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From the Department of Pharmacobiology,* Unit of Pharmacology, and the Departments of Human Anatomy and Histology and General and Environmental Physiology,¶ University of Bari, Bari, Italy; the Muscular Pathology Unit, National Neurological Institute Carlo Besta, Milan, Italy; and MyoContract Limited, Liestal, Switzerland
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B, and to apoptotic pathways, via activation of the proapoptotic Bcl-2 member BAD.16,17
Furthermore, CsA can exert calcineurin-independent effects on mitochondrial permeability pore, a mechanism effective in contrasting apoptosis in collagen VI-deficient muscular dystrophy and potentially important in view of the metabolic sufferance of DMD muscles.5,18
However, the pharmacological inhibition of calcineurin may have controversial outcome because of the physiological roles of the enzyme in skeletal muscle. Calcineurin controls calcium- and activity-dependent differentiation of the slow-twitch phenotype and plays a role in muscle growth, hypertrophy, and regeneration.19-21
Transgenic mdx mice overexpressing activated calcineurin show a better progression of the pathology, through a twofold increase in expression of the dystrophin-replacing protein utrophin.22
However, two studies dealing with the effects of CsA treatment in mdx mice led to controversial results. In both cases, 2-week-old mdx mice were used to match the first round of spontaneous degeneration; however, whereas the administration of CsA at 2 mg/kg/day for 30 days was without significant effect, a worsening of the pathology was reported by Stupka and colleagues24
on the use of much higher dose (30 mg/kg, i.p.) for 2 weeks.23,24
In this latter study, the dose used was in the range for total inhibition of calcineurin activity in skeletal muscle,21,24
a situation of particular hazard for still developing muscles. Because of the narrow therapeutic index of this drug and the recognized role of its target for proper muscle development, in the present study we evaluated the effects of therapeutic doses of CsA on dystrophic mdx mice after the first month of postnatal life. The oral administration route was chosen being less invasive in long-term use. Possible variability in oral absorption, and thus the maintenance of therapeutic dose, was taken into account by using a dose of 10 mg/kg/day, well within the effective dose range (3 to 15 mg/kg).15,18,19
We also considered the available pharmacokinetic data that reported a longer clearance time for CsA in rodents versus humans and a three-compartment kinetic model, for the extensive tissue distribution, leading to longer elimination times during chronic therapies.25-27
The CsA treatment has been performed on 4- to 5-week-old mdx mice for 4 to 8 weeks throughout a period of exercise on treadmill, a protocol that worsens the murine pathology by contrasting the spontaneous regeneration and allows to monitor the effectiveness of drug therapies both in vivo and ex vivo.9,28,29
In vivo we evaluated the ability of CsA to prevent the exercise-induced forelimb muscle strength loss. Creatine kinase level, histological analysis, and markers of fibrosis as well as the level of utrophin expression were determined as indexes of the ability of CsA treatment to contrast muscle damage. To gain insight in the mechanism of drug action, we recorded electrophysiologically the state of cellular parameters linked to the disease, such as the impairment of chloride channel function occurring spontaneously in diaphragm (DIA) muscle or induced by exercise in the hindlimb extensor digitorum longus (EDL) muscle.9
In addition calcium homeostasis was monitored functionally by electrophysiological recordings of the voltage threshold for mechanical activation and by FURA-2 imaging. The data provided evidence that CsA treatment contrasts, in a calcium-independent manner, some of the functional and morphological disease-related alterations, suggesting that the CsA targets contribute to the pathology progression in muscular dystrophy. |
Materials and Methods |
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In Vivo Experiments
Animal Groups and Drug Treatment
Twenty-two mdx and ten wild-type (WT) (C57/BL10) male mice 4 to 5 weeks old (IFFA Credo, France, and Jackson Laboratories, Bar Harbor, ME) were used. Initially, all of the animals were weighed and forelimb force was measured by means of a grip strength meter (see below). Each group was then subdivided into two further groups: exercised and sedentary. The group of exercised mdx mice was further subdivided into seven untreated and eight CsA-treated mice. The CsA treatment (10 mg/kg orally) started a few days before the beginning of the exercise protocol, and continued until the day of sacrifice. CsA was given with drug-enriched chow (0.01% in weight). The daily amount of food eaten by each animal ranged between 2 to 4 g. Thus the drug-enriched chow was given daily (for 6 days a week) up to reach the daily amount for the dose of 10 mg/kg. Then, the mice received the standard drug-free chow to avoid CsA overdose. Both standard and drug-enriched chow made under request was purchased from Eberle Nafag AG (Gossau, Switzerland). One of the CsA-treated mice died few days after the beginning of the treatment. This event also occurred for one of the mdx mice belonging to the sedentary group and was therefore considered unrelated to the treatment and rather because of the degenerative phase of the disease at this age. During the CsA treatment, the mdx mice did not show any abnormal behavior or difference in macroscopic vital functions versus untreated groups. The six mdx mice, taken as sedentary, were left free to move in the cage, without additional exercise. The sedentary mdx mice as well as the exercised WT animals were monitored for in vivo and in vitro studies at the same time points of exercised counterparts, according to the experimental need.
