Thyroid hormone receptor function in maturing ovine cardiomyocytes

Abstract

Key points: Plasma thyroid hormone (tri-iodo-l-thyronine; T₃ ) 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 T₃ 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 T₃ 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 (T₃ ) in fetal sheep leads to the inhibition of proliferation and promotion of maturation in cardiomyocytes. The present study was designed to determine whether these T₃ -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 T₃ (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). T₃ reduced IGF-1 stimulated proliferation by ∼50% in 100 dGA and by ∼75% in 135 dGA 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 100 dGA cardiomyocytes in 135 dGA 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 135 dGA cardiomyocytes. In conclusion, TRs are required for normal proliferation and T₃ signalling in fetal ovine cardiomyocytes, with the sensitivity to TR blockade being age-dependent.

Keywords: Cardiomyocyte proliferation; Fetal; NH3; Thyroid hormone.

Figure 1. TRα1 is the predominant thyroid receptor in the sheep heart

Ontogeny of TRα1 (solid bars) and TRβ1 (open bars) protein expression by western blot analysis in sheep myocardium from 95 dGA (n = 8), 135 dGA (n = 7), early neonates (n = 8) and adult ewes (n = 7). Comparisons by ANOVA and Tukey's multiple comparison post hoc test. Bars that do not share lowecase letters are significantly different (P < 0.05). ^*Statistically significant difference between TRα1 and TRβ1 expression (P < 0.05). Values are expressed as the mean (SD).

Figure 2. NH3 effectively blocks T₃'s inhibition of proliferation

BrdU uptake as an index of proliferation in cultured cardiomyocytes from 100 dGA (A) and 135 dGA (B) sheep fetuses treated with hormones and/or TR blocker. Comparisons by ANOVA and Tukey's multiple comparison post hoc test. Bars that do not share lowercase letters are significantly different (P < 0.05) (n = 7). Values are expressed as the mean (SD).

Figure 3. Control of proliferation is more sensitive to TR availability in near‐term fetal cardiomyocytes

Protein expression of p21 in cultured cardiomyocytes from 100 dGA (A) and 135 dGA (B) sheep fetuses treated with hormones and/or TR blocker. Comparisons by ANOVA and Tukey's multiple comparison post hoc test. Bars that do not share lowercase letters are significantly different (P < 0.05) (n = 7). Values are expressed as the mean (SD).

Figure 4. NH3 does not alter ERK or Akt signaling

Effect of a specific thyroid receptor α1 and β1 blocker (NH3) on ERK (A) and Akt (B) phosphorylation in 135 dGA fetal cardiomyocytes (n = 7). Values are expressed as the mean (SD).

Figure 5. TR blockade (NH3) reverses T₃‐stimulated activation of ERK and Akt in the presence of IGF‐1

Age‐effect on ERK (A and C) and Akt (B and D) phosphorylation in cultured cardiomyocytes from 100 dGA (A and B) and 135 dGA (C and D) sheep fetuses treated with hormones and/or thyroid receptor blocker. Comparisons by ANOVA and Tukey's multiple comparison post hoc test. Bars that do not share lowercase letters are significantly different (P < 0.05) (n = 7). Values are expressed as the mean (SD).

Figure 6. Transfection efficiency using siRNA reduction of TRα

TRα1 gene expression following siRNA TRα1/α2 transfection in cultured cardiomyocytes from 100 dGA (A) and 135 dGA (B) sheep fetuses. Cells were treated with control siRNA (CN si; 0.08 pmol), transfection reagent control (TSF) or siRNA TRα1/α2 (TR si; 0.08 pmol). In both ages, transfection results in a 50% reduction in TRα1 gene expression. Comparisons by ANOVA and Tukey's multiple comparison post hoc test. Bars that do not share lowercase letters are significantly different (P < 0.05) (n = 5). Values are expressed as the mean (SD).

Figure 7. Loss or TRα1 reverses normal T₃‐induced inhibition of cardiomyocyte proliferation

BrdU uptake in cultured cardiomyocytes from 100 dGA (A) and 135 dGA (B) sheep fetuses following siRNA knockdown of thyroid receptor α1/α2. Cells were treated with control siRNA (CN si; 0.08 pmol) or siRNA TRα1/α2 (TR si; 0.08 pmol). A subset of cells in the TR siRNA group were also treated with T₃ and/or IGF‐1. Comparisons by ANOVA and Tukey's multiple comparison post hoc test. Bars that do not share lowercase letters are significantly different (P < 0.05) (n = 5). Values are expressed as the mean (SD).

Figure 8. Loss of TRα1 in younger fetal cardiomyocytes

ERK (A) and Akt (B) phosphorylation and TRα1 protein expression (C) in cultured cardiomyocytes from 100 dGA sheep fetuses following siRNA knockdown of TRα1/α2. Cells were treated with control siRNA (CN si; 0.08 pmol) or siRNA TRα1/α2 (TR si; 0.08 pmol). A subset of cells in the TR si group were also treated with T₃ and/or IGF‐1. TRα1 protein level was not detectable by western blotting following knockdown (C). Comparisons by ANOVA and Tukey's multiple comparison post hoc test. Bars that do not share lowercase letters are significantly different (P < 0.05) (n = 5). Values are expressed as the mean (SD).

Figure 9. Loss of TRα1 in near‐term fetal cardiomyocytes

ERK (A) and Akt (B) phosphorylation and TRα1 protein expression (C) in cultured cardiomyocytes from 135 dGA sheep fetuses following siRNA knockdown of TRα1/α2. Cells were treated with control siRNA (CN si; 0.08 pmol) or siRNA TRα1/α2 (TR si; 0.08 pmol). A subset of cells in the TR siRNA group were also treated with T₃ and/or IGF‐1. TRα1 protein level was not detectable by western blotting following knockdown (C). Comparisons by ANOVA and Tukey's multiple comparison post hoc test. Bars that do not share letters are significantly different (P < 0.05) (n = 5). Values are expressed as the mean (SD).