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The beneficial effect of myostatin deficiency on maximal muscle force and power is attenuated with age
Experimental Gerontology 48 (2013) 183–190
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Experimental Gerontology journal homepage: www.elsevier.com/locate/expgero
The beneficial effect of myostatin deficiency on maximal muscle force and power is attenuated with age E. Schirwis a, O. Agbulut b, Nathalie Vadrot b, E. Mouisel a, C. Hourdé a, A. Bonnieu c, G. Butler-Browne a, H. Amthor a, d, A. Ferry a, d,⁎ a
Université Pierre et Marie Curie-Paris6, Sorbonne Universités, UMR S794, INSERM, U974, CNRS UMR7215, Institut de Myologie, Paris F-75013, France Université Paris Diderot, Sorbonne Paris Cité, CNRS EAC4413, Unit of Functional and Adaptive Biology, Laboratory of Stress and Pathologies of the Cytoskeleton, Paris F-75013, France INRA, UMR 866 Dynamique Musculaire et Métabolisme, Université Montpellier 1, F 34060 Montpellier, France d Université Paris Descartes, Sorbonne Paris Cité, Paris F-75006, France b c
a r t i c l e
i n f o
Article history: Received 21 June 2012 Received in revised form 9 November 2012 Accepted 20 November 2012 Available online 29 November 2012 Section Editor: Christiaan Leeuwenburgh Keywords: Aging Sex Skeletal muscle Myostatin deficiency Maximal force Maximal power
a b s t r a c t The prolonged effect of myostatin deficiency on muscle performance in knockout mice has as yet been only poorly investigated. We have demonstrated that absolute maximal force is increased in 6-month old female and male knockout mice and 2-year old female knockout mice as compared to age- and sex-matched wildtype mice. Similarly, absolute maximal power is increased by myostatin deficiency in 6-month old female and male knockout mice but not in 2-year old female knockout mice. The increases we observed were greater in 6-month old female than in male knockout mice and can primarily result from muscle hypertrophy. In contrast, fatigue resistance was decreased in 6-month old knockout mice of both sexes as compared to age- and sex-matched wildtype mice. Moreover, in contrast to 2-year old female wildtype mice, aging in 2-year old knockout mice reduced absolute maximal force and power of both sexes as compared to their younger counterparts, although muscle weight did not change. These age-related decreases were lower in 2-year old female than in 2-year old male knockout mice. Together these results suggest that the beneficial effect of myostatin deficiency on absolute maximal force and power is greater in young (versus old) mice and female (versus male) mice. Most of these effects of myostatin deficiency are related neither to changes in the concentration of myofibrillar proteins nor to the slow to fast fiber type transition. © 2012 Elsevier Inc. All rights reserved.
1. Introduction Myostatin, a transforming growth factor family member, is a negative regulator of skeletal muscle growth during both development and in the adult. In the two strains of myostatin knockout mice (KO) skeletal muscle undergoes hypertrophy (Grobet et al., 2003; McPherron et al., 1997), for example the extensor digitorum longus (EDL) muscle increases its weight by 66–121% in male KO mice (Amthor et al., 2007; Mendias et al., 2006; Ploquin et al., 2012). The muscle hypertrophy observed in KO mice results more from hyperplasia than muscle fiber hypertrophy (Baligand et al., 2010; Gay et al., 2012; Gentry et al., 2011; Gilson et al., 2007; McPherron et al., 1997; Mendias et al., 2006; Siriett et al., 2006). Since absolute maximal force is roughly proportional to muscle cross-sectional area, it should be expected that absolute maximal force is greater in KO mice as compared to control mice. Indeed this was observed in the study of Mendias et al. (2006) since they found that absolute maximal force of EDL muscle was increased by 34% in male KO mice. Similarly, ⁎ Corresponding author at: G.H. Pitié-Salpétrière, 47, bld de l'Hôpital, Bâtiment Babinski, INSERM U974, 75651 Paris cedex 13, France. E-mail address: [email protected] (A. Ferry). 0531-5565/$ – see front matter © 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.exger.2012.11.008
two studies also reported an increased absolute maximal force in male KO mice (Baligand et al., 2010; Wagner et al., 2002). However four other studies found that absolute maximal force was not increased in KO mice (Amthor et al., 2007; Gentry et al., 2011; Matsakas et al., 2012; Ploquin et al., 2012), one of them even showed that the absolute maximal force was decreased by approximately 50% (Matsakas et al., 2012). These latter studies would suggest that the reduction in specific maximal force (absolute maximal force relative to CSA or muscle weight) theoretically is strongly decreased by 66% in KO mice, which seems surprising in view of what is observed in murine models of neuromuscular diseases (Durieux et al., 2010; Fugier et al., 2011; Lynch et al., 2001; Risson et al., 2009; Trollet et al., 2010). For example, in the mdx mouse a murine model of Duchenne muscular dystrophy in which the limb muscles are hypertrophied by 28%, specific maximal force is only decreased by 13% (Lynch et al., 2001). Fatigue resistance is another important muscle performance parameter since on many occasions the muscle will have to contract repeatedly. This value is lower in male KO mice (Hennebry et al., 2009; Ploquin et al., 2012). It has also been reported that the force drop following lengthening contractions is also greater in male KO mice (Mendias et al., 2006), indicating that contraction-induced muscle injury (fragility) is increased. To our knowledge no studies have measured
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absolute maximal power, a key determinant of muscle performance during movement, in KO mice. Since absolute maximal power increases with muscle size and the percentage of fast-type powerful muscle fibers, it seems likely that absolute maximal power would also increase in KO mice. In order to better understand the role of myostatin in muscle performance (absolute maximal force, absolute maximal power, fatigue resistance), we examined both young (6 month old) and old (20–22 month old) adult KO mice of both sexes. To our knowledge, the muscle performance of aged KO mice has not yet been studied. With aging, there is a decrease in both muscle mass and performance whereas muscle fragility is increased at least in control mice (Chan and Head, 2010; Lynch et al., 2001). In this study, mice of both sexes were studied because sexrelated factors such as ovarian and testicular hormones are known to modulate muscle performance and size and possibly declined differentially with aging (Chambon et al., 2010; Greising et al., 2011; Hourde et al., 2009). Our results reveal that myostatin deficiency has a beneficial effect on some aspects of muscle performance that persists for a prolonged period of life, although it eventually decreases with aging. 2. Materials and methods 2.1. Animals All procedures were performed in accordance with national and European legislations. Female and male myostatin KO mice (KO) were used at 6–8 (6-month old) and 20–22 (2-year old month of age) (n= 8, n = 8, n = 20, n = 12, respectively). KO mice founder breeding pairs on a C57BL/6 background were a kind gift from Se-Jin Lee (McPherron et al., 1997). We also used 6-month and 2-year old female C57BL/6 mice (n= 22, n = 10) and 6-month old male C57BL/6 mice (n= 14) as controls (WT). It should be noted that we studied mice older than 6 months of age to avoid the confounding factor of postnatal growth, since during the first months there is a rapid change in myonuclear domain size (White et al., 2010) and specific maximal force (Gokhin et al., 2008; Goodman et al., 2008), two important parameters in the KO mice (see Discussion section). The age of 20–22 months of age was selected because it has been reported that the regular estrous cycle ceases prior to 20 months of age in female mice (Felicio et al., 1984; Greising et al., 2011). Consequently, the 2 year-old female mice were most probably in a phase of post-ovarian failure.
