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Mice with RyR1 mutation (Y524S) undergo hypermetabolic response to simvastatin

Mice with RyR1 mutation (Y524S) undergo hypermetabolic response to simvastatin Background: Statins are widely used drugs for the treatment of hyperlipidemia. Though relatively safe, some individuals taking statins experience rhabdymyolysis, muscle pain, and cramping, a condition termed statin-induced 2+ myopathy (SIM). To determine if mutations in the skeletal muscle calcium (Ca ) release channel, ryanodine receptor type 1 (RyR1), enhance the sensitivity to SIM we tested the effects of simvastatin, the statin that produces the highest incidence of SIM in humans, in mice with a mutation (Y524S, ‘YS’) in RyR1. This mutation is associated with malignant hyperthermia in humans. Exposure of mice with the YS mutation to mild elevations in environmental temperature produces a life-threatening hypermetabolic response (HMR) that is characterized by increased oxygen consumption (VO ), sustained muscle contractures, rhabdymyolysis, and elevated core body temperature. Methods: We assessed the ability of simvastatin to induce a hypermetabolic response in the YS mice using indirect 2+ calorimetry and to alter Ca release via RyR1 in isolated flexor digitorum brevis (FDB) fibers from WT and YS mice 2+ using fluorescent Ca indicators. We also tested the ability of 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) to protect against the simvastatin effects. Results: An acute dose of simvastatin triggers a hypermetabolic response in YS mice. In isolated YS muscle fibers, 2+ 2+ simvastatin triggers an increase in cytosolic Ca levels by increasing Ca leak from the sarcoplasmic reticulum (SR). 2+ With higher simvastatin doses, a similar cytosolic Ca increase occurs in wild type (WT) muscle fibers. Pre-treatment of YS and WT mice with AICAR prevents the response to simvastatin. Conclusions: A mutation in RyR1 associated with malignant hyperthermia increases susceptibility to an adverse 2+ response to simvastatin due to enhanced Ca release from the sarcoplasmic reticulum, suggesting that RyR1 mutations may underlie enhanced susceptibility to statin-induced myopathies. Our data suggest that AICAR may be useful for treating statin myopathies. Keywords: Statin-induced myopathy, Simvastatin, RyR1, Myopathy, Calcium signaling Background results in a condition termed statin-induced myopathy Statins (3-hydroxy-3-methylglutaryl coenzyme-A (HMG- (SIM) [2,3]. A mechanism to explain the underlying cause CoA) reductase inhibitors) are cholesterol-lowering drugs of SIM has yet to be elucidated. that have proven effective in decreasing low-density lipo- One emerging theory of SIM has centered on statins’ 2+ protein (LDL) levels and improving overall health [1]. For potential to modulate intramyofiber calcium (Ca ) the majority of patients, statins are well tolerated with few homeostasis [4-6]. This theory stems in part from the side effects. However, up to 10% of patients on a statin regi- finding that the direct application of simvastatin to healthy men display muscle-related symptoms including soreness, human myofibers triggers a significant increase in cyto- 2+ 2+ fatigue, and an increase in circulating levels of muscle- solic Ca [7]. The sudden release of Ca in response to specific proteins (for example creatine kinase (CK)) that direct application of statins in vitro has been suggested to originate from both mitochondria and the sarcoplasmic 2+ * Correspondence: susanh@bcm.edu reticulum (SR) [5,8,9], the predominant Ca storage or- Equal contributors ganelle within the myofiber. The potential involvement of Department of Molecular Biology and Biophysics, Baylor College of Medicine, 1 Baylor Plaza, Houston, TX 77030, USA © 2013 Knoblauch 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. Knoblauch et al. Skeletal Muscle 2013, 3:22 Page 2 of 7 http://www.skeletalmusclejournal.com/content/3/1/22 2+ the SR in statin-induced Ca release is particularly studies involving injection into mice for indirect calor- intriguing given the recent findings that mutations in imetry, simvastatin powder was dissolved in dimethyl sulf- 2+ ryanodine receptor type 1 (RyR1), the Ca release oxide (DMSO). For single-fiber perfusion work, a 12 mM channel of the SR, may underlie some instances of SIM simvastatin stock was prepared in 10% EtOH similar to [10,11]. Mutations in RyR1 are known to produce malig- previous studies [14]. After adjusting the pH to 7.0, the nant hyperthermia (MH), a life-threatening condition solution was brought up to 12 mM concentration in 2+ where uncontrolled release of Ca within the myofiber Tyrode’s solution containing 121 mM NaCl, 5 mM KCl, is triggered by exposure to certain volatile inhalants, 1.8 mM CaCl ,500 μM MgCl ,400 μMNaH PO ,100 2 2 2 4 elevated temperature, or exercise [12,13]. This uncon- μM EDTA, 5.5 mM glucose, and 24 mM NaHCO .Separ- 2+ trolled release of Ca results in sustained muscle contrac- ately, a vehicle-only stock was prepared identically but tions, elevated core temperature, rhabdomyolysis and, if without the addition of simvastatin. These prepared stocks unabated, death [12]. were aliquoted and frozen at −80°C until use. At present our understanding of the link between RyR1 mutations and statin myopathies has been limited Indirect calorimetry monitoring of VO max to in vitro work with muscle biopsies. Metterlein et al. Those YS and WT mice used to determine the effects found that biopsied muscle from MH-sensitive swine ex- of statin dosing in vivo were removed from their cage, hibit contraction upon exposure to statins in vitro [10]. weighed, and injected IP with an 30-80 mg/kg dose of ei- Similarly, Guis et al. found that muscle biopsies from ther simvastatin dissolved in DMSO or DMSO alone (‘ve- seven of nine human subjects exhibiting the signs of SIM hicle’). The mice were then returned to their cages for expressed abnormal in vitro contracture tests (IVCT) used 30 minutes, after which they were placed individually to screen for susceptibility to MH [11]. into an environmental chamber at 32°C containing indir- These in vitro findings combined with evidence that ect calorimetry chambers (Oxymax System, Columbus 2+ simvastatin modifies Ca homeostasis suggest that Instruments, Columbus, OH, USA), which allowed for RyR1 mutations may underlie enhanced susceptibility to monitoring of maximum oxygen consumption (VO SIM. We developed a mouse model (Y524S, ‘YS’) with a max (mL/kg/min)). Separately, to evaluate the effect- RyR1 knock-in mutation of tyrosine 524 to serine [13], iveness of a pharmaceutical agent shown previously to which in humans (Y522S) is associated with MH [13]. prevent heat-induced HMR response in YS mice [15], Mice homozygous for the mutation die at birth, while additional YS mice were injected IP with a 600 mg/kg heterozygous YS mice exhibit a hypermetabolic response dose of 5-aminoimadazole-4-carboxamide ribonucleo- (HMR) to elevated (37°C) temperature, volatile anes- side (AICAR) 20 minutes after simvastatin injection. thetics, or exercise