Dysregulation of calcium homeostasis in muscular ... · Dysregulation of calcium homeostasis in muscular dystrophies Ainara Vallejo-Illarramendi1,2,3,*, Ivan Toral-Ojeda1,2, Garazi

Dysregulation of calcium homeostasis

in muscular dystrophies

Ainara Vallejo-Illarramendi1,2,3,*, Ivan Toral-Ojeda1,2,Garazi Aldanondo1 and Adolfo López de Munain1,2,4,5

Muscular dystrophies are a group of diseases characterised by the primarywasting of skeletal muscle, which compromises patient mobility and in themost severe cases originate a complete paralysis and premature death.Existing evidence implicates calcium dysregulation as an underlying crucialevent in the pathophysiology of several muscular dystrophies, such asdystrophinopathies, calpainopathies or myotonic dystrophy among others.Duchenne muscular dystrophy is the most frequent myopathy in childhood,and calpainopathy or LGMD2A is the most common form of limb-girdlemuscular dystrophy, whereas myotonic dystrophy is the most frequentinherited muscle disease worldwide. In this review, we summarise recentadvances in our understanding of calcium ion cycling through the sarcolemma,the sarcoplasmic reticulum and mitochondria, and its involvement in thepathogenesis of these dystrophies. We also discuss some of the clinicalimplications of recent findings regarding Ca2+ handling as well as novelapproaches to treat muscular dystrophies targeting Ca2+ regulatory proteins.

IntroductionCa2+ ions are involved in the regulation of avariety of cellular processes such as musclecontraction, secretion, proliferation or cell death(Ref. 1). Therefore, there is a need of precisetemporal and spatial control of Ca2+ fluxesand Ca2+ concentration within the cell. Indeed,Ca2+ concentration is tightly controlled in alleukaryotic cells by complex interactions among

voltage sensors, Ca2+ transporters, Ca2+ channels,Ca2+ exchangers, Ca2+-binding proteins, ionpumps and ion exchangers (Refs. 2, 3, 4). Thisfine regulation of Ca2+ homeostasis allows thision to act as one of the most important secondmessengers in signal transduction.

In skeletal muscle fibres, the status ofthe excitation–contraction–relaxation cycle isdetermined by the cytosolic Ca2+ levels: high

1Neuroscience Area, Biodonostia Institute, San Sebastian, Spain2Centro de Investigación Biomédica en Red para Enfermedades Neurodegenerativas (CIBERNED),Instituto de Salud Carlos III, San Sebastian, Spain3Euskampus, University of the Basque Country (UPV-EHU), San Sebastian, Spain4Department of Neuroscience, University of the Basque Country (UPV-EHU), San Sebastian, Spain5Department of Neurology, Donostia Hospital, San Sebastian, Spain

*Corresponding author: Ainara Vallejo-Illarramendi, Instituto Biodonostia, Po Dr Begiristain s/n,20014 San Sebastian, Spain. E-mail: [email protected]

expert reviewshttp://www.expertreviews.org/ in molecular medicine

1Accession information: doi:10.1017/erm.2014.17; Vol. 16; e16; October 2014

© Cambridge University Press 2014

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mailto:[email protected]

Ca2+ levels are crucial for triggering musclecontraction, while low levels are critical forinitiating muscle relaxation. Hence, a fastCa2+-cycling mechanism and an efficient storageprocess are absolutely essential in muscle fibres.During normal contractions, local cytosolic Ca2+

concentration is greatly increased reaching up to20 μM (Refs. 5, 6). In addition to these Ca2+

transients associated with muscle contraction,smaller but prolonged increases in intracellularCa2+ levels associated with long-duration fatiguehave been described (Ref. 7). Additionally, smallbut persistent increases in basal intracellular Ca2+

concentration likely occur during certain musclediseases and in ageing (Ref. 8). Since changes incytosolic Ca2+ levels are particularly frequentand diverse in the skeletal muscle, a tight controlin Ca2+ homeostasis is most significant in thissystem. In this regard, small changes in the Ca2+

handling apparatus in the skeletal muscle mightresult in major pathophysiological consequences.Indeed, abnormal expression patterns of ion-regulatory proteins have been reported repeatedlyin muscular dystrophy (Refs. 9, 10, 11, 12, 13).Existing evidence implicates Ca2+ dysregulation

as a common underlying event in thepathophysiology of muscular dystrophies, agroup of diseases characterised by a primarydegeneration of skeletal muscle tissue that resultsin progressive muscle weakness. This reviewexamines Ca2+ handling in muscular dystrophies,with a special focus on dystrophinopathies,calpainopathies and myotonic musculardystrophies. We also summarise recent advancesin our understanding of Ca2+ ion cycling throughthe sarcolemma, the sarcoplasmic reticulum (SR)and mitochondria, and its involvement in thepathogenesis of these dystrophies. Finally, wediscuss some of the clinical implications of thesefindings, and in particular, novel pharmacologicaltherapeutic approaches for muscular dystrophiesthat target Ca2+ regulatory proteins.

Calcium handling in the skeletal muscleStructure and physiology of the skeletalmuscleThe skeletal muscle is under the control of thesomatic nervous system through innervation ofmuscle fibres by motoneurons. Muscle fibres arelong, cylindrical multinucleated cells thatoriginate from the fusion of myoblasts duringembryonic development and behave as a singleunit. Each fibre contains several bundles of

myofibrils, with inter-digitating myosin (thick)and actin (thin) filaments arranged longitudinallyin repeating units known as sarcomeres. Thisparticular arrangement of the cytoskeletalelements accounts for the banding pattern,characteristic of skeletal fibres. Actin filaments arebound to the Z line, which forms the borders ofthe sarcomere. At this location, deepinvaginations of the sarcolemma (T-tubules)permit the conduction of the electrical impulses tothe cell interior (Fig. 1).

A single motoneuron innervating one or moremuscle fibres with similar functional propertiesconstitutes a motor unit. A muscle is formed bymany motor units, each one bringing its ownspecific contribution (Ref. 14). Motor units differin the size of motoneurons and also in the typeof innervated muscle fibres. They are dividedinto three categories: (a) slow; (b) fast fatigable;and (c) fast fatigue-resistant motor units (Ref. 15).

(a) Slow motor units: Constituted by type I, slowtwitch muscle fibres, are essential forsustained muscular contraction. Type I fibrescontract very slowly and generate smallforce, but they are very resistant to fatigue.They contain high mitochondria density andthey generate ATP by the aerobic metabolism.

(b) Fast fatigable motor units: Constituted by typeIIB fibres, or type IIX in humans, are usedfor tasks that require large brief bursts ofenergy such as sprinting. Type IIB/IIX orfast glycolytic fibres have a fast contractionvelocity but they fatigue easily. They containfew mitochondria and they generate ATPmainly by the anaerobic system.

(c) Fast fatigue-resistant motor units:Constituted bytype IIA, fast twitch A fibres or fast oxidativefibres, which contain many mitochondria and,thus, can generate ATP by aerobicmetabolism.They have a fast contraction velocity and theyare resistant to fatigue, although not as muchas type I fibres (Ref. 16).

