Measurement of solubility product in a model condensate reveals the interplay of small oligomerization and self-association.

Cellular condensates often consist of 10s to 100s of distinct interacting molecular species. Because of the complexity of these interactions, predicting the point at which they will undergo phase separation into discrete compartments is daunting. Using experiments and computation, we therefore studied a simple model system consisting of 2 proteins, polySH3 and polyPRM, designed for pentavalent heterotypic binding. We tested whether the peak solubility product, the product of dilute phase monomer concentrations, is a predictive parameter for the onset of phase separation. Titrating up equal total concentrations of each component showed that the maximum solubility product does approximately coincide with the threshold for phase separation in both the experiments and models. However, we found that measurements of dilute phase concentration include contributions from small oligomers, not just monomers; therefore, a quantitative comparison of the experiments and models required inclusion of small oligomers in the model analysis. We also examined full phase diagrams where the model results were almost symmetric along the diagonal, but the experimental results were highly asymmetric. This led us to perform dynamic light scattering experiments, where we discovered a weak homotypic interaction for polyPRM; when this was added to the computational model, it was able to recapitulate the experimentally observed asymmetry. Thus, comparing experiments to simulation reveals that the solubility product can be predictive of phase separation, even if small oligomers and low affinity homotypic interactions preclude experimental measurement of monomer concentration.

Optogenetic confirmation of transverse-tubular membrane excitability in intact cardiac myocytes.

T-tubules (TT) form a complex network of sarcolemmal membrane invaginations, essential for well-co-ordinated excitation-contraction coupling (ECC) and thus homogeneous mechanical activation of cardiomyocytes. ECC is initiated by rapid depolarization of the sarcolemmal membrane. Whether TT membrane depolarization is active (local generation of action potentials; AP) or passive (following depolarization of the outer cell surface sarcolemma; SS) has not been experimentally validated in cardiomyocytes. Based on the assessment of ion flux pathways needed for AP generation, we hypothesize that TT are excitable. We therefore explored TT excitability experimentally, using an all-optical approach to stimulate and record trans-membrane potential changes in TT that were structurally disconnected, and hence electrically insulated, from the SS membrane by transient osmotic shock. Our results establish that cardiomyocyte TT can generate AP. These AP show electrical features that differ substantially from those observed in SS, consistent with differences in the density of ion channels and transporters in the two different membrane domains. We propose that TT-generated AP represent a safety mechanism for TT AP propagation and ECC, which may be particularly relevant in pathophysiological settings where morpho-functional changes reduce the electrical connectivity between SS and TT membranes. KEY POINTS: Cardiomyocytes are characterized by a complex network of membrane invaginations (the T-tubular system) that propagate action potentials to the core of the cell, causing uniform excitation-contraction coupling across the cell. In the present study, we investigated whether the T-tubular system is able to generate action potentials autonomously, rather than following depolarization of the outer cell surface sarcolemma. For this purpose, we developed a fully optical platform to probe and manipulate the electrical dynamics of subcellular membrane domains. Our findings demonstrate that T-tubules are intrinsically excitable, revealing distinct characteristics of self-generated T-tubular action potentials. This active electrical capability would protect cells from voltage drops potentially occurring within the T-tubular network.

Di-5-ANEQ(F)PTEA Offers Better Performance than Di-4-ANEQ(F)PTEA for In-Situ Cardiac Optical Mapping.

Cardiac optical mapping has traditionally been performed in ex-vivo, motion-arrested hearts. Recently, in-situ cardiac optical mapping has been made possible by both motion correction techniques and long-wavelength voltage sensitive dyes (VSDs). However, VSDs have been observed to wash out quickly from blood-perfused in-situ hearts. In this study, we evaluate the performance of a newly developed VSD, di-5-ANEQ(F)PTEA, relative to an earlier VSD, di-4-ANEQ(F)PTEA. We find that di-5-ANEQ(F)PTEA persists over 3 times longer, produces improved signal-to-noise ratio, and does not prolong loading unacceptably.Clinical Relevance-Optical mapping has provided many insights into cardiac arrhythmias, but has traditionally been limited to ex-vivo preparations. The present findings extend the utility of optical mapping in the more realistic in-vivo setting and may eventually enable its use in patients.

Optical mapping of cardiac electromechanics in beating in vivo hearts.

