Fluorescent Voltage Research Articles

Recent Development of Chemigenetic Hybrid Voltage Indicators Enabled by Bioconjugation Chemistry.

Fluorescent voltage indicators enable the optical recording of electrophysiology across large cell populations with subcellular resolution; however, their application is often constrained by a limited photon budget. To address this limitation, advanced bioconjugation methods have been employed to site-specifically attach bright and photostable organic dyes to cell-specific protein scaffolds in live cells. The resulting chemigenetic hybrid voltage indicators enable sustained monitoring of voltage fluctuations with an exceptional signal-to-noise ratio, both in vitro and in vivo. This Viewpoint discusses recent advancements in the development of these indicators through bioconjugation chemistry.

Ex vivo propagation of synaptically-evoked cortical depolarizations in a mouse model of Alzheimer’s disease at 20 Hz, 40 Hz, or 83 Hz

Sensory stimulations at 40 Hz gamma (but not any other frequency), have shown promise in reversing Alzheimer’s disease (AD)-related pathologies. What distinguishes 40 Hz? We hypothesized that stimuli at 40 Hz might summate more efficiently (temporal summation) or propagate more efficiently between cortical layers (vertically), or along cortical laminas (horizontally), compared to inputs at 20 or 83 Hz. To investigate these hypotheses, we used brain slices from AD mouse model animals (5xFAD). Extracellular (synaptic) stimuli were delivered in cortical layer 4 (L4). Leveraging a fluorescent voltage indicator (VSFP) expressed in cortical pyramidal neurons, we simultaneously monitored evoked cortical depolarizations at multiple sites, at 1 kHz sampling frequency. Experimental groups (AD-Female, CTRL-Female, AD-Male, and CTRL-Male) were tested at three stimulation frequencies (20, 40, and 83 Hz). Despite our initial hypothesis, two parameters—temporal summation of voltage waveforms and the strength of propagation through the cortical neuropil—did not reveal any distinct advantage of 40 Hz stimulation. Significant physiological differences between AD and Control mice were found at all stimulation frequencies tested, while the 40 Hz stimulation frequency was not remarkable.

Cl--dependent amplification of excitatory synaptic potentials at distal dendrites revealed by voltage imaging.

The processing of synaptic signals in somatodendritic compartments determines neuronal computation. Although the amplification of excitatory signals by local voltage-dependent cation channels has been extensively studied, their spatiotemporal dynamics in elaborate dendritic branches remain obscure owing to technical limitations. Using fluorescent voltage imaging throughout dendritic arborizations in hippocampal pyramidal neurons, we demonstrate a unique chloride ion (Cl-)-dependent remote computation mechanism in the distal branches. Excitatory postsynaptic potentials triggered by local laser photolysis of caged glutamate spread along dendrites, with gradual amplification toward the distal end while attenuation toward the soma. Tour de force subcellular patch-clamp recordings from thin branches complemented by biophysical model simulations revealed that the asymmetric augmentation of excitation relies on tetrodotoxin-resistant sodium ion (Na+) channels and Cl- conductance accompanied by a more hyperpolarized dendritic resting potential. Together, this study reveals the cooperative voltage-dependent actions of cation and anion conductance for dendritic supralinear computation, which can locally decode the spatiotemporal context of synaptic inputs.

