Hypernitrosylated ryanodine receptor calcium release channels are leaky in dystrophic muscle

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1 Hypernitrosylated ryanodine reeptor alium release hannels are leaky in dystrophi musle Andrew M Bellinger 1, Steven Reiken 1, Christian Carlson 1, Maro Mongillo 1, Xiaoping Liu 1, Lisa Rothman 1, Stefan Mateki,3, Alain Laampagne 3, & Andrew R Marks 1 9 Nature Ameria, In. All rights reserved. Duhenne musular dystrophy is haraterized by progressive musle weakness and early death resulting from dystrophin defiieny. Loss of dystrophin results in disruption of a large dystrophin glyoprotein omplex, leading to pathologial alium (Ca )-dependent signals that damage musle ells 1 5. We have identified a strutural and funtional defet in the ryanodine reeptor (), a saroplasmi retiulum Ca release hannel, in the mouse model of musular dystrophy that ontributes to altered Ca homeostasis in dystrophi musles. isolated from skeletal musle showed an age-dependent inrease in S-nitrosylation oinident with dystrophi hanges in the musle. S-nitrosylation depleted the hannel omplex of FKBP1 (also known as alstabin-1, for alium hannel stabilizing binding protein), resulting in leaky hannels. Preventing alstabin-1 depletion from with S17, a ompound that binds the hannel and enhanes the binding affinity of alstabin-1 to the nitrosylated hannel, inhibited saroplasmi retiulum Ca leak, redued biohemial and histologial evidene of musle damage, improved musle funtion and inreased exerise performane in mie. On the basis of these findings, we propose that saroplasmi retiulum Ca leak via due to S-nitrosylation of the hannel and alstabin-1 depletion ontributes to musle weakness in musular dystrophy, and that preventing the -mediated saroplasmi retiulum Ca leak may provide a new therapeuti approah. Duhenne musular dystrophy (DMD), the most ommon X-linked disorder (affeting 1 in 3,5 male births), typially results in death as a result of respiratory or ardia failure by age 3 (ref. 6). Loss of dystrophin leads to disruption of the dystrophin glyoprotein omplex (DGC) in the sarolemmal membrane that onnets the ytoskeleton and ontratile apparatus of musle ells to the extraellular matrix and basement membrane 7,8. Cytoplasmi alium homeostasis in dystrophi musle fibers is abnormal 1,3,9. Disruption of the DGC impairs sarolemmal membrane integrity and results in inreased influx of Ca into the musle aross the sarolemma, whih has been attributed to Ca leak hannels in the plasma membrane 5, mirosopi membrane tears 3,1, mehanosensitive Ca hannels 11, or store-operated Ca hannels ativated by saroplasmi retiulum Ca depletion 1,13. An elevated ytoplasmi alium onentration ([Ca ] yt ) is impliated in the pathophysiology of protein degradation in musle and ell death 1. A downstream effet of elevated [Ca ] yt is ativation of alpains (Ca -dependent neutral proteases). Transgeni expression of alpastatin in the dystrophi mouse partially resues myofiber damage 16. It has been proposed that elevated [Ca ] yt ontributes to myofiber death at a rate that annot be ompensated for by reruitment of progenitor (satellite) musle ells and regeneration and differentiation of new musle ells 3. Saroplasmi retiulum Ca reuptake is slowed in myofibers, and a saroplasmi retiulum Ca leak of unertain etiology has been reported 5,11,17. The rate of Ca sparks in dystrophi fibers is inreased, further suggesting that a defet in saroplasmi retiulum Ca release may be present in musular dystrophy 18. An approximately 8% redution in neuronal nitri oxide synthase (nnos) messenger RNA and protein levels has been reported in dystrophin-defiient musle 19, and has been impliated in the pathophysiology of musular dystrophy 1. ontains multiple ysteine residues that an be