve alterations in intracellular Ca2+ homeostasis. Con-ceivable, this putative myoplasmic Ca2+ dysregulation mightbe explained by possible alterations in SR Ca2+ uptake, bymeans of an altered function of SR Ca2+ ATPases (SERCAs).Nevertheless, experiments carried-out in the 1980s (on mus-cle samples obtained from MH patients) indicated that SRCa2+ uptake is unaltered[20]. Instead, a higher rate of SRCa2+-induced Ca2+ release was found[21].

Taken together, these findings at the end of the 1980s,strongly suggested MH originates from an altered Ca2+homeostasis, induced by a possible dysregulation on the func-tional properties of SR Ca2+ release channels. This inter-pretation was strengthened over the next 3 years, by resultso l ex-p ts hibith aseds -o SRC ted re-l ility.F na-l . Inp tiblep mo-l MHh feines easeca ug-g diner rveda u-m ewhatp fectsm e thant ouslyt MHa ecep-t oups[ s ast here-a dingt

Fig. 1. Schematic representation of proposed locations for the differentMH/CCD mutations in RyR1. Most of the mutations are distributed alongthree distinct, and relatively delimited regions of RyR1 (see numbers 1–3),termed MH/CCD 1 (amino acids 35–2168), MH/CCD 2 (amino acids2206–2458), and MH/CCD 3 (amino acids 4214–4914). MH/CCD region 3(C-terminal) contains all putative transmembrane segments (including exon102), which are thought to configure the pore-lining and selective-filter do-main, whereas MH/CCD regions 1 and 2 (N-terminal) are located in thelarge cytosolic aspect of RyR1. The topology of transmembrane segmentsin RyR1 is based on Du et al.[70]. MH has been also linked to mutations inthe gene encoding to DHPR (not depicted). Even though MH/CCD regions1 and 2 are separated in the primary structure of RyR1, it is thought thatthese regions might interact to form an important modulation site[71].

this date, at least 39 RyR1 mutations are correlated to MH[10], which are distributed along three relatively delimitedregions of RyR1 (Fig. 1).

Because MHS is often associated to CCD, the corre-sponding association of CCD and RyR1, was almost simul-taneously established[29,30], and two independent groupsreported in 1993 the first RyR1 mutations linked to CCD[31,32]. Quane et al.[31] proposed that CCD might be theresult of extremely overactive RyR1 mutant proteins (leakychannels hypothesis); whereas Zhang et al.[32] hypothesizedthat the skeletal muscle weakness observed in CCD might berelated to a possible uncoupling of electrical stimulus and thesubsequent SR Ca2+ release. Over the last decade, the atten-tion has been focused in discovering new MH and/or CCDmutants, and to elucidate the corresponding functional de-fects. To these days, the most interesting challenge perhaps,is to determine specific functional defects linked to one oranother disease. The fact that certain mutations in RyR1 arealso associated to other two congenital myopathies; termedmultiminicore disease or MmD[33–35] and nemaline rodmyopathy NM[36–38], complicates the issue of how certainmutations in RyR1, might effectively result in these differentclinical phenotypes.

3

ingr ele MHm cen-

btained from both ryanodine binding and single channeeriments. Specifically, Mickelson et al.[22] reported thakeletal muscle samples from MH susceptible pigs exigher specific ryanodine binding, as well as an increensitivity to activation by micromolar Ca2+. Specific ryandine binding is proportional to the open probability ofa2+ release channels, and thus, these results sugges

ease channels linked to MH exhibit a higher open probabill et al. [23,24] reported similar results, by means of a

yzing release channels activity on artificial lipid bilayersarticular, release channels obtained from MH suscepigs exhibited a higher resistant to inactivation by micro

ar Ca2+ [23]. Moreover, release channels obtained fromuman patients exhibited also an abnormally greater cafensitivity[24]. Taken together, these results indicate relhannels linked to MH are more “willing” to release Ca2+,nd unable to close promptly. The results also implicitly sested an overactive behavior of skeletal muscle ryanoeceptors might be responsible for the previously obselterations on intracellular Ca2+ homeostasis, and the han disease. Nevertheless, this conclusion seems somrecipitated, because ryanodine receptor’s functional deight only be a secondary effect, or a consequence, mor