in a warm environment. These mice are a valuable tool for studying some RyR1-associated Fiber isolation 2+ disorders. The purpose of the present study was to de- For mice destined for single-fiber Ca study, the flexor termine whether mice with this RyR1 mutation (Y524S) digitorum brevis (FDB) muscle was removed and imme- display HMR when given simvastatin and to evaluate the diately placed into Dulbecco’s modified Eagle’s medium 2+ effects of simvastatin on intramyofiber Ca homeostasis. (DMEM) containing 3 mg/mL collagenase and 10% (v/v) fetal bovine serum. After a 2-hour incubation at 37°C, Methods whole FDB muscles were transferred to 1 mL of DMEM Animal care and handling and plunged ten times through a 1 mL pipette tip to All procedures were approved by the Institutional separate individual fibers. Next, 150 μL of DMEM Animal Care and Use Committee at Baylor College of containing separated FDB fibers was placed onto a 25 mm Medicine, Houston, TX, USA. As previously described, glass coverslip that had been incubated for 2 hours with Y524S/WT male RyR1 (‘YS’) mice were developed and used 20 μg/mg of laminin in PBS and then subjected to in conjunction with wild type (WT) littermate controls at two washes in PBS and a final wash in DMEM. Prior 8 to 10 weeks of age. Mice were maintained on a 12:12 to use, plated fibers were incubated overnight at 37°C in light:dark cycle, had ad libitum access to water and stand- DMEM containing antibiotic-antimycotic (Gibco, Carlsbad, ard mouse chow, and were limited to normal cage activity CA, USA). only. All mice were sacrificed at the same time of day, consisting of cervical dislocation after anesthetization Isolated fiber preparation and imaging under isoflurane. To assess the sensitivity to simvastatin, after the over- night incubation the fibers were next incubated for 1 Statin preparation hour at room temperature in either DMEM containing Simvastatin was purchased from the manufacturer (LKT (10 μM) Fura-2 acetoxymethyl ester (Fura-2 AM) or 30 Laboratories, St Paul, MN, USA) in powder form. For minutes in DMEM containing (5 μM) Mag-fluo-4, with Knoblauch et al. Skeletal Muscle 2013, 3:22 Page 3 of 7 http://www.skeletalmusclejournal.com/content/3/1/22 (20 μM) contraction-inhibitor 4-methyl-N-(phenylmethyl) the mice to be euthanized prior to a full body contrac- benzenesulfonamide (BTS). Fibers were placed in a tion and death. To determine if statins also trigger an temperature controlled chamber (Dagan Corporation, HMR response, we injected mice with an acute dose of Minneapolis, MN, USA) on the stage of an inverted epi- simvastatin (IP 30 to 80 mg/kg) and placed the mice in fluorescence microscope (Nikon Inc, Melville, NY, USA) the chamber (32°C, a thermoneutral temperature that and warmed to 32°C over a 5-minute period in Tyrode’s does not trigger HMR in the untreated YS mice) of the solution. Fluorescence emission was captured using a indirect calorimeter and measured VO as a function high speed, digital QE CCD camera (TILL Photonics, of time after injection. All YS mice injected with 60 Pleasanton, CA, USA). Each fiber was tested against a sin- or 80 mg/kg simvastatin exhibited subsequent signs of gle dose of simvastatin, and peak fluorescence values were HMR, which included increased VO (Figure 1A), severe averaged across all fibers per group for each concentration. muscle contractures and increased heat production. After injection with simvastatin, a significantly higher peak VO Simvastatin sensitivity and AICAR effectiveness in occurred in YS mice receiving 60 mg/kg (P <0.05) and isolated fibers 80 mg/kg (P <0.001) doses when compared against To determine the effects of simvastatin dosing, YS and YS mice injected with the vehicle. Figure 1B shows WT fibers loaded with Fura-2 AM were perfused for the dose–response curve for peak VO as a function 2 minutes in warmed (32°C) Tyrode’s solution for record- of simvastatin dose in the YS mice. 2+ ing of baseline Ca levels, followed by a 3-minute expos- To determine if the statin-induced HMR event was ure at specified doses of simvastatin. Separately, isolated similar to heat-induced HMR in the YS mice, we fibers used to test AICAR’s effectiveness at preventing the injected the YS mice with 80 mg/kg of simvastatin 2+ statin-modulated change in Ca were pre-incubated in followed by 600 mg/kg of AICAR, which we have previ- 1 mM AICAR in conjunction with the 1-hour incuba- ously shown to prevent temperature-induced HMR in 2+ tion in DMEM/Fura-2 AM before exposure to 500 μM the YS mice by decreasing Ca leak from RyR1 [15]. and 1 mM simvastatin in the YS and WT, respectively. AICAR eliminated the statin-associated HMR in YS Fura-2 fluorescence was recorded and converted to cyto- mice by preventing the significant (P <0.01) increase in 2+ solic Ca values as previously reported [16]. VO that occurs in YS mice not receiving the AICAR treatment (Figure 1C). 2+ 4-CMC-induced Ca store depletion in isolated fibers 2+ To evaluate the effects of simvastatin on SR Ca store 2+ depletion, isolated fibers were exposed to 4-chloro-m- Myofibrillar Ca leak is more sensitive to simvastatin in cresol (4-CmC) immediately after 3 minutes of incuba- YS compared to WT muscle fibers tion in 500 μM simvastatin. 4-CmC was applied to either The strong protective effect of AICAR on the simva- YS or WT fibers at the dose found to induce maximal statin response of the YS mice suggests that statin- 2+ 2+ Ca release without causing death of the individual induced HMR in these mice is likely due to altered Ca fibers, which we determined to be 1 mM in the YS and handling within the myofiber. We tested the effects of 2.5 mM in the WT mice. simvastatin in isolated FDB fibers of YS and WT mice using the fluorescent dye Fura-2 to assess changes in 2+ Statistical analysis cytosolic Ca concentrations. We found that simva- 2+ A Student’s t-test was used for comparison between statin triggered higher cytosolic Ca levels in YS fibers groups to test significance values of P <0.05 (*), P <0.01 at lower concentrations (500 μM(P<0.001) and 750 μM (**), and P <0.001 (***). Dose–response curves were fit (P<0.01)) than in WT FDB fibers (Figure 2). As previ- using 4-parameter (oxygen consumption (VO )) or 3- ously shown with human fibers [7], WT fibers displayed 2+ parameter (single-fiber dose–response) Hill function increased Ca in response to higher doses of simva- curves in SigmaPlot, version 12.0 (Systat Software, statin (1.5 mM (P <0.01)). The concentration response San Jose, CA, USA). YS data was additionally fitted curves in the YS and WT mice were best fit using a Hill with a biphasic function using GraphPad Prism, ver- function (3-parameter) with a resulting EC of 0.6 mM sion 6 (GraphPad Software, La Jolla, CA, USA). in the YS and 0.9 mM in the WT mice. Since the YS fi- 2+ bers are from heterozygous mice, the Ca response re- Results flects the heterogeneous response from a mixture of Simvastatin triggers HMR in YS mice mutant channels (in various combinations of mutation We previously demonstrated that changes in VO could and WT subunits) and WT