12 research outputs found
Stem cells migration during skeletal muscle regeneration - the role of Sdf-1/Cxcr4 and Sdf-1/ Cxcr7 axis
The skeletal muscle regeneration occurs due to the presence of tissue specific stem cells - satellite
cells. These cells, localized between sarcolemma and basal lamina, are bound to muscle fibers and
remain quiescent until their activation upon muscle injury. Due to pathological conditions, such as
extensive injury or dystrophy, skeletal muscle regeneration is diminished. Among the therapies
aiming to ameliorate skeletal muscle diseases are transplantations of the stem cells. In our previous
studies we showed that Sdf-1 (stromal derived factor ÂĄ1) increased migration of stem cells and
their fusion with myoblasts in vitro. Importantly, we identified that Sdf-1 caused an increase in the
expression of tetraspanin CD9 - adhesion protein involved in myoblasts fusion. In the current study
we aimed to uncover the details of molecular mechanism of Sdf-1 action. We focused at the Sdf-1
receptors - Cxcr4 and Cxcr7, as well as signaling pathways induced by these molecules in primary
myoblasts, as well as various stem cells - mesenchymal stem cells and embryonic stem cells, i.e. the
cells of different migration and myogenic potential. We showed that Sdf-1 altered actin
organization via FAK (focal adhesion kinase), Cdc42 (cell division control protein 42), and Rac-1 (Ras-
Related C3 Botulinum Toxin Substrate 1). Moreover, we showed that Sdf-1 modified the
transcription profile of genes encoding factors engaged in cells adhesion and migration. As the
result, cells such as primary myoblasts or embryonic stem cells, became characterized by more
effective migration when transplanted into regenerating muscle
Cell cycle regulation of embryonic stem cells and mouse embryonic fibroblasts lacking functional Pax7
The transcription factor Pax7 plays a key role during embryonic myogenesis and in adult organisms in that
it sustains the proper function of satellite cells, which serve as adult skeletal muscle stem cells. Recently
we have shown that lack of Pax7 does not prevent the myogenic differentiation of pluripotent stem cells.
In the current work we show that the absence of functional Pax7 in differentiating embryonic stem cells
modulates cell cycle facilitating their proliferation. Surprisingly, deregulation of Pax7 function also
positively impacts at the proliferation of mouse embryonic fibroblasts. Such phenotypes seem to be
executed by modulating the expression of positive cell cycle regulators, such as cyclin E
Adhesion proteins--an impact on skeletal myoblast differentiation.
Formation of mammalian skeletal muscle myofibers, that takes place during embryogenesis, muscle growth or regeneration, requires precise regulation of myoblast adhesion and fusion. There are few evidences showing that adhesion proteins play important role in both processes. To follow the function of these molecules in myoblast differentiation we analysed integrin alpha3, integrin beta1, ADAM12, CD9, CD81, M-cadherin, and VCAM-1 during muscle regeneration. We showed that increase in the expression of these proteins accompanies myoblast fusion and myotube formation in vivo. We also showed that during myoblast fusion in vitro integrin alpha3 associates with integrin beta1 and ADAM12, and also CD9 and CD81, but not with M-cadherin or VCAM-1. Moreover, we documented that experimental modification in the expression of integrin alpha3 lead to the modification of myoblast fusion in vitro. Underexpression of integrin alpha3 decreased myoblasts' ability to fuse. This phenomenon was not related to the modifications in the expression of other adhesion proteins, i.e. integrin beta1, CD9, CD81, ADAM12, M-cadherin, or VCAM-1. Apparently, aberrant expression only of one partner of multiprotein adhesion complexes necessary for myoblast fusion, in this case integrin alpha3, prevents its proper function. Summarizing, we demonstrated the importance of analysed adhesion proteins in myoblast fusion both in vivo and in vitro
Changes in expression and localization of adhesion proteins during <i>Soleus</i> muscle regeneration.
<p>A - level of mRNAs encoding adhesion proteins analyzed by sqRT-PCR. Optical densities of representative bands are shown as a percentage of GAPDH band density taken as a 100%. B â localization of integrin alpha3 (red) in intact and in regenerating muscle at day 3, 5, 7, 14. Nuclei â blue. White arrows show degenerating myofibers. Scale bars 50 ”m.C âcolocalization of Pax7, MyoD, myogenin (MG) (red) with integrin alpha3 (green) in intact muscle and at day 1, 3 and 5 of regeneration (intact, d1, d3, d5 respectively). White arrows - myoblasts, pink â muscle fiber nuclei, yellow - MyoD, myogenin and integrin alpha3 negative cells. D â control staing with secondary antibodies only. Nuclei â blue. Scale bars 50 ”m. E - localization of integrin beta1, ADAM12, CD9, or M-cadherin (green) at day 3 of regeneration. Nuclei â blue. Scale bars 50 ”m. F â immunoblotting analysis of M-cadherin (M-cad), integrin beta1, ADAM12 (AD12), CD9 and integrin alpha3 level during muscle regeneration (days 1, 3, 7, 14).</p
Downregulation of integrin alpha3 reduces the C2C12 ability to fuse with control myoblasts.
<p>A â control - myoblasts transfected with scramble siRNA-AlexaRed (red) cultured with myoblasts stained with QTracker (green), experiment+siRNA-alpha3â myoblasts cotransfected with scramble siRNA-AlexaRed (red) and siRNA-alpha3 cultured with control myoblasts stained with QTracker (green). Hybrid myotubes are yellow. Scale bars 20 ”m. B ânumber of hybrid myotubes formed by control myoblasts transfected with scramble siRNA-AlexaRed and control myoblasts stained with QTracker (control) compared with the number of hybrid myotubes formed by myoblasts co-transfected with scramble siRNA-AlexaRed and siRNA-alpha3 and control myoblasts stained with QTracker (siRNA-alpha3) (day 11 of culture). Error bars indicate SEM, results were analyzed by Student's test and differences were considered statistically significant when p<0.05 (marked with asterisks). ** pâ€0.01.</p
Expression of mRNAs encoding integrin alpha3 and other adhesion proteins at 24 and 48 hours after transfection of MPCs-derived myoblasts with siRNA-alpha3. sqRT-PCR analysis.
<p>NT â control, not transfected myoblasts, siRNA-alpha3 â myoblasts transfected with siRNA downregulating the expression of integrin alpha3. Optical densities of representative bands are shown as a percentage of GAPDH band density taken as a 100%.</p
Downregulation of alpha3 integrin expression reduces fusion of MPCs-derived myoblasts.
<p>A - Pappenheim's staining of fusing myoblasts. B â total number of nuclei calculated at day 12 of myoblast culture. C âindex of fusion analyzed at day 12 of culture in control and experimental myoblasts shown as a percentage of myotube nuclei per number of all nuclei. D - proportion of 2â4, 5â7, 8â10 and >11 nuclear myotubes per number of all myotubes, respectively. NT â control, not transfected myoblasts, TR â control myoblasts cultured in medium supplemented with transfection reagent, siRNA-cont - control myoblasts transfected with scramble siRNA, siRNA-alpha3 â myoblasts transfected with siRNA downregulating the expression of integrin alpha3. Error bars indicate SEM, results were analyzed by Kruskal-Wallis test. Kruskal-Wallis One Way Analysis of Variance showed differences between experimental groups which were considered statistically significant when p<0.05 (marked with asterisks). * pâ€0.05.</p