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Mild dystrophic damage in the androgen-sensitive levator ani muscle of the mdx mouse
Neuromuscular Disorders 15 (2005) 48–56 www.elsevier.com/locate/nmd
Mild dystrophic damage in the androgen-sensitive levator ani muscle of the mdx mouse Caden Souccara,*, Maria Do Carmo Gonc¸aloa, Hudson De Sousa Buckb, Maria Teresa R. Lima-Landmana, Antonio Jose´ Lapaa a
Department of Pharmacology, Natural Products Section, Escola Paulista de Medicina, Universidade Federal de Sa˜o Paulo, Rua Treˆs de Maio 100, 04044-020 Sa˜o Paulo, SP, Brazil b Department of Physiological Sciences, Faculdade de Cieˆncias Me´dicas Santa Casa de Sa˜o Paulo, Sa˜o Paulo, SP, Brazil Received 2 February 2004; received in revised form 14 July 2004; accepted 19 October 2004
Abstract The time course of muscular dystrophy on the androgen-sensitive levator ani muscle was compared to that of the diaphragm of dystrophic (mdx) mice aged 1–20 months. Muscle growth, isometric contractile properties and caffeine-induced contractures were determined to assess the hormone myotrophic effect, muscle strength and sarcoplasmic reticulum function, respectively, of both control and dystrophic muscles. Histological analysis of mdx muscles showed variable fiber size, centronucleated cells, infiltration of connective tissue, and necrosis which was less severe in the levator ani than in the diaphragm muscle. Tetanic tension per unit weight in the mdx levator ani was reduced (29%) after aging, while the contraction time remained unchanged. The tetanic tension of the mdx diaphragm muscle decreased with age from 3 to 20 months (20–64%), and the relaxation time was prolonged after aging (22%). Gonadectomy of young adult mdx mice caused atrophy of the levator ani muscle, accelerated muscle wasting, reduced the tetanic force (31%), but it did not affect the contraction time and caffeine responses. The results showed that testosterone does not prevent the progress of muscle disease in the mdx levator ani, but androgen withdrawal accelerates muscle wasting suggesting a normonal beneficial effect. q 2004 Elsevier B.V. All rights reserved. Keywords: Muscle dystrophy; Levator ani muscle; Testosterone; Contraction properties; Caffeine contractures; mdx mouse
1. Introduction The mutant mdx mouse has been frequently used as a model for Duchenne muscular dystrophy (DMD) for presenting mutations of the dystrophin gene with its lack of dystrophin expression [1–3]. Dystrophin deficiency in DMD causes progressive degeneration of the muscle fibers with infiltration of connective and adipose tissues [4,5]. In normal skeletal muscles dystrophin is believed to provide a linkage between costameric actin and the muscle fiber membrane [6], protecting the sarcolemma from mechanical injury induced by muscle contraction and stretches [7,8]. Despite muscular degeneration, the mdx mouse does not present signs of skeletal muscle * Corresponding author. Fax: C55 11 5576 4499. E-mail address: [email protected] (C. Souccar). 0960-8966/$ - see front matter q 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.nmd.2004.10.010
impairment [9,10] except after aging, when the animal exhibits muscle weakness similar to that of the human disease [11–14]. Muscle necrosis in the mdx mouse begins at 3–4 weeks of age and reaches its maximum around 5 and 8 weeks, with a peak regeneration around 10 weeks [15,16]. These stages of muscle disease parallel the prepubertal increase of circulating testosterone (20–40 days) and the animal’s sexual maturity (40–60 days) [17], suggesting a possible relationship between androgen levels and muscle wasting. Accordingly, increased muscle damage and serum creatine kinase activity were reported after a 3-week treatment of prepubertal mdx mice with the anabolic steroid, nandrolone decanoate [18]. In contrast, continuous infusion of a synthetic derivative of testosterone, stanozolol, did not affect the size and muscle strength of hind limb muscles from sedentary male mice [19].
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In view of these observations, the present study assessed the influence of circulating androgens on the evolution of muscular distrophy in the testosterone sensitive levator ani (LA) muscle of mdx mice, at different stages of the muscle disease from prepuberty to senescence. For comparison, the diaphragm (DIA) muscle of the same animals was used, the muscle most responsive to dystrophin absence [11,14]. The LA muscle of the rat [20] and mouse [21,22] is a sexually dimorphic muscle involved in the male copulatory behavior [23] that depends on testosterone for its growth and maintenance. Both the muscle fibers [24] and its motor neurons [25] present testosterone receptors that mediate genomic activation of protein synthesis [26]. Muscle growth and isometric contractile properties of the LA were assessed at the onset of muscle degeneration (1-month-old), during ongoing muscle degeneration and regeneration (2 to 4-month-old), after stabilization of muscle degeneration and regeneration at low levels (12month-old) [15,16], and in aged (20-month-old) mdx mice. LA muscles from 3-month-old gonadectomized mdx mice were used to evaluate the role of testosterone after maximal muscle degeneration. Caffeine-induced contractures in the same muscles were recorded to assess the sarcoplasmic reticulum function integrity. The results showed that in gonadally intact mdx mice, the testosterone-dependent LA muscle presented milder muscle wasting than that developed by the DIA. Hormonal deprivation of mdx mice accelerated deterioration of the LA muscle fibers, but it did not influence the DIA muscle disease. 2. Materials and methods 2.1. Animals Normal and mutant mdx mice from the C57BL/10 strain were obtained from the State University of Sa˜o Paulo at Botucatu, and from the Instituto de Cieˆncias Biome´dicas of the University of Sa˜o Paulo, respectively, and bred in our animal facilities. Control and dystrophic males were kept separate from females from the 25th postnatal day until the experiments. All animals were housed under controlled 12/12 h light/dark cycle and temperature, with free access to food and water. The experiments were conducted according to the institutional ethical guidelines. The intact control and mdx male mice were killed at age 1 month (prepubertal), 2, 3, 4 (young adult), 12 (adult) and 20 (aged) months. Gonadectomy was done under ether anesthesia through the scrotum in the 3-month-old control and the mdx mice, and the experiments were performed after 30 days. Agematched sham-operated animals were used for control. 2.2. Isometric tension recordings The mice were killed by cervical dislocation under ether anesthesia and both the DIA and LA muscles were rapidly
