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Degenerative muscle fiber accelerates adipogenesis of intramuscular cells via RhoA signaling pathway
ARTICLE IN PRESS Differentiation 77 (2009) 350–359
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Degenerative muscle fiber accelerates adipogenesis of intramuscular cells via RhoA signaling pathway Tohru Hosoyama, Naomi Ishiguro, Keitaro Yamanouchi , Masugi Nishihara Department of Veterinary Physiology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan
a r t i c l e in f o
a b s t r a c t
Article history: Received 14 August 2008 Received in revised form 13 November 2008 Accepted 14 November 2008
In some pathological conditions such as Duchenne muscular dystrophy, it has been known that a fatty infiltration in skeletal muscle is often observed and that is also one of primary factors to induce marked decline of muscular strength. However, the mechanism of fatty infiltration, cellular origin of accumulated adipocytes and its significance are not fully understood. The fact that persistent degenerative muscle fibers are present on dystrophic muscle leads us to hypothesize that muscle fiber condition affects fatty infiltration in skeletal muscle. We employed a single fiber culture system to determine whether fiber condition affects an appearance of adipocytes on the fibers. Artificially hypercontracted muscle fibers (HCF), generated from isolated intact fibers (IF) of rat extensor digitrum longus muscle, were maintained as non-adherent cultures for 5–7 days. Interestingly, there appeared to be considerable numbers of mature adipocytes on HCF, whereas no adipocytes were seen on IF, indicating that cells on HCF spontaneously differentiated into mature adipocytes. Activation of RhoA signaling by the addition of thrombin decreased the number of adipocytes on HCF in a dose-dependent manner, whereas the number of MyoD-positive myoblasts increased. In contrast, Y-27632, a specific inhibitor of Rho kinases (ROCK), induced adipogenic differentiation of cells derived from IF. In addition, administration of Y-27632 into mouse regenerating muscle resulted in fat accumulation in the muscle. Taken together, the present studies clearly demonstrated that muscle fiber condition affects fat accumulation in skeletal muscle and that is possibly mediated by the RhoA signaling pathway. & 2008 International Society of Differentiation. Published by Elsevier Ltd. All rights reserved.
Keywords: Adipogenesis Single fiber culture Rho Rho kinase (ROCK) Thrombin Y-27632
1. Introduction In adult skeletal muscle, there are several types of stem cells such as satellite cell, muscle side-population (SP) cells and muscle-derived stem cells (MDSCs), and these intramuscular stem cells are able to differentiate into adipogenic and osteogenic cells in addition to myogenic cells when they are cultured in each optimal conditions (Lee et al., 2000; Asakura et al., 2001, 2002; Wada et al., 2002; Uezumi et al., 2006). Fat accumulation in skeletal muscle is often seen in patients with X-chromosome-linked recessive muscular dystrophy such as Duchenne muscular dystrophy (DMD), and is one of the primary factors leading to marked decline of muscular strength (Tyler, 2003). Fragility of muscle fibers caused by loss of dystrophin induces rapid influx of Ca2+ into skeletal muscle fiber, resulting in hypercontraction of muscle fibers and activation of proteases such as Calpain (Carpenter and Karpati, 1979). Moreover, repeated degeneration and regeneration in dystrophic muscle finally results in loss of muscle fibers, and adipocytes fill up in the area
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E-mail address: [email protected] (K. Yamanouchi).
where muscle fibers are lost (Cullen and Mastaglia, 1980). Similarly, fat accumulation in skeletal muscle is also observed in atrophic muscle of elderly people and experimentally denervatedmuscle (Dulor et al., 1998; Song et al., 2004). In general, maintenance and fate specification of embryonic and somatic stem cells are affected by components of their surrounding microenvironments such as soluble factors, neighboring cells and extracellular matrices (ECM), and particular microenvironments are necessary for each stem cell type to be specified appropriately (Schofield, 1978). In Drosophila, for example, Decapentaplegic (Dpp), a BMP2/4 homolog, is required to maintain germline stem cells in the ovary and regulates their division, and an anchoring germline stem cell to a neighboring cap cells in the ovary mediated by DE-cadherin is important for stem cell maintenance and function (Song et al., 2002; Xie and Spradling, 1998). In addition, substrate elasticity of ECM also affects fate specification of human mesenchymal stem cells (Engler et al., 2006). Csete et al. (2001) demonstrated that oxidative stress induces adipogenic differentiation of muscle fiber associating cells using single muscle fiber culture. More recently, furthermore, Brack et al. (2007) demonstrated that aged microenvironment induces a conversion of satellite cells from myogenic to fibrogenic lineage, and this conversion is mediated by
0301-4681/$ - see front matter & 2008 International Society of Differentiation. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.diff.2008.11.001
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modification of Wnt signaling. Taken together, these results clearly indicate that microenvironment in skeletal muscle affects lineage specification of intramuscular cells. RhoA is a member of the small GTPase Rho family including Rho, Rac, and Cdc42, and it is widely known that RhoA is associated with organization of the cytoskeleton (Hall, 1998; Ridley and Hall, 1992; Ridley et al., 1992). The RhoA signaling pathway is regulated via cell surface receptors such as G-protein coupled receptors (GPCRs), and is activated by the interaction between the receptors and ECM and/or soluble factors (Ren et al., 1999). In myogenic cell lines such as C2C12 and L6 myoblasts, the RhoA signaling pathway participates in myotube formation (Carnac et al., 1998; Castellani et al., 2006; Charrasse et al., 2006; Reuveny et al., 2004; Wei et al., 1998). These observations suggest that RhoA functions in myogenic cells. However, whether RhoA is also expressed in intramuscular stem cells including satellite cells and plays a role in fate specification of these cells is not clear so far. Recent studies showed that inhibition of the Rho signaling pathway by a chemical inhibitor or a microgravity condition induces adipogenic differentiation of human mesenchymal stem cells (McBeath et al., 2004; Meyers et al., 2005). In addition, mouse embryo-derived fibroblasts (MEFs) lacking p190-B RhoGAP, a Rho inhibitory protein, do not undergo adipogenic differentiation. On the other hand, deletion of p190-B RhoGAP in MEFs induces differentiation into myogenic cells in response to IGF-I (Sordella et al., 2003). These observations suggest that the Rho signaling pathway is involved in myogenesisadipogenesis determination of mesenchymal cells. Although the mechanisms of fatty infiltration in skeletal muscle and the cellular origin of accumulated adipocytes in some pathological conditions are unclear, the fact that degenerative or atrophic fibers are persistently present in fat accumulated-muscle indicates the possibility that muscle fiber condition would be one of the niches affecting lineage specification of intramuscular cells. So, we hypothesize that there is the interaction between muscle fiber condition and fat accumulation in muscle, and this interaction is mediated by RhoA signaling pathway.