the first 200 ms. Then, the muscle shortened during the last 300 ms against the load. Each contraction was separated by a 1 min rest period. The (peak) shortening velocity was measured during the first 20 ms of the shortening period. Absolute power was calculated from the force– velocity data and absolute maximal power was reported (mW). Specific maximal power (mW/g) was calculated by dividing maximal power by muscle weight. Fragility or fatigue resistance (TA muscle) was then measured. Fragility was estimated from the force drop resulting from lengthening contraction-induced injury. Nine lengthening contractions of the muscle were performed, each separated by 1 min. This protocol was able to induce force drop in the mdx mice (Hoogaars et al., 2012; Trollet et al., 2010), a murine model for muscular dystrophy. The sciatic nerve was stimulated for 700 ms (125 Hz). A maximal isometric contraction of the TA muscle was initiated during the first 500 ms. Then, muscle lengthening (10% L0) at a velocity of 5.5 mm/s was imposed during the last 200 ms. Maximal isometric force was measured 1 min after each lengthening contraction and expressed as a percentage of the initial maximal isometric force. The fatigue protocol consists of 40 repeated isometric contractions (100 Hz for 500 ms, evoked every 2 s). Absolute maximal force and power were determined following the fatigue protocol, in the first 6 min of recovery. After contractile measurements, the animals were killed with an overdose of pentobarbital and muscles were weighed and flash frozen in liquid nitrogen. 2.3. Gel electrophoresis The muscles were extracted on ice for 60 min in 4 volumes of extracting buffer (pH 6.5), as previously described (Butler-Browne and Whalen, 1984). After centrifugation, the supernatants were diluted 1:1 (vol/vol) with glycerol and stored at − 20 °C. MHC isoforms were separated on 8% polyacrylamide gels, which were made in the Bio-Rad mini-Protean II Dual slab cell system, as described previously (Agbulut et al., 2003). The gels were migrated for 31 h at 72 V (constant voltage) at 4 °C. For the quantification of the relative concentration of MHC and actin, 1 μg of total proteins was loaded and 12% polyacrylamide gels were used. The silver stained gels were scanned using a video acquisition system and bands were quantified by densitometric software (Multi Gauge, Fujifilm). 2.4. Statistical analysis
2.2. Muscle performance and fragility Force-generating capacity and fragility were evaluated by measuring the in situ tibialis anterior (TA) muscle contraction in response to nerve stimulation, as described previously (Koo et al., 2011; Mouisel et al., 2006). Mice were anesthetized using pentobarbital (60 mg/kg intraperitoneally). Body temperature was maintained at 37 °C using radiant heat. The knee and foot were fixed with pins and clamps and the distal tendon of the muscle was attached to a lever arm of a servomotor system (305B, Dual-Mode Lever, Aurora Scientific) using a silk ligature. The sciatic nerve was proximally crushed and distally stimulated by a bipolar silver electrode using supramaximal square wave pulses of 0.1 ms duration. We measured the absolute maximal force that was generated during isometric contractions in response to electrical stimulation (frequency of 75–150 Hz, train of stimulation of 500 ms). Absolute maximal force was determined at L0 (length at which maximal tension was obtained during the tetanus). Absolute maximal force was normalized to the muscle mass as an estimate of specific maximal force, i.e. force-generating capacity. Force–velocity data (TA muscle) were then obtained by eliciting contractions along the sciatic nerve (500 ms, 125 Hz) at 6 different afterloads (over the range of approximately 10–50% absolute maximal force). The sciatic nerve was stimulated for 700 ms (125 Hz). A maximal isometric contraction of the muscle was initiated during
Groups were compared using 2 way-analysis (gender × age, gender× genotype, genotype× age) of variance. If necessary (significant interaction), contrast (Fisher PLSD) analysis was also performed. In some rare cases, only 1 way-analysis of variance analysis could be carried out because of the absence of a 2-year old male WT group, followed if necessary by Tukey's post hoc test. We also analyzed the percentage of variation relative to control (WT, 6 month-old…). For groups that did not pass tests of normality and equal variance, non-parametric tests were used (Kruskal Wallis and Wilcoxon). Values are means ±SEM. A p b 0.05 is considered significant. 3. Results 3.1. Effect of myostatin deficiency The muscle weights of 6-month old female KO mice, 2-year old female KO mice and 6-month old male KO mice were heavier by 79, 61 and 53% as compared to sex- and age-matched WT mice (Fig. 1A,B, p b 0.05). The increase in muscle weight did not differ significantly with sex or age (p > 0.05). The absolute maximal force was increased in 6-month old female KO mice, 2-year old female KO mice and 6-month old male KO mice, as compared to sex- and age-matched WT mice (p b 0.05, Fig. 2A). The
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increase in absolute maximal force was greater in 6-month old female KO mice (+77%) than in 2-year old female KO mice (+ 40%) and 6-month old male KO mice (+32%) (p b 0.05). Specific maximal force was decreased in 2-year old female KO mice (− 10%) and 6-month old male KO mice (− 13%), but not in 6-month old female KO mice, as compared to sex- and age-matched WT mice (p b 0.05, Fig. 2B). The reduced specific maximal force was not related to a decrease in concentrations of either MHC or actin (Fig. 3A,B) or a change in the ratio of MHC to actin (Fig. 3C). Moreover, the reduced specific maximal force was also not associated with a decrease in the percentage of high specific force producing fibers, i.e. fiber expressing MHC-2b, since the expression of MHC-2b was increased in 2-year old female KO mice and 6-month old male KO mice (Fig. 4A,B). 6-month old female KO mice and 6-month old male KO mice but not 2-year old female KO mice had a greater absolute maximal power as compared to sex- and age-matched WT mice (pb 0.05, Fig. 5A), with a greater increase in absolute maximal power in 6-month old female KO mice (+101%) than in 6-month old male KO mice (+33%) (p b 0.05). Specific maximal power was reduced in 2-year old female KO mice
Fig. 2. Absolute maximal force (A) and specific maximal force (B) in TA muscle from KO mice. a: KO different from corresponding WT (pb 0.05). b: 2 year-old (2y) different from 6 month-old (6 m) (pb 0.05), (b): 2 year-old (2y) tended to be different from 6 month-old (6 m) (p=0.08). c: Female different from corresponding male (pb 0.05). n=8–22/group for female and n=8–14/group for male.
Fig. 1. TA muscle weight (A) and TA muscle weight normalized to body weight (B) in KO mice. a: KO different from corresponding WT (p b 0.05). b: 2 year-old (2y) different from 6 month-old (6 m) (p b 0.05). c: Female different from corresponding male (p b 0.05). n = 8–22/group for female and n = 8–14/group for male.
(−22%) and 6-month old male KO mice (−13%), but not in 6-month old female KO mice, as compared to sex- and age-matched WT mice (pb 0.05, Fig. 5B). 6-month old female and male KO mice had a lower fatigue resistance as compared to 6-month old sex-matched WT mice (p b 0.05, Fig. 6), with no significant difference between sex concerning this effect (p > 0.05). Indeed myostatin deficiency decreased the fatigue resistance by 35 and 40% in 6-month old female and male KO mice respectively, as assessed by absolute maximal force loss (p b 0.05, Fig. 6A). Moreover, it reduced to a greater extent absolute maximal power following the fatigue protocol (−69% and − 80%, p b 0.05, for 6-month old female and male KO muscles respectively, Fig. 6B). The reduced fatigue resistance was related to a greater relative number of low fatigue resistant fiber since the percentage of fibers expressing MHC-2b increased in 6-month old female and male KO mice (Fig. 4A, B). Finally, we found that fragility was not affected by myostatin deficiency in 2-year old female and male KO mice. There was no significant drop in force following the lengthening contractions in 2-year old female and male KO mice, similarly to 2-year old female WT mice (p > 0.05, Fig. 7).
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in 2-year old female KO mice (−17%) than in 2-year old male KO mice (−30%) (pb 0.05, Fig. 2A). Similarly with advancing age, specific maximal force was reduced (pb 0.05), but to a smaller extent in 2-year old female KO mice (−10%) than in 2-year old male KO mice (−30%) (pb 0.05, Fig. 2B). The decline in specific maximal force was not related to a change in myofibrillar proteins (Fig. 3A–C). It should be also noted that the percentage of high specific force producing fibers decreased in 2-year old female KO mice whereas it increased in 2-year old male KO mice (Fig. 4A,B). Concerning absolute maximal power, it was equally decreased in 2-year old female KO mice (−38%) and 2-year old male KO mice (−43%) as compared to 6-month old sex-matched KO mice (pb 0.05, Fig. 5A). Moreover, aging reduced specific maximal power in both 2-year old female and male KO mice (pb 0.05), with a smaller reduction in 2-year old female KO mice (−22%) than in 2-year old male KO mice (−44%) (pb 0.05, Fig. 5B). We also found that fatigue resistance was not globally altered by aging in 2-year old female and male KO mice (p> 0.05, Fig. 6A,B). 4. Discussion 4.1. Increased absolute maximal force and power
Fig. 3. MHC concentration (A), actin concentration (B), and ratio of MHC to actin (C) in TA muscle from KO mice. a: KO different from corresponding WT (p b 0.05). b: 2 year-old (2y) different from 6 month-old (6 m) (p b 0.05). c: Female different from corresponding male (p b 0.05). n = 4/group.
