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We report LFC-plus, a simultaneous multicolor volumetric cytometry platform for high-throughput single-cell analysis. 

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. 

The epigenetic landscape and the responses to pharmacological epigenetic regulators in each human are unique. Classes of epigenetic writers and erasers, such as histone acetyltransferases, HATs, and histone deacetylases, HDACs, control DNA acetylation/deacetylation and chromatin accessibility, thus exerting transcriptional control in a tissue- and person-specific manner. Rapid development of novel pharmacological agents in clinical testing—HDAC inhibitors (HDACi)—targets these master regulators as common means of therapeutic intervention in cancer and immune diseases. The action of these epigenetic modulators is much less explored for cardiac tissue, yet all new drugs need to be tested for cardiotoxicity. To advance our understanding of chromatin regulation in the heart, and specifically how modulation of DNA acetylation state may affect functional electrophysiological responses, human-induced pluripotent stem-cell-derived cardiomyocyte (hiPSC-CM) technology can be leveraged as a scalable, high-throughput platform with ability to provide patient-specific insights. This review covers relevant background on the known roles of HATs and HDACs in the heart, the current state of HDACi development, applications, and any adverse cardiac events; it also summarizes relevant differential gene expression data for the adult human heart vs. hiPSC-CMs along with initial transcriptional and functional results from using this new experimental platform to yield insights on epigenetic control of the heart. We focus on the multitude of methodologies and workflows needed to quantify responses to HDACis in hiPSC-CMs. This overview can help highlight the power and the limitations of hiPSC-CMs as a scalable experimental model in capturing epigenetic responses relevant to the human heart. 

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 2018 1 , 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.