he source of the disease. In 1990 (i.e. almost simultaneo the studies of Fill et al.) however, a linkage betweennd the gene encoding the skeletal muscle ryanodine r

or was independently established by three different gr25–27]. Thus, an important role for ryanodine receptorhe causal agent for MH was definitely accepted. Soon tfter, the first nucleotide substitution in the gene enco

he skeletal muscle ryanodine receptor was found[28] and to

. Intracellular resting Ca2+

Because MH-only mutants were overactive followyanodine binding determinations[22] and single channxperiments[23,24], it seems reasonable to propose thatutations in RyR1 could significantly increase the con

G. Avila / Cell Calcium 37 (2005) 121–127 123

tration of cytosolic free Ca2+ ([Ca2+]i ). This topic, however,remains controversial[4]. This is because experimentalresults suggest [Ca2+]i is either significantly increased[39,40] or practically unaltered[41–43], in response to theexpression of MH-only mutations. Conceivably, the differentmethodologies used (e.g. Ca2+-sensitive electrodes versusCa2+-sensitive fluorophores) and expression systems (e.g.homologous versus heterologous) might contribute to ex-plain this controversy. Another potential source of variation,might be attributable to misleading identification of addi-tional mutations in RyR1, which can be present within thesame individual. Support for this interpretation comes fromthe discovery that two CCD patients co-express an MH-onlyRyR1 mutation (R614C), with a most recently identified mu-tation (G215E). Moreover, mutations L4650P and K4724Q,were also found to coexist in another CCD patient[44].

In contrast to the controversy on possible effects of MH-only mutations on resting [Ca2+]i , it is well established thatcertain MH + CCD mutations distributed along all threeMH/CCD regions in RyR1 (Fig. 1), can effectively produceseveral degrees of augmentation in [Ca2+]i [43,45,46]. Inaddition, HEK-293 cells exhibit significant reductions in rel-ative levels of ER Ca2+ content, following expression ofboth MH + CCD and MH-only mutants. The MH + CCDmutants, however, produced the highest degrees of store de-p lyp g ex-p fromR r,t CCD( thatr ffec-tT atef velym de-pi ousc ativee oresa logyo -ai al-t icalc a-t i.e.e ngri lls( ion,e ati orefd sidee CD.

Moreover, the absence of mitochondria in core regions per se,may not represent an important pathophysiologic factor forCCD either, since does nor appear to be a direct relationshipbetween the extent of central cores and the severity of thedisease[3]. In fact, two individuals exhibiting muscle weak-ness did not present cores, and a biopsy study was needed ina third family member, to diagnostic CCD[52]. Conversely,it has been proposed that∼40% of patients exhibiting coresmight be clinically normal[3,53].

4. Excitation–contraction (EC) coupling

Another interesting line of research is how release chan-nels linked to MH and/or CCD, might be affecting theexcitation–contraction (EC) coupling mechanism. In skeletalmuscle, EC coupling depends on a unique, physical interac-tion [54] between RyR1s and the principal subunit of L-typevoltage-gated Ca2+ channels, or dihydropyridine receptors(DHPRs;Fig. 1), located at the sarcolemma[55]. In responseto an electrical depolarization of the sarcolemma, the voltagesensor of DHPRs undergo conformational changes, whichresult in activation of nearby RyR1s (orthograde coupling),and a subsequent release of SR Ca2+ [56]. The resultingvoltage-gated SR Ca2+ release, transitorily elevates [Ca2+]i( 2+ oft en-h n asC g theb icali

tov andC thatm re-l tion,h mus-c ichi de-c onset eR SRC con-fi -outi tion[ 5C-e ss ials,i am-p ass pling,s max-i gradec

