Get 20M+ Full-Text Papers For Less Than $1.50/day. Start a 14-Day Trial for You and Your Team.

Learn More →

Leaky ryanodine receptors in β-sarcoglycan deficient mice: a potential common defect in muscular dystrophy

Leaky ryanodine receptors in β-sarcoglycan deficient mice: a potential common defect in muscular... Background: Disruption of the sarcolemma-associated dystrophin-glycoprotein complex underlies multiple forms of muscular dystrophy, including Duchenne muscular dystrophy and sarcoglycanopathies. A hallmark of these disorders is muscle weakness. In a murine model of Duchenne muscular dystrophy, mdx mice, cysteine-nitrosylation of the calcium release channel/ryanodine receptor type 1 (RyR1) on the skeletal muscle sarcoplasmic reticulum causes depletion of the stabilizing subunit calstabin1 (FKBP12) from the RyR1 macromolecular complex. This results in a sarcoplasmic reticular calcium leak via defective RyR1 channels. This pathological intracellular calcium leak contributes to reduced calcium release and decreased muscle force production. It is unknown whether RyR1 dysfunction occurs also in other muscular dystrophies. Methods: To test this we used a murine model of Limb-Girdle muscular dystrophy, deficient in β-sarcoglycan (Sgcb−/−). Results: Skeletal muscle RyR1 from Sgcb−/− deficient mice were oxidized, nitrosylated, and depleted of the stabilizing subunit calstabin1, which was associated with increased open probability of the RyR1 channels. Sgcb−/− deficient mice exhibited decreased muscle specific force and calcium transients, and displayed reduced exercise capacity. Treating Sgcb−/− mice with the RyR stabilizing compound S107 improved muscle specific force, calcium transients, and exercise capacity. We have previously reported similar findings in mdx mice, a murine model of Duchenne muscular dystrophy. Conclusions: Our data suggest that leaky RyR1 channels may underlie multiple forms of muscular dystrophy linked to mutations in genes encoding components of the dystrophin-glycoprotein complex. A common underlying abnormality in calcium handling indicates that pharmacological targeting of dysfunctional RyR1 could be a novel therapeutic approach to improve muscle function in Limb-Girdle and Duchenne muscular dystrophies. Keywords: Muscular dystrophy, Ryanodine receptor, Calstabin1, Calcium Background proteins that includes dystrophin and the sarcoglycan Muscular dystrophies (MD) comprise a group of in- proteins (α-, β-, δ-, and γ-sarcoglycan), which maintain herited disorders affecting striated muscles that are fiber integrity and protect from contraction-induced characterized by progressive weakness and muscle de- muscle damage [1,2]. Mutation-induced disruption of generation. The dystrophin-glycoprotein complex (DGC) sarcoglycan proteins leads to limb-girdle muscular dys- is a macromolecular structure of membrane-associated trophy (LGMD) [3-5]. A null mutation in one of the sarcoglycans results in loss of the whole sarcoglycan * Correspondence: arm42@columbia.edu complex but not of dystrophin [4,6]. However muta- Department of Physiology and Cellular Biophysics, Columbia University tions in dystrophin, which cause the most common College of Physicians and Surgeons, New York, NY 10032, USA form of muscular dystrophy, Duchenne muscular dys- Department of Medicine, Columbia University College of Physicians and Surgeons, New York, NY 10032, USA trophy (DMD), also lead to loss of the sarcoglycans Full list of author information is available at the end of the article © 2012 Andersson et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Andersson et al. Skeletal Muscle 2012, 2:9 Page 2 of 9 http://www.skeletalmusclejournal.com/content/2/1/9 [7]. This points to the loss of sarcoglycans as the cen- Methods tral upstream event in muscular dystrophies. Disrup- Animals tion of the DGC is associated with oxidative stress, Homozygous β-sarcoglycan deficient mice (Strain: B6.129- 2+ tm1Kcam activation of Ca -dependent neutral proteases (cal- Sgcb /1 J; in this article referred to as Sgcb−/−) were 2+ pains) [8], mitochondrial Ca overload, and apoptosis obtained from The Jackson Laboratory (Bar Harbor, ME, 2+ [9,10]. Moreover, pathological Ca signaling has been USA) [3,24]. The Sgcb−/− mice were backcrossed for attributed to MDs [11-17]. several generations into C57Bl/6 background and aged- Skeletal muscle contraction is regulated by a process matched C57Bl/6 mice were used as controls. All ex- known as excitation-contraction (E-C) coupling. A crit- periments with animals were approved by Columbia 2+ ical feature of this process is the release of Ca from University’s Institutional Animal Care and Use Committee. 2+ the sarcoplasmic reticulum (SR) via the intracellular Ca release channel/ryanodine receptor type 1 (RyR1). To ini- Voluntary exercise and S107 treatment tiate E-C coupling, depolarization of the cell membrane At the beginning of each experiment mice were trans- activates L-type calcium channels (Ca 1.1) on the trans- ferred to individual cages equipped with running wheels verse tubule, which then activates RyR1 through the dir- and exercise was recorded using a data acquisition sys- ect interaction between the two ion channels, causing tem (Respironics). The mice were acclimated to the run- 2+ release of Ca from the SR into the cytoplasm. The in- ning wheels for 7 to 9 days and were randomized into 2+ crease in Ca enables the actin-myosin cross-bridge for- two treatment groups. The first group received S107 mation and sarcomere shortening that results in muscle (25 mg/100 mL) in the drinking water and the second contraction [18]. group received water only. S107 (S107-HCl, FW 245.77) RyR1 is a macromolecular complex with associated was synthesized as previously described [25-27]. The regulatory proteins including kinases, phosphatases, structure and purity of S107 were confirmed by NMR, and the peptidyl-propyl-cis-trans-isomerase FK506 MS, and elemental analysis [25]. The specificity of S107 binding protein 12 (FKBP12, also known as calstabin1). was assessed against a panel of >250 channels, receptors, Calstabin1 binds to RyR1 and stabilizes the closed state phosphatases, and kinases [25]. Mice drank approxi- of the channel, thereby preventing a potentially patho- mately 9 mL/day (water bottle and body weight were 2+ logical Ca leakage from the SR [19]. RyR1 has multi- recorded to monitor consumption) for a daily dose of ple cysteine residues that can be S-nitrosylated and S107 of approximately 1.5 mg. There was no difference S-glutathionylated at physiological pH [20]. These modi- in daily water consumption between the treatment fications can destabilize the closed state of the RyR1, groups (mean ± SEM: control, 9.9 ± 0.6 mL, S107, 9.3 ± 2+ which results in a pathological cytoplasmic Ca ‘leak’ 0.9 mL; n =5, P = NS). Mice were sacrificed using CO [21]. The RyR1 is, moreover, susceptible to oxidation- followed by cervical dislocation and muscles were har- dependent modifications and we have recently shown vested for functional and biochemical analyses. Investi- 2+ that SR Ca ‘leak’ contributes to age-dependent muscle gators performing all aspects of the studies were blinded weakness [22]. Furthermore, inhibition of this intracellu- to the treatment groups. 2+ lar Ca leak with a novel drug that stabilizes the RyR 2+ (S107) [22,23] reduces SR Ca leak and improves muscle Muscle function function in aged mice [22] and in the mdx mouse model Extensor digitorum longus (EDL) muscles were dissected of DMD [23]. from hind limbs. Stainless steel hooks were tied to the In the present study we show that β-sarcoglycan- tendons of the muscles using nylon sutures and the deficient mice (Sgcb−/− mice; an established murine muscles were mounted between a force transducer (Har- model of LGMD) [3], display RyR1 phosphorylation, S- vard Apparatus) and an adjustable hook. The muscles 2+ nitrosylation and oxidation, Ca leak through RyR1, re- were immersed in a stimulation chamber containing 2+ duced tetanic Ca , and specific force in isolated fast O /CO (95/5%) bubbled Tyrode solution (in mM: NaCl 2 2 twitch EDL muscles. Treatment with S107 reduced the 121, KCl 5.0, CaCl 1.8,3 MgCl 0.5, NaH PO 0.4, 2 2 2 4 2+ 2+ Ca leak, increased muscle Ca release, force pro- NaHCO 24, EDTA 0.1, glucose 5.5). Muscles were sti- duction, and improved voluntary exercise capacity in mulated to contract using an electrical field between two Sgcb−/− mice. Disruption of the DGC leads to a platinum electrodes (Aurora Scientific). At the start of common molecular pathophysiological mechanism in each experiment the muscle length (L ) was adjusted to both DMD and LGMD that involves maladaptations yield the maximum force. The force-frequency relation- 2+ of the RyR1 and Ca leak. Furthermore, this disease ships were determined by triggering contraction using 2+ phenotype is likely to respond to therapy with a Ca incremental stimulation frequencies (EDL: 0.5 ms pulses leak-reducing compounds and thus presents new pharma- at 2 to 150 Hz for 350 ms at supra-threshold voltage). ceutical strategies in treating muscular dystrophies. The muscles were allowed to rest between every force- Andersson et al. Skeletal Muscle 2012, 2:9 Page 3 of 9 http://www.skeletalmusclejournal.com/content/2/1/9 frequency stimulation for >1 min. At the end of the pellet, containing the SR fractions, was resuspended and force measurement, the L and weight of the muscles aliquoted using the following solution: 250 mM sucrose, were measured and the muscles were snap frozen in li- 10 mM MOPS (pH 7.4), 1 mM EDTA, and protease quid N . To quantify the specific force, the absolute inhibitors. Samples were frozen in liquid nitrogen and force was normalized to the muscle cross-sectional area, stored at −80°C. calculated as the muscle weight divided by the length -3 using a muscle density constant of 1.056 kg*m [28]. Single-channel recordings SR vesicles containing RyR1 were fused to planar lipid Muscle fatigue protocol bilayers formed by painting a lipid mixture of phosphati- After force-frequency measurements, the EDL muscle dylethanolamine and phosphatidylcholine (Avanti Polar was fatigued. The fatigue protocol for the EDL muscle Lipids) in a 3:1 ratio in decane; across a 200-μm hole in consisted of 50 tetanic contractions (70 Hz, 350 ms dur- polysulfonate cups (Warner Instruments) separating two ation) given at 2-s intervals. chambers. The trans chamber (1.0 mL), representing the intra-SR (luminal) compartment, was connected to the RyR1 immunoprecipitation and immunoblotting head stage input of a bilayer voltage clamp amplifier. EDLs were isotonically lysed in 0.5 mL of a buffer con- The cis chamber (1.0 mL), representing the cytoplasmic taining 50 mM Tris–HCl (pH 7.4), 150 mM NaCl, compartment, was held at virtual ground. Solutions used 20 mM NaF, 1.0 mM Na VO , and protease inhibitors. were the following: (in mM): 1 mM EGTA, 250/125 mM 3 4 An anti-RyR antibody (4 μg 5029 Ab) was used to im- Hepes/Tris, 50 mM KCl, 0.54 mM CaCl , pH 7.35 as cis munoprecipitate RyR1 from 250 μg of tissue homogen- solution, and 53 mM Ca(OH) , 50 mM KCl, 250 mM ate. The samples were incubated with the antibody in Hepes, pH 7.35 as trans solution. The concentration of 2+ 0.5 mL of a modified RIPA buffer (50 mM Tris–HCl pH free Ca in the cis chamber was calculated with Win- 7.4, 0.9% NaCl, 5.0 mM NaF, 1.0 mM Na VO , 1% Tri- MaxC program (version 2.50; www.stanford.edu/~cpat- 3 4 ton-X100, and protease inhibitors) for 1 h at 4°C. The ton/maxc.html). SR vesicles were added to the cis side immune complexes were incubated with protein A and fusion with the lipid bilayer was induced by making Sepharose beads (Sigma, St Louis, MO, USA) at 4°C for the cis side hyperosmotic by the addition of 400 to 1 h and the beads were washed three times with buffer. 