Gene Dose Influences Cellular and Calcium Channel Dysregulation in Heterozygous and Homozygous T4826I-RYR1 Malignant Hyperthermia-susceptible Muscle

Gene Dose Influences Cellular and Calcium ChannelDysregulation in Heterozygous and HomozygousT4826I-RYR1 Malignant Hyperthermia-susceptible Muscle*□S

Received for publication, September 26, 2011, and in revised form, November 16, 2011 Published, JBC Papers in Press, December 2, 2011, DOI 10.1074/jbc.M111.307926

Genaro C. Barrientos‡1, Wei Feng‡1, Kim Truong‡, Klaus I. Matthaei§, Tianzhong Yang¶, Paul D. Allen¶,José R. Lopez¶, and Isaac N. Pessah‡2

From the ‡Department of Molecular Biosciences, School of Veterinary Medicine, University of California, Davis, California 95616,the §John Curtin School of Medical Research, Australian National University, Canberra, ACT 0200, Australia, and the ¶Departmentof Anesthesiology, Brigham and Women’s Hospital, Boston, Massachusetts 02115

Background:Muscle fromheterozygous and homozygous T4826I-RYR1MH-susceptiblemice is investigated for biochem-ical and cellular abnormalities.Results:T4826I-RYR1 gene dose determines severity of [Ca2�]rest, mitochondrial, EC coupling, andCa2� channel impairments.Conclusion: T4826I-RYR1 channel dysfunction is regulated in vivo but imparts susceptibility to environmental triggers.Significance: T4826I-RYR1 is sufficient to confer MHS strongly dependent on gene dose.

Malignant hyperthermia susceptibility (MHS) is primarilyconferred by mutations within ryanodine receptor type 1(RYR1). Here we address how theMHSmutation T4826I withinthe S4-S5 linker influences excitation-contraction coupling andresting myoplasmic Ca2� concentration ([Ca2�]rest) in flexordigitorum brevis (FDB) and vastus lateralis prepared fromheterozygous (Het) and homozygous (Hom) T4826I-RYR1knock-in mice (Yuen, B. T., Boncompagni, S., Feng, W., Yang,T., Lopez, J. R., Matthaei, K. I., Goth, S. R., Protasi, F., Franzini-Armstrong, C., Allen, P. D., and Pessah, I. N. (2011) FASEB J.doi:22131268). FDB responses to electrical stimuli and acutehalothane (0.1%, v/v) exposure showed a rank order of Hom >>Het >> WT. Release of Ca2� from the sarcoplasmic reticulumand Ca2� entry contributed to halothane-triggered increases in[Ca2�]rest in Hom FDBs and elicited pronounced Ca2� oscilla-tions in �30% of FDBs tested. Genotype contributed signifi-cantly elevated [Ca2�]rest (Hom > Het > WT) measured in vivousing ion-selective microelectrodes. Het and Hom oxygen con-sumption ratesmeasured in intactmyotubes using the SeahorseBioscience (Billerica,MA) flux analyzer andmitochondrial con-tent measured with MitoTracker were lower thanWT, whereastotal cellular calpain activity was higher thanWT.Musclemem-branes did not differ in RYR1 expression nor in Ser2844 phosphor-ylation among the genotypes. Single channel analysis showedhighly divergent gating behavior with Hom and WT favoringopen and closed states, respectively, whereas Het exhibited het-erogeneous gating behaviors. [3H]Ryanodine binding analysisrevealed a gene dose influence on binding density and regula-tion by Ca2�, Mg2�, and temperature. Pronounced abnormali-

ties inherent in T4826I-RYR1 channels confer MHS and pro-mote basal disturbances of excitation-contraction coupling,[Ca2�]rest, and oxygen consumption rates. Considering thatboth Het and Hom T4826I-RYR1 mice are viable, the remarka-ble isolated single channel dysfunction mediated through thismutation in S4-S5 cytoplasmic linker must be highly regulatedin vivo.

Fulminant malignant hyperthermia (MH)3 is a pharmacoge-netic, life-threatening syndrome triggered in susceptible indi-viduals by volatile general anesthetics, depolarizing neuromus-cular blocking agents, and heat stress (2). An MH episode ischaracterized by increased expired CO2, rapid onset of meta-bolic acidosis, elevated core temperature, and sustainedmusclecontraction. Such episodes are often associated with ventricu-lar tachycardia and cardiac arrest. The prevalence of MH hasbeen estimated as 1 in 10,000 anesthetic procedures; however,this is likely to be an underestimate of MH susceptibility in thegeneral population, which has been estimated as high as 1:2,000(3–5).Although seven genomic loci have been linked to the disor-

der (6, 7), mutations in only two skeletal muscle proteins havebeen confirmed to conferMH susceptibility. At least 250muta-tions within the RYR1 locus (19q13.1) that encodes for the type1 ryanodine receptor (RYR1), a Ca2� channel that localizeswithin skeletal muscle junctional sarcoplasmic reticulum (SR),have been associated with human MH susceptibility (4, 8–10).RYR1 mutations currently account for more than 50% of thefamilies identified (4). More recently, a small number of muta-tions in CACNA1S (1q32) that encode for the pore-forming

* This work was supported, in whole or in part, by National Institutes of HealthGrants 1P01 AR52354 and 3R01 AR043140 (to P. D. A. and I. N. P.) and 1R01ES014901 (to I. N. P.). This work was also supported by a grant from the J. B.Johnson Foundation.

□S This article contains supplemental Movie 1.1 Both authors contributed equally to this work.2 To whom correspondence should be addressed: Molecular Biosciences, Uni-

versity of California Davis, One Shields Ave., Davis, CA 95616. Tel.: 530-752-6696; Fax: 530-752-4698; E-mail: [email protected].

3 The abbreviations used are: MH, malignant hyperthermia; MHS, MH sus-ceptibility; SR, sarcoplasmic reticulum; EC, excitation-contraction; Het,heterozygous; Hom, homozygous; OCR, oxygen consumption rate; FCCP,carbonyl cyanide p-trifluoromethoxyphenylhydrazone; t-Boc, tert-Boc-L-leucyl-L-methionine amide; Ry, ryanodine; [Ca2�]rest, resting Ca2� concen-tration; RC, respiratory capacity; PP1, protein phosphatase 1; DHPR, dihy-dropyridine receptor; MHS, BLM, bilayer lipid membrane.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 287, NO. 4, pp. 2863–2876, January 20, 2012© 2012 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A.

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subunit (CaV1.1; �1sDHPR) of the L-type voltage-dependentCa2� channels that localize within the skeletal muscle T-tubulemembrane were also confirmed to confer MH susceptibility.Confirmed cases include three families with R1086H (11, 12),one family with R1086S (13), and another with the R174Wmutation (14). One theory currently being tested is that MHmutations in either CaV1.1 or RYR1 alter the fidelity of bidirec-tional signaling across T-tubule SR junctions that is essentialfor normal skeletal muscle excitation-contraction (EC) cou-pling (15) and regulation of SR Ca2� leak (16).

Knock-in mice heterozygous (Het) for missense mutationR163C-RYR1 (17) or Y522S-RYR1 (18), two of the more com-mon mutations conferring MH susceptibility in humans,exhibit fulminant MH when exposed to either an inhaled vola-tile general anesthetic (e.g. halothane) or heat stress. Homozy-gous (Hom)R163C-RYR1 andY522S-RYR1mice are not viable,whereas their Het counterparts maintain MH susceptibilitythroughout a normal life span. Both mouse models have con-tributed valuable information about howN-terminalmutationsaffect basal RYR1 channel dysfunction and alter pharmacolog-ical responses of intact muscle cells (19, 20). Y522S-RYR1miceshow temporal development of skeletal muscle lesions resem-bling central core disease in humans (21), whereas Het R163C-RYR1 mice appear to have minimal muscle pathology.4 Theseobservations in mice are not consistent with clinical evidenceindicating that both analogous mutations cause MH suscepti-bility in humans and that both are associated with central coredisease, although the onset and patterns of muscle damage candiffer (7, 22). Importantly muscle cells expressing either RYR1mutation led to three fundamentally important findings aboutMH susceptibility: 1) both show evidence of altered patterns ofbidirectional signaling between CaV1.1 and RYR1, with activa-tion of L-type Ca2� current shifted to more negative potentials(23–25); 2) both have chronically elevated cytoplasmic restingCa2� measured both in vitro (26) and in vivo (19, 27); and 3)both have basal alterations in mitochondrial functions thatincrease production of reactive oxygen species (20, 28). WhyY522S-RYR1 and R163C promote different patterns of skeletalmuscle damage is not understood.Recently, we completed phenotyping a new MHS mouse

expressing a mutation within the C-terminal region of RYR1,T4826I-RYR1 (1), a mutation first described in a New ZealandMaori pedigree withMH susceptibility but no clinical evidenceof central core disease (29). T4826I-RYR1 mice have severalnotable phenotypic differences comparedwithmice expressingN-terminal mutations. Both Het and Hom T4826I-RYR1 micesurvive to maturity and showmarked genotype and gender dif-ferences in susceptibility to triggered MH with halothaneand/or heat stress throughout their life span. Electron micro-graphic assessment of soleus indicates late onset alterations,including abnormally distributed and enlarged mitochondria,deeply infolded sarcolemma, and frequent z-line streamingregions, which are more severe in males (1). Collectively, thesedata indicate that the location of a mutation within the RYR1sequence influences not only the penetrance and gender

dependence of MH susceptibility but also the patterns of basalchanges inmuscle bioenergetics and the progression and extentof muscle damage.Here we report a detailed analysis of basal functional differ-

ences in EC coupling and resting cytoplasmic Ca2� that conferheightened sensitivity to halothane in single FDB fibers pre-pared from Het and Hom T4826I-RYR1 knock-in mice andhow these differences relate to inherent biochemical and bio-physical alterations in RYR1 Ca2� channels. We also find thatintactmyotubes derived from either Het orHomT4826I-RYR1have altered oxygen consumption rates when compared withWT.

EXPERIMENTAL PROCEDURES

Animals—WT, Het, and Hom skeletal muscle tissue, FDBfibers, and myotubes were obtained from Het � Het crosses ofthe T4826I-RYR1 knock-in mouse line (back-bred at least 10generations to theC57BL/6 line) as described previously (1). Allcellular and biochemical experiments were performed from tis-sues collected from mice whose genotype was determined byPCR screening. The PCR primer sequences were as follows:T4826I-RYR1 (forward), TTT GGA GAC ACG GAA ACAGAA; T4826I-RYR1 (reverse), AGG GAG GTA CCT GGCACT CA; WT-RYR1 (forward), TCT CAC TGT CCA TAGCTG; WT-RYR1 (reverse), ATC CAG CTT CTC CTA CAG.All experiments on animals and collections of animal tissues forthe studies were conducted using protocols approved by theinstitutional animal care and use committees at the Universityof California at Davis.Preparation of Primary Myotubes and Adult FDB Fibers—

Primary skeletal myoblast lines were isolated from newbornWT mice or mice verified Het or Hom for T4826I-RYR1 asdescribed previously (30–32). Themyoblasts were expanded in10-cm cell culture-treated Corning dishes coated with collagen(Calbiochem) and were plated onto 96-well �-clear plates(Greiner) coated with Matrigel (BD Biosciences) for detectionof calpain or mitochondrial content or were alternativelyseeded onto proprietary 12-well plates for measuring oxygenconsumption (Seahorse Biosciences). Upon reaching �80%confluence, growth factors were withdrawn, and the cells wereallowed to differentiate into myotubes over a period of 3 daysfor these analyses (see below).Flexor digitorum brevis (FDB) muscles were dissected from

male 3–6-month-oldmice verified by PCR asWT, Het or HomT4826I-RYR1 genotype. Single intactmyofibers were enzymat-ically isolated as described previously (19, 33). In order toreduce stress-activated SR Ca2� release during isolation, espe-cially in T4826I-RYR1 fibers, 10 �M dantrolene was included inthe initial dissociation medium. After isolation, the fibers wereplated on Matrigel-coated plates (BD Biosciences) and main-tained in Dulbecco’s modified Eagle’s medium (Invitrogen)supplementedwith 10% fetal bovine serum (ThermoFisher Sci-entific, Waltham, MA) and 0.1 mg/ml penicillin-streptomycin(Sigma) in the absence of dantrolene. Fibers were kept over-night in a 5% CO2 incubator, and experiments were conductedwithin 12–24 h of plating.Ca2� Imaging—FDB fibers were loaded with Fluo-4/AM (10

�M; 40 min at room temperature) in normal Ringer solution4 C. Franzini-Armstrong, personal communication.

Functional Abnormalities in Het and Hom T4826I-RYR1 MHS Muscle

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containing 146 mM NaCl, 4.7 mM KCl, 0.6 mM MgSO4, 6 mM

glucose, 25mMHEPES, 2mMCaCl2, and 0.02%Pluronic�F-127(Invitrogen). The cells were thenwashed three timeswith Ring-er’s solution and transferred to the stage of an IX71 invertedmicroscope equippedwith a 40� 0.9 numerical aperture objec-tive (Olympus, Center Valley, PA) and illuminated at 494 nm toexcite Fluo-4 with a DeltaRam wavelength-selectable lightsource. Fluorescence emission at 510 nm was captured fromindividual fibers. Electrical field stimuli were applied using twoplatinum electrodes fixed to opposite sides of the well and con-nected to an A.M.P.I. Master 8 stimulator set at 4 V, 0.5-msbipolar pulse duration over a range of frequencies (1–20 Hz;10-s pulse train duration). Fluo-4 fluorescence emission wasmeasured at 30 frames/s using a Cascade Evolve 512 camera(Photometrics, Tucson, AZ). The images were acquired usingthe Easy Ratio Pro software (PTI). The datawere analyzed usingOrigin 7 software (OriginLab Corp.). Transients were normal-ized to the florescence base line (Fo) of each individual fiber, andthe integrated area within the evoked responses was calculatedfrom the number of fibers indicated in the figure legend. Statis-tical comparisons were performed with an unpaired t test.In Vivo Recording of Vm and [Ca2�]rest—Measurements were

performed on mice sedated with non-triggering ketamine/xy-lazine (100/5 mg/kg), and core temperature was maintainedeuthermic with an automated heating system (ATC1000WPI).Once anesthesia was confirmed by a loss of tail pinch response,micewere intubatedwith a tracheal cannula and connected to aventilator (Harvard Minivent, M-845, Holliston, MA) set at astroke volume of 200 �l, 180 stokes/min, and ventilated withmedical air. Small incisions were made to expose the vastuslateralis muscle of the left leg, and muscle fibers were impaledwith the double-barreled microelectrode as described previ-ously (19). Potentials were recorded via a high impedanceamplifier (WPI FD-223, Sarasota, FL). The potential from the3 M KCl barrel (Vm) was subtracted electronically fromVCaE, toproduce a differential Ca2�-specific potential (VCa) that repre-sents the [Ca2�]rest. Vm and VCa were filtered (30–50 kHz) toimprove the signal/noise ratio and stored in a computer forfurther analysis.Halothane Exposure—Dissociated fibers were perfused with

0.1% (v/v) halothane freshly prepared in the imaging solution.The halothane concentration was confirmed by mass spec-trometry (23).Seahorse XF-24 Metabolic Flux Analysis—Myoblasts were

cultured on Matrigel-coated Seahorse XF-24 plates (SeahorseBiosciences) at a density of 30,000 cells/well using the sameprocedure described above for Ca2� imaging experiments.The oxygen consumption rate (OCR) and extracellular acidifi-cation rate were evaluated after 3 days of differentiation. Beforethe experiment, themyotubeswere equilibrated inDMEMsup-plemented with 1 mM pyruvate and 1 mM GlutaMAX (runningmedium) during 1 h at 37 °C. The oligomycin (10 �g/ml finalconcentration), FCCP (1�M final concentration), and rotenone(0.1 �M final concentration) were dissolved in the runningmedium. The respiratory capacity is defined like the OCR afterthe FCCP addition. To compare the respiratory capacity amongthe three genotypes, we calculated the area under the curve.Values were normalized using the protein content of each well.

