Characterization and temporal development of coresin a mouse model of malignant hyperthermiaSimona Boncompagnia,b, Ann E. Rossic, Massimo Micaronid,e, Susan L. Hamiltonf, Robert T. Dirksenc,Clara Franzini-Armstrongb,1,2, and Feliciano Protasia,1,2

aCentro Scienze dell’Invecchiamento, Department of Basic and Applied Medical Sciences, Interuniversity Institute of Myology, Universita Gabrieled’Annunzio, I-66013 Chieti, Italy; cDepartment of Pharmacology and Physiology, University of Rochester Medical Center, Rochester, NY 14642; dDepartmentof Cell Biology and Oncology, Telethon Electron Microscopy Core Facility, Consorzio Mario Negri Sud, I-66030 Santa Maria Imbaro (CH), Italy; eDepartmentof Molecular Biology, Institute for Molecular Bioscience, University of Queensland, Brisbane QLD 4072, Australia; fDepartment of Molecular Physiology andBiophysics, Baylor College of Medicine, Houston, TX 77030; and bDepartment of Cell and Developmental Biology, University of Pennsylvania School ofMedicine, Philadelphia, PA 19104

Contributed by Clara Franzini-Armstrong, October 21, 2009 (sent for review July 31, 2009)

Malignant hyperthermia (MH) and central core disease are relatedskeletal muscle diseases often linked to mutations in the type 1ryanodine receptor (RYR1) gene, encoding for the Ca2� releasechannel of the sarcoplasmic reticulum (SR). In humans, the Y522SRYR1 mutation is associated with malignant hyperthermia suscep-tibility (MHS) and the presence in skeletal muscle fibers of coreregions that lack mitochondria. In heterozygous Y522S knock-inmice (RYR1Y522S/WT), the mutation causes SR Ca2� leak and MHS.Here, we identified mitochondrial-deficient core regions in skeletalmuscle fibers from RYR1Y522S/WT knock-in mice and characterizedthe structural and temporal aspects involved in their formation.Mitochondrial swelling/disruption, the initial detectable structuralchange observed in young-adult RYR1Y522S/WT mice (2 months),does not occur randomly but rather is confined to discrete areastermed presumptive cores. This localized mitochondrial damage isfollowed by local disruption/loss of nearby SR and transversetubules, resulting in early cores (2–4 months) and small contracturecores characterized by extreme sarcomere shortening and lack ofmitochondria. At later stages (1 year), contracture cores are ex-tended, frequent, and accompanied by areas in which contractileelements are also severely compromised (unstructured cores).Based on these observations, we propose a possible series ofevents leading to core formation in skeletal muscle fibers ofRYR1Y522S/WT mice: Initial mitochondrial/SR disruption in confinedareas causes significant loss of local Ca2� sequestration that even-tually results in the formation of contractures and progressivedegradation of the contractile elements.

central core disease � excitation-contraction coupling � muscle disease �ryanodine receptor � mitochondria

Mutations in the sarcoplasmic reticulum (SR) Ca2� releasechannel [type 1 ryanodine receptor (RYR1)] represent

the primary cause of malignant hyperthermia (MH) (1), alife-threatening pharmacogenetic disorder affecting as many as1:2,000 individuals (2). MH crises are triggered by exposure tohalogenated anesthetics (e.g., halothane, isoflurane) and depo-larizing muscle relaxants (e.g., succinylcholine) during anesthe-sia (3). A subset of MH mutations in RYR1 also result in centralcore disease (CCD), a congenital myopathy characterized byhypotonia and proximal muscle weakness with a slow or non-progressive clinical course (4). Multi-minicore disease (MmD)has also been linked to RYR1 mutations in a small percentageof MmD families (5).

Hallmarks of CCD are the presence of amorphous coreregions devoid of mitochondria and oxidative enzyme activity,which are often centrally located, and type I (slow-twitch) fiberpredominance (6). EM of cores in biopsies from CCD patientsreveals a wide variety of structural alterations affecting thecontractile elements, SR, and transverse tubules (T-tubules)(7–11). Cores vary in the degree of damage/disruption to themyofibrils, including heavily contracted regions (contracture

cores), disorganized and damaged regions (structured cores),and regions lacking contractile filaments (unstructuredcores) (11, 12).

