## 4 Discussion
We seeded cells at different dilutions with 4 and 24 h incubations and found that 24 h incubation significantly increased the mRNA expression of phospholamban compared to 4 h incubation when cells were seeded at high concentrations, but when the cell concentration was lowered, the mRNA expression levels dropped to the levels of cells incubated for 4 h (Supplementary Figure S1). NKA is a heterodimer that consists of a catalytic \(\alpha\)-subunit and a glycoprotein \(\beta\)-subunit. In the heart, \(\alpha\)1, \(\alpha\)2, \(\alpha\)3, and \(\beta\)1 are expressed, while small amounts of \(\beta\)2 have also been detected [53]. In H9c2 cells, NKA\(\alpha\)1 predominates (Figure 2), just like in mature rat and human cardiac tissue [54]. It was shown before that electroporation can affect the function of NKA in erythrocytes [33] and recently also that the silencing of genes for NKA\(\alpha\)1 (ATP1A1) can attenuate the effects of nanopulses on membrane disruption in U937 human monocytes [55], but as with SERCAs, there are no studies that exploring how electroporation with different pulse protocols would affect the expression and activity of NKA. Our results show that treatment with high electric fields caused a downregulation in the protein content of NKA\(\alpha\)1 24 and 4 h after treatment with monopolar (Figure 5G) and H-FIRE pulses (Figure 5), respectively. On the other hand, we observed an upregulation in the protein content of NKA\(\alpha\)2 24 h after treatment with both explored pulse parameters (Figure 5H,K). It is known that NKA\(\alpha\)1 is nearly maximally activated at physiological ion levels [56; 57], while NKA\(\alpha\)2 is much more active when the membrane is depolarized and is a more important regulator of cardiac contractility, even though it is far less abundant than NKA\(\alpha\)1. This may be of importance, as electroporated cells are in an extremely depolarized state due to nonselective membrane permeability allowing for the transport of ions through the plasma membrane [58; 59; 25]. NKA \(\beta\)-subunits are essential for the assembly, maturation, and function of \(\alpha\)/\(\beta\)-heterodimers [60]. While we did see an upregulation in NKA\(\beta\)1 at the mRNA level 4 h after electroporation (Figure 6A,D), this trend was not seen at the protein level. On the other hand, NKA\(\beta\)3, which is also highly expressed in H9c2 cells due to their embryonic origin, was downregulated at the protein level 4 h after electroporation with both pulse parameters (Figure 6,L), but was upregulated 24 h after electroporation in cells exposed to H-FIRE pulses (Figure 6). Due to the NKA\(\beta\) subunit's importance in regulating the assembly of NKA heterodimers, its upregulation might play a role in more heterodimers reaching the cellular membrane, which could increase the cell's pumping capacity. NKAs are regulated by proteins of the FXYD family, of which phospholemman (FXYD1) regulates NKA in heart and skeletal muscles [61; 62]. While unphosphorylated FXYD1 inhibits NKA activity, its phosphorylated form increases NKA activity [62]. When we normalized the phosphorylated form of FXYD1 to total FXYD1 protein content, our results demonstrated that 24 h after electroporation, monopolar pulses may cause an inhibition in NKA activity (Figure 7), while H-FIRE pulses only show a non-significant negative trend in the phosphorylation of FXYD1 (Figure 7). NKA activity is also regulated by the phosphorylation and glutathionylation of NKA itself, which affects its activity and its translocation to and from the sarcolemma [63]. We investigated the phosphorylation rates of NKA\(\alpha\)1 on Tyrosine 10 residue, an under-researched phosphorylation site that may play an important role in the hormonal regulation of NKA. Here, we obtained divergent effects between two explored pulse parameters. Monopolar pulses did not seem to affect the phosphorylation of NKA\(\alpha\)1 on Tyr10 (Figure 7,B), while H-FIRE pulses increased it, even when normalized to total NKA\(\alpha\)1 (Figure 7,E).