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MUSCLE CELL BIOLOGY AND CELL MOTILITY
Section of Oral Biology, The Ohio State University College of Dentistry, Columbus, Ohio
Submitted 29 November 2006 ; accepted in final form 24 March 2007
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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, and the myogenic gene induction was examined over a period of 72 h. Exposure of cells to CTS resulted in a significant upregulation of mRNA expressions and synthesis of myogenic regulatory factors, MYOD1, myogenin (MYOG), MEF2A, and cyclin-dependent kinase inhibitor 1A (CDKN1A; p21) as well as muscle structural proteins like myosin heavy chain (MYHC) isoforms (MYH1, MYH2, and MYH4) and -tropomyosin (TPM1), eventually leading to an increase in myotube formation. Contrarily, TNF- suppressed the expression of all of the above differentiation-inducing factors in C2C12 cells. Further results revealed that simultaneous exposure of C2C12 cells to CTS and TNF- abrogated the TNF--mediated downregulation of myogenic differentiation. In fact, the mRNA expression and protein synthesis of all myogenic factors (Myod1, Myog, Mef2a, Cdkn1a, Myh1, Myh2, Myh4, and Tpm1) were increased in stretched C2C12 cells despite the sustained presence of TNF-. These results demonstrate that mechanotransduction regulates multiple signaling molecules involved in C2C12 cell differentiation. On one hand, these signals are potent transducers of myotube phenotype in myoblasts; on the other, these signals counteract catabolic actions of proinflammatory cytokines like TNF- and allow the expression of myogenic genes to upregulate muscle cell differentiation. myogenesis; myoblasts; inflammation; biomechanical signals
Muscle development is a complex process in which undifferentiated myoblasts exit the cell cycle and undergo fusion to form myotubes. In vivo, skeletal muscles are populated with satellite cells that serve as predecessors of myoblastic cells and undergo differentiation into myotubes in a highly specific spatial and temporal sequence of molecular events. However, the majority of investigations have used cell lines of a myoblastic phenotype, such as murine C2C12 cells, to experimentally delineate the multistep process of muscle cell differentiation (7, 9). Expression of the basic helix-loop-helix (bHLH) family of transcription factors and MADS box transcription enhancer factor 2 (MEF2) family of proteins are required for the regulation of sequential steps in myogenesis. The first muscle-specific transcription factor expressed in this process is MYOD1, which binds to the E box located in the consensus sequences of the regulatory region of muscle-specific genes (4, 29, 30). MYOD1 induces myogenin (Myog), another early target gene recognized as a biomarker, indicating the commitment of myoblastic cells to differentiation (31, 35). Thereafter, the expression of proteins belonging to the MEF2 family and their synergistic interaction with MYOD1 results in the induction of muscle-restricted target genes (39, 40). Concurrently, induction of cyclin-dependent kinase inhibitor 1A (Cdkn1a) mRNA expression and CDKN1A protein synthesis leads to the irreversible exit of cells from the cell cycle and their commitment to the differentiation program (9, 21). These events are followed by transcriptional upregulation of skeletal muscle-specific genes such as myosin heavy chain Myh1, Myh2, and Myh4, as well as tropomyosin 1 (Tpm1), troponin I (Tnni), and troponin T (Tnnt) and other genes, which are required for muscle assembly and function (22, 25, 28). In parallel, differentiating myoblastic cells in the close proximity fuse to form multinuclear myotubes. The entire process of myogenesis is carefully controlled at each step, and any disturbances critically modulate its outcome.
Muscle inflammation has been shown to initiate degradation of intrinsic muscle proteins as well as inhibit myogenesis by suppressing the expression of differentiation-associated molecules and eventual myotube formation in C2C12 cells in vitro (15). Treatment of C2C12 myoblast-like cells with a proinflammatory cytokine, TNF-, leads to the loss of MYOD1, MYOG, CDKN1A, MYHC, and TPM1 proteins, resulting in the eventual inhibition of myotube formation (1, 15). TNF- inhibits skeletal muscle differentiation via the induction of nitric oxide (NO) synthase 2A (NOS2A) and NO production in a NF-
B dependent manner, which subsequently leads to Myod1 mRNA degeneration (6, 14). Furthermore, several genetic approaches have demonstrated that TNF- negatively regulates the process of myogenesis in vitro (14, 20). Conversely, mechanical loading of chronically or acutely inflamed muscles has been shown to regain muscle strength and function in vivo by mechanisms as yet little understood.