500 mM KCl. After the appearance of potassium and Proteins were separated on SDS-PAGE gels (6% for chloride channels, the cis side was perfused with the cis RyR1, 15% for calstabin1) and transferred onto nitrocel- solution. Single-channel currents were recorded at 0 mV lulose membranes for 1 h at 200 mA (SemiDry transfer by using a Bilayer Clamp BC-525 C (Warner Instru- blot, Bio-Rad). After incubation with blocking solution ments), filtered at 1 kHz using a Low-Pass Bessel Filter 8 (LICOR Biosciences, Lincoln, NE, USA) to prevent non- Pole (Warner Instruments), and digitized at 4 kHz. To specific antibody binding, immunoblots were developed confirm RyR identity, 5 μM of ryanodine and/or 20 μM with anti-RyR (Affinity Bioreagents, Bolder, CO, USA; of ruthenium red were added at the end of each ex- 1:2,000), and anti-Cys-NO antibody (Sigma, St Louis, MO, periment. All experiments were performed at room USA; 1:2,000), or an anti-calstabin antibody (1:2,500). temperature (23°C). Po was determined over 2 min of To determine channel oxidation the carbonyl groups on continuous recording using the method of 50% threshold the protein side chains were derivatized to 2,4- dinitro- analysis [29]. The recordings were analyzed by using phenylhydrazone (DNP-hydrazone) by reaction with 2,4 Clampfit 10.1 (Molecular Devices) and Sigma Plot soft- dinitrophenylhydrazine (DNPH). The DNP signal on ware (ver. 10.0, Systat Software), and Prism (ver.5.0, RyR1 was detected by immunoblotting with an anti-DNP GraphPad). antibody. All immunoblots were developed and quanti- 2+ fied using the Odyssey Infrared Imaging System (LICOR Ca imaging in FDB muscle fibers Biosystems, Lincoln, NE, USA) and infrared-labeled sec- Single FDB fibers were obtained by enzymatic dissoci- ondary antibodies. ation as previously described [30]. FDB muscles from both hind limbs were incubated for approximately 2 h at SR vesicle preparation 37°C in approximately 4 mL Dulbecco’s Modified Eagles About 100 mg of isolated mouse EDL muscle was Medium (DMEM) containing 0.3% collagenase 1 (Sigma) homogenized using a tissue mizer (Fisher Scientific) at and 10% fetal bovine serum. The muscles were trans- the highest speed for 1 min with two volumes of: ferred to a culture dish containing fresh DMEM (ap- 20 mM Tris-maleate (pH 7.4), 1 mM EDTA, and prote- proximately 4 mL) and gently triturated using a 1,000 ase inhibitors (Roche). Homogenate was centrifuged at μL pipette until the muscles were dissociated. The cell 4,000 g for 15 min at 4°C and the following supernatant suspension was stored in an incubator at 37°C/5% CO was centrifuged at 40,000 g for 30 min at 4°C. The final until the start of the experiment. FDB fibers were loaded Andersson et al. Skeletal Muscle 2012, 2:9 Page 4 of 9 http://www.skeletalmusclejournal.com/content/2/1/9 2+ with the fluorescent Ca indicator Fluo-4 AM (5 μM, examined under an electron microscope (JEM-1200 Invitrogen/Molecular probes) for 15 min in RT. The EXII, JEOL) and images were taken using an ORCA-HR cells were allowed to attach to a laminin-coated glass digital camera (Hamamatsu) and recorded with an AMT cover slip that formed the bottom of a perfusion cham- Image Capture Engine. ber. The cells were then superfused with tyrode solution (in mM: NaCl 121, KCl 5.0, CaCl 1.8, MgCl 0.5, 2 2 Results and discussion NaH PO 0.4, NaHCO 24, EDTA 0.1, glucose 5.5; 2 4 3 Muscular dystrophy is accompanied by abnormal muscle bubbled with O /CO (95/5%)). The fibers were trig- 2 2 morphology, including fiber degeneration and focal ne- gered to tetanic contraction using electrical field stimu- crosis, which are associated with an enhanced regenerative lation (pulses of 0.5 ms at supra-threshold voltage, at activity in the muscle [3,31-33]. To confirm the dystrophic 70 Hz for 350 ms) and Fluo-4 fluorescence was moni- phenotype in the Sgcb−/− mice, we examined histopatho- tored using confocal microscopy (Zeiss LSM 5 Live, 40x logical changes in EDL muscles from β-sarcoglycan- oil immersion lens, excitation wavelength was 488 nm deficient mice compared to WT (Figure 1A-E). A majority and the emitted fluorescence was recorded between (approximately 75%) of the muscle fibers from Sgcb−/− 495 nm and 525 nm) in linescan mode. Only cells that mice displayed centrally localized nuclei as opposed to the were firmly attached to the glass bottom dish through- subsarcolemmal nuclei that are normally found in the out the tetanic stimulation were included in the analysis. healthy WT muscle (Figure 1C). This finding is consistent After subtraction of background fluorescence, the change with regenerative activity in the muscle and has previously in fluorescent signal during the tetanus (peak–resting been reported in β-, and δ-sarcoglycan-deficient muscle (ΔF)) was divided by the resting signal (ΔF/F ). All ex- [3,32,33]. Moreover, the Sgcb−/− muscle displayed overt periments were performed at RT (approximately 20°C). histopathological changes, with a high prevalence of The investigators were blinded to the genotype and degenerated and necrotic fibers and a larger variability treatment of subjects. in the muscle fiber size (Figure 1B, D, and E). These morphological changes are typical for muscular dys- Histology trophy [3,32,33]. Mitochondrial abnormalities have also The EDL samples were fixed with formalin, embedded been described in patients [34] and murine models [9,31] in paraffin wax, and sliced at 5 μm thickness. The sec- of muscular dystrophy. Accordingly, ultrastructural ana- tions were deparaffinized, stained with hematoxylin and lysis of EDL muscles from Sgcb−/− mice revealed many eosin (H&E staining, Sigma-Aldrich Co., St Louis, MO, fibers with abnormal mitochondrial morphology, such as USA) and observed using light microscopy. The images swelling and loss of cristae structure (Figure 1F, G). How- were captured using a SPOT RT slider camera (Diagnostic ever, the sarcomere ultrastructure appeared normal in Instruments Inc., Sterling Heights, MI, USA). For mor- the Sgcb−/− muscle fibers (Figure 1F, G). phological analysis, images were taken randomly from A hallmark of muscular dystrophies is limb muscle each section using a computer controlled motorized weakness [7]. We used extensor digitorum longus (EDL) stage. Then each image was analyzed by Image-Pro Plus muscles from 4- to 6-month-old Sgcb−/− mice and aged- software (Media Cybernetics, Inc., Bethesda, MD, USA). matched wild-type controls (WT) to examine muscle The judgment of qualitative parameters was performed force production. Isolated EDL muscles were electrically by a clinical pathologist blinded to the mouse genotype. stimulated to contract and force production was mea- Degenerated fibers were defined as having weaker eosin sured. EDL muscle from Sgcb−/− mice displayed reduced staining, which was furthermore confirmed by weaker absolute force compared to WT (mean tetanic force at Gomori Trichrome staining (examples weak eosin stain- 70 Hz stimulation ± SEM: Sgcb−/−, 280 ± 24 mN, vs.WT, ing is indicated by asterisks in Figure 1B). Necrotic fibers 420 ± 30 mN; n = 9 (Sgcb−/−), n = 6 (WT); P <0.01 (t-test)). were defined as a swollen/degraded fiber with loss of When the force was normalized to muscle cross- eosin stain, with or without inflammatory cell infiltration sectional area (specific force) the Sgcb−/− EDL muscles (example is indicated by a circle in Figure 1B). exhibited reduced specific force (mean force at 70 Hz -1 stimulation ± SEM: Sgcb−/−, 200 ± 20 kNm , vs.WT, Transmission electron microscopy -1 440 ± 30 kNm ; n = 9 (Sgcb−/−), n = 6 (WT); P <0.01 EDL muscles were fixed in 2.5% glutaraldehyde in 0.1 M (T-test)), indicating defective force generation that is Sorenson’s buffer (PH 7.2) followed by 1 h of post- independent of muscle size. A pathognomonic sign in fixation with 1% OsO4 in Sorenson’s buffer. After dehy- LGMD is the presence of pseudo-hypertrophy. Indeed, dration the tissue samples were embedded in Lx-112 the EDL muscle mass was increased in Sgcb−/− (Sgcb−/−, (Ladd Research Industries) and 60 nm sections were cut 18 ± 1 mg, vs. WT, 13 ± 0.4; n = 9 (Sgcb−/−), n = 6 (WT); using an ultramicrotome (MT-7000). The sections were P <0.001), as previously published [6,35]. then stained with uranyl acetate and lead citrate and Andersson et al. Skeletal Muscle 2012, 2:9 Page 5 of 9 http://www.skeletalmusclejournal.com/content/2/1/9 Figure 1 EDL muscles from β-sarcoglycan deficient mice exhibit dystrophic morphology and abnormal mitochondrial morphology. (A, B) EDL muscle cross-sections from wild-type (WT) and β-sarcoglycan mice stained with hematoxylin and eosin. (C) Percentage of fibers with the nucleus localized in the center (average ± SEM). (D) Percentages of normal, degenerated (weak eosin staining, examples indicated by asterisk) and necrotic (loss of eosin stain and swollen fiber, example indicated by a circle) muscle fibers. (E) Fiber size was more variable in Sgcb−/− EDL. This is indicated by the difference in the frequency distribution of fiber cross-sectional area. The inset in (E) is an expansion of the region indicated by the dashed rectangle in the main graph. Data were obtained from four mice and > 600 fibers in each group. The scale bar in images (A) and (B) indicate 250 μm. Representative electron microscopy images of EDL muscle from (F) WT and (G) Sgcb−/− mice. Arrows indicate normal mitochondria (F) or mitochondria with abnormal morphology, including low cristae density (G). Images from 11 fibers and two mice in each group were investigated under blinded conditions. The sample is magnified at × 25,000. Scale bar indicates 500 nm. To determine whether the observed reductions in the animals were sacrificed and biochemistry and muscle muscle specific force were associated with remodeling of function were assayed. Immunoprecipitation and im- the RyR1 macromolecular complex, RyR1 were immu- munoblotting of RyR1 indicated that there was increased noprecipitated and immunoblotted to assay for post- calstabin1 bound to RyR1 in the S107 treated Sgcb−/− translational modifications [23]. Skeletal muscle RyR1 mice (Figure 2A, B). channels from Sgcb−/− mice exhibited significantly in- Preserved RyR1-calstabin1 interaction is associated 2+ 2+ creased phosphorylation, oxidation, and nitrosylation with reduced SR Ca leak, improved Ca release, mus- (Figure 2A, B). Moreover, phosphorylation, oxidation, cle function, and