Thyroid hormone receptor function in maturing ovine cardiomyocytes

Key points

Plasma thyroid hormone (T₃) concentrations rise near the end of gestation and are known to inhibit proliferation and stimulate maturation of cardiomyocytes before birth. Thyroid hormone receptors are required for thyroid hormone action in fetal cardiomyocytes. Loss of TRα1 abolishes T₃ signalling through ERK and AKT in fetal cardiomyocytes. The expression of thyroid receptors alpha 1 (TRα1) and beta 1 (TRβ1) in ovine fetal myocardium increases with age, though TRα1 levels always remain higher than those of TRβ1. Near term fetal cardiac myocytes are more sensitive than younger myocytes to thyroid receptor blockade by antagonist, NH3, and to effects of TRα1/α2 siRNA. While T₃ is known to abrogate ovine cardiomyocyte proliferation stimulated by IGF‐1, this effect is mediated through genomic action of thyroid hormone receptors, with little evidence for non‐genomic mechanisms.

Abstract

We have previously shown that the late‐term rise in tri‐iodo‐L‐thyronine, (T₃) in fetal sheep, leads to inhibition of proliferation and promotion of maturation in cardiomyocytes. This study was designed to determine whether these T₃‐induced changes are mediated through thyroid receptors (TR) or by non‐genomic mechanisms. Fetal cardiomyocytes were isolated from 102 ± 3 and 135 ± 1 days gestational age (dGA) sheep (n = 7/age; term ∼145dGA). Cells were treated with T₃ (1.5 nm), IGF‐1 (1μg/ml) or a combination in the presence of TR antagonist NH3 (100 nm) or following siRNA knockdown of TRα1/α2. Proliferation was quantified by BrdU uptake (10μM). Western blots measured protein levels of ERK, AKT, TRα1/β1, and p21. Age specific levels of TRα1/β1 were measured in normal hearts from fetuses (95dGA (n = 8), 135dGA (n = 7)), neonates (n = 8), and adult ewes (n = 7). TRα1 protein levels were consistently > 50% more than TRβ1 at each gestational age (p < 0.05). T₃ reduced IGF‐1 stimulated proliferation by ∼50% in 100dGA and by ∼75% in 135dGA cardiomyocytes (p < 0.05). NH3 blocked the T₃+IGF‐1 reduction of BrdU uptake without altering the phosphorylation of ERK or AKT at both ages. NH3 did not suppress T₃‐induced p21 expression in 100dGA cardiomyocytes though in 135dGA cardiomyocytes, NH3 alone reduced BrdU uptake (−28%, p < 0.05) as well as T₃‐induced p21 (−75%, p < 0.05). In both ages, siRNA knockdown of TRα1/α2 blocked the T₃+IGF‐1 reduction of BrdU uptake and dramatically reduced ERK and AKT signalling in 135dGA cardiomyocytes. In conclusion, TRs are required for normal proliferation and T₃ signalling in fetal ovine cardiomyocytes, and the sensitivity to TR blockade is age‐dependent.

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