channels. Using a 2-site 2+ be used to detect the HMR response in the YS mice ex- model, we obtain EC s of 0.4 and 0.9 mM. Ca con- posed to elevated environmental temperatures [15]. This centrations were calculated from the Fura-2 fluorescence approach allows early detection of the HMR and allows as described in Methods. Knoblauch et al. Skeletal Muscle 2013, 3:22 Page 4 of 7 http://www.skeletalmusclejournal.com/content/3/1/22 Figure 1 An acute dose of simvastatin at 32°C results in higher peak VO levels in YS compared to WT mice. (A) IP injection of simvastatin triggers significantly higher peak VO values at 60 mg/kg (P<0.05) and 80 mg/kg (P <0.001) compared to vehicle-only injection. (B) Curve-fit of increasing simvastatin doses in YS mice. (C) Pre-treatment with 600 mg/kg AICAR results in significantly (P<0.01) lower peak VO values when administered 20 minutes after simvastatin treatment in YS mice. (D) Representative VO tracings of YS mice receiving 80 mg/kg of simvastatin show increasingly higher VO values than YS mice treated with both 80 mg/kg simvastatin and 600 mg/kg AICAR, vehicle-only (DMSO), or WT mice treated with 80 mg/kg simvastatin. AICAR, 5-aminoimidazole-4-carboxamide ribonucleoside; DMSO, dimethyl sulfoxide; VO , oxygen consumption; WT, wild type; YS, Y524S. 2+ Simvastatin depletes SR Ca stores in FDB fibers isolated from YS mice 2+ Ca stores in YS FDB fibers are decreased by exposure to elevated temperatures [17]. To determine if a reduc- 2+ tion in Ca stores occurs with simvastatin, we used 2+ Mag-fluo-4, a low-affinity Ca indicator, and 4-CmC to 2+ assess the readily releasable SR Ca stores [18]. 4-CmC was applied to isolated fibers immediately after a 3- minute incubation with simvastatin. We found a signifi- 2+ cant (P <0.05) decrease in the readily releasable Ca stores in YS fibers exposed to 500 μM simvastatin com- pared with YS fibers exposed to vehicle-only (Figure 3), while no difference was found in WT fibers at this con- centration of simvastatin. This finding suggests that the 2+ increased cytosolic Ca levels in the YS mice that occur 2+ after exposure to simvastatin are due to SR Ca leak 2+ leading to SR Ca store depletion. Figure 2 Isolated fibers from YS mice exhibit increased We assessed the ability of AICAR to regulate the 2+ sensitivity to simvastatin compared to WT mice. Dose–response simvastatin-induced increase in Ca leak in the YS fi- curves from isolated WT and YS FDB fibers incubated for 3 minutes bers. Isolated YS fibers were incubated with 1 mM in respective doses of simvastatin. Data points reflect peak cytosolic 2+ AICAR prior to incubation with 500 μM simvastatin. As Ca change from baseline, indicating that fibers from YS mice 2+ shown in Figure 4A, Ca stores were protected from respond to simvastatin at lower doses than WT fibers. Fibers were used only at a single simvastatin concentration. Each data point the simvastatin-induced depletion by prior administra- 2+ represents the mean cytosolic Ca response from a minimum of tion of AICAR (P <0.01). We determined if AICAR 2+ three fibers taken from three separate mice. Ca , calcium; FDB, 2+ could also prevent the simvastatin-induced Ca release flexor digitorum brevis; WT, wild type; YS, Y524S. at higher simvastatin doses in WT mice (Figure 4B). Knoblauch et al. Skeletal Muscle 2013, 3:22 Page 5 of 7 http://www.skeletalmusclejournal.com/content/3/1/22 2+ Figure 3 Pre-incubation with simvastatin decreases the 4-CmC-modulated cytosolic Ca response in isolated FDB fibers from YS compared to WT mice. Represented as the change (Δ) from baseline to peak values, (A) shows that upon exposure to 1 mM 4-CmC those 2+ YS fibers incubated for 3 minutes in 500 μM simvastatin (YS-Sim) release significantly less Ca from the SR than YS fibers receiving vehicle-only (YS-Veh) incubation (P <0.01) and from YS fibers incubated in 1 mM AICAR followed by 500 μM simvastatin (YS-Sim + AICAR) (P <0.05). Numbers represent total fibers used per group from a minimum of three mice. (B) YS and (C) WT show representative Mag-fluo-4 fluorescence tracings in single fibers exposed to either simvastatin or vehicle. Arrows indicate the time point at which 4-CmC was applied to the 2+ fibers. AICAR, 5-aminoimidazole-4-carboxamide ribonucleoside; 4-CmC, 4-chloro-m-cresol; Ca , calcium; FDB, flexor digitorum brevis; SR, sarcoplasmic reticulum; WT, wild type; YS, Y524S. When WT fibers were incubated with 1 mM simvastatin, Discussion we found that AICAR pre-treatment also greatly de- Despite the prevalence of statin myopathies, a mechan- 2+ creased Ca release in WT fibers (P<0.001), suggesting ism to explain the underlying trigger has remained elu- 2+ that statins have the potential to trigger Ca release in sive. The current study’s objective was to determine normal fibers but require higher simvastatin concentra- whether a MH-associated defect in RyR1 increased sen- tions than YS fibers and that AICAR may be a useful sitivity to simvastatin and whether AICAR, which pre- intervention for SIM even in patients without RyR1 vents heat-induced HMR in the YS mice, blocked the mutations. response to simvastatin. We show that the YS mice 2+ 2+ Figure 4 Pre-treatment with AICAR reduces the cytosolic Ca response to simvastatin. Pre-incubation with AICAR prevents Ca release in (A) YS fibers exposed to 500 μM simvastatin (P <0.01) and in (B) WT fibers exposed to 1 mM simvastatin (P <0.001). (C) YS and (D) WT show 2+ change in Ca concentration for AICAR-treated (dashed) and untreated (solid) fibers after exposure to either 500 μM (YS) or 1 mM (WT) 2+ simvastatin at 2 minutes . AICAR, 5-aminoimidazole-4-carboxamide ribonucleoside; Ca , calcium; WT, wild type; YS, Y524S. Knoblauch et al. Skeletal Muscle 2013, 3:22 Page 6 of 7 http://www.skeletalmusclejournal.com/content/3/1/22 display an MH-like response (elevated VO , sustained lower simvastatin concentrations in the presence of a muscle contractures, elevated body temperature) to an RyR1 mutation associated with MH in humans. The acute dose of simvastatin, and the degree of response is prevalence of genetic abnormalities capable of causing 2+ dose-dependent. Simvastatin also enhances SR Ca leak MH has been estimated to be as low as 1:3,000 [12]. 