Toxicology and Applied Pharmacology 259 (2012) 263–268

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Toxicology and Applied Pharmacology

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Muscle mitochondrial metabolism and calcium signaling impairment in patientstreated with statins

P. Sirvent a,b,c,⁎, O. Fabre a,b, S. Bordenave a,b, D. Hillaire-Buys b, E. Raynaud De Mauverger a,b,A. Lacampagne a,b, J. Mercier a,b

a U1046, INSERM, Université Montpellier 1 & Université Montpellier 2, 34295 Montpellier, Franceb CHRU Montpellier, 34295 Montpellier, Francec Clermont Université, Université Blaise Pascal, EA 3533, Laboratoire des Adaptations Métaboliques à l'Exercice en conditions Physiologiques et Pathologiques (AME2P), BP 80026,F-63171 Aubière cedex, France

Abbreviations: LDL, low density lipoprotein; CK, crereticulum; HRmax, maximal heart rate; Pmax, theoretical mrespiration rate; ACR, acceptor control ratio.⁎ Corresponding author at: Université Blaise Pascal

Métaboliques à l'Exercice en conditions PhysiologiqueBat Biologie B, 24 avenue des Landais, BP 80026, 6317173405062.

E-mail address: pascal.sirvent@univ-bpclermont.fr (

0041-008X/$ – see front matter © 2012 Elsevier Inc. Alldoi:10.1016/j.taap.2012.01.008

a b s t r a c t

a r t i c l e i n f o

Article history:Received 9 November 2011Revised 4 January 2012Accepted 9 January 2012Available online 17 January 2012

Keywords:Skeletal muscleStatinsCalcium homeostasisMitochondriaMuscle pain

The most common and problematic side effect of statins is myopathy. To date, the patho-physiologicalmechanisms of statin myotoxicity are still not clearly understood. In previous studies, we showed thatacute application in vitro of simvastatin caused impairment of mitochondrial function and dysfunction ofcalcium homeostasis in human and rat healthy muscle samples. We thus evaluated in the present study,mitochondrial function and calcium signaling in muscles of patients treated with statins, who present or notmuscle symptoms, by oxygraphy and recording of calcium sparks, respectively. Patients treated with statinsshowed impairment of mitochondrial respiration that involved mainly the complex I of the respiratory chainand altered frequency and amplitude of calcium sparks. Themuscle problems observed in statin-treated patientsappear thus to be related to impairment of mitochondrial function and muscle calcium homeostasis, confirmingthe results we previously reported in vitro.

© 2012 Elsevier Inc. All rights reserved.

Introduction

Statins are reversible and competitive inhibitors of themicrosomalenzyme 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase. Byblocking this rate-limiting step in the mevalonate pathway, endoge-nous de novo synthesis of cholesterol is prevented and low densitylipoprotein (LDL) cholesterol uptake into cells is promoted (Goldsteinand Brown, 1990). Clinical trials have demonstrated a reduction incardiovascular-related morbidity and mortality in patients with orwithout coronary artery disease treated with statins (Anon, 1993,1998; Sacks et al., 1996). In addition to lowering cholesterol, statinsalso present cholesterol-independent effects such as improvement ofendothelial function, enhanced stability of atherosclerotic plaques,decrease of inflammation and oxidative stress and inhibition of thethrombogenic response in the vascular wall (Davignon, 2004; Halcoxand Deanfield, 2004; Liao, 2002; Takemoto et al., 2001).

Usually, statins are well tolerated (Anon, 1994; Bellosta et al.,2004; Pedersen et al., 2000; Sacks et al., 1996), but side effects that

atine kinase; SR, sarcoplasmicaximal power; Vmax, maximal

, Laboratoire des Adaptationss et Pathologiques (AME2P),, Aubière, France. Fax: +33 4

P. Sirvent).

rights reserved.