Here, we hypothesized that biomechanical signals regulate myogenesis despite the presence of inflammatory molecules and upregulate expression of the bHLH and MEF families of myogenic transcription factors, which eventually lead to increased myoblastic differentiation and resultant myotubes formation. Hence, we determined the biomechanical signal-dependent spatial and temporal expression of myogenic transcription factors and muscle structural proteins in C2C12 myoblasts in vitro. Additionally, the effects of biomechanical signals on the expression of differentiation-associated genes were assessed in the presence of the proinflammatory molecule TNF-. These biomechanical signal-induced events act as potent anti-inflammatory signals that suppress TNF--induced NOS2A mRNA and protein expression in C2C12 cells. We show that biomechanical forces upregulate myogenic gene expression as well as promote the progression of differentiation by increased synthesis of myogenic proteins in an environment primed for inflammation.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Application of cyclic tensile strain.
C2C12 cells were seeded in GM at 3 x 105 cells/well on Bioflex collagen type I-coated six-well plates (Flexcell) and grown to 70% confluence (2–3 days). The medium was replaced with DM, and cells were subsequently subjected to cyclic tensile strain (CTS) at an optimal magnitude and frequency using a Flexcell 4000T/FlexLink Cell Culture System in the presence or absence of recombinant human (rh)TNF- (5 ng/ml). The effects of CTS were examined following a 24-, 48-, or 72-h exposure to CTS. Control (untreated) cells and cells exposed to rhTNF- alone in each assay were cultured in DM on Bioflex collagen type I-coated six-well plates but not exposed to CTS.
RNA purification and real-time PCR.
Total RNA was isolated from cells using the RNeasy Mini kit (Invitrogen), subjected to DNase I digestion (Ambion), and stored in DNase-/RNase-/Protease-free water at –80°C. The concentration, purity, and integrity of RNA were assessed using a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies) or an Experion automated electrophoresis system (Bio-Rad Laboratories). One microgram of total RNA was reverse transcribed with 0.5 µg oligo d(T)12–18, 1x First-Strand Synthesis Buffer, 5 mM DTT, 200 U/µl Superscript III reverse transcriptase, and 40 U/µl RNaseOUT inhibitor (Invitrogen) for 60 min at 55°C. Real-time RT-PCR was performed using TaqMan gene expression assays as recommended by the manufacturer (Applied Biosystems), and data were acquired using an iCycler iQ Multicolor Real-Time PCR Detection System (Bio-Rad Laboratories). The following prevalidated TaqMan assays were obtained from Applied Biosystems: Myod1 (Mm00440387_m1), Myog (Mm00446194_m1), Myh1 (Mm01332500_gH), and Cdkn1a (Mm00432448_m1). Custom TaqMan assays were developed as necessary using Primer Express software (Applied Biosystems). SYBR green-based real-time PCR primers were designed using OLIGO software (Molecular Biology Insights). iQ SYBR Green PCR Supermix (Bio-Rad Laboratories)-containing assays were performed at optimized annealing temperatures as determined empirically for each primer set. Primers were designed to span exon/intron junctions to diminish the chances of spurious genomic DNA amplification. The SYBR green real-time primer sequences used were as follows: Mef2a, sense 5'-CAG GCT TCA GCC TGG CAG CAG-3' and antisense 5'-GCT GGA GCT GCT CAG ACT GTC CAC-3'; Myh2, sense 5'-AGC AGA CGG AGA GGA GCA GGA AG-3' and antisense 5'-CTT CAG CTC CTC CGC CAT CAT G-3'; Myh4, sense 5'-CAC CTG GAG CGG ATG AAG AAG AAC-3' and antisense 5'-GTC CTG CAG CCT CAG CAC GTT-3'; Tpm1, sense 5'-ATC ATC GAG AGC GAC CTG GAA CG-3' and antisense 5'-CTT CTT TGG CAT GGG CCA CTT TC-3'; and ribosomal protein S18 (Rps18), sense 5'-GGA AAA TAG CCT TCG CCA TCA CTG-3' and antisense 5'-GCC AGT GGT CTT GGT GTG CTG AC-3'. The data obtained by real-time RT-PCR were analyzed by the comparative threshold cycle (CT) method. In this method, the amount of the experimental target, normalized to a housekeeping gene transcript (Rps18) and relative to a calibrator (either control untreated sample or rhTNF--stimulated cells), is given by the estimation of 2–
CT, where CT = CT (sample) – CT (calibrator) and CT is the CT of the target gene subtracted from the CT of Rps18.