2+ and SR Ca store depletion in FDB fibers from both YS Whereas the incidence of SIM is relatively low (approxi- and WT mice but the response in WT mice requires mately 10%) among the millions of statin users, it is higher concentrations of simvastatin. In FDB fibers from highly possible that those individuals exhibiting signs both YS and WT mice, the response to simvastatin was and symptoms of SIM are harboring an underlying RyR1 prevented by AICAR, suggesting that even in WT fibers myopathy. Guis et al. showed that seven of nine individ- the effect of simvastatin involves RyR1. uals exhibiting symptoms of severe statin myopathy were AICAR is a known activator of the energy sensing kin- found to have a positive IVCT, indicative of an under- ase, AMP-activated protein kinase (AMPK). We recently lying RyR1 abnormality [11]. Therefore, further research demonstrated, however, that AICAR also has a direct ef- is needed to determine whether individuals experiencing fect on RyR1 and rescues the YS mice from heat-induced SIM also have mutations in RyR1. If true, drugs such as sudden death independent of AMPK activation [15]. We AICAR that modulate RyR1 activity can be investigated now demonstrate that treatment of YS mice with AICAR, as a potential therapy for these individuals, which may 2+ which decreases Ca leak in the presence of cellular levels allow continued statin use without the side effects asso- of ATP [15], prevents the simvastatin-associated increases ciated with SIM. in VO and heat production as well as greatly attenuates 2+ Ca leak from the SR upon exposure of FDB fibers to Conclusions simvastatin. AICAR also largely eliminates the statin- The YS mutation in RyR1 increases the sensitivity to the 2+ induced Ca release in healthy WT mice. These results cholesterol-lowering medication simvastatin. This sensi- suggest that AICAR might also be a potential therapeutic tivity is marked by systemic increases in VO , muscle intervention to prevent statin myopathies associated with contractures and heat production due to a temporal re- 2+ RyR1 mutations in sensitive individuals and protect lease of Ca into the cytosol from the SR. Pharmaceutical 2+ against myopathies arising from high statin doses in indi- interventions that decrease Ca leak from RyR1 (such as viduals without RyR1 mutations. AICAR) prevent both the systemic manifestation of SIM 2+ and the statin-induced Ca release from the SR in single YS mutation explains clinical symptoms of SIM fibers. We show that RyR1 mutation increases sensitivity 2+ Alterations in Ca signaling with simvastatin could ex- to SIM, suggesting that individuals affected by SIM could plain many of the symptoms associated with SIM in harbor underlying RyR1 mutations and that AICAR may humans including muscle fatigue, cramping, and increased be an effective therapeutic intervention. levels of circulating CK. Depletion of stores contributes to 2+ fatigue, while increased resting Ca is known to trigger Abbreviations 2+ 4-CmC: 4-chloro-m-cresol; AICAR: 5-aminoimidazole-4-carboxamide Ca release and muscle contraction, giving rise to muscle ribonucleoside; AMPK: AMP-activated protein kinase; BTS: 4-methyl-N- cramping similar to that which occurs in Brody disease. 2+ (phenylmethyl)benzenesulfonamide; Ca : Calcium; CK: Creatine kinase; Brody disease results from a reduction in the number DMEM: Dulbecco’s modified Eagle’s medium; DMSO: Dimethyl sulfoxide; 2+ EC : Half maximal effective concentration; EDTA: Ethylenediaminetetraacetic and activity of sarco/endoplasmic reticulum Ca -ATPase acid; EtOH: Ethanol; FDB: flexor digitorum brevis; Fura-2 AM: Fura-2 (SERCA) proteins in skeletal muscle, which inhibits the acetoxymethyl ester; HMG-CoA: 3-hydroxy-3-methylglutaryl coenzyme-A; 2+ re-uptake of cytosolic Ca during muscle activity [19,20]. HMR: Hypermetabolic response; IP: Intraperitoneal; IVCT: In vitro contracture test; LDL: Low-density lipoprotein; MH: Malignant hyperthermia; Individuals afflicted with Brody disease complain of fa- 2+ RyR1: Ryanodine receptor type 1; SERCA: Sarco/endoplasmic reticulum Ca − tigue as well as muscle cramping that is exacerbated dur- ATPase; SIM: Statin-induced myopathy; SR: Sarcoplasmic reticulum; ing periods of increased activity such as exercise [19]. VO : Oxygen consumption; VO max: Maximum oxygen consumption; 2 2 WT: Wild type; YS: Y524S. These symptoms reflect those commonly reported among individuals experiencing SIM. Separately, elevated circu- Competing interests lating CK levels among individuals experiencing SIM can The authors declare that they have no competing interests. also be explained by rhabdomyolysis triggered by the 2+ statin-modulated increase in cytosolic Ca levels and acti- Authors’ contributions vation of calpains [21,22]. Elevated CK levels are com- MK conceived and developed the study, conducted indirect calorimetry 2+ monly experienced by individuals experiencing SIM. experiments, assisted with Ca imaging experiments, performed statistical 2+ analyses, and prepared the draft manuscript. ADA conducted Ca imaging experiments, assisted with analyses, prepared data, and assisted with Clinical relevance manuscript preparation. SLH created and maintained the YS mouse line, 2+ An acute dose of simvastatin increases cytosolic Ca assisted with study design and data interpretation, and assisted with levels within the myofiber and this increase occurs at manuscript preparation. All authors read and approved the final manuscript. Knoblauch et al. Skeletal Muscle 2013, 3:22 Page 7 of 7 http://www.skeletalmusclejournal.com/content/3/1/22 Acknowledgements G, van Engelen B: Brody syndrome: a clinically heterogeneous entity This was supported by NIH grants 5R01AR041802 and 5R01AR053349 to SLH. distinct from Brody disease: a review of literature and a cross-sectional A.D.A. was supported by a postdoctoral fellowship from the Mexican Council clinical study in 17 patients. Neuromuscul Disord 2012, 22:944–954. of Science and Technology (186607). We would like to thank the Mouse 21. Belcastro A, Shewchuk L, Raj D: Exercise-induced muscle injury: a calpain Phenotyping Core at Baylor College of Medicine, Houston, TX, USA, for the hypothesis. Mol Cell Biochem 1998, 179(1–2):135–145. use and assistance with the Oxymax indirect calorimetry system. 22. 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Durham W, Aracena-Parks P, Long C, Rossi A, Goonasekera S, Boncompagni S, Galvan D, Gilman C, Baker M, Shirokova N, Protasi F, Dirksen R, Hamilton and take full advantage of: S: RyR1 S-nitrosylation underlies environmental heat stroke and sudden death in Y522S RyR1 knockin mice. Cell 2008, 133:53–65. • Convenient online submission 18. Herrmann-Frank A, Richter M, Lehmann-Horn F: 4-Chloro-m-cresol: a • Thorough peer review specific tool to distinguish between malignant hyperthermia-susceptible and normal muscle. Biochem Pharmacol 1996, 52:149–155. • No space constraints or color figure charges 19. Vattemi G, Gualandi F, Oosterhof A, Marini M, Tonin P, Rimessi P, Neri M, • Immediate publication on acceptance Guglielmi V, Russignan A, Poli C, van Kuppevelt T, Ferlini A, Tomelleri G: • Inclusion in PubMed, CAS, Scopus and Google Scholar Brody disease: insights into biochemical features of SERCA1 and identification of a novel mutation. J Neuropathol Exp Neurol 2010, • Research which is freely available for redistribution 69(3):246–252. 20. Voermans N, Laan A, Oosterhof A, van Kuppevelt T, Drost G, Lammens M, Submit your manuscript at Kamsteeg E, Scotton C, Gualandi F, Guglielmi V, van den Heuvel L, Vattemi www.biomedcentral.com/submit http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Skeletal Muscle Springer Journals

Mice with RyR1 mutation (Y524S) undergo hypermetabolic response to simvastatin

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Springer Journals
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Copyright © 2013 by Knoblauch et al.; licensee BioMed Central Ltd.