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removed and kept in Tyrode solution with the following composition (in mM): NaCl 135; NaHCO3 15; KCl 5, CaCl2 2, MgCl2 1, NaH2PO4 1 and glucose 11, pH 7.3–7.4 after bubbling with 95% O2C5% CO2. The gonads and seminal vesicle (SV) weights were used as an index of androgen plasma levels. The LA was freed from the bulbocavernosus muscle and connective tissue, sectioned at the central tendon [27] and mounted under 1 g tension in organ bath containing 2.5 mL of Tyrode solution at 30 8C, continuously gassed with carbogen. One end of the tendon was attached to a force transducer (model FT03, Grass) and the other end was tied to a holder fixed in the organ bath. Muscle strips (3–5 mm wide) of the DIA were mounted under the same conditions, with the central tendon attached to a force transducer and the costal end tied to the organ bath holder [28]. Muscle twitches were elicited by direct stimulation using a S88 Grass stimulator (2 ms, 0.1 Hz, supramaximal voltage) through a pair of platinum electrodes placed on either side of each muscle. The muscles were adjusted to optimum length and isometric twitches were recorded on a polygraph (model R411 Beckman). After 30 min stabilization, the resting tension was readjusted and contraction properties were recorded in the presence of D-tubocurarine (10 mM) to block neuromuscular transmission. Twitches (0.1 Hz) and tetanus (100 Hz) tensions were displayed on an oscilloscope and recorded on a video camera for late measurements of the following parameters: Twitch (TwT) and tetanus (TT) tensions as the maximum tensions developed to a single and tetanic stimuli, respectively, expressed per gram of muscle weight. The time to peak tension (TPT) as the interval between the initiation and maximal tension developed during a single twitch; half relaxation time (HRT) as the time for the maximal tension to fall to half maximum. 2.3. Caffeine contractures Following recordings of the tetanic tension response, the muscles were exposed to increasing concentrations of caffeine (3–100 mM) and the developed contracture tension was recorded. The amplitudes of caffeine contractures were measured and expressed as percent of the maximal tetanic tension (% TT) developed at 100 Hz pulses. The mean effective concentration (EC50) for caffeine was determined for each concentration–response relationship. At the end of the tension recordings, the muscles were blotted dry and weighed. 2.4. Histological studies LA and DIA muscles from intact and gonadectomized (GDX) 4-month-old, and intact 20-month-old mice from both strains were excised under ether anesthesia, rapidly frozen in liquid nitrogen cooled isopentane and stored at K80 8C. Transverse serial cryostat sections 10–12 mm thick were stained with haemathoxylin and eosin (H and E)
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and visualized on a Nikon Optiphot-2 light microscope. Cross-sectional areas of at least 180 fibers in each muscle were measured using a computerized image processing system (Leica Quantimet Q 500 IW). 2.5. Statistical analysis The results were presented as meansGSEM. EC50 values were expressed as geometric means and 95% confidence limits (CL). Differences between control and mdx C57BL/10 strains and between groups at different ages were determined by a two-way analysis of variance followed by the Dunnet test. Distributions of muscle fiber areas from age-matched control and mdx groups were compared using the Kolmogorv–Smirnov two-sample test [29]. The data were considered different at a probability value of P!0.05.
3. Results 3.1. Animal and muscle growth During the first year of life, the body weight of control mice increased by 2.6-fold (from 11G1 to 29G1 g), while the LA and DIA muscle weights increased by six-fold (from 4.1G0.4 to 24.7G1.2 mg) and 2.4-fold (from 17.9G1.2 to 43.0G2.8 mg), respectively (Fig. 1). The gonadal and SV
Fig. 1. Body weight (BW, right ordinate), gonadal weight (G, left ordinate) in (a) levator ani (LA) and diaphragm (DIA) muscles weights in (b) from control (hollow symbols) and mdx (closed symbols) mice aged 1–20 months. The symbols and vertical bars are meansGSEM of 6–9 animals in each group. *Different from age-matched control group (P!0.05).
weights increased by six-fold (from 33.5G4.7 to 200.2G 6.9 mg) and 34-fold (from 1.6G0.4 to 54.7G5.5 mg), respectively. At 20 months, the body and DIA muscle weights remained the same, while the LA muscle, gonads (Fig. 1a and b) and SV weights decreased by 22, 16 and 46%, respectively, compared to corresponding values at 12 months. The body weights of mdx mice aged 1–20 months did not differ from those determined in age-matched control groups. The LA and DIA muscle weights increased in parallel with the animal growth; at 2 months of age, however, the DIA was 27% smaller and the LA was 52% greater in mdx than in age-matched control (Fig. 1b). At 1 month, the gonadal and SV weights were two times greater in mdx than in control mice. In older mdx groups, the gonadal weight was always lower (by 20–40%) than in age-matched control (Fig. 1a), while the SV weight did not differ from control. At 20 months, the body as well as the DIA and LA muscle weights of mdx mice were not significantly changed; the gonads and SV weights were, respectively, 24 and 41% lower than those values determined at 12 months (Fig. 1). 3.2. Histological studies Cross-sections of LA and DIA muscles from intact control animals presented normal structure with relatively uniform muscle fibers size and peripheral nuclei. In the control strain small infiltration of mononuclear inflammatory cells and eosinophilic cells were observed in sections of LA muscles from aged mice, while fiber atrophy and a few centronucleated cells were observed in LA sections from GDX animals (Fig. 2a–c). The LA and DIA of mdx mice exhibited histological changes similar to those described for other dystrophic skeletal muscles [15] characterized by variable fiber size, infiltration of mononuclear inflammatory cells and connective tissue, presence of necrotic and centronucleated fibers. Morphological changes in the LA muscle fibers were less severe than those observed in the DIA, but they were intensified after aging or gonadectomy (Fig. 2d–f). The frequency distribution of LA fiber areas of 4-monthold mdx mice was shifted significantly rightward compared to that of control. The mean LA fiber area at this age was 23% greater in mdx than in control (Fig. 3, Table 1). Neither the distribution nor the mean LA fiber areas differed among control and dystrophic aged mice. Compared to 4-monthold groups, however, the mean LA fiber areas of control and mdx aged mice were increased by 32 and 14%, respectively (Fig. 3, Table 1). The DIA fiber area distribution of young adult mdx mice was also shifted rightward compared to that of control, with a mean value 15% greater in dystrophic than in control group (Fig. 3, Table 1). After aging, the frequency distribution of mdx DIA fiber areas was shifted leftward with a mean value 31% smaller than in control (Fig. 3, Table 1). Compared to corresponding young adult groups,
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Fig. 2. Transverse sections of the levator ani muscle stained with H and E from young adult (a, d), aged (b, e) and gonadectomized (c, f) control (left) and mdx (right) mice. A cluster of small centronucleated cells (arrow) amidst normal muscle fibers, infiltration of inflammatory cells and connective tissue (arrows) can be observed in LA muscle sections from young adult mdx mouse (d). Clusters of eosinophilic muscle fibers and interstitial mononuclear cells (arrow) are shown in LA muscle section from control-aged mouse (b). Degenerating muscle fibers and infiltration of mononuclear inflammatory cells (arrow) in LA muscle from aged mdx mouse (e). LA muscle sections from gonadectomized young adult mouse from control (c) and mdx (f) strains. Notice the presence of centronucleated cells in (c) (arrow), intense cell disorganization and infiltration of phagocytic cells in (f) (arrow). Scale barZ50 mm.
the mean DIA fiber size was increased by 39% in control, and reduced by 17% in the dystrophic strain (Table 1). 3.3. Contraction properties The TwT of control LA muscles recorded at 1 month (324.0G92.0 g/g muscle weight) did not significantly change with age. The TT of the same muscles increased by two-fold from 1 (1018G270 g/g muscle weight) to 3 months of age, decreasing thereafter by 25% (Fig. 4a and b). The TPT (43.3G2.2 ms) and HRT (29.5G2.1 ms) recorded at 1 month were slightly shortened in adult mice, recovering the initial values after aging. In mdx mice, the TwT and TT of LA muscles did not differ up to 4 months compared to age-matched control.
