cyclosporin a treatment increases rat soleus muscle oxidative capacities

ABSTRACT: Previous studies suggested that administration of cyclosporinA (CsA), an immunosuppressive agent, contributes to the increased fatiga-bility of heart transplant recipients. The aim of this study was to investigatewhether CsA itself, without vehicle, affects the function of mitochondriamaintained in situ, in rats treated with CsA (25mg/kg/day) dissolved inethanol and olive oil. Treatment with CsA induced a 16% decrease in slowmyosin heavy chain (MHC) associated with a 225% increase in fast MHCIIa.The proportion of fibers expressing type IIa MHC increased as a result ofCsA treatment. Soleus from the CsA-treated animals showed an increase inboth basal (�85%) and maximal (�37%) mitochondrial respiration (P �0.001), consistent with a 24% increase in citrate synthase activity, whereasthe apparent Km for adenosine diphosphate was unchanged. By itself, CsAhas no deleterious effects on muscle oxidative capacity but induces alter-ations in energy metabolism in accordance with the increased proportion offast-twitch oxidative fibers.

Muscle Nerve 28: 324–329, 2003

CYCLOSPORIN A TREATMENT INCREASESRAT SOLEUS MUSCLE OXIDATIVE CAPACITIES

HERVE SANCHEZ, PhD,1 BENOIT N’GUESSAN, BS,2 FLORENCE RIBERA, PhD,2

RENEE VENTURA-CLAPIER, PhD,3 and XAVIER BIGARD, MD, PhD1

1 Departement des facteurs humains, Centre de Recherches du Service de Sante desArmees, BP 87, 38702 La Tronche, France2 Service de Physiologie Clinique et des Explorations Fonctionnelles, Hopitaux Universitaires,Strasbourg, France3 U-446 INSERM, Cardiologie Cellulaire et Moleculaire, Universite Paris-Sud,Chatenay-Malabry, France

Accepted 11 April 2003

Introduction of cyclosporin A (CsA) as an immuno-suppressive therapy was a decisive step in the devel-opment of organ transplantation. However, despitesome improvement, heart transplant recipients stillcomplain of abnormal fatigue and have a limitedexercise capacity.11 Factors other than the limitedincrease in denervated heart rate play a role in theexercise limitation of heart transplant patients.7 Im-paired oxygen utilization by muscle has been sug-gested in heart transplant patients by the high levelof blood lactate and leg fatigue that occur duringsubmaximal exercise.16,17 Moreover, the relationshipbetween the total mitochondrial volume density ofskeletal muscle and peak VO2 is lost for heart trans-

plant recipients, suggesting that immunosuppressivetherapy is at least partly responsible for persistentmuscle fatigability.8,20 Cyclosporin A treatment hasbeen reported to affect adversely several tissues, suchas skeletal muscle, and to induce mitochondrial dys-function.2 Indeed, treatment of rats with pharmaceu-tical preparation of CsA induces a decrease in thesubmaximal endurance exercise time, associatedwith a decrease in respiration rate of skeletal musclemitochondria in vitro, suggesting that this immuno-suppressive agent exerts a toxic effect on skeletalmitochondria.6,10

We recently showed that administration of apharmaceutical formulation of CsA in rats induced adecrease in oxidative capacity of skeletal muscles.14

However, this effect was related mainly to the vehicle(cremophor and ethanol) of CsA, which primarilyaffected complex I but also affected complex IV ofthe respiratory chain.15 Cyclosporin A directly inhib-its calcineurin, a serine threonine phosphatase withthe ability to dephosphorylate the transcription factorNFAT (nuclear factor of activated T cells), recentlyinvolved in the expression of slow muscle pheno-type.3,12 Comparison of rats treated with CsA or vehicle

Abbreviations: ACR, acceptor control ratio; ADP, adenosine diphosphate ;CS, citrate synthase; CsA, cyclosporin A; KmADP, apparent Km for ADP;MHC, myosin heavy chain; NFAT, nuclear factor of activated T cells; V0, basalmitochondrial respiration; VADP, ADP-stimulated mitochondrial respiration;Vmax, maximal respiration rate (VADP�V0)Key words: cyclosporin A; mitochondria; muscle fatigue; myosin heavychain; skeletal muscle; vehicleCorrespondence to: H. Sanchez; e-mail: [email protected]

© 2003 Wiley Periodicals, Inc.