2. Materials and methods 2.1. Animals Male Wistar-Imamichi rats (2–4 months old) were purchased from the Institute for Animal Reproduction (Ibaraki, Japan). Male C57BL/6J mice (8-weeks old) were purchased from Charles River Laboratories (Yokohama, Japan). They were housed in a temperature-controlled room with a 14 h light and 10 h dark cycle (light on at 5:00 am). Food and water were provided ad libitum. All animal experiments in this study were performed according to the Guideline for the Care and Use of Laboratory Animals, The University of Tokyo. 2.2. Single fiber culture Single fiber culture was carried out according to Bischoff (1986), and Shefer and Yablonka-Reuveni (2005). Briefly, single muscle fibers with attached satellite cells were isolated from the hindlimb extensor digitrum longus (EDL) muscle of adult male rats. The EDL muscle was carefully removed at the tendon and treated with 0.2% (w/v) collagenase type I (Sigma, MO) reconstituted in Dulbecco’s Eagle Medium (DMEM) (Invitrogen, CA) at 37 1C for 90 min with slight shaking. After several gentle triturations with a wide-bore pipette, the muscle was further treated with enzyme at 37 1C for 10 min. The muscle was transferred into fresh
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10% FBS/DMEM, and fine fibers were liberated from the muscle by gentle trituration with a wide-bore pipette. Then, fine fibers were treated with vigorous pipetting to induce contraction of fibers. Fibers were sequentially washed three times in DMEM to remove interstitial and endothelial cells. Hereafter, fine fibers and contracted fibers are referred to as intact fibers (IF) and hypercontracted fibers (HCF), respectively. Both fibers were maintained as non-adherent cultures in 10% FBS/DMEM on 24-well noncoated culture plates (IWAKI, Tokyo, Japan) for 5–7 days, and then stained for specific markers. 2.3. Primary antibodies The following primary antibodies were used: Anti-Pax7, mouse monoclonal (1:250 dilution; Developmental Studies Hybridoma Bank (DSHB), IA); anti-MyoD1, mouse monoclonal (1:100; 5.8A, Novocastra, Newcastle upon Tyne, UK); anti-PPARg2 mouse monoclonal (1:200; Santa Cruz, CA); anti-laminin, rabbit polyclonal (1:1000; Sigma); anti-myosin heavy chain (1:20; MF20, DSHB), anti-RhoA, mouse monoclonal (1:500; 26C4, Santa Cruz); anti-M-cadherin antibody rabbit polyclonal (1:10 000; kindly gifted from Dr. Shin’ichi Takeda, National Center of Neurology and Psychiatry, Tokyo, Japan). 2.4. Immunocytochemistry Cells on fibers and culture dishes were fixed with 4% paraformaldehyde (PFA) in phosphate buffed saline (PBS) for 15 min at room temperature (RT). Cells were washed with PBS and incubated in 5% normal goat serum (NGS; Zymed, CA) and 0.1% Triton-X (Sigma) in PBS to block non-specific binding of antibodies. After cells were washed in PBS, primary antibodies were applied, and cells were incubated overnight at 4 1C. After washing cells with PBS, Alexafluor 594 or 488 conjugated antimouse IgG (1:400 diluted with 5% NGS in PBS; Invitrogen) was added and incubated for 1 h at RT. Cell nuclei were counterstained with Hoechst 33258. In some experiments, mature adipogenic cells were visualized by Oil-Red O (OR) staining after immunostaining. Briefly, cells fixed with 4% PFA were stained in OR solution (Sigma, 0.5% (w/v)) for 7 min at RT, then washed in PBS. Quantitative analyses of cells detached from single muscle fiber were performed. Total number of cells and number of cells on the dish positive for anti-PPARg antibody, MyoD antibody and OR, obtained from seven randomly chosen different microscopic fields using a 20 objective, were counted. Cell number and percentage of positive cells were averaged for the triplicate culture wells. Alternatively, number of positive cells for antiMyoD and -PPARg antibodies, and number of OR-positive cells on fiber obtained from 10 fibers chosen randomly per well, were counted for the triplicate wells. 2.5. Thrombin and Y-27632 administration Thrombin (Sigma) was dissolved in DMEM and added into 10% FBS/DMEM medium (1, 5 and 10 units/500 ml of medium). HCF were cultured in thrombin-containing medium during the first 3 days and then cultured in 10% FBS/DMEM for 4 additional days. Number of MyoD-positive myogenic cells and OR-positive adipogenic cells on fibers and dish were counted at the end of culture (day 7). Fusion index was estimated from number of nuclei in MHC-positive cells per all nuclei in the field at day 10. Y-27632 solution (10 mM, Calbiochem, CA), a specific inhibitor of ROCK (Ishizaki et al., 2000), was added to 10% FBS/DMEM during the first 3 days to inhibit intracellular RhoA signaling, and the fiber was removed from the well on day 4. Cells detached from
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IF were cultured in 10% FBS/DMEM for 7 days and stained with anti-PPARg2 antibody and OR. Thrombin and Y-27632 were concurrently added to HCF culture during the first 3 days in order to contradict an effect of thrombin administration, and then HCF was cultured for 7 days. Number of OR-positive cells and MyoD-positive cells on HCF were counted at the end of culture (day 7). 2.6. Y-27632-injection into mouse muscle Y-27632 dihydrochloride monohydrate (Sigma) was reconstituted with sterile saline. Fifty microliters of 0.5% bupivacainehydrochloride (BPVC, Sigma) were injected into the TA muscle of both legs of 8-week-old mice to induce injury and activation of satellite cells. Immediately after BPVC injection, Y-27632 (20 ml of 41.7 mg/ml) or equivalent volume of saline was injected into the injured muscle. Y-27632 injection was performed three times (0, 24 and 48 h after BPVC injection). Muscle from each leg was dissected at day 7 after BPVC injection and snap-frozen in an isopentane bath cooled by liquid nitrogen. Frozen sections (7-mm thick) prepared from Y-27632-injected and saline-injected TA muscle were stained with hematoxylin and OR. The degree of fat accumulation was quantitatively analyzed by measuring the OR-stained fat area within the regenerating area. Regenerating area was defined by the presence of newly generated fibers. Quantification of the fat area was done using NIH Image software (Ver. 1.62, NIH). 2.7. Statistical analysis The data are expressed as means 7SE. Statistical analyses were performed using commercial software (Statview, Ver. 4.5, Abacus Concepts, Inc, Barkley, CA). Student’s t-test was used to evaluate
statistical differences between control and treated groups. P values less than 0.05 were considered as statistically significant.
3. Results 3.1. Muscle fiber condition affects lineage specification of intramuscular cells We used single fiber culture system to examine whether muscle fiber condition induces adipogenic differentiation of cells on muscle fibers. Although proteolytically isolated fine muscle fibers are free of connective tissue, capillaries, there are quiescent satellite cells beneath the basement membrane (Bischoff, 1986). Single muscle fibers were isolated from rat EDL muscle, and fine fibers (Intact Fiber; IF) were selected among the isolated fibers. Subsequently, collected IF were contracted by pipetting (hypercontracted fibers; HCF) to evaluate the influence of muscle fiber condition on lineage specification of intramuscular cells. Both IF and HCF were maintained as non-adherent cultures in 10% FBS/ DMEM (Fig. 1A) and cultured for 5 days. Pax7-positive satellite cells were present on both IF and HCF just after plating (Fig. 1B). Immunocytochemistry of laminin, one of the major components of basal lamina, was performed to confirm the structural condition of basal lamina. Laminin was distributed on the entire surface of IF, whereas it was scattered on HCF, indicating that the proper structure of basal lamina was disrupted on HCF (Fig. 1C). When IF and HCF were stained with OR on day 5 of culture, ORstained mature adipocytes were only seen in abundance on HCF (Fig. 1D). In order to examine changes over time, number of MyoD-positive cells and PPARg-positive cells on both IF and HCF were counted every day after plating. MyoD-positive cells were continuously present on IF during the culture period, while there
Fig. 1. Cells on hyper-contracted fibers spontaneously differentiate into adipogenic cells: (A) Isolated single muscle fibers (intact fiber; IF) from rat EDL muscle were contracted by pipetting (hyper-contracted fiber; HCF) and maintained as non-adherent cultures. Scale bar, 200 mm. (B) Immunocytochemistry of Pax7 on day 0. Arrowheads indicate Pax7-positive satellite cells on both IF and HCF. Scale bar, 400 mm. (C) Laminin localization of both IF and HCF. Both fibers were stained with anti-laminin antibody on day 0. Scale bar, 200 mm. (D) Fibers were stained with Oil-Red O (OR) on day 5. Many mature adipocytes were present on HCF but not on IF. Boxes indicate OR-negative non-adipogenic cells (IF) and OR-positive adipogenic cells (HCF), respectively. Scale bar, 400 mm.
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Fig. 2. Time-dependent change of number of myogenic and adipogenic cells on muscle fibers: Number of myogenic (MyoD-positive) and adipogenic (PPARg-positive) cells on both IF (left panel) and HCF (right panel) were counted on days 0, 1, 2, 3, 4 and 5. Graphs represent MyoD (~) and PPARg (&), respectively. At each time point, n ¼ 20 (IF) and n ¼ 30 (HCF), respectively.