A small increase (27%) of both parameters was observed at 12 months, while significant decrease of TT (29%) was detected only in aged dystrophic mice (Fig. 4a and b). The TPT and HRT did not change among age-matched control and mdx mice. The TwT of control DIA muscles recorded at 1 month of age (405G79 g/g muscle weight) was reduced at 2 months (38%), stabilizing thereafter. The TT (1553G159 g/g muscle weight) did not change up to 20 months (Fig. 4c and d). The TPT (32.4G1.7 ms) recorded at 1 month of age was slightly shortened (12–20%) from 2 to 20 months, while the HRT (15.47G2.38 ms) was not changed with age. In dystrophic mice, the TwT developed by the DIA muscle was reduced by 57% after aging. The TT was reduced with age from 3 months and up by 20–64% of
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Fig. 3. Fiber area distributions in the levator ani (left) and diaphragm (right) muscles of 4-month-old intact (top) and gonadectomized (GDX, bottom), and 20month-old intact mice (middle) from control (white bars) and mdx (gray bars) strains. Both muscles presented increased proportion of larger fibers in intact young adult mdx mice that persisted after aging in the levator ani. A greater proportion of small size fibers, probably regenerated fibers, predominated in diaphragm muscles of aged mdx mice. Significant leftward shift of fiber area distribution was observed in levator ani of gonadectomized mdx mice. The data represent the number of fibers measured (540–570 fibers) in three muscles for each group.
control values (Fig. 4c and d). The TPT did not differ among age-matched control and mdx groups, while the HRT was prolonged (22%) in aged mdx mice. 3.4. Effect of Gonadectomy Gonadectomy of young adult control and mdx mice decreased the LA muscle weight by 40 and 56% of age-matched sham groups, respectively (Table 2).
The frequency distribution of LA fiber areas from gonadectomized (GDX) mdx mice was shifted toward lower values relatively to that of control GDX group. Compared to gonadally intact groups, the mean LA fiber areas of control and mdx mice were reduced by 28 and 50%, respectively (Fig. 3c, Table 1). Despite muscle atrophy, the TwT developed by the LA from control GDX mice was increased by 32%, while the TT was reduced by 34% leading to increase of
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Table 1 Fiber areas of control and dystrophic levator ani and diaphragm muscles from 4-month-old intact (adult) or gonadectomized (GDX), and 20-month-old (aged) intact mice Group
Adult Aged GDX
Levator ani fiber area (mm2)
Diaphragm fiber area (mm2)
Control
Dystrophic
Control
Dystrophic
684.0G15.9 (561) 901.0G20.2†(556) 490.5G12.1†(575)
844.6G17.0*(564) 966.1G20.3†(572) 426.2G11.1*†(560)
817.2G26.1(550) 1132.0G40.4†(540) 730.9G26.9(550)
935.7G28.7*(555) 781.3G22.2*†(547) 685.8G23.5†(547)
Data are meansGSEM of the number of fibers in parenthesis in three muscles for each group. *Different from age-matched control group (P!0.05).†Different from adult group of the same animal strain (P!0.05).
the twitch/tetanus ratio (61%). The TPT was prolonged by 24%, whereas the HRT remained unchanged (Table 2). In dystrophic GDX mice TwT of the LA muscle did not significantly change, but TT was 31% lower than in sham group leading the twitch/tetanus ratio to increase by 53%. The contraction time in its turn, was not altered (Table 2). Gonadectomy of 3-month-old control and mdx mice did not affect the body and DIA muscle weights or the contraction properties. The frequency distribution of DIA fiber areas did not differ among control and GDX mice. The mean DIA fiber area was not changed in the control strain, but it was reduced in mdx mice by 27% compared to corresponding gonadally intact groups (Fig. 3f, Table 1). 3.5. Caffeine contracture Exposure of control LA muscles to caffeine (3–100 mM) produced maximal contractures greater in prepubertal
(60.4G5.8% TT) than in young adult (23.3G2.2% TT) and aged (34.3G2.3% TT) mice (Fig. 5a). The corresponding EC50 values were 24.4 mM (CL: 10.7–55.5 mM), 48.2 mM (41.8–56.2 mM), and 38.5 mM (25.4–58.2 mM), respectively, not different among all three groups. The responses to caffeine of mdx LA muscles did not differ from those obtained in age-matched control (Fig. 5c). The maximal response of DIA muscles to caffeine did not differ among prepubertal, young adult and aged (35.0G 3.4% TT) control mice. The EC50 values in these groups ranged between 32 and 35 mM (Fig. 5b). In dystrophic mice, the maximum response to caffeine of the DIA was increased in young adult (59.3G9.3% TT) and aged (63.8G 8.7% TT) mice (Fig. 5d), without significant changes in EC50 values compared to age-matched control. After gonadectomy of control mice, the maximal LA muscle response to caffeine was increased (39.5G2.2% TT) with no change of EC50 (24.8 mM, CL: 21.0–29.2 mM).
Fig. 4. Twitch (a, c) and tetanus (b, d) tensions developed per gram of muscle wet weight, by the levator ani (left) and diaphragm (right) muscles from control (hollow symbols) and mdx (filled symbols) mice at various ages. The symbols and vertical bars are meansGSEM of 7–11 animals in each group. *Different from age-matched control group (P!0.05).
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Table 2 Isometric contractile properties of the levator ani (LA) muscle from 4-month-old control and dystrophic (mdx) mice, sham-operated or gonadectomized (GDX) Control
Muscle weight (mg) TwT(g/g wet weight) TT (g/g wet weight) Twitch/Tetanus TPT (ms) HRT (ms)
Dystrophic
Sham (10)
GDX (6)
Sham (11)
GDX (6)
23.6G1.5 258G15 1720G141 0.161G0.010 33.0G1.2 25.1G1.6
14.1G1.5* 341G80** 1139G121* 0.259G0.036* 40.9G2.3** 24.3G1.6
20.4G1.1 289G22 1620G120 0.172G0.013 36.2G1.1 26.0G1.2
9.0G1.1* 291G22 1110G93* 0.264G0.016* 37.9G0.9 29.4G0.8
Data are meansGSEM of the number of determinations in parenthesis. TwT, twitch tension; TT, tetanus tension; TPT, time to peak tension; HRT, half relaxation time. Statistic differences between age-matched sham-operated and GDX group (*P!0.05; **P!0.001).
Gonadectomy of mdx mice, however, did not affect the caffeine-induced contractures in either the LA or DIA muscles.
4. Discussion This study presents evidences of the influence of androgens on the mild dystrophic damage of the hormonedependent LA muscle of mdx mice. Muscle degeneration in mdx mice neither affected the body weight gain nor influenced the normal pubertal surge of plasma androgens [17], indicated by the weight gain of LA muscle and seminal vesicle at 1–2 months of age.