concern skeletal muscle may arise. These can range from transientincreases in creatine kinase (CK), muscle pain and cramps, to myositisand the potentially life threatening rhabdomyolysis (Bellosta et al.,2004; Farmer and Torre-Amione, 2000; Rosenson, 2004). Physicalexercise predisposes patients who take statins to develop muscle-specific side effects (Sinzinger and O'Grady, 2004; Thompson et al.,1997). This is also true for some forms of co-medication (Bellostaet al., 2004). Indeed, the concomitant administration of inhibitors ofCytochrome P450 3A4 (e.g., macrolide antibiotics, cyclosporin A, orazole antimycotics) puts patients at a higher risk (Bellosta et al., 2004;Farmer and Torre-Amione, 2000; Neuvonen et al., 2006; Rosenson,2004). While fatal rhabdomyolysis is a rare event (i.e., 0.15 deaths permillion of prescriptions), deleterious muscle manifestations such asmyalgia and myositis are much more common with an estimatedoccurrence of 1-7% (Thompson et al., 2003). Moreover, these muscleside effects do not correlate with the extent of the cholesterol loweringeffect of the drug used (Baer et al., 2007; Christopher-Stine, 2006).Taking into account the large number of statin-treated patients, whichwill increase in the next few years, it is crucial to understand themechanisms of statin-induced myotoxicity in order to prevent thisadverse effect.

Our group has recently shown that acute application of relativelyhigh doses of simvastatin to human and rat skinned skeletal musclesamples triggers large release of Ca2+ from the sarcoplasmic reticulum(SR) following early alteration of the mitochondrial function (Sirventet al., 2005b). Specifically, we demonstrated that a specificmodification

264 P. Sirvent et al. / Toxicology and Applied Pharmacology 259 (2012) 263–268

of the complex I of the mitochondrial respiratory chain represents theearly step of a cascade of deleterious cellular mechanisms induced bysimvastatin that lead to Ca2+ homeostasis deregulation, which couldbe responsible of the statin-induced myotoxicity in vivo (Sirvent et al.,2005a). Other studies in rat skeletal muscles (Liantonio et al., 2007;Nadanaciva et al., 2007) and human skeletal muscle cells (Sacher etal., 2005) confirmed that mitochondria and calcium homeostasis aredisturbed following acute application of statins.

The present study has been designed to assess in the clinicalsetting and with therapeutic doses of statins whether a similar impair-ment of mitochondrial respiration and/or alteration in Ca2+ homeosta-sis in skeletal muscle could also explain themuscular deleterious effectsfrequently observed in patients treated with statins.

To this aim we recruited three different groups of subjects:controls, patients treated with statins who do not present musclesymptoms (AS) and statin-treated patients with muscle symptoms(SS). The distinction between AS and SS was based on clinical (musclepain) as well as biological (creatine kinase and lactate dehydrogenaseactivities) parameters. We investigated their exercise response duringan incremental andmaximal cycloergometer test. Finally, we analyzedmitochondrial respiration and Ca2+ sparks in skeletal muscle fibersfrom controls, AS and SS patients.

Material and methods

Subjects. 28 male subjects, aged between 40 and 60 years, partici-pated in the study. They did not take any medication known to poten-tially interfere with muscle metabolism (such as fibrates, β-blockers,laxatives, diuretics, bronchodilatators) and they did not exercisemore than two hours per week (exclusion criteria). Individuals witha family history of muscle symptoms were also excluded. Informedconsent was obtained from all the subjects after explanation ofthe protocol and of the potential risks. The study was approved bythe local Ethic Committee (CCP 040710) and was conform to the Dec-laration of Helsinki regarding experimentation with human subjects.Patients were distributed in three groups: 9 controls, 10 subjectstreated with statins, without clinical and biological expression ofmuscle symptoms (normal CK and LDH activities: CK b 200 UI/l, andLDH b 450 UI/l) (Asymptomatic with Statins, AS), 9 subjects treatedwith statins, who present clinical (muscle pain) and/or biologicalexpression of muscular symptoms (CK activity > 200 UI/l and LDH >450 UI/l) (Symptomatic Statin, SS). Study population characteristics(age, BMI, daily physical activity) are described in Table 1. AS and SSindividuals took one of the four following statins for less than twoyears: atorvastatin (n=4; 2 AS, 2 SS), fluvastatin (n=4; 2 AS, 2 SS),pravastatin (n=4; 1 AS, 3 SS), simvastatin (n=6; 4 AS, 2 SS) androsuvastatin (n=1; 1 AS). Daily physical activity was estimated withthe Voorrips questionnaire (Voorrips et al., 1991) and the scoresobtained were not significantly different in the three groups.