Some of the fibre-type differences in twitchkinetics are also likely due to differences in Ca2+

handling. Indeed, the amount of Ca2+ releasedin type II fibres is up to fourfold larger than intype I fibres, there is a faster decline of thecalcium transient in type II fibres (Ref. 14) andthe proportion of Ca2+ that binds troponin toactivate contraction is substantially smaller(Ref. 5). On the other hand, type I fibres showincreased affinity for SR Ca2+ uptake, higher




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relative levels of SRCa2+ loading and increased SRCa2+ leakage compared with type II fibres (Refs.17, 18). Muscle fibres display a high plasticity,being able to adapt to changing demands bymodifying size or fibre-type composition(Ref. 19). Specific types of muscle fibres arepreferentially affected in different forms ofmuscular dystrophies. In Duchenne musculardystrophy, for example, type IIB fibres areprogressively replaced by type I and IIA fibres(Ref. 20). Recently, we have also found incollaboration with Dr Spencer’s group that slow

fibres are preferentially involved in Limb-girdle muscular dystrophy-type 2A (LGMD2A)dystrophic patients (Ref. 21). Interestingly, somevery specific muscle types present in extraocular muscles show great resistance to damagein Duchenne muscular dystrophy, partially dueto an enhanced Ca2+ buffering (Ref. 22).

The Ca2+ cycle duringexcitation–contraction (E–C)Skeletal muscle contraction is initiated bydepolarisation of the sarcolemma in response to

Sarcolemma

Mitochondrion

SR

NCLX

ATP

ATP

PTP

Sarcomere

Glycolysis

SERCA

Ca2+Ca2+

Ca2+

Ca2+

Calstabin

Ca2+

MCU

NCX

Ca2+

Na+

Na+

Ca2+DHPR

PMCA

Ca2+CSQ

T-tu

bul

e

RYR1

RYR1

STIM1SR

TRPC

TRPC

Orai1

Relaxation

Contraction

Representation of calcium signalling dynamics in the muscle fibreExpert Reviews in Molecular Medicine © 2014 Cambridge University Press

Figure 1. Representation of calcium signalling dynamics in the muscle fibre. Signalling begins whensarcolemmal depolarisation reaches the T-tubules. In these structures, DHPR respond with a conformationalchange that activates closely apposed RyR1, resulting in Ca2+ release from the sarcoplasmic reticulum(SR). Ca2+ diffuses out to activate contraction by binding with Troponin C. Relaxation occurs as Ca2+ isreturned into the SR by SERCA ATPase or is pumped out of the cell by NCX or PMCA. A portion of thisCa2+ reaches the mitochondria and driven by the negative membrane potential, it enters through MCU andLETM1 importers, where it stimulates the metabolism to provide the ATP required for maintainingcontraction. Ca2+ can be released from mitochondria through NCLX exchanger and the mitochondrial PTP.Low SR Ca2+ levels are detected by STIM1 and activate SOCE, an extracellular Ca2+ influx through Orai1or TRPC channels to refill intracellular Ca2+ stores.




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acetylcholine release from motoneurons. Thesarcolemmal depolarisation propagates down theT-tubular system and activates dihydropyridinereceptors (DHPRs) (Refs. 5, 23). The resultingconformational change of DHPRs activatesclosely apposed ryanodine receptors (RyRs),which are Ca2+ release channels localised in theSR membranes (Ref. 24). As these releasechannels open, Ca2+ moves from the SR into thecytosol, driven by a steep Ca2+ concentrationgradient. Afterwards, Ca2+ diffuses throughoutthe sarcomere and binds to various cytosolicconstituents or Ca2+ buffers. Binding of Ca2+ totroponin triggers the contractile response of themyofibrils: Ca2+-bound troponin undergoes aconformational change that leads to themovement of tropomyosin, which unmasksmyosin-binding sites on actin filaments andallows myosin and actin ATP-dependent cross-bridge cycling and shortening of the muscle.Lastly, relaxation is initiated by Ca2+ diffusionfrom the myofilaments to the cytosol and Ca2+

transfer back to the SR by the sarco/endoplasmicreticulum Ca2+ pump SERCA (Ref. 25). Thesarcolemmal Na+/Ca2+-exchanger (NCX) andthe plasmalemmal Ca2+-ATPase (PMCA) areother mechanisms involved in relaxation, sincethey remove cytosolic Ca2+ to the extracellularspace (Ref. 26). Figure 1 illustrates the majorintracellular movements of Ca2+ taking placeduring E–C coupling in skeletal muscle fibres.

Cytosolic Ca2+ buffersCytosolic Ca2+ buffering is the rapid binding ofcytosolic incoming Ca2+ ions to different cellularbinding sites, and constitutes a critical processimplicated in the precise regulation of Ca2+

signalling and homeostasis. Ca2+ buffers arecytosolic Ca2+-binding proteins that modulateintracellular Ca2+ transients by controllingdiffusion of free Ca2+ ions within the cytosol. Thisprocess occurs at the sub-second scale, much fasterthan Ca2+ sequestration into intracellular stores.Concentration of Ca2+ buffers and otherparameters, such as mobility and Ca2+-bindingaffinity, are important for determining theirspecific role in modulation of Ca2+ signalling(Ref. 27).In skeletal muscle fibres, the most significant

Ca2+ buffers in order of binding occurrence intime are, first, troponin C is a component of thinfilaments that allows interaction of actin andmyosin and initiates contractile activation when

its regulatory sites are bound to Ca2+. Differenttypes of muscle fibres have specific versions oftroponin C with different Ca2+-binding capacities.Second, ATP acts as a cytosolic mobile buffer forCa2+ and Mg2+ (Ref. 5). Then parvalbumin playsan important role in regulating the speed ofrelaxation in mice (Ref. 28), although it isexpressed at very low concentrations in humanskeletal muscle (Ref. 29). Afterwards, Ca2+ bindsto calmodulin, contributing to the regulation ofcontractile function via myosin light-chain kinase.It is also conducive to the activation of Ca2+-dependent signalling pathways involved inmuscle gene regulation. Lastly, Ca2+ binds toSERCA pumps for Ca2+ sequestration into the SR.

Contribution of sarcolemma to Ca2+

homeostasisIn skeletal muscle, Ca2+ entry through thesarcolemma has been shown to play animportant role in store repletion (Ref. 30),limiting fatigue under conditions of extensiveexercise (Ref. 31), activation of NFATtranscription factor signalling (Refs. 32, 33) andmuscle differentiation (Ref. 34). The sarcolemmahas a very low permeability to Ca2+ and a veryparticular organisation with the majority of thesarcolemma internalised as the tubular system,which allows for tightly regulated Ca2+ trans-sarcolemmal fluxes. Aside from voltage-gatedchannels, Ca2+ may enter the muscle fibre viaSOCE or store-operated Ca2+ entry (Ref. 30);ECCE, or excitation-coupled Ca2+ entry (Ref. 35);SAC or stretch-activated cation channels (Ref. 36);SIC or stretch-inhibitable cation channels (Ref. 37);and leak channels (Ref. 38). Previous studies haveshown that dysregulation of Ca2+ entry may leadto severe muscle pathologies. In particular, severalgenetic mouse models of muscular dystrophypoint towards an enhanced Ca2+ influx throughSOCE or SAC that leads to Ca2+-dependentapoptosis and muscle degeneration (Refs. 39, 40).On the other hand, Ca2+ extrusion from cells mayalso play an important role in maintaining Ca2+

homeostasis. In muscle fibres, Ca2+ is pumpedout of the cell by the Na+/Ca2+ exchangersNCX1-3 and the Ca2+-ATPase PMCA isoforms 1,3 and 4, localised at the sarcolemma (Ref. 14).