3.2. Effect of aging Similarly to 2 year-old female WT mice, no sarcopenia occurred in 2-year old female and male KO mice (Fig. 1A,B). However, in contrast to female 2 year-old WT mice, we found an effect of aging on muscle performance in KO mice of both sexes. Absolute maximal force decreased in 2 year-old female and male KO mice as compared to 6-month old sex-matched KO mice (pb 0.05), with a smaller reduction
In the present study we confirm that myostatin deficiency induced increased muscle weight (Amthor et al., 2007; Gentry et al., 2011; Mendias et al., 2006; Wagner et al., 2005; Wang and McPherron, 2012). The muscle hypertrophy (53–79%) can explain the increased absolute maximal force (32–77%) in both 6-month old female and male KO mice and 2-year old female KO mice, despite the modestly reduced specific maximal force (see below). Our results also confirm and extend those of Mendias et al. (2006) showing that absolute maximal force was increased by 34% in the EDL muscle from male KO mice (+32% in 6-month old male KO mice). The increased absolute maximal force is apparently due to fiber hyperplasia because the absolute maximal force of skinned muscle fibers did not differ between KO mice and WT mice (Mendias et al., 2011; Qaisar et al., 2012). It should be noted that postnatal myostatin deficiency or blockade also increases absolute maximal force in WT mice (Akpan et al., 2009; Goncalves et al., 2010; Murphy et al., 2010; Personius et al., 2010; Whittemore et al., 2003). The reasons why absolute maximal force is not increased in KO mice from other studies (Amthor et al., 2007; Gentry et al., 2011; Matsakas et al., 2012; Ploquin et al., 2012) are not related to 1) the muscle studied (Mendias et al., 2006; Gentry et al., 2011), 2) the type of muscle force measurement (in situ versus in vitro; the present study; Amthor et al., 2007; Gentry et al., 2011; Mendias et al., 2006), 3) sex and age (the present study; Gentry et al., 2011). The origin of this discrepancy concerns the effect of myostatin deficiency on specific maximal force (see below) since muscle hypertrophy is similar between studies. Increased muscle weight can also explain the increased maximal power (33–101%) in 6-month old female and male KO mice. Since maximal power is increased, the effect of hyperplasia exceeds the effect of the reduced specific maximal power of skinned muscle fibers from KO mice (Mendias et al., 2011). It should be noted that the gain in maximal power was not markedly greater than that of absolute maximal force suggesting that the fiber type transition from slow/low to fast/powerful-type induced by myostatin deficiency, as shown in the present and previous studies (Amthor et al., 2007; Hennebry et al., 2009), had little impact on this parameter. 4.2. Reduced specific maximal force and power We also found that specific maximal force and specific maximal power were moderately reduced in 2-year old female KO mice and 6-month old male KO but not in 6-month old female KO mice, indicating
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Fig. 4. Representative images of electrophoretic separation of MHC proteins (A) and MHC-2b distribution (B) in TA muscle from KO mice. a: KO different from corresponding WT (p b 0.05). b: 2 year-old (2y) different from 6 month-old (6 m) (p b 0.05). c: Female different from corresponding male (p b 0.05). n= 4–5/group.
that the maximal force and power generation capacities of the muscle are somewhat reduced by myostatin deficiency. It was recently reported that specific maximal force and specific maximal power of skinned muscle fibers were reduced in KO mice, indicating that myofibrillar dysfunction is responsible for this defect at the level of the intact muscle (Mendias et al., 2011; Qaisar et al., 2012). Two mechanisms have been recently discussed to explain this reduction in the intrinsic qualities of the muscles. Firstly, a deficient ubiquitin proteasome system in KO mice could lead to an accumulation of damaged non functional contractile proteins (Mendias et al., 2011). Secondly, an increased volume of cytoplasm per nucleus (myonuclear domain) in the KO mice could exceed the capacity of the nucleus to maintain myofibrillar protein content (Qaisar et al., 2012). However, our results concerning the myofibrillar protein concentrations did not support the latter hypothesis. In addition, we exclude the possibility that a smaller percentage of high specific force producing fibers, i.e. fibers expressing MHC-2b (D'Antona et al., 2007), was responsible for the reduced specific maximal force. It should be noted that the reductions in specific maximal force and specific maximal power (−13 to −18%, the present study; Mendias et al., 2006) in young male KO mice are apparently consistent with those observed in skinned muscle fibers (−21%, (Mendias et al., 2011; Qaisar et al., 2012)), but in opposition to those observed in other studies showing a 40–75% reduction in muscle specific maximal force from young male KO mice (Amthor et al., 2007; Gentry et al., 2011; Matsakas et al., 2012; Ploquin et al., 2012). The discrepancy could result from differences in myonuclear domain and/or the ubiquitin
proteasome system between the different KO models (McPherron et al., 1997 versus Grobet et al., 2003), or to the different genetic backgrounds of the strains (C57BL6 versus FVB/N-C57BL6) or colonies separated for many years. It should be noted that the decline in muscle quality observed in these studies (Amthor et al., 2007; Gentry et al., 2011; Matsakas et al., 2012; Ploquin et al., 2012) is to our knowledge rarely observed in the murine models for neuromuscular disorders (Durieux et al., 2010; Fugier et al., 2011; Lynch et al., 2001; Risson et al., 2009; Trollet et al., 2010). 4.3. Reduced fatigue resistance Myostatin deficiency is also very detrimental to fatigue resistance in 6-month old KO mice of both sexes. After 40 contractions, absolute maximal force (− 35 to 40%) and more importantly absolute maximal power (− 69 to − 80%) were reduced to a greater extent in 6-month old female and male KO mice than in 6 month-old sex-matched WT mice. This result confirms and extends previous studies (Hennebry et al., 2009; Ploquin et al., 2012) showing that young male KO mice have a lower fatigue resistance. Since it had been shown that aging did not reduce fatigue resistance in WT mice of both sexes (Chan and Head, 2010), it is likely that fatigue resistance is still reduced in 2 year-old KO mice as compared to 2-year old WT mice. The dramatic effect of myostatin deficiency on power during repeated contractions can explain the reduced exercise tolerance observed in the KO mice (Savage and McPherron, 2010). The reduction in the percentage of
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Fig. 5. Absolute maximal power (A) and specific maximal power (B) of TA muscle from KO mice. a: KO different from corresponding WT (pb 0.05). (a): KO tended to be different from corresponding WT (p = 0.07). b: 2 year-old (2y) different from 6 month-old (6 m) (p b 0.05). c: Female different from corresponding male (p b 0.05). n = 8–22/ group for female and n = 8–14/group for male KO mice.
fatigue resistant fibers, mitochondrial depletion, reduced oxidative metabolism and capillarity (Amthor et al., 2007; Baligand et al., 2010; Hennebry et al., 2009; Matsakas et al., 2012; Ploquin et al., 2012) can explain the greater muscle fatigue in KO mice. Another contributing factor is linked to muscle hypertrophy. The increase in muscle blood flow induced by exercise may be no longer sufficient, limiting oxygen blood supply and washout. These results illustrate the well-known negative relationship between muscle size and fatigue resistance/oxidative metabolism (Degens and Veerkamp, 1994; van Wessel et al., 2010).
Fig. 6. Fatigue resistance as a decreased in absolute maximal force (A) and absolute maximal power (B) after 40 contractions of TA muscle from KO mice. a: KO different from corresponding WT (p b 0.05). b: 2 year-old (2y) different from 6 month-old (6 m) (p b 0.05). c: Female different from corresponding male (pb 0.05). n = 8–22/ group for female and n = 8–14/group for male KO mice.
4.4. Changes with aging For the first time, we are able to report important age-related changes in the muscle performance of KO mice of both sexes. Absolute maximal force (−17 to −30%) and maximal power (−38 to −43%) were both reduced by aging in 2-year old female and male KO mice, whereas they did not change in fast muscle from 2-year old WT mice of both sexes (the present study; Hakim and Duan, 2012; Lynch et al., 2001; Moran et al., 2005). Interestingly, these results are not related to muscle atrophy but can be explained by a reduction in specific maximal force and power with aging in both 2-year old female and male KO
Fig. 7. Fragility, i.e., force drop following lengthening contractions of TA muscle from KO mice. a: KO different from corresponding WT (p b 0.05). b: 2 year-old (2y) different from 6 month-old (6 m) (p b 0.05). c: Female different from corresponding male (p b 0.05). n = 8–22/group for female and n= 8–14/group for male KO mice.