Mitochondria Content—Mitochondria content was quanti-fied by staining myotubes with MitoTracker Green (Invitro-gen), which preferentially accumulates inmitochondria regard-less of the of the mitochondrial membrane potential andprovides an accurate assessment of total mitochondrial mass(34, 35). Briefly, myotubes were loaded with 100 nM Mito-Tracker Green for 30 min at 37 °C. The myotubes weretrypsinized and washed by centrifugation, and the associatedfluorescence was measured (excitation/emission 516/490 nm).The fluorescence values were normalized using the proteinconcentration.CalpainActivity—The peptidase activity was evaluated using

the synthetic substrate tert-Boc-L-leucyl-L-methionine amide(t-Boc) (Invitrogen) (36). Skeletal myotubes were loaded with10 �M t-Boc for 30 min at 37 °C. After cleavage by peptidases,the product produces blue fluorescence with excitation andemissionmaxima of�351 and 430 nm, respectively. In order tocompare the fluorescence among the different genotypes, weperformed the assay using the Quantum View feature of anEvolve 512 digital camera, permitting electron counting of eachpixel in the image. Measurements were captured from regionsof interest of identical size for each myotube, and the intensityvalues were averaged for each genotype under identical illumi-nation conditions on the same day.Preparations of Membrane Fractions from Mouse Skeletal

Muscle—Skeletal muscles collected frommaleWT-RYR1, Het,or Hom T4826I-RYR1 mice (2–3 animals/preparation, 3–6months of age) were either prepared from freshly isolated tissueor tissue flash-frozen and stored in liquid nitrogen and stored at�80 °C. Fresh tissue was minced on ice (frozen tissue was pul-verized) and placed in ice-cold buffer containing 300 mM

sucrose, 5 mM imidazole, 0.1 mM PMSF, and 10 �g/ml leupep-tin, pH 7.4, and homogenized with three sequential bursts (30 seach) of a PowerGen 700D (Fisher), at 9,000, 18,000, and 18,000rpm. Homogenates were centrifuged at 10,000 � g for 20 min.Supernatants were saved, whereas the pellets were subjected toa second round of homogenization and centrifugation at thesame settings described above. The remaining pellets werediscarded, and the supernatants were combined and pouredthrough four layers of cheesecloth. The filtrate was centri-fuged at 110,000 � g for 60 min at 4 °C. Pellets were resus-pended in 300 mM sucrose, 10 mM Hepes, pH 7.4, aliquotedinto microcentrifuge tubes (100 �l/sample), and eitherstored at �80 °C for biochemical analyses or subjected tofurther purification to obtain membranes enriched in junc-tional SR as described previously by Saito et al. (37). Proteinconcentration for each preparation was determined usingthe DC protein assay kit (Bio-Rad).Measurements of [3H]Ryanodine Binding—The apparent

association or equilibrium binding of [3H]ryanodine ([3H]Ry)to RYR1-enriched membrane preparation (0.1–0.15 mg/ml)was measured at 25 or 37 °C for 0–3 h with constant shaking inbuffer consisting of 2–5 nM [3H]Ry (PerkinElmer Life Sciences),250 mM KCl, 20 mM HEPES, pH 7.4 (38), and defined free[Ca2�] as indicated in each specific experiment. Free Ca2� wasobtained by the addition of EGTA calculated according to thesoftware Bound-and-Determined (39). RYR1 channel modula-tors Ca2� and/orMg2� were titrated in specific experiments as



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described in the figure legends.Nonspecific [3H]Ry bindingwasdetermined in the presence of a 1,000-fold excess of unlabeledryanodine. Bound and free ligand were separated by rapid fil-tration throughWhatman GF/B glass fiber filters using a Bran-del cell harvester (Whatman, Gaithersburg, MD) with threewasheswith 5ml of ice-cold buffer (250mMKCl, 20mMHEPES,15mMNaCl, and 50�MCa2�, pH 7.4). [3H]Ry retained in filterswas quantified by liquid scintillation spectrometry using a scin-tillation counter (Beckman model 6500). Each experiment wasperformed on at least two independent skeletal muscle prepa-rations, each in triplicate. Linear or non-linear curve fitting wasperformed using Origin� software (Northampton, MA).Measurements and Analyses of Single RYR1 Channels in

Bilayer Lipid Membrane (BLM)—Skeletal muscle membranepreparation was used in order to induce fusion with the planarBLM. BLM was formed by 30 mg/ml phosphatidylethanol-amine/phosphatidylserine/phosphatidylcholine in decane (5:3:2(w/w/w); Avanti Polar Lipids, Inc., Alabaster, AL). A 10-foldCs� gradient was built from cis to trans (500 to 50 mM). Therecording baths were buffered to pH 7.4 by 20 mM Hepes. Theholding potential was held at �40 mV by bilayer clamp BC525C (Warner Instruments, Hampden, CT) on the trans side,where cis was virtually grounded. The cis chamber was wheremembrane protein was added and thus was actually the cytoso-lic face of the incorporated channel. The acquired current sig-nals, filtered at 1 kHz (low pass Bessel Filter 8 Pole; WarnerInstruments) were digitized and acquired at a sampling rate of10 kHz (Digidata 1320A, Molecular Devices (Sunnyvale, CA)).All current recordings were made with Axoscope 10 software(Molecular Devices) for at least 1 min under each definedexperimental condition. The channel open probability (Po),mean open and closed dwell times (�o and �c), and current his-tograms were calculated and obtained using Clampfit, pClampsoftware version 10.0 (Molecular Devices). The number ofchannels recorded under each condition was specified in therespective figure legends. Differences in the WT-RYR1, Het,and HomT4826I-RYR1 Po values were tested for statistical sig-nificance using unpaired Student’s t tests.Western Blotting—Skeletal muscle (genders either pooled or

separated by genotype) was homogenized, and crude mem-brane fractions were prepared and denatured in SDS-PAGEsample buffer (Bio-Rad) containing 5% 2-mercaptoethanol at80 °C for 5 min. Protein (5, 10, or 15 �g/lane) was loaded ontoTris acetate 4–12% or 7% acrylamide gradient SDS-polyacryl-amide gels (Invitrogen), electrophoresed, blotted onto PVDF,labeled using secondary antibodies conjugated to infrared dyes(emission 700 or 800 nm), and imaged using a LI-COR imageras described previously (19). Total RYR1 was detected withantibody 34C (40) at 1:1,000 dilution (Developmental StudiesHybridoma Bank, University of Iowa, Iowa City). Phospho-epitope-specific antibody that recognizes mouse RYR1 Ser2844,a PKA phosphorylation site (41), was purchased from Abcam(ab59225) and used at 1:2,000 dilution. Band intensities ofRYR1 were normalized to glyceraldehyde 3-phosphate dehy-drogenase (GAPDH) as a measure of total RYR1 protein, andintensity of the Ser(P)2844-RYR1/total RYR1 ratio was analyzedusing Odyssey version 3.0 software. Protein phosphatase 1(PP1; NewEngland Biolabs) was utilized to dephosphorylate SR

protein according to the vendor’s instructions. The reactionmixture contained 50 mM HEPES, 100 mM NaCl, 2 mM DTT, 1mM MnCl2, 0.01% Brij 35, pH 7.5, 4 mg/ml membrane protein(WT, Het, and Hom T4826I-RYR1), and 200 units/ml PP1,which was incubated at 30 °C for 10 min. Then the sampleswere diluted into SDS-PAGE sample buffer for Western blot-ting or into [3H]Ry binding buffer. [3H]Ry binding assay wasperformed with 5 nM [3H]Ry, 1 �M free Ca2�, 100 �g/ml pro-teins in binding buffer containing 250 mM KCl and 20 mM

Hepes, pH 7.4, at 37 °C for 3 h.

RESULTS

T4826I-RYR1 FDB Fibers Display Enhanced Ca2� TransientProperties and Heightened Sensitivity to Halothane—Differ-ences in EC coupling in FDB fibers isolated frommaleWTmiceand knock-in Het or Hom T4826I-RYR1 were measured byimaging electrically evoked Ca2� transients with Fluo-4. FDBfibers were sequentially stimulated at frequencies ranging from1 to 20Hz, and themagnitude of the Ca2� transients was quan-tified as the integrated area of the response. Fig. 1A shows rep-resentative traces of electrically evoked Ca2� transients mea-sured inWT and HomT4826I-RYR1myotubes superimposed.

FIGURE 1. T4826I knock-in fibers display an enhanced frequencyresponse compared with the WT fibers. Fibers dissociated from WT-RYR1,Het T4826I-RYR1, or Hom T4826I-RYR1 mice were loaded with Fluo-4 andtested for electrically evoked EC coupling in the Ca2�-replete external buffer([Ca2�]e � 2 mM). A, representative responses of FDB to electrical pulse trainsapplied at 1–20 Hz for a 10-s duration isolated from WT (black trace) and HomT4826I-RYR1 (gray trace) mice. For clarity, the Het T4826I-RYR1 trace is notsuperimposed on the traces. B, summary data for all three genotypes relatingintegrated areas of the Ca2� transient at each stimulus frequency. WT-RYR1results are mean data � S.E. from n � 162 fibers from five animals; Het resultsare from n � 180 fibers from three animals, and Hom results are from n � 203fibers from four animals. ***, p � 0.001 compared with WT. **, p � 0.01 com-pared with WT. *, p � 0.05 compared with WT.



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Compared with WT-RYR1, both Het and Hom T4826I-RYR1FDB fibers consistently displayed accentuated responses to a10-s pulse train of electrical stimuli �10 Hz. Hom, but not Het,exhibited significantly larger Ca2� transients with pulses deliv-ered at 20 Hz (Fig. 1B).Fig. 2 shows three representative traces from each WT (A),

Het (B), and Hom (C) FDB fiber responding to electrical pulsetrains and subsequent responses to perfusion of 0.1% halo-

thane. Although perfusion of halothane had negligible influ-ence on [Ca2�]rest inWT, the anesthetic induced a pronouncedrise in [Ca2�]rest in Het (92% responded, n � 39 fibers tested)and a much larger rise in Hom (100% response, n � 31 fiberstested) T4826I-RYR1 FDBs. The massive rise in [Ca2�]restobservedwithHomT4826I-RYR1 fibers exposed to continuoushalothane perfusion was invariably transitory and returnedtoward the base line, whereas the rise in resting Ca2� was

FIGURE 2. Het and Hom T4826I-RYR1 FDB fibers show heightened sensitivity to halothane. Fibers dissociated from WT-RYR1 (A), Het T4826I-RYR1 (B), orHom T4826I-RYR1 (C) mice were loaded with Fluo-4 and tested for electrically evoked EC coupling in the Ca2� replete external buffer ([Ca2�]e � 2 mM). Fiberswere then challenged with halothane dissolved in the same external buffer ([Ca2�]e � 2 mM) in the absence of electrical stimuli. A–C, traces from individualfibers from each genotype. Similar results were obtained from 39 and 28 Het and Hom fibers, respectively, from three independent fiber isolations for eachgenotype. D, halothane can produce marked elevation in base-line Ca2� and regenerative Ca2� waves that were observed in eight of 28 Hom T4826I-RYR1fibers tested. Oscillations abated, and the base line gradually was restored upon removal of halothane by perfusion. Halothane-triggered oscillations were notobserved in fibers isolated from either WT-RYR1 or Het T4826I-RYR1 mice.



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slower and sustained with Het T4826I-RYR1 fibers. In 29% ofthe Hom T4826I-RYR1 fibers (eight fibers), halothane perfu-sion initially induced a massive Ca2� rise that was followed byregenerative Ca2� waves having a mean � S.D. frequency of3.7 � 2.1 min�1 (Fig. 2D and supplemental Movie 1). NeitherWT nor Het T4826I-RYR1 FDBs showed oscillatory behaviorin response to halothane (not shown). In the absence of halo-thane, spontaneous Ca2� waves were not observed in 180 Hetand 203 Hom T4826I fibers analyzed.To further evaluate the contribution of Ca2� entry to halo-

thane-triggered Ca2� dysregulation, we first verified the integ-rity of electrically evoked EC coupling of Hom T4826I-RYR1FDB fibers in the external solution repletewithCa2� ([Ca2�]e � 2mM) and then exchanged the external solution with [Ca2�]e �0.1 mM in the presence of 0.1% halothane (Fig. 3A). Fig. 3Bshows that halothane-triggered peak elevation of myoplasmicresting Ca2� is attenuated�3.5-fold in [Ca2�]e � 0.1mM, indi-cating that significant Ca2� entry contributes to the halothaneresponse in resting Hom T4826I-RYR1 FDB fibers.Chronically Elevated Cytoplasmic [Ca2�]rest in Adult

T4826I-RYR1 Fibers—We previously reported that RYR null1B5 myotubes transduced with T4826I-RYR1 cDNA results insignificantly elevated [Ca2�]rest compared with WT (42). Herewemeasured [Ca2�]rest in the vastus lateralis of ketamine/xyla-zine-anesthetized WT, Het, and Hom T4826I-RYR1 mice(results from both sexes combined). Compared with WTmus-cles, in vivo microelectrode measurements showed that myo-plasmic [Ca2�]rest was 2.4- and 3.0-fold higher thanWT in Hetand Hom T4826I-RYR1 muscle fibers (mean � S.D. [Ca2�]rest:114 � 3.8, 278 � 21, and 337 � 20 nM, respectively, from n �20–34 fibers/genotype; p � 0.001 among the three genotypes).In contrast to [Ca2�]rest, the corresponding Vm of these vastuslateralis fibers did not differ among the three genotypes (WT��82 � 1.3; n � 20 from two mice; Het � �82 � 1.4; n � 34from five mice; Hom � �82 � 1.5; n � 25 from three mice).

Collectively, these data indicate that adult FDB fibers isolatedfromHet andHomT4826I-RYR1mice exhibit heightened sen-sitivity to halothane, whose magnitude is a function of genedose, consistent with their relative sensitivities to triggeringfulminantMH in vivo (1).Moreover, bothMH-susceptible gen-otypes have two basal abnormalities: 1) enhanced gain of EC

coupling and 2) chronically elevated [Ca2�]rest. We thereforeinvestigated if chronic dysregulation of cellular Ca2� dynamicsdue to the T4826I-RYR1 mutation in the absence of triggeringagents affected mitochondrial bioenergetics and calpain activ-ity, biomarkers known to mediate muscle damage.Reduced Mitochondrial Respiration and Higher Calpain

Activities in Resting T4826I-RYR1 Myotubes—We tested ifT4826I-RYR1 mutations influenced OCR and respiratorycapacity (RC) in intact muscle cells using a Seahorse XF-24metabolic flux analyzer. Technical challenges, including incon-sistent plating density, precluded making these measurementson FDB fibers; therefore, primary myotubes were used as analternative. Myotubes from all three genotypes were plated onseparate wells of a Seahorse plate at a density of 30,000 cells/well and differentiated into myotubes for 3 days, at which timeOCR and RC were measured. In parallel cultures, MitoTrackerwas used to measure mitochondrial mass. Fig. 4A shows a rep-resentative OCR experiment performed on the three geno-types. The OCR values are expressed as a percentage of therespective base lines (before the addition of oligomycin). BothHet and HomT4826I-RYR1myotubes displayed a significantlylower basal OCR (p � 0.001) compared withWT (Fig. 4B), andbasal OCR was significantly lower with Hom compared withHet T4826I-RYR1myotubes (p � 0.0167). In order to compareRC across the three genotypes (OCR values after FCCP injec-tion), the results were expressed as area under the curve (cor-rected for non-mitochondrial rotenone-insensitive OCR). Themean � S.E. respiratory capacities of Het and Hom T4826I-RYR1 myotubes were significantly lower than that of WT (Fig.4C). Interestingly, the Hom T4826I-RYR1 RC was higher thanthat of Het (p � 0.01).MitoTracker green, a dye shown to bind mitochondria

regardless of membrane potential and a quantitative methodfor measuring total mitochondrial mass (34, 35), was used totest if the lower RC of the myotubes expressing T4826I-RyR1was associated with altered mitochondrial content. Fig. 4Dshows that Het and Hom T4826I-RYR1 myotubes have signif-icantly lower mitochondrial mass than WT myotubes (83.0 �1.9 and 67.5 � 6.6%, respectively).t-BOC fluorescence was used to determine if chronically ele-

vated myoplasmic [Ca2�]rest levels could promote calpain

FIGURE 3. [Ca2�]e contributes to halothane-triggered peak Ca2� rise in Hom T4826I-RYR1 FDB. Fibers dissociated from WT-RYR1 and Hom T4826I-RYR1mice were loaded with Fluo-4 and tested for electrically evoked EC coupling in Ca2�-replete external buffer ([Ca2�]e � 2 mM). Fibers were then challenged withhalothane in the same external buffer whose [Ca2�]e was reduced with EGTA ([Ca2�]e � 0.1 mM). A, representative traces from two individual Hom T4826I-RYR1fibers. B, comparison of the halothane-triggered peak Ca2� amplitude (mean � S.E. (error bars)) for Hom T4826I-RYR1 fibers with [Ca2�]e � 2 mM (0.9 � 0.2; n �8) and [Ca2�]e � 0.1 mM (3.7 � 0.7; n � 4) (p � 0.001).