The decreased gonadal weight observed in adult mdx mice has so far not been reported, and may be related to the lack of dystrophin and shorter isoforms that are expressed in nonmuscle tissues, including the testes [30]. Although, the functional role of these proteins in non-muscle tissues is still unknown, their absence in the testes did not affect male fertility in our dystrophic colony, which is in agreement with normal testosterone serum levels reported in adult mdx mice [31]. The mdx mouse is known to adapt to muscle degeneration with muscle fiber hypertrophy and activation of satellite cells [13,32]. Hypertrophy of dystrophic LA was also observed at 2 months of age, when maximal degeneration is reported to occur in most mdx skeletal
Fig. 5. Amplitudes of the caffeine contractures expressed as percent of tetanic tension of diaphragm strips (a, b) and levator ani (c, d) muscles from control (hollow symbols) and mdx (filled symbols) mice at various ages. The symbols and vertical bars are meansGSEM of 7–11 animals in each group.
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muscles [16]. The response was accompanied by degenerative histological changes that were intensified with age in both the LA and DIA, as described for other mdx skeletal muscles [13,15]. The deterioration of the LA muscle fibers, however, was always milder than that observed in the DIA of the same animal. Skeletal muscles with small fiber diameter like the extraocular and denervated muscles were reported as more resistant to necrosis in the mdx mouse [33]. The determined mean LA muscle fiber area was slightly smaller (16%) than that of the DIA, indicating that the moderate muscle degeneration is apparently unrelated to the fiber size. Furthermore, gonadectomy of mdx mice caused atrophy of the LA to 44% of its original size and intensified deterioration of the muscle fibers, suggesting a protective action of testosterone against degeneration of the LA muscle fibers. The activation and proliferation of satellites cells induced by testosterone were reported in the rat LA [34], which is likely to be involved in the regenerative LA muscle responses of dystrophic mice. According to the data, such hormonal influence appears to be exerted at a lesser extent on other skeletal muscles as well, as suggested by the decrease in DIA fiber areas of gonadectomized mdx mice. Significant decrease of LA muscle strength was detected in aged mdx mice, which was coincident with the low plasma androgen levels associated with aging [35], indicated by the decrease in seminal vesicle and gonadal weights in both animal strains. In contrast, the DIA muscle, the most responsive to dystrophic injury, developed a decrease in tetanic force in young adult mdx mice. These data alone suggested a possible influence of androgens on the progress of muscle wasting in the LA. Gonadectomy of young adult mice, however, reduced the LA muscle strength in control and more intensely, in dystrophic mice indicating that the effects are mainly related to androgen deprivation. Thus, despite the evidence of a hormonal beneficial influence, testosterone does not appear to interrupt the progress of muscle disease in dystrophic LA. In agreement with our data, treatment with the anabolic steroid oxandrolone did not cause significant changes in the muscle strength of boys with DMD despite an increase in their linear growth [36]. Muscle necrosis in both DMD and in the mdx mouse has been related to accumulation of intracellular free calcium concentration ([Ca2C]i) caused by sarcolemmal lesion [6,7] and abnormal calcium channel activity [37,38]. Dysfunctions of the sarcoplasmic reticulum (SR) Ca2C handling mechanisms have also been implied in the abnormal Ca2C homeostasis in dystrophic muscles [39,40]. To evaluate the SR function in dystrophic muscles, caffeine-induced contractures were recorded in those groups presenting changes in the contraction properties. Caffeine is known to produce muscle contractures by inducing Ca2C release and inhibition of Ca2C uptake by the SR [41]. The age-related increase in the amplitudes of caffeine contractures observed in mdx DIA muscle was consistent with the reported alteration of SR Ca 2C handling
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mechanisms in dystrophic muscles [40,42,43]. Such an effect, however, was not observed in LA muscles from adult and older mdx mice, indicating that the SR function remained apparently unaffected. The greater caffeine responses observed in LA muscles from prepubertal mice are characteristic of immature skeletal muscles and probably related to poor muscle fiber development and low capacity of the SR for Ca2C storage [44]. Androgen deprivation of LA muscles prolonged the active state duration in both animal strains, while it increased caffeine responses only in the control group, suggesting changes in the SR Ca2C release and uptake. Alternatively, the results may be secondary to muscle atrophy induced by gonadectomy and unrelated to changes in the SR Ca2C handling mechanisms. Ultrastructural studies of androgen-deprived LA muscle fibers revealed intense loss of contractile elements with a relatively preserved sarcoplasmic reticulum in both the rat [45] and mouse [21], which may explain the increased caffeine contractures and twitch tension expressed per muscle weight in control gonadectomized mice. To summarize, the data presented showed that muscle wasting in the testosterone-dependent LA muscle was milder than that observed in the DIA of mdx mice. Hormone deprivation in mdx mice did not affect muscle degeneration in the DIA, but it intensified deterioration of the LA fibers, decreased the muscle strength and modified the contraction kinetics. In conclusion, testosterone may not prevent the progress of muscle disease in the mdx LA, but androgen deprivation accelerates muscle wasting suggesting a hormonal beneficial effect.
Acknowledgements The authors thank Dr V.B. Valero for the care of the dystrophic and control mice colonies, Mrs H.S. Reis for technical assistance in the histological studies, and A.C.C. Vallin for her help in some experiments. Thanks are also due to Drs E. Haapalainen and F.P. Faria, Department of Electron Microscopy, for the use of the imaging processing system, and Dr A.N. Gordan, Department of Pathology, UNIFESP, for the histopathological analysis. This work was supported by grants from Fundac¸a˜o de Amparo a` Pesquisa do Estado de Sa˜o Paulo (FAPESP) and Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´gico (CNPq).
