Muscle injury is characterized by degeneration of muscle fibers and rupture of vessels, accounting for decreased perfusion of the affected tissue (1). A significant reduction in functional capillary density with increased microvascular permeability rapidly evolves edema formation (2). Once a significant amount of muscle cell injury has occurred, the release of intracellular components together with endothelial dysfunction participates in organ dysfunction, often causing the death of patients (3). Milder injury directly passes to muscle regeneration starting with necrosis of damaged muscle fibers and activation as well as invasion of immune cells (4). Neutrophils are the first inflammatory cells recruited to the injured muscle by chemokines such as monocyte chemoattractant protein 1 (MCP-1), followed by macrophages that can be detected around 48 h after injury (4). Both cell types phagocytose cell remnants and secrete cytokines, chemokines, and growth factors that participate in muscle regeneration. This phase is characterized by activation of myogenic cells and replacement of the damaged muscle fibers (4). However, wound-healing studies in several organs showed that repair is even faster in the absence of neutrophils as long as conditions are sterile (5). Depletion of macrophages under similar conditions resulted in wound healing accompanied by diminished scar formation, but reduced clearance of cell and matrix debris at the wound site (5).
Upon injury, activation of satellite cells is crucial for regeneration. They either migrate to adjacent myofibers if the basal lamina is destroyed or migrate under the basal lamina to the site of injury (1). Different stimuli such as extracts from injured fibers (6-9), molecules released by invading macrophages (10), and soluble factors from connective tissue (4) have been proposed as initiators of satellite cell activation. Regeneration after trauma involves an extensive number of trophic factors, including fibroblast growth factor and insulin-like growth factor (IGF) family members in maintaining a balance between growth and differentiation of myoblasts to restore normal muscle architecture (4). In mouse models of contusion injury, IGF-1 and basic fibroblast growth factor improved healing of the muscle (11), suggesting that the administration of appropriate growth factors increases satellite cell activation and improves muscle regeneration. Application of satellite cells or other myogenic cells into the injured muscle favors the formation of new muscle tissue and subsequently improves muscle regeneration (1).
Because of the destruction of vessels, hypoxia is a prominent feature at the injury site. The most important transcription factor activated by hypoxia is hypoxia-inducible factor (HIF). Hypoxia-inducible factor is a heterodimer of an α- and β-subunit, which both are members of the family of basic-helix-loop-helix/PER, ARNT, SIM (PAS) transcription factors (12). Abundance and activity of HIF-1α and the homologous HIF-2α are instantaneously increased upon hypoxia, whereas steady-state levels of the HIF-1β protein are not affected by changes in oxygen tension (12). Stabilized HIF-α subunits translocate to the nucleus, bind to their β-subunit, and induce expression of several target genes (12). Increased transcriptional activity of HIF in muscle tissue elevated the expression of glycolytic, proangiogenic, and proerythropoietic factors such as adrenomedullin (ADM) (13, 14). It is well established that HIF upregulates target genes such as vascular endothelial growth factor during ischemia-reperfusion injury. Moreover, chemical activation of HIF during ischemia-reperfusion enhances regeneration of the affected tissue (13, 15). Unfortunately, nothing is known about the role of HIF in muscle crush injury.
Out aim was to analyze HIF activation in muscle cells after blunt trauma. Therefore, we used an in vitro approach of crushed myotubes. Recently, we showed ischemic HIF-1 activation in both undifferentiated (e.g., satellite cell-like) myoblasts and differentiated myotubes (16). Although HIF activation is thought to be protective for cells in hypoxic or ischemic conditions, prolonged ischemic HIF-1 activation did not improve survival of myotubes. In this study, to simulate blunt trauma, myotubes were scraped from the culture dish and thereby mechanically destroyed. Applying this cell debris to intact cells, we experimentally separated muscle cell reactions toward necrotic cells from hypoxic activation and thus analyzed the impact of muscle cell debris on HIF-1 activation. Furthermore, we analyzed whether genes important for regeneration (MCP-1, ADM, IGF, and metallopeptidase 9 [MMP9]) were induced in skeletal muscle cells by HIF-1.
