Muscle progenitor cells proliferation doesn’t sufficiently contribute to maintaining stretched soleus muscle mass during gravitational unloading

Acta Astronautica 63 (2008) 706 – 713 www.elsevier.com/locate/actaastro

Muscle progenitor cells proliferation doesn’t sufficiently contribute to maintaining stretched soleus muscle mass during gravitational unloading M.V. Tarakinaa , O.V. Turtikovaa , T.L. Nemirovskayaa , A.A. Kokontcevb , B.S. Shenkmana,∗ a SRC, Institute for Biomedical Problems RAS, 76a Khoroshevskoe Shosse, 123007 Moscow, Russia b Russian Scientific Center of Rentgenoradiology, 86 Profsojuznaya Street, 117997 Moscow, Russia

Received 12 July 2007; received in revised form 27 January 2008; accepted 25 February 2008 Available online 23 April 2008

Abstract Skeletal muscle work hypertrophy is usually connected with muscle progenitor satellite cells (SC) activation with subsequent incorporation of their nuclei into myofibers. Passive stretch of unloaded muscle was earlier established to prevent atrophic processes and is accompanied by enhanced protein synthesis. We hypothesized that elimination of SC proliferation capacity by -irradiation would partly avert stretched muscle fiber capability to maintain their size under the conditions of gravitational unloading. To assess the role of muscle progenitor (satellite) cells in development of passive stretch preventive effect SC proliferation was suppressed by local exposing to ionized radiation (2500 rad), subsequent hindlimb suspension or hindlimb suspension with concomitant passive stretch were carried out. Reduction of myofiber cross-sectional area and decrease in myonuclei number accompanying unloaded muscle atrophy were completely abolished by passive stretch both in irradiated and sham-treated animals. We conclude that SC did not make essential contribution to passive stretch preventive action under the conditions of simulated weightlessness. © 2008 Elsevier Ltd. All rights reserved. Keywords: Gravitational unloading; Myofibers; Immunohistochemistry; m. soleus; Passive stretch; Myonuclei; Satellite cells

1. Introduction Under microgravity the atrophic alterations in skeletal muscle, such as the reduction of muscle fiber (MF) cross-sectional area (CSA) and the decrease in muscle protein content are observed [1,2]. It was shown previously, that gravitational unloading leads to the decrease in protein synthesis activity and ∗ Corresponding author.

E-mail address: [email protected] (B.S. Shenkman). 0094-5765/$ - see front matter © 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.actaastro.2008.02.013

proteolysis enhancement [3,4]. The atrophic alterations are supposed to be caused to some extent by the myonuclei number reduction, which results from apoptosis [5,6]. The only source of additional nuclei in the muscles could be mononuclear satellite cells (SC). Being reproduced they can fuse with each other and with adjacent MFs favoring the muscles growth [7], their repair and regeneration [8,9]. Resistive exercise or stretching of a skeletal muscle combined with gravitational unloading diminishes or fully prevents the atrophic alterations development

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[10,11]. Goldspink [12] and Jaspers [13] showed that muscle stretching combined with unloading prevented protein synthesis inhibition and amino acids utilization in m. soleus. Chronic muscle stretching usually leads to the enhancement of the muscle proteins synthesis [14]. It is hypothesized that this effect is caused by the general enhancement of anabolic processes in the fibers, and by the SC proliferation and fusion with the MFs. The hypertrophy provoked by the functional overloading and muscle stretching stimulates active proliferation of SC and their fusion with the adjacent fibers [15,16]. The ionizing -radiation at a dose that leads to DNA rupture, but does not significantly injure myofibers, is usually used to suppress the proliferative properties of the progenitor cells [17]. The results obtained on exercised and irradiated muscles under normal gravity are quite contradictory. Thus, in the series of works on rodents Rosenblatt and colleagues showed that tenotomy of synergist muscles did not lead to the hypertrophy of m. extensor digitorum longus and to the increase in myonuclei number during 4 weeks. At the same time, the fast-to-slow shift of myosin heavy chains expression (that is usually observed under overloading) was not influenced by the irradiation [18–20]. Phelan and Gonyea also showed that ankle muscle irradiation prevented the compensatory hypertrophy of m. soleus developed after synergist muscles ablation during 4 weeks and completely suppressed fiber neoformation [21]. In the work of Adams and his group the hypertrophy of functionally loaded m. plantaris was not observed during 3 months after irradiation [17]. At the same time, in the experiment of Lowe and Alway on stretching of irradiated muscles of Japanese quail wings the SC reproduction was inhibited, while the hypertrophy effect of stretching decreased slightly [16]. In conformity with gravitational unloading, the irradiation is known to lead to the incomplete muscle recovery after the atrophy caused by tail suspension [22]. However, the question if the stretched muscle under simulated microgravity, would maintain its mass when the SC activity is suppressed, still remains unclear. Consequently, the aim of our study was to reveal the contribution of the SC nuclei to the maintenance of MF size under stretching combined with the gravitational unloading. 2. Protocol and methods 2.1. Animal procedures Forty three male Wistar rats of 2.5 months old with the average weight about 270 g were used in the exper-

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Fig. 1. Rat hindlimb suspending (according to Novikov, Ilyin).