Abstract We099: Antisense Oligonucleotide Treatment of Calmodulinopathy

Introduction: Antisense Oligonucleotides (ASOs) are an emerging class of drugs that target RNAs to impede their production, stability, and translation. Calmodulinopathies are severe cardiac arrhythmias caused by dominant mutations in CALM1 , CALM2 , or CALM3 . Each of these genes encodes the identical calmodulin (CaM) protein. Leveraging this genetic redundancy, we hypothesized that ASO-mediated knockdown of the affected CALM gene would treat arrhythmia while functional redundancy of the other CALM genes would maintain CaM protein level and prevent toxicity. Methods: Cells from a patient expressing the heterozygous CALM1 - F142L mutation were reprogrammed into induced pluripotent stem cells (iPSCs; P-CALM1 F142L/+ ). Additionally, the variant or a nonsense codon were introduced into the wild-type reference line, WTC11, to yield isogenic disease (E-CALM1 F142L/+ ) and CALM1 null ( CALM1 Δ/Δ ) lines respectively. iPSCs were differentiated into cardiomyocytes (iPSC-CMs) to assess their action potentials and Ca 2+ transients using multielectrode arrays and fluorescent voltage- and calcium-sensitive dyes. Calm1 N98S/+ mice, previously shown to develop catecholamine-induced ventricular tachycardia, were used for in vivo studies. Using these model systems, we tested ASOs that selectively depleted human or murine CALM1 . Results: Both E-CALM1 F142L/+ and P-CALM1 F142L/+ iPSC-CMs exhibited prolonged action potentials, consistent with QT prolongation observed in the proband. CALM1 Δ/Δ iPSC-CMs expressed normal levels of CaM protein and exhibited no change in action potential duration. ASO that selectively depleted CALM1 transcripts by ~50% did not alter CaM protein level but normalized action potential and Ca 2+ transient duration in CALM1 F142L/+ iPSC-CMs. In Calm1 N98S/+ mice, ASO selectively depleted Calm1 transcripts by ~90% and prevented catecholamine-induced ventricular tachycardia. Calm1 ASO did not alter CaM protein level, cardiac function, or treadmill endurance, or cause significant histopathological changes. Conclusion: ASO-based therapy safely and effectively treated arrhythmias caused by dominant CALM1 variants, suggesting that ASOs are a promising potential therapy for calmodulinopathy.

Orientation and Membrane Partition Free Energy of PeT-Based Voltage-Sensitive Dyes from Molecular Simulations.

Voltage measurement via small-molecule fluorescent indicators is a valuable approach in deciphering complex dynamics in electrically excitable cells. However, our understanding of various physicochemical properties governing the performance of fluorescent voltage sensors based on the photoinduced electron transfer (PeT) mechanism remains incomplete. Here, through extensive molecular dynamics and free energy calculations, we systematically examine the orientation and membrane partition of three PeT-based voltage-sensing VoltageFluor (VF) dyes in different lipid environment. We show that the symmetry of the molecular scaffold and the net charge of the hydrophilic headgroup of a given VF dye dominate its orientation and membrane partition, respectively. Our work provides a mechanistic understanding of the physical properties contributing to the voltage sensitivity, signal-to-noise ratio, as well as membrane distribution of VF dyes and sheds light onto rational design principles of PeT-based fluorescent probes in general.

Functional coupling between Piezo1 and TRPM4 influences the electrical activity of HL-1 atrial myocytes.

