Figure 1.
Model of signaling pathways involved in muscle regeneration.
The satellite cell activating protein synthesis signaling (blue) and the interplay between myogenesis signaling (green) and myostatin-mediated growth control and protein degradation (red) which may form a negative feed-back loop to control growth or regeneration.
Figure 2.
Hepatocyte growth factor activates protein synthesis pathway and increases protein expression of myogenic factors.
In order to establish if HGF initiated the protein synthesis and myogenesis pathways and the timeframe in vivo, we injected a total of 21 normoxic mice (N = 3 for each time point) with hepatocyte growth factor (20 ng HGF/g body weight) and sacrificed them at time points between 0 and 48 h. A western blot of components of the protein synthesis and myogenesis pathways demonstrates how the signal activates the different proteins with time and eventually leads to an increased expression of myogenic factors (A). Two mice were given a 10 times higher dose of hepatocyte growth factor (200 ng HGF/g body weight) and sacrificed after 60 min ( = 60 minH). Western blots of tibialis anterior homogenates (N = 3 for each time point) shows activation of protein synthesis pathway through phosphorylation of PDK1 (B)/mTOR (C)/Akt (D)/p70S6K (E), and increased protein expression of myogenic factors MyoD (F) and myogenin (G). A 200 ng HGF/g body weight dose of hepatocyte growth factor yields a diminished response compared to a 20 ng HGF/g body weight dose RU = Relative Units. Error bars are SD. Thin dividing lines on the blot are for clarification only whereas the bold red line symbolizes that samples were run on two separate gels. One-way ANOVA with Bonferroni post hoc correction for multiple comparisons was used to assess differences among groups for each dependent variable. *denotes significant (P<0.05). Data are representative of one independent experiment.
Figure 3.
Hepatocyte growth factor increases mRNA expression of myogenic factors and myostatin.
Total RNA was purified from the same samples as used in figure 2. RT-qPCR analysis of mRNA expression levels in tibialis anterior of shows hepatocyte growth factor increases myogenic factors MyoD, myogenin (A,B). Muscle negative regulation and protein breakdown pathway myostatin, MAFbx and MuRF1 (C-E) were increased following a single injection with hepatocyte growth factor. RU = Relative Units. N = 3 for all time points, except for 60 minH ( = a 10 times higher dose of hepatocyte growth factor [200 ng HGF/g body weight] sacrificed after 60 min) where N = 2. Error bars are SD; One-way ANOVA with Bonferroni post hoc correction for multiple comparisons was used to assess differences among groups for each dependent variable. *denotes significant (P<0.05). Data are representative of four independent experiments.
Figure 4.
The hypoxic protocol and verification of atrophy model.
In order to create a muscle atrophy model, we let the mice stay in a chamber with a gradually more hypoxic atmosphere until it reaches approx. 7.5%. Oxygen content in the hypoxic chamber was monitored during the hypoxic protocol (•) and time points for alternating intraperitoneal injections (▴) with hepatocyte growth factor = H and leukemia inhibitory factor = L (A). Hypoxia did not change body composition (Lean/fat) measured by quantitative MRI of PBS-injected normoxic mice (N = 8, light blue), PBS-injected hypoxic mice (N = 8, dark blue) (B). Hypoxia did not change muscle water content based on dry weight: wet weight ratio in tibialis anterior (TA) of PBS injected normoxic mice (N = 8, light blue), PBS injected hypoxic mice (N = 8, dark blue) (C). Hypoxia induced loss of muscle protein measured as total soluble protein in muscle homogenates per wet weight of PBS-injected normoxic mice N = 8 (light blue) and PBS injected hypoxic mice (N = 8, dark blue) (D). Error bars are SD; Statistical significance was determined by a two-tailed Student's t-test. *denotes significant (P<0.05). Data are representative of one independent experiment.
Figure 5.
Effect of alternating hepatocyte growth factor and leukemia inhibitory factor treatment on bodyweight and muscle mass during hypoxia.
Hypoxia induced loss of mean bodyweight of both control PBS injected mice group (N = 14, dark blue) and HGF/LIF treated hypoxic mice group (N = 13, red) (A,D). PBS injected normoxic mice (N = 8, light blue) are included for comparison (D-G). Daily food and water intake per mouse during hypoxic protocol in the HGF/LIF treated group (N = 13, red) and in the control group (N = 14, dark blue) (B,C). Compared to PBS injected normoxic mice (N = 8, light blue) hypoxia induced loss of wet weight muscle mass of both tibialis anterior (TA) and extensor digitorum longus (EDL) (E,F). Alternating treatment of hypoxic mice with HGF/LIF (N = 13, red) increased muscle mass compared to PBS injected hypoxic mice (N = 14, dark blue) (E,F). Hypoxia induced loss of midbelly cross-sectional area of EDL of PBS injected hypoxic mice (N = 14, dark blue) compared PBS injected normoxic mice (N = 8, light blue). Alternating treatment of hypoxic mice with HGF/LIF increased cross-sectional area of EDL compared to PBS injected hypoxic mice (N = 14, dark blue) (G). Error bars are SD; Statistical significance was determined by a two-tailed Student's t–test. *denotes significant (P<0.05). Data are representative of one independent experiment.
Figure 6.
Hepatocyte growth factor and leukemia inhibitory factor treatment increases the mitosis of satellite cells in vivo during hypoxia.
