Human iPSC-Cardiomyocytes as an Experimental Model to Study Epigenetic Modifiers of Electrophysiology

Abstract

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.

Keywords: DNA acetylation; HDAC inhibitors; cardiac electrophysiology; cardiac epigenetics; histone acetyltransferases; histone deacetylases; human iPSC-CMs.

Figure 1

Three main methods of extracting epigenetic information surrounding drug introduction: (A) Animal experiments allow chronic observation and biomarker measurement upon drug administration, culminating in postmortem sample collection. (B) Multigroup drug introduction in humans is followed by periodic observation and biomarker collection culminating in biomarker analysis and in-person follow-up. (C) Patient-derived hiPSC-CMs are used to observe pharmacological cardiac effects in long-term, high-throughput optical and chemical modalities with feedback ability from existing in vitro assays for the direct improvement of future therapy. Created with Biorender.com.

Figure 2

HATs and HDACs epigenetically regulate gene expression through reversible (de)acetylation of histone proteins. Activity of HAT and HDAC enzymes controls chromatin conformation, loosening (HAT) or condensing (HDAC) chromatin structure. Small-molecule HDACi pharmaceuticals disrupt this system and promote transcriptional enhancement. Effects on key cardiac ion channels are observable through functional electrophysiological experiments, e.g., contractility assays or measurements of transmembrane potential (V_m) and calcium (Ca²⁺) transients. Representative V_m and Ca²⁺ transients modified from [15]. Created with Biorender.com.

Figure 3

HDAC inhibitor interventions in ongoing US clinical trials (194 total; (A)) and their investigated conditions/diseases (B), retrieved from clinicaltrials.gov on 22 November 2021. Completed, withdrawn, and/or terminated studies were excluded.

Figure 4

Adverse cardiac cases observed in patients being treated with HDACi (total number of adverse cases reported adjacent to each bar), retrieved from the FDA Adverse Event Reporting System (FAERS) Public Dashboard (fda.gov) on 22 November 2021. FDA approval year, when applicable, is listed in parentheses. * indicates possible TdP risk according to crediblemeds.org [144], accessed on 31 December 2021.

Figure 5

Methods for quantifying effects of HDACi. (A) (i) HDACs promote condensed chromatin structure and are counteracted by small-molecule HDACi pharmaceuticals. (ii) HDAC inhibition assay indirectly measures HDAC enzymatic activity. Aminoluciferin (AML), aminoluciferase (AMLase). (B) (i) Chromatin shifts from condensed (pink) to loose (blue) structure as histone acetylation increases. Chromatin accessibility can be measured by assay for transposase-accessible chromatin using sequencing (ATAC-seq) (ii) and chromatin immunoprecipitation followed by sequencing ChIP-seq (iii). Transcription factor (TF), Tn5 transposase (Tn5). (iv) Histone acetylation levels assayed with western blot (WB). “Ac” indicates acetylated histones. (v) ATAC-seq and ChIP-seq maps aligned to genome-wide association study (GWAS) maps to reveal areas of open chromatin where TFs of interest and phenotypes of interest are enriched. (C) (i) RNA quantification reflects gene expression. (ii) qPCR is a low-throughput fluorescence-based assay. (iii) Microarray assays are a high-throughput alternative to qPCR, allowing simultaneous detection and quantification of thousands of genes. cDNA indicates complementary DNA. (iv) Next-generation sequencing (NGS) is a high-throughput deep sequencing tool. (D) Functional behavior is assessed through contractility assays as well as all-optical electrophysiology recordings. Transmembrane potential (V_m), calcium transients (Ca²⁺). Representative V_m and Ca²⁺ transients modified from [15]. Created with Biorender.com.

Figure 6

(A) Volcano plot illustrates time-dependent transcriptional changes between treatments defined as “toxic” (0.5 mM dacinostat, 0.1 mM and 0.01 mM panobinostat, and 5 mM and 0.2 mM vorinostat) and treatments defined as relatively “safe” (0.3 mM entinostat, 1 mM tubastatin-a, and 0.1 mM vorinostat). Top 10 (based on p-value and log ratios, possibly overlapping) differential genes are in black while genes related to cardiac contractility and function are in red. (B) Time-dependent changes in beat rate reveal toxic effects of tested HDACi. Red line indicates applicable C_eff. * p < 0.05. Reproduced with permission from [133].