siRNA delivery into cultured primary human myoblasts – optimization of electroporation parameters and theoretical analysis

Abstract

Introduction of genetic material into muscle tissue has been extensively researched, including isolation and in vitro expansion of primary myoblasts as a potential source of cells for skeletal and heart muscle tissue engineering applications. In this study, we optimized the electroporation protocol for introduction of short interfering ribonucleic acid (siRNA) against messenger RNA for Hypoxia Inducible Factor 1α (HIF-1α) into cultured primary human myoblasts. We established optimal pulsing protocol for siRNA electro transfection, and theoretically analyzed the effect of electric field and pulse duration on silencing efficiency and electrophoretic displacement of siRNA. Silencing of HIF-1α was determined with quantitative polymerase chain reaction and Western Blot. The most efficient silencing (71% knockdown) was achieved with 8 × 2 ms pulses, E = 0.6 kV/cm. Viability was determined immediately, 1 h and 48 h after electroporation. In general, there was a trade-off between efficient silencing and preserved viability. Electric field and pulse duration are crucial parameters for silencing, since both increase membrane permeabilization and electrophoretic transfer of siRNA. Short-term viability showed immediate toxicity of pulses due to membrane damage, while indirect effects on cell proliferation were observed after 48 h. Presented results are important for faster optimization of electroporation parameters for ex vivo electrotransfer of short RNA molecules into primary human myoblasts.

Ouabain Suppresses IL-6/STAT3 Signaling and Promotes Cytokine Secretion in Cultured Skeletal Muscle Cells

“…To examine this possibility, we treated myotubes with 50 nM ouabain for 20 h in Advanced MEM supplemented with 2% FBS. This was followed by a 4-h treatment in serum-free Advanced MEM with 50 nM ouabain with or without 250 μM CoCl 2 , which prevents normoxic degradation of HIF-1α ( Figure 7A ) and was shown to effectively induce HIF-1α in human myoblasts ( Pirkmajer et al, 2010 ; Lojk et al, 2015 ). Recombinant human IL-6 was added during the last 15 min of the experiment ( Figures 7B–F ).…”

Quantitative Proteomic Analysis of Cell Responses to Electroporation, a Classical Gene Delivery Approach

“…However, difficulties have yet to be addressed before expanding the application of EP. For instance, the traditional parameter optimizing method is to exhaust electrical conditions, including voltage, pulse duration, and interval between pulses, for physically investigating the behaviors of both cell membrane and molecular . However, this method is still inefficient, or even invalid, for many primary cells, such as neuron cells and stem cells.…”

“…For instance, the traditional parameter optimizing method is to exhaust electrical conditions, including voltage, pulse duration, and interval between pulses, for physically investigating the behaviors of both cell membrane [9] and molecular. [10] However, this method is still inefficient, or even invalid, for many primary cells, such as neuron cells and stem cells. The lack of comprehensive understanding of cell responses to electrical stimulation at protein level could be an important reason.…”

Electrotransfer of siRNA to Silence Enhanced Green Fluorescent Protein in Tumor Mediated by a High Intensity Pulsed Electromagnetic Field

“…Thus, the local delivery approach, i.e., electroporation, may have advantages over systemic delivery in accessible tumors. Successful gene silencing in different cells and tissues was obtained by using conventional electroporation as non-viral delivery approach of siRNA targeting reporter or therapeutic genes [5,21,[39][40][41][42][43][44]. Silencing therapeutic CD105 (endoglin), VEGF and Rho GTPase Rac1 genes demonstrated good antitumor and antivascular effects [5,21,45].…”