modified at physiologial ph by either S-nitrosylation or S-glutathionylation 3 5. Cyli GMP independent, nitri oxide mediated modifiation of RyRs inreases hannel ativity in single-hannel measurements.exogenouss-nitrosylation of has been shown to redue the affinity of alstabin-1 binding to purified saroplasmi retiulum vesiles 6. We hypothesized that defets in the maromoleular omplex ontaining may ontribute to the abnormalities in [Ca ] yt in musular dystrophy. To test this hypothesis, we assessed the omposition of the maromoleular omplex 7,8 from hind limb extensor digitorum longus (EDL) musle of mie. Histologial evidene of musular dystrophy is evident by 35 d of age. In mie at this age, there was a signifiant inrease in S-nitrosylation of ysteine residues in from mie ompared to age-mathed wild-type () littermates (Fig. 1a,b). Inreased S-nitrosylation orrelated with depletion of alstabin-1 from the omplex (Fig. 1a,b). No differenes in S-nitrosylation, protein kinase A (PKA) phosphorylation of (at Ser8) or alstabin-1 bound to the 1 Clyde and Helen Wu Center for Moleular Cardiology, Departments of Physiology and Cellular Biophysis, and Mediine, Columbia University College of Physiians and Surgeons, 63 West 168th Street, New York, New York 13, USA. Institut National de la Santé et de la Reherhe Médiale, ERI 5, F-395 Montpellier, Frane. 3 Unité de Formation et de Reherhe de Médeine, Université Montpellier 1, F-395 Montpellier, Frane. Institut National de la Santé et de la Reherhe Médiale, U 637, F-395 Montpellier, Frane. Correspondene should be addressed to A.R.M. (arm@olumbia.edu). Reeived July 8; aepted 9 Deember 8; published online 8 February 9; doi:1.138/nm.1916 NATURE MEDICINE VOLUME [ NUMBER 3 [ MARCH 9 35

2 9 Nature Ameria, In. All rights reserved. a 5 5 b 5 IP IP IgG -ps8 / 7 d Age (d) Age (d) Age (d) 37 1 d 35 d 18 d Cys-NO / / GAPDH d -pser8 Cys-NO omplex were observed between mie and littermates at 7 and 1 d of age (Fig. 1a,b). Moreover, there was no inrease in PKA phosphorylation of at Ser8 in mie at any age examined (Fig. 1a,b). Total amounts of alstabin-1 in whole musle lysate were not altered in musle at any age, indiating that the redued alstabin-1 binding to is due to redued binding to rather than altered expression of alstabin-1 (Fig. 1). Immunopreipitation of was speifi and effiient, leaving little in the voided fration (Supplementary Fig. 1 online). Thus, inreased S- nitrosylation and depletion of alstabin-1 orrelated with musular dystrophy in mie. Exogenous S-nitrosylation of with nitri oxide donors resulted in depletion of alstabin-1 from the hannel (Fig. a), as previously reported 6. To identify the basis for the inreased S-nitrosylation of, we determined the abundane of nitri oxide synthase isoforms by immunoblotting and EDL musles for induible NOS (), endothelial NOS (enos) and neuronal NOS (nnos) expression (Fig. b). amounts were signifiantly inreased in EDL musles from mie 35 d and older and were essentially undetetable in musle. enos expression was Figure immunopreipitates and o-loalizes with, and S-nitrosylation of depletes the hannel of alstabin-1. (a) Immunoblot of immunopreipitated and bound alstabin-1 from skeletal saroplasmi retiulum mirosomes S-nitrosylated in vitro with the NO donors Nor-3 or No-1. (b) Immunoblot showing expression of the three NOS isoforms (, enos and nnos) in EDL whole-musle lysates from and mie at the indiated ages. indiates positive ontrol tissues: (mouse marophage), enos (human endothelial ells) and nnos (rat pituitary). n ¼ 3and mie at eah age. () and were separately immunopreipitated from 5 mg of EDL musle lysate, SDS-PAGE separated and probed for and. 5 mg of EDL lysate was loaded as a positive ontrol. As a negative ontrol, anti- antibody was pre-inubated with a 1-fold