The transient receptor potential melastatin 4 (TRPM4) channel contributes extensively to cardiac electrical activity, especially cardiomyocyte action potential formation. Mechanical stretch can induce changes in heart rate and rhythm, and the mechanosensitive channel Piezo1 is expressed in many cell types within the myocardium. Our previous study showed that TRPM4 and Piezo1 are closely co-localized in the t-tubules of ventricular cardiomyocytes and contribute to the Ca2+-dependent signalling cascade that underlies hypertrophy in response to mechanical pressure overload. However, there was no direct evidence showing that Piezo1 activation was related to TRPM4 activation in situ. In the present study, we employed the HL-1mouse atrial myocyte-like cell line as an in vitro model to investigate whether Piezo1-TRPM4 coupling can affect action potential properties. We used the small molecule Piezo1 agonist, Yoda1, as a surrogate for mechanical stretch to activate Piezo1 and detected the action potential changes in HL-1 cells using FluoVolt, a fluorescent voltage sensitive dye. Our results demonstrate that Yoda1-induced activation of Piezo1 changes the action potential frequency in HL-1 cells. This change in action potential frequency is reduced by Piezo1 knockdown using small intefering RNA. Importantly knockdown or pharmacological inhibition of TRPM4significantly affected the degree to which Yoda1-evoked Piezo1 activation influenced action potential frequency. Thus, the present study provides in vitro evidence of a functional coupling between Piezo1 and TRPM4 in a cardiomyocyte-like cell line. The coupling of a mechanosensitive Ca2+ permeable channel and a Ca2+-activated TRP channel probably represents a ubiquitous model for the role of TRP channels in mechanosensory transduction. KEY POINTS: The transient receptor potential melastatin 4 (TRPM4) and Piezo1 channels have been confirmed to contribute to the Ca2+-dependent signalling cascade that underlies cardiac hypertrophy in response to mechanical pressure overload. However, there was no direct evidence showing that Piezo1 activation was related to TRPM4 activation in situ. We employed the HL-1mouse atrial myocyte-like cell line as an in vitro model to investigate the effect of Piezo1-TRPM4 coupling on cardiac electrical properties. The results show that both pharmacological and genetic inhibition of TRPM4significantly affected the degree to which Piezo1 activation influenced action potential frequency in HL-1 cells. Our findings provide in vitro evidence of a functional coupling between Piezo1 and TRPM4 in a cardiomyocyte-like cell line. The coupling of a mechanosensitive Ca2+ permeable channel and a Ca2+-activated TRP channel probably represents a ubiquitous model for the role of TRP channels in mechanosensory transduction in various (patho)physiological processes.

Reporter Coxsackievirus A5 Expressing iLOV Fluorescent Protein or Luciferase Used for Rapid Neutralizing Assay in Cells and Living Imaging in Mice.

Coxsackievirus A5 (CV-A5) is a re-emerging enterovirus that causes hand, foot, and mouth disease in children under five years of age. CV-A5-M14-611 is a mouse-adapted strain that can infect orally and lead to the death of 14-day-old mice. Here, recombinants based on CV-A5-M14-611 were constructed carrying three reporter genes in different lengths. Smaller fluorescent marker proteins, light, oxygen, voltage sensing (iLOV), and nano luciferase (Nluc) were proven to be able to express efficiently in vitro. However, the recombinant with the largest insertion of the red fluorescence protein gene (DsRed) was not rescued. The construction strategy of reporter viruses was to insert the foreign genes between the C-terminus of VP1 and the N-terminus of 2A genes and to add a 2A protease cleavage domain at both ends of the insertions. The iLOV-tagged or Nluc-tagged recombinants, CV-A5-iLOV or CV-A5-Nluc, exhibited a high capacity for viral replication, genetic stability in cells and pathogenicity in mice. They were used to establish a rapid, inexpensive and convenient neutralizing antibody assay and greatly facilitated virus neutralizing antibody titration. Living imaging was performed on mice with CV-A5-Nluc, which exhibited specific bioluminescence in virus-disseminated organs, while fluorescence induced by CV-A5-iLOV was weakly detected. The reporter-gene-tagged CV-A5 can be used to study the infection and mechanisms of CV-A5 pathogenicity in a mouse model. They can also be used to establish rapid and sensitive assays for detecting neutralizing antibodies.

Near-infrared voltage-sensitive dyes based on chromene donor

Voltage-sensitive dyes (VSDs) are used to image electrical activity in cells and tissues with submillisecond time resolution. Most of these fast sensors are constructed from push-pull chromophores whose fluorescence spectra are modulated by the electric field across the cell membrane. It was found that the substitution of naphthalene with chromene produces a 60 to 80 nm red-shift in absorption and emission spectra while maintaining fluorescence quantum efficiency and voltage sensitivity. One dye was applied to ex vivo murine heart with excitation at 730 nm, by far the longest wavelength reported in voltage imaging. This VSD resolves cardiac action potentials in single trials with 12% ΔF/F per action potential. The well-separated excitation spectra between these long-wavelength VSDs and channelrhodopsin (ChR2) enabled monitoring of action potential propagation in ChR2 hearts without any perturbation of electrical dynamics. Importantly, by employing spatially localized optogenetic manipulation, action potential dynamics can be assessed in an all-optical fashion with no artifact related to optical cross-talk between the reporter and actuator. These new environmentally sensitive chromene-based chromophores are also likely to have applications outside voltage imaging.

