Voltage-sensitive Fluorescent Dye Research Articles

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

AbstractThe 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‐1 mouse 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 TRPM4 significantly 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. imageKey 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‐1 mouse 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 TRPM4 significantly 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.

The antimicrobial effect and mechanism of the Artemisia argyi essential oil against bacteria and fungus.

Artemisia argyi is a traditional Chinese herb with antibacterial, antifungal, and antitumor activities. The essential oil of Artemisia argyi was extracted using the steam distillation method in this study. The chemical composition of the essential oil was analyzed using the gas chromatography-mass spectrometry method. Agar disc diffusion and double-broth dilution assays were used to detect the antimicrobial activity of the essential oil. Subsequently, the antimicrobial mechanisms were explored through cytomembrane permeability assay and electron microscopy. Based on gas chromatography-mass spectrometry analysis, 25 compounds were detected, including 13.76% cineole, 6.77% terpinen-4-ol, 6.68% 3-dione, 1,7,7-trimethyl-, 4.07% 3-cyclohexen-1-ol, 4-methyl-1-(1-methylethyl)-acetate, 3.58% 1-isopropyl-2-methylbenzene, and 1.58% g-terpinene. The essential oil was tested for antimicrobial activity, and the IC50 values for Staphylococcus aureus, Escherichia coli, Bacillus subtilis, Listeria monocytogenes, Pseudomonas aeruginosa, Streptococcus pneumoniae, and Candida albicans were determined to be 25.51 ± 2.29, 49.53 ± 0.86, 52.40 ± 1.49, 52.76 ± 1.60, 73.99 ± 1.38, 65.52 ± 0.95, and 214.98 ± 3.27μgmL-1, respectively. For essential oil interaction with cytoderm, the microorganisms treated by 1 × IC50 and 2 × IC50 concentration of essential oil both represented positive test results. Additionally, the alkaline phosphatase levels showed a direct correlation with concentration and treatment duration (range from 0 to 8h). The interaction between essential oils and the cytomembrane was investigated by examining samples containing one of three test strains (Staphylococcus aureus, Escherichia coli, and Candida albicans), essential oil, and voltage-sensitive fluorescent dye disc35. The results demonstrated a significant increase in fluorescence levels within the solution upon introduction of the essential oil-treated strains. The findings of our research suggest that the essential oil disrupts the cytoderm and cytomembrane, thereby exhibiting antimicrobial activity.

Simulating the Voltage-Dependent Fluorescence of Di-8-ANEPPS in a Lipid Membrane.

Voltage-sensitive fluorescent dyes such as di-8-ANEPPS (di-8-aminonaphthylethylenepyridinium propylsulfonate) are powerful tools to study biological membranes. Its fluorescence is affected by changes in the membrane potential and other factors, requiring extensive calibration to extract meaningful quantitative results. The amphiphilic di-8-ANEPPS molecule is expected to bind at the membrane-solution interface. However, atomic-level information is sparse about its position and orientation in the membrane, especially in regards to how the latter dynamically fluctuates to affect the observed fluorescence. In the present work, molecular dynamics simulations of the ground and excited states of di-8-ANEPPS embedded in a DPPC membrane as represented by classical force fields were used to investigate how the fluorescence is affected by externally applied potential. The calculations reproduce the shifts in the wavelength of emission as a function of voltage that are observed experimentally, indicating that the approach can help better understand the various factors that can affect the fluorescence of membrane-bound dyes.

PO-02-174 CHARACTERIZATION OF PAPILLARY MUSCLE ABLATION WITH OPTICAL MAPPING AT DIFFERENT TRANSMURAL PENETRATION DEPTHS IN LIVE HUMAN HEART