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activities associated with muscle damage and degeneration(43). Fig. 5 (left) shows representative micrographs, and bargraphs (Fig. 5, right) show that both Het and Hom T4826I-RYR1 myotubes have elevated calpain activities that are 170and 145% of WT (mean t-BOC fluorescence of 100 myotubes/genotype normalized to WT).

Expression and Phosphorylation of T4826I-RYR1 ChannelsDo Not Account for Increased [3H]Ry Binding Capacity—Theinfluence of PKA phosphorylation of RyR1 on channel activityhas been debated in the literature (19, 41, 44, 45). Our recentstudy of R163C-RYR1 isolated from Het mouse muscle indi-cated higher Ser2844 phosphorylation (�31%) compared with

FIGURE 4. Myotubes isolated from T4826I-RYR1 mice have impaired oxygen consumption and respiratory capacity and lower mitochondrial content.The basal OCR and RC were evaluated before and after sequential injection of oligomycin, FCCP, and rotenone into the wells of a Seahorse Biosciences fluxanalyzer. WT, Het, and Hom 4826I-RYR1 myoblasts were plated at 30,000 cells/well and differentiated to myotubes 3 days prior to measuring OCR and RC. Arepresentative experiment is shown in A, where OCR was measured simultaneously from the three genotypes before and after sequential injection ofmitochondrial inhibitors (oligomycin (O), FCCP (F), and rotenone (R)). Each trace represents the mean � S.E. (error bars) OCR of 5 wells/genotype. The valueswere normalized to basal OCR (before oligomycin injection). B, mean � S.E. OCR obtained from 25 wells from four independent experiments, indicating thatHet and Hom T4826I-RYR1 myotubes have significantly lower basal OCR and RC compared with WT myotubes. The mean � S.E. basal OCR values were Het �81.4 � 3.2 (p � 0.01) and Hom � 70.8 � 3.1% (p � 0.01) of WT. C, RC measured as OCR after injecting FCCP to uncouple electron transport (after FCCP in A). Themean � S.E. RC values were significantly lower in Het and Hom T4826I-RYR1 compared with WT (59.8 � 11.3% (p � 0.034) and 64.8 � 8.7% (p � 0.026),respectively). D, intact myotubes were loaded with 100 nM MitoTracker Green, and the cellular fluorescence was quantitatively measured as described under“Experimental Procedures.” The fluorescence values were normalized to protein concentration. Each bar represents the mean � S.E. of n � 4 independentexperiments, each performed with five replicates. **, p � 0.01; *, p � 0.05.

FIGURE 5. T4826I mutants show enhanced intracellular calpain activity. A, myotube peptidase activity was measured quantitatively using the syntheticsubstrate t-BOC, which is cleaved by intracellular calpain to a fluorescent product using Quantview as described under “Experimental Procedures.” B, calpainactivity summarized, where each bar represents the mean � S.E. (error bars) of 100 myotubes/genotype relative to WT (***, p � 0.001).



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WT-RYR1, although dephosphorylationwith protein phospha-tase did not restore R163C-RYR1 channel behavior to thatobserved with WT-RYR1 channels (19). We therefore meas-ured the level of total RYR1 expression and the degree of phos-phorylation of Ser2844 (Ser(P)2844) in whole membrane frac-tions from skeletal muscle prepared from WT, Het, and HomT4826I-RYR1. Fig. 6A shows results from a typical Westernblot of skeletal muscle membranes probed with monoclonalantibody 34C that recognizes RYR1, indicating that no signifi-cant differences were detected in the level of RYR1 proteinexpression (normalized to GAPDH) among the three geno-types, regardless of whether male and female muscle sampleswere pooled or separated prior to preparing membranes forWestern blotting (Fig. 6A, left). Blots were probed with both

34C and an antibody that recognizes Ser(P)2844, which showedthat unlike in muscles expressing R163C-RYR1, there were nosignificant differences among the three genotypes in the degreeto which RYR1 was phosphorylated (Fig. 6, B (top) and C (barplot of densitometry); n � 16 blots from five differentpreparations).Het and Hom T4826I-RYR1 preparations display a signifi-

cantly higher capacity to specifically bind [3H]Ry than WT-RYR1 preparations under defined assay conditions (Fig. 6B,bottom), which is likely to reflect their inherently higher openprobabilities (see below). Although muscle preparations fromT4826I-RYR1 mice show similar levels of total RYR1 proteinand Ser(P)2844 as those fromWT, we determined if the level ofphosphorylation influences the capacity to bind [3H]Ry to agreater extent in T4826I-RYR1 than WT by exposing mem-brane preparations to PP1 (see “Experimental Procedures”).Western blotting with and without PP1-treated membranesshowed nearly complete dephosphorylation of Ser(P)2844 in allthree genotypes (Fig. 6B, top), although the level of [3H]Rybinding remained unchanged regardless of the degree of phos-phorylation (Fig. 6B, bottom).Het and Hom T4826I-RYR1 Channels Have Inherently

Higher Open Probability than WT—WT-RYR1, Het, or HomT4826-RYR1 channels were incorporated into BLMby inducedfusion of SR vesicles. In the presence of 1 �M free cytosolic (cis)Ca2�, 2mMNa2ATP, and 100�M free luminal (trans) Ca2�, thesingle channel activity was recorded at a holding potential of�40 mV (applied to trans). Fig. 7A shows representative cur-rent traces from a WT-RYR1 (top trace), a Het (middle trace),and a Hom (bottom trace) channel. Compared withWT-RYR1,the Het T4826I-RYR1 channel exhibited 9-fold higher Po, with2.5-fold greater �o and �6-fold shortened �o. Hom T4826-RYR1 channels demonstrated even greater deviations com-pared with WT-RYR1. For example, the Hom T4826-RYR1shown in Fig. 7A displayed�15-fold higher Po,�5-fold greater�o, and 10.5-fold shortened �o compared with WT-RYR1.

As shown in Fig. 7, B andC, channels reconstituted fromHetT4826I-RYR1 mice produced more heterogeneous gatingbehavior with a broadly scattered Po, ranging from 0.19 to 0.71(mean Po � 0.41; n � 5; statistical analysis was not done due tolimited data points). This behavior would be expected fromrandom association of WT- and T4826I-RYR1 monomers intofunctional tetramers as recently described for channels isolatedfrom Het R163C mice (19). Hom T4826-RYR1 channelsshowed invariantly high Po behavior (mean Po � 0.82; n � 10),which was significantly different from WT-RYR1 (mean Po �0.10; n � 12; p � 0.0001).

Analyses of current amplitude distributions from represen-tative channels reconstituted from each genotype are shown inFig. 8. The most frequent transitions of WT-RYR1 were cen-tered about zero current, the closed channel state (top). Inmarked contrast, the Hom T4826I-RYR1 channel (bottom)exhibited most transitions centered about the maximal unitarycurrent amplitude, the full open channel state. Het T4826I-RYR1 channels exhibited an intermediate and broader currentamplitude distribution compared with WT-RYR1 and HomT4826I-RYR1 channels (Fig. 8,middle).

FIGURE 6. No significant differences in total RYR1 expression or phospho-rylation of Ser2844-RYR1 in preparations from WT, Het, or Hom T4826I-RYR1 mice. A, representative Western blot showing the expression of RYR1probed with monoclonal 34C (total RYR1; green channel). The bar graphshows mean � S.E. (error bars) densitometry results for n � 17 blots fromseven membrane preparations where muscle was pooled from males andfemales. No differences among the three genotypes for total RYR1 proteinexpression were detected. In separate preparations, skeletal muscle frommales and females were collected, and membranes prepared and blottedseparately. Bar graphs show mean � S.E. densitometry results for n � 6 blotsfrom two separate membrane preparations. No differences between genderor among genotypes for total RYR1 protein expression were detected. B, rep-resentative Western blot showing Ser(P)2844-RYR1 levels before and aftertreatment with PP1. The bottom panel summarizes the amount of specific[3H]Ry binding to muscle membranes isolated from WT, Het, and Hom T4826Imice with and without PP1 treatment. PP1 effectively dephosphorylatesSer(P)2844-RYR1 but has no effect on [3H]Ry binding levels, which remain sig-nificantly higher in preparations from Het and Hom mutants (also see Fig. 9).C, densitometry shows that the relative levels of Ser(P)2844-RYR1/total RYR1did not significantly differ among the three genotypes (mean � S.E. for n � 16blots from five membrane preparations) or between genders (mean � S.E. forn � 6 blots from two membrane preparations).



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T4826I-RYR1 Exhibits Altered Sensitivities to Regulation byCa2� and Mg2�—Ca2� and Mg2� are physiological modula-tors of RYR1 channel activity (46–48). Using [3H]Ry as a probeto RYR1 channel conformational status, we investigated howthe T4826I-RYR1 mutation alters Ca2� regulation of [3H]Rybinding through interactions with high affinity activation sites,and both Ca2� and Mg2� interact with allosterically coupledlow affinity sites (49, 50). Fig. 9A shows the concentration-ef-fect relationship across a range of Ca2� concentrations from10�8 to 10�2 M. The maximum level of [3H]Ry binding at opti-mal Ca2� was consistently 4- and 9-fold higher for Het andHom T4826I-RYR1 compared with WT, respectively (Fig. 9A,left) (p � 0.01). Analysis of the Ca2� curves normalized to theirrespective maxima (Fig. 9A, right) revealed that both Het andHomT4826I-RYR1 exhibit higher sensitivity toCa2� activatingat high affinity sites, but only Hom has reduced sensitivity toCa2� inhibition through low affinity sites (Fig. 9, A and C).Interestingly, Hom and Het T4826I-RYR1 had reduced sensi-tivity to Mg2� inhibition through low affinity sites comparedwith WT (Fig. 9, B and D) (p � 0.05).Augmented Response of T4826I-RYR1 to Temperature—Ful-

minant MH can be triggered in Het and Hom T4826I-RYR1mice in response to halothane and/or heat stress (1).We there-fore investigated whether temperature differentially influencesthe kinetics of [3H]Ry binding to skeletal muscle prepared fromthe three genotypes. Fig. 10 shows that the observed rate of[3H]Ry binding (kobs) depends significantly on genotype, withthe rank order Hom � Het � WT, and temperature (25 versus37 °C) (A and B). Fig. 10C shows that the relative increase inbinding rate (normalized toWT) is greater at 37 °C than it is at

25 °C (Fig. 10C) and that Hom is more responsive to tempera-ture than Het.

DISCUSSION

As is the case with most RYR1 MHS mutations, heterozygo-sis for T4825I-RYR1 in humans is sufficient to conferMHS, andindividuals homozygous for T4826I-RYR1 have not beendescribed. Although very rare, individuals homozygous forRYR1mutation C35R (51) and R614C (52) have been describedin families with a history of MHS, and muscle biopsies fromHom individuals have increased sensitivity to halothane andcaffeine compared with Het individuals (52). Recently, wedescribed that Het and Hom T4826I-RYR1 mice are viable butdisplay distinctly different phenotypic penetrance for trigger-ing fulminant MH with halothane and heat stress. This differ-ence allowed us to uncover a genotype- and sex-dependent sus-ceptibility to pharmacological and environmental stressors thattrigger fulminant MH and promote myopathy (1). In thisregard, T4826I-RYR1mice provide unique insights into under-standing the genotype-phenotype relationships of MHS muta-tions in vivo and the mechanisms influencing muscle dysfunc-tion and fulminant MH in adult skeletal muscle in vivo and invitro.One important outcome of channel dysfunction in adult

muscle fibers expressing T4826I-RYR1 is a chronically elevatedmyoplasmic [Ca2�]rest under basal (non-triggered) conditions.In this regard, there is a clear gene dose influence on myoplas-mic [Ca2�]rest with WT � Het � Hom. Recently Murayama etal. (53) did not detect a difference in cytoplasmic [Ca2�]rest inHEK 293 cells transiently transfected with T4826I-RYR1 com-

FIGURE 7. Hom T4826I-RYR1 channels are uniformly hyperactive in BLM. Single channels were incorporated in BLM in the presence of 1 �M free Ca2�, 2 mM

Na2ATP in cis chamber, 100 �M Ca2� in trans. A, representative 10-s continuous recording for a WT-RYR1 (top trace), a Het T4826I-RYR1 (middle trace), and HomT4826I-RYR1 channel currents (bottom trace) with their corresponding Po, �o, and �c. O, maximal single channel current amplitude. B and C, summary of Po datafor n � 13 WT-RYR1, n � 5 Het T4826I-RYR1, and n � 10 Hom T4826I-RYR1 channels (B, scatter plot; C, mean � S.D. (error bars)). Channels were reconstitutedfrom three different paired skeletal muscle membrane preparations. ***, p � 0.0001.



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pared with those expressing WT-RYR1. Several significantmethodological differences could account for these divergentresults. First, in vitro heterologous expression of RyRs in a sys-tem where there are no DHPRs to provide negative regulationversus measurements made in vivo in adult muscle fibers willdecrease any difference due to fact that increased numbers ofWT-RYR1 protomers will be in the leak conformation (16).This is compounded by the use of Fura 2 ratios with the inher-ent inaccuracy caused by using an EGTA-based buffer as anindicator versus measurements of [Ca2�]rest using calibrateddouble barreled microelectrodes. Nevertheless, the presentresults are consistent with those previously reported in adultR163C-RYR1 Het muscle fibers (19) and indicate that chroni-cally elevated [Ca2�]rest may be a common outcome of MHSmutations that promote RYR1 conformations that are leaky(26).Arguably, the most significant finding in the present study is

the uniformly high single channel Po and high capacity to bind[3H]Ry of Hom T4826I-RYR1 preparations isolated from adultmice when compared with WT. Although the unitary currentlevel and full gating transitions of HomT4826I-RYR1 channelsremain intact, the mutation concomitantly destabilizes the fullclosed state and stabilizes the full open state of the channel toachieve remarkably high Po with very tight current distribu-tions, consistent with the formation of a uniform population ofT4826I-RYR1 tetramers. Channel gating behavior, rather thandifferences in RYR1 protein expression or phosphorylation, is

responsible for the significantly higher level of [3H]Ry bindingobserved in Hom muscle preparations when assayed in thepresence of optimal Ca2� and is achieved because the inher-ently stable open channel conformation also stabilizes all[3H]Ry binding sites in their high affinity conformation (54).This interpretation is consistent with the fact that Het T4826I-RYR1 channels display more heterogeneous gating behaviorconsistent with random assembly of chimeric (WT/T4826I)tetramers and achieve a [3H]Ry occupancy level intermediatebetween those of WT and Hom. This behavior is similar tothose recently described for preparations isolated from HetR163C-RYR1 MHS mice (19). Both Het R163C and T4826Imutations enhance sensitivity to activation by Ca2� (3- and2-fold compared with WT, respectively), but only the lattermutation confers decreased sensitivity to inhibition byMg2� inboth Het and Hom preparations (�1.5-fold compared withWT) and a decreased sensitivity to Ca2� inactivation in theHom preparation. Cytoplasmic Mg2� is a physiological nega-tive control of RYR1 channel activity under resting and activat-ing conditions, and impaired regulation by Mg2� has beenimplicated as an important contributor to MHS (42, 55–57).However, the current results indicate that subtle shifts in regu-lation by physiological cations cannot fully explain the pro-found dysfunction of Het and Hom T4826I-RYR1 channelsobserved here and may involve altered nitrosylation (20) orother covalent modifications.

FIGURE 8. Het and Hom T4826I-RYR1 channels show distinct gating behaviors. Representative channel current amplitude histograms were obtained forWT-RYR1, Het, and Hom T4826I-RYR1 under identical conditions described in the legend to Fig. 7. WT-RYR1 and T4826I-RYR1 channels show uniform currentamplitudes that strongly favor the closed and full open channel states, respectively. Het T4826I-RYR1 channels exhibited broader intermediate states consis-tent with random association of WT-T4826I RYR1 monomers to form chimeric tetrameric channels. The insets show 1.5 s of representative current trace for eachgenotype.