Because of obvious limitations in obtaining muscle samplesfrom CCD (and MmD) patients, many unanswered questionsremain regarding the mechanisms and temporal aspects of coreformation as well as the extent to which changes in musclestructure correlate with a particular RYR1 mutation. To beginto address this critical gap in knowledge, a knock-in mousecarrying a mutation in RYR1 (Y522S in human RYR1 corre-sponding to Y524S in mouse RYR1) that results in malignanthyperthermia susceptibility (MHS) with a high incidence ofcentral cores and type 1 fiber predominance in humans (13) hasbeen generated and extensively examined (14–16). HomozygousY522S knock-in mice (RYR1Y522S/Y522S) exhibit severe skeletaland muscular defects and die during embryonic development[embryonic day 17]. Heterozygous mice (RYR1Y522S/WT) areviable, reproductive, and exhibit fulminate MH-like responses toboth heat stress and halothane exposure (14, 15). At the mo-lecular level, the Y522S mutation enhances SR Ca2� leak andreleases channel sensitivity to activation by both voltage andRYR1 agonists (14, 17–19), initiating a cascade of events thatenhance oxidative and nitrosative stress (15).

Here, we identified mitochondrial-deficient core regions inskeletal muscle fibers from RYR1Y522S/WT knock-in mice andcharacterized the structural and temporal aspects involved intheir formation

ResultsMitochondrial Damage, the Earliest Detectable Alteration inRYR1Y522S/WT Mice, Is Usually Confined to Subcellular Regions TermedPresumptive Cores (as Early as 2 Months). To confirm and extendour previous description of altered mitochondrial structure inskeletal muscle of RYR1Y522S/WT mice (15), we characterizedmitochondrial alterations in f lexor digitorum brevis (FDB),extensor digitorum longus (EDL), and Soleus muscles fromWT and RYR1Y522S/WT mice using EM and electron tomog-raphy (ET) [Fig. 1 and supporting information (SI) Fig. S1].Although the majority of mitochondria in fibers from 2-month-old RYR1Y522S/WT mice exhibit a normal shape (Fig. 1 A andFig. S1 A–D), a significant fraction are swollen (Fig. S1 E–H),

Author contributions: S.B., R.T.D., C.F.-A., and F.P. designed research; S.B., A.E.R., andC.F.-A. performed research; M.M. and S.L.H. contributed new reagents/analytic tools; S.B.,R.T.D., C.F.-A., and F.P. analyzed data; and S.B., A.E.R., R.T.D., C.F.-A., and F.P. wrote thepaper.

The authors declare no conflict of interest.

1C.F-A. and F.P. contributed equally to this work.

2To whom correspondence may be addressed. E-mail: fprotasi@unich.it orarmstroc@mail.med.upenn.edu.

This article contains supporting information online at www.pnas.org/cgi/content/full/0911496106/DCSupplemental.

21996–22001 � PNAS � December 22, 2009 � vol. 106 � no. 51 www.pnas.org�cgi�doi�10.1073�pnas.0911496106

Dow

nloa

ded

by g

uest

on

May

27,

202

0

misshapen, and/or disrupted (Fig. 1B and Table S1). Impor-tantly, we report here that swollen/damaged mitochondria arenot randomly distributed throughout the fiber but rather areconfined to focal subcellular domains (Fig. 1B). We refer tothese discrete subcellular domains of mitochondrial damage aspresumptive cores. As a result, the RYR1Y522S/WT fiberscontain two populations of mitochondria: structurally normalmitochondria similar to those observed in WT fibers (Fig. 1 Aand Fig. S1 A–D) and presumptive core mitochondria showingsignificant swelling, cristae remodeling (Fig. 1 B–F and Fig. S1E–H), and irregularities of the outer membrane resulting inincreased variability of the organelle shape (Fig. 1 E and F,arrowheads). Mitochondria in muscles from RYR1Y522S/WT

mice also exhibit increased size, with mitochondria in normalregions exhibiting a significantly increased diameter relative toWT and mitochondria in presumptive core regions being evenlarger (Table S1).

In adult muscle, mitochondria are usually closely apposed tocalcium release units (CRUs), also known as triads (20), which arespecialized intracellular junctions formed by the close apposition ofT-tubules and SR. In RYR1Y522S/WT fibers, we also detected a smallbut significant increase in the average distance between mitochon-dria and triads: this increase occurs throughout the fiber but islarger in presumptive cores (Fig. S2 and Table S1).