Transparent and Stretchable Au–Ag Nanowire Recording Microelectrode Arrays (Adv. Mater. Technol. 10/2023)

Microelectrode Arrays In article number 2201716 by Matthew W. Kay, Luyao Lu, and co-workers, a microelectrode array (MEA) is laminated on the heart to show its stretchability. Another stretched MEA is placed in the light path to show the capability for co-localized optical mapping. Green light represents stimulation light. Yellow and red lights represent the resulting calcium and voltage fluorescence while blue light represents the electrical signals from the MEA at the same site.

Expanding the family of genetically encoded voltage indicators with a candidate Heliorhodopsin exhibiting near-infrared fluorescence

Genetically encoded voltage indicators, particularly those based on microbial rhodopsins, are gaining traction in neuroscience as fluorescent sensors for imaging voltage dynamics with high-spatiotemporal precision. Here we establish a novel genetically encoded voltage indicator candidate based on the recently discovered subfamily of the microbial rhodopsin clade, termed heliorhodopsins. We discovered that upon excitation at 530 to 560nm, wildtype heliorhodopsin exhibits near-infrared fluorescence, which is sensitive to membrane voltage. We characterized the fluorescence brightness, photostability, voltage sensitivity, and kinetics of wildtype heliorhodopsin in HEK293T cells and further examined the impact of mutating key residues near the retinal chromophore. The S237A mutation significantly improved the fluorescence response of heliorhodopsin by 76% providing a highly promising starting point for further protein evolution.

Sensitivity optimization of a rhodopsin-based fluorescent voltage indicator

The ability to optically image cellular transmembrane voltages at millisecond-timescale resolutions can offer unprecedented insight into the function of living brains in behaving animals. Here, we present a point mutation that increases the sensitivity of Ace2 opsin-based voltage indicators. We use the mutation to develop Voltron2, an improved chemigeneic voltage indicator that has a 65% higher sensitivity to single APs and 3-fold higher sensitivity to subthreshold potentials than Voltron. Voltron2 retained the sub-millisecond kinetics and photostability of its predecessor, although with lower baseline fluorescence. In multiple invitro and invivo comparisons with its predecessor across multiple species, we found Voltron2 to be more sensitive to APs and subthreshold fluctuations. Finally, we used Voltron2 to study and evaluate the possible mechanisms of interneuron synchronization in the mouse hippocampus. Overall, we have discovered a generalizable mutation that significantly increases the sensitivity of Ace2 rhodopsin-based sensors, improving their voltage reporting capability.

Understanding the voltage-sensitivity of Arch3-based microbial rhodopsins.

The visualization of membrane potentials in living cells allows new insights into the function of neurons and, in particular, the brain. Although a lot of effort has been made during the last years, this task still remains very challenging facing problems such as the low signal intensity. The recently reported voltage-dependent fluorescence in archaerhodopsin-3 (Arch3) offered a new very encouraging basis to develop new optogenetic sensors. Both voltage-sensitivity and fluorescence quantum yields, however, are very low which makes the tuning of this property necessary for application in living rodents. Current approaches focused on this aim used high-throughput techniques without taking the mechanistic understanding on an atomic level into account. In our study, we predicted the voltage response of known Arch3 derivatives such as Arch2, Arch3, Archon1 and its mutants using the atomistic molecular dynamics (MD) based computational electrophysiology approach at the nanosecond to microsecond time scale. The simulations indicated a significant rearrangement of the hydrogen bonding network including the retinal Schiff base and water molecules upon voltage. Based on these findings, so far unrecognized point mutants interrupting the hydrogen bonding network showed an alternative voltage-sensing mechanism. Instead of the well-known arginine R92 adjacent to the counter ions, an intracellular aspartic acid D125 in helix D changed its orientation as response to changes in the applied voltage. The side-chain orientation of D125 was in turn modulated by the voltage-dependent dynamics of the intracellular part of helix D. The mutation D125N which interrupted the aforementioned reorientation in the MD simulations was validated experimentally and showed a higher voltage-sensitive fluorescence. This knowledge on the molecular level will be used in the future to further deepen the understanding of the voltage-sensing mechanism and to rationally predict new generation of fluorescence voltage sensors with improved optogenetic properties.

