Muhammed Sönmez, Tim Stüdemann, Christoph Manthey, Anita Covic, Nancy Shehata, Junsoo Im, Barbora Schwarzova, Judith Rössinger, Rajiven Srikantharajah, Aya Shibamiya, Edzard Schwedhelm, Thomas Eschenhagen, Torsten Christ, Florian Weinberger
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引用次数: 0
Abstract
Transient perturbation of electrical activity is used in neuroscience to study the impact of specific neuronal cell populations on brain function. Similarly, cardiomyocyte (CM) physiology can be controlled by the activation of artificially expressed ion channels. Pharmacologically selective actuator modules (PSAMs) are engineered ligand-gated ion channels that can be activated with small molecules. We aimed to use the ‘inhibitory’ PSAMs, (i) PSAML141F,Y115F-GlyR (PSAM-GlyR) and (ii) PSAML131G,Q139L,Y217F (ultrapotent PSAM4-GlyR), which consist of modified α7-nicotinergic acetylcholine receptor ligand binding domains and the ion pore domain of the glycine receptor, to modulate CM physiology with chloride currents. We employed CRISPR/Cas9 to integrate PSAM-GlyR and PSAM4-GlyR in induced pluripotent stem cells, differentiated CMs and generated engineered heart tissue (EHT). Video optical force recordings, sharp microelectrode action potential measurements and patch-clamp technique were used to characterize PSAM-GlyR and PSAM4-GlyR CMs. PSAM-GlyR and PSAM4-GlyR activation allowed titration of chloride currents in a reversible manner. We found that chloride currents modulated action potential characteristics. Patch clamp recordings showed that channel activation resulted in chloride-driven currents that depolarized the cell. In EHT, this resulted in a stop of contractility that was fully reversible after wash-out. We provide a comprehensive characterization of the chemogenetic tools PSAM-GlyR and PSAM4-GlyR in CMs, demonstrating their utility to modulate CM activity in vitro (PSAM-GlyR and PSAM4-GlyR) but also potential for in vivo applications (PSAM4-GlyR).
Key points
Pharmacologically selective actuator modules (PSAMs) are engineered ligand-gated ion channels that can be activated with small molecules.
These chemogenetic tools have been applied in neuroscience to inhibit neuronal activity.
Chemogenetic tools can also be used to modulate cardiomyocyte physiology.
Activation of the PSAMs, PSAM-GlyR and PSAM4-GlyR depolarized cardiomyocytes and thus stopped cardiac contractility.
Our study characterizes novel tools that can be used to modulate cardiomyocyte physiology in vitro and in vivo.
期刊介绍：
The Journal of Physiology publishes full-length original Research Papers and Techniques for Physiology, which are short papers aimed at disseminating new techniques for physiological research. Articles solicited by the Editorial Board include Perspectives, Symposium Reports and Topical Reviews, which highlight areas of special physiological interest. CrossTalk articles are short editorial-style invited articles framing a debate between experts in the field on controversial topics. Letters to the Editor and Journal Club articles are also published. All categories of papers are subjected to peer reivew.
The Journal of Physiology welcomes submitted research papers in all areas of physiology. Authors should present original work that illustrates new physiological principles or mechanisms. Papers on work at the molecular level, at the level of the cell membrane, single cells, tissues or organs and on systems physiology are all acceptable. Theoretical papers and papers that use computational models to further our understanding of physiological processes will be considered if based on experimentally derived data and if the hypothesis advanced is directly amenable to experimental testing. While emphasis is on human and mammalian physiology, work on lower vertebrate or invertebrate preparations may be suitable if it furthers the understanding of the functioning of other organisms including mammals.
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