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[25] Breedlove SM, Arnold AP. Hormonal accumulation in a sexually dimorphic motor nucleus in the rat spinal cord. Science 1980;210: 564–6. [26] Gobinet J, Poujol N, Sultan Ch. Molecular action of androgens. Mol Cell Endocrinol 2002;198:15–24. [27] Souccar C, Lapa AJ, Valle JR. Influence of castration on the electrical excitability and contraction properties of the rat levator ani muscle. Exp Neurol 1982;75:576–88. [28] Souccar C, Gonc¸alo MC, Lapa AJ, Troncone LR, Lebrun I, Magnoli F. Blockade of acetylcholine release at the motor endplate by a polypeptide from the venom of Phoneutria nigriventer. Br J Pharmacol 1995;116:2817–23. [29] Sokal RR, Rohlf FJ. In: Biometry: the principle and pratice of statistics in biological research. 2nd ed. New York: W.H. Freeman; 1981. [30] Tokarz SA, Duncan NM, Rash SM, Sadeghi A, Dewan AK, Pillers DA. Redefinition of dystrophin isoform distribution in mouse tissue by RT-PCR implies role in nonmuscle manifestations of duchenne muscular dystrophy. Mol Genet Metab 1998;65:272–81. [31] Hirota M, Furukawa Y, Shinoda I, et al. Changes in nerve growth factor content of the submaxillary gland in the genetically dystrophic (mdx) mouse. J Neurol Sci 1994;121:176–82. [32] Hawke TJ, Garry DJ. Myogenic satellite cells: physiology to molecular biology. J Appl Physiol 2001;91:534–51. [33] Karpati G, Carpenter S, Prescott S. Small-caliber skeletal muscle fibers do not suffer necrosis in mdx mouse dystrophy. Muscle Nerve 1988;11:795–803. [34] Joubert Y, Tobin C. Testosterone treatment results in quiescent satellite cells being activated and recruited into cell cycle in rat levator ani muscle. Dev Biol 1995;169:286–94. [35] Coquelin A, Desjardins C. Luteinizing hormone and testosterone secretion in young and old male mice. Am J Physiol 1982;243: E257–E63. [36] Fenichel GM, Griggs RC, Kissel J, et al. A randomized efficacy and safety trial of oxandrolone in the treatment of Duchenne dystrophy. Neurology 2001;56:1075–9. [37] Alderton JM, Steinhardt RA. How calcium influx through calcium leak channels is responsible for the elevated levels of calciumdependent proteolysis in dystrophic myotubes. Trends Cardiovasc Med 2000;10:268–72. [38] Mallouk N, Jacquemond V, Allard B. Elevated subsarcolemmal Ca2C in mdx mouse skeletal muscle fibers detected with Ca2C-activated KC channels. Proc Natl Acad Sci USA 2000;97:4950–5. [39] Gailly P. New aspects of calcium signaling in skeletal muscle cells: implications in Duchenne muscular dystrophy. Biochim Biophys Acta 2002;1600:38–44. [40] Divet A, Huchet-Cadiou C. Sarcoplasmic reticulum function in slowand fast-twitch skeletal muscles from mdx mice. Pflugers Arch 2002; 444:634–43. [41] Weber A, Herz R. The relationship between caffeine contracture of intact muscle and the effect of caffeine on reticulum. J Gen Physiol 1968;52:750–9. [42] Takagi A, Kojima S, Ida M, Araki M. Increased leakage of calcium ion from the sarcoplasmic reticulum of the mdx mouse. J Neurol Sci 1992;110:160–4. [43] Kargacin ME, Kargacin GJ. The sarcoplasmic reticulum calcium pump is functionally altered in dystrophic muscle. Biochim Biophys Acta 1996;1290:4–8. [44] Leberer E, Hartner KT, Pette D. Postnatal development of Ca2Csequestration by the sarcoplasmic reticulum of fast and slow muscles in normal and dystrophic mice. Eur J Biochem 1988;174:247–53. [45] Gori Z, Pellegrino C, Pollera M. The castration atrophy of the dorsal bulbocavernosus muscle of rat: an electron microscopic study. Exp Mol Pathol 1967;6:172–98.
Mild dystrophic damage in the androgen-sensitive levator ani muscle of the mdx mouse Caden Souccara,*, Maria Do Carmo Gonc¸aloa, Hudson De Sousa Buckb, Maria Teresa R. Lima-Landmana, Antonio Jose´ Lapaa a
Department of Pharmacology, Natural Products Section, Escola Paulista de Medicina, Universidade Federal de Sa˜o Paulo, Rua Treˆs de Maio 100, 04044-020 Sa˜o Paulo, SP, Brazil b Department of Physiological Sciences, Faculdade de Cieˆncias Me´dicas Santa Casa de Sa˜o Paulo, Sa˜o Paulo, SP, Brazil Received 2 February 2004; received in revised form 14 July 2004; accepted 19 October 2004
Abstract The time course of muscular dystrophy on the androgen-sensitive levator ani muscle was compared to that of the diaphragm of dystrophic (mdx) mice aged 1–20 months. Muscle growth, isometric contractile properties and caffeine-induced contractures were determined to assess the hormone myotrophic effect, muscle strength and sarcoplasmic reticulum function, respectively, of both control and dystrophic muscles. Histological analysis of mdx muscles showed variable fiber size, centronucleated cells, infiltration of connective tissue, and necrosis which was less severe in the levator ani than in the diaphragm muscle. Tetanic tension per unit weight in the mdx levator ani was reduced (29%) after aging, while the contraction time remained unchanged. The tetanic tension of the mdx diaphragm muscle decreased with age from 3 to 20 months (20–64%), and the relaxation time was prolonged after aging (22%). Gonadectomy of young adult mdx mice caused atrophy of the levator ani muscle, accelerated muscle wasting, reduced the tetanic force (31%), but it did not affect the contraction time and caffeine responses. The results showed that testosterone does not prevent the progress of muscle disease in the mdx levator ani, but androgen withdrawal accelerates muscle wasting suggesting a normonal beneficial effect. q 2004 Elsevier B.V. All rights reserved. Keywords: Muscle dystrophy; Levator ani muscle; Testosterone; Contraction properties; Caffeine contractures; mdx mouse
1. Introduction The mutant mdx mouse has been frequently used as a model for Duchenne muscular dystrophy (DMD) for presenting mutations of the dystrophin gene with its lack of dystrophin expression [1–3]. Dystrophin deficiency in DMD causes progressive degeneration of the muscle fibers with infiltration of connective and adipose tissues [4,5]. In normal skeletal muscles dystrophin is believed to provide a linkage between costameric actin and the muscle fiber membrane [6], protecting the sarcolemma from mechanical injury induced by muscle contraction and stretches [7,8]. Despite muscular degeneration, the mdx mouse does not present signs of skeletal muscle * Corresponding author. Fax: C55 11 5576 4499. E-mail address: [email protected] (C. Souccar). 0960-8966/$ - see front matter q 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.nmd.2004.10.010
impairment [9,10] except after aging, when the animal exhibits muscle weakness similar to that of the human disease [11–14]. Muscle necrosis in the mdx mouse begins at 3–4 weeks of age and reaches its maximum around 5 and 8 weeks, with a peak regeneration around 10 weeks [15,16]. These stages of muscle disease parallel the prepubertal increase of circulating testosterone (20–40 days) and the animal’s sexual maturity (40–60 days) [17], suggesting a possible relationship between androgen levels and muscle wasting. Accordingly, increased muscle damage and serum creatine kinase activity were reported after a 3-week treatment of prepubertal mdx mice with the anabolic steroid, nandrolone decanoate [18]. In contrast, continuous infusion of a synthetic derivative of testosterone, stanozolol, did not affect the size and muscle strength of hind limb muscles from sedentary male mice [19].