Exercise Protocol and in Vivo Studies
The WT and mdx (both untreated and treated) mice belonging to the exercised groups underwent 30 minutes of running on an horizontal treadmill (Columbus Instruments, USA) at 12 meters/minute, twice a week, for 4 to 8 weeks.9,28 The training protocol started at the mouse age of 4 to 5 weeks. Approximately half of the mdx mice showed an avoidance behavior with respect to exercise, with a higher tendency to fatigue and had to be gently stimulated, or should be left resting, to complete the 30 minutes of running session. This behavior, never observed in WT animals, was not modified by either exercise or drug treatment. Every week all of the exercised mice were monitored for body weight and compared with related sedentary counterparts. The force of exercised mice (both controls and mdx) was evaluated before each training section by means of a grip strength meter (Columbus Instruments, USA). For this measurement the mice were allowed to grasp a triangular ring connected to a force transducer and then gently pulled away until the grip was broken. The transducer saved the force value at this point, which was a measure of the maximal resistance the animal can use with its forelimbs. Five measurements were taken from each animal within 2 minutes and the maximum values were used for statistical analysis.9 The sedentary mice, both WT and mdx, were monitored for muscle strength every week. At the end of the fourth week of exercise the electrophysiological in vitro experiments were started. The animals continued to be exercised until the day of sacrifice.
In Vitro Studies
Muscle Preparations
The ex vivo experiments were performed on different muscles collected from mice belonging to the various groups starting after the fourth week of either exercise or exercise plus treatment. The sedentary mice were used at corresponding time points. Thus the age of the animals at the time of experiment was 8 to 12 weeks. The animals were anesthetized with 1.2 g/kg urethane. Extensor digitorum longus (EDL) of one hindlimb was removed and rapidly placed in the recording chamber for the electrophysiological recordings. The contralateral EDL muscle was either placed in normal physiological solution for surgical dissection to be used for fura-2 calcium imaging, or rapidly frozen in isopentane cooled with liquid nitrogen for immunohistochemical determination of fiber phenotyping. Gastrocnemius (GC) muscles were removed to be used for histological and/or biochemical experiments. To these aims the GC muscle of one hindlimb was dissected by surrounding tissue and fixed in 4% paraformaldehyde in phosphate-buffered saline or in a modified acetate-free Bouin fluid for 4 hours and then was routinely processed and paraffin wax-embedded. The contralateral GC muscle was washed in phosphate-buffered saline (PBS) and rapidly frozen in liquid nitrogen-cooled isopentane and stored at –80°C until use. Diaphragms (DIA) were rapidly removed, placed in normal physiological solution, and rapidly cleaned from connective tissue. The right-half side was used for the electrophysiological recordings, whereas the remaining half was rinsed in PBS, dried, and rapidly frozen in liquid N2. Samples were stored at –80°C until use for determination of utrophin content.
Morphometrical Analysis
Six-µm transversally cut sections of GC muscles were stained by hematoxylin and eosin (H&E), toluidine blue, and Azan-Mallory techniques, which allow to distinguish between healthy myofibers, showing peripheral nuclei (peripherally nucleated fibers), regenerating/regenerated myofibers, showing central nuclei (centrally nucleated fibers), and degenerating fibers, often present in areas where also small regenerating fibers and cell infiltrates were visible. Morphometrical analysis was performed on 10 cross-sections from each experimental group by using Image Analysis software (Olympus Italia, Rozzano, Italy). The following parameters were evaluated: 1) area and perimeter of peripherally and centrally nucleated fibers, 2) percentage of peripherally and centrally nucleated fibers referred to the total number, 3) percentage of total nonmuscle area (fibrotic or adipose tissue), and 4) percentage of degeneration tissue area (necrotic fibers and small regenerating/mononuclear inflammatory cells infiltrate).
Evaluation of Markers of Fibrosis
Area of Connective Tissue:
The extent of connective tissue was measured on photographs of H&E-stained sections of GC muscles at x20 magnification using the NIH Image software. At least three fields from each animal were analyzed, the mean area was calculated for each group, and extracellular matrix was evaluated as percentage.
Immunohistochemical Evaluation of Collagen Types:
First of all 5-µm-thick consecutive transverse GC muscle sections, were fixed in ice-cold acetone, incubated with a peroxidase block solution (DAKO, Glostrup, Denmark) for 5 minutes and then treated with a protein block solution (DAKO) for 10 minutes to reduce nonspecific bindings. All incubation steps were performed in humid chambers. For the evaluation of type I and type III collagens, sections were incubated with two commercial polyclonal antibodies (Cedarlane and Abcam, respectively). The sections were incubated for 90 minutes, then rinsed in PBS. Samples were developed using secondary horseradish peroxidase-conjugated antibodies (60 minutes, DAKO), rinsed in PBS and successively developed with diaminobenzidine (DAKO) and counterstained with Mayer hematoxylin for 1 minute.