MATERIALS AND METHODS
If not indicated otherwise, chemicals were purchased from Sigma-Aldrich (Taufkirchen, Germany). The mouse myoblast cell line C2C12 (LGC Promochem, Wesel, Germany) was grown in Dulbecco's modified Eagle medium high glucose supplemented with 20% fetal calf serum, 0.11 mg/mL sodium pyruvate, 100 U/mL penicillin, and 100 μg/mL streptomycin (all from PAA Laboratories, Cölbe, Germany) in a humidified atmosphere of 19% O2, 73% N2, and 8% CO2 (by vol). C2C12 myoblasts were cultured to ∼80% of cell density before the experiments, which corresponds to 1.5 × 104 cells/cm2. Myotubes were differentiated from confluent myoblast cultures (approximately 4 × 104 cells/cm2) by replacement of fetal calf serum with 2% heat-inactivated horse serum (PAA Laboratories) for 6 days. Cell culture inserts were purchased from Greiner Bio One (Solingen-Wald, Germany) and used as recommended by the supplier.
Cell debris was prepared as recently described (17). Shortly, myotubes were differentiated on 100-mm culture dishes and mechanically destroyed in 4.5-mL fresh medium using a cell scraper. For RNA, extraction cells were grown on six-well plates, and 2 mL of the cell debris solution was used, whereas 12 mL of cell debris was used for nuclear protein isolation from 100-mm culture dishes. Cells were incubated for the periods indicated in the figure legends. Supernatant and pellet of cell debris were prepared by centrifugation (2,300g for 5 min at room temperature). Pellets were resuspended in the same volume used before. Heat inactivation was performed by boiling at 100°C for 10 min and afterward cooling to 37°C. Alternatively, cell debris was pretreated with ultrasound for 30 s or exposed to 3 freeze-thaw cycles using liquid N2. Experiments with superoxide dismutase (SOD; 500 U/mL) and catalase (500 U/mL) were carried out by addition of the enzyme to the medium before the cells were mechanically removed; thus, the enzymes were present during the whole process of cell debris preparation. For controls, instead of cell debris, cells were also incubated with 5% albumin or 25 μg/mL PolyIC InvivoGen (Cayla-InvivoGen, Toulouse, France). Lipopolysaccharide (LPS; 1 μg/mL), 100 U interferon γ (IFN-γ), and 5 μM IL-1 receptor-associated kinase (IRAK) 1/4 inhibitor N-(2-morpholiylethyl)-2-(3-nitrobenzoylamido)-benzimidazole were added at the beginning of the experiments. To avoid pericellular hypoxia due to diffusion-limited O2 supply to the cells, cell 60-mm culture dishes with gas permeable bottom were used (Greiner Bio One), and cells were incubated with 4-mL medium or cell debris.
Lentiviruses containing shRNA against HIF-α and a nontarget sequence, which were analyzed as a control, were produced by HEK293T cells. Transduction of C2C12 cells followed protocols supplied by Sigma-Aldrich. Five micrograms of puromycin was used for positive selection.
Reverse transcription and quantitative real-time polymerase chain reaction
Total RNA was isolated with phenol/chloroform extraction from six-well dishes using peqGold RNAPure, and RNA amount was calculated using Nanodrop ND1000 spectrometer (Peqlab, Erlangen, Germany). One microgram of total RNA was transcribed into cDNA with iScript cDNA Synthesis Kit (BioRad Laboratories, Munich, Germany). Subsequently, cDNAs were quantified by real-time polymerase chain reaction (PCR) using Absolute QPCR SYBR Green Fluorescein Mix (Abgene, Darmstadt, Germany) and the MyiQ Single-Color Real-Time Detection System (BioRad Laboratories). The PCR reactions were set up in a final volume of 20 μL with 0.4 μL cDNA, in 1× reaction buffer with SYBR Green I, 10 pmol forward, and 10 pmol reverse primer. The primers used were as follows: ADM cgc agt tcc gaa aga agt gg (forward) and cca gtt gtg ttc tgc tcg tcc (reverse); MMP9 cgt cgt gat ccc cac tta ct (forward) aac aca cag ggt ttg cct tc (reverse) IGF-2 aca ggc att gtg gat gag tg (forward), cct ttg cag ctt cgt ttt ct (reverse); IGF-1eb aca ggc att gtg gat gag tg (forward) cct ttg cag ctt cgt ttt ct (reverse); MCP-1 gat gat ccc aat gag tag gc (forward), ggt tgt gga aaa ggt agt gg (reverse); S16 ribosomal protein aga tga tcg agc cgc gc (forward) gct acc agg gcc ttt gag atg ga (reverse); HIF-1α gaa atg gcc cag tga gaa aa (forward), ctt cca cgt tgc tga ctt ga (reverse). All samples were quantified in triplicate from RNA from three separate culture dishes.