324 Cyclosporin A and Skeletal Muscle MUSCLE & NERVE September 2003

showed a slow-to-fast shift of the myosin heavy chain(MHC) composition of the soleus muscle (a slow-twitch muscle), with a simultaneous increase in mark-ers of oxidative metabolism such as citrate synthase(CS) and the H isoform of lactate dehydrogenase.1

The aim of this study was to examine the effect ofCsA on mitochondrial respiration in situ, indepen-dently of the side effects of vehicle. Thus, we studiedthe effects of chronic treatment with the moleculeCsA on mitochondrial respiration parameters andthe regulation of oxidative phosphorylation by ADP,as well as on fiber-type composition in a slow-twitchmuscle of rats.

METHODS

Animals and Experimental Design. Young maleWistar rats (�210 g) were randomly assigned to oneof two experimental groups. For 3 weeks, animalsreceived by oral gavage either a daily dose of 25mg/kg CsA dissolved in ethanol (CsA group, n � 6)or an equivalent dose of ethanol (control group, n �8). Both preparations were diluted in 0.5 ml of oliveoil, and doses were adjusted according to weightgain. This investigation was carried out in accor-dance with the Helsinki Accords for Humane Treat-ment of Animals during Experimentation.

Tissue Processing. At the end of the 3-week exper-imental treatment, animals were anesthetized with so-dium pentobarbital (50 mg/100 g body weight) admin-istered intraperitoneally. Right soleus muscles werekept for mitochondrial experiments, whereas left so-leus muscles were weighed and immediately frozen forbiochemical determinations. Blood samples for CsAlevels were taken from the abdominal aorta of animals,and blood CsA concentrations were determined byusing a whole-blood fluorescence polymerization im-munoassay (Aventis Behring, Paris, France).

Functional Properties of Mitochondria. Respiratoryparameters of the total mitochondrial populationwere studied in situ in fresh saponin-skinned fibers(50 �g/ml saponin for 30 min) and determined witha Clark electrode (Hansatech Instruments, Norfolk,England) in an oxygraphic cell as previously de-scribed.13 Respiration rates were expressed as �molO2/min/g dry weight. Respiration solution con-tained (in mmol/L) EGTA-CaEGTA buffer 10 (freeCa2� concentration 100 nmol/L), MgCl2 1, taurine20, dithiothreitol 0.5, imidazole 20 (pH 7.1), ionicstrength 160 (potassium methane sulfonate), gluta-mate 5, malate 2, phosphate 3 and 2 mg/ml fattyacid–free bovine serum albumin. Basal oxygen con-sumption without ADP (V0) was recorded, and in-

creasing amounts of ADP were added until maximalrespiration was reached. After measurements, fiberswere carefully removed, dried, and weighed. TheADP-stimulated respiration (VADP) above basal oxy-gen consumption (V0) was plotted as a function of[ADP]. The apparent Km values for ADP and VADP

were calculated with a nonlinear fit of the Michaelis–Menten equation. The maximal respiration rate(Vmax) was (VADP�V0). The acceptor control ratio(ACR) was Vmax/V0. Three determinations weremade for each muscle sample.

