## 4 Discussion  
In this study, we show that the electroporation of H9c2 cells using two different waveforms, i.e., monopolar and H-FIRE pulses, at electric fields where a high percentage of permeabilization and low cell viability caused a dose-dependent downregulation in the expression of the most crucial isoforms of ion pump machinery. However, the only highly expressed subunits that were upregulated in electroporated samples were NKA\(\alpha\)2 24 h after electroporation with both monopolar and H-FIRE pulses (Figure 5H,K) and NKA\(\beta\)3 in cells treated with H-FIRE pulses (Figure 6L). There are several isoforms of SERCA proteins expressed in mammalian tissues. They are coded by three genes, encoding for SERCA1, SERCA2, and SERCA3. Further variability is generated by alternative splicing [50]. In the mammalian heart, SERCA2a largely predominates, while SERCA2b represents a minor share [51]. SERCA2a activity is regulated  
Figure 8: Effects of electroporation with monopolar and bipolar pulses on phosphorylation and protein expression of AMPK and ACC in H9c2 cells. (**A–L**) Immunoblotting was used to estimate pAMPK Thr172, AMPK, phosphorylation of AMPK relative to total protein, pACC Ser79, ACC, and phosphorylation of ACC relative to total protein. 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. Values on the graphs are means \(\pm\) SD of at least three independent experiments (n = 4–8) and results that are statistically significant are indicated (\(\star\), \(p<0.05\); \(\#\)\(p<0.05\) 4 h vs. 24 h; two-way ANOVA with [PERSON]’s post hoc adjustment). by two proteins, phospholamban (PIn) and sarcolipin (SLN). Both Pln and SLN inhibit the activity of SERCA2a, but it is known that the phosphorylation of Pln at two distinct sites disrupts the physical interaction of Pln with SERCA2a, which increases SERCA2a's activity [52]. Previous studies hypothesized that low SERCA and PMCA expression levels in some kinds of tumor cells may play a role in their high susceptibility to treatment with calcium electroporation, as their ability to excrete Ca\({}^{2+}\) out of the cells or transport it into their ER/SR is limited. To our knowledge, no study has been published on how electroporation may affect the expression and activity of SERCA in cells after electroporation. Our results show that electroporation with both monopolar and H-FIRE pulses decreased the expression of SERCA1 and SERCA2. SERCA1 was decreased both at the mRNA and protein levels (Figure 4A,D,G,J), while SERCA2 downregulation was only observed at the protein level (Figure 4H,K). The presence of the SERCA1 isoform might be explained by the embryonic origin of H9c2 cells and their ability to differentiate into a skeletal muscle phenotype, as SERCA1 is normally not present in cardiac tissue, especially in adult ones [50]. We also investigated the expression of phospholamban. Unfortunately, we were only able to evaluate phospholamban at the mRNA level as we were unable to detect any bands at the protein level; thus, we could not determine how SERCA activity may be regulated through changes in the phosphorylation of phospholamban. Nevertheless, the exposure of cells to high electric fields with both monopolar and H-FIRE pulses decreased the expression of phospholamban 24 h after treatment (Figure 4L). Interestingly, phospholamban was significantly upregulated after 24 h compared to 4 h when looking at cells that underwent no treatment (basal), but exposure to high electric fields decreased mRNA expression to the basal levels of cells that were incubated for 4 h (Figure 4L). Due to surprising differences in basal mRNA levels between 4 and 24 h incubated cells, we tested whether the confluence of cells may play a role in the mRNA expression levels of phospholamban, as only a fraction of cells that are treated with high electric fields survive, which leads to lower confluency of seeded cells.