Western blot analysis
Nuclear protein extracts were prepared from 100-mm dishes of cells as described previously (16). Shortly, cells were lysed in 100 μL modified extraction buffer and incubated for 20 min on ice followed by centrifugation (5,900g at 4°C for 8 min). The supernatant was used as cytosolic fraction, and the pellet was resuspended and lysed in the second buffer described previously (16) using a magnetic stirrer for 30 min on ice. Extracts were centrifuged (16,100g at 4°C for 10 min). Supernatants were used as the nuclear fraction. The protein concentration was determined with a commercial protein assay (BioRad Laboratories). Sixty micrograms of the nuclear extracts were loaded onto a 7.5% sodium dodecyl sulfate-polyacrylamide gel. Separated proteins were blotted onto nitrocellulose membranes (Whatman, Dassel, Germany), followed by incubation with a rabbit polyclonal antibody (diluted 1: 750) for mouse HIF-1α (Novus Biologicals Acris, Hiddenhausen, Germany) and a monoclonal mouse anti-tubulin antibody (diluted 1: 2,000). Horseradish peroxidase-conjugated goat anti-rabbit IgG and anti-mouse IgG (1:100,000) were used as secondary antibodies. The ECL Advanced Western blotting system (Amersham Biosciences, Freiburg, Germany) was used for detection.
Enzyme-linked immunosorbent assay
The content of MCP-1 in cell culture supernatants was determined by means of enzyme-linked immunosorbent assay (ELISA; R&D Systems, Wiesbaden, Germany) as recommended by the manufacturer. The detection limit was 15 pg/mL.
RNA and ELISA experiments were performed in triplicate and repeated at least three times. Data were expressed as means ± SEM. Statistic relevance was assessed by comparison of means of ANOVA and Bonferroni test. The P values are given in the figure legends.
Mechanically destroyed muscle fibers induced gene expression in myoblasts and myotubes
We used the skeletal muscle cell line C2C12 and incubated nondifferentiated myoblasts and differentiated myotubes in the presence of mechanically destroyed myotubes (cell debris) for 6 h and analyzed expression of genes involved in muscle regeneration. Adrenomedullin mRNA was significantly upregulated in myoblasts and myotubes (Fig. 1A), whereas IGF-2 (Fig. 1B) and IGF-1 (Fig. 1C) were upregulated only in myoblasts. Myotubes constitutively expressed 330-fold and 30-fold higher levels of IGF-2 and IGF-1, respectively, compared with myoblasts. Cell debris did not further increase the expression of these genes. Metallopeptidase 9 was significantly upregulated in myoblasts and myotubes after contact with cell debris (Fig. 1D). In addition, the chemokine MCP-1 was induced (Fig. 1E) after contact with mechanically destroyed cells. This demonstrated that muscle cells themselves upregulated some growth factors, chemokines, and enzymes involved in tissue regeneration after their exposure to mechanically injured cells.
ADM induction reflected increased transcription
As the addition of cell debris increased the total RNA amount of samples, we wanted to exclude unspecific effects of exogenous RNA on gene induction. Therefore, we plotted ADM expression of myoblasts (Fig. 2A) and myotubes (Fig. 2B) against total RNA amount. Induction of ADM was used for this analysis because it showed the highest increase. Adrenomedullin expression and RNA amount were higher after incubation with cell debris, but no correlation between ADM expression and RNA amount was observed. In addition, we analyzed expression of S16 ribosomal protein mRNA as a housekeeping gene from the same samples and found no increase (Fig. 2C). Apparently, the increase in ADM expression and presumably that of other genes induced by debris were caused by increased transcription after contact to cell debris.