Biochemical and Myosin Determinations. Frozen tis-sue samples were placed into an ice-cold homogeni-zation buffer (30 mg/ml) containing (in mmol/L)Hepes 5 (pH 8.7), EGTA 1, dithiothreitol 1, MgCl2 5,and 0.1% TritonX100 and incubated for 60 min at0°C to ensure complete enzyme extraction. Citratesynthase activity was determined according toSrere.19 The MHC content of soleus muscles wasdetermined as previously described.21

Histology and Immunocytochemistry. Serial trans-verse sections (10 �m thick) were cut from the mid-belly portion in a cryostat maintained at �20°C andstained with hematoxylin and eosin. Three mousemonoclonal antibodies directed against specificMHC isoforms were used in this study. Serial tissuecross-sections were incubated for 30 min at 37°C in ablocking solution consisting of normal horse serum.Sections were subsequently incubated for 1 h at 37°Cin a humid chamber in working solutions of mousemonoclonal antibodies which reacted either withslow type I (Novocastra, reference NCL-MHCS, New-castle-upon-Tyne, UK), or all adult fast and develop-mentally regulated epitopes but not with slow myo-sin (MY-32, Sigma Chemical, St. Louis, Missouri), orfast type IIa (SC-71). The avidin–biotin immunohis-tochemical procedure was used for the localizationof the antigen–antibody binding (Vector Laborato-ries, Burlingame, California). Negative control slideswith omission of the primary antibodies were ran-domly included in the immunostaining procedures.Fibers were classified according to their staining pro-file with the aid of a microscope linked to a comput-er-based image analysis system (Biocom Visiolab 200,Les Ulis, France).

Statistical Procedures. All data are presented asmean � SEM. One-way analysis of variance(ANOVA) was used to determine the global effect oftreatment. When appropriate, differences betweengroups were tested with a Newman–Keuls post hoctest. Statistical significance was accepted at P � 0.05.

Cyclosporin A and Skeletal Muscle MUSCLE & NERVE September 2003 325

RESULTS

Anatomical Data. Mean CsA level in blood was2712� 650 ng/ml and was concordant with ourprevious results.1 Although the body weight was sim-ilar at the beginning of the experiment, CsA animalshad a body weight 19% lower than control rats (P �0.001) at the end of the study, without alteration ofskeletal growth, as shown by similar values for tibialength (Table 1). Treatment with CsA induced a sig-nificant decrease in absolute and relative weight of thesoleus (respectively, 28% and 27%, P � 0.001).

Histological Aspects of Soleus Muscles. Hematoxy-lin and eosin staining showed that in soleus musclesof control or CsA-treated rats, the majority of fiberswere homogeneous in size, with a polygonal shape(Fig. 1). No ragged fibers were detected. The histo-logical aspect of muscles from CsA group was similar

to that of muscles from control animals. The appar-ently lower fiber cross-sectional area reported in so-leus from the CsA group, in comparison with controlrats, is consistent with the decrease in absolute andrelative weight of muscle in these animals (Table 1).

MHC Distribution and Immunohistochemical Analysis.

Administration of CsA affected the MHC isoformcontent of soleus muscle (Fig. 2; Table 1). The per-centage of MHCI was 16% lower in soleus of CsA-treated than in control animals (P � 0.001), and aconcomitant increase in the percentage of MHCIIawas observed (P � 0.001). Interestingly, the MHCIIbisoform was not detected in soleus of the CsA group,and the MHCIIx isoform represented only 0.3% ofthe total myosin content.

In comparison with soleus muscles from controlrats, CsA treatment resulted in a significant increasein the percentage of fibers expressing at least one of

Table 1. Morphological data and MHC distributionin soleus muscles.

Control group(n � 8)

CsA group(n � 6)

Final body weight (g) 325 � 6 264 � 8*Tibia length (cm) 3.89 � 0.02 3.86 � 0.03Absolute soleus weight (mg) 138 � 5 99 � 4*Relative soleus weight (mg/cm)† 35.4 � 1.0 25.7 � 1.1*MHC distribution in soleus (%)

MHCI 93.6 � 1.1 78.9 � 2.7*MHCIIa 6.4 � 1.1 20.8 � 2.9*MHCIIx ND 0.3 � 0.3

Values are mean � SEM.*Significant difference from control group, P � 0.001.Absolute soleus weight values normalized by the tibia length.ND, not detected.

