2018 - Transported by light: optogenetic control of NCX1
SURFE²R N1 poster, Biophysics Annual Meeting 2018 (2.2 MB)
This poster was presented by Axxam S.p.A in collaboration with Nanion (co-authors).
The cardiac Na+-Ca2+ exchanger (NCX1) is one of the key modulators of the cardiomyocytes’ Ca2+ homeostasis and its reverse-mode has been related to several disorders (e.g. heart failure). It therefore represents an important target for both cardiac safety and drug discovery. Robust high-throughput screening (HTS) assays are required to allow early stage of drug discovery, but the so far available assays are poorly specific and involve complex solution exchange protocols.
Our aim was to develop a new HTS-compatible strategy to trigger the NCX1 reverse-mode activity by the use of optogenetics in HEK-293 cells and to validate this approach in human induced pluripotent stem (iPS) cells-derived cardiomyocytes assays, in order to have a more physiological human background. As a proof of concept, we generated a pure HEK-293 clone stably expressing both Channelrhodopsin (ChR2) and NCX1, and we confirmed their functionality with manual patch clamp in voltageclamp mode and standard fluorescence protocols at FLIPRTETRA, measuring intracellular Ca2+ changes or membrane depolarization. Our rationale is that following the ChR2 light stimulation we promote conditions that favour the reverse-mode function of NCX1, which can be evaluated by monitoring intracellular Ca2+. The same “Opto-NCX1” protocol was used in iPS-derived cardiomyocytes co-cultured with ChR2-expressing HEK-293 cells. The contribution of NCX1 to the global Ca2+ cycling of the resulting syncytia was evaluated by pharmacological tools at FLIPRTETRA. Our results indicate that ChR2 activation by blue light is successful in triggering NCX1 reverse-mode function. This is confirmed by the effect of specific blockers, such as KB-R7943. Thus, we have developed a new optical tool suitable to run HTS, looking for novel molecules acting on NCX1, and to evaluate potential cardiotoxicity of compound, in the early phase of drug discovery campaigns. This approach can be extended to both patient-specific and heterologous backgrounds.