In 2- to 4-month-old RYR1Y522S/WT mice, presumptive coresare seen in all muscle types analyzed (FDB, EDL, and Soleus),

but they are relatively infrequent (average of �2/100 �m of fiberlength in FDB muscle), they do not usually extend transverselyacross the fiber diameter (occupying 18 � 12% of the sectionedprofile of the fiber), and their size varies considerably (50–187�m2 in area in thin longitudinal sections, 3–15 sarcomeres alongthe longitudinal axis of the fiber). When more widespread andsevere forms of damage are present at later ages (e.g., 1 year),presumptive cores are not typically observed.

Severe Mitochondrial Damage Is Often Accompanied by the Formationof Focal Areas of Sarcomeric and SR/T-Tubule Disruption Termed EarlyCores (in 2–4-Month-Old RYR1Y522S/WT Mice). Individual mitochondriaand CRUs (or triads) exhibit a specific sarcomere-related disposi-tion in skeletal muscle. CRUs are usually transversely oriented,located at the junction between the A and the I band of thesarcomere (i.e., the A–I junction) and often closely associated withelongated mitochondria positioned between the triad and the Z line(20). In a small subset of presumptive cores containing severelydamaged mitochondria, sarcomere and triad structures are alsoaffected (Fig. 2). We refer to these focal regions of alteredmitochondria, myofilaments, and triads as early cores (Fig. 2 A andB). In early cores, triads are often misshapen and/or damaged andchange from mostly transverse to oblique or longitudinal in orien-tation; individual SR and T-tubule elements may be vesiculated anddilated (Fig. 2 C and D).

Confined areas of SR disorganization are also detectable at thelight microscopic level when triad position is marked with anti-RYR1 antibody (Fig. 2 E and G). In WT fibers as well as themajority of 2- to 4-month-old RYR1Y522S/WT fibers, RyR1-positivefoci (triads) are precisely arranged in double-transversely orientedrows (Fig. 2E). However, the position of triads is slightly disar-ranged in confined regions of fibers from RYR1Y522S/WT mice,presumably corresponding to early cores (Fig. 2G). Areas of alteredtriad positioning are often located adjacent to myonuclei, extendingfor approximately equal longitudinal distances from the nearestnucleus (Fig. 2G, star). Changes in T-tubule disposition from aprecise transverse orientation at the A-I junction in normal domainsand presumptive cores to an oblique orientation with displacedpositioning within early cores are emphasized by selective ‘‘stain-ing’’ for EM (Fig. 2 F and H).

In FDB fibers from 2–4-month-old RYR1Y522S/WT mice, earlycores are found in �65% of all fiber segments analyzed (3muscles and 33 segments) and are less frequent along the fiberlength than presumptive cores (average of �0.2/100 �m of fiberlength). Early cores are observed in all types of muscles exam-ined (FDB, EDL, and Soleus).

Regions of Sarcomere Shortening, or Contracture Cores, Characterizedby Sarcomere Shortening and Lack of Mitochondria and SR, Representa Secondary Stage of Core Development (3-Month-Old to 1-Year-OldRYR1Y522S/WT Mice). Confined areas of extreme sarcomere short-ening, termed contracture cores, are detected by EM and lightmicroscopy in all muscles starting at 3 months of age. Con-traction cores are relatively infrequent and small in size(occupying only few sarcomeres in length) in muscle from3-month-old RYR1Y522S/WT mice (Fig. S3 A and B) but in-crease considerably in frequency and size with age (Fig. S3C–E and Fig. 3). In fact, at 1 year, 45% and 41% of fibersegments examined from EDL and Soleus, respectively, exhibitone or more well-delimited contractures involving a largeproportion of the total segment length (46 � 36% and 51 �40%, respectively; Table S2). At 2–4 months, although deter-mination of contracture frequencies is less accurate as a resultof potentially missing very small contracture areas, �10% offiber segments were found to exhibit small, and often barelydetectable, contractures.