FIGURE 1. Transverse sections of soleus muscles from control (A) or CsA-treated rats (B), stained with hematoxylin and eosin(calibration bar � 100 �m).

FIGURE 2. Electrophoretic separation of MHCs in soleus of CsAand control animals and in plantaris muscle of an animal withouttreatment. The four adult isoforms of MHC were expressed inplantaris muscle.


FIGURE 3. Immunohistochemical detection of MHCI (A and B), MHCIIa (C and D), and MHCfast total (E and F) within soleus musclesfrom rats treated with either ethanol (control group, A, C, E) or CsA (CsA group, B, D, F). Note the marked decrease in fibers expressingMHCI in soleus muscles of the CsA group, balanced by the increase in the percentage of fibers expressing MHCIIa. Arrowheads denotefibers coexpressing MHCI with MHCIIa. (Calibration bar in E �200 �m.) Asterisk denotes one fiber coexpressing MHCI and one of thefast MHC isoforms other than type IIa, likely the type IIx isoform.


the fast MHC isoforms (Fig. 3). Nearly all fibersreacting positively with the antibody against the fastMHC isoforms also reacted with the antibody againsttype IIa MHC. A marked increase in the proportionof fibers coexpressing slow and type IIa MHC iso-forms was observed as a result of CsA administration.Very few fibers stained positively with both the anti-body specific for slow and that specific for fast MHCisoforms and did not react with the antibody specificfor type IIa MHC (Fig. 3). These fibers coexpresstype I and one of the fast MHC isoforms other thantype IIa, likely the type IIx isoform. This finding isconsistent with the very small percentage of type IIxMHC in soleus muscles of CsA-treated rats (Table 1).

Mitochondrial Function. Because in permeabilizedfibers the whole mitochondrial population participatesin the measured oxygen consumption, when normal-ized to fiber dry weight, V0 corresponds to restingrespiration rate, which is related to noncoupled respi-ration, whereas Vmax with ADP represents the wholeoxidative capacity of the tissue. Oxygen consumptionof soleus from the CsA group was higher than in con-trol rats and was increased by 85% and 37% for V0 andVmax, respectively (Fig. 4; Table 2). The ACR values ofboth control and CsA groups were higher than 4,indicating that mitochondria were functionally intactin these preparations and not significantly affected bythe CsA treatment. The well-known low sensitivity ofrespiration for extramitochondrial ADP, as estimatedby the apparent Km for ADP in soleus, was unchangedby treatment with CsA (Table 2).

Mitochondrial content, estimated by CS activity,was significantly higher in the CsA group than in thecontrol group (24%, P � 0.05; Table 2).

DISCUSSION

The principal finding of the current study is thatchronic administration of CsA in rats leads to anincreased oxidative capacity of slow-twitch musclewithout changing the regulation of mitochondrialrespiration by external ADP. Two hypotheses wouldexplain the increase in both V0 and Vmax: eitherincreased respiratory chain elements in mitochon-dria or an increased amount of mitochondria inmuscle. The associated rise of CS activity, usuallytaken as an index of mitochondrial content, is con-sistent with the second hypothesis. Because type IIafibers are known to have higher oxidative potentialthan type I fibers in rats,18 the increase in both theoxidative potential and the content of the fast MH-CIIa isoform supports the contention that CsA ad-ministration induces a transition from a slow oxida-tive to fast oxidative fiber type in rat soleus muscle.1This is consistent with involvement of the cal-cineurin–NFAT pathway in the maintenance of theslow phenotype in adult muscle.1,3,12

Our results explain the apparent protective effectof CsA itself, previously observed when comparingmilder effects of its clinical formulation (Sandim-mun; Novartis, Basel, Switzerland) with the moremarked deleterious effects of its vehicle alone (eth-anol and cremophor) on mitochondrial respiration(Table 2). Because both CsA in ethanol (currentresults) and Sandimmun increased soleus CS activityabove their respective control values,1 this findingadds support to our earlier conclusion that the toxiceffect of immunosuppressive treatment on mito-chondrial respiration mainly arises from vehicle.14,15

