Importance of the electrophoresis and pulse energy for siRNA-mediated gene silencing by electroporation in differentiated primary human myotubes

Background Electrotransfection is based on application of high-voltage pulses that transiently increase membrane permeability, which enables delivery of DNA and RNA in vitro and in vivo. Its advantage in applications such as gene therapy and vaccination is that it does not use viral vectors. Skeletal muscles are among the most commonly used target tissues. While siRNA delivery into undifferentiated myoblasts is very efficient, electrotransfection of siRNA into differentiated myotubes presents a challenge. Our aim was to develop efficient protocol for electroporation-based siRNA delivery in cultured primary human myotubes and to identify crucial mechanisms and parameters that would enable faster optimization of electrotransfection in various cell lines. Results We established optimal electroporation parameters for efficient siRNA delivery in cultured myotubes and achieved efficient knock-down of HIF-1α while preserving cells viability. The results show that electropermeabilization is a crucial step for siRNA electrotransfection in myotubes. Decrease in viability was observed for higher electric energy of the pulses, conversely lower pulse energy enabled higher electrotransfection silencing yield. Experimental data together with the theoretical analysis demonstrate that siRNA electrotransfer is a complex process where electropermeabilization, electrophoresis, siRNA translocation, and viability are all functions of pulsing parameters. However, despite this complexity, we demonstrated that pulse parameters for efficient delivery of small molecule such as PI, can be used as a starting point for optimization of electroporation parameters for siRNA delivery into cells in vitro if viability is preserved. Conclusions The optimized experimental protocol provides the basis for application of electrotransfer for silencing of various target genes in cultured human myotubes and more broadly for electrotransfection of various primary cell and cell lines. Together with the theoretical analysis our data offer new insights into mechanisms that underlie electroporation-based delivery of short RNA molecules, which can aid to faster optimisation of the pulse parameters in vitro and in vivo. Supplementary Information The online version contains supplementary material available at 10.1186/s12938-024-01239-7.

where d denotes membrane thickness, R cell radius and  is the angle measured with respect to the electric field direction.For physiological conditions where the membrane thickness is much smaller then size of the cells d << R and σm << σe, σi , Schwan equation simplifies into: where R cell radius and  is the angle measured with respect to the electric field direction.Um represents the potential drop (induced transmembrane voltage) across the cell membrane.We can define the critical electric field Ec as E where c = 0, therefore: The electric field governs the area of the cell membrane Sc, which is exposed to the above-critical transmembrane voltage (brighter shaded region in Fig. S1).From the above equations we can obtained the permeabilized surface area: where S 0 is the total surface area of the cell.Clearly, the local electric field E is the critical parameter since it defines the permeabilized area of the membrane S c (E) and through which molecular transport occurs.
We can extend this to spheroidal shape of the cells (e.g.myotubes).The generalized Schwan's equation for arbitrary oriented ellipsoid can be written: where Li are depolarizing factor in the x, y and z direction and ri is the vector of the point T(x, y, z) at the surface of the spheroid.Here we shall limit ourselves only on axially symmetrical prolate spheroid as a model of myotube R1>> R2 = R3.Depolarizing factor for prolate spheroid along the symmetry axis is The depolarizing factors in the other two directions can be calculated from: . (S7) If the z axis of the coordinate system is parallel to the symmetry axis of the spheroid then the solution for the parallel and perpendicular orientations are: Without loss of generality we can always choose so that the vector of the electric field lies in the xz plane.The induced transmembrane potential Um on an arbitrary oriented spheroid can be therefore obtained.The full expression is given in [58], which in cartesian coordinate system simplifies in: where the angle α defines the angle between the electric field direction and the symmetry axis of the spheroidal cell.From the solution for the induced potential in parallel and perpendicular orientation, one can calculate Um on an arbitrarily oriented spheroid by means of linear combination of the two solutions.The elongated myotubes can be approximated as prolate ellipsoid with long radius R1 ≅ 400 μm and short diameter 20 μm, from which we obtain the polarization factors Lz ≅ 0 and Lx = Ly ≅ 0.5.Therefore, the maximal induced transmembrane potential is at the poles of the myotubes oriented parallel with E ( = 0) will be: and if we set that Umax = Uc then we obtain that : Consequently the longer myotubes oriented parallel to E will be first electroporated.In case that Uc of myotubes would be similar to myoblasts then Ec in myoblast would be lower for the factor of R1mytoubes/R1myoblats if all other conditions of the electroporation protocol are the same.
For elongated myotubes the permeabilized surface area Sc increases with the applied voltage/electric field similarly as in Eq.S4 but the function is more complex, details are given in [58]: where 1, 2, 1 and 2 are borders of integration defined with the condition that U = Uc.

Figure S1 :
Figure S1: Representation in 2D of a spherical cell exposed to the external electric field.The bright

Figure S2 :
Figure S2: Western blot images of blots of three independent experiments (N1, N2, N3) for different parameters of electric pulses: trains of 8 x 2 ms and 8 x 5 ms pulses with different voltages.Actin bands are shown as the loading control.SI -siRNA against HIF-1α mRNA, SCRnon-targeting scrambled siRNA.