Congenital myopathy results from misregulation of amuscle Ca2+ channel by mutant Stac3Jeremy W. Linsleya,b, I-Uen Hsub,1, Linda Groomc,1, Viktor Yarotskyyc,1, Manuela Lavoratod, Eric J. Horstickb,e,Drew Linsleyf, Wenjia Wangb, Clara Franzini-Armstrongd,1,2, Robert T. Dirksenc,2, and John Y. Kuwadaa,b,2

aCell and Molecular Biology Program, University of Michigan, Ann Arbor, MI 48109; bDepartment of Molecular, Cellular and Developmental Biology,University of Michigan, Ann Arbor, MI 48109; cDepartment of Pharmacology and Physiology, University of Rochester Medical Center, Rochester, NY 14642;dDepartment of Cell and Developmental Biology, University of Pennsylvania School of Medicine, Philadelphia, PA 19104; eDivision of DevelopmentalBiology, Eunice Kennedy Shriver National Institute of Child Health and Human Development, Bethesda, MD 20892; and fDepartment of Cognitive,Linguistic, and Psychological Sciences, Brown University, Providence, RI 02912

Contributed by Clara Franzini-Armstrong, November 23, 2016 (sent for review July 25, 2016; reviewed by Derek R. Laver and Jerome Parness)

Skeletal muscle contractions are initiated by an increase in Ca2+ releasedduring excitation–contraction (EC) coupling, and defects in EC couplingare associated with human myopathies. EC coupling requires commu-nication between voltage-sensing dihydropyridine receptors (DHPRs) intransverse tubule membrane and Ca2+ release channel ryanodine re-ceptor 1 (RyR1) in the sarcoplasmic reticulum (SR). Stac3 protein (SH3and cysteine-rich domain 3) is an essential component of the EC cou-pling apparatus and amutation in human STAC3 causes the debilitatingNative Americanmyopathy (NAM), but the nature of how Stac3 acts onthe DHPR and/or RyR1 is unknown. Using electron microscopy, electro-physiology, and dynamic imaging of zebrafish muscle fibers, we findsignificantly reduced DHPR levels, functionality, and stability in stac3mutants. Furthermore, stac3NAM myofibers exhibited increasedcaffeine-induced Ca2+ release across a wide range of concentrationsin the absence of altered caffeine sensitivity as well as increasedCa2+ in internal stores, which is consistent with increased SR luminalCa2+. These findings define critical roles for Stac3 in EC coupling andhuman disease.

zebrafish | skeletal muscle | excitation–contraction coupling |dihydropyridine receptor | Native American myopathy

Contraction of skeletal muscle is mediated by the sliding ofmyofilaments that is initiated by an increase in cytosolic Ca2+

released from the intracellular organelle, the sarcoplasmic re-ticulum (SR). Ca2+ release from the SR is a voltage-dependentprocess called excitation–contraction (EC) coupling that occurs atjunctions between the SR and invaginations of the sarcolemmacalled transverse (T) tubules that project into the interior of themuscle fiber called triads (1). Defects in EC coupling are the causeof congenital muscle myopathies labeled “triadopathies” that arecharacterized by defects in Ca2+ homeostasis and muscle weak-ness, for which there are few effective therapies (2).EC coupling in skeletal muscle is mediated by a triadic complex

that includes the dihydropyridine receptor (DHPR) and ryanodinereceptor 1 (RyR1), which are both Ca2+ channels (3, 4). DHPRslocated in the T tubule are voltage-gated, L-type channels that actas the voltage sensor for EC coupling. DHPRs are thought to directlyinteract with RyR1s in the SR membrane to rapidly trigger Ca2+ re-lease from the SR at triads upon depolarization of the T-tubulemembrane (5–7). Despite a wealth of knowledge of how DHPRs andRyR1 interact, the precise mechanisms by which this protein in-teraction is coordinated and modulated are poorly understood (8).Several congenital myopathies and the pharmacogenic disorder ma-lignant hyperthermia (MH), a potentially lethal response to volatileanesthesia that affects between 1:5,000 and 1:50,000 of the generalpopulation (9), are caused by defects in EC coupling. However, pre-cisely how genetic defects in proteins of the EC coupling complexcontribute to disease pathogenesis is incompletely understood.Recently, the cytosolic protein Stac3 was identified as an essential

component for skeletal muscle EC coupling in zebrafish (10) andmice (11). Stac3 also regulates hypertrophy and fiber-type composi-tion, and mutations in which it is responsible for impaired contrac-

tility in mouse muscles (12). Stac3 is expressed selectively in skeletalmuscle, colocalizes and biochemically associates with DHPR andRyR1 at triads, and is required for normal release of Ca2+ from theSR. Coexpression of Stac3 with DHPR in cultured nonmuscle celllines promotes the trafficking of the channel to the membrane,suggesting a role for Stac3 in trafficking and/or stabilization of theDHPR in the membrane (13). Furthermore, a hereditary triadopathycalled Native American myopathy (NAM) is caused by a missensemutation of STAC3 (10). NAM, an autosomal-recessive disorderfound within the Lumbee Native American population, is charac-terized by clinical features including congenital onset of muscleweakness, multiple joint contractures, dysmorphic facial features, andsusceptibility to MH, with 36% of afflicted individuals dying by theage of 18 (14). Analysis of the analogous mutation in zebrafish stac3showed that stac3NAM leads to a partial loss of Ca2+ release in musclefibers (10), yet the mechanism for how Stac3 and Stac3NAMmodulateEC coupling has remained undefined. Because there are currently noeffective therapeutic agents to treat congenital triadopathies, a bettermechanistic understanding of how mutations in EC components re-sult in myopathy could lead to the discovery of new therapies.