Optical mapping has been widely used in the study of cardiac electrophysiology in motion-arrested, ex vivo heart preparations. Recent developments in motion artifact mitigation techniques have made it possible to optically map beating ex vivo hearts, enabling the study of cardiac electromechanics using optical mapping. However, the ex vivo setting imposes limitations on optical mapping such as altered metabolic states, oversimplified mechanical loads, and the absence of neurohormonal regulation. In this study, we demonstrate optical electromechanical mapping in an in vivo heart preparation. Swine hearts were exposed via median sternotomy. Voltage-sensitive dye, either di-4-ANEQ(F)PTEA or di-5-ANEQ(F)PTEA, was injected into the left anterior descending artery. Fluorescence was excited by alternating green and amber light for excitation ratiometry. Cardiac motion during sinus and paced rhythm was tracked using a marker-based method. Motion tracking and excitation ratiometry successfully corrected most motion artifact in the membrane potential signal. Marker-based motion tracking also allowed simultaneous measurement of epicardial deformation. Reconstructed membrane potential and mechanical deformation measurements were validated using monophasic action potentials and sonomicrometry, respectively. Di-5-ANEQ(F)PTEA produced longer working time and higher signal/noise ratio than di-4-ANEQ(F)PTEA. In addition, we demonstrate potential applications of the new optical mapping system including electromechanical mapping during vagal nerve stimulation, fibrillation/defibrillation. and acute regional ischemia. In conclusion, although some technical limitations remain, optical mapping experiments that simultaneously image electrical and mechanical function can be conducted in beating, in vivo hearts.

Recent advances and current limitations of available technology to optically manipulate and observe cardiac electrophysiology.

Optogenetics, utilising light-reactive proteins to manipulate tissue activity, are a relatively novel approach in the field of cardiac electrophysiology. We here provide an overview of light-activated transmembrane channels (optogenetic actuators) currently applied in strategies to modulate cardiac activity, as well as newly developed variants yet to be implemented in the heart. In addition, we touch upon genetically encoded indicators (optogenetic sensors) and fluorescent dyes to monitor tissue activity, including cardiac transmembrane potential and ion homeostasis. The combination of the two allows for all-optical approaches to monitor and manipulate the heart without any physical contact. However, spectral congestion poses a major obstacle, arising due to the overlap of excitation/activation and emission spectra of various optogenetic proteins and/or fluorescent dyes, resulting in optical crosstalk. Therefore, optogenetic proteins and fluorescent dyes should be carefully selected to avoid optical crosstalk and consequent disruptions in readouts and/or cellular activity. We here present a novel approach to simultaneously monitor transmembrane potential and cytosolic calcium, while also performing optogenetic manipulation. For this, we used the novel voltage-sensitive dye ElectroFluor 730p and the cytosolic calcium indicator X-Rhod-1 in mouse hearts expressing channelrhodopsin-2 (ChR2). By exploiting the isosbestic point of ElectroFluor 730p and avoiding the ChR2 activation spectrum, we here introduce a novel optical imaging and manipulation approach with minimal crosstalk. Future developments in both optogenetic proteins and fluorescent dyes will allow for additional and more optimised strategies, promising a bright future for all-optical approaches in the field of cardiac electrophysiology.

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.

Non-invasive electromechanical assessment during atrial fibrillation identifies underlying atrial myopathy alterations with early prognostic value.

Electromechanical characterization during atrial fibrillation (AF) remains a significant gap in the understanding of AF-related atrial myopathy. This study reports mechanistic insights into the electromechanical remodeling process associated with AF progression and further demonstrates its prognostic value in the clinic. In pigs, sequential electromechanical assessment during AF progression shows a progressive decrease in mechanical activity and early dissociation from its electrical counterpart. Atrial tissue samples from animals with AF reveal an abnormal increase in cardiomyocytes death and alterations in calcium handling proteins. High-throughput quantitative proteomics and immunoblotting analyses at different stages of AF progression identify downregulation of contractile proteins and progressive increase in atrial fibrosis. Moreover, advanced optical mapping techniques, applied to whole heart preparations during AF, demonstrate that AF-related remodeling decreases the frequency threshold for dissociation between transmembrane voltage signals and intracellular calcium transients compared to healthy controls. Single cell simulations of human atrial cardiomyocytes also confirm the experimental results. In patients, non-invasive assessment of the atrial electromechanical relationship further demonstrate that atrial electromechanical dissociation is an early prognostic indicator for acute and long-term rhythm control.