Western blot analysis.
Immediately after termination of the experiment, Bioflex plates were kept on ice, washed three times with ice-cold PBS, and subjected to lyses with RIPA buffer (Santa Cruz Biotechnology) containing protease inhibitors (Roche Diagnostics). The expression of each protein was analyzed by subjecting 20 µg of whole cell lysate to 10% SDS-PAGE. Proteins were electrophoretically transferred to a nitrocellulose membrane (New England Nuclear) and reacted with blocking buffer containing PBS with 0.02% Tween 20 (PBS-T) and 5% nonfat milk for 60 min. Blots were then reacted overnight at 4°C with monoclonal/polyclonal primary antibody recognizing a specific protein in the blocking buffer at a predetermined dilution. The antibodies used were rabbit anti-MyoD IgG, rabbit anti-myogenin IgG, rabbit anti-MEF2A IgG, and rabbit anti p21 IgG (Santa Cruz Biotechnology). Monoclonal mouse anti-MYHC IgG (MF-20) and mouse anti--TPM (CH1) were obtained from the Developmental Studies Hybridoma Bank. Subsequently, blots were washed three times with PBS-T and incubated in the dark with IRDye 680- or IRDye 800CW-conjugated goat anti-rabbit or goat anti-mouse IgG (LI-COR Biosciences) for 60 min at room temperature. Thereafter, membranes were washed three times with PBS-T and imaged under an ODYSSEY imager (LI-COR), and quantitatively analyzed using ODYSSEY application software (version 2.1). Results are expressed as means ± SD from three independent experiments.
Immunofluorescence. Immediately following each experiment, C2C12 cells to be analyzed by immunofluorescence were washed with ice-cold PBS, fixed using 2% paraformaldehyde (pH 8.0) for 20 min, and permeabilized with 0.2% Triton X-100 for 30 min. Thereafter, the flexible silicone elastomer membranes from the BioFlex plates were excised and divided into six equal pie-shaped pieces. Membranes were reacted with blocking buffer (Vector Labs) and incubated overnight with primary antibodies as appropriate (described above) at 4°C. Membranes were washed three times with PBS-T and then incubated with CY3-conjugated goat anti-rabbit or goat anti-mouse IgG for 45 min at room temperature. Subsequently, cells were counterstained with FITC-conjugated phalloidin (Invitrogen) to visualize F-actin and 4',6-diamidino-2-phenylindole (Sigma) to visualize nuclei. Membranes were subsequently mounted using Vectashield Mounting Medium (Vector Labs) and examined under epifluorescence using a Zeiss Axioplan microscope, and immunofluorescence images were captured with Zeiss imaging software. Numbers of MYHC-positive cells were enumerated in four different areas of 4.84 x 104 µm2, fluorescence was estimated with the use of Axiovision software, and means and SDs were calculated.
Statistical analysis. Results are expressed as means ± SD from three independent experiments performed in triplicate. Statistical analysis was performed using two-way ANOVA and a post hoc Tukey test using the SPSS statistical package (version 13.0). A value of P < 0.05 was deemed significant.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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-induced Nos2 expression in a magnitude-dependent manner.
Most myopathies involve inflammation induced by proinflammatory cytokines like TNF- that initiate muscle degradation through the activation of multiple proinflammatory genes, including Nos2 (6). Therefore, we first examined the effects of rhTNF- on C2C12 cells, where Nos2 mRNA induction was used as a biomarker to estimate the effects of CTS. Since the dose-response curve showed a robust Nos2 mRNA induction by 5 ng/ml rhTNF- (Fig. 1A, inset), this concentration of rhTNF- was used in subsequent experiments. Although exposure of C2C12 cells to CTS resulted in the suppression of TNF--induced Nos2 mRNA expression and NOS2 protein synthesis between the magnitudes of 3% and 18%, the maximal suppression was observed at magnitudes of 3% and 6% (Fig. 1, A and B). Determination of the effects of various frequencies of CTS (0.25, 0.05, 0.01, or 0.0016 Hz) revealed that frequencies of 0.25–0.05 Hz maximally attenuated rhTNF--induced Nos2 mRNA and protein expression (data not shown). Therefore, in subsequent experiments, CTS at a magnitude of 3% at 0.05 Hz was used to examine the effects of CTS on myogenic differentiation in C2C12 cells.