Determination of Transforming Growth Factor (TGF)-ß1 Level and Transcript:
Total TGF-ß1 was determined by an enzyme-linked immunosorbent assay (ELISA) (R&D Systems).30
For determination of TGF-ß1 transcript, total RNA was extracted from 10 to 20 mg of muscle biopsy using RNAzol B reagent (Ambion Inc., Austin, TX). One µg of total RNA was reverse-transcribed according to the manufacturer’s instructions and the cDNA stored at –20°C pending polymerase chain reaction (PCR) amplification. Transcripts of TGF-ß1 were assayed by the following semiquantitative method: a constant amount of cDNA (corresponding to 100 ng of total RNA) was amplified in a PCR reaction containing 1x PCR buffer (Finnzymes), 0.1 mmol/L of each dNTP (PE Applied Biosystems), 1 U of DynaZyme DNA polymerase (Finnzymes), and 1 µmol/L each of the two specific primers. Primer sequences were the following: TGF-ß1: forward 5'-AGTGTGGAGCAACATGTGGA-3', reverse 5'-GTGAGCGCTGAACGAAAG-3' (amplified fragment length, 181 bp). Amplification was performed with 35 cycles of 94°C for 30 seconds, 55°C for 30 seconds, and 72°C for 30 seconds. The PCR product was separated by agarose gel electrophoresis and the fluorescence of the product quantitated using the Kodak Digital Science software. The expression of TGF-ß1 was normalized to that of ß-actin. The results were expressed as ratios of band intensities and grouped according to the different animal subtypes.
Immunohistochemical Determination of Fiber Phenotype
Five-µm cryostat sections of EDL muscles, mounted on silaned microscope slides, were kept in PBS for 10 minutes and preincubated with 0.1% gelatin in PBS for 15 minutes as blocking step. Then, sections were incubated with myosin skeletal slow (type I) or fast (both type IIa and IIb) mouse monoclonal antibodies (Sigma, St. Louis, MO) for 1 hour at room temperature. After being washed with PBS-gelatin, sections were incubated for 1 hour with fluorescein isothiocyanate-coupled goat anti-mouse antibody (diluted 1:1000, Molecular Probes) and then washed again. Sections were finally mounted in PBS/glycerol (1:1) containing 1% n-propylgallate, pH 8, and examined with a Leica DMRXA photomicroscope equipped for epifluorescence. Digital images were obtained with a Nikon DMX 1200 camera. For fiber quantification, 10 photographs of each section, taken at x16 magnification were randomly chosen for control and mdx EDL muscles. The number of slow and fast positive fibers was counted, and the results were expressed as a percentage of the stained cells on total cells on the photograph.
Evaluation of Utrophin Content
Muscle tissue was homogenized and proteins extracted as follows: frozen tissue specimens were disrupted in ice-cold extraction buffer (1% digitonin, 5 mmol/L ethylenediaminetetraacetic acid, 50 mmol/L Tris-HCl, pH 6.8, 500 mmol/L NaCl, supplemented with the protease inhibitors phenylmethyl sulfonyl fluoride, aprotinin, and leupeptin) using a cell disrupter device (2 pulses for 30 seconds at maximum speed; BIO 101, Savant). After disruption, debris were eliminated by centrifugation for 10 minutes at 14,000 rpm in a tabletop centrifuge at 4°C, and the supernatant was stored at –20°C. Protein concentration was determined by the BCA protein assay according to the manufacturer’s recommendations (Pierce).
The utrophin protein content was measured by a two-site ELISA. For this, Costar EIA/RIA plates were coated overnight at 4°C with the rabbit anti-utrophin antibody anti-UT-31, directed against the mid-rod domain of utrophin (epitope comprising the amino acids 1754 to 2091, provided by Professor Schaub, University of Zurich, Zurich, Switzerland) in carbonate buffer (50 mmol/L, pH 9.6) and blocked with 2% fetal calf serum, 1% bovine serum albumin in PBS containing 0.1% Triton X-100). After washing, wells were incubated with the equivalent of 100 µg of total protein extract for 6 hours at 4°C followed by repeated washing. Detection of antibody-bound utrophin protein was performed by reacting with a monoclonal anti-utrophin antibody (NCL-DRP2, diluted 1:1000 in blocking solution, overnight incubation at 4°C; NovoCastra) recognizing an epitope associated with amino acids 1 to 261 of utrophin. After additional washings, the wells were incubated with a peroxidase-conjugated AffiniPure donkey anti-mouse antibody (diluted 1:5000 in blocking solution; Jackson ImmunoResearch, West Grove, PA). On washing the amount of utrophin protein in muscle extracts was determined by fluorometric detection using the QuantaBlue kit (Pierce) according to the manufacturer’s recommendations. As positive control and to determine the dynamic range of the assay and for calibration an extract of HEK cells expressing full-length mouse utrophin [amino acids 1 to 3429; NM_011682 (gi: 46575907)] was used in parallel.