Exercise testing. Subjects performed an incremental and maximalexercise test on an electromagnetically braked cycle ergometer(Ergoline Bosh 500) connected to a breath-by-breath sampling device(ZAN 600) for gas exchange measurements (VO2 and VCO2). Bloodpressure and electrocardiographic recordings were controlled before

Table 1Study population characteristics.

Controls (n=9) StatinAsymptomatic (n=10)

StatinSymptomatic (n=9)

Age (years) 46.9±3.9 55.0±3.8 ** 53.6±4,4 **BMI (kg/m2) 22.9±2.0 28.1±2,1 ** 27.5±3.5 *Voorips score 6.7±3.4 9.5±4.3 5.8±2.5

Data are presented as the mean±SEM. *pb0.05; **pb0.01 compared to controls.

and throughout the test. Pedaling rate was set between 50 and70 rev/min throughout the test. After 3-min warm-up at 20 W, thepower output increased every minute until physical exhaustion.Workload increases were set for each volunteer at 10% of the theoret-ical maximal power (Pmax) determined according to the formulaproposed by Wasserman et al. (30). Exercise test was considered asbeing maximal when the following conditions were fulfilled: constantVO2 despite increment increase; maximal heart rate (HRmax) near thetheoretical HRmax [HRmax=220 – age (year)], respiratory quotient>1.1and exhaustion of the subject. All tests were performed between8 and 10 AM after overnight fast.

Blood samples. Blood samples were drawn through a 32-mm cathe-ter placed into a superficial forearm vein. Venous blood samples,used for the determination of biochemical and hormonal parameters,were collected in Vacutainer® tubes containing heparin at differenttime points: at rest, at 50% of Pmax (corresponding to the ventilatorythreshold), at maximal exercise test and 20 min after the end of thetest.

To estimate muscle damage, CK (Olympus AU 2700, DiaSys CKNAC FS kit), LDH (Olympus AU 2700, Olympus kit) and myoglobin(chemiluminescence immunoassay, Bekman Coulter DXI) were alsomeasured at rest and at maximal exercise.

Muscle biopsy. Muscle biopsies were performed 3 days after thelast exercise testing, in the inferior third level of the vastus lateralismuscle under local anesthesia (xylocain) with a Bergström needle.Biopsies were divided into two portions. For imaging experiments,one half of each biopsy was immediately placed in the internal-likemedium (140 mol/L K-glutamate, 10 mol/L Hepes, 20 mol/L phospho-creatine, 5 mol/L Na2ATP, 4.53 mol/L MgCl2, 1 mol/L EGTA, 0.29 mol/LCaCl2 and 2 mol/L Malate, pH 7.0). For in situ mitochondrial respira-tion studies, the other half of the biopsies was placed in ice-coldrelaxing solution (10 mM EGTA–calcium buffer: 100 nM free Ca2+,20 mM imidazole, 3 mM KH2PO4, 1 mM MgCl2, 20 mM Taurine,0.5 mM DTT, 5 mM MgATP and 15 mM Phosphocreatine, pH 7.1).

