Cells were used for assays at 3 days postransduction. Viral supernatant was concentrated, as described above, and administered to myoblasts. Because migR plasmids facilitate coexpression of green fluorescent protein GFP , transduction efficiency was evaluated based on GFP positivity by immunofluorescence IF. After 48 h, cells were changed to differentiation conditions.
Western blot analysis. Representative Western blotting images of multiple independent experiments are presented below. Femoral artery ligation FAL studies.
In 8- to week-old mice maintained on a mixed B6; background , hind limb ischemia was induced by ligating the left femoral artery as previously described Briefly, the femoral artery was exposed at the hip and separated from the femoral vein and nerve. Silk suture was passed under the artery and tied to occlude it. Limb perfusion measurements were taken before surgery, immediately following surgery, and 48 h later using diffuse correlation spectroscopy DCS DCS measurements were performed using a home-built instrument with two continuous-wave, long coherent nm lasers CrystaLaser Inc.
Data collection was performed simultaneously in both limbs, via four detectors distributed symmetrically along one single source positioned at the center.
This allowed for two source detector separations 0. In order to compare flow from the same region over the two different positions, we measured 3 different points along the bottom portion of the DCS probe, symmetrically positioned in each limb. In addition, mice were imaged before and immediately after surgery using a laser doppler imager Moor Instruments, United Kingdom.
Data collection and image generation were conducted using Moor LDI software. At 48 h after ligation, extensor digitorum longus muscles were harvested from the nonligated right and ligated left limb and homogenized into TRIzol for mRNA analysis or into RIPA buffer for protein analysis.
IF and microscopy. Myoblasts were cultured in well dishes and differentiated. A ratio was generated for each field in an experimental group, and an average ratio was determined. The average number of nuclei per field was generated for each experimental group.
Stem Cells and Cell Signalling in Skeletal Myogenesis - Google книги
After image analysis and data collection were complete, fluorescence images were processed i. Hypoxia inhibits primary and immortalized myoblast differentiation in vitro. To evaluate the impact of O 2 availability on muscle progenitor differentiation, we used established cell culture models of skeletal muscle development: the C2C12 murine myoblast cell line and primary adult mouse myoblasts. Myoblasts can be stimulated to terminally differentiate into multinucleated myotubes, signified by expression of MHC The differentiation conditions recapitulated features of ischemia-induced muscle regeneration: reduced availability of serum factors and local compensatory induction of IGFs 17 , 45 , 56 , 60 , Consistent with previous reports 15 , 19 , 25 , 51 , 64 , culturing C2C12 cells under low-O 2 conditions 0.
The decreased numbers of differentiated cells were not due to increased cell death, as exposure of C2C12 cells to 0. We also examined the expression of muscle regulatory factors MYOD and myogenin. These data indicate that hypoxia inhibits the myogenic transcriptional program and terminal differentiation of C2C12 myoblasts. We extended these analyses to primary skeletal myoblasts, obtained from the hind limb muscles of 8- to week-old mice. We reproducibly found that differentiating primary adult skeletal myoblasts at 0. In addition, myogenin protein levels were also reduced in hypoxic myoblasts Fig.
Therefore, hypoxia negatively regulates the differentiation program of skeletal muscle progenitors in multiple systems. Ischemia correlates with reduced MRF expression in vivo.
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In mouse models of PAD, the femoral artery providing blood to the hind limb muscles is ligated, producing acute skeletal muscle injury 7 , 24 , Skeletal muscle progenitors as well as damaged muscle fibers experience O 2 and nutrient deprivation before the formation of new blood vessels and terminally differentiated muscle 7 , 24 , We hypothesized that following ligation, hypoxic stress in skeletal muscle impedes progenitor differentiation until the revascularization process has restored nutrient availability.
To evaluate this possibility, we surgically occluded the left femoral artery in 8- to week-old adult mice and followed limb perfusion using both laser doppler imaging and diffuse correlation spectroscopy Blood flow within the ligated limb was significantly reduced immediately following surgery Fig.
Myogenin protein levels were also reduced in ischemic muscle Fig. These data suggest that ischemic stress negatively regulates the myogenic program in vivo , which correlates with the effects of hypoxia on myoblast differentiation in vitro. Skeletal muscle ischemia correlates with reduced MRF expression in vivo.