exess antigeni peptide before immunopreipitation (bloked IP). (d) Immunopreipitation and immunoblotting of and enos from EDL lysate as in. IgG negative ontrol immunopreipitation is also shown. (e) After immunopreipitation of from and EDL lysates at the indiated ages, immunoblots were used to detet and. indiates positive ontrols as indiated in b. For a e, data are representative of three independent experiments. (f) Immunohistohemistry showing oloalization of and in mouse EDL skeletal musle from but not mie. n ¼ per group; representative of three or more setions from eah EDL musle. Figure 1 is S-nitrosylated and depleted of alstabin-1 in mie. (a) After immunopreipitation (IP) of from EDL musle of mie (n ¼ 3 for eah time point) and littermates (n ¼ 3 for eah time point) at 7, 1, 35 and 18 d after birth, immunoblots were used to detet total, phosphorylated by PKA at Ser8 (-pser8), S-nitrosylation of ysteine residues on (Cys-NO) and alstabin-1 bound to. Positive (immunopreipitation of from skeletal musle lysate) and negative (omission of antibody to ; immunopreipitation with ontrol IgG) ontrol immunopreipitations were performed from 7-dayold skeletal musle. Blots are representative of three independent experiments. (b) Quantifiation of the levels of PKA-phosphorylated, S-nitrosylated and alstabin-1-bound to relative to total levels of. Data are presented as means ± s.e.m. P o.5, t-test. () Immunoblot for total alstabin-1 in whole EDL musle lysate (5 mg) from (n¼3) and (n¼3) mie at the indiated ages. Glyeraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a loading ontrol. dereased in skeletal musle ompared to littermates. nnos expression was dereased in tissue. and (but not enos) o-immunopreipitated from EDL musle (Fig.,d), but was not deteted in immunopreipitates from musle generated with antibodies to either or (data not shown). immunopreipitated with at 35 and 18 d of age (Fig. e). Moreover, o-loalized with in EDL musle, whereas was not deteted in EDL musle (Fig. f). These data suggest that is a omponent of the maromoleular omplex in mie, but not in mie. We have reported that RyR Ca release hannel stabilizers, whih we propose alling ryals, inhibit depletion of alstabin-1 from hyperphosphorylated by PKA 8,9. We hypothesized that treatment with S17, a stable, ell-permeable ryal 8, begun as early as possible in mie, would redue the -mediated saroplasmi retiulum Ca leak indued by S-nitrosylation of and alstabin-1 depletion and partially protet against musle damage due to [Ca ] yt - mediated alpain ativation. We randomized 5-week-old male mie into groups reeiving treatment with either S17 or vehile (H O) via a subutaneous osmoti pump. After weeks of treatment, forelimb grip strength was assessed. There was signifiant improvement in grip strength in a 5 5 d No-1 Nor-3 Control µm 1, µm 5 µm 5 µm f Lysate IP IP enos IP IgG Cys-NO enos e b d 7 d 1 d 35 d 18 d 5 1 Merge 1 d 35 d 18 d Lysate IP IP Bloked IP µm enos nnos 36 VOLUME [ NUMBER 3 [ MARCH 9 NATURE MEDICINE

3 a Grip strength (ponds) f Abundane relative to total (AU) BW (g) 3 1 -veh -S17 - ps8 Cys- NO -veh -S PDED3 b 6 Grip strength / BW (ponds per g BW) g 1 n = 9 veh n = 1 - S17 n = 1 -veh -S17 1 enos nnos h Creatine kinase (U l 1 ) 8,, -veh, n = 1 veh - S17 n = 1 n = 1 d Calpain ativity (U µg 1 ) -S17 3,, 1, 1 µm 1 µm n = 3 i veh n = 3, - S17 n = 3 e veh -S17 IP IP IgG -ps8 Cys-NO PDED3 IgG -veh -S17 1 µm 1 µm 1 µm 9 Nature Ameria, In. All rights reserved. Figure 3 S17 treatment prevents alstabin-1 depletion from the omplex, improves grip strength and redues musle damage. (a) Forelimb grip strength in sedentary mie after two weeks of treatment with S17 administered via an osmoti pump ( mie given S17 (-S17), n ¼ 1) or vehile ( mie given vehile (-vehile), n ¼ 1) and ompared