Papillary muscles (PMs) are known triggers of ventricular arrhythmia, especially nonsustained ventricular tachycardia (VT) and sustained recurrent VT, and may play a role as triggers of ventricular fibrillation. Catheter ablation of PM is challenging and results in high VT recurrence rates. Clinical characterization of PM electrophysiology is challenging due to the limited spatial resolution of electrode arrays, motion, and distal fields. Thus, ablation procedures are limited in two ways: inaccurate determination of target ablation zones and inaccurate characterization of lesion profiles. Characterization of PM before and after ablation can be accomplished with deep infrared optical mapping toward a better understanding of the role of the PM. Using optical mapping and near-infrared voltage-sensitive fluorescent dyes, transmembrane potentials were recorded from the endocardial side of a live explanted human right ventricle free-wall (transplant recipient). We paced the myocardium at different cycle lengths (CLs) before and after ablation, and transmembrane potential maps were obtained using excitation light bands of different penetration depths to characterize the ablated zones transmurally. The PM exhibited alternans in action potential duration (APD) at longer CLs than wall tissue in non-ablated tissue, indicating a highly heterogeneous myocardium. The figure shows relative conduction velocity maps for subsequent beats before and after ablation. While dissection of the tissue appeared to show a transmural burn, optical mapping with different penetration depths, from green (surface) and near-infrared (NI, transmural) illumination, revealed that the transmural electrophysiology profile was ablated, only at the surface. The presence of conduction velocity alternans using NI light indicated incomplete ablation at the deeper subsurfaces, even though it appeared burnt during dissection. Contemporary electro-mapping systems used in clinics often achieve limited electrophysiological characterization in the depth of ablated areas. Such measurements would yield results similar to optical mapping with the green light excitation band (surface only), suggesting successful ablation that may be incomplete and may result in recurrent VT. We demonstrate that optical mapping with NI light is a useful tool for quantitative characterization of ablation parameters.

Does Enhanced Structural Maturity of hiPSC-Cardiomyocytes Better for the Detection of Drug-Induced Cardiotoxicity?

Human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs) are currently used following the Comprehensive in vitro Proarrhythmic Assay (CiPA) initiative and subsequent recommendations in the International Council for Harmonization (ICH) guidelines S7B and E14 Q&A, to detect drug-induced cardiotoxicity. Monocultures of hiPSC-CMs are immature compared to adult ventricular cardiomyocytes and might lack the native heterogeneous nature. We investigated whether hiPSC-CMs, treated to enhance structural maturity, are superior in detecting drug-induced changes in electrophysiology and contraction. This was achieved by comparing hiPSC-CMs cultured in 2D monolayers on the current standard (fibronectin matrix, FM), to monolayers on a coating known to promote structural maturity (CELLvo™ Matrix Plus, MM). Functional assessment of electrophysiology and contractility was made using a high-throughput screening approach involving the use of both voltage-sensitive fluorescent dyes for electrophysiology and video technology for contractility. Using 11 reference drugs, the response of the monolayer of hiPSC-CMs was comparable in the two experimental settings (FM and MM). The data showed no functionally relevant differences in electrophysiology between hiPSC-CMs in standard FM and MM, while contractility read-outs indicated an altered amplitude of contraction but not changes in time course. RNA profiling for cardiac proteins shows similarity of the RNA expression across the two forms of 2D culture, suggesting that cell-to-matrix adhesion differences may explain account for differences in contraction amplitude. The results support the view that hiPSC-CMs in both 2D monolayer FM and MM that promote structural maturity are equally effective in detecting drug-induced electrophysiological effects in functional safety studies.

Spectral properties of the voltage-sensitive dye di‑4-ANBDQBS.