exercise capacity [18]. To assess the 2+ and nitrosylation cause loss of calstabin1 from the RyR1 presence of RyR1-dependent Ca leak, we measured complex [22,23,36] and Sgcb−/− muscle RyR1 were single channel open probability (P ) of the RyR1 using depleted of calstabin1 (Figure 2A, B). SR membranes from fast twitch muscles that were fused Treatment with the 1,4-benzothiazepine derivative, to planar lipid bilayers. Experimental conditions mimick- 2+ S107, inhibits calstabin1 depletion from the RyR1 com- ing resting skeletal muscle (90 nM Ca on the cis, ‘cyto- plex, stabilizes the closed state of the RyR1 channel, and solic’ side) were used. The P of RyR1 from the Sgcb−/− improves muscle strength in mdx mice as well as in mice was increased (Figure 3A, B, and D), and S107 24-month-old mice with age-related muscle weakness treatment resulted in a significant reduction in RyR1 P [22,23]. We therefore examined whether S107 could in- (Figure 3C, D). These data are consistent with ‘leaky’ 2+ hibit the loss of muscle function in Sgcb−/− mice by ran- RyR1 [22,23]. To study SR Ca release, we loaded iso- domizing Sgcb−/− mice to receive drinking water without lated fast twitch flexor digitorum brevis (FDB) muscle 2+ (n = 6) or with S107 (25 mg/100 mL, n = 6). The treat- fibers with the fluorescent Ca indicator Fluo-4 AM ment persisted for approximately 4 weeks after which and electrically stimulated the fibers to produce tetanic Andersson et al. Skeletal Muscle 2012, 2:9 Page 6 of 9 http://www.skeletalmusclejournal.com/content/2/1/9 In the present study we show that RyR1 in dystrophic muscle are oxidized, cysteine-nitrosylated, phosphory- lated, and depleted of calstabin1, resulting in ‘leaky’ channels, decreased fast twitch muscle force, and im- paired exercise capacity. Furthermore, we show that treating β-sarcoglycan-deficient mice with the RyR sta- bilizing drug, S107, preserves RyR1-calstabin1 binding, 2+ increases SR Ca release, fast twitch muscle force, and improves voluntary exercise capacity. Mutations in components of the DGC or in DGC- associated proteins cause several different muscular dystro- phies, including DMD, the congenital muscular dystrophies, 2+ Figure 2 RyR1 in β-sarcoglycan deficient muscle is cysteine- and LGMD [4]. Previous studies have shown that SR Ca nitrosylated, oxidized, and depleted of calstabin1. (A) release is reduced in muscle from the dystrophic mdx Representative immunoblot of immunoprecipitated RyR1 from wild- mouse model [15-17]. Moreover, it was recently reported type (WT) and β-sarcoglycan deficient (Sgcb−/−) EDL muscles. 2+ that mdx muscle display increased Ca spark frequency Antibodies against RyR1-S2844 phosphorylation (P*RyR1), cysteine- nitrosylated (Cys NO) proteins, calstabin1, and the protein oxidation [23,38]. This is consistent with increased RyR1-mediated 2+ marker 2,4- dinitrophenylhydrazone (DNP) was used. The muscle Ca leak. In the present study, leaky RyR1 was seen in from a mouse treated with S107 is marked (+). (B) Bar graph Sgcb−/− muscle as evidenced by increased RyR1 open showing average band intensities normalized to RyR1 expression probability (Figure 3A-D). Interestingly, it was recently (mean ± SEM, n = 3 for all groups). 2+ shown that overexpression of the SR Ca ATPase (SERCA) in dystrophic mice could rescue the pathological phenotype 2+ in themusclebyeffectively pumpingexcessCa back into 2+ contractions. Ca transients were reduced in FDB myo- the SR [33]. Taken together, these data indicate that intra- 2+ cytes from Sgcb−/− mice (Figure 3E, F). The S107-treated cellular Ca leak is a prominent, but reversible, patho- 2+ Sgcb−/− displayed increased Ca transients compared to logical mechanism in muscular dystrophies. It is possible 2+ untreated Sgcb−/− (mean tetanic F/F ± SEM: WT, 17 ± that cessation of Ca leak would lead to reduction of di- 0.8 (n = 6); Sgcb−/−, 10 ± 0.6 (n = 20); Sgcb−/− S107, 13 ± verse pathogenic signals in muscular dystrophy, including 0.8 (n = 26); cells were taken from three mice per group, those affecting gene expression, protease activity, or redox 2+ P < 0.05 (ANOVA); Figure 3E, F). homeostasis. For instance, the activity of Ca -dependent We next measured force production in isolated EDL proteases such as the calpains are increased in muscular muscles. There was a significant increase in EDL specific dystrophy and have been attributed a role in the breakdown force in the S107-treated Sgcb−/− mice (mean tetanic of myofillament proteins [33,39]. Inhibition of this process forces at 70 Hz stimulation ± SEM: Sgcb−/− S107, 320 ± has been suggested as a therapeutic strategy in myopathies -1 -1 2+ 20 kNm ,Sgcb−/− control 200 ± 20 kNm ; n=9 (Sgcb−/−), [8]. In addition to improving SR Ca release, S107 treat- n = 6 (Sgcb−/− S107), P <0.001 (t-test); Figure 4A). A ment could potentially lead to increased muscle force by 2+ marked feature of skeletal muscle is its susceptibility to preventing Ca -dependent remodeling of the myofilaments. fatigue and recovery. EDL muscles from S107-treated Electron micrographs from Sgcb−/− EDL muscles dis- and untreated Sgcb−/− mice were repeatedly stimulated played abnormal mitochondrial morphology (Figure 1F, to tetanic contractions. The degree of force reduction G). Mitochondrial defects have previously been described during fatigue as well as the recovery was similar in both in both patients [34] and murine models [9,31] of muscu- groups (Figure 4B, C). However, the EDL from S107- lar dystrophy. Ultrastructural analysis of diaphragm treated mice exhibited increased force production prior muscle from α-sarcoglycan-null mice revealed disrupted 2+ to the fatigue protocol. Therefore the EDL from S107- and swollen mitochondria [31]. Furthermore, Ca over- treated mice exhibited higher force production through- load leading to mitochondrial dysfunction has been linked out the fatigue protocol and would likely sustain higher to activation of cell death pathways in δ-sarcoglycan de- levels of work in vivo [30,37]. To determine whether the ficient mice [9], and we have recently reported that improvements in muscle function corresponded to in- mitochondrial ROS dependent oxidation of RyR1 creates 2+ creased exercise capacity, voluntary running performance a vicious cycle of SR Ca leak via RyR1 causing mito- 2+ was recorded in S107-treated and untreated Sgcb−/− chondrial Ca overload and exacerbating mitochondrial mice. S107-treated Sgcb−/− mice ran longer and faster ROS production in muscle aging [22]. (mean daily running distance after 5 weeks ± SEM: WT, Cardiomyopathy is a common symptom of muscle 6.2 ± 0.4 km, Sgcb−/− S107, 3.8 ± 0.3 km, Sgcb−/− 1.5 ± dystrophy [40,41] and improved cardiac function is seen 0.4 km, n =8–5, P <0.05 (ANOVA); Figure 4D, E). following S107 treatment of heart failure (post-myocardial Andersson et al. Skeletal Muscle 2012, 2:9 Page 7 of 9 http://www.skeletalmusclejournal.com/content/2/1/9 2+ Figure 3 β-sarcoglycan deficient muscle displays RyR1 dysfunction and defective SR Ca release that is restored by S107 treatment. (A-C) Representative RyR1 single channel current traces in samples from WT (A), Sgcb−/− (B), and Sgcb−/− S107 (C) treated mice. Channel 2+ activity was measured at 90 nmol/L (nM) free cytosolic [Ca ]. Channel openings are shown as upward deflections; the closed (c -) state of the channel is indicated by horizontal bars in the beginning of each tracing. For each group, channel activity is illustrated by four different traces, each of 5 s length as indicated by dimension bars. The single channel open probability (Po), To (mean open time) and Tc (mean closed time) 2+ were calculated from a 2 min recording under 90 nmol/L free cytosolic [Ca ] are shown above the upper trace. (D) Bar graph summarizing RyR1 2+ single channel Po under 90 nmol/L free cytosolic [Ca ] from WT (n = 4; white bar), Sgcb−/− (n = 3; black bar), and Sgcb−/− + S107 (n = 4; red bar) 2+ samples. Data presented as mean ± S.E.M; * P <0.05; ** P <0.01 (ANOVA). (E) Representative tetanic Ca transients (normalized Fluo-4 fluorescence) in FDB muscle fibers from wild-type (WT), β-sarcoglycan-deficient control (Sgcb−/−), and S107-treated β-sarcoglycan-deficient 2+ (Sgcb−/− S107) mice. (F) Average Ca transient amplitudes (±SEM, n = 6 (WT) n =20 (Sgcb−/−), n = 26 (Sgcb−/− S107) cells from three mice in each group, * P <0.05, ** P <0.01 (ANOVA)). Figure 4 S107 treatment increases muscle force and exercise capacity in β-sarcoglycan deficient mice. (A) Force-frequency curves of EDL muscle from WT control, β-sarcoglycan-deficient (Sgcb−/−), and S107-treated Sgcb−/− (Sgcb−/− S107) mice. (B) Fatigue stimulation (50 tetani; each tetanic stimulation had a duration of 350 ms and was produced by stimulating the muscle with 0.5 ms pulses at 70 Hz frequency) on the same muscles as (A). (C) Relative decline in force production during fatigue in (B). EDL force measurements are presented as mean ± SEM, n =6–9. (D, E) Exercise capacity in Sgcb−/− mice is improved by S107. Daily voluntary running distance (D) and average running speed (E). Pooled data are presented as mean ± SEM, n =8–5, * P <0.05 (ANOVA). Andersson et al. Skeletal Muscle 2012, 2:9 Page 8 of 9 http://www.skeletalmusclejournal.com/content/2/1/9 infarction) and in mdx mice [41,42]. Sgcb−/− mice that affiliation: Faculty of Medicine, Masaryk University, Brno, Czech Republic. Department of Medicine, Columbia University College of Physicians and were treated with S107 displayed improved exercise cap- Surgeons, New York, NY 10032, USA. Current affiliation: Department of acity, measured as voluntary running distance and speed. Medicine, Karolinska Institutet, Stockholm, Sweden. Clyde and Helen Wu Exercise capacity is a compound measure that involves Center for Molecular Cardiology, New York, NY 10032, USA. the function of several organ systems. Therefore, it is Received: 7 March 2012 Accepted: 9 May 2012 possible that improved cardiac function in Sgcb−/− mice Published: 28 May 2012 following S107 treatment could contribute to the im- proved running capacity, this is unlikely however since References the cardiac function was normal by echocardiography in 1. Lim LE, Campbell KP: The sarcoglycan complex in limb-girdle muscular dystrophy. Curr Opin Neurol 1998, 11:443–452. these mice (data not shown). Moreover, muscle function 2. Petrof BJ, Shrager JB, Stedman HH, Kelly AM, Sweeney HL: Dystrophin is a central determinant of exercise capacity [37] and the protects the sarcolemma from stresses developed during muscle 2+ reduced tetanic Ca and impaired muscle specific force contraction. Proc Natl Acad Sci U S A 1993, 90:3710–3714. 3. Durbeej M, Cohn RD, Hrstka RF, Moore SA, Allamand V, Davidson BL, that is seen in Sgcb−/− were improved by fixing the skel- Williamson RA, Campbell KP: Disruption of the beta-sarcoglycan gene 2+ etal muscle SR Ca leak with S107 and these features reveals pathogenetic complexity of limb-girdle muscular dystrophy type were associated with improved voluntary exercise. 2E. Mol Cell 2000, 5:141–151. 4. Durbeej M, Campbell KP: Muscular dystrophies involving the dystrophin-glycoprotein complex: an overview of current mouse Conclusions models. Curr Opin Genet Dev 2002, 12:349–361. 