Cardiac Cellular Coupling and the Spread of Early Instabilities in Intracellular Ca2+

AbstractRecent experimental and modeling studies demonstrate the fine spatial scale, complex nature, and independent contribution of Ca2+ dynamics as a proarrhythmic factor in the heart. The mechanism of progression of cell-level Ca2+ instabilities, known as alternans, to tissue-level arrhythmias is not well understood. Because gap junction coupling dictates cardiac syncytial properties, we set out to elucidate its role in the spatiotemporal evolution of Ca2+ instabilities. We experimentally perturbed cellular coupling in cardiac syncytium in vitro. Coupling was quantified by fluorescence recovery after photobleaching, and related to function, including subtle fine-scale Ca2+ alternans, captured by optical mapping. Conduction velocity and threshold for alternans monotonically increased with coupling. Lower coupling enhanced Ca2+ alternans amplitude, but the spatial spread of early (<2 Hz) alternation was the greatest under intermediate (not low) coupling. This nonmonotonic relationship was closely matched by the percent of samples exhibiting large-scale alternans at higher pacing rates. Computer modeling corroborated these experimental findings for strong but not weak electromechanical (voltage-Ca2+) coupling, and offered mechanistic insight. In conclusion, using experimental and modeling approaches, we reveal a general mechanism for the spatial spread of subtle cellular Ca2+ alternans that relies on a combination of gap-junctional and voltage-Ca2+ coupling

Sex-dependent transcription of cardiac electrophysiology and links to acetylation modifiers based on the GTEx database

Development of safer drugs based on epigenetic modifiers, e.g., histone deacetylase inhibitors (HDACi), requires better understanding of their effects on cardiac electrophysiology. Using RNAseq data from the genotype-tissue-expression database (GTEx), we created models that link the abundance of acetylation enzymes (HDAC/SIRT/HATs), and the gene expression of ion channels (IC) via select cardiac transcription factors (TFs) in male and female adult human hearts (left ventricle, LV). Gene expression data (transcripts per million, TPM) from GTEx donors (21–70 y.o.) were filtered, normalized and transformed to Euclidian space to allow quantitative comparisons in 84 female and 158 male LVs. Sex-specific partial least-square (PLS) regression models, linking gene expression data for HDAC/SIRT/HATs to TFs and to ICs gene expression, revealed tight co-regulation of cardiac ion channels by HDAC/SIRT/HATs, with stronger clustering in the male LV. Co-regulation of genes encoding excitatory and inhibitory processes in cardiac tissue by the acetylation modifiers may help explain their predominantly net-neutral effects on cardiac electrophysiology. ATP1A1, encoding for the Na/K pump, represented an outlier—with orthogonal regulation by the acetylation modifiers to most of the ICs. The HDAC/SIRT/HAT effects were mediated by strong (+) TF regulators of ICs, e.g., MEF2A and TBX5, in both sexes. Furthermore, for male hearts, PLS models revealed a stronger (+/-) mediatory role on ICs for NKX25 and TGF1B/KLF4, respectively, while RUNX1 exhibited larger (-) TF effects on ICs in females. Male-trained PLS models of HDAC/SIRT/HAT effects on ICs underestimated the effects on some ICs in females. Insights from the GTEx dataset about the co-expression and transcriptional co-regulation of acetylation-modifying enzymes, transcription factors and key cardiac ion channels in a sex-specific manner can help inform safer drug design

Computational Optogenetics: Empirically-Derived Voltage- and Light-Sensitive Channelrhodopsin-2 Model

Channelrhodospin-2 (ChR2), a light-sensitive ion channel, and its variants have emerged as new excitatory optogenetic tools not only in neuroscience, but also in other areas, including cardiac electrophysiology. An accurate quantitative model of ChR2 is necessary for in silicoprediction of the response to optical stimulation in realistic tissue/organ settings. Such a model can guide the rational design of new ion channel functionality tailored to different cell types/tissues. Focusing on one of the most widely used ChR2 mutants (H134R) with enhanced current, we collected a comprehensive experimental data set of the response of this ion channel to different irradiances and voltages, and used these data to develop a model of ChR2 with empirically-derived voltage- and irradiance- dependence, where parameters were fine-tuned via simulated annealing optimization. This ChR2 model offers: 1) accurate inward rectification in the current-voltage response across irradiances; 2) empirically-derived voltage- and light-dependent kinetics (activation, deactivation and recovery from inactivation); and 3) accurate amplitude and morphology of the response across voltage and irradiance settings. Temperature-scaling factors (Q10) were derived and model kinetics was adjusted to physiological temperatures. Using optical action potential clamp, we experimentally validated model-predicted ChR2 behavior in guinea pig ventricular myocytes. The model was then incorporated in a variety of cardiac myocytes, including human ventricular, atrial and Purkinje cell models. We demonstrate the ability of ChR2 to trigger action potentials in human cardiomyocytes at relatively low light levels, as well as the differential response of these cells to light, with the Purkinje cells being most easily excitable and ventricular cells requiring the highest irradiance at all pulse durations. This new experimentally-validated ChR2 model will facilitate virtual experimentation in neural and cardiac optogenetics at the cell and organ level and provide guidance for the development of in vivo tools

Integration of Engineered “Spark-Cell” Spheroids for Optical Pacing of Cardiac Tissue