Voltage-sensitive fluorescent dyes allow non-invasive recording of action potentials in excitable cells. The far-red-shifted voltage-sensitive dye, di-4-ANBDQBS (CytoVolt1), is stable for hours and produces optical-voltage changes through the Stark effect. However, the properties and precise voltage responses of the CytoVolt1 emission spectrum in living cells are still unclear. To better understand these changes, HEK293 cells were loaded with CytoVolt1, and emission scanning was performed using a confocal system at several excitation wavelengths. The average emission wavelength peak was 689+/- 2.3 (n = 21 cells;+/- SEM) at an excitation of 528 nm. In addition, peak emission wavelengths changed according to the excitation wavelength, with average peaks at 692+/- 3.3 nm and 705+/- 2.8 nm (n = 9, 9; p < 0.05) for 480 nm and 640 nm excitation wavelengths, respectively. We next wanted to know whether the voltage-dependence of fluorescence (as determined by the Stark shift) was linear at different excitation/emission wavelengths. We simultaneously voltage-clamped the CytoVolt1-stained HEK293 cells and measured fluorescence response at 720 nm (long-pass) for a physiological range of Vm values. With an excitation wavelength of 660 nm, the fluorescence-voltage relationship was linear (R2 = 0.995) with a slope of −0.0051 mV−1 (n = 6). Similarly, when the excitation wavelength was changed to 528 nm, the slopes for the fluorescence-voltage relations were 0.0013 mV−1 (R2= 0.996) and −0.0007 mV−1 (R2 = 0.998) for emissions at 650 nm and 769 nm (bandpass) (n = 10 cells), respectively. Dividing the two emission signals (650 nm/769nm) increased the slope to 0.0021 mV−1 and retained a high degree of linearity (R2 = 0.999). The data demonstrate that CytoVolt1 shows an emission shift corresponding to the excitation wavelength. CytoVolt1 also shows linear fluorescence-voltage relationships at different excitation and emission wavelengths, giving it utility as a voltage-sensitive fluorescent dye with different experimental applications.

Hyperglycaemia-induced impairment of the autorhythmicity and gap junction activity of mouse embryonic stem cell-derived cardiomyocyte-like cells.

Diabetes mellitus with hyperglycaemia is a major risk factor for malignant cardiac dysrhythmias. However, the underlying mechanisms remain unclear, especially during the embryonic developmental phase of the heart. This study investigated the effect of hyperglycaemia on the pulsatile activity of stem cell-derived cardiomyocytes. Mouse embryonic stem cells (mESCs) were differentiated into cardiac-like cells through embryoid body (EB) formation, in either baseline glucose or high glucose conditions. Action potentials (APs) were recorded using a voltage-sensitive fluorescent dye and gap junction activity was evaluated using scrape-loading lucifer yellow dye transfer assay. Molecular components were detected using immunocytochemistry and immunoblot analyses. High glucose decreased the spontaneous beating rate of EBs and shortened the duration of onset of quinidine-induced asystole. Furthermore, it altered AP amplitude, but not AP duration, and had no impact on neither the expression of the hyperpolarisation-activated cyclic nucleotide-gated isoform 4 (HCN4) channel nor on the EB beating rate response to ivabradine nor isoprenaline. High glucose also decreased both the intercellular spread of lucifer yellow within an EB and the expression of the cardiac gap junction protein connexin43 as well as upregulated the expression of transforming growth factor beta1 (TGF-β1) and phosphorylated Smad3. High glucose suppressed the autorhythmicity and gap junction conduction of mESC-derived cardiomyocytes, via mechanisms probably involving TGF-β1/Smad3 signalling. The results allude to glucotoxicity related proarrhythmic effects, with potential clinical implications in foetal diabetic cardiac disease.

Abstract 11027: SGK1 Inhibition and Attenuation of the Action Potential Duration in Re-Engineered Heart Cell Models of Drug-Induced QT Prolongation