Tibialis anterior sections were incubated with antibodies specific for satellite cell marker Pax7, proliferation marker Ki67 and DNA synthesis marker BrdU. Treatment of hypoxic mice with hepatocyte growth factor alone or in combination with leukemia inhibitory factor increased ratio of satellite cells undergoing divisions in following one HGF injection (N = 11, pink) compared to PBS (N = 12, blue) and multiple HGF/LIF (N = 13, red) injections compared to multiple PBS (N = 14, dark blue) or (A,C). Treatment of hypoxic mice with hepatocyte growth factor in combination with leukemia inhibitory factor (N = 13, red) increased number of cells with incorporated BrdU following DNA synthesis in following compared to multiple injections with PBS (N = 14, dark blue) (B,D). Bar in picture is 50 µm. Error bars are SD; Statistical significance was determined by a two-tailed Student's t–test. *denotes significant (P<0.05). Data are representative of one experiment.
Figure 7.
Effect of alternating hepatocyte growth factor and leukemia inhibitory factor treatment on protein synthesis pathway during hypoxia.
Western blots of tibialis anterior homogenates from hypoxia induced atrophic mice shows activation of protein synthesis pathway after repeated alternating injections with hepatocyte growth factor and leukemia inhibitory factor (N = 13, red) compared to PBS (N = 14, dark blue). This is based on evaluation of the phosphorylation of components from the protein synthesis pathway; pPI3K [p55α] (A), pPDK1 (B), pmTOR (C), pp70S6K (D), p4E-BP1 (E) and peIF4E (F). RU = Relative Units. Error bars are SD; Statistical significance was determined by a two-tailed Student's t–test. *denotes significant (P<0.05). Data are representative of one experiment.
Figure 8.
Alternating hepatocyte growth factor and leukemia inhibitory factor treatment increases mRNA expression of myostatin and MAFbx during hypoxia.
Total RNA was purified from the same samples as used in figure 7. RT-qPCR analysis of mRNA expression levels in tibialis anterior of shows elevated mRNA expression levels of myostatin, MAFbx and (C,D) after repeated injections with alternating HGF/LIF (N = 13, red) in hypoxia induced atrophic mice compared to PBS (N = 14, dark blue). Normoxic PBS treated mice (N = 8, light blue) were included for comparison. mRNA expression levels of MyoD, myogenin and MuRF1 was unchanged by treatment (A,B,E). RU = Relative Units. Error bars are SD; One-way ANOVA with Bonferroni post hoc correction for multiple comparisons was used to assess differences among groups for each dependent variable. *denotes significant (P<0.05). Data are representative of four independent experiments.
Figure 9.
Alternating hepatocyte growth factor and leukemia inhibitory factor treatment increases muscle mass and decreases protein breakdown pathway in normoxic myostatin deficient mice.
Alternating injections of normoxic B10 mice with hepatocyte growth factor and leukemia inhibitory factor (N = 8, yellow) did not change wet weight muscle mass of Tibialis anterior = TA and extensor digitorum longus = EDL (compared to PBS injected mice (N = 8, light blue) (A,B). Alternating injections of normoxic Mstnln/ln mice with hepatocyte growth factor and leukemia inhibitory factor increased TA muscle weight compared to PBS injected Mstnln/ln mice (N = 8, light grey). Western blot data of tibialis anterior homogenates (C-F) showed no change in muscle negative regulation and protein breakdown pathway (myostatin, MAFbx, MuRF1 and ubiquitin) after repeated alternating injections with HGF/LIF in normoxic B10. Repeated alternating injections with HGF/LIF in Mstnln/ln mice decreased expression of protein breakdown pathway MAFbx, MuRF1 and ubiquitin (D-F). RU = Relative Units. Statistical significance was determined by a two-tailed Student's t–test. *denotes significant (P<0.05). Data are representative of one independent experiment.
Figure 10.
Alternating hepatocyte growth factor and leukemia inhibitory factor treatment increases satellite cells undergoing mitotic divisions and decreases mRNA expression levels of protein breakdown pathway components in normoxic myostatin deficient mice.
Tibialis anterior sections were incubated with antibodies specific for satellite cell marker Pax7, proliferation marker Ki67. Alternating injections of normoxic B10 mice with hepatocyte growth factor and leukemia inhibitory factor (N = 8, yellow) did not change ratio of satellite cells undergoing mitotic divisions compared to PBS injected B10 mice (N = 8, light blue) (A). Ratio of satellite cells undergoing mitotic divisions was significantly increased in normoxic Mstnln/ln mice after repeated alternating injections with HGF/LIF (N = 8, dark grey) compared to PBS injected Mstnln/ln mice (N = 8, light grey). RT-qPCR analysis of mRNA expression levels yielded no difference in levels of MyoD was observed between groups (B). Alternating injections of normoxic Mstnln/ln mice with hepatocyte growth factor and leukemia inhibitory factor lead to increased levels of myogenin and decreased levels of myostatin, MAFbx and MuRF1 compared to treated normoxic B10 mice (B-F). RU = Relative Units. Error bars are SD; One-way ANOVA with Bonferroni post hoc correction for multiple comparisons was used to assess differences among groups for each dependent variable. *denotes significant (P<0.05). Data are representative of one independent experiment (A) and four independent experiments (B-F).