MolClustPy: a Python package to characterize multivalent biomolecular clusters.

SUMMARY: Low-affinity interactions among multivalent biomolecules may lead to the formation of molecular complexes that undergo phase transitions to become supply-limited large clusters. In stochastic simulations, such clusters display a wide range of sizes and compositions. We have developed a Python package, MolClustPy, which performs multiple stochastic simulation runs using NFsim (Network-Free stochastic simulator); MolClustPy characterizes and visualizes the distribution of cluster sizes, molecular composition, and bonds across molecular clusters. The statistical analysis offered by MolClustPy is readily applicable to other stochastic simulation software, such as SpringSaLaD and ReaDDy. AVAILABILITY AND IMPLEMENTATION: The software is implemented in Python. A detailed Jupyter notebook is provided to enable convenient running. Code, user guide, and examples are freely available at https://molclustpy.github.io/.

Beyond analytic solution: Analysis of FRAP experiments by spatial simulation of the forward problem.

Fluorescence redistribution after photobleaching is a commonly used method to understand the dynamic behavior of molecules within cells. Analytic solutions have been developed for specific, well-defined models of dynamic behavior in idealized geometries, but these solutions are inaccurate in complex geometries or when complex binding and diffusion behaviors exist. We demonstrate the use of numerical reaction-diffusion simulations using the Virtual Cell software platform to model fluorescence redistribution after photobleaching experiments. Multiple simulations employing parameter scans and varying bleaching locations and sizes can help to bracket diffusion coefficients and kinetic rate constants in complex image-based geometries. This approach is applied to problems in membrane surface diffusion as well as diffusion and binding in cytosolic volumes in complex cell geometries. In addition, we model diffusion and binding within phase-separated biomolecular condensates (liquid droplets). These are modeled as spherical low-affinity binding domains that also define a high viscosity medium for exchange of the free fluorescently labeled ligand with the external cytosol.

Optogenetic manipulation of cardiac repolarization gradients using sub-threshold illumination.

Introduction: Mechanisms underlying cardiac arrhythmias are typically driven by abnormalities in cardiac conduction and/or heterogeneities in repolarization time (RT) across the heart. While conduction slowing can be caused by either electrophysiological defects or physical blockade in cardiac tissue, RT heterogeneities are mainly related to action potential (AP) prolongation or abbreviation in specific areas of the heart. Importantly, the size of the area with altered RT and the difference between the short RT and long RT (RT gradient) have been identified as critical determinators of arrhythmogenicity. However, current experimental methods for manipulating RT gradient rely on the use of ion channel inhibitors, which lack spatial and temporal specificity and are commonly only partially reversible. Therefore, the conditions facilitating sustained arrhythmia upon the presence of RT heterogeneities and/or defects in cardiac conduction remain to be elucidated. Methods: We here employ an approach based on optogenetic stimulation in a low-intensity fashion (sub-threshold illumination), to selectively manipulate cardiac electrical activity in defined areas of the heart. Results: As previously described, subthreshold illumination is a robust tool able to prolong action potentials (AP), decrease upstroke velocity as well as slow cardiac conduction, in a fully reversible manner. By applying a patterned sub-threshold illumination in intact mouse hearts constitutively expressing the light-gated ion channel channelrhodopsin-2 (ChR2), we optically manipulate RT gradients and cardiac conduction across the heart in a spatially selective manner. Moreover, in a proof-of-concept assessment we found that in the presence of patterned sub-threshold illumination, mouse hearts were more susceptible to arrhythmias. Hence, this optogenetic-based approach may be able to mimic conduction slowing and RT heterogeneities present in pathophysiological conditions.

The maximum solubility product marks the threshold for condensation of multivalent biomolecules.