Fig. 3. Forced activation of the RhoA signaling pathway by thrombin administration inhibits adipogenesis of intramuscular cells on HCF: Thrombin (Thr; 1, 5, 10 U/ 500 ml) was added to HCF culture medium during the first 3 days, and HCF were stained with OR on day 7 after plating. (A) Arrowheads indicate OR-positive adipogenic cells. Scale bar, 200 mm. (B) OR-positive cells on fibers were counted and graphed for each group. Each experimental group was prepared with t wells, and 10 fibers per well were randomly chosen. Number of positive cells was counted (n ¼ 10 3 wells). Values represent mean 7SE. *Po0.05 vs control (Ctl).
were very few or no PPARg-positive cells (Fig. 2A). In contrast, very few, if any, MyoD-positive cells were seen on HCF throughout the culture period, while PPARg-positive cells first appeared on
Fig. 4. Forced activation of the RhoA signaling pathway by thrombin administration induces myogenesis of intramuscular cells on HCF: (A) MyoD-positive cells on HCF were increased dose-dependently by thrombin administration. Arrowheads indicate MyoD expressing cells. Scale bar, 200 mm. (B) Graphs represent number of MyoD-expressing cells on fibers. Values represent mean7SE (n ¼ 10 3 wells). *Po0.05 vs control (Ctl).
day 3, and their number increased as the culture period progressed (Fig. 2B). These results indicate that fate specification of cells on muscle fibers could be altered by muscle fiber
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condition, and cells on hyper-contracted fibers spontaneously differentiate into adipogenic cells.
3.2. RhoA signaling pathway is associated with lineage specification of intramuscular cells To elucidate the signaling pathway mediating the fate specification of cells on muscle fibers affected by muscle fiber condition, we focused on the RhoA signaling pathway since this pathway may act as a molecular switch for fate specification of mesenchymal cells (McBeath et al., 2004; Meyers et al., 2005; Sordella et al., 2003). We hypothesized that the Rho signaling pathway is associated with fate specification of intramuscular cells, and this pathway is inhibited in cells on HCF, resulting in adipogenic differentiation of these cells. Because the RhoA signaling pathway is activated by thrombin (Ren et al., 1999), we examined whether activation of the RhoA signaling pathway with thrombin could alter the fate specification of cells on HCF. HCF were treated with thrombin for the initial 3 days, the cells on the fibers were stained with anti-MyoD antibody and OR on day 7 of culture. Thrombin dose-dependently inhibited the appearance of OR-positive cells on HCF (Fig. 3). On the other hand, thrombin administration increased MyoD-positive cells on HCF in a dose-dependent manner (Fig. 4). These results indicate that forced activation of the RhoA signaling pathway inhibits adipogenesis of cells on HCF, and conversely accelerates myogenesis. Also, cells on culture dish migrated from HCF were promoted to myogenesis by thrombin administration, whereas adipogenesis were inhibited (Fig. 5). To confirm whether myoblasts on the dish which were generated by thrombin administration has an ability of myotube formation, furthermore, we carried out
immunocytochemistry for myosin heavy chain (MHC) at day 10 after HCF plating. There were a lot of MHC-positive cells on the dish treated with thrombin, indicating that myoblasts resulted from forced activation of RhoA signaling has normal myogenic property and are able to proceed terminal myogenic differentiation (Fig. 6). Activated RhoA (RhoA-GTP) affects its downstream target molecules such as Rho kinases (ROCK) and mDia, resulting in the progression of later signaling pathways (Matsui et al., 1996; Watanabe et al., 1997, 1999). In addition, activation of the RhoA signaling pathway is shown to promote myogenic specification of mesenchymal stem cells via ROCK-dependent pathway (McBeath et al., 2004; Sordella et al., 2003). Therefore, we administered ROCK inhibitor, Y-27632, to examine whether inhibition of the RhoA-ROCK signaling pathway in cells could have any influence on fate specification of cells derived from IF. IF were treated with Y-27632 for the first 3 days, and cells that detached from IF and migrated onto the culture dish were stained with anti-PPARg antibody and OR on day 7. In our experiment, Y-27632-treated IF were often unexplainably hyper-contracted after day 4 of culture, and it was impossible to maintain IF in the presence of Y-27632 treatment. Therefore, fibers were removed from culture on day 4, and only cells that had detached from IF and migrated onto the dish before day 4 of culture were examined for further analysis. Treatment of IF with Y-27632 resulted in the appearance of ORpositive cells, while there were almost no OR-positive cells in the control culture (Fig. 7). A small portion of cells that migrated from IF in the control culture were slightly positive for PPARg, but they were not stained with OR. On the other hand, in the Y-27632 treated culture, PPARg staining was much more intense than in the control culture, and the number of PPARg-positive cells was increased. In addition, some PPARg-positive cells were co-stained
Fig. 5. Forced activation of RhoA signaling pathway accelerates myogenesis and inhibits adipogenesis of cells migrated off into the dish: (A) MyoD-positive cells migrated from HCF into the dish were increased dose-dependently by thrombin administration. Arrowheads indicate same cells. Scale bar, 200 mm. (B) OR-positive cells on the dish were decreased dose-dependently thrombin administration. Scale bar, 200 mm. (C) Graphs represent percentage of MyoD-positive cells (white bars) and OR-positive cells (black bars) on the dish. Each experimental group was prepared with triplicate wells. Values represent mean 7SE (n ¼ 3 wells). *,]Po0.05 vs control (Ctl).
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Fig. 6. Cells treated with thrombin normally pass through myotube formation on the dish: (A) Cells treated with thrombin (10 U) were cultured for 10 days and stained with anti-myosin heavy chain antibody (MHC). Scale bar, 200 mm. (B) Graphs represent fusion index after 10 days culture. Values represent mean 7SE (n ¼ 3 wells). * Po0.05 vs control.
with OR (Fig. 7). These results suggest that inhibition of ROCK, one of the downstream signaling pathways of RhoA, stimulates cells on muscle fibers to enter adipogenesis. Furthermore, we carried out co-administration of Y-27632 and thrombin to HCF in order to confirm whether RhoA signaling pathway is actually concerned with fate specification of intramuscular cells. Both Y-27632 and thrombin were concurrently added to HCF culture for the first 3 days, and cells on HCF were stained with OR and anti-MyoD antibody at day 7 after plating. Co-administration of Y-27632 and thrombin resulted in a significantly increase of OR-positive cells on HCF as compared to single thrombin administration (Fig. 8A). On the other hand, however, number of MyoD-positive cells on HCF was not changed by co-administration (Fig. 8B). These results suggest that effect of thrombin administration is negated by the specific ROCK inhibitor at least in case of adipogenic differentiation of the cells and RhoA signaling pathway through ROCK is participated in fate specification of intramuscular cells.
3.3. Effect of in vivo administration of Y-27632 on regenerating muscle The above findings indicate that fate specification of cells in skeletal muscle could be altered by modulating the RhoA signaling pathway, and inhibition of ROCK leads to spontaneous adipogenesis of these cells. Direct injection of Y-27632 into mouse regenerating muscle was performed in order to examine if this could be mimicked in vitro. Mouse TA muscles were injured by
BPVC injection, and Y-27632 was injected into the injured-region. Muscles were collected at day 7 post-injury and stained with OR. Fat accumulation was evident in the regenerating area of Y-27632treated muscle, whereas it was barely detected in the saline control (Fig. 9A and B). These results suggest that inhibition of ROCK leads to alteration of differentiation fate of intramuscular cells and induces their spontaneous adipogenesis in vivo.