Dual-polarity voltage imaging of the concurrent dynamics of multiple neuron types.

Genetically encoded fluorescent voltage indicators are ideally suited to reveal the millisecond-scale interactions among and between targeted cell populations. However, current indicators lack the requisite sensitivity for in vivo multipopulation imaging. We describe next-generation green and red voltage sensors, Ace-mNeon2 and VARNAM2, and their reverse response-polarity variants pAce and pAceR. Our indicators enable 0.4- to 1-kilohertz voltage recordings from >50 spiking neurons per field of view in awake mice and ~30-minute continuous imaging in flies. Using dual-polarity multiplexed imaging, we uncovered brain state-dependent antagonism between neocortical somatostatin-expressing (SST+) and vasoactive intestinal peptide-expressing (VIP+) interneurons and contributions to hippocampal field potentials from cell ensembles with distinct axonal projections. By combining three mutually compatible indicators, we performed simultaneous triple-population imaging. These approaches will empower investigations of the dynamic interplay between neuronal subclasses at single-spike resolution.

Theoretical investigation of the photocycle dynamics of the Archaerhodopsin 3 based fluorescent voltage sensor Archon2

Previously the retinal photocycle dynamics of the fluorescent voltage sensor Archon2, a mutant of Archaerhodopsin 3, in pH 8 Tris buffer was studied experimentally at room temperature by photo-excitation (excitation wavelengths 590 nm and 632.8 nm) of the protonated retinal Schiff base (PRSB) Ret_586 (absorption maximum at λmax = 586 nm) and following the absorption spectra development during and after light exposure.In this paper the experimental results are simulated numerically with developed photocycle schemes and relevant dynamics parameters are extracted.Photo-excitation of fresh thawed (dark-adapted) Archon2 (Archon2da) caused photoisomerization of Ret_586 (Ret_586da) to Ret_553 (Ret_535da, main component, absorption maximum at 553 nm) and to Ret_485 (Ret_485da, minor component) in a primary excitation process (primary photocycle). Ret_485 does not absorb at λexc = 590 nm or 632.8 nm and therefore is not involved in further photo-excitation. Continued light exposure transformed Archon2da to light-adapted Archon2la with secondary photocycle dynamics changing Ret_553 (Ret_535da) to Ret_535 (Ret_535la, absorption maximum at 535 nm). Ret_535la backward photo-isomerized to Ret_586 in light-adapted state (named Ret_586la) and partly deprotonated to neutral retinal Schiff base (RSB) Ret_372 in light adapted state (named Ret_372la, same isomer form as Ret_535).After excitation light switch-off Archon2la recovered in the dark to Archon2da with recovery of Ret_372la to Ret_372da, Ret_535la to Ret_535da, and Ret_586la to Ret_586da. Ret_372da slowly re-protonated to Ret_535da, and Ret_535da slowly isomerized back to Ret_586da. The long-time dynamics in the dark at room temperature changed over to thermal Archon2 denaturation accompanied with protonated retinal Schiff base deprotonation.

QuasAr Odyssey: the origin of fluorescence and its voltage sensitivity in microbial rhodopsins

Rhodopsins had long been considered non-fluorescent until a peculiar voltage-sensitive fluorescence was reported for archaerhodopsin-3 (Arch3) derivatives. These proteins named QuasArs have been used for imaging membrane voltage changes in cell cultures and small animals. However due to the low fluorescence intensity, these constructs require use of much higher light intensity than other optogenetic tools. To develop the next generation of sensors, it is indispensable to first understand the molecular basis of the fluorescence and its modulation by the membrane voltage. Based on spectroscopic studies of fluorescent Arch3 derivatives, we propose a unique photo-reaction scheme with extended excited-state lifetimes and inefficient photoisomerization. Molecular dynamics simulations of Arch3, of the Arch3 fluorescent derivative Archon1, and of several its mutants have revealed different voltage-dependent changes of the hydrogen-bonding networks including the protonated retinal Schiff-base and adjacent residues. Experimental observations suggest that under negative voltage, these changes modulate retinal Schiff base deprotonation and promote a decrease in the populations of fluorescent species. Finally, we identified molecular constraints that further improve fluorescence quantum yield and voltage sensitivity.