ResultsStac3 Is Necessary for Normal Levels of DHPRα. As a first step, thedistribution of DHPRs and RyRs was assayed quantitatively indissociated skeletal muscle fibers from wild-type (WT) and

Significance

Skeletal muscle contractions are regulated by a process calledexcitation–contraction (EC) coupling, and defects in it are asso-ciated with numerous human myopathies. Recently, stac3 (SH3and cysteine-rich domain 3) was identified as a key regulator ofEC coupling and a STAC3 mutation as causal for the debilitatingNative American myopathy (NAM). We now show that Stac3controls EC coupling by regulating Ca2+ channels in muscles.Both the NAM mutation and a mutation that leads to the loss ofStac3 decrease the amount, organization, stability, and voltagesensitivity of Ca2+ channels. Furthermore, we find evidence thatthe NAM allele of STAC3 is linked to malignant hyperthermia, acommon pharmacogenic disorder. These findings define criticalroles for Stac3 in muscle contraction and human disease.

Author contributions: J.W.L., R.T.D., and J.Y.K. designed research; J.W.L., I.-U.H., L.G., V.Y.,M.L., E.J.H., W.W., and C.F.-A. performed research; J.W.L., I.-U.H., and D.L. contributednew reagents/analytic tools; J.W.L., I.-U.H., L.G., V.Y., D.L., C.F.-A., R.T.D., and J.Y.K. ana-lyzed data; and J.W.L., C.F.-A, and J.Y.K. wrote the paper.

Reviewers: D.R.L., University of Newcastle; and J.P., Children’s Hospital of Pittsburgh.

The authors declare no conflict of interest.1I.-U.H., L.G., V.Y., and C.F.-A. contributed equally to this work.2To whom correspondence may be addressed. Email: armstroc@mail.med.upenn.edu,robert_dirksen@urmc.rochester.edu, or kuwada@umich.edu.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1619238114/-/DCSupplemental.

E228–E236 | PNAS | Published online December 21, 2016 www.pnas.org/cgi/doi/10.1073/pnas.1619238114

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stac3−/− (null) embryos. Whereas there was no difference in RyR1expression between WT and stac3−/− muscle (Fig. 1 A and B andFig. S1A), a significant reduction in DHPRα was observed withintriadic junctions when assayed with mAb1 (15), which recognizes acytoplasmic epitope in DHPRα (30% reduction; Fig. 1 C and D,Left), fluorophore-conjugated antibodies to minimize potential stericeffects of secondary antibodies within the compact triad (24% re-duction; t test, P < 0.05, n > 35), and an anti-DHPRα that recognizesan extracellular epitope (34% reduction; t test, P < 0.0001, n > 70)without detergent (Fig. S1 C–E). DHPRα expression was alsoreduced in stac3−/− fibers expressing stac3NAM-EGFP in comparisonwith those expressing stac3WT-EGFP (35% reduction; t test, P<0.0001, n > 53) (Fig. 1E). Hence, WT Stac3 was required for normalDHPRα, but not RyR1, expression within the triad.To assay whether the decrease in triadic DHPRα was due to

decreased synthesis of DHPRα, transcription of DHPR subunitswas examined by quantitative PCR. No differences were detected

in transcription levels of genes encoding for DHPRα1, DHPRβ1,and RyR1 [cacna1sa, cacna1sb, cacnb1, ryr1a, and ryr1b (16)] be-tween stac3−/− embryos and WT siblings (Fig. S2). Thus, althoughStac3 was required for normal levels of DHPR expression, this wasnot due to regulation of DHPRα1 or DHPRβ1 transcription.A defect in DHPR trafficking to the T tubule is one potential

mechanism for the observed reduced triadic DHPR levels in stac3mutants. In fact, a recent study showed that Stac3 was sufficientto promote DHPR trafficking to the plasma membrane in non-muscle cell lines (13). To more directly test this mechanism, weexamined the role of Stac3 for trafficking of DHPRα with fluo-rescence recovery after photobleaching (FRAP) analysis ofEGFP-DHPRα–expressing WT and stac3−/− muscles. In dhprβ1-null (relaxed) fibers, which exhibit defective DHPRα trafficking(17), EGFP-DHPRα was not localized to triads as expected (Fig.S1E). However, in stac3−/−muscle fibers, EGFP-DHPRα localizedto triads just as it does in WT sibling fibers (Fig. 1E, Left). Tracesof fluorescence from WT siblings and stac3−/− muscles showed thatthe recovery of fluorescence was temperature-sensitive andoccurred in the first 30 min after bleaching with about 30% ofthe fluorescence being mobile at room temperature (Fig. 1F andFig. S3). Although the mobile fraction was not different betweenstac3−/− and WT siblings (t test, P = 0.88) (Fig. 1F), the diffusionrate of EGFP-DHPRα fluorescence to the plateau was signifi-cantly higher in triads of stac3−/− muscle (Fig. 1 G and H). Thus,the loss of Stac3 appears not to prevent trafficking of DHPRα tothe triad in skeletal muscles.

Stac3 Is Required for Normal Triadic DHPR but Not for Linkage to RyRs.The positioning of DHPRs within surface membrane/T tubules ofcalcium release units (CRUs) in skeletal muscle is determined bytheir interaction with RyR1 in the adjoining SR (18). Each DHPRoccupies one corner of the four subunits of the RyR1 homote-tramer. In freeze-fracture images of mature CRUs, the four cor-ners of most RyRs are fully occupied by DHPR particles identifiedby their characteristic size signature (18) such that the DHPRs arearranged in groups of four particles called tetrads. The tetrads arealigned along the T-tubule axis, as dictated by their linkage toRyRs, yet interestingly positioned over alternate RyR1.In freeze-fracture images of T tubules in tails of ∼96-h post-

fertilization (hpf) larvae (Fig. 2 A and A′), the locations of tetradswere confirmed because the distance between the centers of tet-rads was twice the distance between the feet (RyRs) that occupiedthe junctional SR membrane facing the T tubules (Fig. S4A).Tetrads were often incomplete, with one or more DHPR particlesmissing. Only 62% of tetrads were complete in WT larvae. Fur-thermore, T tubules contained junctional segments with tetradsand tetrad-free segments (purple in Fig. 2). In WT, the tetrad-freesegments were directly related to discontinuities in the junctionalSR membrane at triads, as the junctional SR membranes do notcover the entire T-tubule length (Fig. S4A). On the average, thejunctional T-tubule segments made up ∼65% of the total T-tubulelength (Table 1) and the average number of particles per arbitrarylength of T tubule was 2.2 in WT larvae (Fig. 2G). The number ofcomplete tetrads for the same length of T tubule was 0.4 (Fig. 2H).In stac3−/−, T tubules contained junctional and tetrad-free seg-