Clustering of weakly interacting multivalent biomolecules underlies the formation of membraneless compartments known as condensates. As opposed to single-component (homotypic) systems, the concentration dependence of multicomponent (heterotypic) condensate formation is not well understood. We previously proposed the solubility product (SP), the product of monomer concentrations in the dilute phase, as a tool for understanding the concentration dependence of multicomponent systems. In this study, we further explore the limits of the SP concept using spatial Langevin dynamics and rule-based stochastic simulations. We show, for a variety of idealized molecular structures, how the maximum SP coincides with the onset of the phase transition, i.e., the formation of large clusters. We reveal the importance of intracluster binding in steering the free and cluster phase molecular distributions. We also show how structural features of biomolecules shape the SP profiles. The interplay of flexibility, length, and steric hindrance of linker regions controls the phase transition threshold. Remarkably, when SPs are normalized to nondimensional variables and plotted against the concentration scaled to the threshold for phase transition, the curves all coincide independent of the structural features of the binding partners. Similar coincidence is observed for the normalized clustering versus concentration plots. Overall, the principles derived from these systematic models will help guide and interpret in vitro and in vivo experiments on the biophysics of biomolecular condensates.

MolClustPy: A Python Package to Characterize Multivalent Biomolecular Clusters.

S ummary: Low-affinity interactions among multivalent biomolecules may lead to the formation of molecular complexes that undergo phase transitions to become extra-large clusters. Characterizing the physical properties of these clusters is important in recent biophysical research. Due to weak interactions such clusters are highly stochastic, demonstrating a wide range of sizes and compositions. We have developed a Python package to perform multiple stochastic simulation runs using NFsim (Network-Free stochastic simulator), characterize and visualize the distribution of cluster sizes, molecular composition, and bonds across molecular clusters and individual molecules of different types. A vailability and implementation: The software is implemented in Python. A detailed Jupyter notebook is provided to enable convenient running. Code, user guide and examples are freely available at https://molclustpy.github.io/. C ontact: achattaraj007@gmail.com , blinov@uchc.edu. S upplementary information: Available at https://molclustpy.github.io/.

A Complete and Low-Cost Cardiac Optical Mapping System in Translational Animal Models.

Clinicians, biologists, physicists, engineers, and computer scientists are coming together to better understand heart disease, which is currently the leading cause of death globally. Optical mapping, a high-speed fluorescence imaging technique that visualizes and measures key cardiac parameters such as action potentials, cytosolic calcium transients, and fibrillation dynamics, is a core research tool that has arisen from such interdisciplinary collaborations. In an effort to broaden its use, especially among clinical scientists and students, we developed a complete and low-cost optical mapping system, including a constant-flow Langendorff perfusion system, which minimizes the economic threshold to widespread use of this powerful tool in cardiac electrophysiology research. The system described here provides high spatiotemporal resolution data about action potentials, intracellular calcium transients and fibrillation wave dynamics in isolated Langendorff-perfused hearts (pigs and rabbits), relevant for translational research. All system components and software elements are fully disclosed with the aim of increasing the use of this affordable and highly versatile tool among clinicians, basic scientists and students wishing to tackle their own research questions with their own customizable systems.

Hyperexcitable Phenotypes in Induced Pluripotent Stem Cell-Derived Neurons From Patients With 15q11-q13 Duplication Syndrome, a Genetic Form of Autism.

BACKGROUND: Chromosome 15q11-q13 duplication syndrome (Dup15q) is a neurogenetic disorder caused by duplications of the maternal copy of this region. In addition to hypotonia, motor deficits, and language impairments, patients with Dup15q commonly meet the criteria for autism spectrum disorder and have a high prevalence of seizures. It is known from mouse models that synaptic impairments are a strong component of Dup15q pathophysiology; however, cellular phenotypes that relate to seizures are less clear. The development of patient-derived induced pluripotent stem cells provides a unique opportunity to study human neurons with the exact genetic disruptions that cause Dup15q. METHODS: Here, we explored electrophysiological phenotypes in induced pluripotent stem cell-derived neurons from 4 patients with Dup15q compared with 6 unaffected control subjects, 1 patient with a 15q11-q13 paternal duplication, and 3 patients with Angelman syndrome. RESULTS: We identified several properties of Dup15q neurons that could contribute to neuronal hyperexcitability and seizure susceptibility. Compared with control neurons, Dup15q neurons had increased excitatory synaptic event frequency and amplitude, increased density of dendritic protrusions, increased action potential firing, and decreased inhibitory synaptic transmission. Dup15q neurons also showed impairments in activity-dependent synaptic plasticity and homeostatic synaptic scaling. Finally, Dup15q neurons showed an increased frequency of spontaneous action potential firing compared with control neurons, in part due to disruption of KCNQ2 potassium channels. CONCLUSIONS: Together, these data point to multiple electrophysiological mechanisms of hyperexcitability that may provide new targets for the treatment of seizures and other phenotypes associated with Dup15q.