to mie (n ¼ 9). Data are presented as a satter plot of absolute grip strength versus body weight (BW). Least-square fit lines are overlaid. (b) Grip strength normalized to BW in sedentary -S17, -veh and mie. P o., t-test with Bonferroni adjustment, -S17 versus -veh. () Serum reatine kinase abundane in the same three groups of mie as in b (P o. versus ; P o. -S17 versus -veh; t-tests with Bonferroni adjustment). (d) EDL tissue alpain ativity in sedentary -S17, -veh and mie (P o. versus ; P o. -S17 versus -veh; t-tests with Bonferroni adjustment). (e) After immunopreipitation of from EDL musle of sedentary -S17, -veh and mie, immunoblots were used to detet total, S-nitrosylated (Cys-NO), -ps8, -bound PDED3 and -bound alstabin-1. (f) Quantifiation of the results in e showing levels of -ps8, S-nitrosylated, -bound PDED3 and -bound alstabin-1 normalized to the total amount of. AU, arbitrary units. Data are presented as means ± s.e.m. (P o. for Cys-NO omparing versus ; P o. for -bound alstabin-1 for -S17 versus -vehile mie). (g) Immunoblots for, enos and nnos in EDL whole-musle lysates from, -veh, and -S17 mie. For panels e g, n ¼ 3 mie per group. (h) Representative images of DAPI-stained musle setions from Evans blue dye injeted mie to assess musle damage in ontrol and S17-treated mie. (i) Representative H&E-stained images from diaphragm of mie, mie treated with vehile or with S17 for weeks as in h. For panels h and i, n ¼ 3 or more mie per group; at least three setions from eah musle were analyzed. the S17-treated group (n ¼ 1; P o.1 versus vehile ontrols (n ¼ 1); Fig. 3a). Normalized for body weight, grip strength was signifiantly improved in mie treated with S17 (P o.1 versus vehile ontrols, t-test analysis of two independent, pairmathed and blinded ohorts; Fig. 3b). The serum onentration of reatine kinase, a marker of musle nerosis, was signifiantly redued by S17 treatment in mie, suggesting a redution in musle damage (Fig. 3). Calpain levels in EDL hind limb musle (determined by measuring the enzymati ativity of ativatable alpain in the musle) were also redued by S17 treatment (Fig. 3d), suggesting that inhibition of -mediated saroplasmi retiulum Ca leak may redue Ca -ativated proteolyti enzyme ativity, leading to protetion of dystrophi musle against damage. Eentri (lengthening) exerise suh as downhill running is partiularly diffiult for mie 3. S17-treated mie ompleted a 3-min downhill run at a higher rate than did vehile-treated mie (nine of eleven mie versus three of ten, P o.5). Creatine kinase abundane was redued in these mie by treatment with S17 (6, versus 13,3 U l 1 in vehile-treated ontrols, P o.5), providing further evidene that S17 an improve funtion and redue reatine kinase leak in musle. Calpain ativation in EDL hind limb musle in these mie was redued by S17 treatment (1,5 U mg 1 versus,1 U mg 1 in vehile-treated ontrols, P o.5), suggesting that stabilization of redued Ca leak and Ca -ativated proteolyti enzyme ativity. S17 treatment prevented depletion of alstabin-1 from S-nitrosylated without affeting PKA phosphorylation of, S-nitrosylation of or the levels of phosphodiesterase D3 (PDED3; Fig. 3e,f) in the maromoleular omplex. NOS isoform expression in musle was not altered by S17 treatment (Fig. 3g). Treatment of mie with S17 via osmoti pump beginning at 5 weeks of age and ontinuing for up to weeks resulted in improvement in the histologial hallmarks of dystrophy. In the tibialis anterior hind limb musle, there was a redution in Evans blue dye positive fibers (1.1 ±.8% in mie treated with S17 (n ¼ 3) versus 6.3 ± 5.3% in vehile-treated mie (n ¼ 3, P o.5; Fig. 3h)). Comparison of diaphragmati musle from mie treated with S17 (n ¼ 5) to mie treated with vehile alone (n ¼ ) showed a 8% redution in the number of