H+ 5H,

letion [43]. The fact that MH + CCD mutants effectiveroduce store depletion has been corroborated followinression in skeletal muscle cells (i.e. myotubes derivedyR1 “knock-out” ordyspedicmice)[45,46]. Taken togethe

hese results suggest that the coincidence of MH plusi.e. MH + CCD) depends on specific mutations in RyR1esult in extremely overactive release channels, which eively deplete the SR Ca2+ content, and increase [Ca2+]i .he MH-only phenotype, on the other hand, might origin

rom mutations that produce channels exhibiting a relatioderated overactive behavior, which is insufficient tolete SR Ca2+, and increase [Ca2+]i . The diagnostic of CCD

s based on identification of type I fibers, with amorphentral areas (cores) that lack mitochondria and oxidnzyme activity. It is not clear, however, how these cre formed, and their possible role in the pathophysiof CCD. For instance, the higher [Ca2+]i induced by overctive MH + CCD RyR1 mutants[43,45,46], could be an

mportant step in core formation, by means of producingerations in skeletal muscle fiber function and/or biochemomposition[3]. According to this, certain CCD only mutions located within a sub-region of MH/CCD region 3 (xon 102;Fig. 1), which contains the putative pore-formiegion of the release channel[47,48], increase also [Ca2+]in lymphocytes[49,50]. However, in skeletal muscle cei.e. dyspedic myotubes) CCD mutations of the pore regxert no effect on [Ca2+]i [46,51], strongly suggesting th

ncreases in [Ca2+]i are not an absolute requirement for cormation. It thus seems likely that the higher [Ca2+]i in-uced often by CCD mutations might just represent affect, more than a central player in the development of C

termed Ca transient), which in turn activates proteinshe contractile machinery. Interestingly, RyR1 proteinsance also the capability of DHPRs to properly functioa2+ channels (retrograde coupling), thus demonstratini-directional signaling nature of the DHPR/RyR1 phys

nteraction[57,58].Certain MH/CCD mutants exhibit a higher sensitivity

oltage activation, in agreement to the notion that MHCD result from overactive release channels. The factutations leading to the MH-only phenotype produce

ease channels with a higher sensitivity to voltage activaas been thoroughly documented. For example, skeletalle bundles from pigs carrying the R615C mutation (whs equivalent to the R614C human mutation), exhibit areased threshold for activation of contraction, in respo potassium depolarizations[59,60]. This suggests that th615C mutation significantly alters voltage control ofa2+ release. Soon thereafter, this interpretation wasrmed in voltage clamp experiments, which were carriedn porcine myotubes homozygous for the R615C muta61]. Specifically, compared to control myotubes, R61xpressing myotubes exhibited SR Ca2+ release, which wahifted∼15 mV toward more negative membrane potent

n the absence of significant alterations in the maximallitude of voltage-gated SR Ca2+ release. The effect welective on the voltage-dependence of orthograde couince no robust alterations were detected in L-channelsmal conductance and voltage-dependence (i.e. retrooupling)[61].

In addition to the MH-only R615C mutation, certain MCCD mutations (i.e. R163C, Y522S, R2163H, R243

124 G. Avila / Cell Calcium 37 (2005) 121–127

and Y4796C) exhibit also a higher sensitivity to voltage ac-tivation, as determined by simultaneous determinations ofCa2+-transients and L-currents, in dyspedic myotubes sub-jected to the whole cell patch-clamp technique[45,46]. Morerecently, the following mutations were also shown to releaseCa2+ more readily in response to potassium depolarizations:R163C, V2168M (MH + CCD); R614C, G341R, R2163C,R2458H, and T4826I (MH-only)[62].

As mentioned in the previous section, dyspedic myotubesexpressing certain MH + CCD mutations (i.e. R163C, Y522S,R2163H, R2435H, and Y4796C) exhibit also different de-grees of SR Ca2+ store depletion, and parallel but oppositeincreases in [Ca2+]i [45,46]. Interestingly, store depletionsinduced by the MH + CCD mutations result in proportionalreductions in maximal voltage-gated SR Ca2+ release, at dif-ference of the MH-only R614C mutation, which did not affectthis parameter[61]. Taken together, these results support thenotion that, within an skeletal muscle-like environment, re-lease channels produced by MH + CCD mutations in RyR1,are more overactive than the corresponding mutant proteinsresulting from MH-only mutations, in a manner that only MH+ CCD mutants significantly increase myoplasmic Ca2+, de-plete the SR Ca2+ content, and consequently reduce the max-imal amplitude of voltage-gated SR Ca2+ release.