A FAL was performed on 8- to week-old mice. Perfusion heat maps are displayed. B FAL was performed as described for panel A. B C2C12 cells were cultured as for panel A. C C2C12 myoblasts were cultured as for panel A, and protein lysates were harvested. F C2C12 cells were cultured as described for panel E. We then evaluated expression of the myogenic program. Incubating either control or knockdown cells under low-O 2 conditions also caused a reduction in myogenin Fig. Terminal differentiation was also evaluated at 48 h. We initially evaluated this model by measuring the effect of hypoxia on genes regulated by NOTCH transcriptional activity Averages of 5 independent experiments are shown.
We also measured Hey2 levels in response to combined hypoxia and JAG1 treatment. Therefore, we directly assessed whether NOTCH signaling contributes to hypoxic inhibition of myoblast differentiation. Our data suggest that O 2 availability can regulate muscle progenitor differentiation through HIF-independent mechanisms.
Moreover, while AKT generates crucial responses to extracellular growth factors, this pathway is also sensitive to intracellular stress signals 12 , 42 , To assess this possibility, we measured levels of signal transduction downstream of PI3K Fig. Cells were harvested at different times over 24 h for protein lysates. H C2C12 myoblasts were cultured as described for panel F. This effect was detectable within 12 to 16 h of O 2 deprivation Fig.
Myoblast fusion confusion: the resolution begins
These results indicate that O 2 affects AKT activity toward a broad group of substrates. We next examined if AKT signaling was sensitive to O 2 levels in primary myoblasts. This suggests that O 2 controls AKT activity in several models of muscle progenitor differentiation. Treating differentiating myoblasts with either of these reagents or hypoxia resulted in similar decreases in P-AKT S and myogenin levels after 24 h Fig.
We observed that differentiating myoblasts transduced with myrAKT exhibited high levels of AKT activity irrespective of O 2 tension, in contrast to cells expressing the empty vector Fig. These results indicate that elevating AKT activity through constituitive membrane recruitment is sufficient to restore myoblast differentiation in hypoxia.
C C2C12 myoblasts were cultured as described for panel B. G C2C12 myoblasts were cultured as described for panel F. O 2 availability influences IGF-I receptor sensitivity to growth factors. Previous studies of cancer cells and fibroblasts have suggested that hypoxia can promote endoplasmic reticulum ER stress However, incubating myoblasts at 0. D C2C12 myoblasts were cultured as described for panel B. E C2C12 myoblasts were cultured as described for panel B.
In response to increased ER stress or negative feedback signals e. However, in C2C12 cells cultured under low O 2 conditions for 24 h, we observed that IRS1 and IRS2 protein levels were unchanged and that serine residues in IRS1 were hypophosphorylated, suggesting these proteins were not destabilized in hypoxia Fig. In response to growth factors, RAS stimulates mitogen-activated protein kinases i. This suggests that hypoxia does not modulate AKT through effects on RAS and that O 2 affects select growth factor-dependent pathways in myoblasts.
Total receptor levels were, again, unaffected Fig. Determining what factors regulate these precursors will facilitate their use in muscle repair 22 , 32 , 34 , In the present study, we investigated how the differentiation of skeletal muscle progenitors is influenced by O 2 deprivation—a key feature of peripheral arterial disease 4 , 7 , 24 , 26 , 33 , We found that low O 2 inhibits terminal differentiation of both immortalized and primary myoblasts.
Expression of the key muscle regulatory factors MYOD and myogenin is repressed by hypoxia in vitro and ischemia in vivo. Early reports linking O 2 to myoblasts did not evaluate if the HIFs were required for the effects of hypoxia 15 , 25 , These results were unexpected, for O 2 has been shown to control many developmental processes in a variety of lineages through HIF-dependent mechanisms It suggests that O 2 may influence muscle development and regeneration in vivo through pathways other than HIF.
While this is consistent with a recent report 51 , our study provides multiple additional insights into O 2 -dependent AKT inactivation. We also defined the kinetics and O 2 range for these effects and presented evidence that O 2 -dependent regulation of AKT occurs in primary myoblasts.