Background: Drug-induced QT prolongation (DI-QTP) is a clinical entity in which administration of a HERG/I Kr blocker such as dofetilide prolongs the cardiac action potential duration (APD) at the cellular level and the QT interval clinically, and increases the risk for a potentially lethal, QT-triggered ventricular arrhythmia. There may be a role for inhibition of I NaL to counter DI-QTP. Recently, we have shown that inhibition of serum and glucocorticoid regulated kinase-1 (SGK1) reduces the APD90 in induced pluripotent stem cell derived cardiomyocytes (iPSC-CM) from patients with LQT3 and ameliorates increases in I NaL . Objective: To test the efficacy of novel SGK1 inhibitors (SGK-I) in re-engineered cardiomyocyte models of dofetilide-induced APD prolongation. Methods: Normal iPSC-CMs were treated with dofetilide [5 nM] to produce a DI-QTP iPSC-CM model. The SGK1-I’s therapeutic efficacy for shortening the dofetilide-induced APD90 prolongation was compared to mexiletine. The APD90 values were recorded 4 hours after treatment using FluoVolt, a voltage-sensitive fluorescent dye. Results: The APD90 was significantly prolonged in normal iPSC-CMs treated with dofetilide (673 ± 8 ms with dofetilide vs 436 ± 4 ms with DMSO, p&lt;0.0001). While 10 μM mexiletine shortened the average APD90 of dofetilide-treated normal iPSC-CMs from 673 ± 4 ms to 563 ± 8 ms (46% attenuation, p&lt;0.0001), 30 nM of SGK-I shortened the APD90 from 673 ± 4 ms to 478 ± 10 ms (82% attenuation, p&lt;0.0001). Conclusions: Therapeutically inhibiting SGK1 effectively shortens the cardiomyocyte APD in a human heart cell model of DI-QTP. The novel SGK1 inhibitor normalized the pathological APD prolongation almost fully (&gt; 80%) in the iPSC-CM model treated with dofetilide. This pre-clinical data supports further development of this therapeutic strategy to counter and neutralize the threat of DI-QTP.

A guide for membrane potential measurements in Gram-negative bacteria using voltage-sensitive dyes.

Transmembrane potential is one of the main bioenergetic parameters of bacterial cells, and is directly involved in energizing key cellular processes such as transport, ATP synthesis and motility. The most common approach to measure membrane potential levels is through use of voltage-sensitive fluorescent dyes. Such dyes either accumulate or are excluded from the cell in a voltage-dependent manner, which can be followed by means of fluorescence microscopy, flow cytometry, or fluorometry. Since the cell's ability to maintain transmembrane potential relies upon low and selective membrane ion conductivity, voltage-sensitive dyes are also highly sensitive reporters for the activity of membrane-targeting antibacterials. However, the presence of an additional membrane layer in Gram-negative (diderm) bacteria complicates their use significantly. In this paper, we provide guidance on how membrane potential and its changes can be monitored reliably in Gram-negatives using the voltage-sensitive dye 3,3'-dipropylthiadicarbocyanine iodide [DiSC3(5)]. We also discuss the confounding effects caused by the presence of the outer membrane, or by measurements performed in buffers rather than growth medium. We hope that the discussed methods and protocols provide an easily accessible basis for the use of voltage-sensitive dyes in Gram-negative organisms, and raise awareness of potential experimental pitfalls associated with their use.

Gastric pacing response evaluated with simultaneous electrical and optical mapping.

Gastric pacing is an attractive therapeutic approach for correcting abnormal bioelectrical activity. While high-resolution (HR) electrical mapping techniques have largely contributed to the current understanding of the effect of pacing on the electrophysiological function, these mapping techniques are restricted to surface contact electrodes and the signal quality can be corrupted by pacing artifacts. Optical mapping of voltage sensitive dyes is an alternative approach used in cardiac research, and the signal quality is not affected by pacing artifacts. In this study, we simultaneously applied HR optical and electrical mapping techniques to evaluate the bioelectrical slow wave response to gastric pacing. The studies were conducted in vivo on porcine stomachs ( n=3) where the gastric electrical activity was entrained using high-energy pacing. The pacing response was optically tracked using voltage-sensitive fluorescent dyes and electrically tracked using surface contact electrodes positioned on adjacent regions. Slow waves were captured optically and electrically and were concordant in time and direction of propagation with comparable mean velocities ([Formula: see text]) and periods ([Formula: see text]). Importantly, the optical signals were free from pacing artifacts otherwise induced in electrical recordings highlighting an advantage of optical mapping. Clinical Relevance- Entrainment mapping of gastric pacing using optical techniques is a major advance for improving the preclinical understanding of the therapy. The findings can thereby inform the efficacy of gastric pacing in treating functional motility disorders.

Chemical Targeting of Rhodol Voltage-Sensitive Dyes to Dopaminergic Neurons.