Creatine Kinase Determination
Blood was collected from ventricular camera soon after animal death in ethylenediaminetetraacetic acid-rinsed centrifuge tubes. The blood was centrifuged at 3000 x g for 10 minutes and plasma was separated and stored at –20°C. Creatine kinase determination was performed by standard spectrophotometric analysis by using diagnostic kit (Sigma-Aldrich, Milan, Italy) within 7 days from plasma preparation.
Electrophysiological Recordings by Intracellular Microelectrodes
EDL muscle and diaphragm (DIA) were placed in the recording chamber at 30 ± 1°C and superfused with normal and chloride-free physiological solutions. The normal physiological solution had the following composition (in mmol/L): NaCl, 148; KCl, 4.5; CaCl2, 2.0; MgCl2, 1.0; NaHCO3, 12.0; NaH2PO4, 0.44; and glucose, 5.55. The chloride-free solution was made by equimolar substitution of methyl sulfate salts for NaCl and KCl and nitrate salts for CaCl2 and MgCl2. The solutions were continuously gassed with 95% O2 and 5% CO2 (pH = 7.2 to 7.4).31
The two intracellular microelectrode current clamp method was used to measure the membrane electrical properties of muscle fibers by evaluating the attenuation of the electrotonic potential in response to standard hyperpolarizing current pulse at two distances between the recording and the current-injecting electrode, according to the cable equation.32
Assuming constant value of fiber input resistance (Rin) of 140 and 200 
* cm for EDL and DIA muscle fibers,31
respectively, it was possible to calculate membrane cable parameters, among which was membrane resistance (Rm). The total membrane conductance (gm) was calculated as 1/Rm in normal physiological solution, whereas 1/Rm calculated in a chloride-free solution was the potassium conductance gK. Chloride conductance (gCl) was calculated as the mean gm minus the mean gK.31,32
The mechanical threshold (MT) of the fibers was determined in EDL muscle fibers in normal physiological solution using a two microelectrode point voltage clamp method as previously described.9,33
In brief, the two microelectrodes were inserted within 5 µm of each other into the central region of a randomly selected superficial fiber that was continuously viewed using a stereomicroscope (x100 magnification). The holding potential was set at –90 mV and depolarizing command pulses of variable duration were given at a rate of 
0.3 Hz. Tetrodotoxin (3 µmol/L) was continuously present during recordings to prevent action potential generation.9,33
As a standard protocol the command-pulse duration was usually set sequentially to each of the following values: 500, 50, 5, 200, 20, 100, and 10 ms. At each duration, the command voltage was increased using an analogue control until contraction was just visible, and then backed down until the contraction just disappeared. A digital sample-and-hold millivoltmeter stored the value of the threshold membrane potential at this point. We estimated the uncertainty of any single measurement for a given fiber to be 1 to 2 mV.9,33
The threshold membrane potential V (mV) for each fiber was averaged at each pulse duration t (ms) and then mean values plotted against duration giving us a strength-duration relationship. A fit estimate of the rheobase voltage (R) and of the rate constant (1/
) to reach the rheobase was obtained by nonlinear least square algorithm using the following equation: V = [H – R exp (t/) ]/[1 – exp (t/) ] where H is the holding potential (mV), R, is the rheobase (mV), and is the time constant. In the fitting algorithm, each point was weighed by the reciprocal of the variance of that mean V and the best fit estimates of the parameters R and 1/ were made.9,33
We used this procedure to be able to incorporate all of our determination points and their associated errors into our estimate of R under each condition.
Fura-2 Microfluorescence Analyses
Small bundles of 10 to15 EDL muscle fibers arranged in a single layer were dissected length-wise, tendon to tendon, with the use of microscissors, as described elsewhere.29,34 Fluorescent measurements were made using a QuantiCell 900 integrated imaging system (VisiTech International Ltd., Sunderland, UK). Calcium measurements were performed using the membrane-permeant Ca2+ indicator fura 2-acetoxymethyl ester (fura2-AM, Molecular Probes). The loading of muscle fibers was performed for 1 hour at room temperature (23°C), in normal physiological solution containing 2.5 µmol/L fura2-AM and 0.02% Pluronic-F127 (Molecular Probes).29,34 Fura-2-loaded muscle fibers were mounted in a glass-bottomed RC-27NE experimental chamber (Warner Instrument Corp., Hamden, USA), modified by the authors. Tendons of the muscle preparations were attached by the means of hair loops, one extremity was clamped to a fixed tube and the other to a mobile one.29,34 The experimental chamber was placed on the stage of an inverted Eclipse TE300 microscope with a x40 Plan-Fluor objective (Nikon, Japan).