The transition between the shortened sarcomeres of contrac-ture cores and normal sarcomere spacing in adjacent areas, both

A

C

B

D E

F

Fig. 1. Abnormal mitochondria are confined to discrete subcellular domainstermed presumptive cores in FDB fibers from 2–4-month-old RYR1Y522S/WTmice.(A and B) Two different areas of the same muscle fiber are shown: one withapparently normal mitochondrial disposition and morphology (A, black arrows)and another, within a presumptive core region, containing swollen and structur-ally disrupted mitochondria (B, white arrows). (C) Representative tomographicsliceofanabnormalmitochondrion. (D–F)Respective3Dreconstructions inwhichshading is used to provide depth. White arrowheads in E and F point to irregu-larities (grooves and indentation) of the outer membrane.

Boncompagni et al. PNAS � December 22, 2009 � vol. 106 � no. 51 � 21997

PHYS

IOLO

GY

Dow

nloa

ded

by g

uest

on

May

27,

202

0

in series and in parallel, is remarkably abrupt in Soleus of1-year-old RYR1Y522S/WT mice (Fig. 3 and Fig. S3). Further-more, sarcomeres directly in series with contracture core regionsappear stretched to variable degrees. Long fiber segments maybe occupied either by a single long contracture or by multipleshort contractures separated by greatly overstretched sarco-meres. Contractures of the very short and discrete variety aremore prevalent in EDL than in Soleus (Fig. S3 D and E): 75 �24% vs. 41 � 4% of total contractures. A quantitative analysisof the incidence and length of contractures in EDL and Soleusmuscles from 1-year-old RYR1Y522S/WT mice is reported in TableS2. Average fiber length affected by contracted/overstretchedregions is 463 � 362 �m (n � 55 fiber segments, 4 mice) in EDLand 551 � 398 �m (n � 42, 4 mice) in Soleus.

A noticeable feature of contracture cores is the almost com-plete absence of SR, triads, and mitochondria within the inter-myofibrillar spaces (Fig. 3C), whereas these organelles arepresent in immediately adjacent regions (Fig. 3B). This is trueeven for the small discrete contractures observed in fibers from3-month-old RYR1Y522S/WT mice (Fig. S3). The abrupt transi-tion in membrane and organelle content between contracturecores and adjacent areas is emphasized at the light microscopylevel, where contracture cores coincide with sharply delimitedbands devoid of immunocytochemical markers for mitochondria(apoptosis-inducing factor) and the SR (RYR1) (Fig. 3 D–F).

Regions of Severe Damage or Loss of Structural Components, TermedUnstructured Cores, Reflect a Late Stage of Core Development (>1Year). On average, about 80–90% of EDL and Soleus fibers from1-year-old RYR1Y522S/WT mice exhibit at least one significantstructural alteration within each examined section. In addition tocontracture cores, which likely result as a progression frompresumptive and early cores, multiple areas of severe myofibriland organelle disruption are frequently observed (Fig. 4). Theseunstructured cores fall into distinct categories. Small unstruc-tured cores occupy a limited domain of the fiber between regionsof normal muscle structure (Fig. S4). They are sometimespresent at early stages and are slightly more frequent in EDLthan Soleus. Small unstructured core regions may derive directlyfrom early cores. On the other hand, some unstructured coresare much larger, occupying most of the fiber cross-sectional area,and are specifically associated with and adjacent to damaged/contracted areas (Fig. 4). Large unstructured cores are observedonly at later stages (1 year) and are also slightly more frequentin EDL than Soleus (Table S2). Specifically, severe degenerationaffecting long fiber segments occurs in 18% of EDL and 10% ofSoleus fiber segments at 1 year. Other less frequent structuralalterations include Z-line streaming in Soleus (Fig. S5A), afeature common to many muscle pathologies, including CCD(21), and inward folding of the external surface membrane inEDL (Fig. S5B), which is often associated with fiber splitting inneurogenic and myopathic disorders (22).

E G

D

A B

F H

C

Fig. 2. Severe mitochondrial damage is associated with local disruption of adjacent SR, T-tubules, and contractile elements within early cores. (A) Large openarrows point to localized areas of damage termed early cores. Black arrows, marking Z lines, highlight local sarcomeric misalignment. The area marked by theasterisk in A is enlarged in B. (B) Early core showing severely damaged mitochondria (white arrows) and disrupted membrane systems (star). (C and D) Significantdistortion and dilation of T-tubules (arrows) and misorientation of triads in two early cores. (E–H) Anti-RYR labeling (E and G) and selective T-tubule staining(F and H) are used to emphasize triad and T-tubule positioning (F, arrows). Arrows in E mark the position of Z lines. The proper positioning of triads along twotransverse bands/sarcomeres (E) and of T-tubules at the A-I junction (F) are disrupted in confined regions of fibers (early cores) from RYR1Y522S/WT mice (G andH, dashed ovals). The star in G marks the position of a nucleus.