4. Discussion The origin of adipocytes in some pathological muscle such as dystrophic muscle and the mechanism that leads to accumulation of adipocytes are not entirely clear. In the present study, we used a single fiber culture system to demonstrate that many adipocytes are present on cultured HCF, whereas very few, if any, adipocytes are seen on IF. Dystrophic muscle is hyper-contracted by the rapid influx of Ca2+ into the muscle fiber (Carpenter and Karpati, 1979), and muscle fiber hypercontraction can be induced experimentally by BPVC treatment (Nonaka et al., 1983). Previously, we demonstrated that cells from regenerating muscle treated with BPVC increase in adipogenicity, due to a lack of Pref-1 expression (Yamanouchi et al., 2006). In addition, intramuscular cells such as satellite cells are able to give rise to adipogenic cells by hormonal stimulation in vitro (Teboul et al., 1995; Yada et al., 2006), and Shefer et al. (2004) recently used in vitro clonal analyses to demonstrate that satellite cells are able to enter a mesenchymal alternative differentiation (MAD) route, resulting in differentiation to
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Fig. 7. Inhibition of the RhoA signaling pathway by ROCK inhibitor induces adipogenesis of satellite cells: ROCK specific inhibitor, Y-27632, was added to IF culture medium during the first 3 days, and fiber was removed from the well on day 4. (A) Cells on dish were stained with anti-PPARg antibody and OR on day 7. Arrowheads indicate PPARgpositive cells. Scale bar, 200 mm. (B) Graphs show percentage of PPARg-positive cells on dish. Each experimental groups were prepared with triplicate wells. Values represent mean7SE (n ¼ 3). *Po0.05 vs control.
Fig. 8. Effect of thrombin is partially inhibited by Rho specific inhibitor: Thrombin (10 U) and a specific inhibitor of ROCK, Y-27632 (10 mM), were concurrently added into HCF culture for first 3 days. OR- and MyoD-positive cells on fibers were counted at day 7 after plating. (A) Graphs represent number of OR-positive cells on fibers. Each experimental group was prepared with 3 wells, and 10 fibers per well were randomly chosen. Number of positive cells was counted. Values represent mean7SE (n ¼ 10 3 wells). *Po0.05 vs Thrombin (Thr). (B) Graphs represent number of MyoD-positive cells on fibers. Values represent mean 7SE (n ¼ 10 3 wells).
non-myogenic lineages such as adipogenic and smooth musclelike cells. Therefore, taken together with these studies, the results obtained in the present study suggest that hypercontraction of
muscle fibers could be the principle signal to induce adipogenesis, and intramuscular cells-derived adipocytes may contribute to fat accumulation in skeletal muscle.
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Fig. 9. Induction of fat accumulation in regenerating mouse muscle by direct injection of Y-27632: Mouse TA muscle was injured by BPVC injection, and Y-27632 was injected into the injured area. On day 7 after injection, regenerating muscle was dissected and stained with OR. (A) Fat accumulation was seen in the regenerating area of Y27632-treated muscle (right panel), whereas it was hardly seen in the saline control (left panel). Nuclei were stained with hematoxylin. Scale bar, 200 mm. (B) Graphs show percentage of fat area with in regenerating muscle. Values represent mean 7SE (n ¼ 3). *Po0.05 vs control.
It should be mentioned that although BPVC treatment is shown to transiently increase the adipogenicity of cells in skeletal muscle (Yamanouchi et al., 2006), BPVC-treated muscle regenerates completely, and fat accumulation is never seen either during or at the end of regeneration. In BPVC-treated muscle, hypercontracted fibers degenerate and are necrotized. These necrotic fibers are removed by macrophages migrating into the degenerated area, resulting in the loss of association between intramuscular cells including satellite cells and degenerated fibers. CC chemokine receptor 2 (CCR2) plays a role in recruitment of macrophages to the injured site, and CCR2-/- mice exhibit altered muscle regeneration and inflammation, as well as are deficient of intramuscular macrophages. Interestingly, fat accumulation is seen in damaged muscle of CCR2-/- mice (Contreras-Shannon et al., 2007). Hence, taken together with our observation that mature adipocytes were seen only when cells were cultured on HCF, the persistent presence of degenerative fibers in damaged muscle may support terminal adipogenic differentiation of intramuscular cells, thus leading to the appearance of mature adipocytes. In the present study, we could not clearly identify the origin of adipocytes on HCF. In this point, however, we suppose that one of origins of adipocytes on HCF is satellite cells, because MyoDpositive myogenic cells were hardly detected on HCF after 5–7 days in spite of the presence of satellite cells on HCF just after plating and this suggests that satellite cells on the fibers did not differentiate into myogenic cells and differentiate into adipogenic cells. We demonstrated using competitive reagents that effect of thrombin was negated by Y-27632 treatment and number of adipogenic cells was increased by an inhibition of thrombin effect, strongly suggesting that thrombin actually activates RhoA signaling pathway via ROCK in cells on HCF and consequently myogenesis was induced. On the other hand, however, number of myogenic cells on HCF was not affected by concurrently administration of Y-27632 and thrombin. In this point, we
suppose that ROCK-independent RhoA signaling pathway such as mDia is also participated in fate specification of intramuscular cells, because knockdown of mDia in C2C12 cells results in decrease of MyoD expression (Gopinath et al., 2007). In addition, our results suggested that muscle fiber condition affects the RhoA signaling pathway in intramuscular cells. And, results from in vivo study that Y-27632 was directly injected to mouse regenerating muscle are consistent with the results from in vitro study. However, the mechanism associating the alteration of muscle fiber condition to changes in RhoA activity and the origin of adipocytes are unknown so far. At that present time, however, we speculate that modification of RhoA signaling might be happened in satellite cells and this resulted in appearance of adipocytes. Quiescent satellite cells express laminin receptor, a7b1-integrin (LaBarge and Blau, 2002), and majority of b1-integrin-positive cells (490%) on single myofiber express Pax7 (Cerletti et al., 2008). Based on immunocytochemial localization of laminin, the structure of basal lamina of HCF is thought to be disrupted. It is widely recognized that the RhoA signaling pathway is activated by integrin via focal adhesion kinase (Palazzo et al., 2004; Zhai et al., 2003), and disruption of basal lamina may induce loss of this interaction if satellite cells directly interact with muscle fiber through laminin-integrin association. In addition, we also confirmed that quiescent satellite cells (M-cadherin-positive cells) express RhoA (Supplementary Fig. S1). Taken together, it is plausible that the direct interaction between satellite cells and muscle fiber through integrin is necessary for satellite cells to differentiate into myogenic lineage, and that satellite cells differentiate into adipogenic lineage when this interaction is disrupted, thus leading to an inactivation of RhoA signaling in satellite cells. However, we have to carry out further examination to prove our hypothesis, because there are no evidences supporting it so far. And also, our in vivo study indicates the possibility that Y-27632 does not directly affects intramuscular
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cells and hypercontraction of muscle fibers by Y-27632 administration results in adipogenesis of the cells on fiber, because Y-27632 may induce hypercontraction of muscle fibers. So, in further examination, we also have to prove whether Y-27632 actually inhibits RhoA signaling pathway in intramuscular cells. In summary, our findings indicate that degenerative muscle fiber accelerates fat accumulation in skeletal muscle. In addition, it was suggested that the RhoA signaling pathway is affected by muscle fiber condition and is associated with adipogenic differentiation of intramuscular cells. Although further examination is necessary to clarify the exact nature of factors that directly connect muscle fiber condition to intracellular RhoA signaling in fate specification of intramuscular cells, the present findings will be important in elucidating the mechanism of fatty infiltration in some pathological muscle conditions, and for therapeutic applications such as muscular dystrophy.
Acknowledgements We thank Dr. Yousuke Murakami (The University of Tokyo) for his helpful advices. The anti-Pax7 and anti-Myosin heavy chain monoclonal antibodies were obtained from the Developmental Studies Hybridoma Bank developed under the NICHD and maintained by The University of Iowa. This work was supported by grants to M.N and K.Y from the Japan Society for the Promotion of Science, and to K.Y from Ministry of Health, Labor and Welfare of Japan and Program for Promotion of Basic Research Activities for Innovative Biosciences (PROBRAIN). This paper is dedicated to late Professor Chikashi Tachi.
Appendix A. Supporting Information Supplementary data associated with this article can be found in the online version at doi:10.1016/j.diff.2008.11.001.
