Abstract

Introduction: Optogenetics can precisely stimulate cardiac tissue, while optical mapping of voltage (Vm) and calcium transients (CaT) can image electrophysiological function with high spatiotemporal resolution. Here, we present a novel method that combines optogenetic actuation with dual Vm/CaT optical mapping. The optogenetic channels and CaT indicator are genetically encoded, while the Vm indicator is an organic dye. Methods: For model tissue, we used cardiac spheroid (600-800 µm in diameter) comprising cardiomyocytes and cardiac fibroblasts that were differentiated from human induced pluripotent stem cells. We used lentivirus to express the optogenetic actuator CheRiff and the calcium reporter jRCaMP1b, and stained with a voltage dye, RH237. CheRiff was optically excited using royal blue LED light (450 nm), and both RH237 and jRCaMP1b were excited using a single green LED light source (560 nm) that excites both indicators. The two indicators emit in different bands; fluorescence was split with a dichroic mirror and directed to two cameras to image Vm and CaT simultaneously without crosstalk (Fig. A). Results: We first used electrical field stimulation to activate the spheroid while simultaneously imaging Vm and CaT. This demonstrated the efficacy of the recording system (Fig. B[I]). We then switched to optogenetic stimulation to demonstrate the all-optical system and acquired similar signals (Fig. B[II]). Conclusions: This relatively simple system enables all-optical actuation and readout of cardiac electrophysiology. The genetically encoded optogenetic actuator and CaT indicator can be expressed in engineered tissue, while the organic Vm indicator can be used to stain both wild-type tissue and engineered tissue. We expect the system to be useful to investigate electrical coupling between engineered tissue grafts and host tissue because it enables actuation and readout of engineered tissue to be unambiguously separated from host tissue.