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: 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 < 0.001), while the cells transduced with the SOD3 gene exhibited an increase in cell fusion under both standard (p < 0.05) and hypoxic conditions (p < 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 < 0.05) and hypoxic (p < 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 < 0.001) and FOXO (p < 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 < 0.0001) and decreased MyoG expression (approximately 2-fold) after transfection (p < 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.
Highlights
Most reactive oxygen species are generated as by-products during mitochondrial electron transport.Due to the presence of unpaired electrons in separate orbits in its outer electron shell, atomic oxygen can readily form radicals
Despite using the identical procedure to transfect human SkMDS/PCs, a greater transfection efficiency was observed for the superoxide dismutase 3 (SOD3) gene than the green fluorescent protein (GFP) control (~58 and ~30%, respectively), which was likely due to differences in the size of the plasmids (Figure 1)
The following cell populations were observed: wild type (WT), untreated myoblasts; vector pIRES2 (Ir), myoblasts transiently transfected with the Ires vector containing either the GFP or GFP-SOD3 sequences
Summary
Most reactive oxygen species are generated as by-products during mitochondrial electron transport. Due to the presence of unpaired electrons in separate orbits in its outer electron shell, atomic oxygen can readily form radicals. The sequential reduction of oxygen leads to the formation of a number of reactive oxygen species (ROS), including superoxide, hydrogen peroxide, hydroxyl radical, hydroxyl ion, and nitric oxide [1]. ROS serve as signaling molecules [2]. The shift in the balance of oxidants and antioxidants in favor of oxidants may lead to phenomenon called “oxidative stress”. ROS are highly reactive molecules that can damage and alter the functions of cell structures, such as carbohydrates, nucleic acids, lipids, and proteins. The regulation of reducing and oxidizing (redox) potential is crucial for cell viability, activation, and proliferation, and for organ function
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