Ca2+-handling proteins in the sarcoplasmicreticulumThe SR, the major reservoir of intracellular Ca2+ inthe muscle fibre, is a highly specialised form of




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endoplasmic reticulum. It is organised as anextensive tubular network surrounding myofibrilswith periodical dilated end sacs or terminalcisternae. A central T-tubule flanked on both sidesby two terminal cisternae from the SR constitutes atriad, a highly specialised anatomical structure thatis the basis of E–C coupling (Ref. 41). In the SR, thebalance between Ca2+ storage, release andreuptake is achieved through the coordinatedaction of three major types of Ca2+-handlingproteins: luminal Ca2+-binding proteins, SR Ca2+

release channels and Ca2+-ATPase pumps for Ca2+

reuptake (Ref. 42). Ca2+-handling proteins areheterogeneously distributed within the SR, whichresults in functionally distinct subdomains whereCa2+ release and reuptake occur in the terminalcisternae and the longitudinal SR, respectively.

(a) Luminal Ca2+-binding proteins: Within the SRlumen, a large proportion of Ca2+ is boundto Ca2+-binding proteins that perform a dualfunction as Ca2+ reservoir components andas endogenous regulators of Ca2+ fluxes.Luminal Ca2+-binding proteins help toreduce the concentration of free Ca2+ withinthe SR, which assists in the reuptake of Ca2+

to the SR. They determine normal andmaximal Ca2+ levels in the SR, incombination with SERCA pump (Ref. 43).Intraluminal Ca2+ levels have been reportedto range between high micromolar and lowmilimolar concentrations, thus, constitutinga steep electrochemical gradient aimed at thecytosol. The most significant Ca2+-bindingproteins in the SR are: calsequestrin (CSQ),which is a moderate-affinity, high-capacityCa2+-binding proteins located in the terminalcisternae region; histidine-rich Ca2+-bindingprotein (HRC), which has been found to bindSERCA and triadin, and therefore, might beinvolved in the regulation of Ca2+ release byRyRs and sequestration (Refs. 44, 45);sarcalumenin, a Ca2+-binding/shuttle proteinlocalised in the longitudinal SR; andcalreticulin. In particular, CSQ is involved inthe SR Ca2+-loading capacity (Ref. 43), whilealso modulating activity of RyR channels(Ref. 46). In the skeletal muscle, CSQ1 is themain isoform, although CSQ2 is alsoexpressed in type I fibres. In fact, CSQ1 hasbeen found to both inhibit and activate RyR1depending on its binding manner to RyR1(Ref. 47). Recent work supports an essential

role of CSQ1 in the mechanism of RyR1channel closure and termination of SR Ca2+

release in mouse skeletal muscle (Ref. 48).

(b) SR Ca2+ release channels: Activation of thesechannels results in Ca2+ release from SR,which contributes to elevation of cytosolicCa2+ levels, whereas Ca2+ sequestration toSR assists termination of Ca2+ signals in thecytosol. RyRs, the largest known ion channels,are responsible for Ca2+ release from thesarco/endoplasmic reticulum and they alsoplay a central role in the regulation ofcytosolic and SR luminal Ca2+ levels. At leastthree isoforms are expressed in mammals,RyR1-3, being RyR1 the predominant isoformexpressed in the skeletal muscle. RyR2 andRyR3 are expressed in cardiac and immatureskeletal muscle, respectively (Refs. 49, 50, 51,52). RyR1 is essential for the E–C coupling inthe skeletal muscle and its gating is strictlycontrolled by DHPR activation. In fact, Ca2+

release through RyR1 is the key determinantof muscle force since skeletal musclecontraction depends on Ca2+ released fromthe SR (Ref. 7). Cytosolic Ca2+ activates RyR1in the low micromolar, but acts as an inhibitorin the low milimolar range. In addition,luminal Ca2+ levels are also able to modulateRyR1 activity, since previous reports showthat high luminal Ca2+ levels enhance RyR1responsiveness to cytosolic agonist (Ref. 49).

Ca2+ release through RyR1 is alsomodulatedby post-translational modifications and a vastvariety of proteins and small molecules, bothin the SR lumen and cytosol. In addition toCa2+ and DHPR channels, other positiveregulators of RyR1 activity in the cytosol areATP, CaMKII and PKA. Calstabin-1 stabilisesthe closed stage of the channel andcalmodulin has a biphasic effect on RyR1,functioning as an activator at the low cytosolicCa2+ levels and as and inhibitor at the highcytosolic Ca2+ levels. On the luminal side,RyR1 interacts with the CSQ–junctin–triadincomplex, which plays a dual role as luminalCa2+ sensor and RyR activity modulator.

RyRs can be phosphorylated by severalkinases, including CaMKII and PKA. A recentstudy has implicated PKA phosphorylation ofRyR1 in skeletal muscle enhancement ofcontractile force (Ref. 53). However, PKAhyper-phosphorylation of RyR1 might lead to




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SR Ca2+ leak under prolonged pathologicalstress (Refs. 54, 55). Finally, RyR1 activity canalso be modulated by oxidative stress throughmodifications of cysteine thiol residues, suchas S-nitrosylation and S-glutathionylation.Although the effect of RyR1 oxidativemodifications may vary depending on whichresidue is modified, exposure of skeletalmuscle to nitric oxide has been reported toincrease RyR1 activity (Refs. 49, 51).Interestingly, RyRs have been found to releaseCa2+ spontaneously under conditions ofluminal Ca2+ overload in a process known asSOICR or Store Overload-Induced Ca2+

Release (Refs. 56, 57). This mechanism hasbeen recently linked to cardiac and musculardisorders, such as malignant hyperthermia orcatecholaminergic polymorphic ventriculartachycardia (Ref. 58).

The other major SR Ca2+ release channels arethe inositol 1,4,5-triphosphate receptors (IP3Rs).These Ca2+ channels are gated by the combinedbinding of IP3 and Ca2+, which initiates a slowwave of internal store Ca2+ release that oftenoccurs at specific subcellular locations(Ref. 59). Whereas in myoblasts IP3Rs have amajor role regulating Ca2+ homeostasis duringskeletal muscle development, the roles thatIP3Rs may play in adult skeletal muscle arecontroversial. A recent study has not foundany evidence of IP3R affecting global Ca2+

levels in adult mouse skeletal muscle (Ref. 60).Conversely, a previous work supports a role ofIP3Rs mainly at the neuromuscular junction(Ref. 61), and recent evidence indicates thatcrosstalk between IP3R1 and RyR1 underliesactivation of stress-induced Ca2+ sparks inadult mice skeletal muscle (Ref. 62).