Previous studies showed on isolated mitochondriathat clinical formulation of CsA alters mitochondrialrespiration in different tissues, such as kidney,2,9 liv-er,2,5 and skeletal muscles.2,6,10 These studies investi-gated mitochondrial function in vitro and could notexamine the total mitochondrial population presentwithin fibers or the regulation of mitochondrial res-

FIGURE 4. Regulation of mitochondrial respiration by ADP ofrepresentative fibers from soleus muscle of one control (�) andone CsA (Œ) rat. Oxygen consumption rates (VO2) were plottedas a function of ADP concentration. Data were fitted using aMichaelis–Menten equation to obtain Km and Vmax values (dot-ted and solid lines, respectively, for control and CsA rats).

Table 2. Functional properties of mitochondria and CS activity.

Control group(n � 8)

CsA group(n � 6)

RespirationV0, �mol O2/min/g dry wt 1.7 � 0.1 3.2 � 0.2*Vmax, �mol O2/min/g dry wt 9.9 � 0.4 13.6 � 0.7†

Acceptor control ratio 6.0 � 0.5 4.6 � 0.5Km for ADP, �mol/L 336 � 27 361 � 28

CS activityIU/g wet wt 38.1 � 1.6 47.2 � 2.3†

Values are mean � SEM.*P � 0.001, significantly different from control group.†P � 0.01, significantly different from control group.


piration by ADP. More importantly, the respectiveeffects of cyclosporin, the immunosuppressive mol-ecule, and its vehicle were not clearly and specificallyexamined in these experiments.

Our experiment showed that in slow oxidativemuscle of rats, CsA itself, by inhibiting the cal-cineurin–NFAT pathway, likely increased theamount of mitochondria and therefore augmentedoxidative capacity. This is in accordance with thehigher expression of the MHCIIa isoform, which isassociated in rats with an elevated oxidative poten-tial. These results suggest that CsA itself has nodeleterious effect on muscle oxidative capacity butinduces a coordinated slow-to-fast oxidative transi-tion of both contractile and metabolic muscle phe-notype. In transplant recipients, immunosuppressivetherapy is complex, including cyclosporin A but alsocorticosteroids. The specific effects of corticoste-roids on muscle mass and energy metabolism needto be taken into account when explaining the musclelimitations in transplant patients. In humans, be-cause type IIa fibers have a lower oxidative potentialthan do type I fibers, the CsA-induced transitionfrom slow-twitch to fast-twitch oxidative fibers wouldlead to a slight decrease in oxidative capacity. Otherside effects of CsA on the microvasculature, andhepatic and renal functions, may be also involved inexercise limitation.4 Furthermore, the side effects ofvehicle present in pharmaceutical forms of CsA onmitochondrial respiration14 could also partly explainthe impaired skeletal muscle energy metabolism re-ported in transplant recipients.8,20

Taken together, the current results show thatCsA itself has no direct deleterious effect on themuscle mitochondrial content but induces a slightand coordinated slow-twitch to fast-twitch conver-sion. In humans, this phenotypic transition, associ-ated with the specific effects of vehicle on mitochon-drial function, together with the effects of otherimmunosuppressive drugs, helps to explain the limitedexercise capacity observed in transplant recipients.

The authors thank Lahoucine Bahi and Bernard Serrurier for theirhelpful contribution to this work and Drs. Vladimir Veksler andBertrand Mettauer for their insightful comments. This work wassupported by a PROGRES grant from INSERM and a grant from theAssociation Francaise contre les Myopathies. Dr. Ventura-Clapier issupported by Centre National de la Recherche Scientifique.

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cyclosporin a treatment increases rat soleus muscle oxidative capacities

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