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and TNF- receptor 1 (TNF-R1), which are known to be upregulated by TNF-. Similar to Nos2 mRNA expression, CTS alone downregulated TNF- and TNF-R1 in C2C12 cells. As shown in Fig. 1C, within 4 h of TNF- treatment, CTS also downregulated TNF--dependent TNF- and TNF-R1 expression significantly.
CTS upregulates Myod1 induction.
During myogenesis, MYOD1 is the first muscle-specific transcription factor upregulated in differentiating myoblasts. Therefore, to assess transcription factors regulated by CTS, we first examined the expression of Myod1 mRNA expression and MYOD1 protein synthesis in differentiating C2C12 cells. C2C12 myoblasts were either untreated (control) or exposed to rhTNF-, CTS, or CTS + rhTNF-. Within 24 h of the initiation of differentiation, C2C12 cells exhibited increased Myod1 mRNA expression (Fig. 2A), and this expression was a >4.2-fold increase in response to CTS. TNF- exposure suppressed >96% of the Myod1 mRNA expression induced by DM. More importantly, concomitant exposure of cells with CTS during treatment with rhTNF- resulted in a complete abrogation of TNF--mediated inhibition of Myod1 mRNA expression (Fig. 2A). Myod1 mRNA expression was highest during the first 24 h, and, during the ensuing 24 h, it gradually decreased in CTS-treated cells, but remained at the same levels under other treatments. Further analysis after 72 h of CTS or CTS + TNF- treatment revealed that its levels did not change significantly. Western blot analysis revealed that MYOD1 was maintained in control cells at similar levels. However, the presence of rhTNF- suppressed its expression over the entire 72-h period. Contrarily, in both C2C12 cells exposed to CTS alone or CTS + rhTNF-, MYOD1 synthesis was at least fourfold greater compared with control cells, suggesting that CTS alone not only upregulated MYOD1 synthesis but markedly abrogated rhTNF--induced inhibition of MYOD1 synthesis (Fig. 2B). Intracellular localization of MYOD1 by immunofluorescence staining demonstrated that the majority of MYOD1 was concentrated in the nuclei of control cells (Fig. 2C). As expected, the presence of MYOD1 in rhTNF--treated cells was negligible in the cytoplasm or nuclei (Fig. 2C). More interesting was the fact that CTS-induced upregulation of MYOD1 was paralleled by its nearly complete nuclear translocation in cells exposed to CTS alone as well as in cells exposed to CTS + rhTNF- concomitantly (Fig. 2C).
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-mediated suppression of Myog expression.
Nuclear translocation of MYOD1 results in the transcriptional activation of Myog gene expression (4). To determine whether the upregulation and nuclear translocation of MYOD1 by CTS was functionally significant, we next performed differential gene expression analysis of Myog by real-time RT-PCR in the absence or presence of rhTNF- treatment. As shown in Fig. 3A, Myog mRNA expression in C2C12 cells subjected to CTS increased 3.8-fold during the first 24 h and continued to increase in both cells exposed to CTS and controls during the ensuing 24 and 48 h. Not surprisingly, rhTNF- exposure inhibited Myog mRNA expression significantly. MYOG protein induction assessed by Western blot analysis revealed that, compared with control cells, CTS induced a 5.1-, 3.2-, and 1.8-fold increase in MYOG accretion at 24, 48, and 72 h, respectively (Fig. 3B). Intracellular localization of MYOG by immunofluorescence analysis revealed that, following its synthesis, >95% of MYOG was translocated to the nucleus (Fig. 3C). Consistent with the inhibition of mRNA expression, rhTNF- inhibited MYOG synthesis during the first 48 h; however, some synthesis of MYOG was observed in rhTNF--treated cells at 72 h (Fig. 3, B and C). However, CTS markedly abrogated the rhTNF--dependent inhibition of MYOG synthesis and allowed Myog mRNA expression above control levels at all time points tested (Fig. 3, B and C). In addition, the elevated presence of MYOG in the nucleus and its absence in the cytoplasmic compartment suggested that upregulation of MYOG synthesis by CTS may be paralleled by its transcriptional activity (Fig. 3C).