Optical imaging of changes in the membrane potential of living cells can be achieved by means of fluorescent voltage-sensitive dyes (VSDs). A particularly challenging task is to efficiently deliver these highly lipophilic probes to specific neuronal subpopulations in brain tissue. We have tackled this task by designing a solubilizing, hydrophilic polymer platform that carries a high-affinity ligand for a membrane protein marker of interest and a fluorescent VSD. Here, we disclose an improved design of polymer-supported probes for chemical, nongenetic targeting of voltage sensors to axons natively expressing the dopamine transporter in ex vivo mouse brain tissue. We first show that for negatively charged rhodol VSDs functioning on the photoinduced electron transfer principle, poly(ethylene glycol) as a carrier enables targeting with higher selectivity than the polysaccharide dextran in HEK cell culture. In the same experimental setting, we also demonstrate that incorporation of an azetidine ring into the rhodol chromophore substantially increases the brightness and voltage sensitivity of the respective VSD. We show that the superior properties of the optimized sensor are transferable to recording of electrically evoked activity from dopaminergic axons in mouse striatal slices after averaging of multiple trials. Finally, we suggest the next milestones for the field to achieve single-scan recordings with nongenetically targeted VSDs in native brain tissue.

Ratiometry for improving the signal-to-noise with the red-shifted voltage dye di-4ANBDQBS

Voltage-sensitive fluorescent dyes allow non-invasive recording of action potentials in single cells. These experiments often have low signal-to-noise ratios (SNR). To improve SNR, ratiometric fluorescence methods have been utilized. In these studies, we used di-4-ANBDQBS (Cytovolt1), a far-red-shifted voltage-sensitive dye with a stable signal that works through the Stark effect. HEK cells were loaded with CytoVolt1 that was excited at 528 nm using our optical system. Fluorescence emission was measured at 720 nm with a long pass filter and 655 nm with a bandpass filter, corresponding to opposite slope regions of the emission spectrum.

Repolarization heterogeneity within the myocardial infarction border zone correlates with variability of myocyte remodeling

Abstract Background Myocardial infarction (MI) results in a regional scar, with a border zone (BZ) of surviving myocytes interspersed with fibrosis providing an anatomical substrate for re-entry. Heterogeneous repolarization within the BZ may add a functional component aggravating re-entrant arrhythmias. Purpose We studied BZ heterogeneity and developed novel methodology for high resolution mapping of local in vivo activation-repolarization intervals (ARI) within the BZ and for studying the relation to cellular action potential (AP) profiles of cells isolated from the BZ. Methods Anterior-septal myocardial infarction was induced in 5 domestic pigs by 120-minute occlusion of the left anterior descending artery followed by reperfusion (18.9±4.7% of the left ventricle). After 1-month, electro-anatomical mapping was performed. Contact mapping was used to define the BZ (bipolar voltage 0.5–1.5mV). A non-contact recording of a 64-electrode array was translated to 2048 non-contact electrograms distributed over the LV. The non-contact electrograms were processed to determine the ARIs using a custom-made algorithm, validated against monophasic action potential recordings. After 2–4 days recovery, single cardiomyocytes were enzymatically isolated from the anterior-septal BZs and remote regions. Cardiomyocytes were field stimulated at 1Hz at 37°C and cellular AP duration (APD) was optically recorded (fluorescent voltage-sensitive dye Di-8-Annepps). Results In vivo, regional ARIs tended to be longer in the BZs than remote. ARI heterogeneity, quantified as the standard deviation of ARIs in a neighborhood of 1cm radius, was increased in the BZ (anterior BZ: 3.4±1.0 ms, P=0.052, septal BZ: 3.6±1.7 ms, P=0.027 vs remote: 2.0±0.5 ms). Cellular APD was measured in large population samples (&amp;gt;100 cells per region in each pig) and was longer in BZ myocytes compared to the remote region. Cellular APD heterogeneity, measured as the standard deviation within cell population samples pooled by region per animal, was increased in the BZ (anterior BZ: 105.9±17.0 ms, P=0.0010; septal BZ: 98.1±20.8 ms, P=0.0127 vs remote: 73.9±8.6 ms). Cell APD correlated to in vivo ARI (R2=0.34, P=0.021) and cellular heterogeneity correlated strongly with in vivo heterogeneity (R2=0.67, P=0.002). Conclusion In the BZ of MI, in vivo regional heterogeneity adds a functional substrate for re-entry that may result from heterogeneous cellular remodeling and increased cell-cell APD variability. Funding Acknowledgement Type of funding sources: Public Institution(s). Main funding source(s): KU Leuven BOF-C1 “Blood pressure induced premature ventricular beats as triggers for ventricular arrhythmia in ischemic cardiomyopathy”