(c) Ca2+-ATPase pumps: Ca2+ reuptake into the SRafter contraction relies on the Ca2+ ATPasepump SERCA, which actively transportsCa2+ from the cytosol to the SR against alarge concentration gradient at the expenseof ATP hydrolysis. Three genes localised ondifferent chromosomes encode for SERCA1,2 and 3 proteins, although isoform diversityis further increased by alternative splicing.Skeletal fast muscle fibres express SERCA1aexclusively, while SERCA2a is expressed inslow fibres and cardiac muscle. Duringdevelopment or regeneration, muscle fibresexpress the neonatal isoform SERCA1b (Refs.

23, 25). SERCA pumps are highly affected bychanges in cell energetics and ATP supply,since they are, together with myosinATPases, major ATP consumers (Ref. 63).SERCA pumps are also a substantialpathway for SR Ca2+ leakage and thisleakage is increased by high cytosolic ADPlevels (Ref. 64). Low levels of freeintraluminal Ca2+ are, thus, necessary sothat Ca2+ leakage does not metabolicallycompromise muscle function (Ref. 43).In addition to ATP, SERCA activity is

modulated by several cytosolic and luminalproteins, and also by post-translationalmodifications. The endogenous inhibitoryproteins phospholamban and sarcolipinmodulate SERCA activity in slow and fast-twitch fibres, respectively (Ref. 65).Phosphorylation by PKA or CaMKII, results inSERCA activation by dissociation ofphospholamban and sarcolipin from the Ca2+

pump. N-glycosylation and other post-translational modifications such asglutathiolation have been found to affectSERCA activity in different ways: SERCAactivity can be reversibly increased byoxidative modifications under normal or mildoxidative stress levels, but high oxidant levelspresent in some pathological conditions mayfurther lead to irreversible SERCAinactivation and degradation (Refs. 66, 67).This particularly susceptibility of SERCA tooxidative and nitrative modifications has ledto the hypothesis of SERCA functioning as asensor of cellular stress (Ref. 68). Moreover,these data suggest a central role of SERCA inenergy production and consumption throughregulation of Ca2+ homeostasis.

Ca2+ in mitochondriaMitochondria are located in close proximity to theSR in the skeletal muscle, and they account forabout 15% of the cytosolic volume in oxidativefibres (Refs. 69, 70). There is a significant interplaybetween the mitochondria and the SR, throughthe mitochondria-associated SR membrane(MAM), which is a structural element essential forcell physiology and Ca2+ homeostasis (Refs. 71,72). Besides playing a central role in musclebioenergetics, mitochondria are also able to storeCa2+ transiently and therefore, they contributesignificantly to Ca2+ homeostasis in the musclefibre. In fact, their ability to rapidly uptake Ca2+




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for later release in response to different stimulimakes them very good cytosolic Ca2+ buffers(Ref. 73). Ca2+ fluxes between mitochondria andSR are believed to help shape mitochondrialmetabolism and ATP synthesis to thephysiological demands of skeletal fibres (Ref. 74).At the same time, mitochondria have been foundto modulate intracellular Ca2+ transients inskeletal muscle during E–C coupling (Ref. 75).The outer mitochondrial membrane is permeableto Ca2+, unlike the inner mitochondrialmembrane. Influx into the mitochondrial matrix isdriven by the negative membrane potential andoccurs through two types of importers: the low-affinity mitochondrial Ca2+ uniporter (MCU)(Ref. 76) and a high-affinity mitochondrial Ca2+/H+ exchanger, LETM1. In skeletal muscle fibres,Ca2+ release from mitochondria can occur via aNa+/Ca2+ exchange protein (NCLX) or via Ca2+-induced–Ca2+-release pathways (Ref. 76). Also, athigh matrix Ca2+ levels mitochondria releaseCa2+ through the permeability transition pore(PTP), a mechanism that can lead to cell deathand might have an important role in musculardystrophies (Refs. 77, 78). Interestingly, inhumans, mutations in the mitochondrial calciumuptake 1, a regulator of MCU, causesdysfunctional Ca2+ uptake and results in clinicaland pathological features that overlap with thoseof mitochondrial myopathies, congenital coremyopathies and muscular dystrophies (Ref. 79).

Calcium dysregulation in musculardystrophies

In this section, we will discuss current knowledgeregarding Ca2+ homeostasis in dystrophinopathies,calpainopathies and myotonic musculardystrophies. The main Ca2+-related pathwaysalteredinthesepathologiesaresummarisedinTable1.

DystrophinopathiesDuchenne muscular dystrophy (DMD) is aninherited X-linked neuromuscular disease thataffects 1 in 3500 male births and it is characterisedby severe and progressive skeletal muscledegeneration, weakness and premature deathcaused by respiratory failure and cardiacdysfunction (Ref. 80). DMD results from deficiencyin the structural protein dystrophin (Ref. 81), a427 kDa protein located beneath the sarcolemmawhose major role is to protect and maintainmuscle fibre integrity by linking intracellularcytoskeletal actin to the extracellular matrix. This

is achieved through its association to a group ofplasma membrane glycoproteins known as thedystrophin–glycoprotein complex (DGC) (Ref. 82).Dystrophin is essential for sarcolemmal stability,and thus dystrophin-deficient muscle fibres areprone to endure recurrent membrane damage(Ref. 83). This sarcolemma fragility, however, doesnot fully explain the onset and progression ofDMD and, up to date, the exact cause of musclefibre death still remains unsolved. Thepathological events most widely accepted to occurin DMD muscle fibres as a result of dystrophindeficiency include membrane fragility andincreased basal intracellular Ca2+ levels, whichleads to calpain activation, protein degradation,mitochondrial PTP opening and, ultimately, fibredeath by necrosis (Refs. 9, 11, 13, 84, 85, 86).Studies performed more than 30 years ago inmuscle biopsies (Ref. 85) and later on, in humanfoetuses and premature infants (Ref. 87), showedincreased Ca2+ accumulation in prenecrotic DMDfibres, which suggests that Ca2+ dysregulation isan early event in the pathophysiology of thisdisorder. Afterwards, many other studies havedemonstrated increased intracellular Ca2+ levelsin dystrophic muscles from DMD mouse models(mdx mice) and Duchenne patients compared tonormal muscles (Refs. 13, 84, 88, 89). Increasedintracellular Ca2+ levels observed in dystrophin-deficient fibres is a complex process that involves,at least, trans-sarcolemmal Ca2+ fluxes, SR Ca2+

leakage and abnormal SR Ca2+ levels.High cytosolic Ca2+ levels may result from

enhanced Ca2+ influx through the dystrophin-deficient sarcolemma (Refs. 90, 91). This abnormalinflux seems to occur mostly through TRPCchannels, a type of mechanosensitive voltage-independent Ca2+ channels with increasedexpression in dystrophic muscle fibres (Ref. 40).Indeed, several groups have found that blockageof TRPC channels in dystrophic mice reduces theabnormal extracellular Ca2+ influx. Furthermore,overexpression of TRPC3 channels in normalmouse skeletal muscle enhances Ca2+ influx andresults in a phenotype that closely resemblesDGC-lacking dystrophic mice (Ref. 86). TRPCchannels can function as both, stretch-activatedand store-operated channels, and therefore theyare also involved in SOCE response, an influx ofextracellular Ca2+ triggered by SR depletion ofCa2+ that is enhanced in dystrophic fibres(Ref. 39). An increased SOCE associated withTRPC1 channels has been reported in dystrophic