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mice, indicating the deterioration of muscle quality. Since specific maximal force and power do not markedly decline in fast muscle from old WT mice of both sexes until 28 months of age (the present study; Hakim and Duan, 2012; Lynch et al., 2001; Moran et al., 2005), our results suggest that the potential detrimental effect of myostatin on specific maximal force and power (see above) increases with aging in both sexes. It is possible that myostatin deficiency accelerates normal aging. Alternatively, the worsening of the reduced muscle quality with aging could reflect the cumulative detrimental effect of myostatin deficiency. With advancing age, there could be an aggravation of the detrimental effect of myostatin deficiency on the proteasome ubiquitin system and on myofibrillar gene transcription controlled by the nucleus (Mendias et al., 2011; Qaisar et al., 2012). However, we found no effect of aging on myofibrillar protein concentrations in 2-year old female and male KO mice. In contrast, there is apparently no change in fatigue resistance with aging in the 2-year old female and male KO mice, despite the changes in MHC-2b distribution. Of note, muscle fragility in the 2-year old KO mice of both sexes was also not affected by aging. An important point of consideration is that these age-related changes result in the fact that the increases in absolute maximal force and power induced by myostatin deficiency at 6 months of age were reduced 2 fold or abolished in 2 year-old female KO mice. Since aging did not alter absolute maximal force and power in male WT mice (Chan and Head, 2010; Hakim and Duan, 2012; Lynch et al., 2001), it can be expected that there is no increase in maximal force and no decrease in absolute maximal power in 2-year old male KO mice as compared to 2-year old male WT (see Fig. 2A and B: compare 2 year-old KO male mice to 6 month-old male WT mice). In short, our results indicate that the beneficial effect of myostatin deficiency on absolute maximal force and maximal power fades with age, at least in females and also most likely in males, prior to the onset of normal muscular senescence (sarcopenia and dynapenia).
4.5. Sex differences Interestingly, we found that the beneficial effects of myostatin deficiency on muscle performance (increased absolute maximal force and power) are greater in 6-month old female KO mice than in 6-month old male KO mice. This sex difference results from a greater muscle hypertrophy and a smaller reduction in specific maximal force and power in 6-month old female as compared to male KO mice. The differential effect of myostatin deficiency is such that it eliminates not only the sexual dimorphism concerning absolute maximal force and power but also concerning muscle size that we observed in 6-month WT mice. A possible explanation might be that myostatin expression is reduced to a greater extent in male as compared to female WT mice. Indeed, it has been demonstrated that processed myostatin protein expression is lower in male WT mice (McMahon et al., 2003). Moreover, it is plausible that myostatin interferes with androgen pathways that are known to modulate specific maximal force (Chambon et al., 2010). Another interesting finding is that the effect of aging was reduced in 2-year old female KO mice as compared to 2-year old male KO mice, although to a greater extent than that of 2-year old female and male WT mice (the present study; Chan and Head, 2010; Hakim and Duan, 2012; Lynch et al., 2001; Moran et al., 2005). Indeed, absolute maximal force (− 17% versus − 30%) and specific maximal force (− 10% versus − 30%) and power (− 22% versus − 44%) declined less with aging in 2-year old female KO mice than in 2-year old male KO mice, for as yet unknown reasons. The protective effect of the female related factor in the context of myostatin deficiency remains to be determined. It also remains to be determined whether the ubiquitin proteasome system is less affected by aging in 2-year old female KO mice compared with 2-year old male KO mice.
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5. Conclusion Our study demonstrates that myostatin deficiency increases absolute maximal force in both 6-month old female and male KO mice and 2-year old female KO mice. Similarly, absolute maximal power is increased by myostatin deficiency in both 6-month old female and male KO mice but not in 2-year old female KO mice. These increases likely result from muscle hypertrophy. They are greater in the 6-month old female KO mice than in the 6-month old male KO mice, likely due to greater muscle hypertrophy and no reductions in specific maximal force and power in female mice. In contrast, myostatin deficiency markedly reduced fatigue resistance in 6-month old KO mice of both sexes. With aging, in contrast to 2-year old WT mice (the present study; Chan and Head, 2010; Hakim and Duan, 2012; Lynch et al., 2001), absolute maximal force and power were reduced in 2-year old KO mice of both sexes, although muscle weight did not change. These age-related decreases are smaller in 2-year old female mice than in 2-year old male KO mice, possibly because there was a less significant decrease in specific maximal force and power in the female mice. Altogether, these results suggest that the beneficial effect of myostatin deficiency on absolute maximal force and power production is greater in female (versus male) and young (versus old) mice. Most of these effects of myostatin deficiency are related neither to changes in the concentration of myofibrillar proteins nor to the slow to fast fiber type transition. Acknowledgments We are grateful to W. Hadj-Said (UMR 974) and A. Chatonnet (UMR 866) for assistance during the experiments. Financial support has been provided by Université Pierre et Marie Curie (UPMC), CNRS, INSERM, University Paris Descartes, ANR-Genopath In-A-Fib, ANR-Blanc Androgluco, the Association Française contre les Myopathies (AFM), MyoAge (EC 7th FP, contract 223576), MyoGrad International Graduate School for Myology (DRK 1631/1), Agence Française de Lutte contre le Dopage, and Association Monegasque contre les Myopathies, Parents Project France. Appendix A. Supplementary data Supplementary data to this article can be found online at http:// dx.doi.org/10.1016/j.exger.2012.11.008. References Agbulut, O., Noirez, P., Beaumont, F., Butler-Browne, G., 2003. Myosin heavy chain isoforms in postnatal muscle development of mice. Biol. Cell 95, 399–406. Akpan, I., Goncalves, M.D., Dhir, R., Yin, X., Pistilli, E.E., Bogdanovich, S., Khurana, T.S., Ucran, J., Lachey, J., Ahima, R.S., 2009. The effects of a soluble activin type IIB receptor on obesity and insulin sensitivity. Int. J. Obes. (Lond) 33, 1265–1273. Amthor, H., Macharia, R., Navarrete, R., Schuelke, M., Brown, S.C., Otto, A., Voit, T., Muntoni, F., Vrbova, G., Partridge, T., Zammit, P., Bunger, L., Patel, K., 2007. Lack of myostatin results in excessive muscle growth but impaired force generation. Proc. Natl. Acad. Sci. U. S. A. 104, 1835–1840. Baligand, C., Gilson, H., Menard, J.C., Schakman, O., Wary, C., Thissen, J.P., Carlier, P.G., 2010. Functional assessment of skeletal muscle in intact mice lacking myostatin by concurrent NMR imaging and spectroscopy. Gene Ther. 17, 328–337. Butler-Browne, G.S., Whalen, R.G., 1984. Myosin isozyme transitions occurring during the postnatal development of the rat soleus muscle. Dev. Biol. 102, 324–334. Chambon, C., Duteil, D., Vignaud, A., Ferry, A., Messaddeq, N., Malivindi, R., Kato, S., Chambon, P., Metzger, D., 2010. Myocytic androgen receptor controls the strength but not the mass of limb muscles. Proc. Natl. Acad. Sci. U. S. A. 107, 14327–14332. Chan, S., Head, S.I., 2010. Age- and gender-related changes in contractile properties of non-atrophied EDL muscle. PLoS One 5, e12345. D'Antona, G., Brocca, L., Pansarasa, O., Rinaldi, C., Tupler, R., Bottinelli, R., 2007. Structural and functional alterations of muscle fibres in the novel mouse model of facioscapulohumeral muscular dystrophy. J. Physiol. 584, 997–1009. Degens, H., Veerkamp, J.H., 1994. Changes in oxidative capacity and fatigue resistance in skeletal muscle. Int. J. Biochem. 26, 871–878. Durieux, A.C., Vignaud, A., Prudhon, B., Viou, M.T., Beuvin, M., Vassilopoulos, S., Fraysse, B., Ferry, A., Laine, J., Romero, N.B., Guicheney, P., Bitoun, M., 2010. A centronuclear myopathy-dynamin 2 mutation impairs skeletal muscle structure and function in mice. Hum. Mol. Genet. 19, 4820–4836.