entral nulei (P o.5), a 5% redution in the number of Evans blue positive musle fibers (P o.5) and a 3% inrease in fiber ross-setional area (P o.5; Fig. 3i). These improvements in the histology of the dystrophi musles orrelated with improved musle funtion and with dereased reatine kinase and alpain levels (Fig. 3a d). We subjeted EDL musle to eentri ontration and determined the resulting fore defiit, whih we defined as the perentage deline in isometri fore after one eentri ontration. The deline in isometri fore served as a funtional indiator of ontration-indued mehanial injury to dystrophi musle, as previously reported 3. Fore prodution during a tetani ontration was reorded from EDL musle in situ in anesthetized mie first in ombination with an eentri lengthening (to 1% of resting musle length); then, after a 1-min rest, fore prodution was reorded during a seond tetani ontration without eentri stress (Fig. a). Eentri ontration in musle resulted in a marked redution in fore prodution ompared to ontrol musles (Fig. b). In mie treated for 7 1 d NATURE MEDICINE VOLUME [ NUMBER 3 [ MARCH 9 37

4 9 Nature Ameria, In. All rights reserved. Figure S17 treatment dereases Ca leak and inreases musle fore and voluntary exerise in mie. (a) Isometri and eentri fore prodution of EDL musle measured in situ in anesthetized mie, plotted as fore (y axis) versus time (x axis). Top, musle stimulation protool. Bottom, a typial reording of fore prodution obtained in an mouse EDL musle, indiating a deline in fore prodution following the mehanial stress (arrow). L o is the musle length at whih peak fore is developed. (b) Quantifiation of the derease in isometri fore after an eentri ontration normalized to the peak fore amplitude (expressed as the perentage derease in fore) in ontrol mie, vehile-treated mie and mie treated with S17 (.5 mg ml 1 in the drinking water administered for 1 d before testing; n ¼ 5 for eah group). ( e) Spontaneous Ca sparks reorded in mie (), vehile-treated mie (d) and mie treated with S17 (e) on the same musles evaluated in b. In e, representative normalized fluoresene intensity (DF/F ) linesan images (top) from fluo-3 loaded EDL musle fibers and the time ourse of fluoresene (bottom) at different loations on the linesan (olored arrowheads) are shown. Colors are arbitrary (see Supplementary Methods for details). Heat diagram indiates hange in fluoresene as the ratio DF/F.(f) Quantifiation of spark frequeny in ontrol mie, vehile-treated mie and mie treated with S17 (three or four fibers per EDL musle from five mie were examined for eah ondition; the numbers of sparks examined were 6,,586 and 883 in mie, vehile-treated mie and mie treated with S17, respetively). Data are expressed as means ± s.e.m. (P o.5 for versus vehile-treated mie or vehile-treated versus S17-treated mie). (g) Effets of S17 treatment (.5 mg ml 1 in the drinking water administered for 1 d before testing) on spontaneous physial ativity of mie. n ¼ 5 mie for eah ondition, P o.5. (h) Speifi fore-frequeny relationship of EDL musle from the same groups as in g. (n ¼ 5 for eah treatment group, P o.1 by a two-way analysis of variane omparing S17-treated versus vehile-treated mie). (i) EDL fore during a fatigue protool (3-Hz, 3-ms trains applied every seond for 3 s). Fore values are normalized to the fore developed during the first train of the protool. n ¼ 5 for eah treatment group, P o.1 by a two-way analysis of variane omparing S17-treated versus vehile-treated mie. with S17 in their drinking water (B37.5 mg kg 1 d 1 ), this defiit in fore prodution after one eentri ontration was restored to the ontrol value (Fig. b). We thus hypothesized that impaired musle funtion ourring during eentri ontrations in mie was linked to defetive hannel funtion, speifially to a leak