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Differentiation journal homepage: www.elsevier.com/locate/diff
Degenerative muscle fiber accelerates adipogenesis of intramuscular cells via RhoA signaling pathway Tohru Hosoyama, Naomi Ishiguro, Keitaro Yamanouchi , Masugi Nishihara Department of Veterinary Physiology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan
a r t i c l e in f o
a b s t r a c t
Article history: Received 14 August 2008 Received in revised form 13 November 2008 Accepted 14 November 2008
In some pathological conditions such as Duchenne muscular dystrophy, it has been known that a fatty infiltration in skeletal muscle is often observed and that is also one of primary factors to induce marked decline of muscular strength. However, the mechanism of fatty infiltration, cellular origin of accumulated adipocytes and its significance are not fully understood. The fact that persistent degenerative muscle fibers are present on dystrophic muscle leads us to hypothesize that muscle fiber condition affects fatty infiltration in skeletal muscle. We employed a single fiber culture system to determine whether fiber condition affects an appearance of adipocytes on the fibers. Artificially hypercontracted muscle fibers (HCF), generated from isolated intact fibers (IF) of rat extensor digitrum longus muscle, were maintained as non-adherent cultures for 5–7 days. Interestingly, there appeared to be considerable numbers of mature adipocytes on HCF, whereas no adipocytes were seen on IF, indicating that cells on HCF spontaneously differentiated into mature adipocytes. Activation of RhoA signaling by the addition of thrombin decreased the number of adipocytes on HCF in a dose-dependent manner, whereas the number of MyoD-positive myoblasts increased. In contrast, Y-27632, a specific inhibitor of Rho kinases (ROCK), induced adipogenic differentiation of cells derived from IF. In addition, administration of Y-27632 into mouse regenerating muscle resulted in fat accumulation in the muscle. Taken together, the present studies clearly demonstrated that muscle fiber condition affects fat accumulation in skeletal muscle and that is possibly mediated by the RhoA signaling pathway. & 2008 International Society of Differentiation. Published by Elsevier Ltd. All rights reserved.
Keywords: Adipogenesis Single fiber culture Rho Rho kinase (ROCK) Thrombin Y-27632
1. Introduction In adult skeletal muscle, there are several types of stem cells such as satellite cell, muscle side-population (SP) cells and muscle-derived stem cells (MDSCs), and these intramuscular stem cells are able to differentiate into adipogenic and osteogenic cells in addition to myogenic cells when they are cultured in each optimal conditions (Lee et al., 2000; Asakura et al., 2001, 2002; Wada et al., 2002; Uezumi et al., 2006). Fat accumulation in skeletal muscle is often seen in patients with X-chromosome-linked recessive muscular dystrophy such as Duchenne muscular dystrophy (DMD), and is one of the primary factors leading to marked decline of muscular strength (Tyler, 2003). Fragility of muscle fibers caused by loss of dystrophin induces rapid influx of Ca2+ into skeletal muscle fiber, resulting in hypercontraction of muscle fibers and activation of proteases such as Calpain (Carpenter and Karpati, 1979). Moreover, repeated degeneration and regeneration in dystrophic muscle finally results in loss of muscle fibers, and adipocytes fill up in the area
Corresponding author. Tel.: +81 3 5841 5387; fax: +81 3 58418017.
E-mail address: [email protected] (K. Yamanouchi).
where muscle fibers are lost (Cullen and Mastaglia, 1980). Similarly, fat accumulation in skeletal muscle is also observed in atrophic muscle of elderly people and experimentally denervatedmuscle (Dulor et al., 1998; Song et al., 2004). In general, maintenance and fate specification of embryonic and somatic stem cells are affected by components of their surrounding microenvironments such as soluble factors, neighboring cells and extracellular matrices (ECM), and particular microenvironments are necessary for each stem cell type to be specified appropriately (Schofield, 1978). In Drosophila, for example, Decapentaplegic (Dpp), a BMP2/4 homolog, is required to maintain germline stem cells in the ovary and regulates their division, and an anchoring germline stem cell to a neighboring cap cells in the ovary mediated by DE-cadherin is important for stem cell maintenance and function (Song et al., 2002; Xie and Spradling, 1998). In addition, substrate elasticity of ECM also affects fate specification of human mesenchymal stem cells (Engler et al., 2006). Csete et al. (2001) demonstrated that oxidative stress induces adipogenic differentiation of muscle fiber associating cells using single muscle fiber culture. More recently, furthermore, Brack et al. (2007) demonstrated that aged microenvironment induces a conversion of satellite cells from myogenic to fibrogenic lineage, and this conversion is mediated by
0301-4681/$ - see front matter & 2008 International Society of Differentiation. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.diff.2008.11.001
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modification of Wnt signaling. Taken together, these results clearly indicate that microenvironment in skeletal muscle affects lineage specification of intramuscular cells. RhoA is a member of the small GTPase Rho family including Rho, Rac, and Cdc42, and it is widely known that RhoA is associated with organization of the cytoskeleton (Hall, 1998; Ridley and Hall, 1992; Ridley et al., 1992). The RhoA signaling pathway is regulated via cell surface receptors such as G-protein coupled receptors (GPCRs), and is activated by the interaction between the receptors and ECM and/or soluble factors (Ren et al., 1999). In myogenic cell lines such as C2C12 and L6 myoblasts, the RhoA signaling pathway participates in myotube formation (Carnac et al., 1998; Castellani et al., 2006; Charrasse et al., 2006; Reuveny et al., 2004; Wei et al., 1998). These observations suggest that RhoA functions in myogenic cells. However, whether RhoA is also expressed in intramuscular stem cells including satellite cells and plays a role in fate specification of these cells is not clear so far. Recent studies showed that inhibition of the Rho signaling pathway by a chemical inhibitor or a microgravity condition induces adipogenic differentiation of human mesenchymal stem cells (McBeath et al., 2004; Meyers et al., 2005). In addition, mouse embryo-derived fibroblasts (MEFs) lacking p190-B RhoGAP, a Rho inhibitory protein, do not undergo adipogenic differentiation. On the other hand, deletion of p190-B RhoGAP in MEFs induces differentiation into myogenic cells in response to IGF-I (Sordella et al., 2003). These observations suggest that the Rho signaling pathway is involved in myogenesisadipogenesis determination of mesenchymal cells. Although the mechanisms of fatty infiltration in skeletal muscle and the cellular origin of accumulated adipocytes in some pathological conditions are unclear, the fact that degenerative or atrophic fibers are persistently present in fat accumulated-muscle indicates the possibility that muscle fiber condition would be one of the niches affecting lineage specification of intramuscular cells. So, we hypothesize that there is the interaction between muscle fiber condition and fat accumulation in muscle, and this interaction is mediated by RhoA signaling pathway.
