## 1 Introduction
There is barely any literature on how the ion pump function may be affected by electroporation [30; 31; 33], especially concerning the details of how they are regulated after the cell membrane is so drastically damaged, i.e., electroporated. In most cases, publications correlate tumor cells' higher susceptibility to calcium electroporation compared to normal cells to their downregulation of SERCA and PMCA ion pumps [30; 31] but do not explore how electroporation itself may influence the expression levels of ion pumps. 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. One of the pathways that may play a major role in regulating the activity and expression of SERCA, NKA, and PMCA proteins is the 5' AMP-activated protein kinase (AMPK) signaling pathway that regulates catabolic and anabolic processes in cells according to the cell's energy status. AMPK is a heterotrimeric complex that consists of the catalytic \(\alpha\)-subunit and the regulatory \(\beta\)- and \(\gamma\)-subunits [34]. It acts as a cellular energy sensor that modulates energy metabolism, cell growth, and cell cycle [35; 36] and was recently also implied in membrane repair [37]. When the energy levels are low, AMP binds to the \(\gamma\)-subunit, which, in turn, causes an allosteric activation of AMPK and increases phosphorylation of the \(\alpha\)-subunit at Thr172 [38]. AMPK stimulates energy-producing processes and inhibits energy-consuming processes and, thus, restores the energy balance. Interestingly, AMPK has been reported to increase both NKA and SERCA activity in some cases, even though they are both large consumers of energy [39; 40], and was activated itself by nanosecond electroporation (nsEP) [41]. Precisely how AMPK activity may relate to SERCA, PMCA, and NKA activity in electroporated cells is unclear. In our study, we explored how the electroporation of H9c2 rat cardiac myoblast cells using different pulse parameters (i.e., two different waveforms) affects the expression of SERCA and NKA pumps and how electroporation might affect their regulation through the phosphorylation of pumps themselves or their regulatory proteins, phospholamban and phospholemman, respectively. We also evaluated how the electroporation of H9c2 cells affects AMPK activity. Figure 1: Schemes of excitation contraction coupling in ventricular cardiomyocytesytes and possible effects of electroporation on cardiomyocyte. (**A**) Action potential spreads along the cell membrane. (1) The rapid depolarization phase is caused by opening of Na\({}^{+}\) channels. (2) Depolarization opens L-type Ca\({}^{2+}\) channels (DHPR) in the T-tubule membrane, which causes small Ca\({}^{2+}\) uptake into cytosol. (3) Ca\({}^{2+}\) binds to Ca\({}^{2+}\) release channels (RyR) in the sarcoplasmic reticulum (SR), which in turn causes (4) release of larger quantities of Ca\({}^{2+}\). (5) Released Ca\({}^{2+}\) binds to the troponin and causes shift of tropomyosin that exposes myosin-binding sites on actin filaments. This enables the contraction. (6) In the relaxation phase some Ca\({}^{2+}\) is pumped back into SR by SERCA or out of the cell by PMCA, some is transported out of the cell by the Na\({}^{+}\)/Ca\({}^{2+}\) exchanger (not shown). Intracellular Na\({}^{+}\) homeostasis is achieved by the Na\({}^{+}\)/K\({}^{+}\)-ATPase. (**B**) Changes and processes that occur in the cell during and after electroporation.