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Amore significant contributor to channel dysfunction is thelocation of the mutation within the cytoplasmic loop linkingtransmembrane segments 4 and 5 (S4-S5). Recently,Murayama

et al. (53) reported results of substitution scan of the N-termi-nal half of the putative S4-S5 linker (Thr4825–Ser4829) of RYR1,including T4825I (rabbit sequence). Consistent with our find-

FIGURE 9. WT, Het, and Hom T4826I-RYR1 differ in their modulation by Ca2� and Mg2�. Equilibrium [3H]Ry binding (2 nM) was performed at 37 °C for 3 hin the presence of defined [Ca2�] (100 nM to 10 mM) (A) or 5 �M Ca2� � Mg2� (0 –30 mM) (B). EC50 and IC50 values obtained from curve fit of Ca2� activation/inactivation and Mg2� inhibition are plotted in C and D, respectively. The data are from 3– 4 different skeletal muscle membrane preparations (100 �g/ml) (n �9 –11 (A and C); n � 8 –9 (B and D)). The significance of difference is denoted as follows: *, p � 0.05; **, p � 0.01.

FIGURE 10. WT, Het, and Hom T4826I-RYR1 differ in sensitivity to temperature. Initial binding of 5 nM [3H]Ry to 100 �g/ml skeletal muscle membranes wereperformed at 25 or 37 °C in the presence of 5 �M free Ca2� and determined at 5, 10, 15, 20, 25, and 30 min. A, the results are plotted as rate lines. B, The initialrates were calculated, and kobs values are plotted as bar graphs. C, the differences in kobs measured with Het and Hom T4826I-RYR1 are plotted relative to kobsWT-RYR1. Statistical analyses indicate significant difference in temperature sensitivity among the three genotypes (***, p � 0.001; n � 4 from two differentskeletal muscle membrane preparations).



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ings with halothane in the current work and our finding ofincreased sensitivity to caffeine and 4-CmC previously (42),HEK 293 cells expressing T4825I-RYR1 exhibited higher sensi-tivity to caffeine and produced higher Po channels reconsti-tuted in BLM compared with those expressingWT (53). In thisregard, our results fromHomT4826I-RYR1 channels preparedfrom adult skeletal muscle show even more profound stabilityof the open state conformation (Po � 0.8 in the presence ofsuboptimal 1 �M cytoplasmic Ca2�) compared with expressedT4825I-RYR1 (Po �0.3 in the presence of optimal 100 �M cyto-plasmic Ca2�) (53).

Although qualitatively similar, several experimental differ-ences could contribute to the quantitative divergence of oursingle channel results from those of Murayama et al. (53), mostnotable are the BLM solutions having pH of 7.4 versus 6.8, 1 �M

versus 100�M cis-Ca2�, and trans-Ca2� of 100�M versus unde-fined, respectively. Our experimental conditions resulted in amean Po for reconstitutedWT channels of 0.12 (Fig. 7C), whichis lower than those reported by Murayama et al. (53) for WTchannels (mean Po �0.19). Nevertheless, our Hom T4826I-RyR1 channels produced ameanPo of 0.81 (nearly 8-fold higherthan WT), whereas the T4825I-RyR1 channels reconstitutedfrom HEK 293 cells by Murayama et al. had a �1.5-fold highermean Po than WT (Po �0.19 for WT and Po �0.29 T4826I-RyR1; Fig. 4B in Ref. 53). Interestingly, our BLM results areconsistent with both the 7–8-fold higher level of [3H]Ry bind-ing reported in our study (Fig. 9A) and the �7–8-fold increasereported byMurayama et al. at 100�MCa2� (Fig. 5B in Ref. 53).This distinction is important because it suggests that in addi-

tion to the inherent dysregulation imparted by the T4826Imutation, the presence of themutation over time in the contextof its native muscle environment may lead to stable covalentmodifications, other than phosphorylation, that contribute toabnormally active channel behavior, and these differences needto be explored in the future.As for the mechanism by which the T4826I mutation causes

RyR1 dysfunction, two possibilities are apparent: 1) impair-ment of the strong negative feedback regulation provided byprotein-protein interactions within the junctional Ca2� releaseunit, especially the DHPR (16), and 2) increased SR Ca2� leak,possibly mediated by the RyR1 leak conformation (58). Muta-tion of the �-helical conformation of the S4-S5 linker may besufficient to impair both of these regulatory mechanism (53).This hypothesis is reasonable given the important role of theS4-S5 linker in gating other ion channels, such asKV1.2 (59, 60).MHS mutations residing within the RYR1 N-terminal region(amino acids 35–609) have been suggested to destabilizedomain-domain interactions (61, 62). However, it is clear fromthe present study thatmutations residingwithin the transmem-brane assembly are likely to disrupt channel gating throughdistinct molecular mechanisms and underscore the allostericinfluence of RYR1 mutations that confer MHS.Ca2� dysregulation triggered by acute halothane exposure of

FDB fibers from Het and Hom T4826I-RYR1 male mice isclearly dependent on gene dose. A new finding is that the aug-mented response to halothane seen in intact Hom FDB fibersinvolves triggered release from SRCa2� stores and a significantcomponent of Ca2� entry. Thus, the fulminant MH syndrome

is likely to be initiated by disinhibition of RYR1 and exaggeratedCa2� excitation-coupled Ca2� entry and store-operated Ca2�

entry as suggested previously (19, 23, 61, 63). A better under-standing of howMHSmutations impair coordinated regulationof Ca2� release units may have novel therapeutic implications.The fact that Hom T4826I-RYR1 mice survive without overt

clinical pathology, although consistent with the human condi-tion when homozygosis has been identified (51, 52), raises sev-eral fundamentally important issues about the remarkable dys-function in their Ca2� release channels. In previous studieswith Het R163C MHS mice, RYR1 channel dysregulation wasassociated with significantly reduced Ca2� transient ampli-tudes evoked by electrical stimuli (i.e.ECcoupling) inmyotubesbut not adult FDB fibers (19). The difference in penetrance ofthe R163C mutation in myotubes and FDB fibers was inter-preted as an indication of tighter negative regulation of mutantRYR1 channels within the context of more developed adultjunctions present in FDB compared with the peripheral junc-tions found inmyotubes. Here we show that Het andHomFDBfibers exhibit subtle differences in EC coupling responsesalthough significantly larger than those of WT, and the degreeof amplification is gene dose-dependent (Hom � Het). Thus,the location and/or amino acid substitution of an MHS muta-tion (R163C versus T4826I) influences the basal physiologicalresponses of adult fibers. More importantly, the viability ofHom T4826I mice clearly indicates that the inherent dysfunc-tion of RYR1 channelsmust be under extremely strong negativeregulation by the DHPR in the context of adult muscle fibersbecause preferential targeting of WT channels to the junctionsis not possible. However, another consequence of the mutationis to increase the vulnerability of disengaging negative regula-tion of the RYR1 channel when exposed to triggering agents.MHSmutations do produce adaptations that lead to chronic

elevations in cytoplasmic [Ca2�]rest and reactive oxygen speciesthat are associated with bioenergetic adaptations in musclemitochondria (20, 28). Such adaptations may promote muscledamagewhose rate of onset,morphologicalmanifestations, andseverity depend on the location of the mutation, zygosity, andother factors, such as sex (1). Understanding the mechanismsthat limit andpromote fulminantMHand the potential damageof highly dysfunctional MHS RYR1 channels is central tounderstanding the etiology of this disorder.

Acknowledgments—We thank IselaT. Padilla andBenjaminYuen forexcellent technical support for [3H]Ry binding, Western blotting, andBLM experiments.

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Ryanodine receptor channelopathies

Matthew J. Betzenhauser and Andrew R. MarksDepartments of Physiology and Cellular Biophysics and Medicine, Clyde and Helen Wu Center forMolecular Cardiology, Columbia University College of Physicians and Surgeons, 630 West 168thStreet, New York, NY 10032, USAAndrew R. Marks: [email protected]

AbstractRyanodine receptors (RyR) are intracellular Ca2+-permeable channels that provide the sarcoplasmicreticulum Ca2+ release required for skeletal and cardiac muscle contractions. RyR1 underlies skeletalmuscle contraction, and RyR2 fulfills this role in cardiac muscle. Over the past 20 years, numerousmutations in both RyR isoforms have been identified and linked to skeletal and cardiac diseases.Malignant hyperthermia, central core disease, and catecholaminergic polymorphic ventriculartachycardia have been genetically linked to mutations in either RyR1 or RyR2. Thus, RyRchannelopathies are both of interest because they cause significant human diseases and provide modelsystems that can be studied to elucidate important structure–function relationships of these ionchannels.

KeywordsRyanodine receptors; Calcium-induced calcium release; Muscle contraction; Arrhythmias; Mutation

Ryanodine receptors and excitation–contraction couplingA regulated rise in intracellular Ca2+ is required for many physiological functions includingmuscle contraction, secretion, regulation of gene expression, and fertilization [10]. IntracellularCa2+ can be elevated via the activation of plasma membrane Ca2+ permeable channels or viathe release of Ca2+ from intracellular stores [22]. Unregulated or deficient Ca2+ signaling canlead to deleterious cellular outcomes, especially in excitable cells such as skeletal and cardiacmyocytes [95]. The rise in Ca2+ required for myocyte contraction is provided by the activationof the sarcoplasmic reticulum (SR) Ca2+ release channels in the ryanodine receptors (RyR)[122]. Given the central role that RyR play in regulating Ca2+ release, it is not surprising thatdysregulation of these channels can lead to severe muscle pathologies. In fact, mutations inRyR1 and RyR2 isoforms that are associated with human diseases have been identified [90,96]. RyR channelopathies include malignant hyperthermia (MH) and central core disease(CCD) in skeletal muscle and catecholaminergic polymorphic ventricular tachycardia (CPVT)in cardiac muscle [17,42,96]. Furthermore, alterations in RyR post-translational modificationsand remodeling of the RyR channel macromolecular complexes are associated with acquiredmuscle pathologies including skeletal muscle fatigue and heart failure (HF) [7,112]. A morecomplete understanding of RyR channelopathies will allow a greater understanding of RyRfunction and may help in the development of therapeutic strategies designed to rescue normalRyR dysfunction.

Correspondence to: Andrew R. Marks, [email protected].

NIH Public AccessAuthor ManuscriptPflugers Arch. Author manuscript; available in PMC 2011 July 1.

Published in final edited form as:Pflugers Arch. 2010 July ; 460(2): 467–480. doi:10.1007/s00424-010-0794-4.

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RyRs are a family of large, homotetrameric intracellular Ca2+ release channels that, uponactivation, allow rapid release of Ca2+ from SR stores into the cytosol (Fig. 1) [34]. There arethree mammalian isoforms (RyR1, RyR2, and RyR3) that exhibit subtype-specific tissueexpression patterns. RyR1 is the predominant isoform expressed in mammalian skeletalmuscle, and RyR2 is the exclusive subtype expressed in cardiac myocytes [34]. While RyR1and RyR2 are required for myocyte contraction in their respective tissues, the role of RyR3 isless clear, although this isoform is present early in skeletal muscle development, and a role forRyR3 in learning and memory has been proposed based on murine knockout studies [5]. Thethree RyR subtypes exhibit a high degree of structural and functional homology. Each monomeris in excess of 560 Kd with the ∼90% of in the channel sequence comprising enormous cytosolicdomains. Indeed, the N-terminal ∼4,100 amino acids comprise the cytosolic domain, and thetransmembrane domain is contained in the C-terminal ∼800 residues [34]. The number of RyRtransmembrane domains has been the subject of much debate, but it is now generally acceptedthat each monomer spans the SR membrane at least six times and possibly as many as eighttimes [28]. The ion conducting pore and the regions required for SR membrane localizationare contained in this region, and the functional Ca2+ release channels are organized intohomotetramers [34].

RyR1 and RyR2 release the SR Ca2+ required for muscle contraction, and as such, they arerequired for excitation–contraction (EC) coupling in skeletal and cardiac muscles, respectively[11,77]. Even though RyR1 and RyR2 are structurally quite similar, they are activated bydivergent mechanisms. Both isoforms are functionally coupled to changes in sarcolemmalmembrane potential through voltage-gated Ca2+ channels (VGCC) of the L-type class, alsoknown as dihydropyridine receptors (DHPR) [103]. DHPR physically interact with RyR1 inskeletal muscle, and conformational changes in the DHPR following membrane depolarizationinduce RyR1 to open [103]. Activation of L-type Ca2+ channels also induces RyR2 activationin cardiac myocytes, but there is no known physical interaction between the two channels.Instead, Ca2+ enters the myocyte through active LTCC and triggers RyR2 activation in aprocess known as Ca2+-induced Ca2+ release (CICR) [11]. All three isoforms exhibit a biphasicresponse to free Ca2+ and can participate in CICR in permeabilized systems. The three isoformsexhibit different sensitivities to Ca2+-dependent activation and inactivation, but Ca2+-dependent RyR activation generally occurs at ∼0.3–10 μM Ca2+, and the channels are typicallyinhibited by millimolar Ca2+ [34]. This Ca2+ sensitivity ensures that RyRs are normally closedat resting cytosolic Ca2+ (50–150 nM) and may also help prevent the sustained activation ofRyR at high Ca2+ concentrations.

RyRs in myocytes are typically part of large macromolecular complexes and are regulated byendogenous and exogenous ligands [34,122]. Endogenous regulatory molecules in addition toCa2+ include Mg2+ ions, which are inhibitory, and adenine nucleotides and cyclic ADP-ribose,which both activate the channels [34,77,78]. Numerous proteins also interact with and regulateCa2+ release from RyR including SR resident and cytosolic proteins. Notably, FKBP12 andFKBP12.6 (also known as calstabin1 and calstabin2) interact with and maintain the stabilityof RyR isoforms in muscle [12,75]. Calstabin–RyR interactions also promote coupled gatingof RyR from skeletal and cardiac muscles [73,74]. Calmodulin binds to RyR1 and RyR2 andlikely participates in Ca2+-dependent regulation of channel activity [6,102]. Detailed studiesof the effects of CaM on RyR1 have revealed that Ca2+-free CaM activates the receptor, whileCa2+-bound CaM inhibits channel function [82,97]. Phosphorylation represents an additionalmode of RyR regulation and kinases (PKA, CamK) and phosphatases (PP1, PP2A); thephosphodiesterase PDE4D3 and the muscle A-kinase anchoring protein are present in RyRmacromolecular complexes [122]. RyR-mediated Ca2+ release may also be controlled by thecellular oxidative state as the channels can be modulated by oxidation and nitrosylation [8,29,32,115]. RyRs are therefore a focal point for regulating Ca2+ release by multiple separateand interacting pathways.

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Exogenous pharmacological agents have long been known to regulate RyR activity and areuseful tools for examining RyR function [30,34,58,78,100]. The identification, purification,and early characterizations of RyR were greatly facilitated by use of the plant alkaloidryanodine, which binds to RyR with high affinity [37,62,88]. Low (micromolar) concentrationsof ryanodine lock RyR into a characteristic open one-half subconductance state [49], and higherconcentrations of ryanodine inhibit RyR channels in a use-dependent manner. 3H-ryanodinebinding, therefore, is a commonly used readout of channel activity [29]. Caffeine and 4-chloro-m-cresol (CmC) activate RyR channels and are thought to facilitate channel opening byincreasing the sensitivity of RyR to Ca2+-dependent activation [100]. Ruthenium red is acommonly used RyR blocker [70], and local anesthetics such as procaine and tetracaine alsoblock RyR channel activity in bilayers and in intact cells [58,101,116]. Additionalpharmacological agents can exert toxic or beneficial effects on muscle function by acting onRyR. For example, volatile anesthetics such as halothane are thought to increase RyR activityand can precipitate MH in individuals carrying MH mutations in RyR1, and the muscle relaxantdantrolene is used to prevent MH crises because it inhibits SR Ca2+-leak via mutant RyR1[60,71]. While the exact mechanism(s) underlying the actions of these pharmacological agentsis not completely understood, RyR activators and inhibitors are routinely used to study RyRfunction.