Fast Optical Investigation of Cardiac Electrophysiology by Parallel Detection in Multiwell Plates.

Current techniques for fast characterization of cardiac electrophysiology employ optical technologies to control and monitor action potential features of single cells or cellular monolayers placed in multiwell plates. High-speed investigation capacities are commonly achieved by serially analyzing well after well employing fully automated fluorescence microscopes. Here, we describe an alternative cost-effective optical approach (MULTIPLE) that exploits high-power LED arrays to globally illuminate a culture plate and an sCMOS sensor for parallel detection of the fluorescence coming from multiple wells. MULTIPLE combines optical detection of action potentials using a red-shifted voltage-sensitive fluorescent dye (di-4-ANBDQPQ) with optical stimulation, employing optogenetic actuators, to ensure excitation of cardiomyocytes at constant rates. MULTIPLE was first characterized in terms of interwell uniformity of the illumination intensity and optical detection performance. Then, it was applied for probing action potential features in HL-1 cells (i.e., mouse atrial myocyte-like cells) stably expressing the blue light-activatable cation channel CheRiff. Under proper stimulation conditions, we were able to accurately measure action potential dynamics across a 24-well plate with variability across the whole plate of the order of 10%. The capability of MULTIPLE to detect action potential changes across a 24-well plate was demonstrated employing the selective Kv11.1 channel blocker (E-4031), in a dose titration experiment. Finally, action potential recordings were performed in spontaneous beating human induced pluripotent stem cell derived cardiomyocytes following pharmacological manipulation of their beating frequency. We believe that the simplicity of the presented optical scheme represents a valid complement to sophisticated and expensive state-of-the-art optical systems for high-throughput cardiac electrophysiological investigations.

Optical mapping of human embryonic stem cell-derived cardiomyocyte graft electrical activity in injured hearts

BackgroundHuman embryonic stem cell-derived cardiomyocytes (hESC-CMs) show tremendous promise for cardiac regeneration, but the successful development of hESC-CM-based therapies requires improved tools to investigate their electrical behavior in recipient hearts. While optical voltage mapping is a powerful technique for studying myocardial electrical activity ex vivo, we have previously shown that intra-cardiac hESC-CM grafts are not labeled by conventional voltage-sensitive fluorescent dyes. We hypothesized that the water-soluble voltage-sensitive dye di-2-ANEPEQ would label engrafted hESC-CMs and thereby facilitate characterization of graft electrical function and integration.MethodsWe developed and validated a novel optical voltage mapping strategy based on the simultaneous imaging of the calcium-sensitive fluorescent protein GCaMP3, a graft-autonomous reporter of graft activation, and optical action potentials (oAPs) derived from di-2-ANEPEQ, which labels both graft and host myocardium. Cardiomyocytes from three different GCaMP3+ hESC lines (H7, RUES2, or ESI-17) were transplanted into guinea pig models of subacute and chronic infarction, followed by optical mapping at 2 weeks post-transplantation.ResultsUse of a water-soluble voltage-sensitive dye revealed pro-arrhythmic properties of GCaMP3+ hESC-CM grafts from all three lines including slow conduction velocity, incomplete host-graft coupling, and spatially heterogeneous patterns of activation that varied beat-to-beat. GCaMP3+ hESC-CMs from the RUES2 and ESI-17 lines both showed prolonged oAP durations both in vitro and in vivo. Although hESC-CMs partially remuscularize the injured hearts, histological evaluation revealed immature graft structure and impaired gap junction expression at this early timepoint.ConclusionSimultaneous imaging of GCaMP3 and di-2-ANEPEQ allowed us to acquire the first unambiguously graft-derived oAPs from hESC-CM-engrafted hearts and yielded critical insights into their arrhythmogenic potential and line-to-line variation.