Gene induction required direct contact to cell remnants
Next, we assessed time responses of ADM induction in myoblasts up to 24 h and found an induction after 3 h, which remained significant until 18 h of incubation (Fig. 3A). Because 6 h of incubation was sufficient to significantly increase ADM mRNA, we used this incubation period for further experiments. To analyze which fraction of the cell debris was relevant for gene induction, cell debris was treated with ultrasound for 30 s to destroy membranes before addition to the cells. This reduced the capacity to induce ADM, but induction still remained significant in myoblasts (Fig. 3B). When cell debris was frozen (in liquid N2) and thawed, only myotubes significantly induced ADM. Heat denaturation of proteins eliminated ADM induction completely in both cell types (Fig. 3B). When cell debris was centrifuged to separate soluble factors (supernatant) from insoluble cell remnants (pellet), only the pellet induced ADM mRNA (Fig. 3C). In addition, the supernatant of myotubes was analyzed for MCP-1 protein release (Fig. 3D). Parallel to mRNA induction, MCP-1 protein release was enhanced in the presence of cell debris. Cells incubated with the supernatant or the pellet of cell debris also revealed a response only to the pellet showing a strong resemblance of ADM mRNA and MCP-1 protein induction. Incubation of cell debris separated from cells by using transwell inserts diminished ADM induction in myoblasts significantly (Fig. 3C). However, myotubes were activated in the presence of the inserts alone. Because the supernatant did not induce ADM mRNA, it is very likely that they also need a direct contact with destroyed cells. This indicates that proteins from destroyed myofibers in direct contact to precursor myoblasts or differentiated myotubes induce ADM and MCP-1 expression.
To evaluate whether ADM induction by cell debris resulted from increased protein or nucleic acid concentration in the supernatant, myoblasts were incubated in the presence of 5% albumin or 25 μg/mL PolyIC (Fig. 3E). Neither albumin nor PolyIC significantly induced ADM gene expression, indicating that specific muscle cell remnants in muscle cell debris are necessary for myoblast activation. Danger signals such as heat shock proteins, possibly a component of cell debris, are often recognized by toll-like receptors (TLRs). We therefore investigated whether TLR signaling was involved in ADM induction. Because MyD88, IRAK1, and IRAK4 are important upstream adaptor proteins for TLR1-2 and TLR4-9, we used an IRAK-1/4 inhibitor. As a positive control, cells were incubated with LPS/IFN-γ for 6 h to activate TLR4/IRAK-1/4 signaling, which results in 3-fold induction of ADM. Addition of the IRAK-1/4 inhibitor reduced LPS/IFN-γ-induced ADM upregulation to only 1.4-fold (data not shown). In contrast, the addition of IRAK-1/4 inhibitor to cell debris had no effect on ADM gene induction excluding MyD88-IRAK-1/4-dependent signaling (Fig. 3E).
Production of reactive oxygen species did not mediate gene induction
Because mechanically damaged myotubes have been shown to produce reactive oxygen species via membrane-bound NADPH oxidases (17), we next evaluated whether the induction of ADM was reactive oxygen species mediated. Incubation of cells in the presence of SOD or catalase alone or in combination did not alter ADM mRNA (data not shown). Addition of SOD or catalase or both had no effect on mRNA induction by cell debris (Fig. 3F).
Myotubes were able to produce an autocrine factor
In addition, we incubated native myoblasts or myotubes with the supernatant of cells incubated in the presence of cell debris for 6 h. The supernatant of these activated cells was used for another 6 h of incubation on fresh cells. Only supernatant of myotubes was able to increase ADM mRNA expression in fresh myotubes (Fig. 3G). Because the supernatant of control cells (data not shown) did not induce ADM, myotubes activated by cell debris released some soluble factors that were able to activate myotubes in an autocrine manner.