Skeletal muscle RyR channelopathiesMH, the first identified RyR channelopathy, is inherited in an autosomal dominant fashion andcontinues to be of major concern for anesthetic-induced deaths in otherwise healthy individuals[98]. Susceptibility estimates for MH range from 1 in 15,000 children to 1 in 50,000 adultsundergoing anesthesia [14]. The exact prevalence of MH susceptibility is difficult to determinesince the syndrome only becomes apparent after exposure to triggering agents such as halothaneand succinylcholine, but as many as 1 in 2,000–3,000 individuals may be susceptible toanesthesia-induced hyperthermic episodes [81]. A related syndrome referred to as porcinestress syndrome (PSS) is found in certain lines of domestic swine where stressed pigs undergostress-induced hyperthermia [71].

MH in humans and PSS in pigs are thought to develop following excessive skeletal musclecontraction that results from excess Ca2+ in the myoplasm following anesthesia in humans orduring stress in pigs [71]. This excessive Ca2+ causes sustained contractions, which accountsfor the rapid onset of muscle rigidity. Continued contraction and elevated Ca2+ exert a severemetabolic demand on myocytes, and ATP levels become depleted. Myocytes respond byincreasing ATP production via oxidative phosphorylation and glycolysis, which leads toacidosis. This prolonged hypermetabolic state generates heat and is thought to underlie theelevated body temperatures observed in MH episodes [71].

MH episodes in both humans and pigs are typically rapid and severe. Individuals whoexperience an episode can reach core body temperatures of 43°C, which leads to organ failureand death if not quickly treated [80]. Anesthetic-induced death rates in excess of 80% wereobserved in MH episodes prior to the discovery of the preventative effects of the musclerelaxant dantrolene [46]. Rapid administration of dantrolene during MH episodes has reducedmortality to less than 10% [99]. Susceptibility can be determined in vitro by measuring thecontractile response to caffeine or halothane in biopsied muscle fibers from humans and pigs[31]. Samples from MH cases exhibit an enhanced sensitivity to these agents. CICR from SRisolated from MH-susceptible pigs exhibited increased sensitivity to Ca2+, caffeine, andhalothane, which implicated abnormal Ca2+ release as the source of MH. Alterations in 3H-ryanodine binding properties in porcine MH samples provided evidence linking RyR1dysfunction to the disease [79]. 3H-ryanodine binding was increased at optimal [Ca2+] as wellas in the presence of inhibiting [Ca2+] indicating an overall gain-of-function defect. Ryanodine-



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sensitive channels in MH samples are also dysfunctional, further implicating altered RyR1function in the etiology of the disease [35,36].

While hyperthermic responses to surgery where known since the early 1900s, the link betweenvolatile anesthetics, family history, and MH was initially described in a letter to Lancet fromDenborough and Lovell in 1960 [25]. Therein, they described a patient who had survived asevere hyperthermic response after receiving halothane in preparation for surgery to repair abroken leg. The patient's family history had numerous instances (ten of 38 individuals whounderwent surgery) of deaths during or shortly following surgeries in which general inhaledanesthetics were administered [24]. MH has since been described as a pharmacogeneticdisorder since the disease is only manifested in humans during or immediately following theapplication of a general anesthetic [98]. While it was long suspected that MH was caused bydefective Ca2+ handling, the critical link between MH and RyR1 was provided by a moleculargenetic study of the condition in swine. Comparison of the cDNA sequence of RyR1 in MHpigs to that of normal controls revealed a missense mutation (C to T) at nucleotide position1,843 leading to a substitution of a cysteine residue for an arginine at position 615 (R615C)[39]. An analogous mutation in human ryr1 (R614C) was identified later [44].

Mutations in ryr1 are also associated with the rare congenital myopathy CCD and the relateddiseases multi-minicore disease, nemaline myopathy, and centronuclear myopathy [54]. Theserare diseases exhibit autosomal dominant and recessive modes of inheritance and have variablepathologies, but the defining characteristic is the presence of cores of metabolically inactivetissue in the center of muscle fibers [54]. These cores develop over time in CCD patients andare devoid of mitochondria and signs of oxidative metabolism. The pathological significanceof these cores is unclear, but the most severe cases of CCD involve pronounced muscleweakness. CCD symptoms are present at a young age, but unlike MH, these symptoms areapparent in the absence of other factors. MH and CCD may result from similar molecularmechanisms since some CCD patients also exhibit MH symptoms [54].

The clinical overlap of MH and CCD and the severity of CCD-related symptoms may relateto the specific set of mutations that patients carry. Significant symptomatic heterogeneity existsin patient populations that carry ryr1 mutations. Dominant mutations in ryr1 that are associatedwith MH are typically localized to MH susceptibility regions 1 and 2 [123]. Conversely, arecent analysis of a group of patients with various core myopathies demonstrated that CCDmutations inherited in a dominant fashion were clustered in the C-terminus and were associatedwith patients who exhibited pronounced cores and a strong CCD phenotype [123]. In contrast,core-associated mutations inherited in a recessive fashion spanned the entire ryr1 gene, andpatients with these mutations exhibited a wider range of clinical severity [123].

Molecular mechanisms of MH and CCDTo date, nearly 200 mutations in ryr1 have been linked to MH and/or CCD (Fig. 1) [96]. Thebulk of the mutations are missense substitutions and are conserved in three “hot spots” locatedin the N-terminal (C35–R614), central (D2129–R2458), and C-terminal regions (I3916–G4942) in the amino acid sequence of RyR1 [96]. As shown in Fig. 1, however, many mutationsare located outside these hot spots, and the apparent clustering in three regions may be theresult of a sequencing bias [76]. Attempts to understand the molecular mechanisms behind MHand CCD have involved the examination of the effects of these mutations on RyR1 function.This initially involved the laborious methodology of obtaining muscle biopsies from affectedpatients or MH pigs and testing the sensitivity of contracture to activating agents such ascaffeine or halothane [55,84]. Sensitivity to halothane, caffeine, and CICR was measured inthese samples, and 3H-ryanodine binding and single channel studies were also conducted[55,79,86]. These studies established that MH and CCD mutations typically increased the



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sensitivity of RyR1 to activation, indicating a gain-of-function. Recently, the use ofrecombinant techniques has rapidly increased the knowledge about the molecular mechanismsunderlying MH and CCD.

The ability to clone and express mutant RyR1 cDNA in heterologous systems has alloweddetailed analyses of RyR1 function and dysfunction. Recombinant RyR1 expressed in C2C12[87], HEK-293 [106], and COS-7 [107] cell lines function as intracellular Ca2+ release channelsand are activated by caffeine, halothane, and CmC. Ca2+ released from the ER via RyR1 canbe monitored in individual cells by using Ca2+-sensing dyes combined with imaging orphotometric detection. Detailed biophysical analyses can be performed on single recombinantRyR1 by isolating microsomes and fusing them to lipid bilayers. Single RyR1 can be measuredunder tightly controlled conditions to measure the sensitivity to Ca2+ activation or inhibitionor the sensitivity to endogenous or pharmacological activators and inhibitors. Similar studieshave been performed using recombinant RyR2.

Early experiments on heterologously expressed RyR1 established that the RyR1–R614Cmutant channels were more sensitive to caffeine and halothane, similar to results from in vitrocontracture tests [87]. Recombinant RyR1–R614C also exhibited an increase in the sensitivityto CICR similar to defects observed in muscle strips and microsomes isolated from MH patientsand MH pigs. Later experiments showed that 15 different MH, CCD, or MH/CCD RyR1mutations showed increased sensitivity to caffeine and halothane when expressed in HEK-293[106]. Using the same methodology, a mutation in the C-terminal hot spot (I4898T) exhibitedno response to caffeine and had severely reduced 3H-ryanodine binding [69]. Co-expressionof the I4898T mutation with wild type RyR1 partially rescued these deficiencies. Responsesto caffeine were reduced when compared to wild type RyR1 expressing cells, but enhancedwhen compared to I4898T expressing cells. These results led the authors to conclude that theI4898T mutation led to a severe gain-of-function effect [69]. This enhanced activity wasthought to promote a constitutive “leak” of Ca2+ from the SR. The reduced 3H-ryanodinebinding was proposed to result from the mutation disrupting the 3H-ryanodine binding site.

Experiments on RyR1 expressed in HEK-293 cells allow rapid and relatively straightforwardanalyses of receptor function, but HEK-293 cells fail to recapitulate the muscle environment.As such, this system does not allow RyR1 to be activated by DHPR activity or regulated byother muscle-specific factors. Expression of wild type and mutated RyR1 in dyspedic (RyR1knockout) myotubes that can be activated by endogenous DHPR recapitulates EC coupling[4,83]. Voltage clamp allows activation of DHPR and simultaneous intracellular Ca2+

measurements can be used to study MH and CCD mutations in a near-physiological setting.Numerous RyR1 mutations have been examined using this system, and in most cases, theresults corroborated prior studies conducted in HEK-293 cells [26]. That is, most MH and CCDmutations led to a gain-of-function effect that promoted “leaky” RyR1 [3,26,27,119]. In MH,this leak is uncovered when volatile anesthetics are administered, while in CCD cases, chronicleak is thought to lead to a reduction in SR store content and result in muscle weakness [68].

A careful examination of CCD mutations in dyspedic myotubes revealed a graded severity ofmutations on voltage-activated Ca2+ release. N-terminal mutations typically reduced theamount of Ca2+ released in response to depolarization. This was coupled with an increase inthe basal Ca2+ levels and a reduction in the SR content, consistent with leaky channels [3]. Theeffects of the C-terminal I4898T mutations were, however, quite different. Myotubesexpressing this mutant showed severely deficient Ca2+ release in response to depolarization,but normal resting Ca2+ and SR store content [4]. These results are consistent with a mutationthat reduces Ca2+ conductance. I4898 is located in the proposed pore region of RyR1, indicatingthat a reduction in permeation is behind the I4898T CCD mutation. The combined results ofthese studies led Avila and Dirksen to propose that CCD mutations could be grouped into two



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classes: leak-inducing mutations and mutations that uncouple RyR from DHPR activation (Fig.2) [26]. In the case of CCD, either mechanism could explain the muscle weakness observed inpatients with the disease.

A crystal structure was recently solved for the N-terminus (residues 1–210) of rabbit RyR1representing the first high resolution (∼2.5 A) structure for any region of RyR [1]. This structurecontains part of the MH susceptibility region 1 of RyR1, and 11 known RyR1 mutations weremapped onto this structure. Further structural analysis demonstrated that three of the mutations(C36R, R164C, and R178C) did not measurably disrupt the folding of the N-terminal domain.These results suggest that the mutations disrupt function through alterations in intramolecularinteractions instead of disrupting the structure immediately surrounding the mutations [1]. Thisis in line with the observations that mutations in divergent regions of RyR1 lead to similarchannel dysfunction.

Cardiac RyR channelopathiesDefects in SR Ca2+ release in cardiac myocytes are also the source of human disease [92].Inheritable arrhythmogenic disorders brought on by emotional or physical stress have beenlinked to mutations in RyR2 and its luminal binding partner calsequestrin2 [61,92]. Thehallmark of these diseases is PVT evident in electrocardiograms (ECGs) of patients in theabsence of any known structural heart defects [17,64]. Patients diagnosed with CPVT exhibitbidirectional VTs during exercise or stress and are at risk for syncope and sudden cardiac death[64]. Early diagnosis is critical since many CPVT mutation carriers die with the first episodeat a young age and current therapeutic strategies involve β-blocker treatment to blunt the effectsof catecholamines [17].

A genetic locus for CPVT was identified in chromosome 1q42–q43. Priori et al. identified fourseparate missense mutations in the gene for RyR2 in this region in DNA from CPVT patients[92]. More than 70 RyR2 mutations have since been identified and, similar to MH and CCDmutations, are spread throughout the entire amino acid sequence of human RyR2(http://www.fsm.it/cardmoc) (Fig. 1). The finding that mutations in the gene for calsequestrin2are also associated with CPVT further demonstrates that altered SR Ca2+ cycling is the rootcause of the arrhythmias [61]. Cytosolic Ca2+ levels are tightly regulated to allow the rapidinitiation and termination of muscle contraction during a heartbeat [11]. The cardiac actionpotential initiates VGCC opening to allow Ca2+ to enter the cell during systole. This Ca2+

rapidly activates RyR2 to flood the cytosol with Ca2+ and allow contraction. At the terminationof the AP, Ca2+ is removed from the cytosol by plasmalemmal sodium calcium exchanger(NCX) and sarcolemmal calcium pump [11].

Under normal circumstances, β-adrenergic stimulation has positive ionotropic, chronotropic,and lusistropic effects [11]. This allows a rapid increase in contractility, heart rate, andsarcomere relaxation required during times of stress and is a major component of thesympathetic “fight-or-flight” response. Signaling events downstream of β-adrenergic receptors(β-AR) after norepinephrine binding facilitate this efficient upregulation. Activation of β-ARon cardiac myocytes stimulates the production of cAMP and subsequent activation of PKA.PKA phosphorylates numerous substrates involved in EC coupling including Cav1.2 [23],RyR2 [113], and phospholamban [59]. The combined effect of these modifications is toincrease the magnitude and the rate of decline of the Ca2+ transient [15]. This allows the SRto be effectively refilled during shorter diastolic periods brought on by increased AP frequency.This normally efficient upregulation in response to catecholamines is disrupted in CPVT. Thebasis of this disruption is thought to be the aberrant Ca2+ release from the SR during diastole[17]. Similar to digitalis intoxication, this elevation in cytosolic Ca2+ is thought to activateNCX to promote electrogenic influx of three Na+ and efflux of one Ca2+ that can result in



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http://www.fsm.it/cardmoc

delayed afterdepolarizations (DADs), which can trigger ventricular tachycardia and suddencardiac death [38,72].

The β-adrenergic cascade is downregulated and desensitized in HF [13]. When cardiac functionbegins to diminish, there is a compensatory increase in circulating catecholamines. This isinitially protective, but becomes maladaptive over time. A major consequence of this prolongedadrenergic signaling is a remodeling of the RyR2 macromolecular complex. RyR2 becomesPKA hyperphosphorylated in HF [75]. The PKA hyperphosphorylation is exacerbated by theloss of PP1 and PP2A phosphatases and PDE4D3 phosphodiesterase from the RyR2 complex[75,94]. Most importantly, the stabilizing subunit FKBP12.6 (calstabin2) is also depleted fromthe complex, which leads to leaky RyR2 channels [75]. Hyperphosphorylated, FKBP12.6-deficient RyR2 channels exhibit subconductance states and increased activity in the presenceof diastolic levels of Ca2+ [75].

Divergent models to explain the molecular basis of CPVTA major remodeling of the RyR2 macromolecular complex occurs during HF due to chronicPKA hyperphosphorylation causing dissociation of FKBP12.6 from the RyR2 complex [75].It was subsequently found that mice with FKBP12.6 deletions (FKBP12.6−/− mice) exhibitedventricular arrhythmias and sudden cardiac death in response to exercise and catecholamineadministration, therefore exhibiting similarities to human CPVT patents [110]. Catecholamine-treated FKBP12.6−/− cardiac myocytes also exhibited DADs during APs evoked at 12 Hz.Cardiac microsomes isolated from exercised FKBP12.6−/− contained RyR2 that exhibitedincreased sensitivity to 150 nM cytosolic Ca2+ and subconductance states (a hallmark of FKBP-depleted channels [12,75]). Similar effects were observed in lipid bilayer studies with CPVT-mutated RyR2, as PKA treatment of three different CPVT-mutated RyR2 resulted in increasedPo at diastolic Ca2+ levels and the appearance of subconductance states, indicating destabilizedchannels [110]. These results suggested that the common link between HF and CPVT wasleaky RyR2 resulting from a loss of FKBP12.6. Consistent with this, all of the CPVT-mutatedRyR2 tested exhibited reduced FKBP12.6 binding (Fig. 3a) [110]. Furthermore, repairing thisinteraction with a small molecule or overexpression of FKBP12.6 repaired the defectivechannels and exercise-induced arrhythmias [47,110]. The potential role of RyR2hyperphosphorylation and FKBP12.6 depletion in the progression of HF and CPVT has comeunder much scrutiny. Some studies support the general hypothesis [40,121], but other studieshave not recapitulated the main findings [41,50,114]. Much of this confusion likely derivesfrom divergent experimental conditions that are employed by the different groups examiningthe problem.