Measurements of mitochondrial respiration. Respiratory parametersof the total mitochondrial population were studied in situ on freshskeletal muscle fibers as previously described (Thomas et al., 2004).Bundles of muscle fibers were manually isolated and saponin-skinned (50 μg/ml saponin for 30 min at 4 °C). Respiration rateswere determined at 27 °C with a Clark electrode (Strathkelvin,Glasgow, Scotland) in an oxygraphic cell containing respiration solu-tion (same composition as the relaxing solution, except that MgATPand phosphocreatine were replaced by 3 mM phosphate and 2 mMfatty acid-free bovine serum albumin). Respiration rates wereexpressed in micromoles of O2 per minute per gram of dry weight.Basal oxygen consumption without ADP (V0), maximal respirationrate (Vmax) with a saturating concentration of ADP and acceptor con-trol ratio (ACR), and the ratio between Vmax and V0, which representsthe level of coupling between oxidation and phosphorylation, weremeasured. At the end of each measurement, the Cytochrome C testwas used to investigate the outer mitochondrial membrane integrity(Saks et al., 1998). Respiration rates were recorded in the presenceof 5 mM glutamate/2 mM malate, 10 mM pyruvate/2 mM malate,40 μM octanoyl-carnitine/2 nM malate, 5 mM glutamate/2 mMmalate, 10 mM succinate/20 μM rotenone, or 5 μM TMPD/2 mMascorbate/6.5 μM antimycin. V0 was first recorded and Vmax was de-termined in the presence of 2 mM ADP. The ADP-stimulated maximalrespiration under succinate feeding, with rotenone as inhibitor ofcomplex I, represents the maximal respiration from complexes II,III and IV of the respiratory chain. The ADP-stimulated maximalrespiration under TMPD-ascorbate feeding, with antimycin as an in-hibitor of complex III, represents the maximal respiration only fromcomplex IV (Cytochrome C Oxydase complex). After the respiratory

Table 2Effect of statin treatment on response to exercise.

Controls(n=9)

StatinAsymptomatic(n=10)

StatinSymptomatic(n=9)

VO2max (ml/min/Kg) 42.9±1.6 31.9±1.1 ** 31.7±2.2 **VO2max (% VO2max th) 122.8±5.8 112.9±2.8 107.1±5.1Pmax (Watts) 218.8±12.3 201.8±11.1 185.1±11.1 *Pmax (% Pmax th) 129.1±6.6 117.3±5.7 107.2±5.6 *HRmax (ppm) 167.1±4.0 163.1±2.3 148.1±4.7 *HRmax (% HRmax th) 96.5±1.8 98.9±1.2 89±2.6 *

Data are presented as the mean±SEM. *pb0.05; **pb0.01 compared to controls.

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measurements, fibers bundles were removed, dried overnight andweighed the next day. Respiration rates were expressed in micro-moles of O2 per minute per gram of dry weight.

Measurements of Ca2+sparks. Fibers were bathed in an internalmedium containing the fluorescent Ca2+ indicator Fluo-3 (50 μMpentapotassium salt; TefLabs, Austin TX, USA). Variations in intracel-lular Ca2+ were measured by time series (1 image/5 s) of x–y confo-cal fluorescent images (Zeiss LSM 510 Meta, 63X objective, NA=1.2,H2O immersion). Fluo-3 was excited with an argon/krypton laserat 488 nm and fluorescence was recorded at ~525 nm. Local Ca2+ re-lease events (i.e., Ca2+ sparks) were measured as previously reported(Sirvent et al., 2005b). Fluorescence images were acquired with theconfocal system operated in line-scan mode (x vs. t, 1.5 ms/line).Potential spark areas were empirically identified using an auto-detection algorithm (Ward et al., 2003). The mean F value for theimage was calculated by adding up and averaging the temporal Fat each spatial location while ignoring potential spark areas. TheF value was then used to create a pixel-to-pixel ΔF/F image. Selectionand analysis of Ca2+ sparks were performed essentially as previouslydescribed (18). Determination of the spatio-temporal propertiesof individual Ca2+ sparks was made on spatial (x) and temporal (t)profiles of sparks centered at the peak amplitude. ΔF/F amplitude aswell as temporal parameters, rise-time and time constant decaywere derived from the temporal profile. The width of the Ca2+

spark (full width at half of the maximum peak amplitude, FWHM)was determined from the spatial profile.

Data analysis and statistical procedures. For spatio-temporal proper-ties of Ca2+ sparks, statistical comparison of the parameters in thethree groups was conducted using a non-parametric Kruskal–Wallistest. Other statistical comparisons were done by variance analysis(ANOVA), using the Sigmastat software. Student's t-test was used todetermine the statistical significance of differences between twogroups. Data are expressed as means±SEM and differences were con-sidered significant when pb0.05.