5. Lim LE, Duclos F, Broux O, Bourg N, Sunada Y, Allamand V, Meyer J, Richard We show here that remodeling of the RyR1 contributes I, Moomaw C, Slaughter C, Tome FMS, Fardeau M, Jackson CE, Beckmann JS, to skeletal muscle weakness and reduced exercise cap- Campbell KP: Beta-sarcoglycan: characterization and role in limb-girdle acity in Sgcb−/− mice, a model of LGMD. This is con- muscular dystrophy linked to 4q12. Nat Genet 1995, 11:257–265. 6. Araishi K, Sasaoka T, Imamura M, Noguchi S, Hama H, Wakabayashi E, sistent with results from a previous study of the mdx Yoshida M, Hori T, Ozawa E: Loss of the sarcoglycan complex and mouse, in which RyR1 were S-nitrosylated, and displayed sarcospan leads to muscular dystrophy in beta-sarcoglycan-deficient 2+ SR Ca leak through the RyR1 [23]. The pathophysio- mice. Hum Mol Genet 1999, 8:1589–1598. 7. Heydemann A, McNally EM: Consequences of disrupting the logical similarities between the two types of muscular dystrophin-sarcoglycan complex in cardiac and skeletal myopathy. dystrophy, which both result from disruption of the DGC, Trends Cardiovasc Med 2007, 17:55–59. 2+ 2+ suggest that RyR1-mediated SR Ca leak is a common 8. Gissel H: The role of Ca in muscle cell damage. Ann N Y Acad Sci 2005, 1066:166–180. mechanism for DGC-related muscular dystrophy. Fur- 9. Millay DP, Sargent MA, Osinska H, Baines CP, Barton ER, Vuagniaux G, thermore, this mechanism can be targeted for treatment Sweeney HL, Robbins J, Molkentin JD: Genetic and pharmacologic with the orally available 1,4-benzothiazepine derivative inhibition of mitochondrial-dependent necrosis attenuates muscular dystrophy. Nat Med 2008, 14:442–447. S107. Thus, the present findings suggest the possibility 10. Brookes PS, Yoon Y, Robotham JL, Anders MW, Sheu SS: Calcium, ATP, and of a novel therapeutic strategy in muscular dystrophy. ROS: a mitochondrial love-hate triangle. Am J Physiol Cell Physiol 2004, 287:C817–C833. Abbreviations 11. Fong PY, Turner PR, Denetclaw WF, Steinhardt RA: Increased activity of DGC: Dystrophin-glycoprotein complex; DMD: Duchenne muscular calcium leak channels in myotubes of Duchenne human and mdx dystrophy; EDL: Extensor digitorum longus; LGMD: Limb-girdle muscular mouse origin. Science 1990, 250:673–676. dystrophy; RyR1: Ryanodine receptor; Sgcb−/−: β-Sarcoglycan deficient mice; 12. Bradley WG, Fulthorpe JJ: Studies of sarcolemmal integrity in myopathic SR: Sarcoplasmic reticulum. muscle. Neurology 1978, 28:670–677. 13. Franco A Jr, Lansman JB: Calcium entry through stretch-inactivated ion Competing interests channels in mdx myotubes. Nature 1990, 344:670–673. ARM is a consultant for a start-up company, ARMGO Pharma Inc., which is 14. Millay DP, Goonasekera SA, Sargent MA, Maillet M, Aronow BJ, Molkentin JD: targeting RyR1 to improve exercise capacity in muscle diseases. Calcium influx is sufficient to induce muscular dystrophy through a TRPC-dependent mechanism. Proc Natl Acad Sci U S A 2009, Authors’ contributions 106:19023–19028. DCA designed experiments, conducted experiments, analyzed data, and 15. Woods CE, Novo D, DiFranco M, Capote J, Vergara JL: Propagation in the wrote the first draft of the paper. ACM conducted single channel studies. SR transverse tubular system and voltage dependence of calcium release in did the biochemistry. MJB performed calcium measurements. AU did muscle normal and mdx mouse muscle fibres. J Physiol 2005, 568:867–880. function studies. TS did the pathology. JD helped design experiments and 16. Woods CE, Novo D, DiFranco M, Vergara JL: The action potential-evoked analyze data. ARM conceived of the study, designed the experiments, sarcoplasmic reticulum calcium release is impaired in mdx mouse analyzed data, and revised the manuscript. All authors read and approved muscle fibres. J Physiol 2004, 557:59–75. the final manuscript. 17. DiFranco M, Woods CE, Capote J, Vergara JL: Dystrophic skeletal muscle fibers display alterations at the level of calcium microdomains. Proc Natl Acknowledgements Acad Sci U S A 2008, 105:14698–14703. This study was supported by NIH grant R01-AR060037 to ARM. DCA was 18. Andersson DC, Marks AR: Fixing ryanodine receptor Ca leak - a novel supported by grants from the Swedish Research Council (Vetenskapsrådet), therapeutic strategy for contractile failure in heart and skeletal muscle. the Swedish Society for Medical Research (SSMF) and the Swedish Heart Drug Discov Today Dis Mech 2010, 7:e151–e157. Lung Foundation (Hjärt-lungfonden). AU was supported by a fellowship 19. Brillantes AB, Ondrias K, Scott A, Kobrinsky E, Ondriasova E, Moschella MC, (AHA 11PRE7810019) from the American Heart Association. Jayaraman T, Landers M, Ehrlich BE, Marks AR: Stabilization of calcium release channel (ryanodine receptor) function by FK506-binding protein. Author details Cell 1994, 77:513–523. Department of Physiology and Cellular Biophysics, Columbia University 20. Sun J, Xu L, Eu JP, Stamler JS, Meissner G: Nitric oxide, NOC-12, and College of Physicians and Surgeons, New York, NY 10032, USA. Current S-nitrosoglutathione modulate the skeletal muscle calcium release Andersson et al. Skeletal Muscle 2012, 2:9 Page 9 of 9 http://www.skeletalmusclejournal.com/content/2/1/9 channel/ryanodine receptor by different mechanisms. An allosteric 40. Cohn RD, Durbeej M, Moore SA, Coral-Vazquez R, Prouty S, Campbell KP: function for O2 in S-nitrosylation of the channel. J Biol Chem 2003, Prevention of cardiomyopathy in mouse models lacking the smooth 278:8184–8189. muscle sarcoglycan-sarcospan complex. J Clin Invest 2001, 107:R1–R7. 21. Wehrens XH, Lehnart SE, Huang F, Vest JA, Reiken SR, Mohler PJ, Sun J, 41. Fauconnier J, Thireau J, Reiken S, Cassan C, Richard S, Matecki S, Marks AR, Guatimosim S, Song LS, Rosemblit N, D’Armiento JM, Napolitano C, Lacampagne A: Leaky RyR2 trigger ventricular arrhythmias in Duchenne Memmi M, Priori SG, Lederer WJ, Marks AR: FKBP12.6 deficiency and muscular dystrophy. Proc Natl Acad Sci U S A 2010, 107:1559–1564. defective calcium release channel (ryanodine receptor) function linked 42. Shan J, Betzenhauser MJ, Kushnir A, Reiken S, Meli AC, Wronska A, Dura M, to exercise-induced sudden cardiac death. Cell 2003, 113:829–840. Chen BX, Marks AR: Role of chronic ryanodine receptor phosphorylation 22. Andersson DC, Betzenhauser MJ, Reiken S, Meli AC, Umanskaya A, Xie W, in heart failure and beta-adrenergic receptor blockade in mice. J Clin Shiomi T, Zalk R, Lacampagne A, Marks AR: Ryanodine receptor oxidation Invest 2010, 120:4375–4387. causes intracellular calcium leak and muscle weakness in aging. Cell Metab 2011, 14:196–207. doi:10.1186/2044-5040-2-9 23. Bellinger AM, Reiken S, Carlson C, Mongillo M, Liu X, Rothman L, Matecki S, Cite this article as: Andersson et al.: Leaky ryanodine receptors in β- Lacampagne A, Marks AR: Hypernitrosylated ryanodine receptor calcium sarcoglycan deficient mice: a potential common defect in muscular dystrophy. Skeletal Muscle 2012 2:9. release channels are leaky in dystrophic muscle. Nat Med 2009, 15:325–330. 24. Crosbie RH, Barresi R, Campbell KP: Loss of sarcolemma nNOS in sarcoglycan-deficient muscle. FASEB J 2002, 16:1786–1791. 25. Bellinger AM, Reiken S, Dura M, Murphy PW, Deng SX, Landry DW, Nieman D, Lehnart SE, Samaru M, LaCampagne A, Marks AR: Remodeling of ryanodine receptor complex causes "leaky" channels: a molecular mechanism for decreased exercise capacity. Proc Natl Acad Sci U S A 2008, 105:2198–2202. 26. Lehnart SE, Mongillo M, Bellinger A, Lindegger N, Chen BX, Hsueh W, Reiken S, Wronska A, Drew LJ, Ward CW, Lederer WJ, Kass RS, Morley G, Marks AR: 2+ Leaky Ca release channel/ryanodine receptor 2 causes seizures and sudden cardiac death in mice. J Clin Invest 2008, 118:2230–2245. 27. Wehrens XH, Lehnart SE, Reiken SR, Deng SX, Vest JA, Cervantes D, Coromilas J, Landry DW, Marks AR: Protection from cardiac arrhythmia through ryanodine receptor-stabilizing protein calstabin2. Science 2004, 304:292–296. 28. Yamada T, Place N, Kosterina N, Ostberg T, Zhang SJ, Grundtman C, Erlandsson-Harris H, Lundberg IE, Glenmark B, Bruton JD, Westerblad H: Impaired myofibrillar function in the soleus muscle of mice with collagen-induced arthritis. Arthritis Rheum 2009, 60:3280–3289. 29. Colquhoun D, Sigworth FJ: Fitting and statistical analysis of single-channel recording.In Single-channel recording. Edited by Sakmann B, Neher E. New York: Plenum; 1983. 30. Aydin J, Andersson DC, Hanninen SL, Wredenberg A, Tavi P, Park CB, 2+ Larsson NG, Bruton JD, Westerblad H: Increased mitochondrial Ca and 2+ decreased sarcoplasmic reticulum Ca in mitochondrial myopathy. Hum Mol Genet 2009, 18:278–288. 31. Jakubiec-Puka A, Biral D, Krawczyk K, Betto R: Ultrastructure of diaphragm from dystrophic alpha-sarcoglycan-null mice. Acta Biochim Pol 2005, 52:453–460. 32. Allikian MJ, Hack AA, Mewborn S, Mayer U, McNally EM: Genetic compensation for sarcoglycan loss by integrin alpha7beta1 in muscle. J Cell Sci 2004, 117:3821–3830. 33. Goonasekera SA, Lam CK, Millay DP, Sargent MA, Hajjar RJ, Kranias EG, Molkentin JD: Mitigation of muscular dystrophy in mice by SERCA overexpression in skeletal muscle. J Clin Invest 2011, 121:1044–1052. 34. Angelin A, Tiepolo T, Sabatelli P, Grumati P, Bergamin N, Golfieri C, Mattioli E, Gualandi F, Ferlini A, Merlini L, Maraldi NM, Bonaldo P, Bernardi P: Mitochondrial dysfunction in the pathogenesis of Ullrich congenital muscular dystrophy and prospective therapy with cyclosporins. Proc Natl Acad Sci U S A 2007, 104:991–996. 35. Sasaoka T, Imamura M, Araishi K, Noguchi S, Mizuno Y, Takagoshi N, Hama H, Wakabayashi-Takai E, Yoshimoto-Matsuda Y, Nonaka I, Kaneko K, Yoshida Submit your next manuscript to BioMed Central M, Ozawa E: Pathological analysis of muscle hypertrophy and and take full advantage of: degeneration in muscular dystrophy in gamma-sarcoglycan-deficient mice. Neuromuscul Disord 2003, 13:193–206. • Convenient online submission 36. Ward CW, Reiken S, Marks AR, Marty I, Vassort G, Lacampagne A: Defects in ryanodine receptor calcium release in skeletal muscle from • Thorough peer review post-myocardial infarct rats. FASEB J 2003, 17:1517–1519. • No space constraints or color figure charges 37. Allen DG, Lamb GD, Westerblad H: Skeletal muscle fatigue: cellular • Immediate publication on acceptance mechanisms. Physiol Rev 2008, 88:287–332. 38. Wang X, Weisleder N, Collet C, Zhou J, Chu Y, Hirata Y, Zhao X, Pan Z, • Inclusion in PubMed, CAS, Scopus and Google Scholar Brotto M, Cheng H, Ma J: Uncontrolled calcium sparks act as a dystrophic • Research which is freely available for redistribution signal for mammalian skeletal muscle. Nat Cell Biol 2005, 7:525–530. 39. Goll DE, Thompson VF, Li H, Wei W, Cong J: The calpain system. Physiol Rev Submit your manuscript at 2003, 83:731–801. www.biomedcentral.com/submit http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Skeletal Muscle Springer Journals

Leaky ryanodine receptors in β-sarcoglycan deficient mice: a potential common defect in muscular dystrophy

Loading next page...