21998 � www.pnas.org�cgi�doi�10.1073�pnas.0911496106 Boncompagni et al.

Dow

nloa

ded

by g

uest

on

May

27,

202

0

DiscussionCCD is one of the most frequent congenital myopathies in humans(23). Biopsies of CCD patient muscle represent a single snapshot ofthe disorder, typically taken well after cores and a symptomatic

myopathy have developed. Thus, analysis of clinical biopsy samplesdoes not permit determination of the complex sequence of eventsthat underlie core formation. Elucidation of the progression andmechanism of core development in CCD requires an animal modelin which these issues can be systematically studied. Here, we providea comprehensive temporal and structural characterization of coredevelopment in RYR1Y522S/WT mice, which possess an RYR1mutation linked to MHS with a high occurrence of cores in humans(13). From these findings, we propose a plausible model for thesequence of events underlying core formation in RYR1Y522S/WT

mice. Both MH and CCD are diseases with a genetically heterog-enous background; thus, the events described here may not applyto all diseases classified under this heading.

Proposed Mechanism for Core Formation in RYR1Y522S/WT Mice. InCCD patients, cores are described as delimited regions of themuscle fiber in which overall ultrastructure is damaged and mito-chondria are missing and/or nonfunctional. The structural alter-ations observed in muscles of RYR1Y522S/WT mice are also confinedto discrete portions of the fiber and involve a progression from earlymitochondrial damage to the eventual loss of mitochondria, triads,and contractile elements.

Comparison of the structural properties and prevalence of pre-sumptive, early, contracture, and unstructured cores at differentages (2–12 months) reveals a likely time course for progressive fiberdamage and core formation. Mitochondrial swelling/damage (pre-sumptive cores, Fig. 1) and subtle changes in SR-mitochondriaspacing (Fig. S2 and Table S1) are the dominant features in all threemuscles types at 2–4 months of age, and thus must be a fairlypersistent feature during that period. Early cores showing initialdecay of SR, T-tubules, and myofibrils (Fig. 2) are also present inthe initial stages, but their frequency is remarkably lower than thatobserved for either presumptive cores or contracture cores. Thisobservation could be explained by early cores being short-livedstructures if SR/T-tubule damage progresses quickly once initiated.Contracture cores and unstructured cores (Figs. 3 and 4) areinfrequent and small at 2–4 months but show a marked increasedin frequency and size with age. Thus, a logical temporal pathwaywould lead from presumptive cores, featuring initial mitochondrialdamage, to transient formation of early cores, in which neighboringorganelles become involved, and then, ultimately, to contracturecores that completely lack mitochondria and triads and have greatlyshortened sarcomeres. Unstructured cores might result from fur-ther progressive damage either within contracture cores or imme-diately adjacent to them.

A mechanism for the observed sequence of events, based onpublished observations, is proposed in Fig. 5. The Y522S mutationenhances SR Ca2� leak (17, 18), which, in turn, increases reactiveoxygen species (ROS)/reactive nitrogen species (RNS) productionand subsequent RYR1 S-nitrosylation and glutathionylation, fur-ther enhancing SR Ca2� leak and release channel heat sensitivity(15). Both Ca2� overload and increased redox stress promoteactivation of the mitochondrial permeability transition pore(mPTP), the opening of which leads to mitochondrial depolariza-tion, Ca2� overload, and swelling (24). Consistent with theseobservations, the changes in mitochondrial structure observed inpresumptive and early cores (e.g., swollen mitochondria, hypodensematrix, absent/altered cristae) are remarkably similar to thosedescribed previously for mPTP-dependent mitochondrial damagethat occurs in Ullrich congenital muscular dystrophy (25). Consid-ering that mitochondria are structurally tethered to the SR next tosites of Ca2� release (20), local Ca2� and ROS/RNS imbalanceattributable to aberrant Ca2� leak resulting from the Y522S RYR1mutation might trigger mPTP activation and subsequent Ca2�-dependent proteolysis and oxidative protein/lipid damage to themitochondria and adjacent CRU.