Chemical reagents. All drugs used were purchased from Sigma, exceptfor Fluo-3 (TefLabs).

Table 3Effect of statin treatment on biological parameters.

Controls (n=9) Statin A

Rest Max Rest

CK (UI/l) 100±15.1 125.4±17.3 115.9±LDH (UI/l) 398.4±26.7 565.2±88 439.9±Myoglobin (μg/l) 28.2±2.1 29.1±2.9 36.5±

Data are presented as the mean±SEM. *pb0.05; **pb0.01 compared to controls. Max, at m

Results

Effect of statin treatment on the response to exercise

During the incremental exercise test (Table 2), maximal VO2 con-sumption (VO2max) was significantly lower in the two groups of pa-tients treated with statins than in controls (pb0.01). This differencewas not confirmed when the VO2max values were expressed as per-centage of the theoretical VO2max. Furthermore, comparison of thevalues of the maximal power (Pmax) expressed in watts and as per-centage of the theoretical Pmax in the three groups showed significantlower outputs in SS patients compared to controls (pb0.05). Finally,the maximal heart rate (HRmax), expressed in beats per minute(bpm) and as percentage of the theoretical HRmax, was significantlydecreased in the SS group compared to controls and AS patients(pb0.05). Conversely, the ventilatory threshold was not affected bystatin treatment (data not shown).

Effect of statin treatment on biological parameters

Many patients with statin-induced myalgia have normal or onlymarginally elevated creatine kinase (CK) levels (Draeger et al.,2006). Similarly, in the SS group (n=9), only one patient had elevatedserum level of CK at rest (> 200 UI/l). This patient also had elevatedlactate dehydrogenase activities (LDH) at rest (> 450 UI/l), withoutmyalgia. No other differences were found concerning these twoparameters in the three groups. At maximal exercise level, serummyoglobin was significantly increased in the AS group comparedto controls and SS patients, despite values within a normal range(Table 3).

Effect of statin treatment on mitochondrial function

Mitochondrial respiration was assessed on bundles of saponin-skinned human skeletal muscle fibers from muscle biopsies of AS, SSand control patients. To explore distinct metabolic pathways, differentsubstrates, such as pyruvate (with malate), which is metabolized bythe pyruvate dehydrogenase complex localized in the mitochondrialmatrix, and octanoate (with malate), which is transferred into the ma-trix and is metabolized in the β-oxidation, were chosen. Respirationratewas significantly inhibited in both AS and SS groups using pyruvate(a substrate of the glucidic pathway) in comparison to controls(AS: 5.14±0.36, SS: 4.85±0.42 vs controls: 7.0±0.62 μmol/min/g;pb0.02). We also observed a significant inhibition of the respirationrate in the SS group using octanoate (a substrate of the lipidic pathway)compared to controls and AS patients (SS: 3.62±0.30 vs controls:4.8±0.33 and vs AS: 4.61±0.24 μmol/min/g; pb0.03) (Fig. 1).

We then used specific substrates and inhibitors to sequentiallyevaluate the effect of statin treatment on the different complexes ofthe respiratory chain. When glutamate and malate, which activatethe complexes I, III and IV of the respiratory chain, were used assubstrates, a significant inhibition of the maximal ADP-stimulatedrespiration rate was observed in both AS and SS groups in comparisonto controls (AS: 5.58±0.37 and SS: 5.03±0.31 vs controls: 7.0±0.56 μmol/min/g; pb0.01 and pb0.05 respectively) (Fig. 2). In the

symptomatic (n=10) Statin Symptomatic (n=9)

Max Rest Max

15.3 138.5±17 121.6±16.5 152.5±18.469.3 453.2±24.2 446.1±46.1 482.7±52.13.9 47.13±4.65 * 31.9±3.3 34.7±3.2

aximal exercise test.

Fig. 1. Effects of statin treatment onmitochondrial function (control n=9, AS n=10, SSn=9). Comparison of ADP-stimulated respiration rates in the three groups when usingpyruvate (+ malate) (A) or octanoate (+ malate) (B) as substrates. *pb0.05; **pb0.01compared to controls. AS, asymptomatic statin-treated patients; SS, symptomaticstatin-treated patients.