Alternative mechanisms for the molecular basis for CPVT have also been proposed usingrecombinant RyR2 expressed in heterologous systems and more recently with the use oftransgenic mice. Unlike the case with dyspedic myotubes, no muscle-specific null backgroundsexist for reconstitution experiments. Recombinant techniques have been employed that allowexpression of mutated RyR2 in HEK-293 cells and atrial tumor HL-1 cells [41,53]. Cellularbased assays, single channel recordings, and 3H-ryanodine binding assays are typically usedfor functional studies. The majority of studies indicate that CPVT mutations induce gain-of-function changes in RyR2 function.

Chen and colleagues performed the first such studies using CPVT-mutated recombinant RyR2[53]. They expressed murine RyR2 harboring the R4496C mutation (equivalent to humanR4497C) in HEK-293 cells and found that the mutated RyR2 exhibited an increase in basalactivity. Specifically, an increase in 3H-ryanodine binding and single channel Po was observedin R4496C samples. HEK-293 cells expressing the mutant receptor were also more likely toundergo spontaneous Ca2+ oscillations and were more sensitive to caffeine stimulation than



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cells expressing the wild type receptor. The effects of β-AR activation were not tested in thesestudies, but the authors proposed a mechanism for CPVT whereby an increase in SR storecontent induced by adrenergic activity would cause spontaneous release of Ca2+ via mutatedRyR2 (Fig. 3b) [53]. Further studies by the same group refined the hypothesis and proposedthat a reduced threshold for store-overload-induced Ca2+ release (SOICR) is the source ofarrhythmias in CPVT [51].

Enhanced RyR2 activity in the face of elevated SR Ca2+ is a well-known phenomenon observedin response to numerous pathological stimuli including digitalis intoxication, elevatedextracellular Ca2+, and ischemia/reperfusion [63]. An increase in myocyte sensitivity to SOICRis thought to occur as a result of altered luminal regulation of mutated RyR2 in CPVT [52].The finding that mutations in calsequestrin can also lead to CPVT has supported the idea thataltered SR luminal Ca2+ is an important determinant of CPVT. A loss of function in CSQ2reduces SR Ca2+ buffering resulting in elevated SR Ca2+ [21]. Further increases in SR Ca2+

induced by adrenergic upregulation of SERCA would lead to luminal activation of RyR2 atresting diastolic cytosolic Ca2+ and arrhythmias.

The sensitivity of mutated RyR2 to various activating agents was also tested after heterologousexpression in HL-1 cells [41]. These conditions more closely mimicked the disease state in thesense that the mutated RyR2 was expressed in cells that contained endogenous, nonmutatedhuman RyR2. These studies allowed the added advantage that RyR2 could be studied incardiomyocyte-derived cells. George et al. found that the cells expressing the mutated RyR2did not exhibit the increased activity at rest that had been observed in HEK-293 cells. Cellsexpressing the mutated receptors were, however, more responsive to caffeine, 4CmC, and thecAMP-mobilizing agents isoproterenol and forskolin (Fig. 3c). The authors also reported adisruption in the RyR2–FKBP12.6 interactions in response to isoproterenol or forskolin, butthe wild type and mutant receptors were indistinguishable in this respect [41]. In subsequentexperiments examining interdomain RyR2 interactions, the same group proposed that CPVTmutations increased the propensity of RyR2 to undergo Ca2+-induced conformational changes[43].

Matsusaki's group has explored the possibility of another mechanism involving alteredinterdomain interactions in CPVT and HF [85,120]. They proposed that mutations causingCPVT induce an “unzipping” of interdomain interactions between the n-terminal (1–600) andcentral (2,000–2,500) domains that destabilizes the receptor at low cytosolic [Ca2+] (Fig. 3d)[85]. They based their hypothesis on the observation that regions in the N-terminal and centraldomains of RyR2 may interact and that a disruption of these domains occurs during channelactivation. A similar scheme has been proposed for RyR1 interdomain interactions duringactivation [117]. Of note, many CPVT-associated RyR2 mutations are located in these twodomains. A synthetic peptide corresponding to residues (G2460–P2495) that bound to thezipper domain in the N-terminus of RyR2 increased Ca2+ leak from cardiac microsomes [85].Based on these results, Matsuzaki and colleagues have proposed that compounds that restoredefective interdomain interactions may protect against pathological Ca2+ leak observed in HF[118].

Transgenic approaches to study RyR channelopathiesMajor advances in our understanding of RyR channelopathies have been provided by thegeneration of MH/CCD and CPVT mouse models. Numerous transgenic mice harboring RyRmutations have been generated in recent years in an attempt to create animal models thatrecapitulate human RyR channelopathies. These mouse models have helped address somequestions about molecular mechanisms, but discrepancies still remain.



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The first transgenic mouse model harboring a disease-related mutation in RyR1 was createdby Susan Hamilton and colleagues in 2006 where they examined mice engineered to expressthe Y522S MH mutation [20]. Mice homozygous for Y522S exhibited severe developmentalabnormalities and died between E17 and P1, but humans with MH are typically heterozygousfor RyR1 mutations. Heterozygous Y522S mice faithfully recapitulated many aspects of humanMH. MH episodes, characterized by overcontraction and elevated core temperatures, wereprecipitated by isofluorane or stress with heat. In vitro contracture tests also demonstrated anincreased sensitivity to caffeine and isofluorane. At the cellular level, heterozygous Y522Smyotubes exhibited a hyperpolarizing shift in voltage-induced Ca2+ release experimentssimilar to prior experiments using recombinant expression systems. These experiments furtherdemonstrated the “leaky” quality of RyR1 harboring Y522S. Unlike earlier experiments,however, Ca2+ stores were unaffected by the mutation in the mouse model [20]. A reductionin SR Ca2+ would be expected from a chronic leak associated with mutated RyR1. A likelyexplanation for this discrepancy is that some other compensatory process is evoked in thetransgenic mice to maintain SR Ca2+ store content. These experiments highlight the importanceof verifying transfection results with in vivo models. Subsequent studies using the RyR1-Y522S heterozygous mice have yielded novel insights into the susceptibility of these mice toheat stroke and also to the contribution to skeletal EC coupling of retrograde regulation ofDHPR by Ca2+ released from RyR1 [2,29].

As described above, RyR1 14895T mutations and other mutations in the C-terminus of RyR1likely exert their effects via uncoupling EC coupling rather than by increasing SR Ca2+ leak.MacClennan and colleagues generated a transgenic mouse harboring the RyR1-14895Tmutation to help determine whether this mutant is indeed uncoupled from DHPR in vivo[124]. Similar to the RyR1-Y522S mice, animals homozygous for the 14895T mutation exhibitsevere developmental defects and die before birth further emphasizing the important role RyR1plays in embryonic development. However, humans heterozygous for I4895T exhibit a severeform of CCD. A more recent study demonstrated that the heterozygous mice did in fact displaya progressive myopathy that was more similar to the human condition [125]. As the mice aged,abnormalities in skeletal muscle structure and function became apparent. Similar to humanCCD, muscles exhibited cores and some of the mice developed severe muscle deficienciesincluding complete hind limb paralysis [125].

Similar transgenic approaches have also been applied to CPVT-associated mutations in RyR2.Four different transgenic models have been generated in recent years to examine the in vivoeffects of the R4496C, R176Q, P2328S, and R2474S RyR2 mutants [16,45,57,65].Comparisons among these three mutant mice allow the opportunity to examine mechanismsbehind arrhythmogenic mutations in three divergent regions of RyR2. Priori's group generatedthe first RyR2 transgenic model in 2005 to test the effects of the R4496C mutation [16]. Thesemice largely recapitulated the main aspects of human CPVT. Mice heterozygous for R4496Cexhibited a tendency towards bidirectional VT upon epinephrine or caffeine injections whileresting ECGs were normal. Later experiments demonstrated that myocytes isolated from themutant mice also exhibited DADs in response to isoproterenol [67]. Unlike the studies on wholeanimals, myocytes from R4496C heterozygotes exhibit some signs of dysfunction includingDADs in the absence of adrenergic upregulation. These results highlight the notion that thechannels exhibit abnormal activity at rest. Similar observations were made in myocytes frommice engineered to express the R176Q or R2474S RyR2 mutations [57,65].

The R176Q mutation may be associated with the inherited cardiomyopathy arrhythmogenicright ventricular dysplasia (ARVD). This mutation is analogous to the R163C RyR1 mutationassociated with MH. ARVD patients typically exhibit progressive ventricular replacement withfibrofatty deposits in addition to CPVT. ARVD patients with RyR2 mutations tend to havemild ARVD symptoms and are classified as ARVD2, although ARVD diagnosis of patients



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with RyR2 mutations is controversial given the lack of severe ARVD symptoms [91].Nevertheless, these patients are at risk for bidirectional VTs and sudden cardiac death. Patientswith R176Q mutations also carry a second mutation of T2504M [105]. Both of these mutationsexhibit increased activity in vitro [104], but the generation of the R176Q mice allowed thedetermination of the contribution of this mutation to human disease. Hearts from miceheterozygous for R176Q were structurally normal. Catecholamines triggered ventriculartachycardias and isoproterenol elicited oscillatory Ca2+ signals in myocytes from these mice[57].

Additional studies with the R4496 mice have indicated that Ca2+ handling in the Purkinje fibersmay be a source of cardiac arrhythmias [18]. Purkinje fibers and isolated Purkinje cells exhibittriggered and spontaneous Ca2+ release events. The physiological role of these signals is notfully understood, but they may be involved in triggering action potentials. The arrhythmogenicactivity of the R4496C mutation was found to originate in the Purkinje fibers in detailed opticalmapping studies with voltage-sensitive dyes on Langendorff-perfused hearts [18]. Epinephrineand caffeine-induced bidirectional VTs in the ECGs from of these mice were also changed tomonophasic VTs upon Purkinje fiber chemical ablation. This study introduced the idea thatthe deadly arrhythmias triggered in CPVT patients may not originate in ventricles.

Transgenic mice expressing the P2328S mutation were generated to examine the effects ofmutating a site in the central domain of RyR2. Mice homozygous for the mutation were viable,which allowed comparisons to be made with heterozygous animals [45]. Myocytes isolatedfrom the homozygous mice exhibited the most severe alterations in Ca2+ handling. Heartsisolated from homozygous mice were also more prone to arrhythmias in Langendorff studies,indicating a gene dosage effect of the P2328S mutation.

Mice engineered to express the R2474S mutation exhibited ventricular tachycardia uponexercise and catecholamine treatment (Fig. 4) [65]. Cells isolated from these mice alsoexhibited aberrant Ca2+ waves, action potentials, and transient inward currents upon treatmentwith isopro-teranol. Isoproterenol treatment also elicited an increase in the diastolic Ca2+ sparkfrequency. Ca2+ sparks represent the spontaneous activity small clusters of RyR2 under restingconditions. An increase in spark frequency is indicative of an increase in diastolic Ca2+ leak.Similar to human disease, these effects were apparent in mice heterozygous for the mutation.The majority of mice homozygous for R2474S mutation died before birth with only 3.5% ofthe animals being born, while the heterozygous mice were born at the Mendelian ratio.

Some CPVT patients also exhibit neurological dysfunctions including epileptic seizures [64,89]. Consistent with this observation, generalized tonic–clonic seizures were identified in theheterozygous R2474S mice [65]. Seizure activity was mapped to hippocampal regions intelemetry recordings, and brain slices isolated from R2474S heterozygotes exhibited increasedspontaneous, ryanodine-sensitive Ca2+ signals. Of note, these defects were observed in theabsence of catecholamine treatment. This is unlike the cardiac phenotype, which was onlyinduced by exercise and catecholamines. One possible reason for this is that neurons are moreprone to abnormal RyR2 dysfunction at rest than are cardiac myocytes. Another possibility isthat there could be an increase in resting cAMP signaling in hippocampal regions that renderthe R2474S channels leaky.

Blocking RyR leak as a therapeutic approachA common feature of RyR channelopathies is that most disease-causing mutations in RyRcause the channel to be active or leaky at low cytosolic Ca2+ levels. Dantrolene may beprotective in MH episodes by inhibiting RyR function, although the exact mechanism ofdantolene is unknown [60]. A protective effect of flecainide in CPVT has been proposed basedon its ability to block leak from RyR2 in CSQ2-deficient mice [109]. Besides MH, CCD, and



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CPVT, RyR leak may also play a role in HF and some forms of muscular dystrophy and epilepsy[8,65]. The design and development of novel therapeutics that fix RyR leak should be a majorgoal of future research to treat these diseases.

Results linking the loss of FKBP isoforms from RyR complexes with increased Ca2+ leak fromRyR have led to the development of small molecules that restore FKBP/RyR interactions.JTV519 (a 1,4-benzothiazepine derivative) improved cardiac function in a canine HF model[120]. A possible mechanism for these protective effects was provided by evidence that JTV519enhanced FKBP12.6 binding to RyR2, even when the receptor was hyperphosphorylated[111]. The functional consequence of this rescued binding was a reduced single channel openprobability of RyR2 at low [Ca2+] in bilayers. Importantly, JTV519 also rescued FKBP12.6binding to RyR2 harboring CPVT mutations [66]. In addition, JTV519 prevented pacing-induced arrhythmias in mice with a haploinsufficiency of calstabin2 (calstabin2+/−), but waswithout effect in mice homozygous for calstabin2 deletion (calstabin2−/−) [111].

Even though JTV519 has therapeutic potential for its ability to stabilize RyR2 function, it alsoblocks Na+, K+, and Ca2+ channels present in cardiac myocytes [56]. RyR2-specific moleculesare required to rescue the RyR2 gain-of-function present in HF without altering other aspectsof cardiac electrophysiology. The synthesis of a novel, orally bioavailable, benzothiazapine(S107) that meets this requirement has recently been described [9,65]. S107 stabilizes RyR–calstabin interactions, but unlike JTV519, exhibits no activity when tested against hundreds ofGPCRs, ion channels, or enzymes [9]. Importantly, S107 was protective in the R2474S mousemodel of CPVT since mice pretreated with S107 failed to develop exercise and catecholamine-induced arrhythmias (Fig. 4) [65]. Furthermore, myocytes isolated from R2474S micepretreated with S107 exhibited fewer of these aberrant Ca2+ release events further underscoringthe protective effects of this compound.

These results support a model whereby JTV519 and S107 exert therapeutic effects throughstabilizing RyR2–calstabin2 interactions that become disrupted by excessive PKAphosphorylation of S2808. It should be noted, however, that other groups have suggestedalternative mechanisms of action for JTV519 [48,120]. Specifically, it has been proposed thatJTV519 can exert inhibitory effects on RyR2 function by limiting SOICR [48] or by stabilizinginterdomain interactions [120]. The impact that S107 may or may not have on these parametershas not been tested, but the compound has been shown to promote RyR–calstabin interactionsin the face of excessive PKA phosphorylation [9,65] or nitrosylation [8,33].

S107 was also effective in blocking Ca2+ leak and dysfunction in neuronal and skeletal muscles.For example, S107 was able to rescue the developmental and neurological effects observed inthe R247S mice [65]. R2474S homozygous mice were born at a higher rate when the drug wasadministered to the dams prenatally. Furthermore, S107 was effective at blunting the seizureactivity evident in these mice. RyR isoforms are present throughout the brain, but thephysiological role of RyR function in learning, memory, and neurological dysfunction islargely unknown. Results from studies of R2474S mice suggest that fixing Ca2+ leak from RyRmay represent a novel treatment strategy for some form of epilepsy. It remains to be determinedif Ca2+ leak is evident in other neurodegenerative diseases, but an upregulation of RyR2 hasbeen proposed as a pathological feature in mouse models of Alzheimer's disease [19].Preventing Ca2+ leak may also hold promise as a treatment for skeletal muscle weakness foundin HF and muscular dystrophy [8,9]. Similar to RyR2, RyR1 in skeletal muscle is PKAhyperphosphorylated, calstabin-depleted, and leaky in HF [93,108]. S107 successfully reducedmuscle fatigue in mice after strenuous exercise indicating Ca2+ leak as a source of muscleweakness [9]. Similarly, muscle weakness was blunted by S107 in a mouse model of musculardystrophy [8]. This was associated with a reduction in Ca2+ spark activity indicating that SRleak inhibition was the mechanism behind the therapeutic effects of S107.