ments as in WT but the overall frequency of DHPR particles in theT tubules was reduced (51% reduction; Fig. 2 D, D′, and G). Nev-ertheless, the structural relationship of DHPRs with RyRs was es-sentially unaltered in stac3−/− as the remaining particles were groupedin tetrads, albeit with many more incomplete tetrads (Table 1).However, within the junctional T-tubule segments, the spacing be-tween the tetrads in stac3−/− was the same as in WT (Fig. S4D;ANOVA, P = 0.07), reflecting unaltered underlying spacing betweenthe RyR feet in stac3−/− embryos (t test, P = 0.11; Fig. S4A–C and E).The major difference between WT and stac3−/− T tubules was anincrease in tetrad-free segments (Fig. 2D′) and thus a decrease in theratio of junctional to total T-tubule length (Table 1), resulting in a

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Fig. 1. DHPRα1 but not RyR1 is reduced in T-tubule striations of stac3 mu-tants. (A) Immunofluorescence labeling of WT sibling and stac3−/− dis-associated myotubes with anti-pan RyR (34c). (B) Mean immunofluorescenceintensity of anti-RyR in stac3−/− compared with WT siblings showing no dif-ference in triadic RyR (t test, P = 0.89, n = 85 WT sibling, n = 50 stac3−/−). a.u.,arbitrary units. (C) Immunofluorescence labeling of WT sibling and stac3−/−

disassociated myotubes with mAb1 1A against a cytoplasmic region ofDHPRα1S (15). (D) Mean mAb1 1A labeling in WT siblings and stac3−/− showinga decrease in triadic DHPRα1S (t test, ***P < 0.0001, n = 216 WT sibling, n = 264stac3−/−). (E) Quantification of the mean of immunofluorescence labeling ofanti-DHPRα1S in stac3−/− expressing stac3NAM (NAM rescue) at triads comparedwith stac3−/− expressing stac3WT (WT rescue) (n = 75 stac3−/−; stac3WT, n = 53stac3−/−; stac3NAM, t test, ****P < 0.0001). (F) Time course for FRAP of EGFP-DHPRα1S expressed in WT siblings and stac3−/− myofibers. Shown are EGFP-DHPRα1S before (prebleach), after photobleaching (T = 0, 5, 34 min), and amaximum projection (stack) of T = 30 to 34 min (Right). (G) Mean quantificationof the time course of FRAP inWT siblings (thick green line and circles) and stac3−/−

(thick red line and circles). Thin lines represent nonlinear regressions from indi-vidual traces of FRAPs from WT siblings (green) and stac3−/− (red). The verticalthick green line depicts bleaching. (H) Histogram showing that the diffusion rate(D) of EGFP-DHPRα1S is higher in stac3−/− (t test, ***P < 0.0001, n = 33 stac3−/−,n = 45 WT siblings). SEMs are indicated. (Scale bars, 2 μm.)

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Fig. 2. DHPR tetrads are reduced and incomplete in stac3mutants. (A–E) Freeze-fracture electron micrographs of 4-d postfertilization larvae showing DHPR particles intriadic clusters of WT (A), stac3−/− expressing stac3WT-EGFP (WT rescue) (B), stac3−/− expressing stac3NAM-mKate2 (NAM rescue) (C), stac3−/− (D), and stac3−/− injected withan antisense morpholino oligonucleotide against stac3 (stac3−/− + MO) (E). (A′–E′) Same as A–E, with yellow dots and purple shading added for clarity to denote, re-spectively, segments of T tubules with tetrad sites of DHPRs and segments of T tubules with no tetrad sites in muscle fibers of WT (A′), WT rescue (B′), NAM rescue (C′),stac3−/− (D′), and stac3−/− +MO (E′). (F) Illustration showing stereotypical DHPR particles in tetrad sites (labeledwith yellow dots) along a T tubule and gaps with no tetradsites (purple) as seen above. (G) Histogram showing that the particles per T-tubule length are decreased in NAM rescue, stac3−/−, and stac3−/− + MO muscles comparedwith WT and WT rescue. (ANOVA Tukey’s; ***P < 0.001, **P < 0.01.) (H) Histogram showing that full tetrads per tetrad site are decreased in NAM rescue, stac3−/−, andstac3−/− + MO muscles compared with WT and WT rescue. ns, not significant. SEMs are indicated. (ANOVA Tukey’s; ***P < 0.001, *P < 0.05.)

E230 | www.pnas.org/cgi/doi/10.1073/pnas.1619238114 Linsley et al.

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significant reduction in the average frequency of particles per unit ofT-tubule length (Fig. 2G). Furthermore, there were fewer completetetrads in stac3−/−, which further depressed the frequency of tetradsper tetrad site (Fig. 2H).Previous analyses showed that stac3−/− muscles exhibited a clear

decrease in contraction but were not immotile due to maternalmessage in stac3−/− embryos, and that knocking down maternalStac3 rendered the stac3−/− embryos immotile (10). To see howthe loss of both maternal and zygotic stac3 affects DHPR particledistribution, an antisense oligonucleotide (MO) against stac3 thateffectively knocks down Stac3 in the absence of off-target effects(9) was injected into stac3−/− embryos. The frequency of DHPRparticles, complete tetrads, and proportion of T-tubule lengthcontaining clusters of particles in MO-injected stac3−/− larvae wereall decreased even more than seen in stac3−/− larvae (Fig. 2 D–Hand Table 1). Furthermore, complete tetrads were extremely rare.Note, however, that vestiges of particle grouping were still visibleeven in MO-treated stac3−/− larvae (Fig. 2E′), indicating that thelink of DHPRs to RyR subunits persists when Stac3 is nearly orcompletely absent.Expression of WT Stac3 in stac3−/− muscles fully restored