Fig. 3. Effects of statin treatment on the spatio-temporal properties of Ca2+ sparks(control n=9, AS n=10, SS n=9). Spatio-temporal analysis of brief (b 100 ms) andlocalized increases in fluorescence, which correspond to Ca2+ sparks, was performedusing a bi-exponential fitting procedure. (A) Comparison of Ca2+ spark frequencyand (B) comparison of Ca2+ spark amplitude in the three groups. *pb0.05 comparedto controls. AS, asymptomatic statin-treated patients; SS, symptomatic statin-treatedpatients.

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presence of N,N,N0,N0-tetramethyl-p-phenylenediamine (TMPD)and ascorbate, which are used to test specifically the activity of thecomplex IV, the maximal ADP-stimulated respiration rate was againdecreased but only in the SS group (SS: 17.53±0.95 vs controls:21.2±1.35 μmol/min/g; pb0.05). No significant differences amonggroups could be seen using succinate (and rotenone), which restrictsthe activity of the respiratory chain to the complexes II, III and IV.

Effect of statin treatment on Ca2+ sparks properties

To characterize the effects of statin treatment on SR Ca2+ release,we quantified the frequency of spontaneous Ca2+ sparks as it reflectsthe behavior of the SR Ca2+ release channels in situ (Lacampagneet al., 1998). Ca2+ spark frequency was significantly decreased inthe SS group in comparison to controls (SS: 1.97±0.15 and controls:2.48±0.37 events/μm/s/103; pb0.05) (Fig. 3A). Amplitude of Ca2+

sparks was increased in both SS and AS groups (Fig. 3B). Conversely,the temporal properties of Ca2+ sparks (rise time, decay) were notaffected by statin treatment (data not shown).

Discussion

In this study, we show that patients treated with statins have animpairment of mitochondrial respiration that involves mainly thecomplex I of the respiratory chain and altered frequency and ampli-tude of calcium sparks. Moreover, our results in patients furthersupport the notion that statin-induced muscular toxicity is associatedwith early alteration of one or more complexes of the mitochondrialrespiratory chain that would lead to impaired mitochondrial functionand deregulation of Ca2+ homeostasis.

A weakness of this study is the low number of subjects and theheterogeneity of the three groups of patients regarding age and BMI

Fig. 2. Effects of statin treatment on the different complexes of the mitochondrial respiratorrates in the three groups when using glutamate (+ malate) (A), succinate (+ rotenone) (B)to controls. AS, asymptomatic statin-treated patients; SS, symptomatic statin-treated patien

(Table 1). Nevertheless, although age and BMI are significantly higherin the AS and SS groups compared to the control group, the differenceisweak (about 7 years and 5 kg/m2, respectively). Therefore, althoughwe cannot exclude an age and/or BMI effect, the differences observedin this study are more probably linked to the treatment with statins.Moreover, daily physical activity, which can decline when age orBMI increase was not significantly different in the three groups asestimated with the Voorrips questionnaire (Voorrips et al., 1991). Inorder to definitely assess whether the differences observed in thisstudy were influenced by age and/or BMI, we performed a secondstatistical analysis in which the AS and SS patients with the highestage and BMI were excluded (data not shown; the figures show theresults of the statistical analysis carried out with all the subjects).This new analysis, with only six patients per group and no differencesin age and BMI between the control group and the groups treatedwithstatins, confirmed the results for the biological, mitochondrial andcalcium homeostasis parameters. Therefore, we can assume that thedifferences between the groups treated with statins and the controlgroup are not linked to differences in age and/or BMI.

In the first part of this study, exercise capacity was evaluated atrest and during exercise in statin-treated patients with or withoutmuscular symptoms and control individuals. Compared to controls,statin-treated patients exhibited a significant decrease of maximalVO2 consumption that was not confirmed when values were expressedas percentage of the theoretical VO2max. This discrepancy could beexplained by the older age and higher body mass index (BMI) of thetwo statin-treated groups in comparison to controls, as these twofactors are known to influence ventilatory parameters. Moreover, thesignificant decrease in maximal power (Pmax) and maximal heartrate (HRmax) (expressed as percentage of the theoretical Pmax and ofthe theoretical HRmax) observed only in the statin-treated patientswith muscle symptoms can be indicative of the development of

y chain (control n=9, AS n=10, SS n=9). Comparison of ADP-stimulated respirationor TMPD and ascorbate (+ antimycin) (C) as substrates. *pb0.05; **pb0.01 comparedts.