 
/lp/springer-journals/leaky-ryanodine-receptors-in-sarcoglycan-deficient-mice-a-potential-NkLLmAjmN5
Publisher
Springer Journals
Copyright
Copyright © 2012 by Andersson et al; licensee BioMed Central Ltd.
Subject
Life Sciences; Cell Biology; Developmental Biology; Biochemistry, general; Systems Biology; Biotechnology
eISSN
2044-5040
DOI
10.1186/2044-5040-2-9
pmid
22640601
Publisher site
See Article on Publisher Site

Abstract

Background: Disruption of the sarcolemma-associated dystrophin-glycoprotein complex underlies multiple forms of muscular dystrophy, including Duchenne muscular dystrophy and sarcoglycanopathies. A hallmark of these disorders is muscle weakness. In a murine model of Duchenne muscular dystrophy, mdx mice, cysteine-nitrosylation of the calcium release channel/ryanodine receptor type 1 (RyR1) on the skeletal muscle sarcoplasmic reticulum causes depletion of the stabilizing subunit calstabin1 (FKBP12) from the RyR1 macromolecular complex. This results in a sarcoplasmic reticular calcium leak via defective RyR1 channels. This pathological intracellular calcium leak contributes to reduced calcium release and decreased muscle force production. It is unknown whether RyR1 dysfunction occurs also in other muscular dystrophies. Methods: To test this we used a murine model of Limb-Girdle muscular dystrophy, deficient in β-sarcoglycan (Sgcb−/−). Results: Skeletal muscle RyR1 from Sgcb−/− deficient mice were oxidized, nitrosylated, and depleted of the stabilizing subunit calstabin1, which was associated with increased open probability of the RyR1 channels. Sgcb−/− deficient mice exhibited decreased muscle specific force and calcium transients, and displayed reduced exercise capacity. Treating Sgcb−/− mice with the RyR stabilizing compound S107 improved muscle specific force, calcium transients, and exercise capacity. We have previously reported similar findings in mdx mice, a murine model of Duchenne muscular dystrophy. Conclusions: Our data suggest that leaky RyR1 channels may underlie multiple forms of muscular dystrophy linked to mutations in genes encoding components of the dystrophin-glycoprotein complex. A common underlying abnormality in calcium handling indicates that pharmacological targeting of dysfunctional RyR1 could be a novel therapeutic approach to improve muscle function in Limb-Girdle and Duchenne muscular dystrophies. Keywords: Muscular dystrophy, Ryanodine receptor, Calstabin1, Calcium Background proteins that includes dystrophin and the sarcoglycan Muscular dystrophies (MD) comprise a group of in- proteins (α-, β-, δ-, and γ-sarcoglycan), which maintain herited disorders affecting striated muscles that are fiber integrity and protect from contraction-induced characterized by progressive weakness and muscle de- muscle damage [1,2]. Mutation-induced disruption of generation. The dystrophin-glycoprotein complex (DGC) sarcoglycan proteins leads to limb-girdle muscular dys- is a macromolecular structure of membrane-associated trophy (LGMD) [3-5]. A null mutation in one of the sarcoglycans results in loss of the whole sarcoglycan * Correspondence: arm42@columbia.edu complex but not of dystrophin [4,6]. However muta- Department of Physiology and Cellular Biophysics, Columbia University tions in dystrophin, which cause the most common College of Physicians and Surgeons, New York, NY 10032, USA form of muscular dystrophy, Duchenne muscular dys- Department of Medicine, Columbia University College of Physicians and Surgeons, New York, NY 10032, USA trophy (DMD), also lead to loss of the sarcoglycans Full list of author information is available at the end of the article © 2012 Andersson et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Andersson et al. Skeletal Muscle 2012, 2:9 Page 2 of 9 http://www.skeletalmusclejournal.com/content/2/1/9 [7]. This points to the loss of sarcoglycans as the cen- Methods tral upstream event in muscular dystrophies. Disrup- Animals tion of the DGC is associated with oxidative stress, Homozygous β-sarcoglycan deficient mice (Strain: B6.129- 2+ tm1Kcam activation of Ca -dependent neutral proteases (cal- Sgcb /1 J; in this article referred to as Sgcb−/−) were 2+ pains) [8], mitochondrial Ca overload, and apoptosis obtained from The Jackson Laboratory (Bar Harbor, ME, 2+ [9,10]. Moreover, pathological Ca signaling has been USA) [3,24]. The Sgcb−/− mice were backcrossed for attributed to MDs [11-17]. several generations into C57Bl/6 background and aged- Skeletal muscle contraction is regulated by a process matched C57Bl/6 mice were used as controls. All ex- known as excitation-contraction (E-C) coupling. A crit- periments with animals were approved by Columbia 2+ ical feature of this process is the release of Ca from University’s Institutional Animal Care and Use Committee. 2+ the sarcoplasmic reticulum (SR) via the intracellular Ca release channel/ryanodine receptor type 1 (RyR1). To ini- Voluntary exercise and S107 treatment tiate E-C coupling, depolarization of the cell membrane At the beginning of each experiment mice were trans- activates L-type calcium channels (Ca 1.1) on the trans- ferred to individual cages equipped with running wheels verse tubule, which then activates RyR1 through the dir- and exercise was recorded using a data acquisition sys- ect interaction between the two ion channels, causing tem (Respironics). The mice were acclimated to the run- 2+ release of Ca from the SR into the cytoplasm. The in- ning wheels for 7 to 9 days and were randomized into 2+ crease in Ca enables the actin-myosin cross-bridge for- two treatment groups. The first group received S107 mation and sarcomere shortening that results in muscle (25 mg/100 mL) in the drinking water and the second contraction [18]. group received water only. S107 (S107-HCl, FW 245.77) RyR1 is a macromolecular complex with associated was synthesized as previously described [25-27]. The regulatory proteins including kinases, phosphatases, structure and purity of S107 were confirmed by NMR, and the peptidyl-propyl-cis-trans-isomerase FK506 MS, and elemental analysis [25]. The specificity of S107 binding protein 12 (FKBP12, also known as calstabin1). was assessed against a panel of >250 channels, receptors, Calstabin1 binds to RyR1 and stabilizes the closed state phosphatases, and kinases [25]. Mice drank approxi- of the channel, thereby preventing a potentially patho- mately 9 mL/day (water bottle and body weight were 2+ logical Ca leakage from the SR [19]. RyR1 has multi- recorded to monitor consumption) for a daily dose of ple cysteine residues that can be S-nitrosylated and S107 of approximately 1.5 mg. There was no difference S-glutathionylated at physiological pH [20]. These modi- in daily water consumption between the treatment fications can destabilize the closed state of the RyR1, groups (mean ± SEM: control, 9.9 ± 0.6 mL, S107, 9.3 ± 2+ which results in a pathological cytoplasmic Ca ‘leak’ 0.9 mL; n =5, P = NS). Mice were sacrificed using CO [21]. The RyR1 is, moreover, susceptible to oxidation- followed by cervical dislocation and muscles were har- dependent modifications and we have recently shown vested for functional and biochemical analyses. Investi- 2+ that SR Ca ‘leak’ contributes to age-dependent muscle gators performing all aspects of the studies were blinded weakness [22]. Furthermore, inhibition of this intracellu- to the treatment groups. 2+ lar Ca leak with a novel drug that stabilizes the RyR 2+ (S107) [22,23] reduces SR Ca leak and improves muscle Muscle function function in aged mice [22] and in the mdx mouse model Extensor digitorum longus (EDL) muscles were dissected of DMD [23]. from hind limbs. Stainless steel hooks were tied to the In the present study we show that β-sarcoglycan- tendons of the muscles using nylon sutures and the deficient mice (Sgcb−/− mice; an established murine muscles were mounted between a force transducer (Har- model of LGMD) [3], display RyR1 phosphorylation, S- vard Apparatus) and an adjustable hook. The muscles 2+ nitrosylation and oxidation, Ca leak through RyR1, re- were immersed in a stimulation chamber containing 2+ duced tetanic Ca , and specific force in isolated fast O /CO (95/5%) bubbled Tyrode solution (in mM: NaCl 2 2 twitch EDL muscles. Treatment with S107 reduced the 121, KCl 5.0, CaCl 1.8,3 MgCl 0.5, NaH PO 0.4, 2 2 2 4 2+ 2+ Ca leak, increased muscle Ca release, force pro- NaHCO 24, EDTA 0.1, glucose 5.5). Muscles were sti- duction, and improved voluntary exercise capacity in mulated to contract using an electrical field between two Sgcb−/− mice. Disruption of the DGC leads to a platinum electrodes (Aurora Scientific). At the start of common molecular pathophysiological mechanism in each experiment the muscle length (L ) was adjusted to both DMD and LGMD that involves maladaptations yield the maximum force. The force-frequency relation- 2+ of the RyR1 and Ca leak. Furthermore, this disease ships were determined by triggering contraction using 2+ phenotype is likely to respond to therapy with a Ca incremental stimulation frequencies (EDL: 0.5 ms pulses leak-reducing compounds and thus presents new pharma- at 2 to 150 Hz for 350 ms at supra-threshold voltage). ceutical strategies in treating muscular dystrophies. The muscles were allowed to rest between every force- Andersson et al. Skeletal Muscle 2012, 2:9 Page 3 of 9 http://www.skeletalmusclejournal.com/content/2/1/9 frequency stimulation for >1 min. At the end of the pellet, containing the SR fractions, was resuspended and force measurement, the L and weight of the muscles aliquoted using the following solution: 250 mM sucrose, were measured and the muscles were snap frozen in li- 10 mM MOPS (pH 7.4), 1 mM EDTA, and protease quid N . To quantify the specific force, the absolute inhibitors. Samples were frozen in liquid nitrogen and force was normalized to the muscle cross-sectional area, stored at −80°C. calculated as the muscle weight divided by the length -3 using a muscle density constant of 1.056 kg*m [28]. Single-channel recordings SR vesicles containing RyR1 were fused to planar lipid Muscle fatigue protocol bilayers formed by painting a lipid mixture of phosphati- After force-frequency measurements, the EDL muscle dylethanolamine and phosphatidylcholine (Avanti Polar was fatigued. The fatigue protocol for the EDL muscle Lipids) in a 3:1 ratio in decane; across a 200-μm hole in consisted of 50 tetanic contractions (70 Hz, 350 ms dur- polysulfonate cups (Warner Instruments) separating two ation) given at 2-s intervals. chambers. The trans chamber (1.0 mL), representing the intra-SR (luminal) compartment, was connected to the RyR1 immunoprecipitation and immunoblotting head stage input of a bilayer voltage clamp amplifier. EDLs were isotonically lysed in 0.5 mL of a buffer con- The cis chamber (1.0 mL), representing the cytoplasmic taining 50 mM Tris–HCl (pH 7.4), 150 mM NaCl, compartment, was held at virtual ground. Solutions used 20 mM NaF, 1.0 mM Na VO , and protease inhibitors. were the following: (in mM): 1 mM EGTA, 250/125 mM 3 4 An anti-RyR antibody (4 μg 5029 Ab) was used to im- Hepes/Tris, 50 mM KCl, 0.54 mM CaCl , pH 7.35 as cis munoprecipitate RyR1 from 250 μg of tissue homogen- solution, and 53 mM Ca(OH) , 50 mM KCl, 250 mM ate. The samples were incubated with the antibody in Hepes, pH 7.35 as trans solution. The concentration of 2+ 0.5 mL of a modified RIPA buffer (50 mM Tris–HCl pH free Ca in the cis chamber was calculated with Win- 7.4, 0.9% NaCl, 5.0 mM NaF, 1.0 mM Na VO , 1% Tri- MaxC program (version 2.50; www.stanford.edu/~cpat- 3 4 ton-X100, and protease inhibitors) for 1 h at 4°C. The ton/maxc.html). SR vesicles were added to the cis side immune complexes were incubated with protein A and fusion with the lipid bilayer was induced by making Sepharose beads (Sigma, St Louis, MO, USA) at 4°C for the cis side hyperosmotic by the addition of 400 to 1 h and the beads were washed three times with buffer. 