On the other hand, a subgroup of CCD mutations lo-c R,R G),w poref lym ni xhibits e ofvr e de-p e-g theD thine ling( se re-s porto lew tionsi thee cel-l ore,s onal“

5

eent RC hasb giono t are

correctly synthesized, targeted to the junctions and physi-cally interact with DHPRs. Support for this view comes fromthe interpretation that the DHPR–RyR1 physical interactionis not significantly altered by the pore mutations, since asmentioned in the previous section, the corresponding mu-tant proteins fully restore the retrograde signal of EC cou-pling (i.e. amplitude and voltage-dependence of L-currents)[10,46,51]. Thus, it is more likely that pore mutations arenot affecting the DHPR–RyR1 orthograde communication.In fact, the pore region of RyR1 is located far away fromthe DHPR (Fig. 1). It follows then, that in order to result inEC uncoupling, the pore mutations must disrupt other essen-tial properties of the channels, as opposed to the orthogradeDHPR–RyR1 communication.

The observations that in dyspedic myotubes certain CCDmutations of the pore are practically unable to release Ca2+ inresponse to voltage and caffeine activation[45,46], combinedwith previous studies showing that related mutations signif-icantly reduce single channel conductance[47,48], stronglysuggest that the EC uncoupling phenotype of these mutationsis due to significant disruption on the ability of release chan-nels to properly permeate Ca2+ [10]. However, while this in-terpretation might eventually be proved for certain mutations,it may not necessarily reflect a general defect. This is becausemutations of the pore behave as overactive release channelsi teri-z llsy aky[ or-t tion� andGC nt form ord-i ownt rmalm

is-re ce ofs ling[ mu-t ererar l.[ he-n thatmt e-i ngt toret ht thint ) area

ated within exon 102 of MH/CCD region 3 (i.e. G48914893W, I4898T, G4899E, G4899R, A4906V, R4914hich contains the putative pore-leaning and selectivity-

orming segments of RyR1 (Fig. 1), are unable to significantodify [Ca2+]i and SR Ca2+ content, following expressio

n dyspedic myotubes. Nevertheless, these mutants eeveral degrees of reduction in both maximal amplitudoltage-gated SR Ca2+ release and caffeine-induced Ca2+elease, again in the absence of significant levels of storletion [46,51]. Moreover, the inhibitory effect on voltagated Ca2+ release cannot be explained by disruption ofHPR–RyR1 physical interaction, since mutations wixon 102 totally restore the retrograde signal of EC coupi.e. L-current amplitude and voltage-dependence). Theults provide compelling experimental evidence, in supf the hypothesis of Quane et al.[31]; that skeletal musceakness observed in CCD, might be related to muta

n RyR1 that could “cause diminished coupling betweenxcitation and contraction process”. A completely new

ular mechanism, termed “EC uncoupling”, was therefuggested to result in CCD, in addition to the conventileaky” channels hypothesis[10,46,51].

. Primary defects linked to EC uncoupling

EC uncoupling stands for a functional uncoupling betwhe electrical stimulus and Ca2+ release from an intact Sa2+ content. To date, the EC uncoupling mechanismeen only observed on mutations harboring the pore ref RyR1. These mutations produce mutant proteins tha

n HEK-293 cells and leukocytes. Specifically, characation of I4898T CCD mutant proteins in HEK-293 ceielded to the conclusion that this mutant is extremely le63]. Likewise, experimental results obtained from immalized lymphoblastoid cells show that expression of dele4863–4869 and mutations R4861H, R4893W, I4898T,4899R result in “unprompted” Ca2+ release[49,50]. Thus,a2+ impermeant channels is not an absolute requiremeutations of the pore to result in EC uncoupling and acc

ng to this, a pore mutation (R4893W) was recently sho exhibit both the EC uncoupling phenotype and a noaximal amplitude of caffeine-induced Ca2+ release[46].Interestingly, mutation E4032A, which is thought to d

upt the RyR1 Ca2+ sensing mechanism of activation[64]xhibits also an EC uncoupling phenotype, in the absenignificant alterations of the retrograde signal of EC coup65,66]. On the other hand, three different EC uncoupledations of the pore (i.e. R4893W, I4898T and G4899E) wecently shown to practically eliminate Ca2+ sensitivity andmplitude of Ca2+-dependent Ca2+ release[67]. Thus, theeduced sensitivity to Ca2+ activation reported by Du et a67] clearly contributes to explain the EC uncoupling potype of these mutations. The apparent contradictionutation R4893W severely disrupts sensitivity to Ca2+ ac-

ivation [67] in the face of a normal amplitude of caffeinnduced Ca2+ release[46] could be explained by proposihat in contrast to voltage, caffeine might be able to reso a normal level the lower Ca2+ sensitivity observed withis mutation. It is not clear, however, how mutations wihe pore (and probably on the luminal side of the proteinble to reduced RyR1 sensitivity to Ca2+ activation. In this