of saroplasmi retiulum Ca via. To further test this hypothesis, we analyzed spontaneous Ca release events, or Ca sparks, in EDL musle fibers from ontrol mie and in mie after one episode of mild eentri ontration. The Ca spark frequeny was signifiantly (P o.5) inreased in musle fibers from mie ompared to ontrol mie (Fig. f). Other spatiotemporal properties of the sparks, inluding amplitude, rise time, deay time onstant or spatial spread (full width at half maximum), were not different between the two groups (data not shown). Of note, there was no differene in spatiotemporal Ca spark properties between ontrol and mie in the absene of mehanial stress (that is, eentri ontration), or between ontrol musle with and without eentri ontration (data not shown). Thus, inhibition of alstabin-1 depletion from the omplex by S17 treatment of mie redued saroplasmi retiulum Ca leak via, as manifested by a redution in Ca spark frequeny (Fig. e,f). We next tested whether treatment with S17 ould improve the voluntary exerise of mie. After alimating mie for 5 d to a wheel plaed in their age, we measured the length of time they spent on the wheel and their average and maximal veloities over 7 h. Mdx mie treated with S17 spent signifiantly more time on the wheel and ahieved B5% higher maximal veloities ompared to mie treated with vehile alone (Fig. g). In addition, as determined by in situ fore measurements of EDL musle, S17 treatment a % of L o b 75 g e Tetanus 1s 6 g F/F = 1.5 Tetanus 5 ms µm F/F 3 f d Sparks µm 1 s 1 Perentage derease in fore veh - S17 veh - S17 Trip time (min h 1 ) 1 h 5 Speifi fore (N m ) i Relative fore Average speed (m min 1 ) signifiantly (P o.1) inreased speifi fore (Fig. h) and resistane to fatigue (determined as relative fore during repeated tetani stimulation; Fig. i) in musle ompared to vehiletreated ontrol values. Taken together, our data show that Ca release hannels are leaky in skeletal musle owing to hypernitrosylation, whih depletes the hannel omplex of the stabilizing subunit alstabin-1. Hypernitrosylation of was assoiated with a marked inrease in the expression of in the musle and formation of an - omplex. -mediated intraellular Ca leak was assoiated with inreased onentrations of the Ca -ativated protease alpain in musle that may ontribute to the observed musle damage, impaired musle fore and dereased exerise apaity in mie. We have previously shown that alstabin-1 stabilizes the losed state of individual hannels 31 and mediates oupled gating between multiple hannels 3. Both stabilization of the losed state and oupled gating are likely to have roles in preventing aberrant saroplasmi retiulum Ca leak through hannels. Treatment with S17, whih inhibits depletion of alstabin-1 from hypernitrosylated hannels in dystrophi musle, redued pathologial saroplasmi retiulum Ca leak and alpain ativation, proteted against musle damage, improved musle fore, redued fatigue and improved grip strength and voluntary exerise in mie. Notably, these improvements were observed after 1 week (improved exerise apaity) or weeks (histologi improvement) of S17 treatment, whih is onsiderably faster than most geneti therapies. This suggests that the -mediated intraellular Ca leak is downstream of the geneti defets that ause musular dystrophy (for example, dystrophin defiieny). V max (m min 1 ) veh - S17 veh - S17 veh - S Frequeny (Hz) -S17 -veh -S17 -veh Time (s) 38 VOLUME [ NUMBER 3 [ MARCH 9 NATURE MEDICINE