DHPR particle (Fig. 2 B, B′, and G) and tetrad (Fig. 2H) fre-quency to WT levels, confirming that Stac3 promotes DHPRparticle localization and organization within the junctional T tu-bule. To examine how Stac3NAM affects particle and tetrad fre-quencies, the distribution of DHPRs in tetrads was examined instac3−/− muscles expressing stac3NAM. In stac3NAM fibers, thenumber and grouping of DHPR particles and complete tetradsand the proportion of T tubule that contained groups of particleswere similar to that in stac3−/− fibers (Fig. 2 C–D′, G, and H andFig. S4) and significantly lower than in stac3−/− fibers express-ing stac3WT (Fig. 2 B–C′, G, and H and Table 1). As was thecase with the stac3−/− fibers, particle groupings and spacing wereunaltered in stac3NAM-expressing muscles, consistent with a normalrelationship and spacing of underlying RyRs despite the reductionin DHPR particle frequency (Fig. S4D). These findings indicatethat the NAM W-to-S substitution in the first SH3 domain ofStac3 (10) decreases the number of DHPR particles within thetransverse tubule membrane but does not affect the positioning ofDHPRs into tetrads above the underlying RyR1 array.

Loss of Stac3 Destabilizes the DHPR Complex at Triads. The reducedlevels of DHPRs in triadic junctions of stac3−/− muscle (Fig. 1)coupled with their continued association with RyRs raised thepossibility that stac3 may regulate the stability of DHPRs attriads. To test this hypothesis, we developed an optical pulse-labeling method in which the gene encoding the DHPRα subunit(cacna1sa) was tagged with a photoconvertible protein, mEos3.2,that irreversibly switches spectral properties (green to red) whenexposed to short-wavelength light (405 nm) (19). Using time-lapse confocal microscopy, we monitored loss of the photoconvertedsignal in triads as an index of the stability of triadic DHPRα. Afterphotoconversion, the red fluorescence of mEos3.2-DHPRα at

triads gradually decreased over the course of an hour in both WTsibling and stac3−/− embryos. The decrease in fluorescence wasnatural log-transformed and fit to a linear regression, and a decay(β) coefficient was calculated (Table S1). Fluorescence decay oftriadic mEos3.2-DHPRα in stac3−/− fibers occurred at a signifi-cantly faster rate than that observed in WT siblings (Fig. 3 A,B, and E). Relative fluorescence of triadic mEos3.2-DHPRα instac3−/− fibers expressing Stac3NAM also decreased faster than thatof triadic mEos3.2-DHPRα in stac3−/− fibers expressing Stac3WT

(Fig. 3 C–E). These results suggest that DHPRα was destabilizedin triads of stac3−/−- and stac3NAM-expressing stac3−/− mus-cles. In contrast, the stability of photoconverted mEos3.2-β-DG, aprotein found at triads not thought to be involved with EC coupling(20), expressed in stac3−/− fibers was equivalent to that of WT fibers(Fig. 3F), suggesting that Stac3 selectively stabilizes DHPR at tri-ads. Thus, Stac3 is necessary for normal stability of DHPRα attriads, which may in part explain the reduction in DHPR particlesper T-tubule length and tetrads per tetrad site (Fig. 2 G and H)observed in stac3-null and Stac3NAM-expressing muscle.Although stac3NAM expression destabilized DHPRα compared

with stac3WT, Stac3NAM protein nevertheless localized to the triad(Fig. 3 C and D). To understand how Stac3NAM increased DHPRαinstability, the stability of Stac3NAM at triads was assayed by FRAPanalysis of Stac3NAM-GFP– and Stac3WT-GFP–expressing stac3−/−

muscle fibers. The mobile fraction of Stac3NAM-GFP expressed instac3−/− fibers was significantly greater than that of Stac3WT-GFPexpressed in stac3−/− fibers, despite the fact that the diffusion rates ofStac3NAM-GFP and Stac3WT-GFP were not different (Fig. 3G–I).Therefore, the Stac3NAM protein is less stable at triads than theStac3WT protein, which correlates with the decreased stability oftriadic DHPRα in stac3−/− expressing Stac3NAM, although thetrafficking rate of Stac3NAM to the triad was unchanged.

Stac3 Enhances DHPR Intramembrane Charge Movement and Voltage-Gated SR Ca2+ Release. Because DHPRs serve as the voltage sensorfor EC coupling, we examined how Stac3 affects the voltage de-pendence of DHPR charge movements and Ca2+ release from theSR. In zebrafish muscles, DHPRs do not pass ionic Ca2+ current,as is observed for DHPRs in mammalian skeletal muscle (16).However, DHPR functionality in fish can be assayed by measuringthe magnitude and voltage dependence of intramembrane chargemovement. Electrophysiological recordings from disassociatedmyotubes indicated an almost complete absence of intramembranecharge movement (Q) in stac3−/− fibers compared with that ob-served for WT fibers (Fig. 4 A and C and Table 1).To test the effect of the stac3NAM mutation on the DHPR charge

movement, stable muscle actin:stac3NAM-EGFP; stac3−/− transgeniclines were generated and compared with muscle actin:stacWT-EGFP;stac3−/− transgenic embryos. Expression ofmuscle actin:stac3WT-EGFPin stac3−/− fibers restored maximum charge movement (Qmax)back to WT sibling levels (Fig. 4 A and C and Table 1). How-ever, in fibers expressing muscle actin:stac3NAM-EGFP myo-tubes, Qmax was significantly decreased compared with muscle