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 K v 11.1 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.

Mechanism of actin filament nucleation.

We used computational methods to analyze the mechanism of actin filament nucleation. We assumed a pathway where monomers form dimers, trimers, and tetramers that then elongate to form filaments but also considered other pathways. We aimed to identify the rate constants for these reactions that best fit experimental measurements of polymerization time courses. The analysis showed that the formation of dimers and trimers is unfavorable because the association reactions are orders of magnitude slower than estimated in previous work rather than because of rapid dissociation of dimers and trimers. The 95% confidence intervals calculated for the four rate constants spanned no more than one order of magnitude. Slow nucleation reactions are consistent with published high-resolution structures of actin filaments and molecular dynamics simulations of filament ends. One explanation for slow dimer formation, which we support with computational analysis, is that actin monomers are in a conformational equilibrium with a dominant conformation that cannot participate in the nucleation steps.

The solubility product extends the buffering concept to heterotypic biomolecular condensates.

Biomolecular condensates are formed by liquid-liquid phase separation (LLPS) of multivalent molecules. LLPS from a single ("homotypic") constituent is governed by buffering: above a threshold, free monomer concentration is clamped, with all added molecules entering the condensed phase. However, both experiment and theory demonstrate that buffering fails for the concentration dependence of multicomponent ("heterotypic") LLPS. Using network-free stochastic modeling, we demonstrate that LLPS can be described by the solubility product constant (Ksp): the product of free monomer concentrations, accounting for the ideal stoichiometries governed by the valencies, displays a threshold above which additional monomers are funneled into large clusters; this reduces to simple buffering for homotypic systems. The Ksp regulates the composition of the dilute phase for a wide range of valencies and stoichiometries. The role of Ksp is further supported by coarse-grained spatial particle simulations. Thus, the solubility product offers a general formulation for the concentration dependence of LLPS.

Transcranial photoacoustic imaging of NMDA-evoked focal circuit dynamics in the rat hippocampus.

OBJECTIVE: We report the transcranial functional photoacoustic (fPA) neuroimaging of N-methyl-D-aspartate (NMDA) evoked neural activity in the rat hippocampus. Concurrent quantitative electroencephalography (qEEG) and microdialysis were used to record real-time circuit dynamics and excitatory neurotransmitter concentrations, respectively. APPROACH: We hypothesized that location-specific fPA voltage-sensitive dye (VSD) contrast would identify neural activity changes in the hippocampus which correlate with NMDA-evoked excitatory neurotransmission. MAIN RESULTS: Transcranial fPA VSD imaging at the contralateral side of the microdialysis probe provided NMDA-evoked VSD responses with positive correlation to extracellular glutamate concentration changes. qEEG validated a wide range of glutamatergic excitation, which culminated in focal seizure activity after a high NMDA dose. We conclude that transcranial fPA VSD imaging can distinguish focal glutamate loads in the rat hippocampus, based on the VSD redistribution mechanism which is sensitive to the electrophysiologic membrane potential. SIGNIFICANCE: Our results suggest the future utility of this emerging technology in both laboratory and clinical sciences as an innovative functional neuroimaging modality.

Recent progress in optical voltage-sensor technology and applications to cardiac research: from single cells to whole hearts.

The first workshop on Novel Optics-based approaches for Cardiac Electrophysiology (NOtiCE) was held in Florence Italy in 2018. Here, we learned how optical approaches have shaped our basic understanding of cardiac electrophysiology and how new technologies and approaches are being developed and validated to advance the field. Several technologies are being developed that may one day allow for new clinical approaches for diagnosing cardiac disorders and possibly intervening to treat human patients. In this review, we discuss several technologies and approaches to optical voltage imaging with voltage-sensitive dyes. We highlight the development and application of fluorinated and long wavelength voltage-sensitive dyes. These optical voltage sensors have now been applied and well validated in several different assays from cultured human stem cell-derived cardiomyocytes to whole hearts in-vivo. Imaging concepts such as dual wavelength ratiometric techniques, which are crucial to maximizing the information from optical sensors by increasing the useful signal and eliminating noise and artifacts, are presented. Finally, novel voltage sensors including photoacoustic voltage-sensitive dyes, their current capabilities and potential advantages, are introduced.