High-throughput optical action potential recordings in hiPSC-derived cardiomyocytes with a genetically encoded voltage indicator in the AAVS1 locus

Abstract

Cardiomyocytes (CMs) derived from human induced pluripotent stem cells (hiPSCs) represent an excellent in vitro model in cardiovascular research. Changes in their action potential (AP) dynamics convey information that is essential for disease modeling, drug screening and toxicity evaluation. High-throughput optical AP recordings utilizing intramolecular Förster resonance energy transfer (FRET) of the voltage-sensitive fluorescent protein (VSFP) have emerged as a substitute or complement to the resource-intensive patch clamp technique. Here, we functionally validated our recently generated voltage indicator hiPSC lines stably expressing CAG-promoter-driven VSFP in the AAVS1 safe harbor locus. By combining subtype-specific cardiomyocyte differentiation protocols, we established optical AP recordings in ventricular, atrial, and nodal CMs in 2D monolayers using fluorescence microscopy. Moreover, we achieved high-throughput optical AP measurements in single hiPSC-derived CMs in a 3D context. Overall, this system greatly expands the spectrum of possibilities for high-throughput, non-invasive and long-term AP analyses in cardiovascular research and drug discovery.

Epicardioid single-cell genomics uncovers principles of human epicardium biology in heart development and disease

“…In vivo, CMs closest to the epicardium generate shorter action potentials than those in the middle of the ventricular wall; this is an evolutionarily conserved feature known as the transmural voltage gradient, which increases the efficiency of ventricular contraction 27 . We assessed action potential dynamics in the myocardial compartment of epicardioids generated from human induced PSCs (hiPSCs) constitutively expressing a FRET-based voltage sensor knocked into the AAVS1 safe harbor locus 28 , 29 (AAVS1-CAG-voltage-sensitive fluorescent protein (AAVS1-CAG-VSFP); Fig. 2f ).…”

“…For optical action potential measurements, 250-µm-thick slices of spheroids derived from the AAVS1-CAG-VSFP hiPSC line 29 (hPSCreg MRI003-A-6) were transferred to Tyrode’s solution (135 mM NaCl, 5.4 mM KCl, 1 mM MgCl 2 , 10 mM glucose, 1.8 mM CaCl 2 and 10 mM HEPES, pH 7.35) before imaging at 100 fps on an inverted epifluorescence microscope (DMI6000B, Leica Microsystems) equipped with a Zyla V sCMOS camera (Andor Technology). The VSFP was excited at 480 nm, and the emitted GFP and RFP fluorescence signals were separated using an image splitter (OptoSplit II, Caim Research).…”

Nanotechnology and Cancer Bioelectricity: Bridging the Gap Between Biology and Translational Medicine

“…These preliminary findings suggest that combining rhodopsin‐based GEVIs with low‐intensity LED stimulation is a potential approach worth further investigation for the long‐term voltage imaging of non‐excitable cells. [ 157 ] Other examples of recently developed GEVIs are JEDI‐2P, which reported light‐evoked responses in Drosophila interneurons and in the mice retinas, as well as at the single‐neuron and neuronal pair level, [ 161 ] and protein indicators for high‐throughput cardiomyocytes recording, [ 162 ] which are extensively reviewed elsewhere. [ 128,154 ] Video‐based screening of pooled libraries can be a powerful approach to target photoselection over a large field of view, enhancing the capabilities of imaging technologies.…”