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Life Sciences; Cell Biology; Developmental Biology; Biochemistry, general; Systems Biology; Biotechnology
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2044-5040
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10.1186/2044-5040-3-22
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24004537
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Abstract

Background: Statins are widely used drugs for the treatment of hyperlipidemia. Though relatively safe, some individuals taking statins experience rhabdymyolysis, muscle pain, and cramping, a condition termed statin-induced 2+ myopathy (SIM). To determine if mutations in the skeletal muscle calcium (Ca ) release channel, ryanodine receptor type 1 (RyR1), enhance the sensitivity to SIM we tested the effects of simvastatin, the statin that produces the highest incidence of SIM in humans, in mice with a mutation (Y524S, ‘YS’) in RyR1. This mutation is associated with malignant hyperthermia in humans. Exposure of mice with the YS mutation to mild elevations in environmental temperature produces a life-threatening hypermetabolic response (HMR) that is characterized by increased oxygen consumption (VO ), sustained muscle contractures, rhabdymyolysis, and elevated core body temperature. Methods: We assessed the ability of simvastatin to induce a hypermetabolic response in the YS mice using indirect 2+ calorimetry and to alter Ca release via RyR1 in isolated flexor digitorum brevis (FDB) fibers from WT and YS mice 2+ using fluorescent Ca indicators. We also tested the ability of 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) to protect against the simvastatin effects. Results: An acute dose of simvastatin triggers a hypermetabolic response in YS mice. In isolated YS muscle fibers, 2+ 2+ simvastatin triggers an increase in cytosolic Ca levels by increasing Ca leak from the sarcoplasmic reticulum (SR). 2+ With higher simvastatin doses, a similar cytosolic Ca increase occurs in wild type (WT) muscle fibers. Pre-treatment of YS and WT mice with AICAR prevents the response to simvastatin. Conclusions: A mutation in RyR1 associated with malignant hyperthermia increases susceptibility to an adverse 2+ response to simvastatin due to enhanced Ca release from the sarcoplasmic reticulum, suggesting that RyR1 mutations may underlie enhanced susceptibility to statin-induced myopathies. Our data suggest that AICAR may be useful for treating statin myopathies. Keywords: Statin-induced myopathy, Simvastatin, RyR1, Myopathy, Calcium signaling Background results in a condition termed statin-induced myopathy Statins (3-hydroxy-3-methylglutaryl coenzyme-A (HMG- (SIM) [2,3]. A mechanism to explain the underlying cause CoA) reductase inhibitors) are cholesterol-lowering drugs of SIM has yet to be elucidated. that have proven effective in decreasing low-density lipo- One emerging theory of SIM has centered on statins’ 2+ protein (LDL) levels and improving overall health [1]. For potential to modulate intramyofiber calcium (Ca ) the majority of patients, statins are well tolerated with few homeostasis [4-6]. This theory stems in part from the side effects. However, up to 10% of patients on a statin regi- finding that the direct application of simvastatin to healthy men display muscle-related symptoms including soreness, human myofibers triggers a significant increase in cyto- 2+ 2+ fatigue, and an increase in circulating levels of muscle- solic Ca [7]. The sudden release of Ca in response to specific proteins (for example creatine kinase (CK)) that direct application of statins in vitro has been suggested to originate from both mitochondria and the sarcoplasmic 2+ * Correspondence: susanh@bcm.edu reticulum (SR) [5,8,9], the predominant Ca storage or- Equal contributors ganelle within the myofiber. The potential involvement of Department of Molecular Biology and Biophysics, Baylor College of Medicine, 1 Baylor Plaza, Houston, TX 77030, USA © 2013 Knoblauch 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. Knoblauch et al. Skeletal Muscle 2013, 3:22 Page 2 of 7 http://www.skeletalmusclejournal.com/content/3/1/22 2+ the SR in statin-induced Ca release is particularly studies involving injection into mice for indirect calor- intriguing given the recent findings that mutations in imetry, simvastatin powder was dissolved in dimethyl sulf- 2+ ryanodine receptor type 1 (RyR1), the Ca release oxide (DMSO). For single-fiber perfusion work, a 12 mM channel of the SR, may underlie some instances of SIM simvastatin stock was prepared in 10% EtOH similar to [10,11]. Mutations in RyR1 are known to produce malig- previous studies [14]. After adjusting the pH to 7.0, the nant hyperthermia (MH), a life-threatening condition solution was brought up to 12 mM concentration in 2+ where uncontrolled release of Ca within the myofiber Tyrode’s solution containing 121 mM NaCl, 5 mM KCl, is triggered by exposure to certain volatile inhalants, 1.8 mM CaCl ,500 μM MgCl ,400 μMNaH PO ,100 2 2 2 4 elevated temperature, or exercise [12,13]. This uncon- μM EDTA, 5.5 mM glucose, and 24 mM NaHCO .Separ- 2+ trolled release of Ca results in sustained muscle contrac- ately, a vehicle-only stock was prepared identically but tions, elevated core temperature, rhabdomyolysis and, if without the addition of simvastatin. These prepared stocks unabated, death [12]. were aliquoted and frozen at −80°C until use. At present our understanding of the link between RyR1 mutations and statin myopathies has been limited Indirect calorimetry monitoring of VO max to in vitro work with muscle biopsies. Metterlein et al. Those YS and WT mice used to determine the effects found that biopsied muscle from MH-sensitive swine ex- of statin dosing in vivo were removed from their cage, hibit contraction upon exposure to statins in vitro [10]. weighed, and injected IP with an 30-80 mg/kg dose of ei- Similarly, Guis et al. found that muscle biopsies from ther simvastatin dissolved in DMSO or DMSO alone (‘ve- seven of nine human subjects exhibiting the signs of SIM hicle’). The mice were then returned to their cages for expressed abnormal in vitro contracture tests (IVCT) used 30 minutes, after which they were placed individually to screen for susceptibility to MH [11]. into an environmental chamber at 32°C containing indir- These in vitro findings combined with evidence that ect calorimetry chambers (Oxymax System, Columbus 2+ simvastatin modifies Ca homeostasis suggest that Instruments, Columbus, OH, USA), which allowed for RyR1 mutations may underlie enhanced susceptibility to monitoring of maximum oxygen consumption (VO SIM. We developed a mouse model (Y524S, ‘YS’) with a max (mL/kg/min)). Separately, to evaluate