Severe SR/mitochondrial damage and dysfunction in earlycores would, in turn, lead to hypercontractures in these regions

A B

D

C

E F

Fig. 3. Contracture cores are frequent at 1 year of age and are characterizedby severe sarcomere shortening in areas lacking mitochondria and SR. (A–C) Alarge contracture core (lower part of the micrograph) in a Soleus fiber from a1-year-old RYR1Y522S/WT mouse (A). Contracture cores are characterized byshort sarcomere length and near-complete lack of mitochondria and SR (C),whereas both mitochondria and triads are present in apparently normaladjacent areas (B). (D–F) Immunolabeling of mitochondria (E, anti-apoptosis-inducing factor) and triads (F, anti-RYR) confirms the absence of SR andmitochondria in contracture cores (arrowheads in D, phase contrast). Otherexamples are shown in SI Text.

A B

Fig. 4. Large unstructured cores define areas of the fiber with near-completedegeneration of myofibrils and intracellular organelles. Large unstructured cores(large open arrows) are common at 1 year of age in both EDL and Soleus,although somewhat more frequent in EDL. They are often in proximity tocontracture cores (lower left corner of A).

Boncompagni et al. PNAS � December 22, 2009 � vol. 106 � no. 51 � 21999

PHYS

IOLO

GY

Dow

nloa

ded

by g

uest

on

May

27,

202

0

attributable to the combined effects of local deficiency in Ca2�

sequestration/extrusion and ATP production and diffusion ofSR Ca2� spillover from adjacent regions attributable to eitherchronic SR Ca2� leak or Ca2� release during excitation-contraction coupling (Fig. 5). The discrete separation betweencontracture core regions (where SR content is greatly reduced)and adjacent relaxed areas (where SR remains) provides astrong testament to the Ca2� sequestration powers of the SR(26). Note also that sarcomere shortening in contracture corescannot be transient in nature, because the contractures persistduring glutaraldehyde fixation, which requires several minutes.

Unstructured cores (Fig. 4) appear to represent a final stageof focal structural decay, possibly arising from the combinedeffects of extreme stretching (produced by contracture cores)and ROS/RNS-induced oxidative damage, proteolytic cleavage,and degradation of myofibrillar proteins.

Why Is Structural Damage Restricted to Discrete Regions of the Fiber?The mechanism by which structural damage in the core regionsis restricted to discrete areas of the fiber remains unresolved. Thediscontinuous nature of the structural alterations observed inRyR1Y522S/WT muscle (i.e., cores interspersed with regions ofnormal structure) would suggest that the severity of the defectis not uniform throughout the fiber, even from inception. Inaddition, the absence of detectable disruptions in the sarco-lemma and absence of signs of significant macrophage infiltra-tion or fat disposition in muscles from RYR1Y522S/WT miceindicate that the observed structural damage remains confinedwithin an otherwise healthy muscle fiber (14). It is possible thatstochastic variations in the relative level of transcription and/ortranslation of the two RYR1 alleles (RYR1WT and RYR1Y522S)within different parts of the fiber, resulting in different degreesof SR Ca2� leak across the fiber length, may contribute to the

Fig. 5. Proposed model for core formation in RYR1Y522S/WT fibers. Local mitochondrial damage within presumptive cores and concomitant disruption ofthe sarcotubular system in early cores occur in young (2– 4 months) RYR1Y522S/WT mice. At later stages (�1 year), contracture cores and unstructured coresare frequently observed. The potential contributions of increased SR Ca2� leak (14) and ROS/RNS stress (15) to the initial mitochondrial/SR damage andsubsequent formation of contracture and unstructured cores observed in muscle from RYR1Y522S/WT mice are shown in gray, because those steps remainto be demonstrated.

22000 � www.pnas.org�cgi�doi�10.1073�pnas.0911496106 Boncompagni et al.

Dow

nloa

ded

by g

uest

on

May

27,

202

0

observed spatial heterogeneity in fiber damage. Alternatively,differential susceptibility to damage downstream of Ca2� dys-regulation that depends on relative mitochondrial oxidativecapacity (27) and/or functional heterogeneity within the inter-myofibrillar mitochondrial population may also contribute to theobserved spatial variability of core formation within a singlefiber. Finally, functional heterogeneity within the intermyofi-brillar mitochondrial population may contribute to the observednonuniformity of mitochondrial structural disruption and someof the minor fiber-type specific differences reported here.