2. Materials and methods 2.1. Animals Male Wistar-Imamichi rats (2–4 months old) were purchased from the Institute for Animal Reproduction (Ibaraki, Japan). Male C57BL/6J mice (8-weeks old) were purchased from Charles River Laboratories (Yokohama, Japan). They were housed in a temperature-controlled room with a 14 h light and 10 h dark cycle (light on at 5:00 am). Food and water were provided ad libitum. All animal experiments in this study were performed according to the Guideline for the Care and Use of Laboratory Animals, The University of Tokyo. 2.2. Single fiber culture Single fiber culture was carried out according to Bischoff (1986), and Shefer and Yablonka-Reuveni (2005). Briefly, single muscle fibers with attached satellite cells were isolated from the hindlimb extensor digitrum longus (EDL) muscle of adult male rats. The EDL muscle was carefully removed at the tendon and treated with 0.2% (w/v) collagenase type I (Sigma, MO) reconstituted in Dulbecco’s Eagle Medium (DMEM) (Invitrogen, CA) at 37 1C for 90 min with slight shaking. After several gentle triturations with a wide-bore pipette, the muscle was further treated with enzyme at 37 1C for 10 min. The muscle was transferred into fresh
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10% FBS/DMEM, and fine fibers were liberated from the muscle by gentle trituration with a wide-bore pipette. Then, fine fibers were treated with vigorous pipetting to induce contraction of fibers. Fibers were sequentially washed three times in DMEM to remove interstitial and endothelial cells. Hereafter, fine fibers and contracted fibers are referred to as intact fibers (IF) and hypercontracted fibers (HCF), respectively. Both fibers were maintained as non-adherent cultures in 10% FBS/DMEM on 24-well noncoated culture plates (IWAKI, Tokyo, Japan) for 5–7 days, and then stained for specific markers. 2.3. Primary antibodies The following primary antibodies were used: Anti-Pax7, mouse monoclonal (1:250 dilution; Developmental Studies Hybridoma Bank (DSHB), IA); anti-MyoD1, mouse monoclonal (1:100; 5.8A, Novocastra, Newcastle upon Tyne, UK); anti-PPARg2 mouse monoclonal (1:200; Santa Cruz, CA); anti-laminin, rabbit polyclonal (1:1000; Sigma); anti-myosin heavy chain (1:20; MF20, DSHB), anti-RhoA, mouse monoclonal (1:500; 26C4, Santa Cruz); anti-M-cadherin antibody rabbit polyclonal (1:10 000; kindly gifted from Dr. Shin’ichi Takeda, National Center of Neurology and Psychiatry, Tokyo, Japan). 2.4. Immunocytochemistry Cells on fibers and culture dishes were fixed with 4% paraformaldehyde (PFA) in phosphate buffed saline (PBS) for 15 min at room temperature (RT). Cells were washed with PBS and incubated in 5% normal goat serum (NGS; Zymed, CA) and 0.1% Triton-X (Sigma) in PBS to block non-specific binding of antibodies. After cells were washed in PBS, primary antibodies were applied, and cells were incubated overnight at 4 1C. After washing cells with PBS, Alexafluor 594 or 488 conjugated antimouse IgG (1:400 diluted with 5% NGS in PBS; Invitrogen) was added and incubated for 1 h at RT. Cell nuclei were counterstained with Hoechst 33258. In some experiments, mature adipogenic cells were visualized by Oil-Red O (OR) staining after immunostaining. Briefly, cells fixed with 4% PFA were stained in OR solution (Sigma, 0.5% (w/v)) for 7 min at RT, then washed in PBS. Quantitative analyses of cells detached from single muscle fiber were performed. Total number of cells and number of cells on the dish positive for anti-PPARg antibody, MyoD antibody and OR, obtained from seven randomly chosen different microscopic fields using a 20 objective, were counted. Cell number and percentage of positive cells were averaged for the triplicate culture wells. Alternatively, number of positive cells for antiMyoD and -PPARg antibodies, and number of OR-positive cells on fiber obtained from 10 fibers chosen randomly per well, were counted for the triplicate wells. 2.5. Thrombin and Y-27632 administration Thrombin (Sigma) was dissolved in DMEM and added into 10% FBS/DMEM medium (1, 5 and 10 units/500 ml of medium). HCF were cultured in thrombin-containing medium during the first 3 days and then cultured in 10% FBS/DMEM for 4 additional days. Number of MyoD-positive myogenic cells and OR-positive adipogenic cells on fibers and dish were counted at the end of culture (day 7). Fusion index was estimated from number of nuclei in MHC-positive cells per all nuclei in the field at day 10. Y-27632 solution (10 mM, Calbiochem, CA), a specific inhibitor of ROCK (Ishizaki et al., 2000), was added to 10% FBS/DMEM during the first 3 days to inhibit intracellular RhoA signaling, and the fiber was removed from the well on day 4. Cells detached from
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IF were cultured in 10% FBS/DMEM for 7 days and stained with anti-PPARg2 antibody and OR. Thrombin and Y-27632 were concurrently added to HCF culture during the first 3 days in order to contradict an effect of thrombin administration, and then HCF was cultured for 7 days. Number of OR-positive cells and MyoD-positive cells on HCF were counted at the end of culture (day 7). 2.6. Y-27632-injection into mouse muscle Y-27632 dihydrochloride monohydrate (Sigma) was reconstituted with sterile saline. Fifty microliters of 0.5% bupivacainehydrochloride (BPVC, Sigma) were injected into the TA muscle of both legs of 8-week-old mice to induce injury and activation of satellite cells. Immediately after BPVC injection, Y-27632 (20 ml of 41.7 mg/ml) or equivalent volume of saline was injected into the injured muscle. Y-27632 injection was performed three times (0, 24 and 48 h after BPVC injection). Muscle from each leg was dissected at day 7 after BPVC injection and snap-frozen in an isopentane bath cooled by liquid nitrogen. Frozen sections (7-mm thick) prepared from Y-27632-injected and saline-injected TA muscle were stained with hematoxylin and OR. The degree of fat accumulation was quantitatively analyzed by measuring the OR-stained fat area within the regenerating area. Regenerating area was defined by the presence of newly generated fibers. Quantification of the fat area was done using NIH Image software (Ver. 1.62, NIH). 2.7. Statistical analysis The data are expressed as means 7SE. Statistical analyses were performed using commercial software (Statview, Ver. 4.5, Abacus Concepts, Inc, Barkley, CA). Student’s t-test was used to evaluate
statistical differences between control and treated groups. P values less than 0.05 were considered as statistically significant.
3. Results 3.1. Muscle fiber condition affects lineage specification of intramuscular cells We used single fiber culture system to examine whether muscle fiber condition induces adipogenic differentiation of cells on muscle fibers. Although proteolytically isolated fine muscle fibers are free of connective tissue, capillaries, there are quiescent satellite cells beneath the basement membrane (Bischoff, 1986). Single muscle fibers were isolated from rat EDL muscle, and fine fibers (Intact Fiber; IF) were selected among the isolated fibers. Subsequently, collected IF were contracted by pipetting (hypercontracted fibers; HCF) to evaluate the influence of muscle fiber condition on lineage specification of intramuscular cells. Both IF and HCF were maintained as non-adherent cultures in 10% FBS/ DMEM (Fig. 1A) and cultured for 5 days. Pax7-positive satellite cells were present on both IF and HCF just after plating (Fig. 1B). Immunocytochemistry of laminin, one of the major components of basal lamina, was performed to confirm the structural condition of basal lamina. Laminin was distributed on the entire surface of IF, whereas it was scattered on HCF, indicating that the proper structure of basal lamina was disrupted on HCF (Fig. 1C). When IF and HCF were stained with OR on day 5 of culture, ORstained mature adipocytes were only seen in abundance on HCF (Fig. 1D). In order to examine changes over time, number of MyoD-positive cells and PPARg-positive cells on both IF and HCF were counted every day after plating. MyoD-positive cells were continuously present on IF during the culture period, while there
Fig. 1. Cells on hyper-contracted fibers spontaneously differentiate into adipogenic cells: (A) Isolated single muscle fibers (intact fiber; IF) from rat EDL muscle were contracted by pipetting (hyper-contracted fiber; HCF) and maintained as non-adherent cultures. Scale bar, 200 mm. (B) Immunocytochemistry of Pax7 on day 0. Arrowheads indicate Pax7-positive satellite cells on both IF and HCF. Scale bar, 400 mm. (C) Laminin localization of both IF and HCF. Both fibers were stained with anti-laminin antibody on day 0. Scale bar, 200 mm. (D) Fibers were stained with Oil-Red O (OR) on day 5. Many mature adipocytes were present on HCF but not on IF. Boxes indicate OR-negative non-adipogenic cells (IF) and OR-positive adipogenic cells (HCF), respectively. Scale bar, 400 mm.
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Fig. 2. Time-dependent change of number of myogenic and adipogenic cells on muscle fibers: Number of myogenic (MyoD-positive) and adipogenic (PPARg-positive) cells on both IF (left panel) and HCF (right panel) were counted on days 0, 1, 2, 3, 4 and 5. Graphs represent MyoD (~) and PPARg (&), respectively. At each time point, n ¼ 20 (IF) and n ¼ 30 (HCF), respectively.