5 9 Nature Ameria, In. All rights reserved. It has been suggested that hannel funtion may be regulated by S-nitrosylation, but, to date, the physiologial onsequenes of this form of regulation in vivo have not been well understood. nnos is the prinipal soure of nitri oxide in skeletal musle. It is loalized at the plasma membrane, where an N-terminal GLGF peptide motif binds to the dystrophin omplex via an interation with syntrophin 19,33. Disruption of the DGC in DMD results in a seletive loss of nnos atalyti ativity that is assoiated with its downregulation at the transriptional level 19,. Reently, dereased abundane of sarolemmal-loalized nnos was shown to be linked to vasoonstrition and dereased physial ativity after mild exerise (1-min downhill run) in mouse models inluding mie, and treatment with a PDE5 inhibitor inreased physial ativity after mild exerise 3. In ontrast, expression is substantially inreased in the skeletal musle of both humans with DMD and mie, and resue of the phenotype by adenoviral-mediated dystrophin or utrophin expression normalizes ativity 35.The onurrent downregulation of nnos and upregulation of suggests that the latter ould be a ompensatory response. However, funtional ompensation may be preluded by differenes in the subellular loalization of the two NOS isoforms. In musular dystrophy, both the effets of inreased nitrosative stress on the omplex and inreased Ca influx aross the plasma membrane due to disruption of the DGC may ativate hannels. Depletion of the stabilizing subunit alstabin-1 (FKBP1) from the hannel due to nitrosative stress may render it partiularly sensitive to Ca -mediated ativation. Therapeuti strategies for musular dystrophy inlude gene therapy to replae dystrophin 36, upregulation of the dystrophin homolog utrophin 37, aeleration of the rate of musle regeneration 38,39 and exon skipping ahieved via virus-mediated expression of antisense sequenes linked to a modified U7 small nulear RNA.Stabilization of by inhibiting saroplasmi retiulum Ca leak with a small moleule may provide an additional strategy to protet against musle damage and improve funtion. METHODS Mie and treatment with S17. We obtained C57BL/1SS-Dmd /J mie, referred to as mie (stok number 181) and C57BL/6J mie, referred to as mie, from Jakson Laboratories and bred them to obtain male and littermate ontrols. We randomized the mie to groups reeiving treatment with either S17 (for additional information on synthesis and speifiity, see Supplementary Methods online and ref. 8) or vehile (H O). In some experiments, we subutaneously implanted osmoti pumps (Alzet model 1, 1 ml totalvolume,.11ml h 1 delivery for B8 d, Duret) filled with H OorS17(8mg ml 1 diluted in H O) on the dorsal surfae of eah mouse by a horizontal inision at the nek for weeks as indiated in the legend to Figure 3. In other experiments, we added S17 to the drinking water (final onentration,.5 mg ml 1 ) as indiated in the legend to Figure. Themie drank B3 ml per day (water onsumption was variable, and we reorded water bottle and body weight to monitor onsumption) for a daily dose of B.75 mg (B37.5 mg kg 1 d 1 ), whih resulted in a plasma S17 onentration of B35 ±1ngml 1 (B1 nm, whih we determined in the early morning to reflet higher water onsumption during the night). We onduted all experiments in aordane with protools approved by the Institutional Animal Care and Use Committees of Columbia University and Université Montpellier. All studies involving drug treatments, inluding analyses of funtion and histologi setions, were onduted by individuals blinded to the treatment status of the mie. Immunoblotting. Membranes were inubated for 1 h at room temperature with primary antibody to (-137, an affinity-purified rabbit polylonal antibody raised against a KLH-onjugated peptide with the amino aid sequene CAEPDTDYENLRRS, orresponding to residues of mouse skeletal, with an additional ysteine residue added to the amino terminus), and affinity purified with the unonjugated peptide. This antibody speifially reognizes, as it does not reat with RyR or RyR3 when used at a 1 in,5 or 1 in 5, dilution