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The beneficial effect of myostatin deficiency on maximal muscle force and power is attenuated with age E. Schirwis a, O. Agbulut b, Nathalie Vadrot b, E. Mouisel a, C. Hourdé a, A. Bonnieu c, G. Butler-Browne a, H. Amthor a, d, A. Ferry a, d,⁎ a
Université Pierre et Marie Curie-Paris6, Sorbonne Universités, UMR S794, INSERM, U974, CNRS UMR7215, Institut de Myologie, Paris F-75013, France Université Paris Diderot, Sorbonne Paris Cité, CNRS EAC4413, Unit of Functional and Adaptive Biology, Laboratory of Stress and Pathologies of the Cytoskeleton, Paris F-75013, France INRA, UMR 866 Dynamique Musculaire et Métabolisme, Université Montpellier 1, F 34060 Montpellier, France d Université Paris Descartes, Sorbonne Paris Cité, Paris F-75006, France b c
a r t i c l e
i n f o
Article history: Received 21 June 2012 Received in revised form 9 November 2012 Accepted 20 November 2012 Available online 29 November 2012 Section Editor: Christiaan Leeuwenburgh Keywords: Aging Sex Skeletal muscle Myostatin deficiency Maximal force Maximal power
a b s t r a c t The prolonged effect of myostatin deficiency on muscle performance in knockout mice has as yet been only poorly investigated. We have demonstrated that absolute maximal force is increased in 6-month old female and male knockout mice and 2-year old female knockout mice as compared to age- and sex-matched wildtype mice. Similarly, absolute maximal power is increased by myostatin deficiency in 6-month old female and male knockout mice but not in 2-year old female knockout mice. The increases we observed were greater in 6-month old female than in male knockout mice and can primarily result from muscle hypertrophy. In contrast, fatigue resistance was decreased in 6-month old knockout mice of both sexes as compared to age- and sex-matched wildtype mice. Moreover, in contrast to 2-year old female wildtype mice, aging in 2-year old knockout mice reduced absolute maximal force and power of both sexes as compared to their younger counterparts, although muscle weight did not change. These age-related decreases were lower in 2-year old female than in 2-year old male knockout mice. Together these results suggest that the beneficial effect of myostatin deficiency on absolute maximal force and power is greater in young (versus old) mice and female (versus male) mice. Most of these effects of myostatin deficiency are related neither to changes in the concentration of myofibrillar proteins nor to the slow to fast fiber type transition. © 2012 Elsevier Inc. All rights reserved.
1. Introduction Myostatin, a transforming growth factor family member, is a negative regulator of skeletal muscle growth during both development and in the adult. In the two strains of myostatin knockout mice (KO) skeletal muscle undergoes hypertrophy (Grobet et al., 2003; McPherron et al., 1997), for example the extensor digitorum longus (EDL) muscle increases its weight by 66–121% in male KO mice (Amthor et al., 2007; Mendias et al., 2006; Ploquin et al., 2012). The muscle hypertrophy observed in KO mice results more from hyperplasia than muscle fiber hypertrophy (Baligand et al., 2010; Gay et al., 2012; Gentry et al., 2011; Gilson et al., 2007; McPherron et al., 1997; Mendias et al., 2006; Siriett et al., 2006). Since absolute maximal force is roughly proportional to muscle cross-sectional area, it should be expected that absolute maximal force is greater in KO mice as compared to control mice. Indeed this was observed in the study of Mendias et al. (2006) since they found that absolute maximal force of EDL muscle was increased by 34% in male KO mice. Similarly, ⁎ Corresponding author at: G.H. Pitié-Salpétrière, 47, bld de l'Hôpital, Bâtiment Babinski, INSERM U974, 75651 Paris cedex 13, France. E-mail address: [email protected] (A. Ferry). 0531-5565/$ – see front matter © 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.exger.2012.11.008
two studies also reported an increased absolute maximal force in male KO mice (Baligand et al., 2010; Wagner et al., 2002). However four other studies found that absolute maximal force was not increased in KO mice (Amthor et al., 2007; Gentry et al., 2011; Matsakas et al., 2012; Ploquin et al., 2012), one of them even showed that the absolute maximal force was decreased by approximately 50% (Matsakas et al., 2012). These latter studies would suggest that the reduction in specific maximal force (absolute maximal force relative to CSA or muscle weight) theoretically is strongly decreased by 66% in KO mice, which seems surprising in view of what is observed in murine models of neuromuscular diseases (Durieux et al., 2010; Fugier et al., 2011; Lynch et al., 2001; Risson et al., 2009; Trollet et al., 2010). For example, in the mdx mouse a murine model of Duchenne muscular dystrophy in which the limb muscles are hypertrophied by 28%, specific maximal force is only decreased by 13% (Lynch et al., 2001). Fatigue resistance is another important muscle performance parameter since on many occasions the muscle will have to contract repeatedly. This value is lower in male KO mice (Hennebry et al., 2009; Ploquin et al., 2012). It has also been reported that the force drop following lengthening contractions is also greater in male KO mice (Mendias et al., 2006), indicating that contraction-induced muscle injury (fragility) is increased. To our knowledge no studies have measured
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absolute maximal power, a key determinant of muscle performance during movement, in KO mice. Since absolute maximal power increases with muscle size and the percentage of fast-type powerful muscle fibers, it seems likely that absolute maximal power would also increase in KO mice. In order to better understand the role of myostatin in muscle performance (absolute maximal force, absolute maximal power, fatigue resistance), we examined both young (6 month old) and old (20–22 month old) adult KO mice of both sexes. To our knowledge, the muscle performance of aged KO mice has not yet been studied. With aging, there is a decrease in both muscle mass and performance whereas muscle fragility is increased at least in control mice (Chan and Head, 2010; Lynch et al., 2001). In this study, mice of both sexes were studied because sexrelated factors such as ovarian and testicular hormones are known to modulate muscle performance and size and possibly declined differentially with aging (Chambon et al., 2010; Greising et al., 2011; Hourde et al., 2009). Our results reveal that myostatin deficiency has a beneficial effect on some aspects of muscle performance that persists for a prolonged period of life, although it eventually decreases with aging. 2. Materials and methods 2.1. Animals All procedures were performed in accordance with national and European legislations. Female and male myostatin KO mice (KO) were used at 6–8 (6-month old) and 20–22 (2-year old month of age) (n= 8, n = 8, n = 20, n = 12, respectively). KO mice founder breeding pairs on a C57BL/6 background were a kind gift from Se-Jin Lee (McPherron et al., 1997). We also used 6-month and 2-year old female C57BL/6 mice (n= 22, n = 10) and 6-month old male C57BL/6 mice (n= 14) as controls (WT). It should be noted that we studied mice older than 6 months of age to avoid the confounding factor of postnatal growth, since during the first months there is a rapid change in myonuclear domain size (White et al., 2010) and specific maximal force (Gokhin et al., 2008; Goodman et al., 2008), two important parameters in the KO mice (see Discussion section). The age of 20–22 months of age was selected because it has been reported that the regular estrous cycle ceases prior to 20 months of age in female mice (Felicio et al., 1984; Greising et al., 2011). Consequently, the 2 year-old female mice were most probably in a phase of post-ovarian failure.
