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. 2017 Dec 12;6:e29330. doi: 10.7554/eLife.29330

Figure 5. Nucleotide metabolism regulates cardiomyocyte maturation.

(A) Glucose-deprived hESC-CMs are cultured in the absence (a, b) or presence (c, d) of 25 mM uridine, and stained for pH3 (a mitosis marker). The addition of uridine restored proliferative activity even in the absence of glucose. Data are representative of three independent experiments. (B) Proliferation rate, presented as number of pH3+ cells/α-actinin+cells, seen in the stained images of Figure 5A. (n = 3, mean ± SD, p<0.01 by t-test.) (C) Relative mRNA expression of TNNT2 in hESC-CMs in 25 mM or 0 mM glucose with 0 or 25 mM uridine. Uridine dose-dependently inhibited the TNNT2 expression level in glucose-deprived conditions. (n = 3, mean ± SD, p<0.0005 by one-way ANOVA test.) See also Figure 5—figure supplement 1A. (D) Relative mRNA expression of TNNT2 and NKX2-5 in hESC-CMs cultured in 0–25 mM of glucose in the presence or absence of thymidine. Thymidine block increases the levels of TNNT2 and NKX2-5 (n = 3, mean ±SD, p<0.01 for a t-test comparing samples with or without 25 mM thymidine in 25 mM glucose. See also Figure 5—figure supplement 1B. (E) Relative mRNA expression of TNNT2 and NKX2-5 in hESC-CMs cultured in 0–25 mM glucose and 0–2 mM hydroxyurea (HU, a ribonucleotide reductase inhibitor). HU dose-dependently induced the expression of TNNT2 and NKX2-5 at 1, 5, and 25 mM glucose. (n = 3, mean ± SD, p-value by one-way ANOVA test.) See also Figure 5—figure supplement 1C.

Figure 5.

Figure 5—figure supplement 1. Nucleotide inhibits hESC-CM maturation.

Figure 5—figure supplement 1.

(A) The relative expression of TNNT2 mRNA measured by qPCR in hESC-CMs in 16 different conditions of 0–25 mM of glucose and 0–25 mM uridine. Uridine recapitulates the effect of glucose by dose-dependently inhibiting TNNT2 expression level. Data are representative of three independent experiments. (B) The relative mRNA expression of TNNT2 and NKX2-5 measured by qPCR in hESC-CMs cultured in 0–25 mM glucose in the presence or absence of thymidine. Thymidine block increases the levels of TNNT2 and NKX2-5. Data are representative of three independent experiments. (C) The relative mRNA expression of TNNT2 and NKX2-5 measured by qPCR in hESC-CMs cultured in 0–25 mM glucose and 0–2 mM hydroxyurea (HU, a ribonucleotide reductase inhibitor). HU dose-dependently induced the expression of TNNT2 and NKX2-5 at 1, 5, and 25 mM glucose. Data are representative of three independent experiments.
Figure 5—figure supplement 2. Nucleotide deprivation, not cell cycle block, is the primary inducer of cardiomyocyte maturation.

Figure 5—figure supplement 2.

(A) The impact of CDK4/6 inhibitor on the relative mRNA expression of TNNT2 and NKX2-5 measured by qPCR. Cell cycle block by CDK4/6 inhibitor did not significantly induce the expression of cardiac markers. Data are representative of three independent experiments. (B) The impact of paclitaxel on the relative mRNA expression of TNNT2 and NKX2-5 measured by qPCR. Cell cycle block by paclitaxel did not significantly induce the expression of cardiac markers. Data are representative of three independent experiments.