the effect- RyR1 knock-in mutation of tyrosine 524 to serine [13], iveness of a pharmaceutical agent shown previously to which in humans (Y522S) is associated with MH [13]. prevent heat-induced HMR response in YS mice [15], Mice homozygous for the mutation die at birth, while additional YS mice were injected IP with a 600 mg/kg heterozygous YS mice exhibit a hypermetabolic response dose of 5-aminoimadazole-4-carboxamide ribonucleo- (HMR) to elevated (37°C) temperature, volatile anes- side (AICAR) 20 minutes after simvastatin injection. thetics, or exercise in a warm environment. These mice are a valuable tool for studying some RyR1-associated Fiber isolation 2+ disorders. The purpose of the present study was to de- For mice destined for single-fiber Ca study, the flexor termine whether mice with this RyR1 mutation (Y524S) digitorum brevis (FDB) muscle was removed and imme- display HMR when given simvastatin and to evaluate the diately placed into Dulbecco’s modified Eagle’s medium 2+ effects of simvastatin on intramyofiber Ca homeostasis. (DMEM) containing 3 mg/mL collagenase and 10% (v/v) fetal bovine serum. After a 2-hour incubation at 37°C, Methods whole FDB muscles were transferred to 1 mL of DMEM Animal care and handling and plunged ten times through a 1 mL pipette tip to All procedures were approved by the Institutional separate individual fibers. Next, 150 μL of DMEM Animal Care and Use Committee at Baylor College of containing separated FDB fibers was placed onto a 25 mm Medicine, Houston, TX, USA. As previously described, glass coverslip that had been incubated for 2 hours with Y524S/WT male RyR1 (‘YS’) mice were developed and used 20 μg/mg of laminin in PBS and then subjected to in conjunction with wild type (WT) littermate controls at two washes in PBS and a final wash in DMEM. Prior 8 to 10 weeks of age. Mice were maintained on a 12:12 to use, plated fibers were incubated overnight at 37°C in light:dark cycle, had ad libitum access to water and stand- DMEM containing antibiotic-antimycotic (Gibco, Carlsbad, ard mouse chow, and were limited to normal cage activity CA, USA). only. All mice were sacrificed at the same time of day, consisting of cervical dislocation after anesthetization Isolated fiber preparation and imaging under isoflurane. To assess the sensitivity to simvastatin, after the over- night incubation the fibers were next incubated for 1 Statin preparation hour at room temperature in either DMEM containing Simvastatin was purchased from the manufacturer (LKT (10 μM) Fura-2 acetoxymethyl ester (Fura-2 AM) or 30 Laboratories, St Paul, MN, USA) in powder form. For minutes in DMEM containing (5 μM) Mag-fluo-4, with Knoblauch et al. Skeletal Muscle 2013, 3:22 Page 3 of 7 http://www.skeletalmusclejournal.com/content/3/1/22 (20 μM) contraction-inhibitor 4-methyl-N-(phenylmethyl) the mice to be euthanized prior to a full body contrac- benzenesulfonamide (BTS). Fibers were placed in a tion and death. To determine if statins also trigger an temperature controlled chamber (Dagan Corporation, HMR response, we injected mice with an acute dose of Minneapolis, MN, USA) on the stage of an inverted epi- simvastatin (IP 30 to 80 mg/kg) and placed the mice in fluorescence microscope (Nikon Inc, Melville, NY, USA) the chamber (32°C, a thermoneutral temperature that and warmed to 32°C over a 5-minute period in Tyrode’s does not trigger HMR in the untreated YS mice) of the solution. Fluorescence emission was captured using a indirect calorimeter and measured VO as a function high speed, digital QE CCD camera (TILL Photonics, of time after injection. All YS mice injected with 60 Pleasanton, CA, USA). Each fiber was tested against a sin- or 80 mg/kg simvastatin exhibited subsequent signs of gle dose of simvastatin, and peak fluorescence values were HMR, which included increased VO (Figure 1A), severe averaged across all fibers per group for each concentration. muscle contractures and increased heat production. After injection with simvastatin, a significantly higher peak VO Simvastatin sensitivity and AICAR effectiveness in occurred in YS mice receiving 60 mg/kg (P <0.05) and isolated fibers 80 mg/kg (P <0.001) doses when compared against To determine the effects of simvastatin dosing, YS and YS mice injected with the vehicle. Figure 1B shows WT fibers loaded with Fura-2 AM were perfused for the dose–response curve for peak VO as a function 2 minutes in warmed (32°C) Tyrode’s solution for record- of simvastatin dose in the YS mice. 2+ ing of baseline Ca levels, followed by a 3-minute expos- To determine if the statin-induced HMR event was ure at specified doses of simvastatin. Separately, isolated similar to heat-induced HMR in the YS mice, we fibers used to test AICAR’s effectiveness at preventing the injected the YS mice with 80 mg/kg of simvastatin 2+ statin-modulated change in Ca were pre-incubated in followed by 600 mg/kg of AICAR, which we have previ- 1 mM AICAR in conjunction with the 1-hour incuba- ously shown to prevent temperature-induced HMR in 2+ tion in DMEM/Fura-2 AM before exposure to 500 μM the YS mice by decreasing Ca leak from RyR1 [15]. and 1 mM simvastatin in the YS and WT, respectively. AICAR eliminated the statin-associated HMR in YS Fura-2 fluorescence was recorded and converted to cyto- mice by preventing the significant (P <0.01) increase in 2+ solic Ca values as previously reported [16]. VO that occurs in YS mice not receiving the AICAR treatment (Figure 1C). 2+ 4-CMC-induced Ca store depletion in isolated fibers 2+ To evaluate the effects of simvastatin on SR Ca store 2+ depletion, isolated fibers were exposed to 4-chloro-m- Myofibrillar Ca leak is more sensitive to simvastatin in cresol (4-CmC) immediately after 3 minutes of incuba- YS compared to WT muscle fibers tion in 500 μM simvastatin. 