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Table

1.Calcium

-related

mec

hanism

saffected

inmus

culardys

trophies

Disea

seProtein

affected

Sub

cellu

lar

loca

lisation

Propose

dmec

hanism

Sys

tem

Referen

ces

Duc

henn

e(D

MD)

Dystrop

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lemma-

cytosk

eleton

Abse

nceca

uses

mem

brane

frag

ility

and

tran

sarcolem

mal

Ca2

+en

try

Mou

semdxan

dhu

man

DMD

myo

tubes

90,9

1

TRPC

chan

nels

Sarco

lemma

Increa

sedex

pressionca

uses

high

Ca2

+

entry(SOCE)

mdxfib

res,

tran

sgen

icmice,

mou

semyo

tubes

39,4

0,86

,107

NCX

Sarco

lemma

Misfunc

tion:

reve

rsal

offunc

tionca

uses

high

Ca2

+en

try

human

myo

tubes

–rat

spinal

cord

cocu

ltures

93

Orai1

Sarco

lemma

Increa

sedex

pressionca

uses

increa

sed

SOCE

mdxmus

cles

96

RyR

1SRtriad,

tran

smem

brane

Misfunc

tionca

uses

SRCa2

+leak

ageto

thecy

toso

lmdxmus

cles

101

IP3R

SRtran

smem

brane

Increa

sedleve

ls,inc

reas

edac

tivity

caus

eshigh

cytoso

licCa2

+.A

lsoinvo

lved

inmito

chon

dria

ldefec

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mdxan

dDMD

myo

tubes

C.e

lega

ns,z

ebrafis

h

105,

106,

107,

110,

111

SERCA1

SRne

twork,

tran

smem

brane

Red

uced

activ

ity(con

trov

ersial)

mdxmus

cles

117,

118,

165

CSQ1,

CSQ-like

proteins

SRlumen

Red

uced

expressionresu

ltsin

low

SR

Ca2

+leve

lsmdxmus

cle

119,

120

sarcalum

enin

SRlumen

Red

uced

expressionresu

ltsin

low

SR

Ca2

+leve

lsmdxfib

res

121

–Mito

chon

dria

Calcium

overload

induc

esap

optosis

Dramatic

frag

men

tatio

nea

rlyin

the

patho

logy

mdxmyo

tubes

C.e

lega

ns,z

ebrafis

h10

9,11

0,11

1

(con

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Table

1.Calcium

-related

mec

hanism

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(con

tinued

)

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141,

142

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triad

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mRNAsp

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and

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cles

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149,

150,

153

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153

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fibres)

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uced

protein

expressionin

DM1an

dDM2mus

cles

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+

conc

entration

DM1an

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153

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ytos

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dDM2mus

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dDM2

mus

cles

153




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fibres, and remarkably, it can be restored to normallevels by inducing expression of minidystrophin, asmaller and partially functional dystrophin variant(Ref.92).Finally,enhancedCa2+ influxindystrophicfibres has also been reported to occur by reversalNCX sarcolemmal channels (Ref. 93) and also bythe Orai1/Stim SOCE pathway. In addition toTRPC channels, other proteins may also beresponsible for increased SOCE in DMD, such asstromal interaction molecule 1 (STIM1), whichacts as an SR Ca2+ sensor that can trigger SOCEthrough both TRPCs and Orai1 channels (Ref. 94).In this regard, it has been recently reportedincreased Orai1 (Refs. 95, 96) and STIM1 (Ref. 95)expression in dystrophic muscles from mdx mice.This indicates that enhanced activation of SOCEpathways may contribute to the disrupted Ca2+

homeostasis in DMD pathology. Several otherproteins have been involved in this enhancedSOCE activity in dystrophin-deficient musclemyotubes, such as phospholipase C, proteinkinase C (Ref. 97), phospholipase A2 (iPLA2) andits derived metabolites (Ref. 98). In this regard,increased iPLA2 activity has been known for morethan 10 years in DMDmuscles (Ref. 99). Therefore,specific inhibitors of these pathways may be ofinterest to reduce Ca2+ influx and subsequentdegeneration of dystrophic muscle (Ref. 100).Another source of elevated cytosolic Ca2+ levels

may involve SR Ca2+ leakage through RyRs orIP3Rs. Regarding RyRs, several studies havepointed to abnormally high S-nitrosylation ofRyR1 cysteine residues in mdx mice causingcalstabin-1 depletion from the RyR complex, thatlead to unstable and leaking RyR1 channels atresting conditions (Refs. 101, 102, 103). Theseabnormal S-nitrosylation levels seem to be due toinducible nitric oxide synthase (iNOS), which wasalso found increased in dystrophic mdx muscles.A recent study has found evidence of significant

Ca2+ leakage through IP3Rs as well as throughRyRs in dystrophic mdx myotubes contributingto the increased intracellular Ca2+ levels(Ref. 104), which suggests that IP3R malfunctionmay also be involved in DMD Ca2+

mishandling. Indeed, previous studies haveshown increased IP3R and IP3 levels, andenhanced IP3R-dependent Ca2+ transients inmyotubes from DMD patients and mdx mice(Refs. 105, 106, 107). Moreover, inducedexpression of minidystrophin in dystrophin-deficient myotubes reverts the IP3R-dependentincrease in Ca2+ release to normal levels

(Ref. 108), which suggests an IP3R involvementin DMD impaired Ca2+ homeostasis. Finally,there seems to be a connection between IP3Rand mitochondria that may play a relevant rolein Ca2+ dysregulation. Previous work in mdxdystrophic myotubes has shown mitochondrialCa2+ overload due to increased SR Ca2+ release(Ref. 109). Interestingly, overexpression of Bcl-2antiapoptotic protein in mdx myotubes preventsthis mitochondrial Ca2+ overload by inhibitingIP3Rs (Ref. 110). More recently, abnormalmitochondrial fragmentation has been observedin Caenorhabditis elegans and zebrafish models ofDMD, where involvement of cytochrome c inmuscle fibre death was demonstrated, acting inpart through an interaction with IP3R (Ref. 111).

In DMD and in many other neuromusculardisorders such as sarcoglinopathies, desminopathies,LGMD2B muscular dystrophy and Pompedisease, the cardiac and respiratory muscles areaffected and, consequently, there is an increasedrisk of respiratory and cardiac failure inthese patients that leads to early mortality(Ref. 84). Abnormal Ca2+ homeostasis andmitochondrial dysfunction have also beenreported in these muscles. In particular,previous works in mdx cardiac myocytes haveshown that stretch-activate channels contributeto abnormal Ca2+ influx (Ref. 112), andexcessive cytosolic Ca2+ signals induced bymechanical stress result in mitochondrialdysfunction (Ref. 113). More recently, highermitochondrial Ca2+ levels have been reported incardiac dystrophic myocytes and activation ofDHPR seems to contribute to abnormalmitochondrial Ca2+ handling (Ref. 114).Evidence for early mitochondrial dysfunction indystrophic cardiomyopathy is further supportedby alterations in mitochondrial citric acid cycle-related parameters observed in ex vivo mdxhearts preceding overt cardiomyopathy (Ref. 115).