Before fluorescence measurements and to check electrical integrity, preparations were stimulated by electric pulses (2-ms duration, 0.33 Hz) of increasing intensity, until visible contraction, with a pair of platinum-wire electrodes, one in the medium surrounding the fibers and the other close to muscle fibers (100 to 200 µm). Fibers that did not contract were not used for analysis. Then, the sarcomere length was set to 2.4 to 2.5 µm as previously described.29
Pairs of background subtracted images of the fura-2 fluorescence (510 nm) excited at 340 nm and 380 nm were acquired at rest and pixel-to-pixel radiometric images were calculated for each muscle fiber. Autofluorescence, determined in unloaded muscle fibers, was <2% of total signal and was not subtracted. Resting ratio values were used to calculate the resting cytosolic calcium using the equation: [Ca2+]i = (R– Rmin)/(Rmax – R) x KD x ß where R is the ratio of fluorescence excited at 340 nm to that excited at 380 nm; KD = 145 nmol/L; ß, Rmin, and Rmax are constants according to Grynkiewicz and colleagues35 and were determined experimentally in situ as previously described.29,35
The manganese quench technique was used to estimate the sarcolemmal permeability to divalent cations.36
Muscle preparations were first perfused for 2 minutes with normal physiological solution containing 0.5 mmol/L Mn2+ as a surrogate of CaCl2 (quenching solution). During the whole quenching protocol the fluorescence of fura-2 excited at 360 nm was acquired at 1 Hz. The quench rates were estimated using linear regression analysis of fluorescence signal and expressed as the decline per minute of the initial fluorescence intensity.
Statistics
All data are expressed as mean ± SEM or ±SD. The SE estimate for gCl were obtained as previously described.9,31
Statistical analysis for direct comparison between two groups of data means was performed by unpaired Student’s t-test, whereas multiple statistical comparison between groups was performed by one-way analysis of variance, with Bonferroni’s t-test posthoc correction for allowing a better evaluation of intra- and intergroup variability and avoiding false positives. The MT values are expressed as both mean ± SEM for the absolute values of voltage threshold at each pulse duration. Fitted rheobase (R) and rate constant (1/) parameters ± SE were determined from the variance-covariance matrix in the nonlinear least square fitting algorithm.9,33
For these fitted parameters, the statistical significance between groups was estimated by using the above tests using these standard errors and a number of degrees of freedom equal to the total number of threshold values determining the curves minus the number of means minus two for the free parameters.9,33
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Results |
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At the beginning of the training protocol the body weight of 4- to 5-week-old mdx mice either belonging to the untreated or to the CsA-treated group did not show significant differences with respect to that of age-matched WT animals (Table 1)
. Four weeks of exercise did not modify the body weight gain of mdx mice versus sedentary counterparts. However in both mdx groups the body weight gain was significantly larger than that observed in WT animals. After 4 weeks of CsA treatment the body weight of mdx mice was lower than that observed in exercised untreated counterparts, being rather similar, as well as the 4-week gain, to that of WT animals. This allowed us to exclude the occurrence of a general detrimental effect of CsA reported by others.24
As expected, no significant differences were observed in forelimb strength of the mice belonging to all groups at the beginning of the experimental section (time 0); however, in line to previous results9
after 4 weeks of exercise, the mdx mice appeared to be significantly weaker than the sedentary counterparts (Figure 1)
. On the absolute values of mouse strength, the treatment with CsA seems to be rather ineffective. To minimize the influence of the different body weight gain observed between groups, for each mouse we normalized the forelimb strength to body weight at the beginning (time 0) and at the end of 4 weeks of exercise (time 4) and we considered the difference as normalized strength increment (Figure 1)
. By this approach it was evident that the 4 weeks of exercise protocol produced a significant drop in strength in mdx mice and that the CsA treatment significantly counteracted the deleterious effect of exercise on mouse force. In fact the normalized strength increment fully overlapped that observed in sedentary mdx and WT mice (Figure 1)
. No significant effect of the treatment was observed on the weight of organs known to be targeted by CsA, ie, liver, kidney, spleen, collected when the animals were sacrificed for ex vivo experiments (Table 1)
. Similarly no difference was observed in the weight of the EDL muscle used for electrophysiological recordings between the various groups of mice (Table 1)
.
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In line with previous observations,9
although the exercise protocol was without damaging effect in WT muscles (Table 2
and Figure 2, A and D
), the GC muscles of exercised mdx mice showed structural alterations that were similar, although quantitatively more serious, compared to sedentary mdx mice. In fact, extensive areas of degeneration, characterized by the presence of necrotic fibers and by their replacement with nonmuscle tissues, likely adipose and fibrotic ones, were observed in exercised mdx muscles (Table 2
and Figure 2; C, E, and G
). Moreover, small centronucleated fibers, isolated or in clusters often nearby necrotic fibers, were present. These areas were also characterized by the presence of several infiltrates, resembling mononuclear inflammatory cells described in dystrophic muscle7
(Figure 2, C and E)
. The treatment of the mdx-exercised mice with CsA, induced a significant reduction of degenerating areas and a reduced percentage of the nonmuscle tissue replacing muscle fibers compared with untreated ones (Figure 2, D and H)
, indicating a greater preservation of viable contractile material. Moreover, the muscle tissue composed by both peripherally and centronucleated myofibers, showed a more regular shape and a more closely arrangement (Figure 2D)
.
