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

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.


Background
Muscular dystrophies (MD) comprise a group of inherited disorders affecting striated muscles that are characterized by progressive weakness and muscle degeneration. The dystrophin-glycoprotein complex (DGC) is a macromolecular structure of membrane-associated proteins that includes dystrophin and the sarcoglycan proteins (α-, β-, δ-, and γ-sarcoglycan), which maintain fiber integrity and protect from contraction-induced muscle damage [1,2]. Mutation-induced disruption of sarcoglycan proteins leads to limb-girdle muscular dystrophy (LGMD) [3][4][5]. A null mutation in one of the sarcoglycans results in loss of the whole sarcoglycan complex but not of dystrophin [4,6]. However mutations in dystrophin, which cause the most common form of muscular dystrophy, Duchenne muscular dystrophy (DMD), also lead to loss of the sarcoglycans [7]. This points to the loss of sarcoglycans as the central upstream event in muscular dystrophies. Disruption of the DGC is associated with oxidative stress, activation of Ca 2+ -dependent neutral proteases (calpains) [8], mitochondrial Ca 2+ overload, and apoptosis [9,10]. Moreover, pathological Ca 2+ signaling has been attributed to MDs [11][12][13][14][15][16][17].
Skeletal muscle contraction is regulated by a process known as excitation-contraction (E-C) coupling. A critical feature of this process is the release of Ca 2+ from the sarcoplasmic reticulum (SR) via the intracellular Ca 2+ release channel/ryanodine receptor type 1 (RyR1). To initiate E-C coupling, depolarization of the cell membrane activates L-type calcium channels (Ca v 1.1) on the transverse tubule, which then activates RyR1 through the direct interaction between the two ion channels, causing release of Ca 2+ from the SR into the cytoplasm. The increase in Ca 2+ enables the actin-myosin cross-bridge formation and sarcomere shortening that results in muscle contraction [18].
RyR1 is a macromolecular complex with associated regulatory proteins including kinases, phosphatases, and the peptidyl-propyl-cis-trans-isomerase FK506 binding protein 12 (FKBP12, also known as calstabin1). Calstabin1 binds to RyR1 and stabilizes the closed state of the channel, thereby preventing a potentially pathological Ca 2+ leakage from the SR [19]. RyR1 has multiple cysteine residues that can be S-nitrosylated and S-glutathionylated at physiological pH [20]. These modifications can destabilize the closed state of the RyR1, which results in a pathological cytoplasmic Ca 2+ 'leak' [21]. The RyR1 is, moreover, susceptible to oxidationdependent modifications and we have recently shown that SR Ca 2+ 'leak' contributes to age-dependent muscle weakness [22]. Furthermore, inhibition of this intracellular Ca 2+ leak with a novel drug that stabilizes the RyR (S107) [22,23] reduces SR Ca 2+ leak and improves muscle function in aged mice [22] and in the mdx mouse model of DMD [23].
In the present study we show that β-sarcoglycandeficient mice (Sgcb−/− mice; an established murine model of LGMD) [3], display RyR1 phosphorylation, Snitrosylation and oxidation, Ca 2+ leak through RyR1, reduced tetanic Ca 2+ , and specific force in isolated fast twitch EDL muscles. Treatment with S107 reduced the Ca 2+ leak, increased muscle Ca 2+ release, force production, and improved voluntary exercise capacity in Sgcb−/− mice. Disruption of the DGC leads to a common molecular pathophysiological mechanism in both DMD and LGMD that involves maladaptations of the RyR1 and Ca 2+ leak. Furthermore, this disease phenotype is likely to respond to therapy with a Ca 2+ leak-reducing compounds and thus presents new pharmaceutical strategies in treating muscular dystrophies.

Animals
Homozygous β-sarcoglycan deficient mice (Strain: B6.129-Sgcb tm1Kcam /1 J; in this article referred to as Sgcb−/−) were obtained from The Jackson Laboratory (Bar Harbor, ME, USA) [3,24]. The Sgcb−/− mice were backcrossed for several generations into C57Bl/6 background and agedmatched C57Bl/6 mice were used as controls. All experiments with animals were approved by Columbia University's Institutional Animal Care and Use Committee.

Voluntary exercise and S107 treatment
At the beginning of each experiment mice were transferred to individual cages equipped with running wheels and exercise was recorded using a data acquisition system (Respironics). The mice were acclimated to the running wheels for 7 to 9 days and were randomized into two treatment groups. The first group received S107 (25 mg/100 mL) in the drinking water and the second group received water only. S107 (S107-HCl, FW 245.77) was synthesized as previously described [25][26][27]. The structure and purity of S107 were confirmed by NMR, MS, and elemental analysis [25]. The specificity of S107 was assessed against a panel of >250 channels, receptors, phosphatases, and kinases [25]. Mice drank approximately 9 mL/day (water bottle and body weight were recorded to monitor consumption) for a daily dose of S107 of approximately 1.5 mg. There was no difference in daily water consumption between the treatment groups (mean ± SEM: control, 9.9 ± 0.6 mL, S107, 9.3 ± 0.9 mL; n = 5, P = NS). Mice were sacrificed using CO 2 followed by cervical dislocation and muscles were harvested for functional and biochemical analyses. Investigators performing all aspects of the studies were blinded to the treatment groups.