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In view of these observations, the present study assessed the influence of circulating androgens on the evolution of muscular distrophy in the testosterone sensitive levator ani (LA) muscle of mdx mice, at different stages of the muscle disease from prepuberty to senescence. For comparison, the diaphragm (DIA) muscle of the same animals was used, the muscle most responsive to dystrophin absence [11,14]. The LA muscle of the rat [20] and mouse [21,22] is a sexually dimorphic muscle involved in the male copulatory behavior [23] that depends on testosterone for its growth and maintenance. Both the muscle fibers [24] and its motor neurons [25] present testosterone receptors that mediate genomic activation of protein synthesis [26]. Muscle growth and isometric contractile properties of the LA were assessed at the onset of muscle degeneration (1-month-old), during ongoing muscle degeneration and regeneration (2 to 4-month-old), after stabilization of muscle degeneration and regeneration at low levels (12month-old) [15,16], and in aged (20-month-old) mdx mice. LA muscles from 3-month-old gonadectomized mdx mice were used to evaluate the role of testosterone after maximal muscle degeneration. Caffeine-induced contractures in the same muscles were recorded to assess the sarcoplasmic reticulum function integrity. The results showed that in gonadally intact mdx mice, the testosterone-dependent LA muscle presented milder muscle wasting than that developed by the DIA. Hormonal deprivation of mdx mice accelerated deterioration of the LA muscle fibers, but it did not influence the DIA muscle disease. 2. Materials and methods 2.1. Animals Normal and mutant mdx mice from the C57BL/10 strain were obtained from the State University of Sa˜o Paulo at Botucatu, and from the Instituto de Cieˆncias Biome´dicas of the University of Sa˜o Paulo, respectively, and bred in our animal facilities. Control and dystrophic males were kept separate from females from the 25th postnatal day until the experiments. All animals were housed under controlled 12/12 h light/dark cycle and temperature, with free access to food and water. The experiments were conducted according to the institutional ethical guidelines. The intact control and mdx male mice were killed at age 1 month (prepubertal), 2, 3, 4 (young adult), 12 (adult) and 20 (aged) months. Gonadectomy was done under ether anesthesia through the scrotum in the 3-month-old control and the mdx mice, and the experiments were performed after 30 days. Agematched sham-operated animals were used for control. 2.2. Isometric tension recordings The mice were killed by cervical dislocation under ether anesthesia and both the DIA and LA muscles were rapidly
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removed and kept in Tyrode solution with the following composition (in mM): NaCl 135; NaHCO3 15; KCl 5, CaCl2 2, MgCl2 1, NaH2PO4 1 and glucose 11, pH 7.3–7.4 after bubbling with 95% O2C5% CO2. The gonads and seminal vesicle (SV) weights were used as an index of androgen plasma levels. The LA was freed from the bulbocavernosus muscle and connective tissue, sectioned at the central tendon [27] and mounted under 1 g tension in organ bath containing 2.5 mL of Tyrode solution at 30 8C, continuously gassed with carbogen. One end of the tendon was attached to a force transducer (model FT03, Grass) and the other end was tied to a holder fixed in the organ bath. Muscle strips (3–5 mm wide) of the DIA were mounted under the same conditions, with the central tendon attached to a force transducer and the costal end tied to the organ bath holder [28]. Muscle twitches were elicited by direct stimulation using a S88 Grass stimulator (2 ms, 0.1 Hz, supramaximal voltage) through a pair of platinum electrodes placed on either side of each muscle. The muscles were adjusted to optimum length and isometric twitches were recorded on a polygraph (model R411 Beckman). After 30 min stabilization, the resting tension was readjusted and contraction properties were recorded in the presence of D-tubocurarine (10 mM) to block neuromuscular transmission. Twitches (0.1 Hz) and tetanus (100 Hz) tensions were displayed on an oscilloscope and recorded on a video camera for late measurements of the following parameters: Twitch (TwT) and tetanus (TT) tensions as the maximum tensions developed to a single and tetanic stimuli, respectively, expressed per gram of muscle weight. The time to peak tension (TPT) as the interval between the initiation and maximal tension developed during a single twitch; half relaxation time (HRT) as the time for the maximal tension to fall to half maximum. 2.3. Caffeine contractures Following recordings of the tetanic tension response, the muscles were exposed to increasing concentrations of caffeine (3–100 mM) and the developed contracture tension was recorded. The amplitudes of caffeine contractures were measured and expressed as percent of the maximal tetanic tension (% TT) developed at 100 Hz pulses. The mean effective concentration (EC50) for caffeine was determined for each concentration–response relationship. At the end of the tension recordings, the muscles were blotted dry and weighed. 2.4. Histological studies LA and DIA muscles from intact and gonadectomized (GDX) 4-month-old, and intact 20-month-old mice from both strains were excised under ether anesthesia, rapidly frozen in liquid nitrogen cooled isopentane and stored at K80 8C. Transverse serial cryostat sections 10–12 mm thick were stained with haemathoxylin and eosin (H and E)
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and visualized on a Nikon Optiphot-2 light microscope. Cross-sectional areas of at least 180 fibers in each muscle were measured using a computerized image processing system (Leica Quantimet Q 500 IW). 2.5. Statistical analysis The results were presented as meansGSEM. EC50 values were expressed as geometric means and 95% confidence limits (CL). Differences between control and mdx C57BL/10 strains and between groups at different ages were determined by a two-way analysis of variance followed by the Dunnet test. Distributions of muscle fiber areas from age-matched control and mdx groups were compared using the Kolmogorv–Smirnov two-sample test [29]. The data were considered different at a probability value of P!0.05.
3. Results 3.1. Animal and muscle growth During the first year of life, the body weight of control mice increased by 2.6-fold (from 11G1 to 29G1 g), while the LA and DIA muscle weights increased by six-fold (from 4.1G0.4 to 24.7G1.2 mg) and 2.4-fold (from 17.9G1.2 to 43.0G2.8 mg), respectively (Fig. 1). The gonadal and SV
Fig. 1. Body weight (BW, right ordinate), gonadal weight (G, left ordinate) in (a) levator ani (LA) and diaphragm (DIA) muscles weights in (b) from control (hollow symbols) and mdx (closed symbols) mice aged 1–20 months. The symbols and vertical bars are meansGSEM of 6–9 animals in each group. *Different from age-matched control group (P!0.05).
weights increased by six-fold (from 33.5G4.7 to 200.2G 6.9 mg) and 34-fold (from 1.6G0.4 to 54.7G5.5 mg), respectively. At 20 months, the body and DIA muscle weights remained the same, while the LA muscle, gonads (Fig. 1a and b) and SV weights decreased by 22, 16 and 46%, respectively, compared to corresponding values at 12 months. The body weights of mdx mice aged 1–20 months did not differ from those determined in age-matched control groups. The LA and DIA muscle weights increased in parallel with the animal growth; at 2 months of age, however, the DIA was 27% smaller and the LA was 52% greater in mdx than in age-matched control (Fig. 1b). At 1 month, the gonadal and SV weights were two times greater in mdx than in control mice. In older mdx groups, the gonadal weight was always lower (by 20–40%) than in age-matched control (Fig. 1a), while the SV weight did not differ from control. At 20 months, the body as well as the DIA and LA muscle weights of mdx mice were not significantly changed; the gonads and SV weights were, respectively, 24 and 41% lower than those values determined at 12 months (Fig. 1). 3.2. Histological studies Cross-sections of LA and DIA muscles from intact control animals presented normal structure with relatively uniform muscle fibers size and peripheral nuclei. In the control strain small infiltration of mononuclear inflammatory cells and eosinophilic cells were observed in sections of LA muscles from aged mice, while fiber atrophy and a few centronucleated cells were observed in LA sections from GDX animals (Fig. 2a–c). The LA and DIA of mdx mice exhibited histological changes similar to those described for other dystrophic skeletal muscles [15] characterized by variable fiber size, infiltration of mononuclear inflammatory cells and connective tissue, presence of necrotic and centronucleated fibers. Morphological changes in the LA muscle fibers were less severe than those observed in the DIA, but they were intensified after aging or gonadectomy (Fig. 2d–f). The frequency distribution of LA fiber areas of 4-monthold mdx mice was shifted significantly rightward compared to that of control. The mean LA fiber area at this age was 23% greater in mdx than in control (Fig. 3, Table 1). Neither the distribution nor the mean LA fiber areas differed among control and dystrophic aged mice. Compared to 4-monthold groups, however, the mean LA fiber areas of control and mdx aged mice were increased by 32 and 14%, respectively (Fig. 3, Table 1). The DIA fiber area distribution of young adult mdx mice was also shifted rightward compared to that of control, with a mean value 15% greater in dystrophic than in control group (Fig. 3, Table 1). After aging, the frequency distribution of mdx DIA fiber areas was shifted leftward with a mean value 31% smaller than in control (Fig. 3, Table 1). Compared to corresponding young adult groups,
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Fig. 2. Transverse sections of the levator ani muscle stained with H and E from young adult (a, d), aged (b, e) and gonadectomized (c, f) control (left) and mdx (right) mice. A cluster of small centronucleated cells (arrow) amidst normal muscle fibers, infiltration of inflammatory cells and connective tissue (arrows) can be observed in LA muscle sections from young adult mdx mouse (d). Clusters of eosinophilic muscle fibers and interstitial mononuclear cells (arrow) are shown in LA muscle section from control-aged mouse (b). Degenerating muscle fibers and infiltration of mononuclear inflammatory cells (arrow) in LA muscle from aged mdx mouse (e). LA muscle sections from gonadectomized young adult mouse from control (c) and mdx (f) strains. Notice the presence of centronucleated cells in (c) (arrow), intense cell disorganization and infiltration of phagocytic cells in (f) (arrow). Scale barZ50 mm.