the first 200 ms. Then, the muscle shortened during the last 300 ms against the load. Each contraction was separated by a 1 min rest period. The (peak) shortening velocity was measured during the first 20 ms of the shortening period. Absolute power was calculated from the force– velocity data and absolute maximal power was reported (mW). Specific maximal power (mW/g) was calculated by dividing maximal power by muscle weight. Fragility or fatigue resistance (TA muscle) was then measured. Fragility was estimated from the force drop resulting from lengthening contraction-induced injury. Nine lengthening contractions of the muscle were performed, each separated by 1 min. This protocol was able to induce force drop in the mdx mice (Hoogaars et al., 2012; Trollet et al., 2010), a murine model for muscular dystrophy. The sciatic nerve was stimulated for 700 ms (125 Hz). A maximal isometric contraction of the TA muscle was initiated during the first 500 ms. Then, muscle lengthening (10% L0) at a velocity of 5.5 mm/s was imposed during the last 200 ms. Maximal isometric force was measured 1 min after each lengthening contraction and expressed as a percentage of the initial maximal isometric force. The fatigue protocol consists of 40 repeated isometric contractions (100 Hz for 500 ms, evoked every 2 s). Absolute maximal force and power were determined following the fatigue protocol, in the first 6 min of recovery. After contractile measurements, the animals were killed with an overdose of pentobarbital and muscles were weighed and flash frozen in liquid nitrogen. 2.3. Gel electrophoresis The muscles were extracted on ice for 60 min in 4 volumes of extracting buffer (pH 6.5), as previously described (Butler-Browne and Whalen, 1984). After centrifugation, the supernatants were diluted 1:1 (vol/vol) with glycerol and stored at − 20 °C. MHC isoforms were separated on 8% polyacrylamide gels, which were made in the Bio-Rad mini-Protean II Dual slab cell system, as described previously (Agbulut et al., 2003). The gels were migrated for 31 h at 72 V (constant voltage) at 4 °C. For the quantification of the relative concentration of MHC and actin, 1 μg of total proteins was loaded and 12% polyacrylamide gels were used. The silver stained gels were scanned using a video acquisition system and bands were quantified by densitometric software (Multi Gauge, Fujifilm). 2.4. Statistical analysis
2.2. Muscle performance and fragility Force-generating capacity and fragility were evaluated by measuring the in situ tibialis anterior (TA) muscle contraction in response to nerve stimulation, as described previously (Koo et al., 2011; Mouisel et al., 2006). Mice were anesthetized using pentobarbital (60 mg/kg intraperitoneally). Body temperature was maintained at 37 °C using radiant heat. The knee and foot were fixed with pins and clamps and the distal tendon of the muscle was attached to a lever arm of a servomotor system (305B, Dual-Mode Lever, Aurora Scientific) using a silk ligature. The sciatic nerve was proximally crushed and distally stimulated by a bipolar silver electrode using supramaximal square wave pulses of 0.1 ms duration. We measured the absolute maximal force that was generated during isometric contractions in response to electrical stimulation (frequency of 75–150 Hz, train of stimulation of 500 ms). Absolute maximal force was determined at L0 (length at which maximal tension was obtained during the tetanus). Absolute maximal force was normalized to the muscle mass as an estimate of specific maximal force, i.e. force-generating capacity. Force–velocity data (TA muscle) were then obtained by eliciting contractions along the sciatic nerve (500 ms, 125 Hz) at 6 different afterloads (over the range of approximately 10–50% absolute maximal force). The sciatic nerve was stimulated for 700 ms (125 Hz). A maximal isometric contraction of the muscle was initiated during
Groups were compared using 2 way-analysis (gender × age, gender× genotype, genotype× age) of variance. If necessary (significant interaction), contrast (Fisher PLSD) analysis was also performed. In some rare cases, only 1 way-analysis of variance analysis could be carried out because of the absence of a 2-year old male WT group, followed if necessary by Tukey's post hoc test. We also analyzed the percentage of variation relative to control (WT, 6 month-old…). For groups that did not pass tests of normality and equal variance, non-parametric tests were used (Kruskal Wallis and Wilcoxon). Values are means ±SEM. A p b 0.05 is considered significant. 3. Results 3.1. Effect of myostatin deficiency The muscle weights of 6-month old female KO mice, 2-year old female KO mice and 6-month old male KO mice were heavier by 79, 61 and 53% as compared to sex- and age-matched WT mice (Fig. 1A,B, p b 0.05). The increase in muscle weight did not differ significantly with sex or age (p > 0.05). The absolute maximal force was increased in 6-month old female KO mice, 2-year old female KO mice and 6-month old male KO mice, as compared to sex- and age-matched WT mice (p b 0.05, Fig. 2A). The
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increase in absolute maximal force was greater in 6-month old female KO mice (+77%) than in 2-year old female KO mice (+ 40%) and 6-month old male KO mice (+32%) (p b 0.05). Specific maximal force was decreased in 2-year old female KO mice (− 10%) and 6-month old male KO mice (− 13%), but not in 6-month old female KO mice, as compared to sex- and age-matched WT mice (p b 0.05, Fig. 2B). The reduced specific maximal force was not related to a decrease in concentrations of either MHC or actin (Fig. 3A,B) or a change in the ratio of MHC to actin (Fig. 3C). Moreover, the reduced specific maximal force was also not associated with a decrease in the percentage of high specific force producing fibers, i.e. fiber expressing MHC-2b, since the expression of MHC-2b was increased in 2-year old female KO mice and 6-month old male KO mice (Fig. 4A,B). 6-month old female KO mice and 6-month old male KO mice but not 2-year old female KO mice had a greater absolute maximal power as compared to sex- and age-matched WT mice (pb 0.05, Fig. 5A), with a greater increase in absolute maximal power in 6-month old female KO mice (+101%) than in 6-month old male KO mice (+33%) (p b 0.05). Specific maximal power was reduced in 2-year old female KO mice
Fig. 2. Absolute maximal force (A) and specific maximal force (B) in TA muscle from KO mice. a: KO different from corresponding WT (pb 0.05). b: 2 year-old (2y) different from 6 month-old (6 m) (pb 0.05), (b): 2 year-old (2y) tended to be different from 6 month-old (6 m) (p=0.08). c: Female different from corresponding male (pb 0.05). n=8–22/group for female and n=8–14/group for male.
Fig. 1. TA muscle weight (A) and TA muscle weight normalized to body weight (B) in KO mice. a: KO different from corresponding WT (p b 0.05). b: 2 year-old (2y) different from 6 month-old (6 m) (p b 0.05). c: Female different from corresponding male (p b 0.05). n = 8–22/group for female and n = 8–14/group for male.
(−22%) and 6-month old male KO mice (−13%), but not in 6-month old female KO mice, as compared to sex- and age-matched WT mice (pb 0.05, Fig. 5B). 6-month old female and male KO mice had a lower fatigue resistance as compared to 6-month old sex-matched WT mice (p b 0.05, Fig. 6), with no significant difference between sex concerning this effect (p > 0.05). Indeed myostatin deficiency decreased the fatigue resistance by 35 and 40% in 6-month old female and male KO mice respectively, as assessed by absolute maximal force loss (p b 0.05, Fig. 6A). Moreover, it reduced to a greater extent absolute maximal power following the fatigue protocol (−69% and − 80%, p b 0.05, for 6-month old female and male KO muscles respectively, Fig. 6B). The reduced fatigue resistance was related to a greater relative number of low fatigue resistant fiber since the percentage of fibers expressing MHC-2b increased in 6-month old female and male KO mice (Fig. 4A, B). Finally, we found that fragility was not affected by myostatin deficiency in 2-year old female and male KO mice. There was no significant drop in force following the lengthening contractions in 2-year old female and male KO mice, similarly to 2-year old female WT mice (p > 0.05, Fig. 7).
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in 2-year old female KO mice (−17%) than in 2-year old male KO mice (−30%) (pb 0.05, Fig. 2A). Similarly with advancing age, specific maximal force was reduced (pb 0.05), but to a smaller extent in 2-year old female KO mice (−10%) than in 2-year old male KO mice (−30%) (pb 0.05, Fig. 2B). The decline in specific maximal force was not related to a change in myofibrillar proteins (Fig. 3A–C). It should be also noted that the percentage of high specific force producing fibers decreased in 2-year old female KO mice whereas it increased in 2-year old male KO mice (Fig. 4A,B). Concerning absolute maximal power, it was equally decreased in 2-year old female KO mice (−38%) and 2-year old male KO mice (−43%) as compared to 6-month old sex-matched KO mice (pb 0.05, Fig. 5A). Moreover, aging reduced specific maximal power in both 2-year old female and male KO mice (pb 0.05), with a smaller reduction in 2-year old female KO mice (−22%) than in 2-year old male KO mice (−44%) (pb 0.05, Fig. 5B). We also found that fatigue resistance was not globally altered by aging in 2-year old female and male KO mice (p> 0.05, Fig. 6A,B). 4. Discussion 4.1. Increased absolute maximal force and power
Fig. 3. MHC concentration (A), actin concentration (B), and ratio of MHC to actin (C) in TA muscle from KO mice. a: KO different from corresponding WT (p b 0.05). b: 2 year-old (2y) different from 6 month-old (6 m) (p b 0.05). c: Female different from corresponding male (p b 0.05). n = 4/group.
