Modulation of cardiomyocyte contractility and action potentials with chemogenetic chloride currents

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) PSAM^{L141F,Y115F-GlyR} (PSAM-GlyR) and (ii) PSAM^{L131G,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.

Keywords: cardiac electrophysiology; cardiac function; cardiomyocyte; chemogenetics; pluripotent stem cells.

Figure 1. Baseline characterization of PSAM‐GlyR and PSAM⁴‐GlyR hiPSC and hiPSC‐cardiomyocytes

A and B, flow cytometry of either PSAM‐GlyR or PSAM⁴‐GlyR human induced pluripotent stem cells (hiPSCs; A) or hiPSC‐CMs (B) stained for SSEA3 or cardiac troponin T. C, longitudinal sections of a PSAM‐GlyR and PSAM⁴‐GlyR EHT stained for GFP and ACTN2. α‐Bungarotoxin was used to label PSAM‐GlyR. D–G, video‐optical contractility measurement of control, PSAM‐GlyR or PSAM‐GlyR⁴ EHT (n = 19/39/31 EHTs from n = 1/3/3 batches). D, contractile force at day 28 of culture. E, beating frequency at day 28 of culture. F, relaxation time at day 28 of culture. G, contraction time at day 28 of culture. Groups were compared using one‐way ANOVA with multiple comparison, and P‐values are provided in the graph. Measurements were performed in culture medium. Plotted are means ± SD. [Colour figure can be viewed at wileyonlinelibrary.com]

Figure 2. PSAM‐GlyR EHT but not PSAM⁴‐GlyR EHT had a less negative maximum diastolic potential

A, action potential traces recorded in control, PSAM‐GlyR and PSAM⁴‐GlyR EHT. B–F, mean values ± SD for various electrophysiological parameters. B, maximum diastolic potential (MDP); C, amplitude of action potential (APA); D, cycle length (CL); E, action potential duration at that 90% repolarization (APD₉₀); F, maximum upstroke velocity (V _max). The number of EHT/number of batches is given as n/n and different shades indicate different batches. Groups (Control, PSAM: PSAM‐GlyR and PSAM⁴: PSAM⁴‐GlyR) are compared using one‐way ANOVA, and P‐values are provided in the graph. Measurements were performed in Tyrode's solution. [Colour figure can be viewed at wileyonlinelibrary.com]

Figure 3. PSAM‐GlyR expression in hiPSC‐CMs associated with some ‘constitutively leak’ current

A, time courses of currents measured at −75 mV in hiPSC‐CMs expressing PSAM‐GlyR (red) and hiPSC‐CMs expressing PSAM⁴‐GlyR (blue). Cells were exposed to 1 mM picrotoxin (PTX) initially and then to 50 nM varenicline (hiPSC‐CMs expressing PSAM⁴‐GlyR) or 50 µM PSEM^89S (hiPSC‐CMs expressing PSAM‐GlyR). B and C, mean values ± SD for increase in current by 50 µM PSEM^89S or 50 nM varenicline in the absence and presence of 1 mM PTX. The number of hiPSC‐CMs/number of EHTs/number of batches are given as n/n/n and different shapes within the groups indicate different batches. Groups are compared using unpaired t test, and P‐values are provided in the graph. [Colour figure can be viewed at wileyonlinelibrary.com]

Figure 4. Charge carrier for PSAM‐GlyR and PSAM⁴‐GlyR was chloride and the agonists PSEM^89S and varenicline showed the expected potency

A, current–voltage relationship of PSAM^89S‐ and varenicline‐evoked currents. The reversal potential and liquid–liquid junction potential (LLJP) are indicated by an arrow. Mean values ± SD for normalized currents. n/n indicates the number of hiPSC‐CMs/number of EHTs. B, time courses of inward currents at −75 mV in hiPSC‐CMs expressing PSAM‐GlyR or PSAM⁴‐GlyR exposed to increasing concentrations of PSEM^89S or varenicline. C, concentration–effect curve for PSEM^89S and varenicline. Basal current density of PSAM and PSAM⁴‐GlyR‐hiPSC‐CMs is compared using unpaired t test, and P‐values are provided in the graph. Mean values ± SD for chloride current density (I _m), and n/n/n indicates the number of hiPSC‐CMs/number of EHTs/number of batches. [Colour figure can be viewed at wileyonlinelibrary.com]

Figure 5. Concentration‐dependent effects of varenicline on action potential characteristics in PSAM⁴‐GlyR EHT

A, action potential traces at baseline and at increasing varenicline concentrations. B, time course of action membrane potential recorded in PSAM⁴‐GlyR EHT exposed to increasing concentration of varenicline in PSAM⁴‐GlyR EHT. AP stopped at a varenicline concentration of 30 nM (indicated by an arrow). The black line after this point represents only maximal diastolic potential (MDP). C, concentration–effect curve for varenicline effect on MDP and maximum upstroke velocity (V _max). Note that plotted EC₅₀ values are calculated from the MDP concentration–response curve. Two out of 11 EHTs stopped beating at 10 nM varenicline and 6 out of 11 EHTs stopped beating at 30 nM varenicline. Mean values ± SD for MDP (black) or V _max (red). The number of EHTs/number of batches are given as n/n. [Colour figure can be viewed at wileyonlinelibrary.com]

Figure 6. Relationship between activated chloride current and AP parameters

A, original action potential traces recorded in PSAM⁴‐GlyR EHTs in the presence of cumulatively increasing varenicline concentrations (1 nM steps up to 20 nM), illustrating the shift of the membrane potential. B, comparison between the chloride current density on the maximal diastolic potential (MDP) and beating rate in PSAM⁴‐GlyR EHT. Data are taken from patch clamp and sharp microelectrode measurements at matching varenicline concentrations. Note that 2 out of 11 EHTs stopped beating at 10 nM varenicline and 6 out of 11 EHTs stopped beating at 30 nM varenicline. Mean values ± SD for MDP (black) or beating rate (red). The number of EHTs/number of batches are given as n/n. C, maximum beating rate at 10 nM varenicline represented by two action potentials in PSAM⁴‐GlyR EHT. [Colour figure can be viewed at wileyonlinelibrary.com]

Figure 7. PSAM‐GlyR and PSAM⁴‐GlyR activation stopped contractility in engineered heart tissue

A, video‐optical contractility measurement of control (n = 7) and PSAM‐GlyR EHT (n = 11). B, video‐optical contractility measurement of control (n = 14) or PSAM⁴‐GlyR EHT (n = 8). C, change in EHT length from diastole to systole and during PSEM^89S or varenicline treatment, respectively. Length was normalized to diastolic length. n = 4/3. One‐way ANOVA with multiple comparisons. P‐values are provided in the graph. D–F, contractility measurement during long‐term silencing by constant PSEM^89S (D and F) after a culture period of (D) ∼1 month and (F) ∼2 months or varenicline application (E; n = 4/3/3 EHTs per group). Mean values ± SD for contractile force (A and B) and EHT length (C). n indicates the number of EHTs. [Colour figure can be viewed at wileyonlinelibrary.com]

Figure A1. Molecular characterization of PSAM⁴‐GlyrR knock‐in

[Colour figure can be viewed at wileyonlinelibrary.com]

Figure A2. PSEM^89S but not varenicline prolonged action potential duration

[Colour figure can be viewed at wileyonlinelibrary.com]

Figure A3. Concentration‐dependent effect of PSEM^89S on AP characteristics in PSAM‐GlyR EHT

[Colour figure can be viewed at wileyonlinelibrary.com]