Uninterrupted monitoring of drug effects in human-induced pluripotent stem cell-derived cardiomyocytes with bioluminescence Ca2+ microscopy

Abstract

ObjectiveCardiomyocytes derived from human-induced pluripotent stem cells are a powerful platform for high-throughput drug screening in vitro. However, current modalities for drug testing, such as electrophysiology and fluorescence imaging have inherent drawbacks. To circumvent these problems, we report the development of a bioluminescent Ca2+ indicator GmNL(Ca2+), and its application in a customized microscope for high-throughput drug screening.ResultsGmNL(Ca2+) gives a 140% signal change with Ca2+, and can image drug-induced changes of Ca2+ dynamics in cultured cells. Since bioluminescence requires application of a chemical substrate, which is consumed over ~ 30 min we made a dedicated microscope with automated drug dispensing inside a light-tight box, to control drug addition. To overcome thermal instability of the luminescent substrate, or small molecule, dual climate control enables distinct temperature settings in the drug reservoir and the biological sample. By combining GmNL(Ca2+) with this adaptation, we could image spontaneous Ca2+ transients in cultured cardiomyocytes and phenotype their response to well-known drugs without accessing the sample directly. In addition, the bioluminescent strategy demonstrates minimal perturbation of contractile parameters and long-term observation attributable to lack of phototoxicity and photobleaching. Overall, bioluminescence may enable more accurate drug screening in a high-throughput manner.Electronic supplementary materialThe online version of this article (10.1186/s13104-018-3421-7) contains supplementary material, which is available to authorized users.

Acute effects of cardiac contractility modulation on human induced pluripotent stem cell–derived cardiomyocytes

“…Additionally, concomitant β‐adrenergic block in the clinical setting does not eliminate the CCM response (Campbell et al, 2020) consistent with our assessment of the β‐adrenergic blocker metoprolol. β‐adrenergic blockers such as metoprolol and propranolol have demonstrated efficacy in uninnervated hiPSC‐CM monocultures suggesting that while the sympathetic stimulation from neurons is absent these cultures may possess an innate level of sympathetic basal tone that can be modulated by β‐adrenergic blockade (Blinova et al, 2018; Grimm et al, 2018; Sirenko et al, 2013; Suzuki et al, 2018).…”

Small Molecule–Protein Hybrid for Voltage Imaging via Quenching of Bioluminescence

“…However, the long photon collection times (0.1–1 s), limit single-cell imaging of rapid neuronal action potentials. In systems where cells have a uniform change in membrane potential, like the cardiomyocyte syncytium, Q-BOLT can readily detect changes and oscillations in membrane potential (Figure ), presaging its utility, for example, in phenotypic assays of hiPSC-CMs, where phototoxicity is often a limiting factor. ,, In these contexts, slow-release or cell impermeant versions of the NLuc substrate, furimazine, may aid long-term imaging and improve the signal-to-noise ratio; tethered DPA-fluorophore pairs may also improve performance. We are currently investigating methods to improve the photon output of Q-BOLT by screening pairs of HaloTag-NLuc fusions that show both enhanced BRET and voltage sensitivity and combining these constructs with even brighter synthetic fluorophore-HaloTag ligands.…”

A Small Molecule – Protein Hybrid for Voltage Imaging via Quenching of Bioluminescence

“…In systems where cells have a uniform change in membrane potential, like the cardiomyocyte syncytium, Q-BOLT can readily detect changes and oscillations in membrane potential (Figure 3), presaging its utility, for example, in phenotypic assays of hiPSC-CMs, where phototoxicity is often a limiting factor. 32,37,38 In these contexts, slow-release or cell impermeant versions 39 of the NLuc substrate, furimazine, may aid long-term imaging, and improve signal-tonoise. We are currently investigating methods to improve the photon output of Q-BOLT by screening 40 constructs that show both enhanced BRET and voltage sensitivity and combining these constructs with even brighter 41 synthetic fluorophore-Hal-oTag ligands.…”