3.2. Effect of aging Similarly to 2 year-old female WT mice, no sarcopenia occurred in 2-year old female and male KO mice (Fig. 1A,B). However, in contrast to female 2 year-old WT mice, we found an effect of aging on muscle performance in KO mice of both sexes. Absolute maximal force decreased in 2 year-old female and male KO mice as compared to 6-month old sex-matched KO mice (pb 0.05), with a smaller reduction
In the present study we confirm that myostatin deficiency induced increased muscle weight (Amthor et al., 2007; Gentry et al., 2011; Mendias et al., 2006; Wagner et al., 2005; Wang and McPherron, 2012). The muscle hypertrophy (53–79%) can explain the increased absolute maximal force (32–77%) in both 6-month old female and male KO mice and 2-year old female KO mice, despite the modestly reduced specific maximal force (see below). Our results also confirm and extend those of Mendias et al. (2006) showing that absolute maximal force was increased by 34% in the EDL muscle from male KO mice (+32% in 6-month old male KO mice). The increased absolute maximal force is apparently due to fiber hyperplasia because the absolute maximal force of skinned muscle fibers did not differ between KO mice and WT mice (Mendias et al., 2011; Qaisar et al., 2012). It should be noted that postnatal myostatin deficiency or blockade also increases absolute maximal force in WT mice (Akpan et al., 2009; Goncalves et al., 2010; Murphy et al., 2010; Personius et al., 2010; Whittemore et al., 2003). The reasons why absolute maximal force is not increased in KO mice from other studies (Amthor et al., 2007; Gentry et al., 2011; Matsakas et al., 2012; Ploquin et al., 2012) are not related to 1) the muscle studied (Mendias et al., 2006; Gentry et al., 2011), 2) the type of muscle force measurement (in situ versus in vitro; the present study; Amthor et al., 2007; Gentry et al., 2011; Mendias et al., 2006), 3) sex and age (the present study; Gentry et al., 2011). The origin of this discrepancy concerns the effect of myostatin deficiency on specific maximal force (see below) since muscle hypertrophy is similar between studies. Increased muscle weight can also explain the increased maximal power (33–101%) in 6-month old female and male KO mice. Since maximal power is increased, the effect of hyperplasia exceeds the effect of the reduced specific maximal power of skinned muscle fibers from KO mice (Mendias et al., 2011). It should be noted that the gain in maximal power was not markedly greater than that of absolute maximal force suggesting that the fiber type transition from slow/low to fast/powerful-type induced by myostatin deficiency, as shown in the present and previous studies (Amthor et al., 2007; Hennebry et al., 2009), had little impact on this parameter. 4.2. Reduced specific maximal force and power We also found that specific maximal force and specific maximal power were moderately reduced in 2-year old female KO mice and 6-month old male KO but not in 6-month old female KO mice, indicating
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Fig. 4. Representative images of electrophoretic separation of MHC proteins (A) and MHC-2b distribution (B) in TA muscle from KO mice. a: KO different from corresponding WT (p b 0.05). b: 2 year-old (2y) different from 6 month-old (6 m) (p b 0.05). c: Female different from corresponding male (p b 0.05). n= 4–5/group.
that the maximal force and power generation capacities of the muscle are somewhat reduced by myostatin deficiency. It was recently reported that specific maximal force and specific maximal power of skinned muscle fibers were reduced in KO mice, indicating that myofibrillar dysfunction is responsible for this defect at the level of the intact muscle (Mendias et al., 2011; Qaisar et al., 2012). Two mechanisms have been recently discussed to explain this reduction in the intrinsic qualities of the muscles. Firstly, a deficient ubiquitin proteasome system in KO mice could lead to an accumulation of damaged non functional contractile proteins (Mendias et al., 2011). Secondly, an increased volume of cytoplasm per nucleus (myonuclear domain) in the KO mice could exceed the capacity of the nucleus to maintain myofibrillar protein content (Qaisar et al., 2012). However, our results concerning the myofibrillar protein concentrations did not support the latter hypothesis. In addition, we exclude the possibility that a smaller percentage of high specific force producing fibers, i.e. fibers expressing MHC-2b (D'Antona et al., 2007), was responsible for the reduced specific maximal force. It should be noted that the reductions in specific maximal force and specific maximal power (−13 to −18%, the present study; Mendias et al., 2006) in young male KO mice are apparently consistent with those observed in skinned muscle fibers (−21%, (Mendias et al., 2011; Qaisar et al., 2012)), but in opposition to those observed in other studies showing a 40–75% reduction in muscle specific maximal force from young male KO mice (Amthor et al., 2007; Gentry et al., 2011; Matsakas et al., 2012; Ploquin et al., 2012). The discrepancy could result from differences in myonuclear domain and/or the ubiquitin
proteasome system between the different KO models (McPherron et al., 1997 versus Grobet et al., 2003), or to the different genetic backgrounds of the strains (C57BL6 versus FVB/N-C57BL6) or colonies separated for many years. It should be noted that the decline in muscle quality observed in these studies (Amthor et al., 2007; Gentry et al., 2011; Matsakas et al., 2012; Ploquin et al., 2012) is to our knowledge rarely observed in the murine models for neuromuscular disorders (Durieux et al., 2010; Fugier et al., 2011; Lynch et al., 2001; Risson et al., 2009; Trollet et al., 2010). 4.3. Reduced fatigue resistance Myostatin deficiency is also very detrimental to fatigue resistance in 6-month old KO mice of both sexes. After 40 contractions, absolute maximal force (− 35 to 40%) and more importantly absolute maximal power (− 69 to − 80%) were reduced to a greater extent in 6-month old female and male KO mice than in 6 month-old sex-matched WT mice. This result confirms and extends previous studies (Hennebry et al., 2009; Ploquin et al., 2012) showing that young male KO mice have a lower fatigue resistance. Since it had been shown that aging did not reduce fatigue resistance in WT mice of both sexes (Chan and Head, 2010), it is likely that fatigue resistance is still reduced in 2 year-old KO mice as compared to 2-year old WT mice. The dramatic effect of myostatin deficiency on power during repeated contractions can explain the reduced exercise tolerance observed in the KO mice (Savage and McPherron, 2010). The reduction in the percentage of
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Fig. 5. Absolute maximal power (A) and specific maximal power (B) of TA muscle from KO mice. a: KO different from corresponding WT (pb 0.05). (a): KO tended to be different from corresponding WT (p = 0.07). b: 2 year-old (2y) different from 6 month-old (6 m) (p b 0.05). c: Female different from corresponding male (p b 0.05). n = 8–22/ group for female and n = 8–14/group for male KO mice.
fatigue resistant fibers, mitochondrial depletion, reduced oxidative metabolism and capillarity (Amthor et al., 2007; Baligand et al., 2010; Hennebry et al., 2009; Matsakas et al., 2012; Ploquin et al., 2012) can explain the greater muscle fatigue in KO mice. Another contributing factor is linked to muscle hypertrophy. The increase in muscle blood flow induced by exercise may be no longer sufficient, limiting oxygen blood supply and washout. These results illustrate the well-known negative relationship between muscle size and fatigue resistance/oxidative metabolism (Degens and Veerkamp, 1994; van Wessel et al., 2010).
Fig. 6. Fatigue resistance as a decreased in absolute maximal force (A) and absolute maximal power (B) after 40 contractions of TA muscle from KO mice. a: KO different from corresponding WT (p b 0.05). b: 2 year-old (2y) different from 6 month-old (6 m) (p b 0.05). c: Female different from corresponding male (pb 0.05). n = 8–22/ group for female and n = 8–14/group for male KO mice.
4.4. Changes with aging For the first time, we are able to report important age-related changes in the muscle performance of KO mice of both sexes. Absolute maximal force (−17 to −30%) and maximal power (−38 to −43%) were both reduced by aging in 2-year old female and male KO mice, whereas they did not change in fast muscle from 2-year old WT mice of both sexes (the present study; Hakim and Duan, 2012; Lynch et al., 2001; Moran et al., 2005). Interestingly, these results are not related to muscle atrophy but can be explained by a reduction in specific maximal force and power with aging in both 2-year old female and male KO
Fig. 7. Fragility, i.e., force drop following lengthening contractions of TA muscle from KO mice. a: KO different from corresponding WT (p b 0.05). b: 2 year-old (2y) different from 6 month-old (6 m) (p b 0.05). c: Female different from corresponding male (p b 0.05). n = 8–22/group for female and n= 8–14/group for male KO mice.