Time-Multiplexed Illumination for simultaneous Structural and Functional Voltage Sensitive Dye Recordings with a single Photo Sensor.

The in-vivo optical imaging of the cortical surface provides the ability to record different types of biophysiological signals, e.g., structural information, intrinsic signals, like blood oxygenation coupled reflection changes as well as extrinsic properties of voltage sensitive probes, like fluorescent voltage-sensitive dyes. The recorded data sets have very high temporal and spatial resolutions on a meso- to macroscopic scale, which surpass conventional multi-electrode recordings. Both, intrinsic and functional data sets, each provide unique information about temporal and spatial dynamics of cortical functioning, yet have individual drawbacks. To optimize the informational value it would thus be opportune to combine different types of optical imaging in a near simultaneous recording.Due to the low signal-to-noise ratio of voltage-sensitive dyes it is necessary to reduce stray light pollution below the level of the camera's dark noise. It is thus impossible to record full-spectrum optical data sets. We address this problem by a time-multiplexed illumination, bespoke to the utilized voltage sensitive dye, to record an alternating series of intrinsic and extrinsic frames by a high-frequency CMOS sensor. These near simultaneous data series can be used to compare the mutual influence of intrinsic and extrinsic dynamics (with regards to extracorporeal functional imaging) as well as for motion compensation and thus for minimizing frame averaging, which in turn results in increased spatial precision of functional data and in a reduction of necessary experimental data sets (3R principle).

Fatal arrhythmias: Another reason why doctors remain cautious about chloroquine/hydroxychloroquine for treating COVID-19

BackgroundEarly during the current coronavirus disease 19 (COVID-19) pandemic, hydroxychloroquine (HCQ) received a significant amount of attention as a potential antiviral treatment, such that it became one of the most commonly prescribed medications for COVID-19 patients. However, not only has the effectiveness of HCQ remained questionable, but mainly based on preclinical and a few small clinical studies, HCQ is known to be potentially arrhythmogenic, especially as a result of QT prolongation.ObjectiveThe purpose of this study was to investigate the arrhythmic effects of HCQ, as the heightened risk is especially relevant to COVID-19 patients, who are at higher risk for cardiac complications and arrhythmias at baseline.MethodsAn optical mapping technique utilizing voltage-sensitive fluorescent dyes was used to determine the arrhythmic effects of HCQ in ex vivo guinea pig and rabbit hearts perfused with the upper therapeutic serum dose of HCQ (1000 ng/mL).ResultsHCQ markedly increased action potential dispersion, resulted in development of repolarization alternans, and initiated polymorphic ventricular tachycardia.ConclusionThe study results further highlight the proarrhythmic effects of HCQ.

Chemical Targeting of Voltage Sensitive Dyes to Specific Cells and Molecules in the Brain.

Voltage sensitive fluorescent dyes (VSDs) are important tools for probing signal transduction in neurons and other excitable cells. The impact of these highly lipophilic sensors has, however, been limited due to the lack of cell-specific targeting methods in brain tissue or living animals. We address this key challenge by introducing a nongenetic molecular platform for cell- and molecule-specific targeting of synthetic VSDs in the brain. We employ a dextran polymer particle to overcome the inherent lipophilicity of VSDs by dynamic encapsulation and high-affinity ligands to target the construct to specific neuronal cells utilizing only native components of the neurotransmission machinery at physiological expression levels. Dichloropane, a monoamine transporter ligand, enables targeting of dense dopaminergic axons in the mouse striatum and sparse noradrenergic axons in the mouse cortex in acute brain slices. PFQX in conjunction with ligand-directed acyl imidazole chemistry enables covalent labeling of AMPA-type glutamate receptors in the same brain regions. Probe variants bearing either a classical electrochromic ANEP dye or state-of-the-art VoltageFluor-type dye respond to membrane potential changes in a similar manner to the parent dyes, as shown by whole-cell patch recording. We demonstrate the feasibility of optical voltage recording with our probes in brain tissue with one-photon and two-photon fluorescence microscopy and define the signal limits of optical voltage imaging with synthetic sensors under a low photon budget determined by the native expression levels of the target proteins. This work demonstrates the feasibility of a chemical targeting approach and expands the possibilities of cell-specific imaging and pharmacology.