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and confirmed the histological observations. In fact, after CsA treatment, in the muscles of exercised mdx mice the number of peripherally nucleated cells as well as the area and perimeter of both normal and centronucleated fibers were significantly increased. Also the percentage of the area occupied by nonmuscle and degenerating tissue was reduced, as compared to the untreated mdx-exercised mice, thus indicating a recruitment of the contractile mass.
The histological evidence of the ability of CsA treatment to reduce the exercise-induced muscle damage in mdx animals has been confirmed by the values of plasma creatine kinase (CK), a well known diagnostic marker for muscle injury (Figure 3)
. As expected, the mdx mice were characterized by high levels of CK and this increase was particularly evident in the exercised group. The increase observed in exercised mdx mice was clearly attributable to the effect of contractile stress on the dystrophic state, because the same protocol of exercise was without effect on plasma CK level of WT mice (Figure 3)
. Interestingly, the plasma levels of exercised mdx mice treated for 4 to 8 weeks with CsA was significantly lower with respect to that of the untreated group and even slightly lower with respect to that of sedentary counterparts.
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CsA, as well as other immunosuppressants, has been reported to exert a profibrotic action in many tissues,30,37,38
a mechanism that may oppose to the amelioration of the histological picture. The connective tissue present in the mdx GC muscles belonging to the various groups was determined in H&E sections. As shown in Table 3
, the area occupied by connective tissue was higher in GC muscle of exercised (11.5% with respect to the area examined) versus sedentary (8.5%) mdx mice. The CsA treatment significantly reduced the area of connective tissue to 5.9% of the standard area analyzed (Table 3)
. The staining for collagen type I was localized in the extracellular matrix between the muscle fibers and no significant difference was observed in the collagen distributions as a result of the drug treatment (data not shown).
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. Although the high intergroup variability did not allow reaching the statistical significance between the groups, the data clearly support the ability of the CsA treatment to decrease rather than increase the progression of fibrotic processes.
Recently, it has been proposed that calcineurin activity, important for maintaining the slow fiber phenotype, may exert a beneficial effect on dystrophic progression in mdx muscle by increasing the expression of utrophin.22,39
Thus it was important to establish the impact of calcineurin inhibition by the CsA treatment on both the level of utrophin and the fiber phenotype. The utrophin levels, detected by ELISA test in DIA of each experimental group are shown in Figure 4A
. As can be seen comparable levels of the protein were detected in all groups. As far as the mdx genotype is concerned, a slight but significant increase in utrophin content was found in both sedentary and exercised DIA muscles versus WT groups. This is in line with earlier observations40,41
and may indicate a compensatory mechanism activated in surviving muscle fibers to withstand both pathology and exercise protocol. Interestingly, the CsA treatment did not increase nor decrease the utrophin content in a significant manner with respect to the mdx groups.
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. The CsA treatment further reduced the fiber population co-expressing the slow MHC (Figure 4B)
. This data, in line with the physiological role of calcineurin in skeletal muscle, is an important evidence that some of the observed effects in treated animals were because of a muscle-based action of CsA. Effect of CsA on Functional Cellular Indexes of Dystrophic Degeneration in Exercised Mdx Mice: Component Ionic Conductances and Calcium Homeostasis
In agreement with previous observations9,31
the main electrophysiological index of degenerative events occurring in dystrophic muscle either spontaneously occurring or triggered by exercise was the decrease of macroscopic chloride conductance gCl, sustained by both activity and expression of muscle ClC-1 chloride channels (Figure 5A)
. The CsA treatment was able to significantly restore gCl of DIA muscle fibers to a value close to that of normal controls. As can be seen in Figure 5A
, the value of gCl after CsA treatment was significantly higher with respect to that of untreated exercised mice (P = 0.0031 by Bonferroni’s t-test) and almost overlapped that of WT DIA muscle fibers. Similarly, the CsA treatment fully counteracted the exercise-induced decrease in gCl occurring in mdx EDL muscle fibers, maintaining gCl to a value not significantly different with respect to that of WT animals. Actually the value of gCl in CsA-treated EDL muscle fibers was higher than that of WT, approaching the gCl value typical of regenerating sedentary mdx EDL muscle fibers (data not shown).31
In exercised mdx muscle fibers, and in particular in DIA, the mean value of potassium conductance (gK) was significantly higher with respect to the value of WT, an alteration not so evident in previously used exercised mdx strains.9
We did not characterize the channel underlying this increase, but we found that the in vitro application of 50 µmol/L glybenclamide, a specific blocker of ATP-sensitive potassium channels, reduced gK of exercised mdx EDL muscle fibers from 459 ± 40 µS/cm2 (n fibers = 14) to 371 ± 31 µS/cm2 (n = 15). A similar 20% decrease was also observed in DIA muscle fibers. Thus, an exercise-induced metabolic sufferance might in part account for the observed increase in gK. No effect of CsA treatment was observed on the increased gK of exercised mdx muscle fibers. This suggests that the alterations of gK and gCl in dystrophic fibers occurs through different pathways differently sensitive to CsA actions.