Table 1. Particles, tetrads, and functionality statistics

TypeNo. of particlesper tetrad site

No. of completetetrads pertetrad site

Junctional T tubule/totalT-tubulelength Qmax, nC/μF ΔF/Fmax ΔF/F V1/2, mV KF, mV

WT 3.40 ± 0.52 (n = 84) 0.62 ± 0.36 0.65 ± 0.13 (n = 42) 8.4 3.87 ± 0.37 −14.6 ± 2.3 6.2 ± 0.5WT rescue 3.04 ± 0.70** (n = 72) 0.45 ± 0.31* 0.68 ± 0.13** (n = 40) 9.5* 2.30 ± 0.21* −21.2 ± 1.8* 7.2 ± 0.8NAM rescue 2.76 ± 0.44**,ns (n = 43) 0.23 ± 0.25*,ns 0.44 ± 0.13**,ns (n = 38) 0.9*,ns 0.28 ± 0.04*,nd −2.3 ± 6.3*,nd 17.2 ± 2.8*,ndstac3−/− 2.52 ± 0.54** (n = 31) 0.23 ± 0.41** 0.42 ± 0.14** (n = 31) 1.1* 0* nd ndstac3−/− MO 2.37 ± 0.44ns (n = 35) 0.08 ± 0.14# 0.26 ± 0.14# (n = 28) nd nd nd nd

Tukey post hoc comparisons: WT rescue vs. stac3−/−. NAM rescue vs. WT rescue (first P value) and NAM rescue vs. stac3−/− (second P value). stac3−/− vs. WT.ANOVA, **P < 0.001, *P < 0.01. t test was used for stac3−/− MO vs. stac3−/−. #t test, P < 0.05. nd, not determined.

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actin:stac3WT-EGFP–expressing stac3−/− and WT sibling fibers,and comparable to that of stac3−/− fibers. These data indicatethat Stac3, and more specifically the W residue in the first SH3domain of Stac3 that is replaced by S in stac3NAM, is requiredfor normal DHPR charge movement in zebrafish muscle.We previously showed that stac3−/− muscle fibers expressing

stac3NAM-mCherry exhibited a partial release of Ca2+ fromthe SR with slower kinetics than usual (10). To get a morecomprehensive characterization of the magnitude, kinetics, andvoltage dependence of Ca2+ release, depolarization-inducedchanges in intracellular Ca2+ were assessed using Fluo-4 AMrecorded simultaneously with charge movement in myofibers ofWT, stac3−/−, and stable transgenic stac3−/− embryos expressingStac3WT or Stac3NAM. In stac3−/− fibers, Ca2+ release was nearlyabsent in response to voltage-clamp depolarizations of the muscleplasma membrane, whereas expression of muscle actin:stac3WT-EGFP restored robust Ca2+ release, exhibiting a sigmoidal voltagedependence (Fig. 4 B and D and Table 1). Despite the near ab-sence of detectable charge movement (Fig. 4 A and C), stac3−/−

fibers expressing muscle actin:stac3NAM-EGFP exhibited a lowlevel of voltage-dependent Ca2+ release that was significantlyhigher than that observed in stac3−/− fibers (Fig. 4 B and D,Insets), indicating that muscle actin:stac3NAM-EGFP expressionrestored some Ca2+ release. In addition, voltage-gated Ca2+ release

in myofibers from muscle actin:stac3NAM-EGFP was significantlyright-shifted (V1/2max −2.3 ± 6.3 mV) and exhibited a dramati-cally shallower slope (slope 17.2 ± 2.8 mV) compared with thatof muscle actin:stac3WT-EGFP (V1/2max −21.2 ± 1.8 mV, slope7.2 ± 0.8 mV) (Table 1). Similar decreases in Ca2+ release wereobserved when delivering field stimulation of 10 Hz to fibers tomimic trains of action potentials exhibited by muscles duringevoked swimming (10, 21) (Fig. S5). Thus, Stac3 was required forboth normal DHPR charge movement and voltage-dependentSR Ca2+ release.

Embryos Expressing Stac3NAM Exhibit Decreased Swimming andIncreased Caffeine Responsiveness. Stac3NAM-expressing myofibersare able to release a low level of Ca2+ in response to voltage-clampdepolarization (Fig. 4D) and electrical stimulation (Fig. S5). Thispredicts that muscle actin:stac3NAM-EGFP; stac3−/− transgenicembryos should exhibit more mobility than stac3−/− embryos. In-deed, muscle actin:stac3NAM transgenic embryos were significantlymore motile than stac3−/− embryos but less motile than stac3−/−

embryos expressing muscle actin:stac3WT (Fig. 5 A and B). Pre-sumably, the lack of swimming in embryos injected with stac3NAM

expression constructs previously reported (9) was due to mosaicand inconsistent expression of Stac3NAM. Thus, the stable andconsistent expression of Stac3NAM within transgenic fish used here

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Fig. 3. DHPRα is less stable in stac3mutants. (A and B) Time course for optical pulse-labeling assay of mEos3.2-DHPRα1S expressed in themyofibers ofWT sibling (A)and stac3−/− (B). Green channel (Top) and red channel (Bottom) before photoconversion (Left), immediately following photoconversion (Middle), and 60 min afterphotoconversion (Right). (C and D) Time course for optical pulse-labeling assay of mEos3.2-DHPRα1S in stac3−/− muscles expressing stac3WT-mKate2 (C) or stac3NAM-mKate2 (D). Blue channel representing the far-red mKate2 fluorescence (Top), green channel (Middle), and red channel (Bottom) for mEos3.2-DHPRα1S fluorescenceas in A and B. (E) Time course of decay of photoconverted mEos3.2-DHPRα1S shows that fluorescence decays faster in stac3−/− (n = 24) compared withWT siblings (n = 24)(t-permutation test, P < 0.001) (Left) and that photoconverted mEos3.2-DHPRα1S decays faster in myofibers of stac3−/− expressing stac3NAM (n = 20) compared withexpressing stac3WT (n = 20) (t-permutation test, P < 0.001) (Right). (F) Time course of decay of photoconvertedmEos3.2-β-dystroglycan inWT siblings (n = 9) and stac3−/−

(n = 9) shows that fluorescence decays at the same rate inWT and stac3−/− (t-permutation test, P = 0.86). (G) FRAP analysis of stac3−/−myofibers expressing stac3WT-EGFP(Top) or stac3NAM-EGFP (Bottom) before photobleaching (Left), immediately after photobleaching (Middle), and 30 min after photobleaching (Right). (H) Mean timecourse of FRAP of stac3−/− myofibers expressing stac3WT (n = 18) and stac3NAM (n = 36). (I, Top) Histogram showing the percentage of the mobile fraction is larger instac3−/−myofibers expressing stac3NAM compared with stac3WT (t test, P < 0.0001). (I, Bottom) Histogram showing that the rate of recovery following photobleaching isunchanged between stac3−/− myofibers expressing stac3WT and stac3NAM (t test, ***P = 0.9). SEMs are indicated. n.s., not significant. (Scale bars, 2 μm.)