Fig. 3. Forced activation of the RhoA signaling pathway by thrombin administration inhibits adipogenesis of intramuscular cells on HCF: Thrombin (Thr; 1, 5, 10 U/ 500 ml) was added to HCF culture medium during the first 3 days, and HCF were stained with OR on day 7 after plating. (A) Arrowheads indicate OR-positive adipogenic cells. Scale bar, 200 mm. (B) OR-positive cells on fibers were counted and graphed for each group. Each experimental group was prepared with t wells, and 10 fibers per well were randomly chosen. Number of positive cells was counted (n ¼ 10 3 wells). Values represent mean 7SE. *Po0.05 vs control (Ctl).
were very few or no PPARg-positive cells (Fig. 2A). In contrast, very few, if any, MyoD-positive cells were seen on HCF throughout the culture period, while PPARg-positive cells first appeared on
Fig. 4. Forced activation of the RhoA signaling pathway by thrombin administration induces myogenesis of intramuscular cells on HCF: (A) MyoD-positive cells on HCF were increased dose-dependently by thrombin administration. Arrowheads indicate MyoD expressing cells. Scale bar, 200 mm. (B) Graphs represent number of MyoD-expressing cells on fibers. Values represent mean7SE (n ¼ 10 3 wells). *Po0.05 vs control (Ctl).
day 3, and their number increased as the culture period progressed (Fig. 2B). These results indicate that fate specification of cells on muscle fibers could be altered by muscle fiber
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condition, and cells on hyper-contracted fibers spontaneously differentiate into adipogenic cells.
3.2. RhoA signaling pathway is associated with lineage specification of intramuscular cells To elucidate the signaling pathway mediating the fate specification of cells on muscle fibers affected by muscle fiber condition, we focused on the RhoA signaling pathway since this pathway may act as a molecular switch for fate specification of mesenchymal cells (McBeath et al., 2004; Meyers et al., 2005; Sordella et al., 2003). We hypothesized that the Rho signaling pathway is associated with fate specification of intramuscular cells, and this pathway is inhibited in cells on HCF, resulting in adipogenic differentiation of these cells. Because the RhoA signaling pathway is activated by thrombin (Ren et al., 1999), we examined whether activation of the RhoA signaling pathway with thrombin could alter the fate specification of cells on HCF. HCF were treated with thrombin for the initial 3 days, the cells on the fibers were stained with anti-MyoD antibody and OR on day 7 of culture. Thrombin dose-dependently inhibited the appearance of OR-positive cells on HCF (Fig. 3). On the other hand, thrombin administration increased MyoD-positive cells on HCF in a dose-dependent manner (Fig. 4). These results indicate that forced activation of the RhoA signaling pathway inhibits adipogenesis of cells on HCF, and conversely accelerates myogenesis. Also, cells on culture dish migrated from HCF were promoted to myogenesis by thrombin administration, whereas adipogenesis were inhibited (Fig. 5). To confirm whether myoblasts on the dish which were generated by thrombin administration has an ability of myotube formation, furthermore, we carried out
immunocytochemistry for myosin heavy chain (MHC) at day 10 after HCF plating. There were a lot of MHC-positive cells on the dish treated with thrombin, indicating that myoblasts resulted from forced activation of RhoA signaling has normal myogenic property and are able to proceed terminal myogenic differentiation (Fig. 6). Activated RhoA (RhoA-GTP) affects its downstream target molecules such as Rho kinases (ROCK) and mDia, resulting in the progression of later signaling pathways (Matsui et al., 1996; Watanabe et al., 1997, 1999). In addition, activation of the RhoA signaling pathway is shown to promote myogenic specification of mesenchymal stem cells via ROCK-dependent pathway (McBeath et al., 2004; Sordella et al., 2003). Therefore, we administered ROCK inhibitor, Y-27632, to examine whether inhibition of the RhoA-ROCK signaling pathway in cells could have any influence on fate specification of cells derived from IF. IF were treated with Y-27632 for the first 3 days, and cells that detached from IF and migrated onto the culture dish were stained with anti-PPARg antibody and OR on day 7. In our experiment, Y-27632-treated IF were often unexplainably hyper-contracted after day 4 of culture, and it was impossible to maintain IF in the presence of Y-27632 treatment. Therefore, fibers were removed from culture on day 4, and only cells that had detached from IF and migrated onto the dish before day 4 of culture were examined for further analysis. Treatment of IF with Y-27632 resulted in the appearance of ORpositive cells, while there were almost no OR-positive cells in the control culture (Fig. 7). A small portion of cells that migrated from IF in the control culture were slightly positive for PPARg, but they were not stained with OR. On the other hand, in the Y-27632 treated culture, PPARg staining was much more intense than in the control culture, and the number of PPARg-positive cells was increased. In addition, some PPARg-positive cells were co-stained
Fig. 5. Forced activation of RhoA signaling pathway accelerates myogenesis and inhibits adipogenesis of cells migrated off into the dish: (A) MyoD-positive cells migrated from HCF into the dish were increased dose-dependently by thrombin administration. Arrowheads indicate same cells. Scale bar, 200 mm. (B) OR-positive cells on the dish were decreased dose-dependently thrombin administration. Scale bar, 200 mm. (C) Graphs represent percentage of MyoD-positive cells (white bars) and OR-positive cells (black bars) on the dish. Each experimental group was prepared with triplicate wells. Values represent mean 7SE (n ¼ 3 wells). *,]Po0.05 vs control (Ctl).
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Fig. 6. Cells treated with thrombin normally pass through myotube formation on the dish: (A) Cells treated with thrombin (10 U) were cultured for 10 days and stained with anti-myosin heavy chain antibody (MHC). Scale bar, 200 mm. (B) Graphs represent fusion index after 10 days culture. Values represent mean 7SE (n ¼ 3 wells). * Po0.05 vs control.
with OR (Fig. 7). These results suggest that inhibition of ROCK, one of the downstream signaling pathways of RhoA, stimulates cells on muscle fibers to enter adipogenesis. Furthermore, we carried out co-administration of Y-27632 and thrombin to HCF in order to confirm whether RhoA signaling pathway is actually concerned with fate specification of intramuscular cells. Both Y-27632 and thrombin were concurrently added to HCF culture for the first 3 days, and cells on HCF were stained with OR and anti-MyoD antibody at day 7 after plating. Co-administration of Y-27632 and thrombin resulted in a significantly increase of OR-positive cells on HCF as compared to single thrombin administration (Fig. 8A). On the other hand, however, number of MyoD-positive cells on HCF was not changed by co-administration (Fig. 8B). These results suggest that effect of thrombin administration is negated by the specific ROCK inhibitor at least in case of adipogenic differentiation of the cells and RhoA signaling pathway through ROCK is participated in fate specification of intramuscular cells.
3.3. Effect of in vivo administration of Y-27632 on regenerating muscle The above findings indicate that fate specification of cells in skeletal muscle could be altered by modulating the RhoA signaling pathway, and inhibition of ROCK leads to spontaneous adipogenesis of these cells. Direct injection of Y-27632 into mouse regenerating muscle was performed in order to examine if this could be mimicked in vitro. Mouse TA muscles were injured by
BPVC injection, and Y-27632 was injected into the injured-region. Muscles were collected at day 7 post-injury and stained with OR. Fat accumulation was evident in the regenerating area of Y-27632treated muscle, whereas it was barely detected in the saline control (Fig. 9A and B). These results suggest that inhibition of ROCK leads to alteration of differentiation fate of intramuscular cells and induces their spontaneous adipogenesis in vivo.