500 mM KCl. After the appearance of potassium and Proteins were separated on SDS-PAGE gels (6% for chloride channels, the cis side was perfused with the cis RyR1, 15% for calstabin1) and transferred onto nitrocel- solution. Single-channel currents were recorded at 0 mV lulose membranes for 1 h at 200 mA (SemiDry transfer by using a Bilayer Clamp BC-525 C (Warner Instru- blot, Bio-Rad). After incubation with blocking solution ments), filtered at 1 kHz using a Low-Pass Bessel Filter 8 (LICOR Biosciences, Lincoln, NE, USA) to prevent non- Pole (Warner Instruments), and digitized at 4 kHz. To specific antibody binding, immunoblots were developed confirm RyR identity, 5 μM of ryanodine and/or 20 μM with anti-RyR (Affinity Bioreagents, Bolder, CO, USA; of ruthenium red were added at the end of each ex- 1:2,000), and anti-Cys-NO antibody (Sigma, St Louis, MO, periment. All experiments were performed at room USA; 1:2,000), or an anti-calstabin antibody (1:2,500). temperature (23°C). Po was determined over 2 min of To determine channel oxidation the carbonyl groups on continuous recording using the method of 50% threshold the protein side chains were derivatized to 2,4- dinitro- analysis [29]. The recordings were analyzed by using phenylhydrazone (DNP-hydrazone) by reaction with 2,4 Clampfit 10.1 (Molecular Devices) and Sigma Plot soft- dinitrophenylhydrazine (DNPH). The DNP signal on ware (ver. 10.0, Systat Software), and Prism (ver.5.0, RyR1 was detected by immunoblotting with an anti-DNP GraphPad). antibody. All immunoblots were developed and quanti- 2+ fied using the Odyssey Infrared Imaging System (LICOR Ca imaging in FDB muscle fibers Biosystems, Lincoln, NE, USA) and infrared-labeled sec- Single FDB fibers were obtained by enzymatic dissoci- ondary antibodies. ation as previously described [30]. FDB muscles from both hind limbs were incubated for approximately 2 h at SR vesicle preparation 37°C in approximately 4 mL Dulbecco’s Modified Eagles About 100 mg of isolated mouse EDL muscle was Medium (DMEM) containing 0.3% collagenase 1 (Sigma) homogenized using a tissue mizer (Fisher Scientific) at and 10% fetal bovine serum. The muscles were trans- the highest speed for 1 min with two volumes of: ferred to a culture dish containing fresh DMEM (ap- 20 mM Tris-maleate (pH 7.4), 1 mM EDTA, and prote- proximately 4 mL) and gently triturated using a 1,000 ase inhibitors (Roche). Homogenate was centrifuged at μL pipette until the muscles were dissociated. The cell 4,000 g for 15 min at 4°C and the following supernatant suspension was stored in an incubator at 37°C/5% CO was centrifuged at 40,000 g for 30 min at 4°C. The final until the start of the experiment. FDB fibers were loaded Andersson et al. Skeletal Muscle 2012, 2:9 Page 4 of 9 http://www.skeletalmusclejournal.com/content/2/1/9 2+ with the fluorescent Ca indicator Fluo-4 AM (5 μM, examined under an electron microscope (JEM-1200 Invitrogen/Molecular probes) for 15 min in RT. The EXII, JEOL) and images were taken using an ORCA-HR cells were allowed to attach to a laminin-coated glass digital camera (Hamamatsu) and recorded with an AMT cover slip that formed the bottom of a perfusion cham- Image Capture Engine. ber. The cells were then superfused with tyrode solution (in mM: NaCl 121, KCl 5.0, CaCl 1.8, MgCl 0.5, 2 2 Results and discussion NaH PO 0.4, NaHCO 24, EDTA 0.1, glucose 5.5; 2 4 3 Muscular dystrophy is accompanied by abnormal muscle bubbled with O /CO (95/5%)). The fibers were trig- 2 2 morphology, including fiber degeneration and focal ne- gered to tetanic contraction using electrical field stimu- crosis, which are associated with an enhanced regenerative lation (pulses of 0.5 ms at supra-threshold voltage, at activity in the muscle [3,31-33]. To confirm the dystrophic 70 Hz for 350 ms) and Fluo-4 fluorescence was moni- phenotype in the Sgcb−/− mice, we examined histopatho- tored using confocal microscopy (Zeiss LSM 5 Live, 40x logical changes in EDL muscles from β-sarcoglycan- oil immersion lens, excitation wavelength was 488 nm deficient mice compared to WT (Figure 1A-E). A majority and the emitted fluorescence was recorded between (approximately 75%) of the muscle fibers from Sgcb−/− 495 nm and 525 nm) in linescan mode. Only cells that mice displayed centrally localized nuclei as opposed to the were firmly attached to the glass bottom dish through- subsarcolemmal nuclei that are normally found in the out the tetanic stimulation were included in the analysis. healthy WT muscle (Figure 1C). This finding is consistent After subtraction of background fluorescence, the change with regenerative activity in the muscle and has previously in fluorescent signal during the tetanus (peak–resting been reported in β-, and δ-sarcoglycan-deficient muscle (ΔF)) was divided by the resting signal (ΔF/F ). All ex- [3,32,33]. Moreover, the Sgcb−/− muscle displayed overt periments were performed at RT (approximately 20°C). histopathological changes, with a high prevalence of The investigators were blinded to the genotype and degenerated and necrotic fibers and a larger variability treatment of subjects. in the muscle fiber size (Figure 1B, D, and E). These morphological changes are typical for muscular dys- Histology trophy [3,32,33]. Mitochondrial abnormalities have also The EDL samples were fixed with formalin, embedded been described in patients [34] and murine models [9,31] in paraffin wax, and sliced at 5 μm thickness. The sec- of muscular dystrophy. Accordingly, ultrastructural ana- tions were deparaffinized, stained with hematoxylin and lysis of EDL muscles from Sgcb−/− mice revealed many eosin (H&E staining, Sigma-Aldrich Co., St Louis, MO, fibers with abnormal mitochondrial morphology, such as USA) and observed using light microscopy. The images swelling and loss of cristae structure (Figure 1F, G). How- were captured using a SPOT RT slider camera (Diagnostic ever, the sarcomere ultrastructure appeared normal in Instruments Inc., Sterling Heights, MI, USA). For mor- the Sgcb−/− muscle fibers (Figure 1F, G). phological analysis, images were taken randomly from A hallmark of muscular dystrophies is limb muscle each section using a computer controlled motorized weakness [7]. We used extensor digitorum longus (EDL) stage. Then each image was analyzed by Image-Pro Plus muscles from 4- to 6-month-old Sgcb−/− mice and aged- software (Media Cybernetics, Inc., Bethesda, MD, USA). matched wild-type controls (WT) to examine muscle The judgment of qualitative parameters was performed force production. Isolated EDL muscles were electrically by a clinical pathologist blinded to the mouse genotype. stimulated to contract and force production was mea- Degenerated fibers were defined as having weaker eosin sured. EDL muscle from Sgcb−/− mice displayed reduced staining, which was furthermore confirmed by weaker absolute force compared to WT (mean tetanic force at Gomori Trichrome staining (examples weak eosin stain- 70 Hz stimulation ± SEM: Sgcb−/−, 280 ± 24 mN, vs.WT, ing is indicated by asterisks in Figure 1B). Necrotic fibers 420 ± 30 mN; n = 9 (Sgcb−/−), n = 6 (WT); P <0.01 (t-test)). were defined as a swollen/degraded fiber with loss of When the force was normalized to muscle cross- eosin stain, with or without inflammatory cell infiltration sectional area (specific force) the Sgcb−/− EDL muscles (example is indicated by a circle in Figure 1B). exhibited reduced specific force (mean force at 70 Hz -1 stimulation ± SEM: Sgcb−/−, 200 ± 20 kNm , vs.WT, Transmission electron microscopy -1 440 ± 30 kNm ; n = 9 (Sgcb−/−), n = 6 (WT); P <0.01 EDL muscles were fixed in 2.5% glutaraldehyde in 0.1 M (T-test)), indicating defective force generation that is Sorenson’s buffer (PH 7.2) followed by 1 h of post- independent of muscle size. A pathognomonic sign in fixation with 1% OsO4 in Sorenson’s buffer. After dehy- LGMD is the presence of pseudo-hypertrophy. Indeed, dration the tissue samples were embedded in Lx-112 the EDL muscle mass was increased in Sgcb−/− (Sgcb−/−, (Ladd Research Industries) and 60 nm sections were cut 18 ± 1 mg, vs. WT, 13 ± 0.4; n = 9 (Sgcb−/−), n = 6 (WT); using an ultramicrotome (MT-7000). The sections were P <0.001), as previously published [6,35]. then stained with uranyl acetate and lead citrate and Andersson et al. Skeletal Muscle 2012, 2:9 Page 5 of 9 http://www.skeletalmusclejournal.com/content/2/1/9 Figure 1 EDL muscles from β-sarcoglycan deficient mice exhibit dystrophic morphology and abnormal mitochondrial morphology. (A, B) EDL muscle cross-sections from wild-type (WT) and β-sarcoglycan mice stained with hematoxylin and eosin. (C) Percentage of fibers with the nucleus localized in the center (average ± SEM). (D) Percentages of normal, degenerated (weak eosin staining, examples indicated by asterisk) and necrotic (loss of eosin stain and swollen fiber, example indicated by a circle) muscle fibers. (E) Fiber size was more variable in Sgcb−/− EDL. This is indicated by the difference in the frequency distribution of fiber cross-sectional area. The inset in (E) is an expansion of the region indicated by the dashed rectangle in the main graph. Data were obtained from four mice and > 600 fibers in each group. The scale bar in images (A) and (B) indicate 250 μm. Representative electron microscopy images of EDL muscle from (F) WT and (G) Sgcb−/− mice. Arrows indicate normal mitochondria (F) or mitochondria with abnormal morphology, including low cristae density (G). Images from 11 fibers and two mice in each group were investigated under blinded conditions. The sample is magnified at × 25,000. Scale bar indicates 500 nm. To determine whether the observed reductions in the animals were sacrificed and biochemistry and muscle muscle specific force were associated with remodeling of function were assayed. Immunoprecipitation and im- the RyR1 macromolecular complex, RyR1 were immu- munoblotting of RyR1 indicated that there was increased noprecipitated and immunoblotted to assay for post- calstabin1 bound to RyR1 in the S107 treated Sgcb−/− translational modifications [23]. Skeletal muscle RyR1 mice (Figure 2A, B). channels from Sgcb−/− mice exhibited significantly in- Preserved RyR1-calstabin1 interaction is associated 2+ 2+ creased phosphorylation, oxidation, and nitrosylation