Optogenetic methods for pacing of cardiac tissue can be realized by direct genetic modification of the cardiomyocytes to express light-sensitive actuators, such as channelrhodopsin-2, ChR2, or by introduction of light-sensitized non-myocytes that couple to the cardiac cells and yield responsiveness to optical pacing. In this study, we engineer three-dimensional “spark cells” spheroids, composed of ChR2-expressing human embryonic kidney cells (from 100 to 100,000 cells per spheroid), and characterize their morphology as function of cell density and time. These “spark-cell” spheroids are then deployed to demonstrate site-specific optical pacing of human stem-cell-derived cardiomyocytes (hiPSC-CMs) in 96-well format using non-localized light application and all-optical electrophysiology with voltage and calcium small-molecule dyes or genetically encoded sensors. We show that the spheroids can be handled using liquid pipetting and can confer optical responsiveness of cardiac tissue earlier than direct viral or liposomal genetic modification of the cardiomyocytes, with 24% providing reliable stimulation of the iPSC-CMs within 6 h and &gt;80% within 24 h. Moreover, our data show that the spheroids can be frozen in liquid nitrogen for long-term storage and transportation, after which they can be deployed as a reagent on site for optical cardiac pacing. In all cases, optical stimulation was achieved at relatively low light levels (&lt;0.15 mW/mm2) when 5 ms or longer pulses were used. Our results demonstrate a scalable, cost-effective method with a cryopreservable reagent to achieve contactless optical stimulation of cardiac cell constructs without genetically modifying the myocytes, that can be integrated in a robotics-amenable workflow for high-throughput drug testing

CellExcite: an efficient simulation environment for excitable cells

Background Brain, heart and skeletal muscle share similar properties of excitable tissue, featuring both discrete behavior (all-or-nothing response to electrical activation) and continuous behavior (recovery to rest follows a temporal path, determined by multiple competing ion flows). Classical mathematical models of excitable cells involve complex systems of nonlinear differential equations. Such models not only impair formal analysis but also impose high computational demands on simulations, especially in large-scale 2-D and 3-D cell networks. In this paper, we show that by choosing Hybrid Automata as the modeling formalism, it is possible to construct a more abstract model of excitable cells that preserves the properties of interest while reducing the computational effort, thereby admitting the possibility of formal analysis and efficient simulation. Results We have developed CellExcite, a sophisticated simulation environment for excitable-cell networks. CellExcite allows the user to sketch a tissue of excitable cells, plan the stimuli to be applied during simulation, and customize the diffusion model. CellExcite adopts Hybrid Automata (HA) as the computational model in order to efficiently capture both discrete and continuous excitable-cell behavior. Conclusions The CellExcite simulation framework for multicellular HA arrays exhibits significantly improved computational efficiency in large-scale simulations, thus opening the possibility for formal analysis based on HA theory. A demo of CellExcite is available at http://www.cs.sunysb.edu/~eha/ webcite

Novel optics-based approaches for cardiac electrophysiology: a review

Optical techniques for recording and manipulating cellular electrophysiology have advanced rapidly in just a few decades. These developments allow for the analysis of cardiac cellular dynamics at multiple scales while largely overcoming the drawbacks associated with the use of electrodes. The recent advent of optogenetics opens up new possibilities for regional and tissue-level electrophysiological control and hold promise for future novel clinical applications. This article, which emerged from the international NOTICE workshop in 20181, reviews the state-of-the-art optical techniques used for cardiac electrophysiological research and the underlying biophysics. The design and performance of optical reporters and optogenetic actuators are reviewed along with limitations of current probes. The physics of light interaction with cardiac tissue is detailed and associated challenges with the use of optical sensors and actuators are presented. Case studies include the use of fluorescence recovery after photobleaching and super-resolution microscopy to explore the micro-structure of cardiac cells and a review of two photon and light sheet technologies applied to cardiac tissue. The emergence of cardiac optogenetics is reviewed and the current work exploring the potential clinical use of optogenetics is also described. Approaches which combine optogenetic manipulation and optical voltage measurement are discussed, in terms of platforms that allow real-time manipulation of whole heart electrophysiology in open and closed-loop systems to study optimal ways to terminate spiral arrhythmias. The design and operation of optics-based approaches that allow high-throughput cardiac electrophysiological assays is presented. Finally, emerging techniques of photo-acoustic imaging and stress sensors are described along with strategies for future development and establishment of these techniques in mainstream electrophysiological research

PI3Ks Maintain the Structural Integrity of T-Tubules in Cardiac Myocytes

Phosphoinositide 3-kinases (PI3Ks) regulate numerous physiological processes including some aspects of cardiac function. Although regulation of cardiac contraction by individual PI3K isoforms has been studied, little is known about the cardiac consequences of downregulating multiple PI3Ks concurrently.Genetic ablation of both p110α and p110β in cardiac myocytes throughout development or in adult mice caused heart failure and death. Ventricular myocytes from double knockout animals showed transverse tubule (T-tubule) loss and disorganization, misalignment of L-type Ca(2+) channels in the T-tubules with ryanodine receptors in the sarcoplasmic reticulum, and reduced Ca(2+) transients and contractility. Junctophilin-2, which is thought to tether T-tubules to the sarcoplasmic reticulum, was mislocalized in the double PI3K-null myocytes without a change in expression level.PI3K p110α and p110β are required to maintain the organized network of T-tubules that is vital for efficient Ca(2+)-induced Ca(2+) release and ventricular contraction. PI3Ks maintain T-tubule organization by regulating junctophilin-2 localization. These results could have important medical implications because several PI3K inhibitors that target both isoforms are being used to treat cancer patients in clinical trials