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. In parallel, a higher level of cytosolic calcium was found by fura-2 calcium imaging (Figure 5C)
. The treatment with CsA did not produce any significant amelioration of the calcium homeostasis. In fact the strength-duration curve was only slightly shifted toward more positive potential with respect to that of untreated exercised EDL muscle fibers (Figure 5B)
. The rheobase voltage was –71.3 ± 0.99 mV, a value very close and not significantly different to that of untreated mdx mice (–72.5 ± 0.8 mV). Thus, the rheobase values of both mdx groups were significantly different with respect to WT value (P < 0.001 by Student’s t-test). The rate constants (1/) were 0.12 ± 0.013 seconds–1 and 0.13 ± 0.01 seconds–1 in CsA-treated and untreated exercised EDL muscle, respectively, both values accounting for a longer time constant to reach the rheobase with respect to WT muscle fibers (1/ = 0.15 ± 0.007 seconds–1, P < 0.05 versus CsA treated mdx mice). Accordingly, the fura-2 microspectrofluorimetric analysis showed a slight but not significantly lower value of resting calcium in CsA-treated versus untreated EDL muscle fibers (Figure 5C)
. In parallel, no significant difference was observed in the sarcolemmal permeability to calcium, estimated by the Mn2+ quench rate, between untreated and CsA-treated fibers (data not shown). |
Discussion |
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CsA Exerts a Protective Effect against Exercise-Induced Muscle Damage
The ability of CsA to decrease muscle damage was evidenced by both the histological analysis and by the reduction of plasma creatine kinase. In agreement with data available in the literature, the high number of fully regenerated and regenerating centronucleated fibers in mdx muscle and its further increase with exercise, is an index of ongoing degeneration-regeneration cycles.7,22 Furthermore, this is accompanied in exercised muscles by a larger area of nonmuscle tissue and by the presence of small mononuclear cells resembling inflammatory infiltrates.7 The ability of CsA to reduce the percentage of centronucleated fiber as well as the areas of degeneration and nonmuscle tissue in exercised mdx muscles is indicative of a protective effect of the treatment against stress-induced worsening of the pathology. Despite the high regenerative potential of mdx mouse muscles, also supported in the present study by the large percentage of centronucleated fibers in all dystrophic groups, it is known that this mechanism can undergo a long-term failure because of an age-dependent fibrosis.1,45 Thus the decrease in connective tissue and in the level and expression of the profibrotic cytokine TGF-ß1 in CsA-treated muscle corroborate a potential ability of the drug to enhance the efficiency of regeneration on chronic treatments. Moreover, the levels TGF-ß1 presently found were, in absolute terms, lower than those reported by others in mdx DIA37 and this is in line with the less severity of the disorder in hindlimb muscles and further support a possible involvement of TGF-ß1 in the exercise-induced fibrotic events occurring in dystrophic animals. It is important to underline that CsA is known to exert a profibrotic activity in many organs by enhancing the expression of TGF-ß.30,37,38 Thus our findings suggest that the CsA-induced reduction of fibrotic events occurs through an indirect mechanism, ie, the reduction of the inflammatory state. Many of the parameters of mdx limb muscles worsened by exercise, including the impairment in chloride channel function observed in EDL muscle fibers (see below), were all similarly ameliorated by CsA treatment, suggesting that their alteration may be because of a general event aggravated by exercise and sensitive to CsA, such as inflammation induced by mechanical stress.42
CsA Exerts Different Effects on Functional Cellular Parameters: A Basis for a Calcium-Independent Action
We observed ex vivo a specific ability of CsA treatment to contrast the impairment of chloride channel function, evidenced by the low gCl values, both in DIA, where this impairment is permanent and exercise-independent, and in EDL muscle, where, as opposite, it is an effect triggered by exercise.9,31 This evidence suggests that CsA is able to beneficially act on the different mechanisms responsible for gCl impairment in the two muscle types.9 Taking into account the pivotal role of calcineurin in maintaining the slow-twitch program and the phenotype-dependent expression of chloride channels,19,46 it is feasible that the CsA treatment can increase gCl by reducing the proportion of fibers belonging to the slow phenotype, as supported by the observation that the low percentage of EDL fibers co-expressing the slow isoform of myosin heavy chain, was further reduced in CsA-treated mice. However, although this mechanism may account for the CsA effect on gCl of mdx DIA, in which a compensatory fast-to-slow phenotype shift has been described, albeit nonunanimously,47,48 it cannot explain the drug effect in mdx EDL muscle because we confirmed that no functionally significant phenotype transition occurs in this fast muscle in response to either genotype or exercise.29 We previously hypothesized,9 that the decrease of gCl induced by exercise in EDL muscle fibers might be related to a short-term action of proinflammatory cytokines, produced in response to the damaging effect of exercise, on the function of chloride channel. Thus, the effect of CsA in EDL muscle can be because of its ability to reduce the immune response and consequently the production of cytokines by injured muscle tissue or proinflammatory cell infiltrates.14,43,44 Another intriguing, although purely speculative, hypothesis to explain the CsA effectiveness in both DIA and EDL muscle is that the expression of functional chloride channel may be controlled by mechanosensitive mitogen-activated protein kinase (MAPK) cascades that are enhanced in dystrophin-deficient muscle fibers and also involves activation of calcineurin.42,49,50 In fact this complex network of kinase/phosphatase pathways modulates different cellular targets either in a short term or through the modulation of gene expression, ie, by enhancing the transcription of inflammatory genes or controlling translational events of ion channels.42,50,51