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is likely sufficient for the low-level swimming observed in thesefish despite low-level Ca2+ transients observed in Stac3NAM mus-cle fibers (Fig. 4 and Fig. S5).Individuals homozygous for the Stac3NAMmutation are susceptible

to malignant hyperthermia, a pharmacogenic disease characterizedby Ca2+ dysregulation in skeletal muscle. Because NAM is found in arelatively homogeneous population, the Lumbee Native Americans,other genetic factors within the population could contribute to theMH susceptibility seen in NAM patients. To determine whetherstac3−/− and/or NAM muscle fibers might exhibit characteristics ofMH, the caffeine responsiveness of fibers of stac3−/− andmuscle actin:stac3NAM-EGFP; stac3−/− embryos was examined, because an in-creased sensitivity to caffeine is observed in muscle fibers exhib-iting MH. Ca2+ release in stac3−/− fibers in response to increasingconcentrations of caffeine was not different from that observed forWT siblings (Fig. 5C). However, myofibers from muscle actin:stac3NAM-EGFP embryos exhibited significantly higher Ca2+

release than fibers from muscle actin:stac3WT-EGFP; stac3−/−

embryos (Fig. 5D) at all concentrations of caffeine tested. Thus,doses of caffeine above saturation (>10 mM) induced significantly

greater Ca2+ release in muscle actin:stac3NAM-EGFP; stac3−/− fi-bers, consistent with higher luminal Ca2+ levels in the SR ofmyofibers from muscle actin:stac3NAM-EGFP; stac3−/− embryos.This idea was also tested by application of a rapid Ca2+ releasemixture (ICE; 10 μM ionomycin, 30 μM cyclopiazonic acid, and100 μM EGTA/0 Ca2+) to deplete intracellular Ca2+ stores as anindex of total Ca2+ store content (22). Using this assay, total re-leasable Ca2+ store content was not different between myofibersfrom WT and stac3−/− embryos (Fig. 5E) but was significantly in-creased in myofibers from muscle actin:stac3NAM embryos com-pared with myofibers from muscle actin:stac3WT transgenics (Fig.5F). These results are consistent with the idea that luminal SRCa2+ is increased in stac3NAM embryos.

DiscussionOur results indicate that a relatively modest reduction (∼24 to34%) in DHPRα1S expression within the triad junction of stac3−/−

and stac3NAM muscle is not sufficient to explain the near-completeloss of depolarization-induced Ca2+ release. In addition, our freeze-fracture results support previous work indicating that complete

WT sibling WT rescue NAM rescue stac3 -/-A20 mV

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Fig. 4. DHPR charge movement and SR Ca2+ release are dramatically reduced in stac3 mutants. (A) Representative DHPR gating currents elicited by testpulses (Vtest) to +20 mV, −10 mV, and −40 mV from a holding potential of −80 mV in myofibers from WT siblings, stac3−/− expressing stac3WT-EGFP (WTrescue), stac3−/− expressing stac3NAM-EGFP (NAM rescue), and stac3−/− zebrafish. Gating currents in WT rescue fibers were comparable to WT fibers butdramatically decreased in stac3−/− and NAM rescue fibers. (B) Representative Fluo-4 fluorescence traces elicited by 200-ms test pulses to −40, −10, and +20 mVin fibers from WT siblings, WT rescue, NAM rescue, and stac3−/−. Fluo-4 transients in WT rescue myofibers were comparable to WT fibers but dramaticallydecreased in stac3−/− and NAM rescue fibers. (Insets) Magnifications (10×) of the Fluo-4 transients (green) in NAM rescue and stac3−/− fibers. (C) The voltagedependence of the integrated ON component of intramembrane DHPR charge movement was comparable between myofibers from WT siblings (n = 10) andWT rescue (n = 14) (ANOVA, ns) but dramatically decreased in fibers from stac3−/− (n = 19) and NAM rescue (n = 12) fibers. Data from stac3−/− fibers were toosmall to be accurately fit (SI Materials and Methods). (D) Voltage dependence of Fluo-4 transients in fibers from WT siblings (n = 6), WT rescue (n = 13), NAMrescue (n = 8), and stac3−/− (n = 6) (ANOVA Tukey’s, P < 0.01). (Inset) Fluo-4 transients were small but clearly detectable in NAM rescue fibers but not in stac3−/−

fibers. SEMs are indicated.

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tetrads represent the critical DHPR structural unit required forfunctional EC coupling (17, 23–25). Indeed, the number of com-plete tetrads and amount of DHPR charge movement are tightlycorrelated in each genotype studied (Table 1). Additionally,knockdown of maternal stac3 message in stac3−/− embryos resultedin few to no complete tetrads (Fig. 2 and Table 1) and completeimmotility (10). These data demonstrate the importance of defectsin tetrads as a fundamental mechanism for altered EC coupling inhuman myopathies.As expected, the number of complete tetrads varies as the

fourth power of DHPR expression in all genotypes examined,suggesting that the decrease in complete tetrads is due to a de-crease in triadic DHPRs in stac3 mutants. Interestingly, DHPRcharge movement and SR Ca2+ release are decreased dispro-portionately to that of complete tetrads and DHPR particles instac3−/− and stac3NAM fibers (Table 1). This suggests that Stac3regulates charge movement and EC coupling in ways beyond

simply regulating the amount of complete DHPRs and tetrads attriads. One possibility is that Stac3 may be required for theproper conformation of DHPRs in T tubules required for volt-age-dependent charge movement. Consistent with this, chargemovement and voltage-gated Ca2+ release are also reduced inmyotubes from Stac3-null mice (26), although the magnitude ofcharge movement reduction in Stac3-null and Stac3NAM-expressingzebrafish fibers was greater than that observed in mouse myotubes.In addition, the lack of proper folding of the DHPRα in theT-tubule membrane has been proposed as the basis for a similarnear-complete absence of DHPR charge movement in theDHPRβ1a-null zebrafish (17, 24).DHPRα is less stable at triads in stac3−/− and stac3NAM fibers.