4-CmC was applied to either The strong protective effect of AICAR on the simva- YS or WT fibers at the dose found to induce maximal statin response of the YS mice suggests that statin- 2+ 2+ Ca release without causing death of the individual induced HMR in these mice is likely due to altered Ca fibers, which we determined to be 1 mM in the YS and handling within the myofiber. We tested the effects of 2.5 mM in the WT mice. simvastatin in isolated FDB fibers of YS and WT mice using the fluorescent dye Fura-2 to assess changes in 2+ Statistical analysis cytosolic Ca concentrations. We found that simva- 2+ A Student’s t-test was used for comparison between statin triggered higher cytosolic Ca levels in YS fibers groups to test significance values of P <0.05 (*), P <0.01 at lower concentrations (500 μM(P<0.001) and 750 μM (**), and P <0.001 (***). Dose–response curves were fit (P<0.01)) than in WT FDB fibers (Figure 2). As previ- using 4-parameter (oxygen consumption (VO )) or 3- ously shown with human fibers [7], WT fibers displayed 2+ parameter (single-fiber dose–response) Hill function increased Ca in response to higher doses of simva- curves in SigmaPlot, version 12.0 (Systat Software, statin (1.5 mM (P <0.01)). The concentration response San Jose, CA, USA). YS data was additionally fitted curves in the YS and WT mice were best fit using a Hill with a biphasic function using GraphPad Prism, ver- function (3-parameter) with a resulting EC of 0.6 mM sion 6 (GraphPad Software, La Jolla, CA, USA). in the YS and 0.9 mM in the WT mice. Since the YS fi- 2+ bers are from heterozygous mice, the Ca response re- Results flects the heterogeneous response from a mixture of Simvastatin triggers HMR in YS mice mutant channels (in various combinations of mutation We previously demonstrated that changes in VO could and WT subunits) and WT channels. Using a 2-site 2+ be used to detect the HMR response in the YS mice ex- model, we obtain EC s of 0.4 and 0.9 mM. Ca con- posed to elevated environmental temperatures [15]. This centrations were calculated from the Fura-2 fluorescence approach allows early detection of the HMR and allows as described in Methods. Knoblauch et al. Skeletal Muscle 2013, 3:22 Page 4 of 7 http://www.skeletalmusclejournal.com/content/3/1/22 Figure 1 An acute dose of simvastatin at 32°C results in higher peak VO levels in YS compared to WT mice. (A) IP injection of simvastatin triggers significantly higher peak VO values at 60 mg/kg (P<0.05) and 80 mg/kg (P <0.001) compared to vehicle-only injection. (B) Curve-fit of increasing simvastatin doses in YS mice. (C) Pre-treatment with 600 mg/kg AICAR results in significantly (P<0.01) lower peak VO values when administered 20 minutes after simvastatin treatment in YS mice. (D) Representative VO tracings of YS mice receiving 80 mg/kg of simvastatin show increasingly higher VO values than YS mice treated with both 80 mg/kg simvastatin and 600 mg/kg AICAR, vehicle-only (DMSO), or WT mice treated with 80 mg/kg simvastatin. AICAR, 5-aminoimidazole-4-carboxamide ribonucleoside; DMSO, dimethyl sulfoxide; VO , oxygen consumption; WT, wild type; YS, Y524S. 2+ Simvastatin depletes SR Ca stores in FDB fibers isolated from YS mice 2+ Ca stores in YS FDB fibers are decreased by exposure to elevated temperatures [17]. To determine if a reduc- 2+ tion in Ca stores occurs with simvastatin, we used 2+ Mag-fluo-4, a low-affinity Ca indicator, and 4-CmC to 2+ assess the readily releasable SR Ca stores [18]. 4-CmC was applied to isolated fibers immediately after a 3- minute incubation with simvastatin. We found a signifi- 2+ cant (P <0.05) decrease in the readily releasable Ca stores in YS fibers exposed to 500 μM simvastatin com- pared with YS fibers exposed to vehicle-only (Figure 3), while no difference was found in WT fibers at this con- centration of simvastatin. This finding suggests that the 2+ increased cytosolic Ca levels in the YS mice that occur 2+ after exposure to simvastatin are due to SR Ca leak 2+ leading to SR Ca store depletion. Figure 2 Isolated fibers from YS mice exhibit increased We assessed the ability of AICAR to regulate the 2+ sensitivity to simvastatin compared to WT mice. Dose–response simvastatin-induced increase in Ca leak in the YS fi- curves from isolated WT and YS FDB fibers incubated for 3 minutes bers. Isolated YS fibers were incubated with 1 mM in respective doses of simvastatin. Data points reflect peak cytosolic 2+ AICAR prior to incubation with 500 μM simvastatin. As Ca change from baseline, indicating that fibers from YS mice 2+ shown in Figure 4A, Ca stores were protected from respond to simvastatin at lower doses than WT fibers. Fibers were used only at a single simvastatin concentration. Each data point the simvastatin-induced depletion by prior administra- 2+ represents the mean cytosolic Ca response from a minimum of tion of AICAR (P <0.01). We determined if AICAR 2+ three fibers taken from three separate mice. Ca , calcium; FDB, 2+ could also prevent the simvastatin-induced Ca release flexor digitorum brevis; WT, wild type; YS, Y524S. at higher simvastatin doses in WT mice (Figure 4B). Knoblauch et al. Skeletal Muscle 2013, 3:22 Page 5 of 7 http://www.skeletalmusclejournal.com/content/3/1/22 2+ Figure 3 Pre-incubation with simvastatin decreases the 4-CmC-modulated cytosolic Ca response in isolated FDB fibers from YS compared to WT mice. Represented as the change (Δ) from baseline to peak values, (A) shows that upon exposure to 1 mM 4-CmC those 2+ YS fibers incubated for 3 minutes in 500 μM simvastatin (YS-Sim) release significantly less Ca from the SR than YS fibers receiving vehicle-only (YS-Veh) incubation (P <0.01) and from YS fibers incubated in 1 mM AICAR followed by 500 μM simvastatin (YS-Sim + AICAR) (P <0.05). Numbers represent total fibers used per group from a minimum of three mice. (B) YS and (C) WT show representative Mag-fluo-4 fluorescence tracings in single fibers exposed to either simvastatin or vehicle. Arrows indicate the time point at which 4-CmC was applied to the 2+ fibers. AICAR, 5-aminoimidazole-4-carboxamide ribonucleoside; 4-CmC, 4-chloro-m-cresol; Ca , calcium; FDB, flexor digitorum brevis; SR, sarcoplasmic reticulum; WT, wild type; YS, Y524S. When WT fibers were incubated with 1 mM simvastatin, Discussion we found that AICAR pre-treatment also greatly de- Despite the prevalence of statin myopathies, a mechan- 2+ creased Ca release in WT fibers (P<0.001), suggesting ism to explain the underlying trigger has remained elu- 2+ that statins have the potential to trigger Ca release in sive. The current study’s objective was to determine normal fibers but require higher simvastatin concentra- whether a MH-associated defect in RyR1 increased sen- tions than YS fibers and that AICAR may be a useful sitivity to simvastatin and whether AICAR, which pre- intervention for SIM even in patients without RyR1 vents heat-induced HMR in the YS mice, blocked the mutations. response to simvastatin. We show that the YS mice 2+ 2+ Figure 4 Pre-treatment with AICAR reduces the cytosolic Ca response to simvastatin. Pre-incubation with AICAR prevents Ca release in (A) YS fibers exposed to 500 μM simvastatin (P <0.01) and in (B) WT fibers exposed to 1 mM simvastatin (P <0.001). (C) YS and (D) WT show 2+ change in Ca concentration for AICAR-treated (dashed) and untreated (solid) fibers after exposure to either 500 μM (YS) or 1 mM (WT) 2+ simvastatin at 2 minutes . AICAR, 5-aminoimidazole-4-carboxamide ribonucleoside; Ca , calcium; WT, wild type; YS, Y524S. Knoblauch et al. Skeletal Muscle 2013, 3:22 Page 6 of 7 http://www.skeletalmusclejournal.com/content/3/1/22 display an MH-like response (elevated VO , sustained lower simvastatin concentrations in the presence of a muscle contractures, elevated body temperature) to an RyR1 mutation associated with MH in humans. The acute dose of simvastatin, and the degree of response is prevalence of genetic abnormalities capable of causing 2+ dose-dependent. Simvastatin also enhances SR Ca leak MH has been estimated to be as low as 1:3,000 [12]. 