The CsA treatment did not ameliorate the alteration of calcium homeostasis that is a typical hallmark of dystrophic fibers and is worsened by exercise.2,4,29
CsA per se may even lead to a further increase in calcium mobilization;52
thus our finding leads to two main considerations: the first one is that the alteration in calcium homeostasis is rather independent on the inflammatory reaction. The second is that CsA acts on the sensitive parameters independently or downstream the alteration of calcium homeostasis. Interestingly, calcineurin is a calcium-dependent enzyme and its activation may also be involved in activation of nuclear factor-B and in apoptotic programs.16,17
Thus, in dystrophic muscle CsA may counteract calcineurin activity, and consequently the pathological cascade related to the enzyme, at a step following its overactivation by the increase in calcium level.
Specificity of CsA Effects: Possible Mechanisms
The lack of beneficial effect of CsA on calcium homeostasis, and thus its inability to contrast the other pathological events linked to this pathway, may explain why the CsA treatment was in fact partially effective. Accordingly, CsA may be ineffective on all of the pathological pathways that are strictly linked to the primary defect, ie, the absence of dystrophin. Another intriguing hypothesis to explain the partial effectiveness of CsA is the possible role that immune response pathways may play in muscle regeneration itself and supported by a recent microarray study showing a mitigated expression of inflammatory genes in DIA versus hindlimb mdx muscles.53
In addition calcineurin pathway may play a role in muscle regeneration20
and may be protective for dystrophic muscle through an enhanced expression of the compensatory protein utrophin.22
Although we cannot totally exclude this possibility, it is important to underline that no decrease in utrophin level has been observed by us as a result of CsA treatment. This data suggests that the positive effects of the CsA treatment observed on various in vivo (forelimb strength) and ex vivo (chloride channel function, CK levels, and morphology) parameters are not because of an utrophin-dependent mechanism, but also rule out a detrimental effect of our CsA treatment on utrophin expression. Furthermore, we did not observe the deleterious effects observed by Stupka and colleagues24
in mdx mice treated with high doses of CsA (30 mg/kg i.p.), likely in relation of the more therapeutic dose used by us and the different ages of the mice undergoing the treatment. In their study 2-week-old mice were treated for 2 weeks and the deleterious effect of CsA was associated to the proposed role of calcineurin in muscle growth and regeneration.21,24
This conclusion does not reconcile with the concomitant finding of a lower rather than higher enzyme level in mdx versus control and with a greater number of myogenin-positive fibers in CsA-treated muscles.24
Interestingly, the overall effects observed by us after CsA treatment were similar to those produced by a treatment with insulin-like growth factor-1.9
Far from proposing that CsA may stimulate regeneration, this observation corroborates the hypothesis that the reduction of immune reaction and, consequently, of fibrosis (see above) may facilitate the action of regeneration-promoting factors. However, we agree that, because of both the physiological role of calcineurin for skeletal muscle fibers and the toxicity of the drug, the dose of CsA to perform a treatment during muscle development, ie, in very young dystrophic patients, should be monitored with attention.
Concluding Remarks
An enhanced immune reaction contributes to the pathology progression in muscular dystrophy. This is supported by the beneficial effect observed by us on dystrophic muscle by CsA, one of first choice drugs for immunosuppression,14 which may also prove beneficial through direct actions on skeletal muscle tissue, ie, the inhibition of calcineurin or the amelioration of impaired mitochondrial metabolism. The effectiveness of CsA for muscle function is an important point because the immunological reaction is also responsible for the rejection of the administered genetic material and a greater success of these approaches have been obtained in dystrophic animals that were immunosuppressed.10-13 Although the narrow therapeutic index of CsA shall be taken into account especially for early treatment of boys with DMD, our data generally strengthen the usefulness for muscle function of therapies addressed against immunoreaction in dystrophic patients.
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Acknowledgements |
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Supported by Telethon-Italy (to project no. 1150) and the Association Français Contre les Myopathies (as part of postdoctoral fellowships to B.F. and J.-F.R.).
Accepted for publication October 6, 2004.
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