G. Avila / Cell Calcium 37 (2005) 121–127 125

regard, a very recent study showing that transmembrane do-mains of cardiac RyRs interact functionally with cytoplamicregion 3722–4610[68], which contains the proposed sensorof Ca2+ activation[69], might represent an intriguing mech-anism to explain how RyR1 mutations of the pore disruptCa2+ sensitivity to activation and EC coupling.

For instance, in addition to mutations of the pore, whichmost likely result in EC uncoupling by reducing RyR1 Ca2+permeability and/or sensitivity to activation by Ca2+, the ECuncoupling phenotype (i.e. reduction of voltage-gated Ca2+release in the absence of store depletion) might originate alsofrom mutations reducing the expression level of RyR1s, thusresulting in a decreased number of SR Ca2+ release units,or mutations that disrupt the physical interaction with thevoltage sensor, either by directly altering the DHPR/RyR1interaction domain(s), or by probably inducing a disruptedtargeting of RyR1 mutant proteins to the junctions. Interest-ingly, a recently discovered out-of-frame homozygous muta-tion was found to result in a massive depletion of the normalRyR1 protein[35]. Thus, it is tempting to speculate that atleast this particular mutation might also induce the EC un-coupling phenotype, in addition to mutations in the region ofthe pore.

Paradoxically, in skeletal muscle cells (i.e. dyspedic my-otubes) the EC uncoupling mechanism arise from RyR1 mu-t 2+ m( ac nels[ rgei ge-g toe

6

op-e ote( ents lationw MHi sisti scleb andc clec d asM esc erna-t , opi-o entivea es,h

ing,t lop-m ntro-l of

blocking Ca2+ efflux thorough RyR1s[3]. Hence by neu-tralizing the overactive behavior of leaky CCD mutations,dantrolene might represent an intriguing candidate to effec-tively restore the normal levels of both SR Ca2+ content andvoltage-gated SR Ca2+ release. Conversely, individuals pos-sessing EC uncoupled CCD mutants would be expected todeteriorate in response to dantrolene, since this drug mightfurther reduce Ca2+ permeability trough RyR1s. Thus, in ad-dition to systematically evaluate the pharmacological effectsof dantrolene on skeletal muscle EC coupling, identificationof specific CCD mutations, and the corresponding associatedmechanisms (i.e. leaky versus EC uncoupling) are probablyessential, before using dantrolene as a potential therapy forCCD.

Acknowledgements

I thank M. in Sc. A. Sandoval for artwork. Research workin the authors’ laboratory is supported by a Conacyt grant(39512-Q).

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lig-adultigree,

andEd.),ed.,

on–

tho-–17.mu-Hum.

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[ ularrol. 7

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. Future directions

MH episodes might be death-threatening if not prrly treated by administration of the appropriate antidi.e. dantrolene). Administration of the MH-triggering aghould be also immediately suspended, and hyperventiith 100% oxygen applied. Presymptomatic diagnosis of

s based on in vitro contracting test (IVCT), which conn determining the contracting response of skeletal muundles, in response to MH triggering agents (halothaneaffeine). According to this, a higher sensitivity of musontraction to both halothane and caffeine, is interpreteH susceptible (MHS). Thanks to the IVCT, MH episod

an be totally prevented by the consequent use of altive anesthetics (e.g. non-depolarizing muscle relaxantsids, barbiturates, benzodiazepines, etc.). These prevctions, together with a properly handling of MH episodave greatly reduced the mortality of MH.

In contrast, while CCD might not be death-threatenhere are not currently effective therapies for core deveent and muscle weakness in CCD. Interestingly, da

ene is thought to decrease SR Ca2+ release, by means

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