the mean DIA fiber size was increased by 39% in control, and reduced by 17% in the dystrophic strain (Table 1). 3.3. Contraction properties The TwT of control LA muscles recorded at 1 month (324.0G92.0 g/g muscle weight) did not significantly change with age. The TT of the same muscles increased by two-fold from 1 (1018G270 g/g muscle weight) to 3 months of age, decreasing thereafter by 25% (Fig. 4a and b). The TPT (43.3G2.2 ms) and HRT (29.5G2.1 ms) recorded at 1 month were slightly shortened in adult mice, recovering the initial values after aging. In mdx mice, the TwT and TT of LA muscles did not differ up to 4 months compared to age-matched control.
A small increase (27%) of both parameters was observed at 12 months, while significant decrease of TT (29%) was detected only in aged dystrophic mice (Fig. 4a and b). The TPT and HRT did not change among age-matched control and mdx mice. The TwT of control DIA muscles recorded at 1 month of age (405G79 g/g muscle weight) was reduced at 2 months (38%), stabilizing thereafter. The TT (1553G159 g/g muscle weight) did not change up to 20 months (Fig. 4c and d). The TPT (32.4G1.7 ms) recorded at 1 month of age was slightly shortened (12–20%) from 2 to 20 months, while the HRT (15.47G2.38 ms) was not changed with age. In dystrophic mice, the TwT developed by the DIA muscle was reduced by 57% after aging. The TT was reduced with age from 3 months and up by 20–64% of
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Fig. 3. Fiber area distributions in the levator ani (left) and diaphragm (right) muscles of 4-month-old intact (top) and gonadectomized (GDX, bottom), and 20month-old intact mice (middle) from control (white bars) and mdx (gray bars) strains. Both muscles presented increased proportion of larger fibers in intact young adult mdx mice that persisted after aging in the levator ani. A greater proportion of small size fibers, probably regenerated fibers, predominated in diaphragm muscles of aged mdx mice. Significant leftward shift of fiber area distribution was observed in levator ani of gonadectomized mdx mice. The data represent the number of fibers measured (540–570 fibers) in three muscles for each group.
control values (Fig. 4c and d). The TPT did not differ among age-matched control and mdx groups, while the HRT was prolonged (22%) in aged mdx mice. 3.4. Effect of Gonadectomy Gonadectomy of young adult control and mdx mice decreased the LA muscle weight by 40 and 56% of age-matched sham groups, respectively (Table 2).
The frequency distribution of LA fiber areas from gonadectomized (GDX) mdx mice was shifted toward lower values relatively to that of control GDX group. Compared to gonadally intact groups, the mean LA fiber areas of control and mdx mice were reduced by 28 and 50%, respectively (Fig. 3c, Table 1). Despite muscle atrophy, the TwT developed by the LA from control GDX mice was increased by 32%, while the TT was reduced by 34% leading to increase of
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Table 1 Fiber areas of control and dystrophic levator ani and diaphragm muscles from 4-month-old intact (adult) or gonadectomized (GDX), and 20-month-old (aged) intact mice Group
Adult Aged GDX
Levator ani fiber area (mm2)
Diaphragm fiber area (mm2)
Control
Dystrophic
Control
Dystrophic
684.0G15.9 (561) 901.0G20.2†(556) 490.5G12.1†(575)
844.6G17.0*(564) 966.1G20.3†(572) 426.2G11.1*†(560)
817.2G26.1(550) 1132.0G40.4†(540) 730.9G26.9(550)
935.7G28.7*(555) 781.3G22.2*†(547) 685.8G23.5†(547)
Data are meansGSEM of the number of fibers in parenthesis in three muscles for each group. *Different from age-matched control group (P!0.05).†Different from adult group of the same animal strain (P!0.05).
the twitch/tetanus ratio (61%). The TPT was prolonged by 24%, whereas the HRT remained unchanged (Table 2). In dystrophic GDX mice TwT of the LA muscle did not significantly change, but TT was 31% lower than in sham group leading the twitch/tetanus ratio to increase by 53%. The contraction time in its turn, was not altered (Table 2). Gonadectomy of 3-month-old control and mdx mice did not affect the body and DIA muscle weights or the contraction properties. The frequency distribution of DIA fiber areas did not differ among control and GDX mice. The mean DIA fiber area was not changed in the control strain, but it was reduced in mdx mice by 27% compared to corresponding gonadally intact groups (Fig. 3f, Table 1). 3.5. Caffeine contracture Exposure of control LA muscles to caffeine (3–100 mM) produced maximal contractures greater in prepubertal
(60.4G5.8% TT) than in young adult (23.3G2.2% TT) and aged (34.3G2.3% TT) mice (Fig. 5a). The corresponding EC50 values were 24.4 mM (CL: 10.7–55.5 mM), 48.2 mM (41.8–56.2 mM), and 38.5 mM (25.4–58.2 mM), respectively, not different among all three groups. The responses to caffeine of mdx LA muscles did not differ from those obtained in age-matched control (Fig. 5c). The maximal response of DIA muscles to caffeine did not differ among prepubertal, young adult and aged (35.0G 3.4% TT) control mice. The EC50 values in these groups ranged between 32 and 35 mM (Fig. 5b). In dystrophic mice, the maximum response to caffeine of the DIA was increased in young adult (59.3G9.3% TT) and aged (63.8G 8.7% TT) mice (Fig. 5d), without significant changes in EC50 values compared to age-matched control. After gonadectomy of control mice, the maximal LA muscle response to caffeine was increased (39.5G2.2% TT) with no change of EC50 (24.8 mM, CL: 21.0–29.2 mM).
Fig. 4. Twitch (a, c) and tetanus (b, d) tensions developed per gram of muscle wet weight, by the levator ani (left) and diaphragm (right) muscles from control (hollow symbols) and mdx (filled symbols) mice at various ages. The symbols and vertical bars are meansGSEM of 7–11 animals in each group. *Different from age-matched control group (P!0.05).