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mice, indicating the deterioration of muscle quality. Since specific maximal force and power do not markedly decline in fast muscle from old WT mice of both sexes until 28 months of age (the present study; Hakim and Duan, 2012; Lynch et al., 2001; Moran et al., 2005), our results suggest that the potential detrimental effect of myostatin on specific maximal force and power (see above) increases with aging in both sexes. It is possible that myostatin deficiency accelerates normal aging. Alternatively, the worsening of the reduced muscle quality with aging could reflect the cumulative detrimental effect of myostatin deficiency. With advancing age, there could be an aggravation of the detrimental effect of myostatin deficiency on the proteasome ubiquitin system and on myofibrillar gene transcription controlled by the nucleus (Mendias et al., 2011; Qaisar et al., 2012). However, we found no effect of aging on myofibrillar protein concentrations in 2-year old female and male KO mice. In contrast, there is apparently no change in fatigue resistance with aging in the 2-year old female and male KO mice, despite the changes in MHC-2b distribution. Of note, muscle fragility in the 2-year old KO mice of both sexes was also not affected by aging. An important point of consideration is that these age-related changes result in the fact that the increases in absolute maximal force and power induced by myostatin deficiency at 6 months of age were reduced 2 fold or abolished in 2 year-old female KO mice. Since aging did not alter absolute maximal force and power in male WT mice (Chan and Head, 2010; Hakim and Duan, 2012; Lynch et al., 2001), it can be expected that there is no increase in maximal force and no decrease in absolute maximal power in 2-year old male KO mice as compared to 2-year old male WT (see Fig. 2A and B: compare 2 year-old KO male mice to 6 month-old male WT mice). In short, our results indicate that the beneficial effect of myostatin deficiency on absolute maximal force and maximal power fades with age, at least in females and also most likely in males, prior to the onset of normal muscular senescence (sarcopenia and dynapenia).
4.5. Sex differences Interestingly, we found that the beneficial effects of myostatin deficiency on muscle performance (increased absolute maximal force and power) are greater in 6-month old female KO mice than in 6-month old male KO mice. This sex difference results from a greater muscle hypertrophy and a smaller reduction in specific maximal force and power in 6-month old female as compared to male KO mice. The differential effect of myostatin deficiency is such that it eliminates not only the sexual dimorphism concerning absolute maximal force and power but also concerning muscle size that we observed in 6-month WT mice. A possible explanation might be that myostatin expression is reduced to a greater extent in male as compared to female WT mice. Indeed, it has been demonstrated that processed myostatin protein expression is lower in male WT mice (McMahon et al., 2003). Moreover, it is plausible that myostatin interferes with androgen pathways that are known to modulate specific maximal force (Chambon et al., 2010). Another interesting finding is that the effect of aging was reduced in 2-year old female KO mice as compared to 2-year old male KO mice, although to a greater extent than that of 2-year old female and male WT mice (the present study; Chan and Head, 2010; Hakim and Duan, 2012; Lynch et al., 2001; Moran et al., 2005). Indeed, absolute maximal force (− 17% versus − 30%) and specific maximal force (− 10% versus − 30%) and power (− 22% versus − 44%) declined less with aging in 2-year old female KO mice than in 2-year old male KO mice, for as yet unknown reasons. The protective effect of the female related factor in the context of myostatin deficiency remains to be determined. It also remains to be determined whether the ubiquitin proteasome system is less affected by aging in 2-year old female KO mice compared with 2-year old male KO mice.
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5. Conclusion Our study demonstrates that myostatin deficiency increases absolute maximal force in both 6-month old female and male KO mice and 2-year old female KO mice. Similarly, absolute maximal power is increased by myostatin deficiency in both 6-month old female and male KO mice but not in 2-year old female KO mice. These increases likely result from muscle hypertrophy. They are greater in the 6-month old female KO mice than in the 6-month old male KO mice, likely due to greater muscle hypertrophy and no reductions in specific maximal force and power in female mice. In contrast, myostatin deficiency markedly reduced fatigue resistance in 6-month old KO mice of both sexes. With aging, in contrast to 2-year old WT mice (the present study; Chan and Head, 2010; Hakim and Duan, 2012; Lynch et al., 2001), absolute maximal force and power were reduced in 2-year old KO mice of both sexes, although muscle weight did not change. These age-related decreases are smaller in 2-year old female mice than in 2-year old male KO mice, possibly because there was a less significant decrease in specific maximal force and power in the female mice. Altogether, these results suggest that the beneficial effect of myostatin deficiency on absolute maximal force and power production is greater in female (versus male) and young (versus old) mice. Most of these effects of myostatin deficiency are related neither to changes in the concentration of myofibrillar proteins nor to the slow to fast fiber type transition. Acknowledgments We are grateful to W. Hadj-Said (UMR 974) and A. Chatonnet (UMR 866) for assistance during the experiments. Financial support has been provided by Université Pierre et Marie Curie (UPMC), CNRS, INSERM, University Paris Descartes, ANR-Genopath In-A-Fib, ANR-Blanc Androgluco, the Association Française contre les Myopathies (AFM), MyoAge (EC 7th FP, contract 223576), MyoGrad International Graduate School for Myology (DRK 1631/1), Agence Française de Lutte contre le Dopage, and Association Monegasque contre les Myopathies, Parents Project France. Appendix A. Supplementary data Supplementary data to this article can be found online at http:// dx.doi.org/10.1016/j.exger.2012.11.008. References Agbulut, O., Noirez, P., Beaumont, F., Butler-Browne, G., 2003. Myosin heavy chain isoforms in postnatal muscle development of mice. Biol. Cell 95, 399–406. Akpan, I., Goncalves, M.D., Dhir, R., Yin, X., Pistilli, E.E., Bogdanovich, S., Khurana, T.S., Ucran, J., Lachey, J., Ahima, R.S., 2009. The effects of a soluble activin type IIB receptor on obesity and insulin sensitivity. Int. J. Obes. (Lond) 33, 1265–1273. Amthor, H., Macharia, R., Navarrete, R., Schuelke, M., Brown, S.C., Otto, A., Voit, T., Muntoni, F., Vrbova, G., Partridge, T., Zammit, P., Bunger, L., Patel, K., 2007. Lack of myostatin results in excessive muscle growth but impaired force generation. Proc. Natl. Acad. Sci. U. S. A. 104, 1835–1840. Baligand, C., Gilson, H., Menard, J.C., Schakman, O., Wary, C., Thissen, J.P., Carlier, P.G., 2010. Functional assessment of skeletal muscle in intact mice lacking myostatin by concurrent NMR imaging and spectroscopy. Gene Ther. 17, 328–337. Butler-Browne, G.S., Whalen, R.G., 1984. Myosin isozyme transitions occurring during the postnatal development of the rat soleus muscle. Dev. Biol. 102, 324–334. Chambon, C., Duteil, D., Vignaud, A., Ferry, A., Messaddeq, N., Malivindi, R., Kato, S., Chambon, P., Metzger, D., 2010. Myocytic androgen receptor controls the strength but not the mass of limb muscles. Proc. Natl. Acad. Sci. U. S. A. 107, 14327–14332. Chan, S., Head, S.I., 2010. Age- and gender-related changes in contractile properties of non-atrophied EDL muscle. PLoS One 5, e12345. D'Antona, G., Brocca, L., Pansarasa, O., Rinaldi, C., Tupler, R., Bottinelli, R., 2007. Structural and functional alterations of muscle fibres in the novel mouse model of facioscapulohumeral muscular dystrophy. J. Physiol. 584, 997–1009. Degens, H., Veerkamp, J.H., 1994. Changes in oxidative capacity and fatigue resistance in skeletal muscle. Int. J. Biochem. 26, 871–878. Durieux, A.C., Vignaud, A., Prudhon, B., Viou, M.T., Beuvin, M., Vassilopoulos, S., Fraysse, B., Ferry, A., Laine, J., Romero, N.B., Guicheney, P., Bitoun, M., 2010. A centronuclear myopathy-dynamin 2 mutation impairs skeletal muscle structure and function in mice. Hum. Mol. Genet. 19, 4820–4836.
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