Fig. 2. Passive stretch of rat soleus muscle. Hind limb is fixed in dorsiflexed position.

iments. Standard chew according to the laboratory animal ratio and water were supplied to the rats ad libitum. All the procedures with the animals were carried out in accordance with the International Animal Care regulations and the Russian Legacy and approved by Physiological Branch of the Russian National Committee on Biological Ethics [23]. The gravitational unloading of the rat hindlimbs was carried out according to the standard Novikov-Ilyin technique of tail suspension [24], so that the hind limbs could not reach the cage floor, while the fore limbs could touch it. The rat body was placed at the angle of 45◦ to the cage floor (Fig. 1). For ankle extensors stretching ankle joints of both rat hindlimbs were casted at the dorsal flexion position of ankle at the angle of 35◦ [10,22] (Fig. 2). The experiment duration was 14 days.

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For the experiment the animals were randomly divided into six groups. Each group included seven animals, except the group “Stretching + Irradiation + Suspension” (SIS) contained eight animals. The animals of the “Control” group (C) were kept in cages throughout the experiment. For blocking of the progenitor cells proliferation we used irradiation at a dose of 2500 rad, [17–20,22,25]. Five days before the experiment both ankles of rats of the groups “Irradiation” (I), “Irradiation + Susupension” (IS), and SIS were -irradiated at a dose of 2500 rad for 15 min on the distant therapeutic gamma-apparatus Rocus-AM (developed by Research and Production Association “Agat”, Moscow; produced by “Ravenstvo” plant, St. Petersburg). Co-60, the radionuclide, was the source of radiation. The dose was controlled by the clinical dosimeter “Keithley-3540”, USA. Before the irradiation the animals were anaesthetized with Nembutal (20 mg/kg, i.p.) and covered with the lead shield, so that only the ankles were exposed to the Co-60 containing radiation source. This irradiation dose, according to the literature [17,18,26], suppresses proliferative properties of the SC without significant injury of myofibers. The rats of “I” group were kept in cages after the irradiation. The animals of the “Suspension” (S) and “IS” groups were tail suspended for 14 days. The animals of “Suspension + Stretching” (SS) and “SIS” groups were suspended with both hindlimbs casted [27,28]. At the day 15 of the experiment the rats were sacrificed with i.p. nembutal injection (50 mg/kg). Both soleus muscle were frozen in liquid isopentane cooled in liquid nitrogen, and stored at −80 ◦ C. The m. soleus 7 m cross-sections were made in cryostat Leica CM 1900 at −20 ◦ C. 2.2. Immunohistochemical analysis of muscle samples Following reagents were used: mouse monoclonal antibodies against the isoforms of myosin heavy chains (MHC) NCL—MHCf and NCL—MHCs, mouse monoclonal antibodies against dystrophin NCL—Dys 2 (Novocastra Laboratories, Great Britain), antibodies against CD 56 (Becton Dickinson), goat polyclonal antibodies against mouse immunoglobulins, conjugated with FITC, f-GAM (IMTEK, Russia); goat antibodies against mouse immunoglobulins IgG (H + L) conjugated with Alexa Fluor 546 (Molecular Probes, USA); biotinilated goat antibodies against mouse immunoglobulins; streptavidine conjugate with hoarse reddish peroxidase (Amersham Bioscienses, UK); normal goat serum (IMTEK, Russia); 4 ,6-diamidino-2-phenilindol dihydrochloride (DAPI, MP Biomedicals, USA); PBS

Fig. 3. Double-labeling of rat soleus muscle by DAPI and dystrophin antibodies.