2+ and SR Ca store depletion in FDB fibers from both YS Whereas the incidence of SIM is relatively low (approxi- and WT mice but the response in WT mice requires mately 10%) among the millions of statin users, it is higher concentrations of simvastatin. In FDB fibers from highly possible that those individuals exhibiting signs both YS and WT mice, the response to simvastatin was and symptoms of SIM are harboring an underlying RyR1 prevented by AICAR, suggesting that even in WT fibers myopathy. Guis et al. showed that seven of nine individ- the effect of simvastatin involves RyR1. uals exhibiting symptoms of severe statin myopathy were AICAR is a known activator of the energy sensing kin- found to have a positive IVCT, indicative of an under- ase, AMP-activated protein kinase (AMPK). We recently lying RyR1 abnormality [11]. Therefore, further research demonstrated, however, that AICAR also has a direct ef- is needed to determine whether individuals experiencing fect on RyR1 and rescues the YS mice from heat-induced SIM also have mutations in RyR1. If true, drugs such as sudden death independent of AMPK activation [15]. We AICAR that modulate RyR1 activity can be investigated now demonstrate that treatment of YS mice with AICAR, as a potential therapy for these individuals, which may 2+ which decreases Ca leak in the presence of cellular levels allow continued statin use without the side effects asso- of ATP [15], prevents the simvastatin-associated increases ciated with SIM. in VO and heat production as well as greatly attenuates 2+ Ca leak from the SR upon exposure of FDB fibers to Conclusions simvastatin. AICAR also largely eliminates the statin- The YS mutation in RyR1 increases the sensitivity to the 2+ induced Ca release in healthy WT mice. These results cholesterol-lowering medication simvastatin. This sensi- suggest that AICAR might also be a potential therapeutic tivity is marked by systemic increases in VO , muscle intervention to prevent statin myopathies associated with contractures and heat production due to a temporal re- 2+ RyR1 mutations in sensitive individuals and protect lease of Ca into the cytosol from the SR. Pharmaceutical 2+ against myopathies arising from high statin doses in indi- interventions that decrease Ca leak from RyR1 (such as viduals without RyR1 mutations. AICAR) prevent both the systemic manifestation of SIM 2+ and the statin-induced Ca release from the SR in single YS mutation explains clinical symptoms of SIM fibers. We show that RyR1 mutation increases sensitivity 2+ Alterations in Ca signaling with simvastatin could ex- to SIM, suggesting that individuals affected by SIM could plain many of the symptoms associated with SIM in harbor underlying RyR1 mutations and that AICAR may humans including muscle fatigue, cramping, and increased be an effective therapeutic intervention. levels of circulating CK. Depletion of stores contributes to 2+ fatigue, while increased resting Ca is known to trigger Abbreviations 2+ 4-CmC: 4-chloro-m-cresol; AICAR: 5-aminoimidazole-4-carboxamide Ca release and muscle contraction, giving rise to muscle ribonucleoside; AMPK: AMP-activated protein kinase; BTS: 4-methyl-N- cramping similar to that which occurs in Brody disease. 2+ (phenylmethyl)benzenesulfonamide; Ca : Calcium; CK: Creatine kinase; Brody disease results from a reduction in the number DMEM: Dulbecco’s modified Eagle’s medium; DMSO: Dimethyl sulfoxide; 2+ EC : Half maximal effective concentration; EDTA: Ethylenediaminetetraacetic and activity of sarco/endoplasmic reticulum Ca -ATPase acid; EtOH: Ethanol; FDB: flexor digitorum brevis; Fura-2 AM: Fura-2 (SERCA) proteins in skeletal muscle, which inhibits the acetoxymethyl ester; HMG-CoA: 3-hydroxy-3-methylglutaryl coenzyme-A; 2+ re-uptake of cytosolic Ca during muscle activity [19,20]. HMR: Hypermetabolic response; IP: Intraperitoneal; IVCT: In vitro contracture test; LDL: Low-density lipoprotein; MH: Malignant hyperthermia; Individuals afflicted with Brody disease complain of fa- 2+ RyR1: Ryanodine receptor type 1; SERCA: Sarco/endoplasmic reticulum Ca − tigue as well as muscle cramping that is exacerbated dur- ATPase; SIM: Statin-induced myopathy; SR: Sarcoplasmic reticulum; ing periods of increased activity such as exercise [19]. VO : Oxygen consumption; VO max: Maximum oxygen consumption; 2 2 WT: Wild type; YS: Y524S. These symptoms reflect those commonly reported among individuals experiencing SIM. Separately, elevated circu- Competing interests lating CK levels among individuals experiencing SIM can The authors declare that they have no competing interests. also be explained by rhabdomyolysis triggered by the 2+ statin-modulated increase in cytosolic Ca levels and acti- Authors’ contributions vation of calpains [21,22]. Elevated CK levels are com- MK conceived and developed the study, conducted indirect calorimetry 2+ monly experienced by individuals experiencing SIM. experiments, assisted with Ca imaging experiments, performed statistical 2+ analyses, and prepared the draft manuscript. ADA conducted Ca imaging experiments, assisted with analyses, prepared data, and assisted with Clinical relevance manuscript preparation. SLH created and maintained the YS mouse line, 2+ An acute dose of simvastatin increases cytosolic Ca assisted with study design and data interpretation, and assisted with levels within the myofiber and this increase occurs at manuscript preparation. All authors read and approved the final manuscript. Knoblauch et al. Skeletal Muscle 2013, 3:22 Page 7 of 7 http://www.skeletalmusclejournal.com/content/3/1/22 Acknowledgements G, van Engelen B: Brody syndrome: a clinically heterogeneous entity This was supported by NIH grants 5R01AR041802 and 5R01AR053349 to SLH. distinct from Brody disease: a review of literature and a cross-sectional A.D.A. was supported by a postdoctoral fellowship from the Mexican Council clinical study in 17 patients. Neuromuscul Disord 2012, 22:944–954. of Science and Technology (186607). We would like to thank the Mouse 21. Belcastro A, Shewchuk L, Raj D: Exercise-induced muscle injury: a calpain Phenotyping Core at Baylor College of Medicine, Houston, TX, USA, for the hypothesis. Mol Cell Biochem 1998, 179(1–2):135–145. use and assistance with the Oxymax indirect calorimetry system. 22. 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