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treatment suppressed both Mef2a mRNA expression and MEF2A protein synthesis. However, CTS inhibited rhTNF--mediated downregulation to allow Mef2a mRNA expression and MEF2A protein synthesis above control levels (Fig. 4, A and B).
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suppressed its expression significantly during the initial 24 h of treatment (Fig. 4C). mRNA levels for Cdkn1a did not change significantly during 24–72 h of treatment. Nevertheless, a marginal upregulation (1.5- to 1.8-fold) of CDKN1A protein synthesis was observed in response to CTS at 48 h (Fig. 4D). rhTNF- exposure consistently inhibited >60% of CDKN1A protein synthesis compared with control cells. CTS also counteracted the rhTNF--induced suppression of CDKN1A, and a minimal upregulation of its synthesis was observed in cells subjected to a simultaneous exposure of CTS + rhTNF- at all time points tested (Fig. 4D).
CTS upregulates skeletal muscle structural proteins.
In a temporal analysis of differentiation, the ultimate consequence of the upregulation of myogenic transcription factors and CDKN1A is increased synthesis of muscle-restricted target proteins like MYHC, TPM1, and TNNI/TNNT. Hence, to determine the functional consequence of CTS-mediated upregulation of bHLH and MEF2 family transcription factors, we next examined the mRNA expression and protein synthesis of the various isoforms of MYHC (Myh1, Myh2, and Myh4) and Tpm1. In C2C12 cells maintained in DM, the expression of Myh1, Myh2, and Myh4 mRNA during the first 24 h of differentiation was negligible, whereas it dramatically increased in the ensuing 24 h, exhibiting a 14-, 73-, and 132-fold increase in Myh1, Myh2 and Myh4 mRNA, respectively (Fig. 5A, inset). Strikingly, compared with controls cells maintained in DM, CTS further induced a 3.8-, 2.8-, and 1.5-fold increase in mRNA expression for Myh1, Myh2, and Myh4 mRNA, respectively (Fig. 5A). In parallel, 72 h after the initiation of differentiation, CTS augmented a 3.2-fold increase in MYHC synthesis above that observed in control cells (Fig. 5B). rhTNF- treatment inhibited 89% of Myh1, 81% of Myh2, and 92% of Myh4 mRNA expression (Fig. 5A). This was also reflected by a 64% reduction of the MYHC synthesis in cells treated with rhTNF-. Simultaneous exposure of cells to CTS abrogated the rhTNF--induced inhibition of Myh1, Myh2 and Myh4 mRNA expression. Nevertheless, levels of Myhc mRNA were not higher than those present in control cells maintained in DM. In parallel, Western blot analysis using the MF-20 monoclonal antibody confirmed the results of the above experiments in that simultaneous exposure of CTS during rhTNF- treatment neutralized the effects of rhTNF-, albeit the presence of MYHC protein did not exceed above that expressed by control C2C12 levels (Fig. 5, A and B). As shown in Fig. 5, D and E, CTS + rhTNF- regulated Tpm1 mRNA expression and TPM1 protein synthesis in a manner similar to MYHC proteins.
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significantly suppressed MYHC expression in C2C12 cells. However, cells simultaneously exposed to CTS + rhTNF- exhibited a complete reversal of rhTNF--induced suppression of MYHC expression to control levels (Fig. 5C). Exposure to CTS alone yielded 2.6-fold greater MYHC-positive cells compared with controls during a 72-h period of differentiation. C2C12 cells transform into myotubes in DM within 72–96 h (4, 29). Consequently, we investigated whether the expression of muscle target proteins is paralleled by phenotypic changes in C2C12 cells in response to CTS and/or rhTNF-. Consistent with our hypothesis, immunofluorescence analysis detected a 2.7-fold increase in MYHC-positive multinucleate myotubes formed by the fusion of cells in cultures exposed to CTS compared with control cells (Fig. 5, F–H). Not surprisingly, cells treated with rhTNF- stained poorly for MYHC and lacked multinucleate cell formation (Fig. 5, E and F). C2C12 cells treated concurrently with CTS + rhTNF- exhibited MYHC-positive cells and multinucleate myotube formation that were equal in numbers to control cells maintained in DM, further confirming that CTS abrogates the rhTNF--induced suppression of phenotypic changes in C2C12 cells. |
DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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, we have emulated the effects of tensile forces imposed on satellite cells during muscle movement in vivo. TNF- inhibits skeletal muscle differentiation via activation of the NF-B signal transduction pathway and its subsequent induction of NOS2 and NO production leading to MYOD1 protein loss (6, 14). Therefore, we used Nos2 mRNA expression as a biomarker to optimize the dose and magnitude of CTS required for the inhibition of inflammatory responses. The results demonstrate that C2C12 myoblast-like cells respond to biomechanical signals in a magnitude-dependent manner, inhibiting rhTNF--induced Nos2 mRNA expression to various degrees between the magnitudes of 3% and 18% of strain. However, the maximal inhibition of rhTNF- actions was observed at magnitudes of 3% and 6%. These magnitudes of strain are similar to those used in an earlier study (18) for in vitro and ex vivo muscle stimulation. Furthermore, in chondrocytes, CTS at these magnitudes also inhibits the expression of several proinflammatory genes controlled by NF-B transcriptional activity, such as Nos2, prostaglandin-endoperoxide synthase 2 (cyclooxygenase 2), matrix metalloproteinase 1, and IL-1
(2, 26). Since TNF- actions on muscle cells are mediated via the NF-B pathway, it is likely that CTS may also inhibit the expression of other proinflammatory genes in addition to Nos2.