(AMRESCO, USA); diaminobenzidine tetrachloride (DAB, ICN, USA); and hematoxylin (Sigma, USA). The frozen muscle sections were kept at room temperature for 1 h and incubated in damp chamber with suitable antibodies diluted in PBS (the antibodies against slow and fast MHC isoforms: 1:30; the antibodies against dystrophin: 1:20) at 37 ◦ C for 1 h. Then, the sections were washed with PBS three times for 5 min and secondary antibodies conjugated with FITC were added. The sections were incubated with the secondary antibodies for 60 min in a dark place at room temperature. For staining myonuclei on the sections marked with antibodies against dystrophin into the solution of secondary antibodies conjugated with Alexa Fluor we added DAPI (2 g/ml of antibody solution). After secondary antibodies ablation with PBS, the sections were mounted with Fluoromount-G. The samples obtained were photographed using the fluorescent microscope Leica Q500MC (Germany) (40×) and digital camera Leica TCM 300F. Photographs obtained were analyzed using Leica software. For determination of myofiber CSA (MF CSA) not less than 100 MFs were analyzed, for measuring of myonuclei number per myofiber perimeter and CSA not less than 200 myofibers were analyzed. To distinguish myonuclei from the mononuclear adjacent cells, the myofibers were outlined with antibodies against subsarcolemmal protein dystrophin [29]. All the nuclei inside the area bounded with immunohistochemical reaction products were

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considered to be the myonuclei (Fig. 3). Only the safe, uninjured myonuclei were taken into account. The same sections were used for average MF CSA measurement. To reveal the SC we used the immunoperoxidase staining against NCAM (CD 56) (Fig. 4) [30–32]. CD56 is similar to the antigen leu-19, the neural cell adhesion molecule (NCAM), the molecule of intercellular recognition. NCAM is expressed in the myoblasts at the early stage of myogenesis and in the SC of adult animals [31]. The transverse 7 m thick sections were thawed, dried at the room air, rehydrated in PBS during 20 min, and incubated in 2% normal goat serum for 40 min at the room temperature. Then, the sections were incubated with primary antibodies against CD-56 (diluted 1:100 in 1% of bovine serum albumin) (Becton Dickinson) for 2 h at 37 ◦ C, and washed three times for 5 min in PBS. After that, the sections were incubated in biotin-conjugated sheep antibodies against mouse

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immunoglobulins (dilution 1:200) at room temperature for 1 h, then, in streptavidin conjugated with horseradish peroxidase (Amersham Biosciences) for 30 min. After each incubation the sections were washed in PBS three times for 5 min. To visualize binding of the primary antibodies we used DAB diluted in PBS containing 3% of hydrogen peroxide. After washing the sections were dyed with hematoxylin for 30 s, then lightened in ascending alcohols, and enclosed into Canada balsam. The SC were visualized under great magnification (objective 100×). Myonuclei and the SC were counted in each fiber. The number of myonuclei was calculated per myofiber, average CSA per 1 myonucleus, and cytoplasm volume per 1 myonucleus. The following formulae [33] X = NL/(1 + d), where X is the number of myonuclei in one myofiber segment, N the myonuclei number in myofiber at the fiber transverse section, L the segment length (was accepted as 1 mm), 1 section thickness, and d myonuclei length, equal to 13.4 m for rat m. soleus [29]. The cytoplasm volume per 1 myonuclei (Y ) was calculated using formulae (2) [34] Y = (CL)/X, where C is the CSA of MF, L the segment length (1 mm), and X the number of myonuclei per one myofiber segment. Microsoft Excel was used for calculations and estimation of average value and standard error. For myonuclei number determination we calculated all the myonuclei in a single fiber, including those destructed by the irradiation. For statistical significance estimation we used one-dimensional ANOVA version and t-Student’s test with the Bonferonni adjustment for the multiple group analysis. 3. Results 3.1. Muscle weight and fiber size

Fig. 4. Immuhistochemical staining against CD 56 (NCAM) (arrows point to labeled cells). NCAM is stained as a brown rim and nuclei stained blue.

As a result of suspension m. soleus mass decreased more than twice as compared to control values (Table 1). Stretching completely prevented m. soleus mass losses

Table 1 Animal weight after the experiment, m. soleus weight, cross-sectional area (CSA) of slow and fast myofibers Group

C

S

SS

I

IS

SIS

m. soleus weight Slow myofibers CSA, sqr. mkm Fast myofibers CSA, sqr. mkm

119 ± 5 2555 ± 138 1853 ± 186

56 ± 2∗ 1291 ± 145∗ 1198 ± 83∗

122 ± 4 3055 ± 145 1864 ± 199

125 ± 4 2652 ± 114 2129 ± 56

60 ± 3# 1340 ± 82# 1162 ± 27#

116 ± 4 2896 ± 175 1900 ± 136

C, control group; S, hindlimb suspension; SS, hindlimb suspension + stretch; I, irradiation; IS, irradiation + hindlimb suspension; SIS, irradiation + hindlimb suspension + stretch. ∗, #—significant difference correspondingly from groups: C, I (P < 0.05).