Another controversial issue is whether SERCAexpression or function is reduced, unchanged orincreased in dystrophin-deficient fibres, withrecent examples in the literature supporting alldifferent possibilities (Refs. 96, 116, 117, 118).Regardless the need for further studies toestablish a clear understanding on this subject,the majority of the studies agree with a reducedSERCA activity in mdx muscles. In this line,nitrative stress could be causing a reducedSERCA activity in dystrophic fibres, sinceSERCA is highly sensitive to reactive nitrogen




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species (Ref. 68) and high iNOS levels have beenreported in mdx fibres (Ref. 101).Ca2+ binding proteins and decreased SR Ca2+

buffering have also been involved in thepathology of dystrophinopathies. In particular,in the SR lumen, reduction of CSQ1, CSQ-likeproteins and sarcalumenin has been observed inmdx dystrophic muscles (Ref. 119, 120, 121). Asignificant reduction of the SR Ca2+ bufferingwould have a direct negative impact on theCa2+ reuptake capacity of SERCA, which inturn, would result in the increased cytosolicCa2+ levels. Remarkably, less affected musclesin the mdx muscular dystrophy mouse model,such as the extraocular and intrinsic laryngealmuscles, exhibit higher expression levels ofseveral Ca2+-handling proteins such as CSQ,calmodulin and/or SERCA (Refs. 122, 123),which suggests that these proteins are playing aprotective role in DMD pathology.

CalpainopathiesLGMDs are a large group of hereditary musculardystrophies characterisedbyprogressiveproximalweakness and a predominant involvement of thescapula, pelvic girdle and trunk muscles withoutaffecting cardiac or facial muscles (Ref. 124).Among the recessive forms of LGMDs,calpainopathy or LGMD2A is the most frequentmuscular dystrophy with a prevalence of82–100% in genetically isolated populations suchas the Basque Country (Refs. 125, 126).LGMD2A is caused by mutations in the geneencoding Calpain 3 (CAPN3), a non-lysosomalcysteine protease necessary for normal musclefunction and regeneration (Refs. 127, 128). The exactpathogenic mechanisms that underlie musculardystrophic features caused by mutations in CAPN3are still unclear, and in view of the lack of effectivetherapies available for LGMD2A patients,understanding these pathological mechanisms isneeded in order to advance in the design ofpotential treatments.In skeletal muscle CAPN3 is present in different

subcellular locations including myofibrils,membrane and cytoplasm. Most of the CAPN3is bound within the contractile filamentnetwork, where it remains in its inactive statethrough its association with titin, a giant proteinthat connects the Z line to the M line in thesarcomere (Refs. 129, 130). Absence of CAPN3from mouse and human muscles results insarcomere disorganisation (Refs. 127, 131),

which suggests that CAPN3 is involved insarcomere repair and maintenance and may alsobe acting as a sensor of its integrity andfunctionality (Ref. 129). Remarkably, sarcomerealignment is preserved in mice expressing aninactive form of CAPN3 (Capn3cs/cs mice),indicating that the structural role of CAPN3 andnot the enzymatic one is involved in sarcomericremodelling (Ref. 132).

In the membranes, CAPN3 is localised in thetriad protein complex and plays an essential rolein preserving its structure and function. CAPN3has been found to interact with RyR1 (Ref. 132),the main SR Ca2+ release channel, and regulateCa2+ release from SR (Ref. 133). CAPN3 has alsobeen found to interact with the Ca2+-handlingprotein CSQ by immunoprecipitation analysis(Ref. 132). This interaction is proposed to occurby means of connecting proteins such as triadin,since CSQ and CAPN3 are localised in differentcellular compartments. In CAPN3 knockoutmice (C3KO, Capn3-/-), reduced expression ofRyR1 is concomitant with the reduced SR Ca2+

release (Refs. 133, 134). Also, it has beenpreviously reported slower Ca2+ reuptake intoSR after SR Ca2+ depletion in CAPN3 mice,although the basis of these finding needs furtheranalysis (Ref. 134). However, loss of CAPN3proteolytic activity in Capn3cs/cs mice does notaffect Ca2+ homeostasis (Ref. 132), whichindicates that the structural role of CAPN3 isa key for maintenance of Ca2+ homeostasis.In this line, we have recently shown in acollaboration study that muscles from LGMD2Apatients with deficient CAPN3 also expressedlower RyR1 protein levels. Moreover, we founda correlation between CAPN3 and RyR1expression levels that supports a structuralfunction of CAPN3 in the preservation of thetriad protein complex (Ref. 21).

Another CAPN3-binding partner in the triadcomplex is aldolase A (AldoA) (Ref. 133), aglycolytic enzyme that may modulate Ca2+

handling in the muscle fibre through multiplemechanisms, including direct regulation of RyRactivity by intermediates and products ofglycolysis, and modulation of SERCA activitythrough local changes of glycolytically derivedATP (Ref. 135). In the skeletal muscle, AldoAdirectly interacts with RyR1 and in vitro it hasbeen shown to modulate RyR1 activity (Refs.133, 136). Interestingly, CAPN3 knockout miceshow decreased levels of AldoA in the




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sarcomere due to impaired recruitment of AldoAto the triads (Ref. 133). Remarkably, mutations inthe gene encoding AldoA result in severe skeletalmuscle damage in humans (Refs. 137, 138).Ca2+ calmodulin kinase II (CaMKII) is another

triad component that is modulated by CAPN3. Inthis regard, our group has found that CaMKIIsignalling is compromised in CAPN3 knockoutmice and, thus, we propose that impairedCa2+-mediated signalling and weakened muscleadaptation are pathogenic mechanisms likelyoperating in CAPN3-deficient muscles (Ref. 21).CaMKII is a multifunctional protein activatedby muscle contraction-induced Ca2+ increasethat regulates several Ca2+-handling proteins(Ref. 7). In slow twitch skeletal fibres, CaMKIImay increase SERCA2 activity through directphosphorylation or by phosphorylation of itsinhibitory protein phospholamban (Refs. 67,139). On the other hand, CaMKII is also able tomodulate Ca2+ release through phosphorylationof related proteins, such as triadin, whichinhibits RyR1 activity when unphosphorylated(Ref. 140). Thus, decreased CaMKII signallinglevels, comparable to the ones observed inCAPN3 deficient muscles, may result inreduction of SR Ca2+ release during contractionsas well as reduced Ca2+ reuptake by SERCA.However, this hypothesis needs verificationthrough experimental analysis.Finally, there seems to be a connection between

CAPN3 and mitochondrial function, sinceLGMD2A patients and CAPN3 knockout miceshow abnormal distribution and structure ofmitochondria in skeletal fibres (Ref. 141).Moreover, CAPN3-deficient muscles show ATPproduction deficits and increase oxidative stresslevels (Ref. 142). Accordingly, muscles withhigher percentage of slow twitch fibres, whichhave higher oxidative metabolism, are the mostaffected in CAPN3 knockout mice (Refs. 21,142). These mitochondrial abnormalities arelikely associated to a severe Ca2+ dysregulationin CAPN3-deficient muscle fibres, althoughfuture experiments are still needed in order toelucidate the pathogenic mechanisms involvedin the mitochondrial dysfunction observed inLGMD2A.

