## 3 Results
### Effects of electroporation on the Expression of NKA \(\alpha\)-Subunits  
As ion imbalance and a loss of ATP also affect the function of NKA (Figure 1B), we were interested in how electroporation with monopolar or H-FIRE pulses affects its expression and function. We first evaluated the expression of catalytic \(\alpha\)-subunits (1-3) at the mRNA and protein levels. Figure 3: Evaluation of plasma membrane permeability and survival of H9c2 cells after electroporation. Cells were treated with either 8 \(\times\) 100 \(\upmu\)s long monopolar pulses (Monopolar; 1 Hz repetition frequency), or 100 bursts of 32 short bipolar pulses (H-FIRE) of 2–2–2–2 \(\upmu\)s (bipolar; 1 Hz repetition frequency, pulses of 2 \(\upmu\)s duration of positive and negative phase, 2 \(\upmu\)s pause between positive and negative phase, 2 \(\upmu\)s pause between bipolar pulses). The uptake of PI, measured by the flow cytometer 3 min after electroporation, was used to evaluate permeability of plasma membrane, while MTS viability assay was used to evaluate cell survival 24 h after electroporation. Results are means \(\pm\) SD (n = 3–4). Figure 4: Effects of different types of electric pulses (monopolar and bipolar) on mRNA and protein expression of SERCA isoforms and phospholamban (PIn) in H9c2 cells. (**A**–**F**) qPCR was used to determine gene expression of SERCA1-3 (_Atp2a1_-3) subunits following electroporation for indicated time periods. (**G**,**H**,**J**,**K**) Immunoblotting was used to estimate protein abundance of SERCA1 and 2 isoforms following electroporation for indicated time periods. Total protein loading was evaluated by Ponceau S staining (shown above the blots). Numbers next to the blots and Ponceau stains indicate molecular weight markers in kDa. Black arrowheads indicate the analyzed bands when unspecific bands were detected, (**L**,**L**) qPCR was used to determine gene expression of phospholamban (_PIn_) following electroporation for indicated time periods. qPCR was performed using rat-specific gene expression assays, normalized to geometric mean of 18S and PPIA endogenous controls. Values on the graphs are means \(\pm\) SD (n = 3–4 for qPCR, n = 6–8 for immunoblotting) and results that are statistically significant are indicated (* \(p\) < 0.05 vs. Basal; # \(p\) < 0.05 4 h vs. 24 h. Two-way ANOVA with [PERSON]’s post hoc adjustment was used for statistical evaluation). At the mRNA level, we observed no effects of electroporation on the expression of NKA\(\alpha\)1 (_Atp1a1_) with both monopolar and H-FIRE pulses (Figure 5A,D). After electroporation with monopolar pulses, we observed a non-significant trend in the downregulation of NKA\(\alpha\)2 (_Atp1a2_) at the mRNA level, after both 4 and 24 h (Figure 5B), as a function of treatment intensity. H-FIRE pulses caused a significant downregulation after 24 h (Figure 5E). We also observed an upregulation in the mRNA expression of NKA\(\alpha\)3 (_Atp1a3_), 24 h after being treated with H-FIRE pulses at 1250 V/cm (Figure 5F). It is important to note, however, that the expression levels of NKA \(\alpha\)3 were the lowest of the 3 alpha subunits, so the relevance of this result might be negligible (Figure 2). At the protein level, we observed a downregulation in the expression of NKA\(\alpha\)1 for monopolar pulses after 24 h at 1250 V/cm (Figure 5G), while H-FIRE pulses caused a downregulation after 4 h and at electric field values from 500 to 1250 V/cm (Figure 5J). H-FIRE pulses caused a significant downregulation in the protein expression of NKA\(\alpha\)2 after 4 h, while both monopolar and H-FIRE pulses increased the expression at high electric fields after 24 h (Figure 5H,K). We also detected a downregulation in the expression of NKA\(\alpha\)3 after 4 h in cells treated with monopolar pulses (Figure 5J), but as at the mRNA level, due to the low overall expression levels of the \(\alpha\)3 subunit in H9c2 cells, these results may not have an important effect on the function of treated cells.