Figure 1.
Effect of suspension culture and TSA treatment on histone acetylation of hESC-CM colonies.
Western blot analyses show increased histone H3 acetylation (AcH3) levels in 35-day-old hESC-CMs (A) following 14 days of suspension culture (Sus) or (B) following treatment with 100 nM TSA for 48 hrs under adhesive (Ad) culture conditions (DMSO treatment had no effect on AcH3 levels). (C) Results from western blot analyses comparing Sus to Ad cultures (n = 9) and TSA to DMSO treatments (n = 13) show increased AcH3 levels in hESC-CMs. (D) Results of qRT-PCR analysis showing that TSA increases cardiac gene expression in hESC-CMs for at least 48 hrs after treatment. The corresponding values for human adult hearts (AH) are shown for each sample. Each graph displays the mean and standard deviation (SD) of three independent experiments. *, p<0.05; **, p<0.01.
Figure 2.
Microarray data for the expression of genes involved in cardiac ion channel function.
Global gene expression analyses were conducted using TSA- or DMSO-treated hESC-CMs under the Ad culture conditions (black bar) or Sus culture conditions (white bar). Genes involved in (A) sodium, (B) calcium and (C) potassium ion channels were analyzed. Each graph displays the mean and SD of two or three independent experiments.
Figure 3.
Heterogeneous responses to the hERG inhibitor, E4031, in MEA tests using early hESC/hiPSC-CM colonies.
(A) Sensitivity to 200 nM E4031 was measured in Sus cultured hiPSC-CM colonies (d35) after an additional 2 days of Ad culture on multi-electrode array (MEA) probes. The hiPSC-CM colonies showed the following heterogeneous responses to the hERG inhibitor: lengthened field potential duration (L-FPD), shortened FPD (S-FPD), and transient FP phenotypes (T-FPs). Red arrows indicate the initiation of FPs and the black arrows show the end-point of the cardiac repolarization phase in the FP signals. The broken line shows the shift in depolarization from 0 nM through 200 nM E4031. (B, C) Dose increment analyses in Sus-cultured (d35) hESC-CM colonies. The colony showing the L-FPD response to 50 nM of E4031 showed an S-FPD response at 200 nM through to a T-FPs response at 100 nM. This indicates that some of the Sus-cultured (d35) hESC-CM colonies were more sensitive to E4031 than others. However, on the basis of the rate-corrected QT (QTc) values, the S-FPD spheroids also show QTc prolongation (red line), because of the cells’ shortened FP rates. The whole bar containing a gray part displays the mean and SD of six FP cycles, and the gray bar displays the mean and SD of FPD.
Figure 4.
TSA enhances electrophysiological function in hESC-CMs.
(A) Experimental scheme: 21-day-old beating colonies obtained from αMHC-EGFP transgenic hESCs were maintained for 13 to 28 days in suspension. Sensitivity to 200 nM E4031 was measured three times for each beating colony using an MEA system on Days 2, 6, and 9 after the colonies were plated on the MEA probes, as shown at the lower panel. DMSO, or 100 nM TSA, was added for 48 hrs between Days 3 and 5 and then removed for 1 day before the second MEA recording to avoid contact with ion channel inhibitors. (B) Sensitivity of EGFP-positive hESC-CMs to E4031 before TSA treatment (Day 2) and after TSA treatment (Day 6) was measured using an MEA system. Representative FPs were demonstrated in hESC-CM spheroids before and after 100 nM E4031 treatment showing phenotypic changes in the response to 100 nM E4031 from S-FPD to L-FPD in TSA-treated hESC-CM spheroids. (C) Dose-dependent QTc prolongation seen in TSA-treated hESC-CM colonies that had shown arrhythmia in response to 100 nM E4031 in the first MEA test. Each value displays the mean and SD of FPDs obtained from six contiguous FP cycles. (D) Sequential MEA analyses of every hESC-CM colony before and after TSA treatment showing that all colonies reproducibly acquired a uniform electrophysiological phenotype with 2 days of TSA treatment (n = 7).
Figure 5.
Responses to the ion channel blockers, E4031, Nifekalant and Sotalol, are improved in TSA-treated hESC-CMs.
Relative FPD values were obtained with the MEA system on Day 2 to Day 6 for each colony in the presence of ion channel inhibitor (200 nM E4031; 20 µM Nifekalant; 200 µM Sotalol). Each graph displays changes in the relative FPD values in each hESC-CM colony after DMSO or TSA treatment and the mean and SD (E4031, n = 5 for DMSO and n = 6 for TSA; Nifekalant, n = 3 for DMSO and n = 4 for TSA; Sotalol, n = 3 for DMSO and n = 3 for TSA). *, p<0.05; †, tendency to differ (p<0.1).
Figure 6.
The TSA-mediated positive effect on MEA testing is transient.
(A) Results of qRT-PCR analyses of cardiac gene expression in hESC-CMs showed that the effect of TSA was transient. Day 5 (d5): immediately after 2 days of TSA treatment; Day 10 (d10): 5 days of culture after TSA washout. Values relative to the DMSO-treated hESC-CMs (DMSO = 100%) are shown (n = 3). Values represent the mean ± SD for each set of measurements. (B) Sequential MEA analyses of all hESC-CM colonies were performed on Days 2, 6, and 9 in the presence of 200 nM E4031. Changes in the relative FPD values on Day 6 or Day 9 from Day 2 for each colony are shown in Figures 5 and 6B, respectively. Values represent the mean ± SD (n = 5 for DMSO, n = 6 for TSA). Significant differences between DMSO and TSA treatment detected on Day 6 were not detected on Day 9.
Figure 7.
TSA enhances electrophysiological function in hiPSC-CMs accompanied by histone H3 acetylation.
(A) Western blot analyses show increased histone H3 acetylation levels in hiPSC-CMs treated with 100 nM TSA for 48 hrs. (B) Results from western blot analyses comparing Sus to Ad cultures (n = 7) and TSA to DMSO treatments (n = 9) show increased AcH3 levels in hiPSC-CMs. (C) Sequential MEA analyses in all hiPSC-CM colonies on Day 2 and Day 6 showed that TSA altered the electrophysiological phenotypes evaluated on the basis of their sensitivity to 200 nM of E4031 (n = 8). (D) Sequential MEA analyses in all hiPSC-CM spheroid on Days 2, 6 and 9 after plating showed electrophysiological phenotypes improved reproducibly following 2 days of TSA treatment in the presence of 200 nM E4031 (n = 7 for DMSO, n = 8 for TSA). Changes in relative FPD values (Day 2 = 1) from Day 2 to Days 6 and 9 and the mean±SD are shown. †, tendency to differ (p<0.1) only on Day 6.