Functional and Transcriptional Characterization of Histone Deacetylase Inhibitor-Mediated Cardiac Adverse Effects in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes

Abstract

Histone deacetylase (HDAC) inhibitors possess therapeutic potential to reverse aberrant epigenetic changes associated with cancers, neurological diseases, and immune disorders. Unfortunately, clinical studies with some HDAC inhibitors displayed delayed cardiac adverse effects, such as atrial fibrillation and ventricular tachycardia. However, the underlying molecular mechanism(s) of HDAC inhibitor-mediated cardiotoxicity remains poorly understood and is difficult to detect in the early stages of preclinical drug development because of a delayed onset of effects. In the present study, we show for the first time in human induced pluripotent stem cell-derived cardiomyocytes (hiPS-CMs) that HDAC inhibitors (dacinostat, panobinostat, vorinostat, entinostat, and tubastatin-a) induce delayed dose-related cardiac dysfunction at therapeutic concentrations associated with cardiac adverse effects in humans. HDAC inhibitor-mediated delayed effects on the beating properties of hiPS-CMs developed after 12 hours by decreasing the beat rate, shortening the field potential duration, and inducing arrhythmic behavior under form of sustained contractions and fibrillation-like patterns. Transcriptional changes that are common between the cardiotoxic HDAC inhibitors but different from noncardiotoxic treatments identified cardiac-specific genes and pathways related to structural and functional changes in cardiomyocytes. Combining the functional data with epigenetic changes in hiPS-CMs allowed us to identify molecular targets that might explain HDAC inhibitor-mediated cardiac adverse effects in humans. Therefore, hiPS-CMs represent a valuable translational model to assess HDAC inhibitor-mediated cardiotoxicity and support identification of better HDAC inhibitors with an improved benefit-risk profile.

Significance: Histone deacetylase (HDAC) inhibitors are a promising class of drugs to treat certain cancers, autoimmune, and neurodegenerative diseases. However, treated patients can experience various cardiac adverse events such as hearth rhythm disorders. This study found that human induced pluripotent stem cell-derived cardiomyocytes (hiPS-CMs) can predict cardiac adverse events in patients caused by HDAC inhibitors. Furthermore, transcriptional changes at the level of gene expression supported the effects on the beating properties of hiPS-CMs and highlight targets that might cause these cardiac adverse effects. hiPS-CMs represent a valuable translational model to assess HDAC inhibitor-mediated cardiotoxicity and to support development of safer HDAC inhibitors.

Keywords: Arrhythmia; Cardiotoxicity; Histone deacetylase inhibitor; Human-induced stem cell-derived cardiomyocytes; Impedance.

Figure 1.

Delayed cardiotoxicity of histone deacetylase (HDAC) inhibitors in human induced pluripotent stem cell-derived cardiomyocytes. Impedance recordings are shown for HDAC inhibitors at 6, 24, and 72 hours after dose compared with vehicles. The different time points represent the same experiment (well) per treatment, where the free peak plasma concentration (C_eff) and approximately 10× C_eff are indicated by red and blue labels, respectively. For tubastatin-a, the C_eff is not available because it is a preclinical drug.

Figure 2.

Histone deacetylase (HDAC) inhibitors decrease the beat rate and cause arrhythmic events in human induced pluripotent stem cell-derived cardiomyocytes (hiPS-CMs). (A–E): Concentration-dependent effect of HDAC inhibitors on the normalized beat rate is shown as a function of time. A concentration range based on the effective concentration (C_eff) was compared with the vehicle treatment. Note that the red line represents the C_eff. Data are presented as mean ± SEM (n = 7–11). ∗, p < .05 (differences between treatments and vehicles). (F): Examples of arrhythmic-like events representing prolonged/sustained contractions and fibrillation-like pattern on hiPS-CMs. Abbreviation: Norm., normalized.

Figure 3.

Histone deacetylase (HDAC) inhibitors shorten the field potential duration in hiPS-CMs. (A): MEA recordings representing an external potential signal of human induced pluripotent stem cell-derived cardiomyocytes (hiPS-CMs). FPD is determined from the depolarization spike to the repolarization peak. Note that the repolarization peak can be either negative or positive. (B): Some HDAC inhibitors shorten the FPD shown by a faster manifestation of the repolarization peak (arrow) at 24 hours. The level of FPD shortening is HDAC inhibitor-dependent, that is, more pronounced for dacinostat and panobinostat than for entinostat and vorinostat. (C, D): Bar plots showing the baseline-normalized FPD and beat rate for vehicle (n = 9), 0.1 µM dacinostat (n = 5), 10 nM panobinostat (n = 4), 0.3 µM entinostat (n = 5), and 0.1 µM vorinostat (n = 5) treatments at 6 and 24 hours after dose. Data are represented as mean ± SEM. ∗, p < .05 (differences between treatments and vehicles). Abbreviations: Daci, dacinostat; Enti, entinostat; FPD, field potential duration; norm., normalized; Pano, panobinostat; Vori, vorinostat.

Figure 4.

Overall transcriptional effects of histone deacetylase inhibitors on human induced pluripotent stem cell-derived cardiomyocytes. (A): Heat map representing the number of significantly differentially expressed genes for each selected treatment and time point (limma adjusted p < .05), illustrating the overall expression changes over time. The figure key is shown as an inset. (B): Sample similarity based on the log ratios (treatment versus vehicle) for all measured genes illustrating the overall effect of the different treatments and time points. Each treatment is represented by at least three biological replicates. Abbreviations: Daci, dacinostat; Enti, entinostat; Pano, panobinostat; PC, principal component; Tuba, tubastatin-a; Vori, vorinostat.

Figure 5.

Gene Ontology (GO)-based enrichment analysis of transcriptional changes related to histone deacetylase inhibitor-mediated cardiotoxicity. For each time point, the five most strongly affected gene sets are shown, as based on three levels: biological processes (GOBP), molecular functions (GOMF), and cellular compartments (GOCC) as defined by GO. The significance determined using the permutation procedure (Materials and Methods) is represented on the y-axis and used to rank the pathways. Pathways are colored by gene set name within the same database. Abbreviations: GOBP, Gene Ontology Biological Process; GOCC, Gene Ontology Cellular Component; GOMF, Gene Ontology Molecular Function.

Figure 6.

Volcano plots of the transcriptional effects between cardiotoxic and noncardiotoxic histone deacetylase inhibitor treatments. For each time point, the top 10 genes based on p values and top 10 genes based on log ratios (possibly identical) are highlighted in black, and genes linked to cardiac contractility and functioning in red. The significance and magnitude are calculated as described in Materials and Methods. Abbreviation: tox, toxic.