The Electromotive Protein Prestin as a Sensitive Core of the Fluorescent Voltage Indicator

The Electromotive Protein Prestin as a Sensitive Core of the Fluorescent Voltage Indicator

Heterogeneous myocyte remodelling and spatial heterogeneity of repolarization within the myocardial infarction border zone

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Fund for Scientific Research-Flanders (FWO) Background Sudden cardiac death due to ventricular arrhythmias is a major cause of mortality after myocardial infarction (MI). The border zone (BZ) surrounding the infarct is the dominant source of arrhythmias. Here a substrate of heterogeneous repolarization is implicated, which could be due to heterogeneous myocyte remodelling. Objective To examine myocyte remodelling within the BZ, in comparison to the remote myocardium, and evaluate the local profile of repolarization of these regions in vivo. Methods MI was induced by 120-minute occlusion of the left anterior descending coronary artery followed by reperfusion in 6 domestic pigs. After 4 weeks, magnetic resonance imaging was performed to assess infarct remodelling and local wall thickness. Within 3 days, electro-anatomical mapping was performed. A non-contact recording of a 64-electrode array was translated to 2048 electrograms distributed over the LV and local activation-recovery-interval (ARI) determined by custom software. After recovery (2-4 days), the pigs were sacrificed, and samples collected from the BZ and remote region for RNA analysis and single cardiomyocyte isolation. Cell dimensions were measured and cellular AP duration (APD) was optically recorded using a fluorescent voltage dye, Di-8-Annepps (stimulation at 1Hz, 37°C). Expression and variability of cardiomyocyte hypertrophy biomarkers were extracted from single nuclear RNA sequencing data (10x Genomics). Results Cardiomyocyte APD in large population samples (> 100 cells per region in each pig) revealed higher heterogeneity in the BZ than the remote region, quantified as the standard deviation (SD) (BZ: 105.9 ± 17.0ms vs remote: 73.9 ± 8.6ms, P = 0.001). Cellular APD heterogeneity correlated strongly with in vivo local ARI heterogeneity, which demonstrated increased heterogeneity in the BZ (R2 = 0.67, P = 0.002). BZ myocytes were hypertrophied with greater increase in cell width than length, and cellular hypertrophy was more heterogeneous by SD in the BZ (BZ: 12.9 ± 2.4μm vs remote: 8.3 ± 1.1μm, P < 0.001). NPPB transcripts reporting on hypertrophic remodelling were higher in BZ than remote (mean lognorm gene expression, BZ: 0.431 ± 0.014 vs remote: 0.107 ± 0.004, P < 0.001), and showed greater heterogeneity in expression between cells by proportion of hypertrophic (NPPB +ve) cells (BZ: 30.86% vs remote: 8.37%, P < 0.001). Wall thickness variance was higher in the BZ compared to the remote region (anterior BZ: 0.15 ± 0.02mm, septal BZ: 0.16 ± 0.04mm vs remote: 0.04 ± 0.02mm, P < 0.001), contributing to increased heterogeneity of local wall stress in BZ. Conclusion Cardiomyocyte remodelling in the BZ is heterogeneous, possibly related to differences in local wall stress, which may contribute to heterogeneous repolarization in vivo and underlie arrhythmia vulnerability within the BZ.

Photocycle dynamics of the Archaerhodopsin 3 based fluorescent voltage sensor Archon2.