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observed under gravitational unloading. In the groups subjected to suspension and stretching (“C”, “S”, and “Stretching”) and groups subjected to suspension and stretching combined with irradiation (“I”, “IS”, and “I + Stretching”) we did not observe significant differences in m. soleus mass. Consequently, the irradiation did not affect the atrophy process development in rat m. soleus and did not influence on the preventive effects of stretching. The data distribution of m. soleus morphometric measurements (Table 1) are similar to those on the muscle mass changes: the MF CSA in the suspended group (“S”) decreased by 50% for slow (type I) MFs, and by 35% for fast (type II) fibers as compared to the control group (“C”). Stretching prevented the MF CSA decrease. Statistically significant differences in CSA of both fiber types between the irradiated and unirradiated groups were not revealed. 3.2. Myonuclei and nuclear domain The 50% decrease in m. soleus mass caused by gravitational unloading was accompanied by myonuclei

Fig. 5. The number of myonuclei per myofiber cross-section of rat soleus muscle. Y -direction is the amount of myonuclei per one myofiber. ∗, #, and $—significant difference appropriately from groups C, S, and SS (P < 0.05).

number reduction. That is, the number of myonuclei per myofiber on the transverse section decreased by 23% (Fig. 5). Muscle stretching combined with suspension led to maintaining of myonuclei number at the control level (shown above). In the “I” group the tendency to the myonuclei number decrement was observed as compared to the “C” group. After the suspension combined with irradiation the myonuclei number decreased by 30%, as compared to the “S” (unirradiated) group. In the “SIS” group the myonuclei number decreased by 50%, as compared to the “SS” group, and by 18%, as compared to the “IS” group. We also analyzed the myonuclei number per 1 mm of myofiber perimeter. Since the myofiber perimeter decreased under gravitational unloading by about 27% (Table 2), the myonuclei number per 1 mm of perimeter after the suspension did not alter, as compared to control. At the same time we observed the significant reduction of this parameter in the stretched group, as compared to the suspended animals, but there was no alteration of this index in comparison with control. The irradiation led to the significant decrease in this parameter in the groups of irradiated rats subjected to suspension and stretching in comparison with the unirradiated groups. The analysis of the sections loaded with DAPI showed that a part of nuclei in the irradiated specimens, first of all in “IS” and “SIS” groups was destructed. Consequently, some destructive effect of stretching does exist and manifests in fragmentation of some part of myonuclei material and marked nuclei destruction under stretching combined with irradiation. As a result of the suspension the CSA/myonucleus and the cytoplasm volume/myonucleus ratios decreased by 36%. The stretching maintained these parameters at the control level (Table 2 and Fig. 6). The irradiation itself did not cause significant changes. At the same time, in the “IS” group the myonuclei domain increased significantly, by 30%, as compared to the group “S”, and diminished by 30% in comparison with the group “I”.

Table 2 Measured and calculated parameters of nuclei—cytoplasm ratio in rat m. soleus C Myofiber CSA, sqr. mkm Myofiber perimeter mkm Myonuclei/mm of perimeter Myonuclei/mm of length CSA, mkm per one myonucleus

2451 ± 101 199 ± 4 11.94 ± 0.82 111 ± 9 1053 ± 76

S ∗

1223 ± 53 1.85 ± 0.13∗ 12.74 ± 0.90 86 ± 6∗ 674 ± 45∗

SS

I

IS

2660 ± 240 202 ± 8 10.06 ± 0.44$ 95 ± 5 1308 ± 87$

2530 ± 114 200 ± 4 10.02 ± 0.41 94 ± 3 1290 ± 71

1158 ± 70 136 ± 4# 9.64 ± 0.42$ 61 ± 3$ 887 ± 50#,$

∗, #, $, @, &—significant difference correspondingly from groups C, I, S, SS, IS (P < 0.05).

SIS #

2682 ± 191 204 ± 7 5.28 ± 0.77@ 50 ± 8@ 2737 ± 355@,&

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4. Discussion

Fig. 6. Cytoplasm volume per one myonucleus in rat soleus muscle. Y -direction is the volume of cytoplasm per one myonucleus, sqr. mkm. ∗, #, $, &, and @—significant difference appropriately from groups C, S, SS, I, and IS (P < 0.05).

Fig. 7. The number of satellite cells for myofiber cross-section. Y -direction is the number of satellite cell per myofiber cross-section. ∗, @, and #—significant difference correspondingly from groups C, SS, and SIS (P < 0.05).