for immunoblotting and at a 1 in 5 dilution for immunopreipitation (data not shown). We also used antibody to alstabin (1 in,5 in bloking buffer, LICOR Biosienes); phospho-epitope speifi antibody to human RyR phosphorylated on Ser89 (1 in 5,) 1, whih detets PKA-phosphorylated mouse (on Ser8) and RyR (on Ser88); antibody to S-nitrosylated ysteine residues (1 in 1,, Sigma); antibody to PDED3 (1 in 1,) ; and antibodies to (1 in,, VWR), enos (1 in,, VWR) and nnos (1 in 1,, VWR). For additional biohemial methods, inluding S-nitrosylation of with Nor-3 and No- 1, see Supplementary Methods. Grip strength. We assessed forelimb grip strength after two weeks of treatment with S17 or vehile. We allowed eah mouse to grab a hold bar attahed to a fore transduer that reords the peak fore generated as the mouse is pulled by the tail horizontally away from the bar (model number 335-M/C-1, TSE Systems). We performed five onseutive pulls separated by -s pauses between eah pull. We alulated the absolute grip strength as the average of the peak fores reorded from the middle three pulls (in ponds, 1 pond ¼ B9.8 mn), and a normalized grip strength, whih is the absolute grip strength divided by the body weight in grams. Creatine kinase and alpain assay. We determined reatine kinase levels on the basis of absorbane hange per minute by a ommerial assay aording to the manufaturer s instrutions (Pointe Sientifi). We diluted EDL musle homogenates to a final onentration of 6 mg ml 1, and we determined the alpain ativity in the homogenate with a ommerial assay aording to the manufaturer s instrutions (Calbiohem). Measurement of extensor digitorum longus resistane to ontrationindued mehanial stress. We anesthetized mie (male, d old) with 13 mg kg 1 ketamine and mg kg 1 xylazine and immobilized them in the supine position on a surgial platform at 37 1C. We exposed the anterior region of the lower hind limb from the ankle to just above the knee and disseted free the distal tendons of the tibialis anterior and EDL. We tied the distal tendon of the EDL with a nylon suture to the lever arm of a fore transduer and length servomotor system (model 35B dual mode; Aurora Sientifi), whih was mounted on a mobile mirometer stage to allow fine inremental adjustments of musle length. We kept the exposed musles moist with a 37 1C isotoni saline drip. We stimulated the EDL indiretly via an eletrode plaed on the belly of the tibialis anterior. For details of the stimulation protools, see Supplementary Methods. Calium sparks. We aquired fluoresene images with a Zeiss LSM 51 META NLO onfoal system equipped with a three-point 63 water immersion objetive (numerial aperture ¼ 1.) operated in line-san mode (1.5 ms per line, 3, lines per san) along the longitudinal axis of the fibers. We exited Fluo-3 at 88 nm with an Argon laser and reorded the emitted fluoresene at 55 nm. Additional details of spark measurement are in the Supplementary Methods. Voluntary exerise measurements. We evaluated voluntary exerise in mie by plaing a 5.5-m-wide by 1-m diameter wheel in a m m m age. Statistial analysis. We presented data as means ± s.e.m. We used an independent t-test with a signifiane level of.5 to test differenes between and mie. When we made multiple omparisons between mie, mie treated with vehile and mie treated with S17, we used a Bonferroni adjustment with a pair-wise signifiane level of.. Note: Supplementary information is available on the Nature Mediine website. ACKNOWLEDGMENTS This work was supported in part by a grant from the Leduq Foundation. We thank J. Shan for assistane with analyses of histologi setions and J. Fauonnier for help with voluntary exerise measurements in mie. NATURE MEDICINE VOLUME [ NUMBER 3 [ MARCH 9 39

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