RyR1Y522S/WT Mouse Is a Powerful Animal Model for Studies of CoreDevelopment in CCD. Reports of cores in human CCD muscleinclude many characteristics of the contracture and unstructuredcores described here in skeletal muscle from RyR1Y522S/WT mice.Specifically, contracture cores that lack mitochondria; exhibitdisintegration of the contractile machinery; and exhibit alteredstructure and content of SR, T-tubules, and mitochondria havepreviously been documented in EM analyses of muscle biopsiesfrom human CCD patients (8, 10, 28). Importantly, lack ofhistological staining of oxidative enzymes in human CCD musclemay well correspond to the mitochondrial-free contracture andunstructured cores observed in muscle from RyR1Y522S/WT mice(Fig. 3). Moreover, the prevalence of a particular type of core(e.g., presumptive, early, contracture, unstructured) is also likelyto be directly related to the disease stage at which the tissue isexamined or to mutation-specific differences in the degree andform of SR Ca2� release channel dysfunction.

In summary, the findings presented here indicate thatRyR1Y522S/WT mice develop a core-like phenotype, and thusprovide a powerful animal model for mechanistic investigations

into the process of core development as well as the pathophys-iology and treatment of core myopathies.

Materials and MethodsRYR1Y522S/WT Mice. Methods used to generate RYR1Y522S/WT knock-in mice aredescribed elsewhere (14, 15).

Preparation and Analysis of Samples for Light Microscopy and EM. WT andRYR1Y522S/WT animals were killed by CO2 overdose followed by cervical dislo-cation (protocols approved by the University Committee on Animal Resources,University of Rochester). FDB muscles were fixed in situ; EDL and Soleusmuscles were dissected, pinned on Sylgard (Dow Corning), and then fixed inglutaraldehyde. Postfixation, embedding, sectioning, and staining for lightmicroscopy and EM followed standard protocols (20). For histology, 1-�mthick sections were sequentially stained with 1% toluidine blue O and 1%(vol/vol) sodium borate tetra for 3 min at 55–60 °C and with 1% basic fuchsinin 50% ethanol for 1 min at room temperature.

Quantitative Analysis. Random EM images were collected for the followingmeasurements: (i) surface areas of mitochondrial profiles in the fiber interiorat a magnification of �17,700, (ii) SR-mitochondrial distances at a magnifica-tion of �105,000 , and (iii) counts of presumptive cores at a magnification of�8,100. Longitudinal sections �1 �m thick from two randomly selected tissueblocks for each sample were visually scored for the presence of contractureareas and measurement of fiber segment length under a light microscope.

ET and 3D Reconstructions. ET was performed on 250-nm thick sections usingeither 5- or 10-nm gold particles as fiduciary markers (20). Tilt series werecollected automatically at 120 kV accelerating voltage and at 1° incrementsover an angular range of �65° to �65° on an FEI Tecnai-12 microscope usingXplore3D software (FEI).

ACKNOWLEDGMENTS. We acknowledge the Telethon Electron MicroscopyCore Facility, Consorzio Mario Negri Sud, Santa Maria Imbaro (CH), Italy. Thisstudy was supported by National Institutes of Health Grants AR044657 (toR.T.D.), AR053349 (to R.T.D. and S.L.H.), and 5P01AR052354 (to R.T.D., S.L.H.,and C.F.A.) and by Telethon Research Grant GGP08153 (to F.P.).

1. Galli L, et al. (2002) Mutations in the RYR1 gene in Italian patients at risk for malignanthyperthermia: Evidence for a cluster of novel mutations in the C-terminal region. CellCalcium 32:143–151.

2. Monnier N, et al. (2002) Presence of two different genetic traits in malignant hyper-thermia families: Implication for genetic analysis, diagnosis, and incidence of malig-nant hyperthermia susceptibility. Anesthesiology 97:1067–1074.

