## 4 Discussion
Finally, we investigated potential AMPK activation by electroporation, an energy sensor that is activated in response to energy stress. It was shown by others that electroporation can lead to increased AMPK activity [41; 49]. It is also known that AMPK can affect NKA and SERCA activity. We show that treatment with both monopolar and H-FIRE pulses at the highest electric fields caused the activation of AMPK in H9c2 cells, both 4 and 24 h after treatment (Figure 8,C,D,F). Whether AMPK activation leads to increased or decreased NKA activity depends largely on the signaling pathway that causes AMPK activation. AMPK activation via the calcium/calmodulin-dependent protein kinase (CaMKII) signaling pathway decreased NKA activity in alveolar epithelial cells [64], while the activation of AMPK with AICAR, an AMP analogue in the rat L6 skeletal muscle cell line, increased NKA activity [39]. A769662, a direct activator of AMPK, also led to increased SERCA activity in freshly isolated vascular smooth muscle cells [40]. It was shown in human Jurkat cells and HeLa S3 cells that nanosecond pulses increase AMPK activity and that this increase can also be achieved via CaMKK [41]. Increased AMPK phosphorylation by CaMKK is achieved when Ca\({}^{2+}\) levels in the cytosol are increased. This is also the case when cells are treated with monopolar and H-FIRE pulses, which may explain why the AMPK activation observed at high electric fields in our experiments correlates mostly with a decreased expression of NKA subunits in our experiments. In the Introduction, we hypothesized that treatment with lower electric fields than lethal may lead to increased expression and activity of ion pumps, while higher electric fields where the majority of cells do not survive would cause a downregulation in expression and activity. Our hypothesis was based on prior studies, which showed that reversible electroporation leads to increased NKA activity in skeletal muscles [65; 66]. We observed no increased expression of ion pumps at lower electric fields than lethal, and we observed no changes in the expression or activation of SERCA and NKA regulators, which may increase the activity of NKA or SERCA proteins. In previous publications, our group showed that the effects of electroporation on cells can last for several hours or even days [32; 46] and cause different types of cell death [16]. In the clinical use of electroporation (which includes PFA), both irreversible and reversible electroporation are present, as cells are exposed to different electroporation intensities, depending on their distance from the electrodes/catheter [67]. In this study, we were especially interested in what happens in cells that are reversibly electroporated and survive. Our protocol was, thus, designed in a way that we only evaluated mRNA and protein expression in cells that were still alive 4 or 24 h after electroporation. Nevertheless, it is possible that the population of cells, alive 4 and 24 h after electroporation, is not the same, which is also evident in some of our results, where the effects of electroporation with both monopolar and H-FIRE pulses are different between cells that were incubated for 4 or 24 h. Overall, no major differences were observed between the effects of monopolar and H-FIRE pulses on the expression of the explored proteins. In the future, it may also be interesting to see how the expression of ion pumps is affected after a shorter incubation. We need to acknowledge some limitations of this study. Firstly, we decided to use the H9c2 rat cardiac myoblast cell line, which has an embryonic origin and is derived from the left ventricle. While the ventricular origin of H9c2 cells can be considered a limitation of our study, we also state in the Introduction that there is a growing interest in using PFA in the treatment of ventricular arrhythmias, which makes the results of our study important for a deeper understanding of that application as well.