Muscle function
Extensor digitorum longus (EDL) muscles were dissected from hind limbs. Stainless steel hooks were tied to the tendons of the muscles using nylon sutures and the muscles were mounted between a force transducer (Harvard Apparatus) and an adjustable hook. The muscles were immersed in a stimulation chamber containing O 2 /CO 2 (95/5%) bubbled Tyrode solution (in mM: NaCl 121, KCl 5.0, CaCl 2 1.8,3 MgCl 2 0.5, NaH 2 PO 4 0.4, NaHCO 3 24, EDTA 0.1, glucose 5.5). Muscles were stimulated to contract using an electrical field between two platinum electrodes (Aurora Scientific). At the start of each experiment the muscle length (L 0 ) was adjusted to yield the maximum force. The force-frequency relationships were determined by triggering contraction using incremental stimulation frequencies (EDL: 0.5 ms pulses at 2 to 150 Hz for 350 ms at supra-threshold voltage). The muscles were allowed to rest between every force-frequency stimulation for >1 min. At the end of the force measurement, the L 0 and weight of the muscles were measured and the muscles were snap frozen in liquid N 2 . To quantify the specific force, the absolute force was normalized to the muscle cross-sectional area, calculated as the muscle weight divided by the length using a muscle density constant of 1.056 kg*m -3 [28].

Muscle fatigue protocol
After force-frequency measurements, the EDL muscle was fatigued. The fatigue protocol for the EDL muscle consisted of 50 tetanic contractions (70 Hz, 350 ms duration) given at 2-s intervals.

SR vesicle preparation
About 100 mg of isolated mouse EDL muscle was homogenized using a tissue mizer (Fisher Scientific) at the highest speed for 1 min with two volumes of: 20 mM Tris-maleate (pH 7.4), 1 mM EDTA, and protease inhibitors (Roche). Homogenate was centrifuged at 4,000 g for 15 min at 4°C and the following supernatant was centrifuged at 40,000 g for 30 min at 4°C. The final pellet, containing the SR fractions, was resuspended and aliquoted using the following solution: 250 mM sucrose, 10 mM MOPS (pH 7.4), 1 mM EDTA, and protease inhibitors. Samples were frozen in liquid nitrogen and stored at −80°C. The concentration of free Ca 2+ in the cis chamber was calculated with Win-MaxC program (version 2.50; www.stanford.edu/~cpatton/maxc.html). SR vesicles were added to the cis side and fusion with the lipid bilayer was induced by making the cis side hyperosmotic by the addition of 400 to 500 mM KCl. After the appearance of potassium and chloride channels, the cis side was perfused with the cis solution. Single-channel currents were recorded at 0 mV by using a Bilayer Clamp BC-525 C (Warner Instruments), filtered at 1 kHz using a Low-Pass Bessel Filter 8 Pole (Warner Instruments), and digitized at 4 kHz. To confirm RyR identity, 5 μM of ryanodine and/or 20 μM of ruthenium red were added at the end of each experiment. All experiments were performed at room temperature (23°C). Po was determined over 2 min of continuous recording using the method of 50% threshold analysis [29]. The recordings were analyzed by using Clampfit 10.1 (Molecular Devices) and Sigma Plot software (ver. 10.0, Systat Software), and Prism (ver.5.0, GraphPad).

Ca 2+ imaging in FDB muscle fibers
Single FDB fibers were obtained by enzymatic dissociation as previously described [30]. FDB muscles from both hind limbs were incubated for approximately 2 h at 37°C in approximately 4 mL Dulbecco's Modified Eagles Medium (DMEM) containing 0.3% collagenase 1 (Sigma) and 10% fetal bovine serum. The muscles were transferred to a culture dish containing fresh DMEM (approximately 4 mL) and gently triturated using a 1,000 μL pipette until the muscles were dissociated. The cell suspension was stored in an incubator at 37°C/5% CO 2 until the start of the experiment. FDB fibers were loaded with the fluorescent Ca 2+ indicator Fluo-4 AM (5 μM, Invitrogen/Molecular probes) for 15 min in RT. The cells were allowed to attach to a laminin-coated glass cover slip that formed the bottom of a perfusion chamber. The cells were then superfused with tyrode solution (in mM: NaCl 121, KCl 5.0, CaCl 2 1.8, MgCl 2 0.5, NaH 2 PO 4 0.4, NaHCO 3 24, EDTA 0.1, glucose 5.5; bubbled with O 2 /CO 2 (95/5%)). The fibers were triggered to tetanic contraction using electrical field stimulation (pulses of 0.5 ms at supra-threshold voltage, at 70 Hz for 350 ms) and Fluo-4 fluorescence was monitored using confocal microscopy (Zeiss LSM 5 Live, 40x oil immersion lens, excitation wavelength was 488 nm and the emitted fluorescence was recorded between 495 nm and 525 nm) in linescan mode. Only cells that were firmly attached to the glass bottom dish throughout the tetanic stimulation were included in the analysis. After subtraction of background fluorescence, the change in fluorescent signal during the tetanus (peak-resting (ΔF)) was divided by the resting signal (ΔF/F 0 ). All experiments were performed at RT (approximately 20°C). The investigators were blinded to the genotype and treatment of subjects.