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mobility restriction and exercise intolerance, both possibly linked tothe presence of muscle pain.

Muscle symptoms in patients treated with statins appear to beindependent from the increase of biological markers of muscle break-down (Christopher-Stine, 2006; Rosenson, 2004). Our results confirmthese observations. Indeed, plasma levels of CK, LDH and myoglobindid not show clear changes in the two groups of patients treatedwith statins at rest or at maximal exercise in comparison to controls.Only one patient, who did not have clinical muscle symptoms,presented slightly elevated levels of CK and LDH. This observationsuggests that elevation of biological markers of muscle breakdownmight appear only occasionally when some cellular defects, such as,for instance, impaired calcium homeostasis, would exceed a certainthreshold.

As previously reported (for review (Golomb and Evans, 2008;Sirvent et al., 2008)), the main mechanism of statin-induced myo-toxicity appears to result from a direct effect of statins on mitochon-dria. Similarly, we have previously demonstrated (Sirvent et al.,2005a, 2005b) that acute application of high doses of simvastatin tohealthy human muscle cells led to impaired mitochondrial function,as indicated by their higher NADH auto-fluorescence and lowermaximal ADP-stimulated respiration rate and membrane potentialin comparison to untreated samples. The deleterious effect of simva-statin was significantly targeted to complex I of the mitochondrialrespiratory chain. We also observed a limited effect on complex IVbut, according also to the observations by Mazat et al. (Mazat et al.,1997), the amplitude of simvastatin-induced inhibition of this com-plex was probably too low to affect global mitochondrial respiration.In the present study, we report that muscles from patients chronicallytreated with statins at therapeutic doses present similar alterationsof mitochondrial respiration. Indeed, comparison of the maximalADP-stimulated respiration rates in the two statin-treated groupsand controls indicates that, like in the in vitro studies, statins targetspecifically complex I of the respiratory chain and, to a less extent,complex IV, as suggested by the inhibition with TMPD/ascorbate weobserved in statin-treated patients with muscle symptoms. Theseresults are in agreement with the suggestion that mitochondria isan early target of statins in vivo (Golomb and Evans, 2008). The effectof statins appears to be specifically targeted to the complex I of therespiratory chain rather than on an overall decrease in mitochondrialvolume. These results contrast with those from a randomizedcontrolled trial showing a reduced activity of complexes 2, 3 and 4of the respiratory chain due to a decrease in mitochondrial volumefollowing treatment with high doses of simvastatin (Paiva et al.,2005; Schick et al., 2007). In this study, the ratio between the activityof complexes 2, 3 and 4 and the activity of citrate synthase was notchanged. However, the activity of complex I of the respiratory chainwas not measured. Thus, a specific impairment of complex 1 of therespiratory chain, as we see in our study, cannot be excluded. Theimpairment of complex 1 of the respiratory chain induced by statinsseems to be an early event in the physio-pathological mechanismof myopathies, maybe caused by a direct effect of statins on thisenzymatic complex. Indeed, in vitro studies showed that, in healthyhuman muscle samples, the impairment of the mitochondrial functionfollowing acute application of statins is rapid (almost immediate)(Sirvent et al., 2005a, 2005b). Moreover, in mitochondria from bovineheart, the acute application of statins on immunocaptured complexesof the respiratory chain could induce inhibition, strongly supportinga direct effect of statins (Nadanaciva et al., 2007). Further studies areneeded to precisely define themechanism(s) conducting to respiratorychain inhibition by statins.