One of the earliest events in muscle cell differentiation is the transcriptional activation and increased synthesis of the bHLH family transcription factors (MYOD1, MYOG, MYF5, and MYF6). These transcription factors by acting at multiple points regulate the skeletal muscle phenotype. During this process, bHLH transcription factors act synergistically with the MEF2 family of cofactors to induce the synthesis of muscle-restricted target genes (39, 40). We examined salient members of the bHLH and MEF2 families of proteins to delineate the mechanisms of CTS-mediated regulation of the skeletal muscle phenotype. CTS, as an extracellular signal, appears to be involved in regenerative responses of skeletal muscle cells (18). At low physiological levels, it activates one of the earliest events in muscle cell differentiation by augmenting rapid and significantly greater expression, synthesis, and nuclear translocation of MYOD1. Furthermore, we observed that nuclear MYOD1 and MYOG levels are upregulated by CTS even in the presence of TNF- and that mRNA expression of these genes is significantly increased. This suggests that CTS-dependent upregulation of their nuclear translocation is paralleled by an increase in their transcriptional activity, even in the presence of TNF-. Muscle cell growth is controlled by two distinct mechanisms. One involves IGF-induced activation of phosphoinositol 3-kinase (PI3K) and its subsequent activation of the mammalian target of rapamycin (mTOR) pathway, and the other, which is independent of PI3K activation, involves calcineurin signaling (15). Multiaxial stretch has been shown to activate PI3K-independent mTOR signaling in differentiating C2C12 cells (18). Whether the mechanically induced transcriptional activation of Myod1 is PI3K dependent or independent is as yet not clear. In either case, eventual transcriptional activation of Myod1 is an absolute requirement for muscle cell differentiation. Therefore, CTS-mediated augmentation of Myod1 expression at levels that are severalfold greater than those induced by the insulin present in DM suggest that biomechanical signals have greater capacity to induce skeletal muscle differentiation.
Subsequent to MYOD1 activation, expression of Myog is the credible molecular biomarker for myoblast commitment to differentiation. In differentiating C2C12 cells, Myog levels follow a temporal expression that exhibits a gradual increase over 24–72 h of differentiation. CTS manifests a marked upregulation of Myog expression that is also temporally regulated in a manner that parallels DM-induced differentiation. The fact that Myog expression corresponds to commitment of cells to myogenic differentiation further indicates that the actions of CTS are myogenic. In parallel to the upregulation of Myog, CTS induces the upregulation of two proteins, MEF2A and CDKN1A. Interestingly, while we observed CDKN1A upregulation at all time points tested, we did not observed similar changes in Cdkn1a mRNA expression. Whether this is due to increased stability of mRNA or increased translation is as yet to be explored. Nevertheless, by augmenting transcriptional activation of Mef2a, CTS upregulates the transcriptional activity of Myod1 and, hence, the ultimate synthesis of skeletal muscle structural proteins (35, 40). Synthesis and activation of CDKN1A are essential for the irreversible exit of C2C12 cells from the cell cycle and commitment to the differentiation program. Interestingly, CTS does not appear to upregulate the transcriptional activity of Cdkn1a above control levels; however, its synthesis is upregulated in cells exposed to CTS, suggesting a likely posttranscriptional control of this protein by CTS.