The reduced stability of DHPRα is presumably responsible forthe decrease in triadic DHPRs observed in these fibers. HowStac3 regulates the stability of DHPRs is unknown. Stability ofDHPRs could be regulated by Stac3 once DHPRs are in the

WT sibling + NAM stac3 -/-

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Fig. 5. Stac3NAM transgenic zebrafish have reduced motility and hallmarks of malignant hyperthermia. (A) Overlaid traces of touch-evoked swimming bytransgenic 72-hpf WT siblings expressing stac3NAM-EGFP (WT sibling + NAM), stac3−/−, transgenic stac3−/−;stac3WT-EGFP (WT rescue), transgenic stac3−/−;stac3NAM-EGFP (NAM rescue), and stac3−/− showing that whereas stac3−/− embryos do not swim, NAM rescue embryos do. (B) Histograms of the speed ofswimming by WT sibling + NAM (n = 55, 11, and 32 at 56, 72, and 96 hpf, respectively), stac3−/− (n = 15, 15, and 15), WT rescue (n = 8, 20, and 91), and NAMrescue (n = 18, 55, and 58) show that NAM rescue zebrafish exhibit partial rescue of swimming compared with WT rescue (ANOVA Tukey multiple com-parisons, ****P < 0.0001, **P < 0.001). (C) Dose–response plots of Ca2+ release as a function of caffeine concentration indicate that stac3−/− muscles do notshow increased Ca2+ release in response to caffeine compared with WT sibling muscles (0.3 mM, n = 30, 30; 1.0 mM, n = 19, 14; 10.0 mM, n = 16, 20; 30.0 mM,n = 18, 29). The data were fit with a sigmoidal with Hill slope of 1. (D) Dose–response plots of Ca2+ release as a function of caffeine concentration show thatNAM rescue transgenic muscles release more Ca2+ compared with WT rescue transgenic muscles. Each point represents the average maximal caffeine responserelative to the baseline immediately before caffeine application (0.3 mM, n = 171, 192; 1.0 mM, n = 24, 20; 10.0 mM, n = 16, 16; 30.0 mM, n = 22, 23) (t-testcomparisons, ****P < 0.0001, **P < 0.01, *P < 0.05). (E) Histogram showing that mean peaks of Ca2+ released from internal stores induced by application ofthe ICE release mixture are comparable between WT sibling (n = 27) and stac3−/− (n = 35) myofibers (t test, P < 0.7). ns, not significant. (F) Histogram showingthat the mean peak of Ca2+ released from NAM rescue fibers (n = 45) is higher than in WT rescue fibers (n = 30, t test, *P = 0.01). SEMs are indicated.

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triadic T-tubule membrane. Another possibility is that Stac3 mayact as a triad-localized chaperone responsible for proper in-sertion of DHPRs into the T-tubule membrane. The absence ofStac3 to properly fold/insert DHPRα1S proteins into the T-tu-bule membrane would increase the fraction of DHPRα1S pro-teins available for degradation, perhaps explaining the increasedDHPR mobility and instability in stac3−/− fibers (Fig. 6).DHPRβ1a-null zebrafish also display reduced DHPR triadic

levels, charge movements, tetrad formation, and voltage-gated Ca2+

release. However, unlike DHPRβ1a, a chaperone that is cotrans-ported with DHPRα1S to the triadic junction, Stac3 is required in-stead for stability of DHPRα1S within the triadic junction (17, 23, 24).Interestingly, whereas both Stac3-null and DHPRβ1a-null fibers showa 90% reduction inQmax, Stac3-null shows a rightward shift in voltagedependence whereas DHPRβ1a-null exhibits a leftward shift. Addi-tional work is needed to further elucidate how DHPRβ1a and Stac3cooperate to facilitate the formation of DHPR into functional tetrads.Although NAM patients are susceptible to MH, a causal role of

Stac3NAM in MH had not been previously established. As a result,STAC3 is not routinely examined as a candidate gene in screensfor MH in the general population (27, 28). The data presentedhere strengthen the link between Stac3 and MH susceptibility andpotentially provide a novel animal model for the study of MHsusceptibility. Furthermore, because zebrafish are amenable tohigh-throughput drug screens, stac3NAM zebrafish have the po-tential to become a powerful tool for new drug discovery in thetreatment of NAM and, potentially, MH susceptibility.Analysis of stac3NAM zebrafish may also contribute to a greater

understanding of the pathogenesis and mechanism of MH. In-triguingly, the mechanics of Ca2+ dysregulation in Stac3NAM-expressing myofibers appears to be unique. Previous studies haveshown that mutations in RyR1(29–31) or DHPRα1S (32, 33)linked to increased MH susceptibility result in an increase in thesensitivity of caffeine-induced Ca2+ release that occurs in theabsence of a change in maximal caffeine-induced Ca2+ release.In contrast, we found that whereas caffeine-induced Ca2+ releasewas unaltered in stac3−/− fibers, stac3NAM fibers exhibited in-creased Ca2+ release in response to all concentrations of caf-feine, suggesting that SR luminal Ca2+ may be higher in stac3NAM

fibers and that this may contribute to higher MH susceptibility.The increased Ca2+ release in response to caffeine observed instac3NAM fibers could in principle be caused by increases inseveral factors, including the number of activated RyR1 chan-nels, RyR1 open-channel probability or single-channel conduc-tance, and/or the chemical driving force Ca2+ release. However,because triadic RyR1 levels were not increased in stac3NAM