Mechanically destroyed muscle fibers activated HIF-1
Because ADM, MMP9, IGF-2, and MCP-1 are reported to be upregulated by HIF, we analyzed HIF-1α expression in myoblasts and myotubes. A slight increase of HIF-1α protein expression in myotubes and myoblasts in the presence of cell debris was observed after 4 h and became more pronounced after 8 h (Fig. 4A). The increase was smaller but still prominent compared with hypoxic accumulation of HIF-1α in these cells and was not caused by increased HIF-1α mRNA expression (Fig. 4B) or reduced expression of PHD2 (data not shown). Thus, HIF-1 was activated in myoblasts and myotubes after contact to crushed muscle cells, presumably by increased protein stability.
HIF-1 upregulated ADM and MCP-1
To evaluate whether upregulation of genes after contact to cell debris was mediated by HIF, we knocked down HIF-1α in C2C12 myoblasts by lentiviral transfection. Hypoxia-inducible factor 1α protein and mRNA expression were reduced to about 40% of controls, whereas transfection with the control virus left HIF-1α expression unaltered (Fig. 5, A and B). We then analyzed mRNA expression of ADM, MMP9, IGF-2, and MCP-1 in wild-type and knockdown myoblasts (KO) after incubation with cell debris. Inductions of ADM and MCP-1 were reduced in knockdown cells (Fig. 5, C and D), whereas MMP9 and IGF-2 inductions were not affected (Fig. 5, E and F). These data show that direct contact to damaged myofibers is able to activate HIF-1 in myotubes and myoblasts, which upregulated ADM and MCP-1. In addition, HIF-unrelated pathways are activated, resulting in elevated expression of MMP9 and IGF-2.
To exclude HIF activation due to decreased pericellular pO2 of the cells incubated with cell debris, myoblasts were cultured on culture dishes with a gas-permeable bottom. Because of the diffusion-limited O2 supply of cells in conventional culture dishes, compounds that increase mitochondrial O2 consumption may cause pericellular hypoxia (18). When we investigated ADM induction comparing conventional and gas-permeable dishes, we observed a reduced ADM expression on gas-permeable dishes even under control conditions and a lower induction by hypoxia (Fig. 5G). Cell debris induced ADM gene expression, although the induction was less compared with conventional dishes. Nevertheless, these experiments showed that cell debris induced ADM gene expression and therefore HIF activation due to protein interaction with C2C12 cells not by inducing pericellular hypoxia.
To study the responses of skeletal muscle cells during blunt trauma, we used an in vitro model of scrape-injured myotubes (17). This model allowed to distinguish between reactions of muscle cells to damaged cells from those toward hypoxia or other environmental modifications. In general, detection of necrotic cell remnants is mediated by molecules such as high-mobility group box 1, nucleic acids, ATP, heat shock proteins, or extracellular matrix components (19, 20). Toll-like receptors seem necessary for this recognition in sterile injuries (19). Because C2C12 myoblasts such as primary skeletal muscle tissue express TLRs and respond to LPS and other TLR ligands with the production of cytokines such as IL-6 (21, 22), it is likely that C2C12 cells detect necrotic cells (cell debris) by TLR ligation. Activation of TLR4 by its ligand LPS activates HIF-1 by multiple pathways in macrophages (23, 24). When we studied HIF-1α expression in myoblasts and myotubes, we noticed accumulation of the HIF-1α protein but not mRNA after their contact with cell debris. Although ischemia-reperfusion settings make HIF activation likely, our experiments indicate a nonhypoxic activation of HIF during blunt trauma. The addition of cell debris with residual oxygen-consuming capacity (17) might have increased overall O2 consumption in the culture dish and decreased pericellular O2 concentrations. Our experiment using gas-permeable culture dishes, however, proved that O2 supply was not limited, and thus, HIF activation and ADM induction was unlikely driven by local hypoxia. Furthermore, the fact that ADM mRNA was not induced by the supernatant of the cell debris ruled out the participation of ATP, growth factors, or other soluble factors. Rather, a direct contact of proteins of the necrotic cells with vital myoblasts and myotubes was necessary for HIF activation and thus ADM upregulation. Although the missing effect of the IRAK-1/4 inhibitor may exclude MyD88/IRAK-dependent signaling involved in gene induction, we cannot rule out TLR/TRIF-dependent activation of HIF after contact to muscle cell debris.
