Iwona Grabowska

Protein histidine methylation is a rare post-translational modification of unknown biochemical importance. In vertebrates, only a few methylhistidine-containing proteins have been reported, including β-actin as an essential example. The evolutionary conserved methylation of β-actin H73 is catalyzed by an as yet unknown histidine N-methyltransferase. We report here that the protein SETD3 is the actin-specific histidine N-methyltransferase. In vitro, recombinant rat and human SETD3 methylated β-actin at H73. Knocking-out SETD3 in both human HAP1 cells and in Drosophila melanogaster resulted in the absence of methylation at β-actin H73 in vivo, whereas β-actin from wildtype cells or flies was > 90% methylated. As a consequence, we show that Setd3-deficient HAP1 cells have less cellular F-actin and an increased glycolytic phenotype. In conclusion, by identifying SETD3 as the actin-specific histidine N-methyltransferase, our work pioneers new research into the possible role of this modification in health and disease and questions the substrate specificity of SET-domain-containing enzymes.

BACKGROUND INFORMATION: The regeneration of skeletal muscles involves satellite cells, which are muscle-specific precursor cells. In muscles, injured either mechanically or as a consequence of a disease, such as muscular dystrophy, local release of the growth factors and cytokines leads to satellite cells activation, proliferation and differentiation of the resulting myoblasts, followed by the formation of new myofibres. Various cell types, such as stem and progenitor cells, originating from other tissues different than the muscle, are also able to follow a myogenic program. Participation of these cells in the repair process depends on their precise mobilisation to the site of the injury. RESULTS: In this study, we showed that stromal-derived factor-1 (Sdf-1) impacts on the mobilisation of CXC chemokine receptor (Cxcr)4-positive cells and improves skeletal muscle regeneration. Analysis of isolated and in vitro cultured satellite cells showed that Sdf-1 did not influence myoblasts proliferation and expression of myogenic regulatory transcription factors but induced migration of the myoblasts in Cxcr4-dependent ways. This phenomenon was also associated with the increased activity of crucial extracellular matrix modifiers, i.e. metalloproteases Mmp-2 and Mmp-9. CONCLUSIONS: Thus, positive impact of Sdf-1 on muscle regeneration is related to the mobilisation of endogenous cells, that is satellite cells and myoblasts, as well as non-muscle stem cells, expressing Cxcr4 and CD34.

The proper functioning of tissues and organs depends on their ability to self-renew and repair. Some of the tissues, like epithelia, renew almost constantly while in the others this process is induced by injury or diseases. The stem or progenitor cells responsible for tissue homeostasis have been identified in many organs. Some of them, such as hematopoietic or intestinal epithelium stem cells, are multipotent and can differentiate into various cell types. Others are unipotent. The skeletal muscle tissue does not self-renew spontaneously, however, it presents unique ability to regenerate in response to the injury or disease. Its repair almost exclusively relies on unipotent satellite cells. However, multiple lines of evidence document that some progenitor cells present in the muscle can be supportive for skeletal muscle regeneration. Here, we summarize the current knowledge on the complicated landscape of stem and progenitor cells that exist in skeletal muscle and support its regeneration. We compare the cells from two model organisms, i.e., mouse and human, documenting their similarities and differences and indicating methods to test their ability to undergo myogenic differentiation.

Changes in the expression of adhesion proteins involved in myoblast differentiation were investigated in monolayer (two-dimensional) and 3D (three-dimensional) cell cultures. The expression of integrin alpha3 subunit, integrin beta1 subunit, ADAM12 (a disintegrin and metalloproteinase 12), tetraspanins CD9 and CD81 and M-cadherin were examined in the murine myoblast cell line C2C12 and in a primary culture of rat satellite cells. Myoblasts in monolayer and 3D cultures showed significant differences in their morphology and cytoskeletal organization. All of the studied proteins participated in myoblast fusion in each culture examined, but differences in their levels of expression were observed. Satellite cell-derived myoblasts exhibited higher expression of adhesion protein mRNAs than C2C12 cells. Also, C2C12 cells from a 3D culture showed slightly higher expression of adhesion protein transcripts than the same cells cultured as a monolayer. Significantly, the levels of adhesion protein mRNAs were found to change in parallel in all cell culture types. Despite this finding, it is important that differences between satellite cell-derived myoblasts and cell line C2C12 grown in monolayer and 3D cultures are taken into account when studying processes of myoblast differentiation in vitro.

BACKGROUND: The skeletal muscle reconstruction occurs thanks to unipotent stem cells, i.e., satellite cells. The satellite cells remain quiescent and localized between myofiber sarcolemma and basal lamina. They are activated in response to muscle injury, proliferate, differentiate into myoblasts, and recreate myofibers. The stem and progenitor cells support skeletal muscle regeneration, which could be disturbed by extensive damage, sarcopenia, cachexia, or genetic diseases like dystrophy. Many lines of evidence showed that the level of oxygen regulates the course of cell proliferation and differentiation. METHODS: In the present study, we analyzed hypoxia impact on human and pig bone marrow-derived mesenchymal stromal cell (MSC) and mouse myoblast proliferation, differentiation, and fusion. Moreover, the influence of the transplantation of human bone marrow-derived MSCs cultured under hypoxic conditions on skeletal muscle regeneration was studied. RESULTS: We showed that bone marrow-derived MSCs increased VEGF expression and improved myogenesis under hypoxic conditions in vitro. Transplantation of hypoxia preconditioned bone marrow-derived MSCs into injured muscles resulted in the improved cell engraftment and formation of new vessels. CONCLUSIONS: We suggested that SDF-1 and VEGF secreted by hypoxia preconditioned bone marrow-derived MSCs played an essential role in cell engraftment and angiogenesis. Importantly, hypoxia preconditioned bone marrow-derived MSCs more efficiently engrafted injured muscles; however, they did not undergo myogenic differentiation.