Irradiation led to increase in myonuclei domain in the group “SIS” two times, as compared to the “SS” group, and three times in comparison with the “IS” group. 3.3. Satellite cells The number of SC (positively stained against NCAM) in the “SS” group increased twice, as compared to the group “S”, and by 72 % in comparison with the “C” group. The number of SC in animals of the “SIS” group was 2.6 higher than in the “IS” group, and 26% lower than in the respective unirradiated group (“SS” group) (Fig. 7).

Passive stretching of the postural soleus muscle allows preventing the atrophy changes development under simulated gravitational unloading [3,11]. In our experiment we showed that more than 50% decrease in m. soleus wet weight and MF CSA under the suspension were fully prevented by m. soleus passive stretching. Among possible mechanisms underlying the increase in protein synthesis intensity under stretching, the most interesting is the possible activation of superincumbent resident progenitor cells (SC). These cells can be drawn to the proliferative cycle and then fuse with a maternal fiber that lead to an increase in the fiber myonuclei pool. Hill and Goldspink [35] demonstrated that such processes (namely, proliferative action of uninuclear cells in the muscle tissue) accompanied the anabolic effects development under stretching of an intact fast muscle. In our experiment we demonstrated that the decrease in MF CSA under suspension is accompanied by a decrement of the myonuclei number per MF (that is, the myonuclear number), which is in agreement with the data of other authors. Thus, the decrease in myonuclear number is observed also under the atrophy caused by the removal of the sensory ganglia isolation [5], by space flight [29,36], and by chronic denervation [37]. We demonstrated that 2 weeks of m. soleus passive stretching did not cause any changes in the myonuclear number, as compared to the control level. Muscle stretching is a component of eccentric exercise, which is accompanied by IGF-1 synthesis. This is the reason why the result obtained by the Allen group [38] which showed that everyday trainings combined with IGF-1 injections significantly delay the decrease in CSA and myonuclei number under suspension. The use of irradiation for satellites proliferation blocking prevents functional hypertrophy [21]. At the same time, Lowe and Alway [25] showed that in spite of the fact of active SC proliferation, hypertrophy caused by stretching of the muscles of Japanese quail wings developed even in the absence of proliferating satellites. Our experiment demonstrated that irradiation did not prevent MF CSA maintenance under chronic stretching combined with suspension. At that, the myonuclear number in the group “SIS” decreased twice, as compared to the unirradiated “SS” group. The mechanical tension combined with irradiation led to development of myofiber nuclear pool destruction. Consequently, we can suppose that the myonuclei number is not the essential factor for myofiber size maintenance under stretching. Probably, there are some other mechanisms based on transcription–translation

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processes take place in this situation. The latter supposition is based on our results demonstrating the twofold increase in nuclear domain after stretching combined with irradiation. The conception of nuclear domain as myofiber cytoplasm volume guided by myonucleus gene expression products was firstly introduced in works of Cheek and colleagues [39]. Regardless of the fact that the nuclear domain conception is quite relative and the fact that the myonucleus gene expression and proteins distribution in the MF depends on many variable values, this term is convenient for muscle plasticity description. So, the muscle hypertrophy development can be caused by nuclear domain increase (at the expense of satellites nuclei incorporation), and by ingenuous myofiber size induced by myofibers nuclei activation. Experimental works based on the morphometric analysis of nucleus/cytoplasm ratio [40] showed that the working and functional hypertrophy is most probably explained by the increase of nuclear domains number (myofiber size is stable), which is caused by satellites activation. The data obtained evidenced that the processes in a stretched m. soleus differed from those developing under working hypertrophy. Under muscle stretching combined with gravitational unloading the nuclear domain size stayed at the control level. Myofiber size maintenance under stretching combined with irradiation is accompanied by myonuclear domain increase. This can be explained by enhancement of protein synthesis in the MF itself. Some authors [16,35] demonstrated the enhancement of proliferative activity of SC under stretching. We observed the increase in the number of cells expressing NCAM (CD 56) under muscle stretching of suspended animals. However, there is no literature data concerning connection of this molecule expression with some specific phase of SC life cycle. In irradiated animals under stretching combined with suspension the number of SC per fiber increased as compared to suspended animals. Probably, we can explain it by the ability of small part of them to accomplish mitosis after the irradiation. These cells could be uninjured by irradiation or could finish the process of DNA repair [41]. Moreover, migration of the progenitor cells from the pool cannot be excluded [42]. It should be noted, that the SC number in rats subjected to suspension combined with stretching of m. soleus after the irradiation was markedly lower in comparison with the unirradiated animals and was not higher than in rats of the control group. Thus, we demonstrated that stretching of rat m. soleus combined with gravitational unloading did not lead to

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