Enhanced activation of HIF in skeletal muscle tissue after ischemic injury increased recovery and enhanced angiogenesis in animal models (15, 25, 26). Here, HIF-1 activation in C2C12 cells after contact to crushed myofibers increased ADM expression. Adrenomedullin has several effects on the vascular system, such as stabilization of the endothelial barrier function with subsequent reduced edema formation during inflammation or ischemia-reperfusion (27). In addition, ADM was shown to enhance angiogenesis by increasing the recruitment of bone marrow-derived cells after ischemia-reperfusion in skeletal muscle (28). Therefore, activation of HIF-1 in muscle cells directly after contact to crushed cells may reflect a mechanism that contributes to muscle regeneration by upregulation of proteins important for angiogenesis.
In addition, HIF-mediated upregulation and release of the chemokine MCP-1 in myotubes and myoblasts after their contact to crushed myofibers might be involved in the recruitment of monocytes to the damaged site. Treatment with anti-MCP-1 serum, knockout of MCP-1, or its receptor reduced monocytes recruitment to the injured muscle, delayed phagocytosis of necrotic tissue, and thus impaired regeneration of injured muscle in animal models (29-31). Overexpression of MCP-1 in heart tissue increased tissue regeneration by increasing macrophage content (32), again demonstrating the importance of macrophages for muscle regeneration. Moreover, MCP-1 receptors are expressed in myoblasts, and stimulation with its ligand resulted in increased proliferation and migration (33).
Mechanically damaged muscles produce factors such as IGF-1 or IGF-2 to enhance proliferation and migration of satellite cells (7-9). The addition of IGF-1 after injury in vivo enhances proliferation and differentiation of myoblasts and reduces fibrosis (11). We showed that especially undifferentiated myoblasts respond to crushed muscle fibers by upregulation of IGF-1 and IGF-2, whereas myotubes already express elevated levels. Upon injury, quiescent satellite cells start to proliferate, fuse to form myotubes, and subsequently differentiate into mature myofibers (34). Insulin-like growth factors 1 and 2 are central in this process and are upregulated during differentiation parallel to activation of MyoD (35). Thus, the upregulation of IGF by myoblasts may reflect activation of these cells to contribute to myofiber replacement.
In addition, the matrix protease MMP9, necessary for tissue remodeling and regeneration, was significantly upregulated in myoblasts and myotubes after contact to crushed muscle cells. Metallopeptidases 9 and 2 are involved in destruction of extracellular matrix that liberates growth factors and are therefore important in muscle regeneration (34). Studies of others so far showed that upregulation of MMPs in muscle tissue after blunt trauma in vivo mostly depends on macrophages (36). Nevertheless, myoblasts are able to upregulate MMP9 after stimulation with proinflammatory cytokines in vitro (37). This response depends on nuclear factor κB and AP-1 activity, which might also be involved in cell debris-induced effects because our data on MMP9 regulation indicate that it was not induced by HIF-1.
Directly after mechanical trauma of muscle tissue, a complex program of tissue regeneration is initiated. In the first phase, damaged cells need to be removed, whereas formation of new fibers is required afterward. Macrophages play an important role in this process, predominantly by phagocytosis of cell remnants. We observed increased MCP-1 production by muscle cells presumably to support phagocyte recruitment. In addition, muscle cells, besides macrophages and other immune cells, produce growth factors such as IGF and ADM and enzymes important for tissue regeneration in response to cell debris. Several factors are HIF-dependently upregulated in muscle cells, which implicates that activation of HIF might be beneficial to overcome blunt muscle trauma and would be a rewarding target for further studies. The fact that HIF is activated directly by dead cells indicates a unique function of HIF during regeneration after blunt trauma independent of hypoxia.
The authors thank Tanja Keppler and Bettina Wenzel for their skillful technical assistance.
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