4. Discussion The origin of adipocytes in some pathological muscle such as dystrophic muscle and the mechanism that leads to accumulation of adipocytes are not entirely clear. In the present study, we used a single fiber culture system to demonstrate that many adipocytes are present on cultured HCF, whereas very few, if any, adipocytes are seen on IF. Dystrophic muscle is hyper-contracted by the rapid influx of Ca2+ into the muscle fiber (Carpenter and Karpati, 1979), and muscle fiber hypercontraction can be induced experimentally by BPVC treatment (Nonaka et al., 1983). Previously, we demonstrated that cells from regenerating muscle treated with BPVC increase in adipogenicity, due to a lack of Pref-1 expression (Yamanouchi et al., 2006). In addition, intramuscular cells such as satellite cells are able to give rise to adipogenic cells by hormonal stimulation in vitro (Teboul et al., 1995; Yada et al., 2006), and Shefer et al. (2004) recently used in vitro clonal analyses to demonstrate that satellite cells are able to enter a mesenchymal alternative differentiation (MAD) route, resulting in differentiation to
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Fig. 7. Inhibition of the RhoA signaling pathway by ROCK inhibitor induces adipogenesis of satellite cells: ROCK specific inhibitor, Y-27632, was added to IF culture medium during the first 3 days, and fiber was removed from the well on day 4. (A) Cells on dish were stained with anti-PPARg antibody and OR on day 7. Arrowheads indicate PPARgpositive cells. Scale bar, 200 mm. (B) Graphs show percentage of PPARg-positive cells on dish. Each experimental groups were prepared with triplicate wells. Values represent mean7SE (n ¼ 3). *Po0.05 vs control.
Fig. 8. Effect of thrombin is partially inhibited by Rho specific inhibitor: Thrombin (10 U) and a specific inhibitor of ROCK, Y-27632 (10 mM), were concurrently added into HCF culture for first 3 days. OR- and MyoD-positive cells on fibers were counted at day 7 after plating. (A) Graphs represent number of OR-positive cells on fibers. Each experimental group was prepared with 3 wells, and 10 fibers per well were randomly chosen. Number of positive cells was counted. Values represent mean7SE (n ¼ 10 3 wells). *Po0.05 vs Thrombin (Thr). (B) Graphs represent number of MyoD-positive cells on fibers. Values represent mean 7SE (n ¼ 10 3 wells).
non-myogenic lineages such as adipogenic and smooth musclelike cells. Therefore, taken together with these studies, the results obtained in the present study suggest that hypercontraction of
muscle fibers could be the principle signal to induce adipogenesis, and intramuscular cells-derived adipocytes may contribute to fat accumulation in skeletal muscle.
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Fig. 9. Induction of fat accumulation in regenerating mouse muscle by direct injection of Y-27632: Mouse TA muscle was injured by BPVC injection, and Y-27632 was injected into the injured area. On day 7 after injection, regenerating muscle was dissected and stained with OR. (A) Fat accumulation was seen in the regenerating area of Y27632-treated muscle (right panel), whereas it was hardly seen in the saline control (left panel). Nuclei were stained with hematoxylin. Scale bar, 200 mm. (B) Graphs show percentage of fat area with in regenerating muscle. Values represent mean 7SE (n ¼ 3). *Po0.05 vs control.
It should be mentioned that although BPVC treatment is shown to transiently increase the adipogenicity of cells in skeletal muscle (Yamanouchi et al., 2006), BPVC-treated muscle regenerates completely, and fat accumulation is never seen either during or at the end of regeneration. In BPVC-treated muscle, hypercontracted fibers degenerate and are necrotized. These necrotic fibers are removed by macrophages migrating into the degenerated area, resulting in the loss of association between intramuscular cells including satellite cells and degenerated fibers. CC chemokine receptor 2 (CCR2) plays a role in recruitment of macrophages to the injured site, and CCR2-/- mice exhibit altered muscle regeneration and inflammation, as well as are deficient of intramuscular macrophages. Interestingly, fat accumulation is seen in damaged muscle of CCR2-/- mice (Contreras-Shannon et al., 2007). Hence, taken together with our observation that mature adipocytes were seen only when cells were cultured on HCF, the persistent presence of degenerative fibers in damaged muscle may support terminal adipogenic differentiation of intramuscular cells, thus leading to the appearance of mature adipocytes. In the present study, we could not clearly identify the origin of adipocytes on HCF. In this point, however, we suppose that one of origins of adipocytes on HCF is satellite cells, because MyoDpositive myogenic cells were hardly detected on HCF after 5–7 days in spite of the presence of satellite cells on HCF just after plating and this suggests that satellite cells on the fibers did not differentiate into myogenic cells and differentiate into adipogenic cells. We demonstrated using competitive reagents that effect of thrombin was negated by Y-27632 treatment and number of adipogenic cells was increased by an inhibition of thrombin effect, strongly suggesting that thrombin actually activates RhoA signaling pathway via ROCK in cells on HCF and consequently myogenesis was induced. On the other hand, however, number of myogenic cells on HCF was not affected by concurrently administration of Y-27632 and thrombin. In this point, we
suppose that ROCK-independent RhoA signaling pathway such as mDia is also participated in fate specification of intramuscular cells, because knockdown of mDia in C2C12 cells results in decrease of MyoD expression (Gopinath et al., 2007). In addition, our results suggested that muscle fiber condition affects the RhoA signaling pathway in intramuscular cells. And, results from in vivo study that Y-27632 was directly injected to mouse regenerating muscle are consistent with the results from in vitro study. However, the mechanism associating the alteration of muscle fiber condition to changes in RhoA activity and the origin of adipocytes are unknown so far. At that present time, however, we speculate that modification of RhoA signaling might be happened in satellite cells and this resulted in appearance of adipocytes. Quiescent satellite cells express laminin receptor, a7b1-integrin (LaBarge and Blau, 2002), and majority of b1-integrin-positive cells (490%) on single myofiber express Pax7 (Cerletti et al., 2008). Based on immunocytochemial localization of laminin, the structure of basal lamina of HCF is thought to be disrupted. It is widely recognized that the RhoA signaling pathway is activated by integrin via focal adhesion kinase (Palazzo et al., 2004; Zhai et al., 2003), and disruption of basal lamina may induce loss of this interaction if satellite cells directly interact with muscle fiber through laminin-integrin association. In addition, we also confirmed that quiescent satellite cells (M-cadherin-positive cells) express RhoA (Supplementary Fig. S1). Taken together, it is plausible that the direct interaction between satellite cells and muscle fiber through integrin is necessary for satellite cells to differentiate into myogenic lineage, and that satellite cells differentiate into adipogenic lineage when this interaction is disrupted, thus leading to an inactivation of RhoA signaling in satellite cells. However, we have to carry out further examination to prove our hypothesis, because there are no evidences supporting it so far. And also, our in vivo study indicates the possibility that Y-27632 does not directly affects intramuscular
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cells and hypercontraction of muscle fibers by Y-27632 administration results in adipogenesis of the cells on fiber, because Y-27632 may induce hypercontraction of muscle fibers. So, in further examination, we also have to prove whether Y-27632 actually inhibits RhoA signaling pathway in intramuscular cells. In summary, our findings indicate that degenerative muscle fiber accelerates fat accumulation in skeletal muscle. In addition, it was suggested that the RhoA signaling pathway is affected by muscle fiber condition and is associated with adipogenic differentiation of intramuscular cells. Although further examination is necessary to clarify the exact nature of factors that directly connect muscle fiber condition to intracellular RhoA signaling in fate specification of intramuscular cells, the present findings will be important in elucidating the mechanism of fatty infiltration in some pathological muscle conditions, and for therapeutic applications such as muscular dystrophy.
Acknowledgements We thank Dr. Yousuke Murakami (The University of Tokyo) for his helpful advices. The anti-Pax7 and anti-Myosin heavy chain monoclonal antibodies were obtained from the Developmental Studies Hybridoma Bank developed under the NICHD and maintained by The University of Iowa. This work was supported by grants to M.N and K.Y from the Japan Society for the Promotion of Science, and to K.Y from Ministry of Health, Labor and Welfare of Japan and Program for Promotion of Basic Research Activities for Innovative Biosciences (PROBRAIN). This paper is dedicated to late Professor Chikashi Tachi.
Appendix A. Supporting Information Supplementary data associated with this article can be found in the online version at doi:10.1016/j.diff.2008.11.001.
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