with reduced SR Ca leak, improved Ca release, mus- (Figure 2A, B). Moreover, phosphorylation, oxidation, cle function, and exercise capacity [18]. To assess the 2+ and nitrosylation cause loss of calstabin1 from the RyR1 presence of RyR1-dependent Ca leak, we measured complex [22,23,36] and Sgcb−/− muscle RyR1 were single channel open probability (P ) of the RyR1 using depleted of calstabin1 (Figure 2A, B). SR membranes from fast twitch muscles that were fused Treatment with the 1,4-benzothiazepine derivative, to planar lipid bilayers. Experimental conditions mimick- 2+ S107, inhibits calstabin1 depletion from the RyR1 com- ing resting skeletal muscle (90 nM Ca on the cis, ‘cyto- plex, stabilizes the closed state of the RyR1 channel, and solic’ side) were used. The P of RyR1 from the Sgcb−/− improves muscle strength in mdx mice as well as in mice was increased (Figure 3A, B, and D), and S107 24-month-old mice with age-related muscle weakness treatment resulted in a significant reduction in RyR1 P [22,23]. We therefore examined whether S107 could in- (Figure 3C, D). These data are consistent with ‘leaky’ 2+ hibit the loss of muscle function in Sgcb−/− mice by ran- RyR1 [22,23]. To study SR Ca release, we loaded iso- domizing Sgcb−/− mice to receive drinking water without lated fast twitch flexor digitorum brevis (FDB) muscle 2+ (n = 6) or with S107 (25 mg/100 mL, n = 6). The treat- fibers with the fluorescent Ca indicator Fluo-4 AM ment persisted for approximately 4 weeks after which and electrically stimulated the fibers to produce tetanic Andersson et al. Skeletal Muscle 2012, 2:9 Page 6 of 9 http://www.skeletalmusclejournal.com/content/2/1/9 In the present study we show that RyR1 in dystrophic muscle are oxidized, cysteine-nitrosylated, phosphory- lated, and depleted of calstabin1, resulting in ‘leaky’ channels, decreased fast twitch muscle force, and im- paired exercise capacity. Furthermore, we show that treating β-sarcoglycan-deficient mice with the RyR sta- bilizing drug, S107, preserves RyR1-calstabin1 binding, 2+ increases SR Ca release, fast twitch muscle force, and improves voluntary exercise capacity. Mutations in components of the DGC or in DGC- associated proteins cause several different muscular dystro- phies, including DMD, the congenital muscular dystrophies, 2+ Figure 2 RyR1 in β-sarcoglycan deficient muscle is cysteine- and LGMD [4]. Previous studies have shown that SR Ca nitrosylated, oxidized, and depleted of calstabin1. (A) release is reduced in muscle from the dystrophic mdx Representative immunoblot of immunoprecipitated RyR1 from wild- mouse model [15-17]. Moreover, it was recently reported type (WT) and β-sarcoglycan deficient (Sgcb−/−) EDL muscles. 2+ that mdx muscle display increased Ca spark frequency Antibodies against RyR1-S2844 phosphorylation (P*RyR1), cysteine- nitrosylated (Cys NO) proteins, calstabin1, and the protein oxidation [23,38]. This is consistent with increased RyR1-mediated 2+ marker 2,4- dinitrophenylhydrazone (DNP) was used. The muscle Ca leak. In the present study, leaky RyR1 was seen in from a mouse treated with S107 is marked (+). (B) Bar graph Sgcb−/− muscle as evidenced by increased RyR1 open showing average band intensities normalized to RyR1 expression probability (Figure 3A-D). Interestingly, it was recently (mean ± SEM, n = 3 for all groups). 2+ shown that overexpression of the SR Ca ATPase (SERCA) in dystrophic mice could rescue the pathological phenotype 2+ in themusclebyeffectively pumpingexcessCa back into 2+ contractions. Ca transients were reduced in FDB myo- the SR [33]. Taken together, these data indicate that intra- 2+ cytes from Sgcb−/− mice (Figure 3E, F). The S107-treated cellular Ca leak is a prominent, but reversible, patho- 2+ Sgcb−/− displayed increased Ca transients compared to logical mechanism in muscular dystrophies. It is possible 2+ untreated Sgcb−/− (mean tetanic F/F ± SEM: WT, 17 ± that cessation of Ca leak would lead to reduction of di- 0.8 (n = 6); Sgcb−/−, 10 ± 0.6 (n = 20); Sgcb−/− S107, 13 ± verse pathogenic signals in muscular dystrophy, including 0.8 (n = 26); cells were taken from three mice per group, those affecting gene expression, protease activity, or redox 2+ P < 0.05 (ANOVA); Figure 3E, F). homeostasis. For instance, the activity of Ca -dependent We next measured force production in isolated EDL proteases such as the calpains are increased in muscular muscles. There was a significant increase in EDL specific dystrophy and have been attributed a role in the breakdown force in the S107-treated Sgcb−/− mice (mean tetanic of myofillament proteins [33,39]. Inhibition of this process forces at 70 Hz stimulation ± SEM: Sgcb−/− S107, 320 ± has been suggested as a therapeutic strategy in myopathies -1 -1 2+ 20 kNm ,Sgcb−/− control 200 ± 20 kNm ; n=9 (Sgcb−/−), [8]. In addition to improving SR Ca release, S107 treat- n = 6 (Sgcb−/− S107), P <0.001 (t-test); Figure 4A). A ment could potentially lead to increased muscle force by 2+ marked feature of skeletal muscle is its susceptibility to preventing Ca -dependent remodeling of the myofilaments. fatigue and recovery. EDL muscles from S107-treated Electron micrographs from Sgcb−/− EDL muscles dis- and untreated Sgcb−/− mice were repeatedly stimulated played abnormal mitochondrial morphology (Figure 1F, to tetanic contractions. The degree of force reduction G). Mitochondrial defects have previously been described during fatigue as well as the recovery was similar in both in both patients [34] and murine models [9,31] of muscu- groups (Figure 4B, C). However, the EDL from S107- lar dystrophy. Ultrastructural analysis of diaphragm treated mice exhibited increased force production prior muscle from α-sarcoglycan-null mice revealed disrupted 2+ to the fatigue protocol. Therefore the EDL from S107- and swollen mitochondria [31]. Furthermore, Ca over- treated mice exhibited higher force production through- load leading to mitochondrial dysfunction has been linked out the fatigue protocol and would likely sustain higher to activation of cell death pathways in δ-sarcoglycan de- levels of work in vivo [30,37]. To determine whether the ficient mice [9], and we have recently reported that improvements in muscle function corresponded to in- mitochondrial ROS dependent oxidation of RyR1 creates 2+ creased exercise capacity, voluntary running performance a vicious cycle of SR Ca leak via RyR1 causing mito- 2+ was recorded in S107-treated and untreated Sgcb−/− chondrial Ca overload and exacerbating mitochondrial mice. S107-treated Sgcb−/− mice ran longer and faster ROS production in muscle aging [22]. (mean daily running distance after 5 weeks ± SEM: WT, Cardiomyopathy is a common symptom of muscle 6.2 ± 0.4 km, Sgcb−/− S107, 3.8 ± 0.3 km, Sgcb−/− 1.5 ± dystrophy [40,41] and improved cardiac function is seen 0.4 km, n =8–5, P <0.05 (ANOVA); Figure 4D, E). following S107 treatment of heart failure (post-myocardial Andersson et al. Skeletal Muscle 2012, 2:9 Page 7 of 9 http://www.skeletalmusclejournal.com/content/2/1/9 2+ Figure 3 β-sarcoglycan deficient muscle displays RyR1 dysfunction and defective SR Ca release that is restored by S107 treatment. (A-C) Representative RyR1 single channel current traces in samples from WT (A), Sgcb−/− (B), and Sgcb−/− S107 (C) treated mice. Channel 2+ activity was measured at 90 nmol/L (nM) free cytosolic [Ca ]. Channel openings are shown as upward deflections; the closed (c -) state of the channel is indicated by horizontal bars in the beginning of each tracing. For each group, channel activity is illustrated by four different traces, each of 5 s length as indicated by dimension bars. The single channel open probability (Po), To (mean open time) and Tc (mean closed time) 2+ were calculated from a 2 min recording under 90 nmol/L free cytosolic [Ca ] are shown above the upper trace. (D) Bar graph summarizing RyR1 2+ single channel Po under 90 nmol/L free cytosolic [Ca ] from WT (n = 4; white bar), Sgcb−/− (n = 3; black bar), and Sgcb−/− + S107 (n = 4; red bar) 2+ samples. Data presented as mean ± S.E.M; * P <0.05; ** P <0.01 (ANOVA). (E) Representative tetanic Ca transients (normalized Fluo-4 fluorescence) in FDB muscle fibers from wild-type (WT), β-sarcoglycan-deficient control (Sgcb−/−), and S107-treated β-sarcoglycan-deficient 2+ (Sgcb−/− S107) mice. (F) Average Ca transient amplitudes (±SEM, n = 6 (WT) n =20 (Sgcb−/−), n = 26 (Sgcb−/− S107) cells from three mice in each group, * P <0.05, ** P <0.01 (ANOVA)). Figure 4 S107 treatment increases muscle force and exercise capacity in β-sarcoglycan deficient mice. (A) Force-frequency curves of EDL muscle from WT control, β-sarcoglycan-deficient (Sgcb−/−), and S107-treated Sgcb−/− (Sgcb−/− S107) mice. (B) Fatigue stimulation (50 tetani; each tetanic stimulation had a duration of 350 ms and was produced by stimulating the muscle with 0.5 ms pulses at 70 Hz frequency) on the same muscles as (A). (C) Relative decline in force production during fatigue in (B). EDL force measurements are presented as mean ± SEM, n =6–9. (D, E) Exercise capacity in Sgcb−/− mice is improved by S107. Daily voluntary running distance (D) and average running speed (E). Pooled data are presented as mean ± SEM, n =8–5, * P <0.05 (ANOVA). Andersson et al. Skeletal Muscle 2012, 2:9 Page 8 of 9 http://www.skeletalmusclejournal.com/content/2/1/9 infarction) and in mdx mice [41,42]. Sgcb−/− mice that affiliation: Faculty of Medicine, Masaryk University, Brno, Czech Republic. Department of Medicine, Columbia University College of Physicians and were treated with S107 displayed improved exercise cap- Surgeons, New York, NY 10032, USA. Current affiliation: Department of acity, measured as voluntary running distance and speed. Medicine, Karolinska Institutet, Stockholm, Sweden. Clyde and Helen Wu Exercise capacity is a compound measure that involves Center for Molecular Cardiology, New York, NY 10032, USA. the function of several organ systems. Therefore, it is Received: 7 March 2012 Accepted: 9 May 2012 possible that improved cardiac function in Sgcb−/− mice Published: 28 May 2012 following S107 treatment could contribute to the im- proved running capacity, this is unlikely however since References the cardiac function was normal by echocardiography in 1. Lim LE, Campbell KP: The sarcoglycan complex in limb-girdle muscular dystrophy. Curr Opin Neurol 1998, 11:443–452. these mice (data not shown). Moreover, muscle function 2. Petrof BJ, Shrager JB, Stedman HH, Kelly AM, Sweeney HL: Dystrophin is a central determinant of exercise capacity [37] and the protects the sarcolemma from stresses developed during muscle 2+ reduced tetanic Ca and impaired muscle specific force contraction. Proc Natl Acad Sci U S A 1993, 90:3710–3714. 3. Durbeej M, Cohn RD, Hrstka RF, Moore SA, Allamand V, Davidson BL, that is seen in Sgcb−/− were improved by fixing the skel- Williamson RA, Campbell KP: Disruption of the beta-sarcoglycan gene 2+ etal muscle SR Ca leak with S107 and these features reveals pathogenetic complexity of limb-girdle muscular dystrophy type were associated with improved voluntary exercise. 