Histology
The EDL samples were fixed with formalin, embedded in paraffin wax, and sliced at 5 μm thickness. The sections were deparaffinized, stained with hematoxylin and eosin (H&E staining, Sigma-Aldrich Co., St Louis, MO, USA) and observed using light microscopy. The images were captured using a SPOT RT slider camera (Diagnostic Instruments Inc., Sterling Heights, MI, USA). For morphological analysis, images were taken randomly from each section using a computer controlled motorized stage. Then each image was analyzed by Image-Pro Plus software (Media Cybernetics, Inc., Bethesda, MD, USA). The judgment of qualitative parameters was performed by a clinical pathologist blinded to the mouse genotype. Degenerated fibers were defined as having weaker eosin staining, which was furthermore confirmed by weaker Gomori Trichrome staining (examples weak eosin staining is indicated by asterisks in Figure 1B). Necrotic fibers were defined as a swollen/degraded fiber with loss of eosin stain, with or without inflammatory cell infiltration (example is indicated by a circle in Figure 1B).

Transmission electron microscopy
EDL muscles were fixed in 2.5% glutaraldehyde in 0.1 M Sorenson's buffer (PH 7.2) followed by 1 h of postfixation with 1% OsO4 in Sorenson's buffer. After dehydration the tissue samples were embedded in Lx-112 (Ladd Research Industries) and 60 nm sections were cut using an ultramicrotome (MT-7000). The sections were then stained with uranyl acetate and lead citrate and examined under an electron microscope (JEM-1200 EXII, JEOL) and images were taken using an ORCA-HR digital camera (Hamamatsu) and recorded with an AMT Image Capture Engine.