The function of bHLH and MEF2 transcription factors, in the context of myogenic differentiation, is to upregulate the induction of skeletal muscle structural proteins. The consistent upregulation of muscle structural proteins in response to CTS suggests that the transcription factors upregulated by CTS are functionally active. One of the most abundant proteins in a mature myotube is MYHC. Of the various isoforms, we examined Myh1 (MYH IIx), Myh2 (MYH IIa), and Myh4 (MYH IIb), which are essentially the fast twitch type of MYHC proteins (32). CTS markedly upregulates the transcriptional activation of Myh1, Myh2, and Myh4 and synthesis of MYHC and TPM1. Our findings further revealed that CTS, within 72 h, augments a >2.6 fold increase in MYHC-positive cells and nearly a 2.7-fold increase in myotube formation compared with C2C12 cells maintained in DM. Collectively, these observations underscore the importance of biomechanical signals in muscle regeneration and suggest that these extracellular signals are converted into biochemical events that can initiate and sustain the differentiation program that converts myoblasts into myotubes.
Muscle pathologies involving inflammation are the major causes of muscle weakness and loss of function. Accumulated evidences suggest that exercise or physical therapies provide increased functionality to inflamed muscle, and their lack results in muscle atrophy (12). To investigate the role played by CTS in preventing muscle inflammation, we examined the actions of CTS on C2C12 cell differentiation in the presence of TNF-. TNF- is a mediator of muscle inflammation. It downregulates the myogenic transcriptional program by activating NF-B, and its subsequent binding to the promoter regions of Myod1 results in reduced synthesis and activity of MYOD1 (1, 14). In our experiments, TNF- suppressed the promyogenic actions of DM at all steps, from the induction and nuclear translocations of MYOD1 and MYOG to the synthesis of MEF2A and CDKN1A. Thus, TNF- repressed the DM-induced exit of C2C12 cells from the cell cycle and commitment to the differentiation. CTS counteracted the actions of TNF- and allowed the expression of bHLH and MEF2 transcription factors as well as CDKN1A despite the presence of inflammatory levels of TNF-. Nevertheless, the levels of these transcription factors were similar or higher than those observed in control cells grown in DM. Since expression of Cdkn1a is one of the key events in the differentiation process that leads to a postmitotic state and fusion of cells to form multinucleated myotubes, CTS actions may be critical in removing the TNF--dependent inhibition of cells to exit from the cell cycle.
By three distinct ways, we examined the consequences of exposure of cells to CTS in the presence of TNF-, i.e., MYHC expression, numbers of MYHC-positive cells, and numbers of myotubes formed. Our findings are consistent with earlier reports (1, 14) that showed that TNF- severely impairs the ability of C2C12 cells to synthesize MYHC and TPM1. Furthermore, our findings provide additional evidence that TNF- inhibits C2C12 cell fusion and myotube formation even in the presence of low levels of MYHC in cells. Contrarily, CTS rescues C2C12 cells from TNF--mediated inhibition of MYHC synthesis, as evidenced by the levels of MYHC in the cells and the numbers of MYHC-positive cells. Interestingly, CTS also upregulates myotube formation in the presence of TNF-. The numbers of myotubes formed were at least similar to those observed in control cells maintained in DM. Thus, it can be concluded that CTS is able to activate the synthesis of skeletal muscle target genes and myotube formation and may thus prevent TNF--induced muscle decay in C2C12 myogenic cells.
Although stretch-induced upregulation of myogenic regulatory factors has been shown in whole muscle models (24, 27) and culture models (34), our findings are the first to demonstrate that CTS is a potent myogenic signal that dramatically upregulates myogenesis despite the presence of a strong proinflammatory environment. Signals generated by CTS are converted into molecular events that dynamically augment the activation and induction of myogenic transcription factors, skeletal muscle structural proteins, and eventual cell fusion to form multinucleate myotubes (Fig. 6). Signals generated by CTS also act as potent inhibitors of the intracellular actions of proinflammatory cytokines like TNF-, on the one hand, and induce muscle cell differentiation in a proinflammatory environment, on the other hand. Thus, biomechanical signals play an essential role in muscle regeneration and pathological muscle repair. In view of the fact that nonsteroidal anti-inflammatory drugs and steroids provide limited success in inflammatory myopathies, use of appropriate biomechanical signals may provide a promising mechanism for the development of therapeutic approaches.
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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P < 0.05 between TNF-
