Thyroid hormone receptor function in maturing ovine cardiomyocytes

Key points: Plasma thyroid hormone (tri-iodo-l-thyronine; T ₃ ) concentrations rise near the end of gestation and is known to inhibit proliferation and stimulate maturation of cardiomyocytes before birth. Thyroid hormone receptors are required for the action of thyroid hormone in fetal cardiomyocytes. Loss of thyroid hormone receptor (TR)α1 abolishes T ₃ signalling via extracellular signal-related kinase and Akt in fetal cardiomyocytes. The expression of TRα1 and TRβ1 in ovine fetal myocardium increases with age, although 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 the effects of TRα1/α2 short interfering RNA. Although T ₃ is known to abrogate ovine cardiomyocyte proliferation stimulated by insulin-like growth factor 1, this effect is mediated via the 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 (T ₃ ) in fetal sheep leads to the inhibition of proliferation and promotion of maturation in cardiomyocytes. The present study was designed to determine whether these T ₃ -induced changes are mediated via thyroid hormone receptors (TRs) or by non-genomic mechanisms. Fetal cardiomyocytes were isolated from 102 ± 3 and 135 ± 1 days of gestational age (dGA) sheep (n = 7 per age; term ∼145 dGA). Cells were treated with T ₃ (1.5 nm), insulin-like growth factor (IGF)-1 (1 μg mL ^–1 ) or a combination in the presence of TR antagonist NH3 (100 nm) or following short interfering RNA (siRNA) knockdown of TRα1/α2. Proliferation was quantified by 5-bromo-2′-deoxyuridine (BrdU) uptake (10 μm). Western blots measured protein levels of extracellular signal-related kinase (ERK), Akt, TRα1/β1 and p21. Age specific levels of TRα1/β1 were measured in normal hearts from fetuses [95 dGA (n = 8), 135 dGA (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). T ₃ reduced IGF-1 stimulated proliferation by ∼50% in 100 dGA and by ∼75% in 135 dGA cardiomyocytes (P < 0.05). NH3 blocked the T ₃  + IGF-1 reduction of BrdU uptake without altering the phosphorylation of ERK or Akt at both ages. NH3 did not suppress T ₃ -induced p21 expression in 100 dGA cardiomyocytes in 135 dGA cardiomyocytes, NH3 alone reduced BrdU uptake (−28%, P < 0.05), as well as T ₃ -induced p21 (−75%, P < 0.05). In both ages, siRNA knockdown of TRα1/α2 blocked the T ₃  + IGF-1 reduction of BrdU uptake and dramatically reduced ERK and Akt signalling in 135 dGA cardiomyocytes. In conclusion, TRs are required for normal proliferation and T ₃ signalling in fetal ovine cardiomyocytes, with the sensitivity to TR blockade being age-dependent.