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

Abstract

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: <i>GFP</i><i>-</i>transfection (using electroporation) and <i>SOD3</i> transduction (using a lentiviral vector). SkMDS/PCs were cultured under different <i>in vitro</i> 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 (<i>SOD1</i>, <i>SOD2</i>, and <i>SOD3</i>), anti-aging (<i>SIRT1</i> and <i>FOXO</i>), anti-apoptotic (<i>BCL2</i>), and myogenic (<i>MyoD</i> and <i>MyoG</i>) 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 <i>in vitro</i> culture conditions, the ability of the differentiated, transfected SkMDS/PCs to form myotubes was greater than that of the wild type (WT) cell population (<i>p</i> < 0.001), while the cells transduced with the <i>SOD3</i> gene exhibited an increase in cell fusion under both standard (<i>p</i> < 0.05) and hypoxic conditions (<i>p</i> < 0.001). In transduced SkMDS/PCs, we observed a positive influence of <i>SOD3</i> overexpression on cell ageing and apoptosis. We observed an increase in the percentage of young cells under standard (<i>p</i> < 0.05) and hypoxic (<i>p</i> < 0.001) <i>in vitro</i> culture conditions, with a notable decrease in the percentage of senescent and advanced senescent cells in the <i>SOD3</i>-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 <i>in vitro</i> culture conditions. In transiently transfected SkMDS/PCs, we observed significantly higher gene expression levels of <i>SOD2</i> (almost 40-fold) (<i>p</i> < 0.001) and <i>FOXO</i> (<i>p</i> < 0.05) (approximately 3-fold) under both normoxic and hypoxic culture conditions and of <i>BCL2</i> under hypoxia compared to those observed in untreated cells (WT). In addition, myogenic genes showed a significant increase in <i>MyoD</i> (almost 18-fold) expression under standard culture conditions (<i>p</i> < 0.0001) and decreased <i>MyoG</i> expression (approximately 2-fold) after transfection (<i>p</i> < 0.05) compared with that detected in the WT skeletal muscle-derived cell control. Taken together, these results demonstrate that <i>SOD3-</i>tranduced skeletal muscle-derived cells may have potential for use in the regenerative treatment of the post-infarction heart.