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An additional therapeutic approach was uncovered in studies examining calsequestrin2-mediated CPVT where increased Ca2+ leak was observed in myocytes isolated from miceexpressing a calsequestrin2 mutation [109]. This leak is thought to result from increasedCa2+ stores caused by the loss of calsequestrin SR Ca2+ buffering resulting in an increase inRyR2 activity. Flecainide was found to block this Ca2+ leak at the cellular level and preventCPVT symptoms in mice with the calsequestrin mutation. Importantly, flecainide was alsoeffective at preventing CPVT episodes in human subjects, one of whom carried a calsequestrinmutation and the other carried a RyR2 mutation. Both were symptomatic even with β-blockadeand Ca2+ channel blockers, demonstrating the utility in treating CPVT by targeting theunderlying molecular cause of the disease [109].

Future outlookMuch work has been done in an attempt to elucidate the molecular mechanisms underlying theRyR channelopathies since the first disease-causing RyR mutations were discovered in 1991[39,44]. A wealth of experimental evidence has suggested that many facets of MH, CCD, andCPVT can be accounted for by an increase in SR Ca2+ leak from mutated RyR. Much workremains to be done in terms of defining the exact molecular basis for this leak. Continued effortswill address how alterations in channel activity lead to cellular and ultimately whole organdysfunction. A complete understanding of the molecular underpinnings of RyRchannelopathies will facilitate the generation of novel therapeutics designed to block leak fromRyR. Additional structure–function studies and transgenic approaches are also needed to helpsolve some of the remaining mysteries concerning RyR regulation by cytosolic and luminalCa2+, gating and permeation, and modulation by protein binding partners and kinases.

AcknowledgmentsDr. Marks is a consultant for ARMGO Pharma, Inc, a start-up company targeting RyR2 to treat HF and cardiacarrhythmias, and is the recipient of funding from the National Institutes of Health (HL 061503, HL 067849, HL 056180,and HL 083418) and from the Fondation Leducq.

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120. Yano M, Kobayashi S, Kohno M, Doi M, Tokuhisa T, Okuda S, Suetsugu M, Hisaoka T, ObayashiM, Ohkusa T, Matsuzaki M. FKBP12.6-mediated stabilization of calcium-release channel(ryanodine receptor) as a novel therapeutic strategy against heart failure. Circulation 2003;107:477–484. [PubMed: 12551874]

121. Yano M, Ono K, Ohkusa T, Suetsugu M, Kohno M, Hisaoka T, Kobayashi S, Hisamatsu Y,Yamamoto T, Noguchi N, Takasawa S, Okamoto H, Matsuzaki M. Altered stoichiometry of



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FKBP12.6 versus ryanodine receptor as a cause of abnormal Ca (2+) leak through ryanodinereceptor in heart failure. Circulation 2000;102:2131–2136. [PubMed: 11044432]

122. Zalk R, Lehnart SE, Marks AR. Modulation of the ryanodine receptor and intracellular calcium.Annu Rev Biochem 2007;76:367–385. [PubMed: 17506640]

123. Zhou H, Jungbluth H, Sewry CA, Feng L, Bertini E, Bushby K, Straub V, Roper H, Rose MR,Brockington M, Kinali M, Manzur A, Robb S, Appleton R, Messina S, D'Amico A, Quinlivan R,Swash M, Muller CR, Brown S, Treves S, Muntoni F. Molecular mechanisms and phenotypicvariation in RYR1-related congenital myopathies. Brain 2007;130:2024–2036. [PubMed:17483490]

124. Zvaritch E, Depreux F, Kraeva N, Loy RE, Goonasekera SA, Boncompagni S, Kraev A, GramoliniAO, Dirksen RT, Franzini-Armstrong C, Seidman CE, Seidman JG, Maclennan DH. AnRyr1I4895T mutation abolishes Ca2+ release channel function and delays development inhomozygous offspring of a mutant mouse line. Proc Natl Acad Sci U S A 2007;104:18537–18542.[PubMed: 18003898]

125. Zvaritch E, Kraeva N, Bombardier E, McCloy RA, Depreux F, Holmyard D, Kraev A, Seidman CE,Seidman JG, Tupling AR, Maclennan DH. Ca2+ dysregulation in Ryr1I4895T/wt mice causescongenital myopathy with progressive formation of minicores, cores, and nemaline rods. Proc NatlAcad Sci U S A 2009;106:21813–21818. [PubMed: 19959667]



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Fig. 1.Disease-related mutations in human RyR1 and RyR2 are clustered in three mutational “hotspots.” a The tetrameric structure of RyR Ca2+ release channels with the membrane topologysuperimposed on one of the subunits. b The distribution of over 200 malignant hyperthermiaand central core disease-causing mutations in human RyR1 and over 70 catecholaminergicpolymorphic ventricular tachycardia-associated mutations in human RyR2



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Fig. 2.Two different effects of central core disease-related mutations on skeletal muscle excitation–contraction coupling. a A cartoon depicting the effects of a leaky RyR1 mutation. This classof mutations exhibits an increased sensitivity to activation by membrane depolarization,Ca2+, caffeine, and halothane. b A cartoon depicting the effects of an uncoupled mutation.Mutations of this class lead to nonfunctional channels and increased sarcoplasmic reticulumCa2+ store content



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Fig. 3.Four different proposed mechanisms to explain how RyR2 mutations lead to catecholaminergicpolymorphic ventricular tachycardia (CPVT). a CPVT mutations are more sensitive to PKAhyperphosphorylation, which leads to depletion of the channel stabilizing protein, calstabin2(FKBP12.6), and sarcoplasmic reticulum (SR) Ca2+ leak. b CPVT mutations lead to an increasein sensitivity to store-overload-induced Ca2+ release that is exacerbated by the increased SRstore content triggered by stressful situations. c CPVT mutations lead to increased RyR2sensitivity to activating agents such as caffeine, 4-CmC, or cAMP-mobilizing agents. d CPVTmutations lead to an unzippering of intramolecular interactions, which leads to increaseddiastolic SR Ca2+ leak



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Fig. 4.Fixing sarcoplasmic reticulum (SR) Ca2+ leak by targeting RyR2–calstabin2 interactions. Thetraces in (a), (b), and (c) are representative telemetric electrocardiogram (ECG) recordingsfrom wild type, R2474S heterozygous (R2474S+/−) mice, and R2474S+/− mice that were treatedwith S107. Mice were subjected to a stress protocol consisting of a treadmill exercise followedby epinephrine injections. Wild type mice did not exhibit irregular ECGs under theseconditions, while a majority of R2474S+/− mice exhibited severe ventricular tachycardias andsudden cardiac death (single asterisk, bidirectional VT; double asterisk, polymorphic VT).Treatment with S107 largely prevented these effects as shown by the regular ECG. Below thetraces are cartoons depicting the condition of the RyR–calstabin2 complex under eachexperimental condition. Data were from Lehnart et al. [65] and reprinted with permission fromthe American Society of Clinical Investigation



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A canine DNM1 mutation is highly associated with thesyndrome of exercise-induced collapseEdward E Patterson1, Katie M Minor2, Anna V Tchernatynskaia1, Susan M Taylor3, G Diane Shelton4,Kari J Ekenstedt5 & James R Mickelson2

Labrador retrievers are the most common dog breed in theworld, with over 200,000 new kennel club registrations peryear. The syndrome of exercise-induced collapse (EIC) in thisbreed is manifested by muscle weakness, incoordinationand life-threatening collapse after intense exercise. Using agenome-wide microsatellite marker scan for linkage inpedigrees, we mapped the EIC locus to canine chromosome 9.We then used SNP association and haplotype analysis to finemap the locus, and identified a mutation in the dynamin 1gene (DNM1) that causes an R256L substitution in a highlyconserved region of the protein. This first documentedmammalian DNM1 mutation is present at a high frequencyin the breed and is a compelling candidate causal mutationfor EIC, as the dynamin 1 protein has an essential role inneurotransmission and synaptic vesicle endocytosis.

EIC is a newly characterized syndrome affecting Labrador retrievers,especially those dogs used for hunting and field trials1,2. Five to fifteenminutes of strenuous exercise causes dogs suffering from this condi-tion to develop a ‘wobbly’ gait, which soon progresses to nonpainful,flaccid paraparesis and a loss of control of the rear limbs. The episodemay progress to all four limbs. Collapse episodes usually last for5–10 min, and after 30 min there is often complete recovery, butepisodes are occasionally fatal. The rectal temperature of dogs duringan episode typically reaches 41.7 1C (from a resting temperature of39 1C); however, the post-exercise rectal temperature is no higher, andthe rate of cooling is no lower, in EIC-affected than in unaffectedLabrador retrievers1,3. EIC has a familial clustering and pattern ofinheritance consistent with autosomal recessive inheritance4, but theunderlying mechanisms responsible for this condition are unknown.

Genome-wide genetic linkage and association approaches havedefined the basis for a number of canine health and diseasetraits5–11. We carried out a genome-wide scan with 459 microsatellitemarkers on six pedigrees of Labrador retrievers in which EIC wassegregating. The initial genome scan of 96 dogs identified a locus at

the 60.4 Mb position of canine chromosome 9 (CFA9) with significantlinkage. Testing of additional CFA9 markers on a total of 252 dogsfrom eight pedigrees (Supplementary Fig. 1 online) confirmed thelocus, with nine lod scores 43.3 and a maximum lod score of 11.4(Supplementary Table 1 online).

We then analyzed SNP markers from the region for association withEIC using 310 Labrador retrievers. Initially, we found multipleassociated markers (Fig. 1a), but after correcting for populationstratification, we identified three significantly associated SNPs in a355-kb region (58.519–58.874 Mb) (Fig. 1b). The most commonhaplotype in EIC-affected dogs (n ¼ 104) extended nearly the entire4.5-Mb segment for which SNPs were analyzed (Fig. 2a), andhomozygosity was observed for a minimum of 929 kb in 89% ofthese dogs. Homozygosity in 10% of the affected dogs was, however,limited to two short haplotype blocks: a 137-kb block and an 87-kbblock, separated by a 184-kb gap (Fig. 2a). These two haplotype blockswere also observed in the unaffected dogs, as would be expected fromtheir unavoidably close relationships to the affected dogs. However,only 11.4% of all 132 unaffected dogs were homozygous for the137-kb segment (significantly different from presumed affected cases,P ¼ 1.01 � 10�38), whereas 56.1% of the unaffected dogs werehomozygous for the 87-kb segment (P ¼ 1.43 � 10�11), suggestingthat the 87-kb region was less likely to contain the EIC-associatedlocus. Furthermore, the AK1 gene was the only plausible positionalcandidate gene in the 87-kb block (Fig. 2b), and we found no exonicAK1 SNPs from our sequencing of affected and control dogs.

The DNM1 gene was clearly the most plausible positional candidatein the 137-kb block (Fig. 2b). We sequenced all DNM1 exons, 220 bpof the 5¢ untranslated region and 79 bp of the 3¢ untranslated regionfrom genomic DNA. The predicted full-length canine dynamin 1protein contains 864 amino acids, and a short form predicted frompossible alternative splicing contains 845 amino acids. Six SNPs werefound within the DNM1 coding sequence. Five of these SNPs weresynonymous and not strongly associated with the EIC phenotype(Supplementary Table 2 and Supplementary Fig. 2 online); however,

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Received 17 March; accepted 1 July; published online 21 September 2008; doi:10.1038/ng.224

1Department of Veterinary Clinical Sciences, College of Veterinary Medicine, University of Minnesota, St. Paul, Minnesota 55108, USA. 2Department of Veterinary andBiomedical Sciences, College of Veterinary Medicine, University of Minnesota, St. Paul, Minnesota 55108, USA. 3Department of Small Animal Clinical Sciences,Western College of Veterinary Medicine, University of Saskatchewan, Saskatoon, Saskatchewan S7N5B4, Canada. 4Comparative Neuromuscular Laboratory,Department of Pathology, University of California San Diego, La Jolla, California 92093, USA. 5Department of Veterinary Population Medicine, College of VeterinaryMedicine, University of Minnesota, St. Paul, Minnesota 55108, USA. Correspondence should be addressed to E.E.P. ([email protected]).

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http://www.nature.com/doifinder/10.1038/ng.224

mailto:[email protected]

http://www.nature.com/naturegenetics/

an exon 6 G to T SNP at coding nucleotide position 767 resulted in thenonconservative conversion of codon 256 from arginine in controldogs to leucine in affected dogs. Alignment of the canine and humanamino acid sequences shows notable conservation, with 860 of the 864residues identical. There was also a high level of conservation of the241–270 residue segment across multiple species, as well as withdynamins 2 and 3 (Fig. 3a). The canineR256L substitution is located in the boundaryregion between the GTPase and dynaminfamily central domains12 (Fig. 3b).

We genotyped the G767T DNM1 SNP inour entire population of Labrador retrievers,

which yielded a lod score of 14.6 at a y of 0.02 (SupplementaryTable 1), and a –log(P value) for association of 32.7 before (Fig. 1a),and 8.2 after (Fig. 1b), correction for population stratification. Whenwe included the DNM1 genotype data in the haplotype analysis(Fig. 2a), we observed the same minimally conserved 137-kb haplo-type of AAGTGGTG (where T represents the DNM1 mutation) over99% of the time in chromosomes containing the T767 allele. Thehomologous unaffected AAGTGGGG haplotype (where G representsthe wild-type allele) was common in control dogs (found 33% of thetime), suggesting that the G767T DNM1 mutation arose on acommon haplotype. We have observed presumed EIC-affected dogsthat are T767 homozygotes in two breeds closely related to Labradorretrievers: Chesapeake Bay retrievers (CBRs) and curly-coated retrie-vers. The haplotype for a CBR T767 homozygote is homozygous overthe 137-kb block (Fig. 2a), suggesting that the allele may be identicalby descent across breeds separated by many generations.

The DNM1 G767T genotype frequency is presented in Table 1 for343 Labrador retrievers within the different classification criteria forEIC. We found that 97% of all dogs that fulfilled the study criteria for

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36a

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Figure 1 CFA9 SNP association analysis. w2 tests comparing allele

frequencies to phenotype were done for 43 CFA9 SNPs and 205 dogs (108

affected, 84 unaffected and 13 of unknown phenotype) (see Methods and

Supplementary Table 6 for details). The negative log of the P value of the w2

results, with Bonferroni correction for multiple testing, is shown. The dotted

horizontal line indicates the threshold for significant association of �log

(P value) 42.0. (a) Association analysis results uncorrected for population

stratification. Thirty-one SNPs had significant –logP values, with the highest

value of 28.9 for SNP BICF2S23143955 (58.51 Mb). (b) Association

analysis results corrected for population stratification. Three SNPs, all in the

58.51- to 58.87-Mb region, remained significant, with the highest �log

(P value) of 5.66 (again for SNP BICF2S23143955). The �log(P value) for

the G767T mutation subsequently found in DNM1 is circled in both panels.

LR1

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COQ4 C9orf119 DNM1 C9orf16 PTGES2 ENGST6GALNAC6

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Figure 2 SNP haplotypes and genes from theregion of CFA9 genetically linked to EIC. (a) SNP

genotypes in the longest EIC haplotype observed

are indicated in the top row. Identities of each

SNP can be found in Supplementary Table 6. The

regions of haplotype conservation relative to the

longest haplotype are shown in blue for selected

Labrador retrievers (LR) with strong evidence of

EIC. The most common SNP haplotype extended

nearly the entire 4.5-Mb segment. However, SNP

haplotype homozygosity was observed for a

minimum of 929 kb (58.279–59.208 Mb) for

89% of all 104 presumed affected dogs

(examples LR1–LR8). Homozygosity for the

remaining 10% of all EIC presumed affected

dogs (examples LR9–LR11) was limited to two

short haplotype blocks, 137 kb from 58.545 to

58.682 Mb, and 87 kb from 58.866 to

58.953 Mb (outlined vertically). The haplotypeof a Chesapeake Bay retriever (CBR) presumed

affected with EIC is also shown. The G767T

mutation subsequently found in DNM1 is in

orange. (b) CFA9 positions (Mb) of ENSEMBL-

annotated genes in the 137-kb and 87-kb blocks.

2 ADVANCE ONLINE PUBLICATION NATURE GENETICS

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presumed EIC affected and 88% of dogs with collapse consistent withEIC but with incomplete documentation were homozygous for theDNM1 T767 allele. Dogs with a lower likelihood of having EIC (thatis, a single reported collapse, atypical collapse or another potentialcause identified) had a decreasing likelihood of being homozygous forthe T767 allele. Genotypes of the 35 parents of T767 homozygous dogsavailable for genotyping were consistent with an autosomal recessivebasis for EIC.