2E. Mol Cell 2000, 5:141–151. 4. Durbeej M, Campbell KP: Muscular dystrophies involving the dystrophin-glycoprotein complex: an overview of current mouse Conclusions models. Curr Opin Genet Dev 2002, 12:349–361. 5. Lim LE, Duclos F, Broux O, Bourg N, Sunada Y, Allamand V, Meyer J, Richard We show here that remodeling of the RyR1 contributes I, Moomaw C, Slaughter C, Tome FMS, Fardeau M, Jackson CE, Beckmann JS, to skeletal muscle weakness and reduced exercise cap- Campbell KP: Beta-sarcoglycan: characterization and role in limb-girdle acity in Sgcb−/− mice, a model of LGMD. This is con- muscular dystrophy linked to 4q12. Nat Genet 1995, 11:257–265. 6. Araishi K, Sasaoka T, Imamura M, Noguchi S, Hama H, Wakabayashi E, sistent with results from a previous study of the mdx Yoshida M, Hori T, Ozawa E: Loss of the sarcoglycan complex and mouse, in which RyR1 were S-nitrosylated, and displayed sarcospan leads to muscular dystrophy in beta-sarcoglycan-deficient 2+ SR Ca leak through the RyR1 [23]. The pathophysio- mice. Hum Mol Genet 1999, 8:1589–1598. 7. Heydemann A, McNally EM: Consequences of disrupting the logical similarities between the two types of muscular dystrophin-sarcoglycan complex in cardiac and skeletal myopathy. dystrophy, which both result from disruption of the DGC, Trends Cardiovasc Med 2007, 17:55–59. 2+ 2+ suggest that RyR1-mediated SR Ca leak is a common 8. Gissel H: The role of Ca in muscle cell damage. Ann N Y Acad Sci 2005, 1066:166–180. mechanism for DGC-related muscular dystrophy. Fur- 9. Millay DP, Sargent MA, Osinska H, Baines CP, Barton ER, Vuagniaux G, thermore, this mechanism can be targeted for treatment Sweeney HL, Robbins J, Molkentin JD: Genetic and pharmacologic with the orally available 1,4-benzothiazepine derivative inhibition of mitochondrial-dependent necrosis attenuates muscular dystrophy. Nat Med 2008, 14:442–447. S107. Thus, the present findings suggest the possibility 10. Brookes PS, Yoon Y, Robotham JL, Anders MW, Sheu SS: Calcium, ATP, and of a novel therapeutic strategy in muscular dystrophy. ROS: a mitochondrial love-hate triangle. Am J Physiol Cell Physiol 2004, 287:C817–C833. Abbreviations 11. Fong PY, Turner PR, Denetclaw WF, Steinhardt RA: Increased activity of DGC: Dystrophin-glycoprotein complex; DMD: Duchenne muscular calcium leak channels in myotubes of Duchenne human and mdx dystrophy; EDL: Extensor digitorum longus; LGMD: Limb-girdle muscular mouse origin. Science 1990, 250:673–676. dystrophy; RyR1: Ryanodine receptor; Sgcb−/−: β-Sarcoglycan deficient mice; 12. Bradley WG, Fulthorpe JJ: Studies of sarcolemmal integrity in myopathic SR: Sarcoplasmic reticulum. muscle. Neurology 1978, 28:670–677. 13. Franco A Jr, Lansman JB: Calcium entry through stretch-inactivated ion Competing interests channels in mdx myotubes. Nature 1990, 344:670–673. ARM is a consultant for a start-up company, ARMGO Pharma Inc., which is 14. Millay DP, Goonasekera SA, Sargent MA, Maillet M, Aronow BJ, Molkentin JD: targeting RyR1 to improve exercise capacity in muscle diseases. Calcium influx is sufficient to induce muscular dystrophy through a TRPC-dependent mechanism. Proc Natl Acad Sci U S A 2009, Authors’ contributions 106:19023–19028. DCA designed experiments, conducted experiments, analyzed data, and 15. Woods CE, Novo D, DiFranco M, Capote J, Vergara JL: Propagation in the wrote the first draft of the paper. ACM conducted single channel studies. SR transverse tubular system and voltage dependence of calcium release in did the biochemistry. MJB performed calcium measurements. AU did muscle normal and mdx mouse muscle fibres. J Physiol 2005, 568:867–880. function studies. TS did the pathology. JD helped design experiments and 16. Woods CE, Novo D, DiFranco M, Vergara JL: The action potential-evoked analyze data. ARM conceived of the study, designed the experiments, sarcoplasmic reticulum calcium release is impaired in mdx mouse analyzed data, and revised the manuscript. All authors read and approved muscle fibres. J Physiol 2004, 557:59–75. the final manuscript. 17. DiFranco M, Woods CE, Capote J, Vergara JL: Dystrophic skeletal muscle fibers display alterations at the level of calcium microdomains. Proc Natl Acknowledgements Acad Sci U S A 2008, 105:14698–14703. This study was supported by NIH grant R01-AR060037 to ARM. DCA was 18. Andersson DC, Marks AR: Fixing ryanodine receptor Ca leak - a novel supported by grants from the Swedish Research Council (Vetenskapsrådet), therapeutic strategy for contractile failure in heart and skeletal muscle. the Swedish Society for Medical Research (SSMF) and the Swedish Heart Drug Discov Today Dis Mech 2010, 7:e151–e157. Lung Foundation (Hjärt-lungfonden). AU was supported by a fellowship 19. Brillantes AB, Ondrias K, Scott A, Kobrinsky E, Ondriasova E, Moschella MC, (AHA 11PRE7810019) from the American Heart Association. Jayaraman T, Landers M, Ehrlich BE, Marks AR: Stabilization of calcium release channel (ryanodine receptor) function by FK506-binding protein. Author details Cell 1994, 77:513–523. Department of Physiology and Cellular Biophysics, Columbia University 20. Sun J, Xu L, Eu JP, Stamler JS, Meissner G: Nitric oxide, NOC-12, and College of Physicians and Surgeons, New York, NY 10032, USA. Current S-nitrosoglutathione modulate the skeletal muscle calcium release Andersson et al. Skeletal Muscle 2012, 2:9 Page 9 of 9 http://www.skeletalmusclejournal.com/content/2/1/9 channel/ryanodine receptor by different mechanisms. An allosteric 40. Cohn RD, Durbeej M, Moore SA, Coral-Vazquez R, Prouty S, Campbell KP: function for O2 in S-nitrosylation of the channel. J Biol Chem 2003, Prevention of cardiomyopathy in mouse models lacking the smooth 278:8184–8189. muscle sarcoglycan-sarcospan complex. J Clin Invest 2001, 107:R1–R7. 21. Wehrens XH, Lehnart SE, Huang F, Vest JA, Reiken SR, Mohler PJ, Sun J, 41. Fauconnier J, Thireau J, Reiken S, Cassan C, Richard S, Matecki S, Marks AR, Guatimosim S, Song LS, Rosemblit N, D’Armiento JM, Napolitano C, Lacampagne A: Leaky RyR2 trigger ventricular arrhythmias in Duchenne Memmi M, Priori SG, Lederer WJ, Marks AR: FKBP12.6 deficiency and muscular dystrophy. Proc Natl Acad Sci U S A 2010, 107:1559–1564. defective calcium release channel (ryanodine receptor) function linked 42. Shan J, Betzenhauser MJ, Kushnir A, Reiken S, Meli AC, Wronska A, Dura M, to exercise-induced sudden cardiac death. Cell 2003, 113:829–840. Chen BX, Marks AR: Role of chronic ryanodine receptor phosphorylation 22. Andersson DC, Betzenhauser MJ, Reiken S, Meli AC, Umanskaya A, Xie W, in heart failure and beta-adrenergic receptor blockade in mice. J Clin Shiomi T, Zalk R, Lacampagne A, Marks AR: Ryanodine receptor oxidation Invest 2010, 120:4375–4387. causes intracellular calcium leak and muscle weakness in aging. Cell Metab 2011, 14:196–207. doi:10.1186/2044-5040-2-9 23. Bellinger AM, Reiken S, Carlson C, Mongillo M, Liu X, Rothman L, Matecki S, Cite this article as: Andersson et al.: Leaky ryanodine receptors in β- Lacampagne A, Marks AR: Hypernitrosylated ryanodine receptor calcium sarcoglycan deficient mice: a potential common defect in muscular dystrophy. Skeletal Muscle 2012 2:9. release channels are leaky in dystrophic muscle. Nat Med 2009, 15:325–330. 24. Crosbie RH, Barresi R, Campbell KP: Loss of sarcolemma nNOS in sarcoglycan-deficient muscle. FASEB J 2002, 16:1786–1791. 25. Bellinger AM, Reiken S, Dura M, Murphy PW, Deng SX, Landry DW, Nieman D, Lehnart SE, Samaru M, LaCampagne A, Marks AR: Remodeling of ryanodine receptor complex causes "leaky" channels: a molecular mechanism for decreased exercise capacity. Proc Natl Acad Sci U S A 2008, 105:2198–2202. 26. Lehnart SE, Mongillo M, Bellinger A, Lindegger N, Chen BX, Hsueh W, Reiken S, Wronska A, Drew LJ, Ward CW, Lederer WJ, Kass RS, Morley G, Marks AR: 2+ Leaky Ca release channel/ryanodine receptor 2 causes seizures and sudden cardiac death in mice. J Clin Invest 2008, 118:2230–2245. 27. Wehrens XH, Lehnart SE, Reiken SR, Deng SX, Vest JA, Cervantes D, Coromilas J, Landry DW, Marks AR: Protection from cardiac arrhythmia through ryanodine receptor-stabilizing protein calstabin2. Science 2004, 304:292–296. 28. Yamada T, Place N, Kosterina N, Ostberg T, Zhang SJ, Grundtman C, Erlandsson-Harris H, Lundberg IE, Glenmark B, Bruton JD, Westerblad H: Impaired myofibrillar function in the soleus muscle of mice with collagen-induced arthritis. Arthritis Rheum 2009, 60:3280–3289. 29. Colquhoun D, Sigworth FJ: Fitting and statistical analysis of single-channel recording.In Single-channel recording. Edited by Sakmann B, Neher E. New York: Plenum; 1983. 30. Aydin J, Andersson DC, Hanninen SL, Wredenberg A, Tavi P, Park CB, 2+ Larsson NG, Bruton JD, Westerblad H: Increased mitochondrial Ca and 2+ decreased sarcoplasmic reticulum Ca in mitochondrial myopathy. Hum Mol Genet 2009, 18:278–288. 31. Jakubiec-Puka A, Biral D, Krawczyk K, Betto R: Ultrastructure of diaphragm from dystrophic alpha-sarcoglycan-null mice. Acta Biochim Pol 2005, 52:453–460. 32. Allikian MJ, Hack AA, Mewborn S, Mayer U, McNally EM: Genetic compensation for sarcoglycan loss by integrin alpha7beta1 in muscle. J Cell Sci 2004, 117:3821–3830. 33. Goonasekera SA, Lam CK, Millay DP, Sargent MA, Hajjar RJ, Kranias EG, Molkentin JD: Mitigation of muscular dystrophy in mice by SERCA overexpression in skeletal muscle. J Clin Invest 2011, 121:1044–1052. 34. Angelin A, Tiepolo T, Sabatelli P, Grumati P, Bergamin N, Golfieri C, Mattioli E, Gualandi F, Ferlini A, Merlini L, Maraldi NM, Bonaldo P, Bernardi P: Mitochondrial dysfunction in the pathogenesis of Ullrich congenital muscular dystrophy and prospective therapy with cyclosporins. Proc Natl Acad Sci U S A 2007, 104:991–996. 35. Sasaoka T, Imamura M, Araishi K, Noguchi S, Mizuno Y, Takagoshi N, Hama H, Wakabayashi-Takai E, Yoshimoto-Matsuda Y, Nonaka I, Kaneko K, Yoshida Submit your next manuscript to BioMed Central M, Ozawa E: Pathological analysis of muscle hypertrophy and and take full advantage of: degeneration in muscular dystrophy in gamma-sarcoglycan-deficient mice. Neuromuscul Disord 2003, 13:193–206. • Convenient online submission 36. Ward CW, Reiken S, Marks AR, Marty I, Vassort G, Lacampagne A: Defects in ryanodine receptor calcium release in skeletal muscle from • Thorough peer review post-myocardial infarct rats. FASEB J 2003, 17:1517–1519. • No space constraints or color figure charges 37. Allen DG, Lamb GD, Westerblad H: Skeletal muscle fatigue: cellular • Immediate publication on acceptance mechanisms. Physiol Rev 2008, 88:287–332. 38. Wang X, Weisleder N, Collet C, Zhou J, Chu Y, Hirata Y, Zhao X, Pan Z, • Inclusion in PubMed, CAS, Scopus and Google Scholar Brotto M, Cheng H, Ma J: Uncontrolled calcium sparks act as a dystrophic • Research which is freely available for redistribution signal for mammalian skeletal muscle. Nat Cell Biol 2005, 7:525–530. 39. Goll DE, Thompson VF, Li H, Wei W, Cong J: The calpain system. Physiol Rev Submit your manuscript at 2003, 83:731–801. www.biomedcentral.com/submit

Journal

Skeletal MuscleSpringer Journals

Published: May 28, 2012

References