Results and discussion
Muscular dystrophy is accompanied by abnormal muscle morphology, including fiber degeneration and focal necrosis, which are associated with an enhanced regenerative activity in the muscle [3,[31][32][33]. To confirm the dystrophic phenotype in the Sgcb−/− mice, we examined histopathological changes in EDL muscles from β-sarcoglycandeficient mice compared to WT ( Figure 1A-E). A majority (approximately 75%) of the muscle fibers from Sgcb−/− mice displayed centrally localized nuclei as opposed to the subsarcolemmal nuclei that are normally found in the healthy WT muscle ( Figure 1C). This finding is consistent with regenerative activity in the muscle and has previously been reported in β-, and δ-sarcoglycan-deficient muscle [3,32,33]. Moreover, the Sgcb−/− muscle displayed overt histopathological changes, with a high prevalence of degenerated and necrotic fibers and a larger variability in the muscle fiber size ( Figure 1B, D, and E). These morphological changes are typical for muscular dystrophy [3,32,33]. Mitochondrial abnormalities have also been described in patients [34] and murine models [9,31] of muscular dystrophy. Accordingly, ultrastructural analysis of EDL muscles from Sgcb−/− mice revealed many fibers with abnormal mitochondrial morphology, such as swelling and loss of cristae structure ( Figure 1F, G). However, the sarcomere ultrastructure appeared normal in the Sgcb−/− muscle fibers ( Figure 1F, G).
Treatment with the 1,4-benzothiazepine derivative, S107, inhibits calstabin1 depletion from the RyR1 complex, stabilizes the closed state of the RyR1 channel, and improves muscle strength in mdx mice as well as in 24-month-old mice with age-related muscle weakness [22,23]. We therefore examined whether S107 could inhibit the loss of muscle function in Sgcb−/− mice by randomizing Sgcb−/− mice to receive drinking water without (n = 6) or with S107 (25 mg/100 mL, n = 6). The treatment persisted for approximately 4 weeks after which the animals were sacrificed and biochemistry and muscle function were assayed. Immunoprecipitation and immunoblotting of RyR1 indicated that there was increased calstabin1 bound to RyR1 in the S107 treated Sgcb−/− mice (Figure 2A, B).
In the present study we show that RyR1 in dystrophic muscle are oxidized, cysteine-nitrosylated, phosphorylated, and depleted of calstabin1, resulting in 'leaky' channels, decreased fast twitch muscle force, and impaired exercise capacity. Furthermore, we show that treating β-sarcoglycan-deficient mice with the RyR stabilizing drug, S107, preserves RyR1-calstabin1 binding, increases SR Ca 2+ release, fast twitch muscle force, and improves voluntary exercise capacity.
Mutations in components of the DGC or in DGCassociated proteins cause several different muscular dystrophies, including DMD, the congenital muscular dystrophies, and LGMD [4]. Previous studies have shown that SR Ca 2+ release is reduced in muscle from the dystrophic mdx mouse model [15][16][17]. Moreover, it was recently reported that mdx muscle display increased Ca 2+ spark frequency [23,38]. This is consistent with increased RyR1-mediated Ca 2+ leak. In the present study, leaky RyR1 was seen in Sgcb−/− muscle as evidenced by increased RyR1 open probability ( Figure 3A-D). Interestingly, it was recently shown that overexpression of the SR Ca 2+ ATPase (SERCA) in dystrophic mice could rescue the pathological phenotype in the muscle by effectively pumping excess Ca 2+ back into the SR [33]. Taken together, these data indicate that intracellular Ca 2+ leak is a prominent, but reversible, pathological mechanism in muscular dystrophies. It is possible that cessation of Ca 2+ leak would lead to reduction of diverse pathogenic signals in muscular dystrophy, including those affecting gene expression, protease activity, or redox homeostasis. For instance, the activity of Ca 2+ -dependent proteases such as the calpains are increased in muscular dystrophy and have been attributed a role in the breakdown of myofillament proteins [33,39]. Inhibition of this process has been suggested as a therapeutic strategy in myopathies [8]. In addition to improving SR Ca 2+ release, S107 treatment could potentially lead to increased muscle force by preventing Ca 2+ -dependent remodeling of the myofilaments.
Electron micrographs from Sgcb−/− EDL muscles displayed abnormal mitochondrial morphology ( Figure 1F, G). Mitochondrial defects have previously been described in both patients [34] and murine models [9,31] of muscular dystrophy. Ultrastructural analysis of diaphragm muscle from α-sarcoglycan-null mice revealed disrupted and swollen mitochondria [31]. Furthermore, Ca 2+ overload leading to mitochondrial dysfunction has been linked to activation of cell death pathways in δ-sarcoglycan deficient mice [9], and we have recently reported that mitochondrial ROS dependent oxidation of RyR1 creates a vicious cycle of SR Ca 2+ leak via RyR1 causing mitochondrial Ca 2+ overload and exacerbating mitochondrial ROS production in muscle aging [22].
Cardiomyopathy is a common symptom of muscle dystrophy [40,41] and improved cardiac function is seen following S107 treatment of heart failure (post-myocardial   infarction) and in mdx mice [41,42]. Sgcb−/− mice that were treated with S107 displayed improved exercise capacity, measured as voluntary running distance and speed. Exercise capacity is a compound measure that involves the function of several organ systems. Therefore, it is possible that improved cardiac function in Sgcb−/− mice following S107 treatment could contribute to the improved running capacity, this is unlikely however since the cardiac function was normal by echocardiography in these mice (data not shown). Moreover, muscle function is a central determinant of exercise capacity [37] and the reduced tetanic Ca 2+ and impaired muscle specific force that is seen in Sgcb−/− were improved by fixing the skeletal muscle SR Ca 2+ leak with S107 and these features were associated with improved voluntary exercise.

Conclusions
We show here that remodeling of the RyR1 contributes to skeletal muscle weakness and reduced exercise capacity in Sgcb−/− mice, a model of LGMD. This is consistent with results from a previous study of the mdx mouse, in which RyR1 were S-nitrosylated, and displayed SR Ca 2+ leak through the RyR1 [23]. The pathophysiological similarities between the two types of muscular dystrophy, which both result from disruption of the DGC, suggest that RyR1-mediated SR Ca 2+ leak is a common mechanism for DGC-related muscular dystrophy. Furthermore, this mechanism can be targeted for treatment with the orally available 1,4-benzothiazepine derivative S107. Thus, the present findings suggest the possibility of a novel therapeutic strategy in muscular dystrophy.
Competing interests ARM is a consultant for a start-up company, ARMGO Pharma Inc., which is targeting RyR1 to improve exercise capacity in muscle diseases.
Authors' contributions DCA designed experiments, conducted experiments, analyzed data, and wrote the first draft of the paper. ACM conducted single channel studies. SR did the biochemistry. MJB performed calcium measurements. AU did muscle function studies. TS did the pathology. JD helped design experiments and analyze data. ARM conceived of the study, designed the experiments, analyzed data, and revised the manuscript. All authors read and approved the final manuscript.