Myotonic dystrophyMyotonic dystrophy type 1 (DM1), the mostfrequent adult form of muscular dystrophy(Ref. 143), is an inherited autosomal dominant

disease caused by an unstable expansion of theCTG triplet repeat located in the 3′ untranslatedregion of DMPK gene that codes for myotonicdystrophy protein kinase. This gene is locatedin the long arm of chromosome 19 and it ispredominantly expressed in skeletal muscle(Ref. 144). DM2 form is a rare disease associatedwith a CCTG tetranucleotide repeat expansion,located in the first intron of the zinc fingerprotein 9 (ZNF9) gene in chromosome 3. Thus,both DM1 and DM2 originate from long non-coding repeat expansions, which cause a similarchronic, slowly progressing, multisystemicdisease with a dominant inheritance pattern.Expanded mRNA molecules in the cell nucleusaffect expression and/or re-allocation of RNA-binding proteins and lead to alteration in RNAprocessing (Refs. 145, 146, 147). The multi-systemic nature of both diseases is thought tobe caused by aberrant splicing affecting anumber of proteins involved in multiple cellularprocesses. It has recently been shown thatmyotonic dystrophy is associated withderegulated alternative splicing of the geneCACNA1S that encodes for DHPR alpha 1Ssubunit (DHPRα1s), a voltage sensor that playsa central role in E–C coupling (Ref. 148). In DM1and DM2, skipping of CACNA1S exon 29 isenhanced, which increases DHPRα1s channelconductance and voltage sensitivity. The long-term consequences are very likely harmful tomuscle, especially when DHPR missplicing iscombined with deregulated alternative splicingof other genes related to Ca2+-handling or E–Ccoupling such as the ones encoding chloridechannel type 1, SERCA and RyR1 (Refs. 149,150). This would likely lead to a chronic Ca2+

overload, a common scenario occurring in otherforms of muscular dystrophies. In fact, severalstudies have reported that Ca2+-signallingpathways are consistently affected in DMmuscles, DM myotubes and mouse models ofthis disease (Refs. 151, 152, 153). These findingssuggest that the combined effect of misregulatedsplicing of several genes involved in Ca2+

regulation and E–C coupling contributes tomuscle degeneration in myotonic dystrophy. Inparticular, a recent study performed on humanDM samples demonstrates abnormal expressionof mRNA and proteins involved in muscle Ca2+

release, reuptake, storage and signalling, such asjunctophilin-1 (JPH1), SERCA2, RyR1, CSQ2and DHPR (Ref. 153). In DM muscles they




Dys

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found reduced protein levels of JPH1, but notmRNA, which is consistent with a generaldysregulation of Ca2+ homeostasis since JPH1undergoes Ca2+-dependent proteolysis whenintracellular Ca2+ levels are elevated for asustained period (Refs. 154, 155). Interestingly,in most cases mRNA levels of Ca2+-handlingmolecules were found increased while theprotein levels were decreased, which suggeststhat the translational block may be one of theunderlying mechanisms of DM.

Therapeutic targeting ofmitochondria andcalcium homeostasis in muscular

dystrophiesOver the years, several pharmacological andmolecular therapies that target Ca2+ homeostasisand/or mitochondrial function have beenproposed as potential treatments for musculardystrophies (summarised in Table 2). Differentstudies have demonstrated beneficial effects ofRyR stabilisers on different animal models ofmuscular dystrophy and heart failure (Refs. 50,101, 156). In particular, treatment of mousemodels of Duchenne and LGMD2E musculardystrophy with the RyR stabiliser S107 results inreduced RyR1 nitrosylation, reversion ofcalstabin-1 depletion and an overall improvementof muscle function and exercise performance(Refs. 101, 156). Interestingly, a recent study hasdemonstrated that S107, as well as the RyRinhibitor dantrolene, synergise with antisense-mediated exon-skipping therapies and increasesmuscle function in mdx mice and human DMDmyotubes (Ref. 157). Also, several Ca2+ channelblockers such as nifedipine, diltiazem andverapamil have shown to improve musclestructure and function in dystrophic mice (Refs.158, 159), but they have failed to prove significantbeneficial effects on DMD patients in clinical trials(Ref. 160). Likewise, pharmacological ormolecular blockage of stretch-activated channels(TRPC, TRPV2) has also shown a beneficial effecton several dystrophic models (Refs. 89, 161).Angiotensin II-receptor blockers, β-blockers and

angiotensin-converting-enzyme inhibitors haveshown beneficial effects for the treatment ofcardiomyopathies in clinical trials and animalmodels (Refs. 162, 163). Interestingly, somepharmacological agents from these groups havealso been reported to increase SERCA2 expressionand/or function, and therefore they are potentialenhancers of cardiac Ca2+ homeostasis (Ref. 164).

In this line, different strategies comprisingoverexpression of SERCA1 or SERCA2 in skeletalmuscle of dystrophic mice, such as treatment withBGP-15, or AAV-mediated gene transfer haveconsistently shown a significant improvement inthe dystrophic phenotype (Refs. 117, 118, 165),which further supports that reduced SERCAactivity is a contributing factor in the pathology ofmuscular dystrophies.

Finally, several therapeutic approaches targetingmitochondrial dysfunction have been shown toimprove muscle function in different dystrophicmouse models. These include treatment withcyclosporine A and cyclophilin D inhibitors(Debio 025) for impaired mitochondrial PTPopening, treatment with pargyline for excessiveaccumulation of reactive oxygen species oroverexpression of peroxisome proliferator-activated receptor γ coactivator 1-gene α (PGC1α) for mitochondrial dysfunction in postnecroticdystrophic muscles (Ref. 166). In this line, anantioxidant that improves mitochondrialrespiratory function, idebenone, has shownprotection of cardiac and skeletal muscle in mdxmice and trends of efficacy in a Phase II studywith DMD patients (Ref. 167).

Conclusions and future addressThe full extent to which intracellular Ca2+

signalling influences the physiopathology ofprimary and secondary muscle diseases is onlybeginning to be understood. In the forthcomingyears, future technological advances, includingnew powerful Ca2+ indicators are neededin order to accelerate and target-specificCa2+-signalling pathways within cells. Somepromising genetically encoded Ca2+ indicatorsfrom invertebrates or algae are already underdevelopment and will likely provide us withnew clues to understanding the role of Ca2+ indegenerative processes. Since these indicatorsare both, incorporated at the genome level andtranslated as fluorescent proteins that modifytheir emission intensity as a function of Ca2+

levels, they can be used for long-termexperiments over days to months and they canalso be introduced into in vivo animal models totest Ca2+ activity over a lifetime (Refs. 168, 169).Furthermore, other imaging technologies suchas fluorescence resonance energy transfer-based(FRET) analysis can demonstrate Ca2+-dependent responses at the single protein level(Ref. 170). In the near future, these




Dys

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Table

2.Trea

tmen

tstargetingca

lcium

homeo

stas

isan

dmitoch

ond

rial

func

tionin

mus

culardys

trophies

Metho

dEffec

tonCa2

+ho

meo

stas

is/

mitoch

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func

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Sys

tem

Clin

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pha

rmac

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use

Commen

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Pha

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K20

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Mou

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heartfailure

Can

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ent.Preve

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onistof

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prove

sca

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andmus

clefunc

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50

S10

7(K20

1an

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r/regu

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mdxan

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b−/−

mice

Preve

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+leak

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Amelioratesmus

cular

dystrop

hicphe

notype

Enh

ance

sex

onsk

ipping

101,

156,

157

Dan

trolen

eRyR

antago

nist

mdxmou

sean

dDMD

myo

tubes

Mus

clerelaxa

nt.T

reatmen

tformaligna

nthy

perthermia

Enh

ance

sex

onsk

ipping

157

Nife

dipine

DHPRca

lcium

block

ermdxmice.

