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In the present study, the genetic modification of human skeletal muscle-derived stem/progenitor cells (SkMDS/PCs) was investigated to identify the optimal protocol for myogenic cell preparation for use in post-infarction heart therapy. We used two types of modifications: GFP  - transfection (using electroporation) and SOD3 transduction (using a lentiviral vector). SkMDS/PCs were cultured under different in vitro conditions, including standard (21% oxygen) and hypoxic (3% oxygen), the latter of which corresponded to the prevailing conditions in the post-infarction heart. Transfection/transduction efficacy, skeletal myogenic cell marker expression (CD56), cellular senescence, and apoptosis, as well as the expression of antioxidant ( SOD1 , SOD2 , and SOD3 ), anti-aging ( SIRT1 and FOXO ), anti-apoptotic ( BCL2 ), and myogenic ( MyoD and MyoG ) genes, were evaluated. The percentage of GFP-positive SkMDS/PCs was determined as an indicator of the efficacy of transfection, which reached 55%, while transduction showed better efficiency, reaching approximately 85% as estimated by fluorescence microscopy. The CD56-positive SkMDS/PCs were present in approximately 77% of the tested cells after transient transfection and approximately 96% after transduction. Under standard in vitro culture conditions, the ability of the differentiated, transfected SkMDS/PCs to form myotubes was greater than that of the wild type (WT) cell population ( p &lt; 0.001), while the cells transduced with the SOD3 gene exhibited an increase in cell fusion under both standard ( p &lt; 0.05) and hypoxic conditions ( p &lt; 0.001). In transduced SkMDS/PCs, we observed a positive influence of SOD3 overexpression on cell ageing and apoptosis. We observed an increase in the percentage of young cells under standard ( p &lt; 0.05) and hypoxic ( p &lt; 0.001) in vitro culture conditions, with a notable decrease in the percentage of senescent and advanced senescent cells in the SOD3 -overexpressing cell population detected compared to that observed for the untransduced muscle-derived cells. A lower percentage of apoptotic cells was observed for transduced SkMDS/PCs than that for WT cells under hypoxic in vitro culture conditions. In transiently transfected SkMDS/PCs, we observed significantly higher gene expression levels of SOD2 (almost 40-fold) ( p &lt; 0.001) and FOXO ( p &lt; 0.05) (approximately 3-fold) under both normoxic and hypoxic culture conditions and of BCL2 under hypoxia compared to those observed in untreated cells (WT). In addition, myogenic genes showed a significant increase in MyoD (almost 18-fold) expression under standard culture conditions ( p &lt; 0.0001) and decreased MyoG expression (approximately 2-fold) after transfection ( p &lt; 0.05) compared with that detected in the WT skeletal muscle-derived cell control. Taken together, these results demonstrate that SOD3- tranduced skeletal muscle-derived cells may have potential for use in the regenerative treatment of the post-infarction heart.

antioxidants

Transient and Stable Overexpression of Extracellular Superoxide Dismutase is Positively Associated with the Myogenic Function of Human Skeletal Muscle-Derived Stem/Progenitor Cells