myofibers, an increase in RyR1s appears not to underlie the

increase in caffeine-induced Ca2+ release. Although a potentialeffect of stac3NAM on RyR1 open-channel probability and single-channel conductance is unclear, we found that total releasableCa2+ store content was increased in rescued stac3NAM fiberscompared with rescued stac3WT fibers. This observation raises thepossibility that increased SR Ca2+ content, and possibly luminalCa2+ regulation of RyR1 activity (34), contributes to the increasedcaffeine responsiveness observed in rescued stac3NAM fibers.Increased luminal Ca2+ levels in rescued Stac3NAM fibers could

be explained by either increased ability to sequester Ca2+ and storeCa2+ within the SR such as through increased sarco/endoplasmicreticulum calcium ATPase (SERCA) activity or calsequestrin ex-pression, or through a reduction of steady-state SR Ca2+ leak.Interestingly, a significant component of myoplasmic Ca2+ ho-meostasis depends on SR Ca2+ leak via RyR1, which is tightlycontrolled through the orthograde interaction between DHPRα1Sand RyR1 (35, 36). DHPRα1S normally suppresses RyR1 Ca2+

leak, and a mutation in DHPRα1S linked to MH (R174W) hasbeen proposed to promote RyR1 in the leak state (37). Thecontinued presence of DHPRα1S and its ability to suppress RyR1opening could also explain how elevated SR Ca2+ in Stac3NAM fibersdoes not induce RyR1 to open at resting state, as has been reportedwhen RyR1 is expressed in HEK293 cells without DHPRα1S (34).Our data indicate that although inefficient, DHPRα1S structural (Fig.2) and functional (Fig. 4) coupling to RyR1 is maintained in rescuedStac3NAM fibers. Thus, the observed increase in Ca2+ store contentand caffeine responsiveness in rescued Stac3NAM fibers could resultin part from increased DHPRα1S-mediated suppression of RyR1 Ca2+

leak that facilitates accumulation of SR luminal Ca2+ levels. Addi-tionally, the marked reduction in voltage-gated Ca2+ releaseobserved in rescued Stac3NAM fibers would further promote anincrease in luminal SR Ca2+ content. The consequences of ex-cessive SR Ca2+ levels and its contribution to the pathology andMH susceptibility of NAM are areas that require furtherinvestigation.

Materials and MethodsAnimal Behavioral Analysis. Zebrafish were bred and maintained according toapproved guidelines of the University Committee on Use and Care of Animals atthe University of Michigan. stac3mi34 (stac3−/−) carriers were raised to 48 hpf andstac3−/−mutant embryos were behaviorally identified as previously described (10).

Disassociation of Zebrafish Myotubes and Quantification of Triadic Fluorescence.Zebrafish myofibers were dissociated with collagenase, and fluorescence wasquantified as previously described (17). Embryos were identified by behaviorand fluorescence, and the heads of embryos were removed for genotypingbefore disassociation of tails. Photoactivation of mEos3.2 and quantification offluorescence are described in SI Materials and Methods.

Measurement of Cav1.1 Gating Charge Movement and Depolarization-InducedCa2+ Transients. The whole-cell patch-clamp technique was used to quantifythe magnitude and voltage dependence of CaV1.1 channel Q movement inisolated zebrafish fibers as described previously (24). Ca2+ transients weremeasured by loading fibers with Fluo-4 AM from the patch pipette. Detailedmethods are in SI Materials and Methods.

Intracellular Ca2+ Measurements in Intact Muscle Fibers. Fibers were loadedwith 5 μM Fluo-4 AM and excited at 480 ± 15 nm, and fluorescence emissiondetected at 535 ± 20 nm was collected at 10 kHz using a photomultipliersystem. Electrically evoked Ca2+ transients were measured in dissociated fi-bers and elicited by electrical field stimulation using a glass electrode filledwith 200 mM NaCl placed adjacent to the cell of interest. Caffeine concen-tration–response curves were obtained by sequential exposure of fibers tovarious concentrations of caffeine applied through a rapid (response time<5 s) local perfusion system (Warner Instruments).

Freeze-Fracture Electron Microscopy. Four-day-old zebrafish larvae were fixedat room temperature with 6% (vol/vol) glutaraldehyde in 0.1 M cacodylatebuffer at neutral pH after removal of tail skin, stored at 4 °C, and shipped atroom temperature to C.F.-A.’s laboratory while in the fixative. Thin-section EMand freeze-fracture analysis were performed as previously described (24, 38).

T TubuleJ-SR

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Fig. 6. Model for the role of Stac3 in DHPR trafficking and maintenance offunctional tetrads. In WT fibers (Left), Stac3 facilitates direct EC coupling be-tween DHPR tetrads and RyR1, allowing normal Ca2+ release (gray arrowhead)and subsequent refilling of Ca2+ SR stores by SERCA. In stac3NAM fibers (Middle),DHPRα and Stac3NAM are unstable, causing DHPRα to enter degradation andrecycling pathways and leaving triadic DHPRα reduced and in incomplete tet-rads. EC coupling is less efficient, reducing Ca2+ release, but resulting in ex-cessive SR Ca2+ buildup. In stac3−/− fibers (Right), DHPRα are unstable, reduced,and in incomplete tetrads, and EC coupling is inhibited.

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ACKNOWLEDGMENTS. We thank Alex Migda, Matthew Lacey, Sean Lowe,Hoaxing Xu, and Richard Hume for technical assistance and discussions. Researchwas supported by the National Institute of Arthritis and Musculoskeletal andSkin Diseases (NIAMS) of the National Institutes of Health Grant R01-AR-063056

(to J.Y.K.); Grants NIAMS AR059646 and AR053349 (to R.T.D.); Grant NIAMSAR060831 (to V.Y.); and Grant NIAMS 2P01 AR 052354-06A1, PI: P. D. Allen(to C.F.-A.). J.W.L. was supported in part by a Rackham Merit Fellowship (Uni-versity of Michigan) and NIGMS (Grant T32 GM007315).

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