We cannot exclude that the mitochondrial damage reported hereis partly caused by decreased muscle ubiquinone. Nevertheless,in our previous in vitro study, the similar defect of mitochondrialfunction (i.e. decreased activity of complex I) was not preventedby mevalonate supplementation (Sirvent et al., 2005a, 2005b). In

addition, the association between the impairment of mitochondriaand decreased concentration of ubiquinone in muscle is not alwaysfound in patients treated with statins (Lamperti et al., 2005).

In vitro data from our precedent studies on the effects of acuteapplication of statin to healthy muscles (Sirvent et al., 2005a,2005b) also demonstrated that altered mitochondrial respiration isan early step of a complex patho-physiological mechanism leadingto muscle damage. Recent data on the effects of statins on primaryhuman skeletal myotubes (decreased maximal rate of mitochondrialrespiration using glutamate+malate and palmitoylcarnitine+malateas substrates, associated with reactive oxygen species over-productionand apoptotis) confirmed these results (Kwak et al., 2011). It has alsobeen reported that acute application of statins on healthy skeletal mus-cle samples induces spontaneous Ca2+ spark behavioral abnormalitiesthat might be a sign of impaired Ca2+ homeostasis. Alteration in theproperties of spontaneous Ca2+ sparks has also been linked withother muscular defects associated with Ca2+-signaling impairment,like diabetes (Yaras et al., 2005) or heart failure (Ward et al., 2003),showing that it is a good marker of Ca2+ homeostasis defects. Accord-ingly, we observed changes in the properties of spontaneous Ca2+

sparks (i.e., decreased frequency and increased amplitude) in patientstreated with statins. Therefore, our present data, combined with previ-ous in vitro results fromour laboratory (Sirvent et al., 2005a, 2005b) andby others (Kwak et al., 2011), strongly support a patho-physiologicalmechanism in which statin-induced alteration of mitochondrial respi-ration finally leads to deregulated Ca2+ homeostasis. This sequenceof events could easily explain all the muscle symptoms frequentlyobserved in patients treated with statins.

One objective of this study was to compare exercise tolerance,mitochondrial function and muscle calcium homeostasis in patientswith and without statin-induced muscle symptoms. The resultsshow little difference between the two groups of patients. However,the maximum power reached during the incremental exercise test,the maximum rate of mitochondrial respiration with octanoate orTMPD+ascorbate as substrates, and the frequency of calcium sparkswere altered significantly in symptomatic patients only. It wouldappear that cellular damages in asymptomatic patients are not signif-icant enough to trigger muscle pain (threshold concept). We canalso make the assumption of differences between the two groups ofpatients in term of pain tolerance, which remains a very subjectiveassessment. Finally, the differences between symptomatic andasymptomatic may also find their origin in different genetic suscepti-bility. Indeed, many studies have recently shown that polymorphismof certain genes (SLCO1B1, UGTs, ABCG2, ABCB1) may have a signifi-cant impact on the pharmacokinetics of statins and their potentialside effects (Maggo et al., 2011).

In conclusion, this study further supports in patients that statin-induced muscular toxicity is associated with early alteration of oneor more complexes of the mitochondrial respiratory chain thatwould lead to impaired mitochondrial function and deregulationof Ca2+ homeostasis. These phenomena could alter the expressionof Ca2+-activated proteins, such as calpains, thus favoring apopto-sis, protein degradation and muscular remodeling. These changescould explain why statin-treated patients develop exercise intoler-ance. Statin-induced muscle disorders are an important cause ofmorbidity, but the risk-benefit profile of these drugs must be putinto perspective. Indeed, statin myotoxicity could be prevented inmany cases by anticipating possible drug-drug interactions and bylimiting physical exercise, especially when high doses of statinsare prescribed and during the first year of treatment (when theabsolute risk of myopathy is greatest) for a safer and more effectivetherapy.

Statement of conflicts of interest

None.

268 P. Sirvent et al. / Toxicology and Applied Pharmacology 259 (2012) 263–268

Acknowledgments

Financial support: This workwas supported by the agence françaisede sécurité sanitaire des produits de santé (AFSSAPS) ; the institutnational de la santé et de la recherche médicale (INSERM) ; the asso-ciation française contre les myopathies (AFM) and the fondationpour la recherche médicale (FRM).

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