3. MacLennan DH, Phillips MS (1992) Malignant hyperthermia. Science 256:789–794.4. Magee KR, Shy GM (1956) A new congenital non-progressive myopathy. Brain 79:610–

621.5. Jungbluth H (2007) Multi-minicore disease. Orphanet J Rare Dis 2:31.6. Dubowitz V, Pearse AG (1960) Oxidative enzymes and phosphorylase in central-core

disease of muscle. Lancet 2:23–24.7. Engel WK, Foster JB, Hughes BP, Huxley HE, Mahler R (1961) Central core disease—An

investigation of a rare muscle cell abnormality. Brain 84:167–185.8. Hayashi K, Miller RG, Brownell AK (1989) Central core disease: Ultrastructure of the

sarcoplasmic reticulum and T-tubules. Muscle Nerve 12:95–102.9. Dubowitz V, Roy S (1970) Central core disease of muscle: Clinical, histochemical and

electron microscopic studies of an affected mother and child. Brain 93:133–146.10. Isaacs H, Heffron JJ, Badenhorst M (1975) Central core disease. A correlated genetic,

histochemical, ultramicroscopic, and biochemical study. J Neurol Neurosurg Psychiatry38:1177–1186.

11. Neville HE, Brook MH (1973) Central core fibers: Structured and unstructured. Pro-ceedings of the Second International Congress on Muscle Diseases, ed Kakukas B(Excerpta Medica, Amsterdam), Part 1, pp 497–511.

12. Gonatas NK, et al. (1965) Central ‘‘core’’ disease of skeletal muscle. Ultrastructural andcytochemical observations in two cases. Am J Pathol 47:503–524.

13. Quane KA, et al. (1994) Mutation screening of the RYR1 gene in malignant hyperther-mia: Detection of a novel Tyr to Ser mutation in a pedigree with associated centralcores. Genomics 23:236–239.

14. Chelu MG, et al. (2006) Heat- and anesthesia-induced malignant hyperthermia in anRyR1 knock-in mouse. FASEB J 20:329–330.

15. Durham WJ, et al. (2008) RyR1 S-nitrosylation underlies environmental heat stroke andsudden death in Y522S RyR1 knockin mice. Cell 133:53–65.

16. Andronache Z, Hamilton SL, Dirksen RT, Melzer W (2009) A retrograde signal from RyR1alters DHP receptor inactivation and limits window Ca2� release in muscle fibers ofY522S RyR1 knock-in mice. Proc Natl Acad Sci USA 106:4531–4536.

17. Avila G, Dirksen RT (2001) Functional effects of central core disease mutations in the cytoplas-mic region of the skeletal muscle ryanodine receptor. J Gen Physiol 118:277–290.

18. Dirksen RT, Avila G (2002) Altered ryanodine receptor function in central core disease:Leaky or uncoupled Ca(2�) release channels? Trends Cardiovasc Med 12:189–197.

19. Dirksen RT, Avila G (2004) Distinct effects on Ca2� handling caused by malignanthyperthermia and central core disease mutations in RyR1. Biophys J 87:3193–3204.

20. Boncompagni S, et al. (2009) Mitochondria are linked to calcium stores in striated muscleby developmentally regulated tethering structures. Mol Biol Cell 20:1058–1067.

21. Carpenter S, Karpati G (1984) in Pathology of Skeletal Muscle (Churchill Livingstone,New York), pp 121–129.

22. Schwartz MS, Sargeant M, Swash M (1976) Longitudinal fibre splitting in neurogenicmuscular disorders—Its relation to the pathogenesis of ‘‘myopathic’’ change. Brain99:617–636.

23. Shuaib A, Paasuke RT, Brownell KW (1987) Central core disease. Clinical features in 13patients. Medicine (Baltimore) 66:389–396.

24. Brookes PS, Yoon Y, Robotham JL, Anders MW, Sheu SS (2004) Calcium, ATP, and ROS:A mitochondrial love-hate triangle. Am J Physiol 287:C817–C833.

25. Palma E, et al. (2009) Genetic ablation of cyclophilin D rescues mitochondrial defectsand prevents muscle apoptosis in collagen VI myopathic mice. Hum Mol Genet18:2024–2031.

26. Podolsky RJ, Costantin LL (1964) Regulation by calcium of the contraction and relax-ation of muscle fibers. Fed Proc 23:933–939.

27. Philippi M, Sillau AH (1994) Oxidative capacity distribution in skeletal muscle fibers ofthe rat. J Exp Biol 189:1–11.

28. Sewry CA, et al. (2002) The spectrum of pathology in central core disease. NeuromusculDisord 12:930–938.

Boncompagni et al. PNAS � December 22, 2009 � vol. 106 � no. 51 � 22001

PHYS

IOLO

GY

Dow

nloa

ded

by g

uest

on

May

27,

202

0