Tri-iodo-l-thyronine promotes the maturation of human cardiomyocytes-derived from induced pluripotent stem cells

Abstract

Background: Cardiomyocytes derived from human induced pluripotent stem cells (hiPSC-CMs) have great potential as a cell source for therapeutic applications such as regenerative medicine, disease modeling, drug screening, and toxicity testing. This potential is limited, however, by the immature state of the cardiomyocytes acquired using current protocols. Tri-iodo-l-thyronine (T3) is a growth hormone that is essential for optimal heart growth. In this study, we investigated the effect of T3 on hiPSC-CM maturation.

Methods and results: A one-week treatment with T3 increased cardiomyocyte size, anisotropy, and sarcomere length. T3 treatment was associated with reduced cell cycle activity, manifest as reduced DNA synthesis and increased expression of the cyclin-dependent kinase inhibitor p21. Contractile force analyses were performed on individual cardiomyocytes using arrays of microposts, revealing an almost two-fold higher force per-beat after T3 treatment and also an enhancement in contractile kinetics. This improvement in force generation was accompanied by an increase in rates of calcium release and reuptake, along with a significant increase in sarcoendoplasmic reticulum ATPase expression. Finally, although mitochondrial genomes were not numerically increased, extracellular flux analysis showed a significant increase in maximal mitochondrial respiratory capacity and respiratory reserve capability after T3 treatment.

Conclusions: Using a broad spectrum of morphological, molecular, and functional parameters, we conclude that T3 is a driver for hiPSC-CM maturation. T3 treatment may enhance the utility of hiPSC-CMs for therapy, disease modeling, or drug/toxicity screens.

Keywords: Cardiomyocyte maturation; Contractile force; Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs); Mitochondria; Tri-iodo-l-thyronine.

Figure 1

T3 treatment leads to hiPSC-CM morphological and molecular changes. Representative control (A) and T3-treated (B) cells were stained with α-actinin (green) and Hoechst 33342 (blue). Scale bar: 25 m. Compared to control hiPSC-CMs, T3-treated hiPSC-CMs exhibited significant changes in cell area (C), circularity index (D), and sarcomere length (E). n>100 per condition. # P<0.001, *P<0.05. T3 treatment led to an increase α-MHC, decreased β-MHC, and increased SERCA2a expression level (F). Gene expression is shown normalized first to HPRT mRNA levels and then normalized to untreated control levels.

Figure 2

Effects of T3 on cardiomyocyte cell cycle activity. Cells were treated with 10 μM BrdU overnight and co-stained with α-actinin (green), Hoechst 44432 (blue), and BrdU (red). Double-positive hiPSC-CMs nuclei are magenta (arrows). Representative images for control (A) and after T3 treatment (B) are shown. Scale bar: 25 μm. (C) Quantitative analysis reveals a significant decrease in BrdU-positive percentage of hiPSC-CMs after T3 treatment. * P<0.05. n> 2000 α-actinin-positive cardiomyocyte nuclei in each group in three separate experiments. (D) Cell cycle inhibitor p21 mRNA transcript expression level compared with control cells (n=3). There is a significant increase in p21 transcript after T3 treatment. (E) Representative immunoblots of p21 and GAPDH in cardiomyocytes from control and T3 groups. (F) Quantitation of western blots demonstrating that p21 protein was significantly elevated in T3-treated cardiomyocytes (n=3). * P<0.05 vs. control.

Figure 3

HiPSC-CMs generate more contractile force after T3 treatment and show enhanced contractile kinetics. (A) representative hiPSC-CM stained for α-actinin (green) and Hoechst 33342 (blue). Microposts that were stained with BSA 594 are shown in red. Scale bar is 10 μm. (B) Representative force traces generated by control and T3-treated hiPSC-CMs. The statistical analysis results are shown in (C). T3 treatment led to significant decrease in time to peak contraction (D), time to 90% relaxation (E), and total twitch time (F). # P<0.001 vs. control. N=44 for control hiPSC-CMs and N=66 for T3-treated hiPSC-CMs.

Figure 4

T3-treated hiPSC-CMs exhibit increased calcium transient kinetics but no change in magnitude. Calcium transients were evaluated by loading the hiPSC-CMs with the intracellular calcium ratiometric indicator fura-2 AM. (A) Representative transients from control and T3-treated hiPSC-CMs. Note the faster upstroke and decay of the Ca²⁺ transient in the T3 cell. (B) The transient amplitude magnitudes were similar, though the calcium kinetics were significantly different in T3-treated hiPSC-CMs, as indicated by increases in maximal upstroke (C) an decay (D) velocities, reduced time to 90% peak [Ca²⁺]i (E) and (F) reduced time to 50% decay. n=10-15 cells per condition. *P<0.05 vs. control hiPSC-CMs.

Figure 5

The effect of T3 on mitochondrial function. Representative traces for control and T3 treated hiPSC-CMs responding to the ATP synthase inhibitor oligomycin, the respiratory uncoupler FCCP, and the respiratory chain blockers rotenone and antimycin A in (A). B shows the statistical analysis of the differences in basal OCR, maximal OCR, respiratory reserve capacity, and non-mitochondrial OCR. * P<0.05, # P<0.001 vs control hiPSC-CMs. n=6 biological replicates.