Rapidly Assaying Voltage-Gated Sodium Channels using Light-Induced Action Potentials and Fluorescent Recordings of the Membrane Potential in an Instrument with a Novel Detector Array

Combining light-induced activation of ion channels with recordings of the cell membrane potential via voltage-sensitive fluorescent dyes potentially provides an economical, easier to use alternative to traditional electrophysiological approaches in drug discovery. However, accurately resolving the signature signals produced by rapidly inactivating voltage-gated ion channel is challenging for optical instrumentation, because of the need for sub-millisecond scale temporal resolution and graded activation. We previously introduced a novel high throughput plate reader instrument, the Photoswitch Bolt that combines the instantaneous response of next generation membrane voltage sensor dyes with a unique light detector array providing voltage readouts at 10 kHz sampling rate. The red-shift in the excitation wavelength of the dye (660nm) allows for optical monitoring of membrane potential without significant contamination from the light used to activate a genetically encoded optical actuator. Using a recombinant HEK293 cell line expressing a channelrhodopsin actuator together with Nav1.5/Nav1.7 and Kir2.1, light-induced action potentials were monitored under different stimulus regimes. Appropriate isoform selectivity and state-and use-dependent pharmacology of the sodium channels will be presented for different tool compounds validating the usefulness of these types of optical assays for large scale screening of ion channel targets.

Low-Noise Fluorescent Infrared Detection of Single-Cell Cardiac Action Potentials

Optical recordings with voltage-sensitive fluorescent dyes enable non-invasive action potential (AP) measurements, which are useful for high-throughput drug screening. However, optical measurements are hampered by dye rundown, photobleaching, and phototoxicity. Long-lasting infrared dyes overcome these limitations, but optical recordings from single-cells are difficult due to low signal-to-noise ratios (SNR). Using the voltage-sensitive infrared dye, Di-4-ANBDQBS (Cytovolt1), we developed an optical system consisting of matched hardware, biological, and dye components to produce a low-noise fluorescent infrared detection system for single-cell AP recording. We combined a high-sensitivity photodiode-based detector, a high-stability LED excitation source, and an optimized optical filter set for enhanced single-cell optical AP recordings. We validated our system by correlating membrane voltage to fluorescence under single-cell patch clamp, and showed the fluorescence signal is highly-linear with membrane voltage. We also optimized dye concentration and staining method along with emission/excitation wavelengths to yield maximum ΔF/F and thus maximum SNR. To correlate membrane voltages to optical voltage signals, we performed simultaneous patch clamp and optical recordings on HEK293 cells and human induced pluripotent stem cell derived cardiomyocytes (hIPSC-CMs). Low-noise recordings, with minimal dye rundown, were obtained from single-cells. Optical signals were recorded in response to a voltage ramp, and averaged R2 values from linear fits of fluorescence versus voltage were 0.88+0.04, 0.97+0.007, 0.97+0.004 and 0.98+0.004 for 50, 75, 100 and 150 μM of Di‑4‑ANBDQBS, respectively, in HEK293 cells. ΔF/F measurements were 10.5+0.3%, 14.9+1.8%, 13.8+2.5% and 13.6+3.5% for 50, 75, 100 and 150 μM of dye, respectively. For hiPSC-CMs, ΔF/F measurements from simultaneous voltage and fluorescence of APs were up to 23.1% per 100 mV. Taken together, these data demonstrate the feasibility of high fidelity single cell voltage measurements in a simple system not requiring photomultiplier tubes or laser excitation.