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Table 2 Isometric contractile properties of the levator ani (LA) muscle from 4-month-old control and dystrophic (mdx) mice, sham-operated or gonadectomized (GDX) Control
Muscle weight (mg) TwT(g/g wet weight) TT (g/g wet weight) Twitch/Tetanus TPT (ms) HRT (ms)
Dystrophic
Sham (10)
GDX (6)
Sham (11)
GDX (6)
23.6G1.5 258G15 1720G141 0.161G0.010 33.0G1.2 25.1G1.6
14.1G1.5* 341G80** 1139G121* 0.259G0.036* 40.9G2.3** 24.3G1.6
20.4G1.1 289G22 1620G120 0.172G0.013 36.2G1.1 26.0G1.2
9.0G1.1* 291G22 1110G93* 0.264G0.016* 37.9G0.9 29.4G0.8
Data are meansGSEM of the number of determinations in parenthesis. TwT, twitch tension; TT, tetanus tension; TPT, time to peak tension; HRT, half relaxation time. Statistic differences between age-matched sham-operated and GDX group (*P!0.05; **P!0.001).
Gonadectomy of mdx mice, however, did not affect the caffeine-induced contractures in either the LA or DIA muscles.
4. Discussion This study presents evidences of the influence of androgens on the mild dystrophic damage of the hormonedependent LA muscle of mdx mice. Muscle degeneration in mdx mice neither affected the body weight gain nor influenced the normal pubertal surge of plasma androgens [17], indicated by the weight gain of LA muscle and seminal vesicle at 1–2 months of age.
The decreased gonadal weight observed in adult mdx mice has so far not been reported, and may be related to the lack of dystrophin and shorter isoforms that are expressed in nonmuscle tissues, including the testes [30]. Although, the functional role of these proteins in non-muscle tissues is still unknown, their absence in the testes did not affect male fertility in our dystrophic colony, which is in agreement with normal testosterone serum levels reported in adult mdx mice [31]. The mdx mouse is known to adapt to muscle degeneration with muscle fiber hypertrophy and activation of satellite cells [13,32]. Hypertrophy of dystrophic LA was also observed at 2 months of age, when maximal degeneration is reported to occur in most mdx skeletal
Fig. 5. Amplitudes of the caffeine contractures expressed as percent of tetanic tension of diaphragm strips (a, b) and levator ani (c, d) muscles from control (hollow symbols) and mdx (filled symbols) mice at various ages. The symbols and vertical bars are meansGSEM of 7–11 animals in each group.
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muscles [16]. The response was accompanied by degenerative histological changes that were intensified with age in both the LA and DIA, as described for other mdx skeletal muscles [13,15]. The deterioration of the LA muscle fibers, however, was always milder than that observed in the DIA of the same animal. Skeletal muscles with small fiber diameter like the extraocular and denervated muscles were reported as more resistant to necrosis in the mdx mouse [33]. The determined mean LA muscle fiber area was slightly smaller (16%) than that of the DIA, indicating that the moderate muscle degeneration is apparently unrelated to the fiber size. Furthermore, gonadectomy of mdx mice caused atrophy of the LA to 44% of its original size and intensified deterioration of the muscle fibers, suggesting a protective action of testosterone against degeneration of the LA muscle fibers. The activation and proliferation of satellites cells induced by testosterone were reported in the rat LA [34], which is likely to be involved in the regenerative LA muscle responses of dystrophic mice. According to the data, such hormonal influence appears to be exerted at a lesser extent on other skeletal muscles as well, as suggested by the decrease in DIA fiber areas of gonadectomized mdx mice. Significant decrease of LA muscle strength was detected in aged mdx mice, which was coincident with the low plasma androgen levels associated with aging [35], indicated by the decrease in seminal vesicle and gonadal weights in both animal strains. In contrast, the DIA muscle, the most responsive to dystrophic injury, developed a decrease in tetanic force in young adult mdx mice. These data alone suggested a possible influence of androgens on the progress of muscle wasting in the LA. Gonadectomy of young adult mice, however, reduced the LA muscle strength in control and more intensely, in dystrophic mice indicating that the effects are mainly related to androgen deprivation. Thus, despite the evidence of a hormonal beneficial influence, testosterone does not appear to interrupt the progress of muscle disease in dystrophic LA. In agreement with our data, treatment with the anabolic steroid oxandrolone did not cause significant changes in the muscle strength of boys with DMD despite an increase in their linear growth [36]. Muscle necrosis in both DMD and in the mdx mouse has been related to accumulation of intracellular free calcium concentration ([Ca2C]i) caused by sarcolemmal lesion [6,7] and abnormal calcium channel activity [37,38]. Dysfunctions of the sarcoplasmic reticulum (SR) Ca2C handling mechanisms have also been implied in the abnormal Ca2C homeostasis in dystrophic muscles [39,40]. To evaluate the SR function in dystrophic muscles, caffeine-induced contractures were recorded in those groups presenting changes in the contraction properties. Caffeine is known to produce muscle contractures by inducing Ca2C release and inhibition of Ca2C uptake by the SR [41]. The age-related increase in the amplitudes of caffeine contractures observed in mdx DIA muscle was consistent with the reported alteration of SR Ca 2C handling
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mechanisms in dystrophic muscles [40,42,43]. Such an effect, however, was not observed in LA muscles from adult and older mdx mice, indicating that the SR function remained apparently unaffected. The greater caffeine responses observed in LA muscles from prepubertal mice are characteristic of immature skeletal muscles and probably related to poor muscle fiber development and low capacity of the SR for Ca2C storage [44]. Androgen deprivation of LA muscles prolonged the active state duration in both animal strains, while it increased caffeine responses only in the control group, suggesting changes in the SR Ca2C release and uptake. Alternatively, the results may be secondary to muscle atrophy induced by gonadectomy and unrelated to changes in the SR Ca2C handling mechanisms. Ultrastructural studies of androgen-deprived LA muscle fibers revealed intense loss of contractile elements with a relatively preserved sarcoplasmic reticulum in both the rat [45] and mouse [21], which may explain the increased caffeine contractures and twitch tension expressed per muscle weight in control gonadectomized mice. To summarize, the data presented showed that muscle wasting in the testosterone-dependent LA muscle was milder than that observed in the DIA of mdx mice. Hormone deprivation in mdx mice did not affect muscle degeneration in the DIA, but it intensified deterioration of the LA fibers, decreased the muscle strength and modified the contraction kinetics. In conclusion, testosterone may not prevent the progress of muscle disease in the mdx LA, but androgen deprivation accelerates muscle wasting suggesting a hormonal beneficial effect.
Acknowledgements The authors thank Dr V.B. Valero for the care of the dystrophic and control mice colonies, Mrs H.S. Reis for technical assistance in the histological studies, and A.C.C. Vallin for her help in some experiments. Thanks are also due to Drs E. Haapalainen and F.P. Faria, Department of Electron Microscopy, for the use of the imaging processing system, and Dr A.N. Gordan, Department of Pathology, UNIFESP, for the histopathological analysis. This work was supported by grants from Fundac¸a˜o de Amparo a` Pesquisa do Estado de Sa˜o Paulo (FAPESP) and Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´gico (CNPq).
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