The retinal photocycle dynamics of the fluorescent voltage sensor Archon2 in pH8 Tris buffer was studied. Archon2 is a mutant of Archaerhodopsin 3 (Arch) from Halorubrum sodomense obtained by a robotic multidimensional directed evolution approach (Archon2=Arch T56P-P60S-T80P-D95H-T99S-T116I-F161V-T183I-L197I-A225C). The samples were photo-excited to the first absorption band of the protonated retinal Schiff base (PRSB) Ret_586 (absorption maximum at λmax=586nm, excitation wavelengths λexc=590nm and 632.8nm). The photocycle dynamics were studied by recording absorption spectra during light exposure and after light exposure. Ret_586 photoisomerized to Ret_535 (main component) and Ret_485 (minor component). Ret_535 backward photoisomerized to Ret_586 in light-adapted state (named Ret_586la) and partly deprotonated to neutral retinal Schiff base (RSB) Ret_372 in light adapted state (named Ret_372la, same isomer form as Ret_535). After excitation light switch-off Ret_372la recovered to Ret_372 in dark-adapted state (Ret_372da) which slowly re-protonated to Ret_535, and Ret_535 slowly isomerized back to Ret_586 in dark-adapted state (Ret_586da). Photocycle schemes and reaction coordinate diagrams are developed and photocycle parameters are determined.

Abstract 14161: Drug Compounds Combination Screening Using Human Induced Pluripotent Stem Cell-Derived Cardiac Tissues

Introduction: The introduction of human-induced pluripotent stem cells derived cardiomyocytes (hiPSC-CMs) technology provided an important tool for the field of safety pharmacology (QT screening). This approach has been limited, however, mostly to the assessment of a single drug. We present a large scale EP phenotyping system comprised of hiPSC-derived cardiac cell sheets (hiPSC-CCSs), fluorescent voltage sensors and actuators, and an optical mapping system; with the aim of performing drug combination screening. Methods and Results: hIPSCs-CMs were seeded as hiPSC-CCSs in multi-well plates. Tissues were treated with varying concentrations of drug compounds’ combinations. Optical signals were acquired following loading of the hiPSC-CCSs with voltage-sensitive dyes or expression of genetically-encoded voltage indicator (ArcLight). EP parameters such as conduction velocity (CV) and action potential duration (APD) were extracted. ATX2 (late sodium current activator) and Dofetilide (IKr blocker) were chosen as representative APD prolonging reagents. Example of the effects of 16 different drug doses combinations on APD (% change) prolongation is depicted in Fig.1a. Similarly, examples of the restitution plots obtained at 4 different drug combinations are presented in Fig.1b-c. Notice that drug compound's effect on prolonging APD showed greater effect as compared to the cumulative effect of each drug alone. Figure 1: (a) Percentage of change in APD 80 15 minutes post treatment (b, c) APD 80 per condition before and after treatment, respectively Conclusion: These results highlight the potential of the hiPSC-CCS as a model for combinational drug screening. This may bring a unique value to the fields of safety pharmacology, pathophysiology testing and drug screening applications.

Linearly polarized excitation enhances signals from fluorescent voltage indicators

Voltage imaging in cells requires high-speed recording of small fluorescent signals, often leading to low signal/noise ratios. Because voltage indicators are membrane bound, their orientations are partially constrained by the plane of the membrane. We explored whether tuning the linear polarization of excitation light could enhance voltage indicator fluorescence. We tested a panel of dye- and protein-based voltage indicators in mammalian cells. The dye BeRST1 showed a 73% increase in brightness between the least and most favorable polarizations. The protein-based reporter ASAP1 showed a 22% increase in brightness, and QuasAr3 showed a 14% increase in brightness. In very thin neurites expressing QuasAr3, improvements were anomalously large, with a 170% increase in brightness between polarization parallel versus perpendicular to the dendrite. Signal/noise ratios of optically recorded action potentials were increased by up to 50% in neurites expressing QuasAr3. These results demonstrate that polarization control can be a facile means to enhance signals from fluorescent voltage indicators, particularly in thin neurites or in high-background environments.