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Maria Barthmes, Andre Bazzone, Ulrich Thomas, Andrea Brüggemann, Michael George, Niels Fertig, Alison Obergrussberger

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Maria Barthmes, Andre Bazzone, Ulrich Thomas, Andrea Brüggemann, Michael George, Niels Fertig, Alison Obergrussberger Label-free analysis of Na+/Ca2+- exchanger (NCX) isolated from iPSC-derived cardiomyocytes Maria Barthmes, Andre Bazzone, Ulrich Thomas, Andrea Brüggemann, Michael George, Niels Fertig, Alison Obergrussberger Nanion Technologies GmbH, Ganghoferstr. 70A, 80339 Munich, Germany, contact: [email protected] Abstract Measuring NCX activity in human iPSC derived The Sodium-Calcium Exchangers (NCX) play an To drive the progress in pharmacological NCX cardiomyocytes important role in the cellular calcium research, new methods to measure NCX Human iPSC derived cardiomyocytes are being with high fluidic speed, the cells detached homeostasis under physiological and function are needed. At the current time, investigated as a model for cardiac safety again, but a sheet of the cell membrane pathological conditions. NCX has been of functional investigation of NCX range from assessment. To measure native NCX in these remains on the sensor. NCX currents can be interest as a pharmacological target for many patch-clamp, calcium flux assays, Langendorff- cardiomyocytes a cell based assay was evoked in these sheets. For a higher NCX signal years, in particular because clinical trials perfused hearts to studies in whole animals. We developed. Cardiomyocytes were detached female cardiomyocytes were used. This method involving inhibitors of the sodium-proton have developed an electrophysiological from the culture dish and added to the lipid enables the efficient investigation of the isolated exchanger, NHE, have delivered mixed results. method to investigate NCX function which is coated SSM sensor. Where the cell connected cardiac NCX current in a native membrane. Inhibition of the reversed mode of NCX is based on the solid supported membrane (SSM) with the lipid layer. Upon rinsing of the sensor thought to be beneficial in ischemia/reperfusion technology. HEK cells overexpressing NCX or injury by reducing cardiac, neuronal and renal human induced pluripotent stem cell-derived infarct areas. Moreover, inhibition of NCX has cardiomyocytes (hiPSC-CMs) were used on a been proposed to exhibit an anti-arrhythmic single-well or 96-well SSM electrophysiology effect and therefore, may provide a novel device and NCX was recorded from these cells. target for the treatment of a variety of NCX was activated using Ca2+ in the buffer and arrhythmic pathologies. So far, a number of inhibited by Cd2+ and other compounds. studies have shown promising results but investigations are limited by the currently available NCX inhibitors such as KB-R7943, SEA- 0400 and SN-6 which are only partially specific. Cor.4U (Ncardia) iPSC derived The cardiomyocytes were The cardiomyocytes were added to The sensor was rinsed in a fluidic cardiomyocytes growing ad- detached using trypsin a lipid coated SSM sensor. The sensor system with high speed. This leads to herent on a cell culture dish. was centrifuged to form a tight tearing of the cells – only a connection of the cells with the lipid membrane sheet remains. This is a layer working hypothesis, further investigation is necessary. Sample preparation Resolving low amplitude ion currents After a rinse with a sodium A B C containing buffer cardiac NCX was activated with 30 µM “Solid Supported Membrane (SSM)-based calcium (A). A transient A electrophysiology” is a method, which allows negative current indicates a the resolution of low amplitude electric events net outward current (B). in biological membranes, bringing the Calcium affinity of NCX advantages of electrophysiology to the recorded from HEK cells transporter field. It enables real-time activity (74 µM) was comparable with measurement of electrogenic SLC-transporters, iCell Cardiomyocytes2 MFS-transporters and ion pumps, localized in the (125 µM) (C). NCX in HEK cells, B plasma membrane, in intracellular or bacterial D E iCell Cardiomyocytes2 and membranes. Cor.4U cells was blocked by Unlike cell-based electrophysiology, a large KB-R7943 in a concentration- capacitive sensor coated with a high amount of dependent manner (D) with membrane vesicles or proteoliposomes is used. similar IC50 values. The IC50 In this way any process that generates an values agreed well with the electrical potential can be registered with high literature (Matsuda et al, amplification. The method was established in 2001). SEA0400 is a specific the 90’s and has only recently been scaled-up blocker of NCX (Matsuda et to a 96-well format. al, 2001). SEA0400 blocked 2+ NCX activated by 30 µM Ca with IC50 in good agreement A: Structure of a SSM sensor. The 3 mm diameter thin layer gold electrode is coated with a SSM consisting of a thiol and a lipid with the literature (Matsuda et monolayer. On top liposomes or membrane vesicles containing the al, 2001) (E). target protein are adsorbed. B: Protocol of one activation cycle. The sensor is perfused with a passive non activating solution followed by an activating solution, containing a transported substrate or ATP. During this step a transient A B current occurs, since the active transporter moves charge into the vesicles and this generates a membrane potential. Instrumentation: We used the SURFE2R N1 (A) and the SURFE2R 96SE Development of a screening tool for NCX (B) instruments in this project. The respective SSM sensors are depicted above the instruments. The SURFE2R N1 is a flexible single To screen compounds for an effect on NCX for safety purposes or during development of novel channel measurement instrument, whereas the SURFE2R 96SE is a robotic device designed for experiments that require higher inhibitors a robust method with a high throughput is required. We employed the 96-well based throughput. C: Membrane proteins successfully studied using SSM- platform SURFE2R 96SE to record NCX activity from stably transfected HEK293 cells. The cells were based electrophysiology cultured, harvested and a membrane preparation was performed. This has the advantage that the C resulting signal amplitudes are higher, no running cell culture is required and it yields a very high reproducibility. The membrane preparation is defrosted and added to a 96-well sensor where it is Transporters ATPases Redox-driven ion pumps Channels and Pores recorded by fluidic activation with calcium. Inorganic ions Amino acids Sugars Organic ions NaK-ATPase Complex I Gramicidine NhaA PEPT1 (Slc15a1) SGLT1/2 (Slc5a1/a2) OCT2 (Slc22a2) HK-ATPase respiratory chain complex I/III P2X2 1 2 3 4 5 6 7 8 9 10 11 12 NhaP YdgR MelB CNT1 (Slc28a1) SERCA respiratory chain complex II/III nAChR A 1 µM 5 µM 10 µM 50 µM 1 µM 5 µM 10 µM 50 µM 1 µM 5 µM 10 µM 50 µM NhaB YhiP/DtpB LacY ANT (Slc25a4) V-ATPase cyctochrome c-oxidase A/M2 Enlarged view on 16 B 1 µM 5 µM 10 µM 50 µM 1 µM 5 µM 10 µM 50 µM 1 µM 5 µM 10 µM 50 µM NCX1 (Slc8a1) PutP FucP AAC FoF1-ATPase respiratory chain complexes I/III/V UCP1 (Slc25a7) selectable wells. NCX C 1 µM 5 µM 10 µM 50 µM 1 µM 5 µM 10 µM 50 µM 1 µM 5 µM 10 µM 50 µM Clc-7 GltP XylE GAT1 (Slc6a1) Kdp-ATPase TRPC5 activation on the EcClc EAAC1 (Slc1a1) GlcP BetP CopA Light-driven ion pumps TRPA1 plate can be D 1 µM 5 µM 10 µM 50 µM 1 µM 5 µM 10 µM 50 µM 1 µM 5 µM 10 µM 50 µM NirC PAT1 (Slc36a1) CHT (Slc5a7) ATP7A/B Bacteriorhodopsin (BR) CFTR repeated. Previous E 1 µM 5 µM 10 µM 50 µM 1 µM 5 µM 10 µM 50 µM 1 µM 5 µM 10 µM 50 µM Amt1-3 ArcD NupC VrPPase Oxyrrhis marina Rhodopsin AQP6 current responses are F 1 µM 5 µM 10 µM 50 µM 1 µM 5 µM 10 µM 50 µM 1 µM 5 µM 10 µM 50 µM AmtB CAT2B (Slc7a2) NacT (Slc13a5) Rhodopsin-2 (KR2) depicted in blue G 1 µM 5 µM 10 µM 50 µM 1 µM 5 µM 10 µM 50 µM 1 µM 5 µM 10 µM 50 µM SulP GlyT1/2 (Slc6a9/a5) Halorhodopsin (HR) H 1 µM 5 µM 10 µM 50 µM 1 µM 5 µM 10 µM 50 µM 1 µM 5 µM 10 µM 50 µM NIS (Slc5a5) Acerhodopsin NaPi2b (Slc34a2) Channelrhodopsin (ChR) 1 2 3 4 5 6 7 8 9 10 11 12 A 10 µM 10 µM 10 µM 10 µM 10 µM 10 µM 10 µM 10 µM 10 µM 10 µM 10 µM 10 µM MntH2 B 10 µM 10 µM 10 µM 10 µM 10 µM 10 µM 10 µM 10 µM 10 µM 10 µM 10 µM 10 µM C 10 µM 10 µM 10 µM 10 µM 10 µM 10 µM 10 µM 10 µM 10 µM 10 µM 10 µM 10 µM D 10 µM 10 µM 10 µM 10 µM 10 µM 10 µM 10 µM 10 µM 10 µM 10 µM 10 µM 10 µM E 10 µM 10 µM 10 µM 10 µM 10 µM 10 µM 10 µM 10 µM 10 µM 10 µM 10 µM 10 µM Overview on 96 parallel recordings F 10 µM 10 µM 10 µM 10 µM 10 µM 10 µM 10 µM 10 µM 10 µM 10 µM 10 µM 10 µM with color coding of sensor quality G 10 µM 10 µM 10 µM 10 µM 10 µM 10 µM 10 µM 10 µM 10 µM 10 µM 10 µM 10 µM H 10 µM 10 µM 10 µM 10 µM 10 µM 10 µM 10 µM 10 µM 10 µM 10 µM 10 µM 10 µM Different compound distributions on the 96 well plate were applied. Either compound 1, 2 and 3 in four Summary and Future prospects different concentrations per compound for IC50 determination or 96 individual compounds each at a We have developed two novel methodic • The developed methods shall be applied in a concentration of 10 µM.
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