Nine percent of dogs without a history of collapse are homozygousfor the DNM1 T767 allele (Table 1), which can be explained by theirnot having been exposed to sufficient exercise or excitement to triggercollapse, or the possibility of genetic and environmental modifyingfactors. The high frequency of heterozygotes in the no-collapsepopulation makes conclusions concerning genotype-phenotype rela-tionships in heterozygotes ambiguous. We suspect that many of thehomozygous GG and heterozygous Labrador retrievers with a collapsephenotype have a different type of collapse syndrome that occurswhen they are excited, but not necessarily exercising intensely, such asa focal seizure disorder or another paroxsymal neurological condition.The T767 allele carrier frequency in a population of 400 Labradorretrievers from field trials conducted in the upper Midwest region of

the USA was 37%, and the homozygote frequency was 3%, suggestingthat this allele is present at a very high frequency in the generalpopulation. We have also found T767 homozygous Labrador retrieverswith recurrent collapse from Europe, the Middle East and Australasia.

Members of the classical dynamin subfamily of GTPases regulateendocytic vesicle formation. Dynamin 1 seems to be almost exclusivelyexpressed in the brain and spinal cord13, where it has a key role insynaptic vesicle fission by assembling into collar-like structures aroundinvaginations of the presynaptic terminal membrane. The twisting andpinching action of dynamin releases new membrane vesicles tocontain neurotransmitter and enable continuous synaptic commu-nication. Dnm1 knockout mice are born alive, but postnatal viability isbrief14. The products of the Dnm2 and Dnm3 genes seem capable ofhandling low-frequency fetal neurological stimulation, but Dnm1expression becomes essential postnatally with heightened neuralactivity. Induced mutations in the central and GTPase domains oforthologous Drosophila and C. elegans genes affect dynamin aggrega-tion and assembly on membranes, and in some cases cause EIC-liketemperature-dependent reversible loss of motor function, which canbe fatal if the temperature remains elevated15–18. Additional study isneeded to unequivocally prove that there are functional differencesbetween the Arg256 and Leu256 forms of canine dynamin 1. At thistime, we believe it is likely that insufficient activity of the Leu256 formof dynamin 1 in homozygotes during high-intensity exercise or highexcitement leads to a lack of sufficient vesicles for sustained synaptictransmission, resulting in a reversible loss of motor function.

Mutations in the central and Pleckstrin homology domains ofDNM2 have been associated with centronuclear myopathy19 andCharcot-Marie-Tooth disease20 in humans (Fig. 3b). Our finding ofa DNM1 mutation highly associated with EIC in the Labradorretriever dog follows closely on the discovery of a SINE insertionmutation in PTPLA, a tyrosine phosphatase–like gene responsible fora form of centronuclear myopathy in this breed21, and furtherdemonstrates the utility of gene mapping in canine models. Theknowledge that a naturally occurring DNM1 mutation could cause acondition of reversible collapse should prove useful in the study ofendocytosis, synaptic transmission and neuropathobiology.

METHODSSample collection. This study was done using protocols approved by the

Institutional Animal Care and Use Committees (IACUC) of the University of

Minnesota and the University of Saskatchewan. Written consent was obtained

from all dog owners. Affected Labrador retriever families were ascertained

through affected offspring by requesting medical records, pedigrees and DNA

from all dogs within two generations of clinically affected dogs. The pedigrees

for linkage analysis were assembled using Cyrillic 2.1 software.

We identified six different phenotypic groups of dogs on the basis of

available medical data and questionnaire information. Group 1 consisted of

presumed EIC-affected dogs; dogs with a well-documented history of more

than one typical collapse episode in which the pelvic limbs became ataxic and

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Dog DNM1

Dog DNM1

241

a

b

241

270

270

Cross-species comparison

Paralog comparison

Dog DNM1 (EIC)Human DNM1

Human DNM2

Human DNM3

GTPase

Ce Dm DmEICCMT or CNM

1001

200

300

400

500

600

700

800

864

Central domain PHD GED

Mouse DNM2

Mouse DNM3

Mouse DNM1

Bovine DNM1Chicken

Danio rerio

Drosophila

C. elegans

Table 1 DNM1 genotypes in phenotyped Labrador retrievers

Phenotype TT GT GG Total TT (%)

Group 1, presumed EIC affected (complete data) 101 0 3 104 97

Group 2, recurrent collapse (incomplete data) 60 3 5 68 88

Group 3, single collapse 5 3 0 8 62

Group 4, atypical collapse 11 6 9 26 43

Group 5, alternative collapse (other cause) 1 2 2 5 20

Group 6, no collapse 12 65 55 132 9

Parents of presumed affected 6 29 0 35 16

Genotypes at coding-nucleotide 767 of canine DNM1 were determined as described in theMethods. Dogs were evaluated on the basis of reported clinical signs and medical dataquestionnaires and placed into one of six phenotype categories as described in the Methods.Groups 1 and 2 were considered affected, groups 3–5 as unknown status, and group 6 asunaffected. In group 5, other potential causes of repeated collapse were cardiac arrhythmia forthe TT genotype, laryngeal paralysis and lactic acidemia for the GT phenotypes, and metabolicmyopathy and cardiac arrhythmia for the GG phenotypes. Not all parents of presumed affecteddogs had enough data to be accurately phenotyped, but four of six homozygous TT parents hadbeen reported to collapse and were also included in group 1.

Figure 3 Dynamin amino acid sequence homologies and substitutions.

(a) Species alignment of dynamin family member amino acid sequences.

The R256L substitution highly associated with EIC is indicated by the arrow.

Underlined sequences are conserved across species or gene families in that

group. (b) Approximate positions of the GTPase, central region, Pleckstrin

homology (PHD) and GTPase effector (GED) domains of dynamin family

members are indicated12. The canine EIC mutation in DNM1 is on the

border between the GTPase and central domains. Mutations in the

orthologous gene (shibire or dynamin) in temperature-sensitive mutants

of Drosophila melanogaster (Dm) and C. elegans (Ce), respectively, as well

as mutations in the DNM2 gene associated with human centronuclear

myopathy (CNM)19 and Charcot-Marie-Tooth disease (CMT)20 are indicated.


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then flaccid, with complete recovery within 30 min, consistent with the

previously established criteria1. In group 2 were dogs showing recurrent

collapse. These dogs had an incomplete description of the collapse episodes

and/or medical history, although these were, for the most part, consistent with

the criteria for presumed affected. Group 3 dogs had a single collapse episode,

and otherwise met the criteria for presumed affected. In group 4 were dogs with

atypical collapse: they showed recurrent episodes of collapse; however, the

description did not entirely match with the criteria for classification of

presumed affected. Group 5 consisted of dogs with collapse due to other

causes. The final group, group 6, consisted of dogs never observed to collapse.

Microsatellite markers. For the initial scan, we chose 459 microsatellite

markers dispersed across all 38 dog autosomes from published canine linkage

and radiation-hybrid maps22,23. Follow-up markers (n ¼ 22) were chosen from

the University of California-Davis canine linkage map (see URLs section below)

and in several cases from the assembled canine genome sequence24 (markers

denoted KM/JM in Supplementary Table 1, with primers listed in Supple-

mentary Table 3 online). A fluorescent primer was included in the PCR

reactions to generate labeled products that were size separated using the

Beckman CEQ (8000) automated DNA-fragment analyzer.

Linkage analysis. We selected 96 dogs (60 of them affected) from the six

pedigrees that contributed most of the statistical power in simulated linkage

analysis for the initial genotyping, and 252 dogs (143 of them affected) from

the same six pedigrees plus two additional pedigrees (Supplementary Fig. 1)

were ultimately included for fine mapping. Inbreeding loops were broken at the

most distant relationship. We confirmed mendelian inheritance and carried out

two-point parametric linkage analysis with FASTLINK25–27 assuming an

autosomal-recessive mode of inheritance. The frequency of the normal allele

was assumed to be 0.80 with the frequency of the affected allele 0.20. See

Supplementary Table 4 online for a summary of the genome scan. The actual

allele frequency and penetrance were not known; however, we used 80%

penetrance for homozygous affected and 0% penetrance for heterozygotes,

knowing that the disease is not 100% penetrant because dogs only collapse after

exposure to certain triggering events. See Supplementary Table 5 online for

details on the modeling of linkage analysis penetrance performed after the

initial linkage analysis. Dogs in phenotype groups 1 and 2 were categorized as

affected, dogs in groups 3–5 as unknown phenotype, and group 6 as unaffected.

We calculated allele frequencies and marker heterozygosity using unrelated

parents in the pedigrees. Significance levels for linkage were based on the

thresholds previously proposed28.

SNP marker association and haplotype analysis. SNP markers within the 56-

to 61-Mb region of CFA9 known to be informative in Labrador retrievers (see

URLs section below) were identified. A subset of these SNPs was genotyped

(Supplementary Table 6 online) using the Sequenom platform; SNP genotype

calls were filtered and examined manually, and aggressive calls were omitted

from the dataset. An intronic PTGES2 SNP was genotyped from a 392-bp PCR

product (Supplementary Table 3), which was digested with restriction enzyme

AvaI. The PTGES2 SNP is included in the statistical analysis, as was the DNM1

exon 6 SNP, for a total of 57 SNPs tested.

Association analysis was conducted with PLINK29 (version 1.01) (see URLs

section below). Pruning of the initial dataset with default parameters (exclusion

for 410% individual genotyping failure, 410% SNP genotyping failure,

and minor allele frequency o1%) resulted in 43 usable SNPs on 205

individuals. This population consisted of 108 affected, 84 unaffected and

13 dogs with unknown phenotypic status. A standard case-control association

analysis was conducted and adjusted for multiple testing (data reported are

Bonferroni-adjusted P values); however, the genomic inflation factor was 54.36,

indicating the existence of strong stratification. We then subjected the data

to identical-by-state clustering to control for stratification, using the pair-

wise population concordance (PPC) test with a 1% cut-off and the additional

criteria that at least one case and one control must be in each cluster.

This excluded any dogs with unknown phenotypic status and resulted in seven

clusters of dogs that could not be merged at the selected PPC cut-off.

We repeated the association analysis using the Cochran-Mantel-Haenszel

association statistic, which tests for disease-SNP association conditional

on the clusters. This lowered the genomic inflation factor to 7.24 and corrected

for a large amount of stratification. We recognized that the PPC test requires

whole genome–level data for ideal accuracy; however, we concluded that

this type of analysis was most appropriate for our dataset. The transmission

disequilibrium test option within PLINK was not used because of the

insufficient number of trios. Statistical significance was considered to be

a Bonferroni-corrected P value o0.01 (�log(P value) 42.0). We inferred

haplotype phase with PHASE version 2.1.1 software using the default

parameters (see URLs section below).

Genomic DNA sequencing. We designed PCR primers to amplify segments of

the positional candidate genes on the basis of the known intron-exon bound-

aries of the human and/or canine gene. In several cases, canine exons were not

well annotated in comparison to other species, and we used our best judgment

as to their correct positioning for PCR primer design and sequencing. Initially,

two affected dogs and two unaffected dogs were sequenced. We aligned

sequences with Sequencher software on a backbone of the assembled canine

genome sequence (CanFam2.0) and the human RefSeq coding-DNA sequences.

Genotyping the DNM1 G767Tmutation. We used intron-based PCR primers

(Supplementary Table 3) to generate a 337-bp fragment that contained all of

exon 6. Restriction enzyme SmlI (9 U with a 3 h incubation at 55 1C) cut the

T767 allele to generate fragments of 165 and 172 bp, which were resolved by

electrophoresis on a 2% agarose gel. More than 400 other Labrador retrievers

from field trials conducted in the upper Midwest, including dogs from 20 states

of the United States and 3 Canadian provinces, were collected and genotyped

for the DNM1 mutation.

Accession codes. For study of amino acid homology, sequences for alignment

were obtained from the following GenBank accession codes: Dog DNM1,

EU682271; Human DNM1, NP_004399.2; Human DNM2, NP_001005360;

Human DNM3, NP_056384.2; Mouse Dnm1, NP_034195.2; Mouse Dnm2,

NP_001034609.1; Mouse Dnm3, NP_001033708.1; Bovine DNM1,

NP_001092839.1; Chicken, XP_001233250.1; Danio rerio, NP_001025299.1;

Drosophila, NP_727910.1.

The canine DNM1 sequence has been deposited at GenBank and assigned

accession codes EU682271 and EU707921. Sequences for the canine AK1,

PTGES2 and SLC2A8 genes generated during this study are assigned accession

numbers EU707922, EU707923 and EU707924, respectively.

URLs. Sources for estimating annual kennel club registrations for Labra-

dor retrievers, http://www.cbc.ca/news/story/2007/01/16/dog.html, http://www.

akc.org/reg/dogreg_stats_2006.cfm, http://www.thekennelclub.org.uk/item/926,

http://www.ankc.org.au/home/default.asp, http://www.aniwa.com.br/en/chien/

Grand_Public/document/101820/108312/index.htm, http://www.cpc.pt/, http://

www.vdh.de/; University of California-Davis canine linkage map, http://www.

vgl.ucdavis.edu/cghg/resources.php; Broad Institute Canine SNP database,

http://www.broad.mit.edu/ftp/pub/papers/dog_genome/snps_canfam2/snp_

lists/chr9_allsnps.txt; PLINK, http://pngu.mgh.harvard.edu/purcell/plink/;

PHASE version 2.1.1, http://www.stat.washington.edu/stephens/instruct2.1.pdf.

Note: Supplementary information is available on the Nature Genetics website.

ACKNOWLEDGMENTSWe acknowledge the assistance of M. McCue and K. Matchett in manuscriptpreparation, S. Dalsen in figure preparation and C. Wade in SNP selection andanalysis. This work was funded in part by grants from the Morris AnimalFoundation to J.R.M., E.E.P. and S.M.T. (D01CA-021), and American KennelClub Canine Health Foundation to J.R.M. and E.E.P. (#352). The contents ofthis publication are solely those of the authors and do not necessarily reflect theviews of the Canine Health Foundation. This work is dedicated to the memoryof Monica C. Roberts, who was a major contributor to the initiation ofthese studies.

AUTHOR CONTRIBUTIONSS.M.T., E.E.P., J.R.M. and G.D.S. were responsible for the project’s conception andinitiation. S.M.T., G.D.S. and E.E.P. developed the phenotypic criteria. S.M.T.,K.M.M., G.D.S. and E.E.P. recruited dogs into the study. K.M.M., A.V.T. andE.E.P. performed the microsatellite genotyping and linkage analysis. K.M.M.,E.E.P., K.J.E. and J.R.M. were responsible for the SNP association and haplotype

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LET TERS

www.cbc.ca/news/story/2007/01/16/dog.html

www.akc.org/reg/dogreg_stats_2006.cfm

www.akc.org/reg/dogreg_stats_2006.cfm

www.thekennelclub.org.uk/item/926

http://www.ankc.org.au/home/default.asp

www.aniwa.com.br/en/chien/Grand_Public/document/101820/108312/index.htm

www.aniwa.com.br/en/chien/Grand_Public/document/101820/108312/index.htm

http://www.cpc.pt/

http://www.vdh.de/

http://www.vdh.de/

http://www.vgl.ucdavis.edu/cghg/resources.php

http://www.vgl.ucdavis.edu/cghg/resources.php

http://www.broad.mit.edu/ftp/pub/papers/dog_genome/snps_canfam2/snp_lists/chr9_allsnps.txt

http://www.broad.mit.edu/ftp/pub/papers/dog_genome/snps_canfam2/snp_lists/chr9_allsnps.txt

http://pngu.mgh.harvard.edu/purcell/plink/

http://www.stat.washington.edu/stephens/instruct2.1.pdf


analysis. K.M.M. and J.R.M. performed and analyzed the DNA sequencing.J.R.M., E.E.P. and S.M.T. were responsible for overall project oversight. E.E.P.and J.R.M. co-wrote the manuscript, which was edited by all co-authors.

COMPETING INTERESTS STATEMENTThe authors declare competing financial interests: details accompany the full-textHTML version of the paper at http://www.nature.com/naturegenetics/.

Published online at http://www.nature.com/naturegenetics/

Reprints and permissions information is available online at http://npg.nature.com/

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