DMDclinical

trial

Antihyp

ertens

ive,

antia

nginal

Improve

sdystrop

hic

mus

clefunc

tion.

No

sign

ifica

nteffect

onDMD

clinical

trials

158,

160

Diltiaze

mVe

rapam

ilNon

-DHPRca

lcium

block

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Inhibito

rSERCA2func

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mdxmice.

DMDclinical

trial

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dystrop

hicfib

res

from

deg

eneration.

No

sign

ifica

nteffectson

DMD

clinical

trials

159,

160,

164

BGP-15

Increa

sesSERCAac

tivity

mdx,

dko

mice

Insu

linse

nsitise

rPha

rmac

olog

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erof

HSP70

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geffect

ondystrop

hicmice

118

Ena

lapril

Increa

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pression

andfunc

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mdxmice.

DMDclinical

trial

Ang

iotens

in-con

verting

enzymeinhibito

rIm

prove

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than

dreduc

esfib

rosis.

Like

lyprese

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cardiac

func

tionin

DMDpatients

162,

164

Carve

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sesSERCA2ex

pression

andfunc

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DMDclinical

trials

β-block

erLike

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cardiac

func

tionin

DMDpatients

163,

164

(con

tinue

don

next

pag

e)




Dys

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Table

2.Trea

tmen

tstargetingca

lcium

homeo

stas

isan

dmitoch

ond

rial

func

tionin

mus

culardys

trophies

(con

tinued

)

Metho

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+ho

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is/

mitoch

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func

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Sys

tem

Clin

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pha

rmac

ological

use

Commen

tsReferen

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Losa

rtan

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sesSERCA2ex

pression

andfunc

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mdxmice

Ang

iotens

inIIrece

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block

er.B

lock

sTG

F-β

sign

alling

Prese

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cardiacfunc

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Improve

sresp

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func

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162,

164

Res

veratrol

Increa

sesSERCA2ex

pression

mdxmice

Antioxidan

tSIRTac

tivator

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Ameliorateingeffect

ondystrop

hicmice

162,

164

Strep

tomyc

inBlock

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(TRPCs)

mdxmice

Amyn

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mdx,

Sgc

b−/−,

Col6a

1(−/−

)

mice

Immun

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tsIm

prove

mus

clefunc

tionin

dystrop

hicmou

semod

els

164,

166

Ideb

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eIm

prove

smito

chon

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lfun

ction

mdx,

DMD

patients

Antioxidan

tAmelioratin

geffect

ondystrop

hicmice.

Tren

dsof

effic

acyin

DMDPha

seII

clinical

stud

y

162,

167

Pargy

line

Mon

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)

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Amelioratin

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hicmice

166

Molec

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-med

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2ex

pression

andfunc

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mdx,

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Amelioratin

geffect

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hicmice

117,

165

Aden

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PV2

Red

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161

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Res

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mdxmice

Amelioratin

geffect

ondystrop

hicmice

166




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developments will contribute to the design of newexperiments with potential applications in basicscience and translational research studies(Ref. 171).The target of these research lines in the field of

muscular dystrophies should be focused in thefirst place on disorders where Ca2+ homeostasisseems to play a central role, such as inmalignant hyperthermia, central core disease orarrythmogenic cardiomyopathies (Ref. 50).Nevertheless, as awareness of Ca2+-signallingdysregulation in other muscular diseasesprogresses, such as LGMD2A, attention shouldbe addressed to specific elements of intracellularCa2+-signalling cascades in these entities. Froma therapeutical point of view, some progress inthese areas is already taking place: the DHPRblocker nifedipine and the SR leaky-channelblockers dantrolene and S-107, for instance, havebeen successfully tested in animal models ofmuscular dystrophy (Refs. 101, 156, 158). It isvery likely that whatever achievements maycome from these on-going research projects, theywill be useful for several other muscular disorderssharing similar underlying Ca2+ pathologicalmechanisms.

Financial supportResearch in our laboratories is supported by theInstituto Carlos III (A.V.-I., grant number PI11/01499), Gobierno Vasco (A.V.-I., grant numbersSAIO12-PR12BN002, 2010111089), Marie CurieInternational Reintegration Grant (A.V.-I., grantnumber IRG256512) and Fundación de EstudiosNeurológicos ILUNDAIN. I.T.-O. and G.A. arefunded by PhD fellowships from Gobierno Vasco.

Conflicts of interestNone.

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Further reading, resources and contacts

Disease pages in OMIM:

Duchenne muscular dystrophy, http://www.omim.org/entry/310200

Becker muscular dystrophy, http://www.omim.org/entry/300376

LGMD2A muscular dystrophy, http://www.omim.org/entry/253600

Myotonic dystrophy 1, http://www.omim.org/entry/160900

Myotonic dystrophy 2, http://www.omim.org/entry/602668

ClinicalTrialsGov offers a complete list of worldwide clinical trials on muscular dystrophies and other medicalconditions: http://clinicaltrials.gov/

The Muscular Dystrophy Association provides information on muscular dystrophies as well as helpful resourcesfor patients: http://mda.org/

Treat Neuromuscular Disorders (TREAT-NMD) provides ample information about muscular dystrophies and newtherapeutic developments: http://www.treat-nmd.eu/

Features associated with this article

FigureFigure 1. Representation of calcium signalling dynamics in the muscle fibre.

TablesTable 1. Calcium-related mechanisms affected in muscular dystrophies.Table 2. Treatments targeting calcium homeostasis and mitochondrial function in muscular dystrophies.

Citation details for this article

Ainara Vallejo-Illarramendi, Ivan Toral-Ojeda,Garazi Aldanondo and Adolfo López de Munain (2014) Dysregulation of calcium homeostasis inmuscular dystrophies. Expert Rev. Mol. Med. Vol. 16, e16, October 2014, doi:10.1017/erm.2014.17




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http://www.omim.org/entry/310200










http://clinicaltrials.gov/

http://clinicaltrials.gov/

http://mda.org/

http://mda.org/

http://www.treat-nmd.eu/

http://www.treat-nmd.eu/

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Dysregulation of calcium homeostasis in muscular ... · Dysregulation of calcium homeostasis in muscular dystrophies Ainara Vallejo-Illarramendi1,2,3,*, Ivan Toral-Ojeda1,2, Garazi