Thyroid hormone receptor-beta is associated with coronary angiogenesis during pathological cardiac hypertrophy

Abstract

Insufficient angiogenesis is one of the causes leading to tissue ischemia and dysfunction. In heart failure, there is increasing evidence showing decreased capillary density in the left ventricle (LV) myocardium, although the detailed mechanisms contributing to it are not clear. The goal of this study was to investigate the role of thyroid hormone receptors (TRs) in the coronary microvascular rarefaction under pathological cardiac hypertrophy. The LV from hypertrophied/failing hearts induced by ascending aortic constriction (AAC) exhibited severe microvascular rarefaction, and this phenomenon was restored by chronic T(3) administration. Coronary endothelial cells (ECs) isolated from AAC hearts expressed lower TRbeta mRNA than control ECs, and chronic T(3) administration restored TRbeta mRNA expression level in AAC hearts to the control level. Among different TR subtype-specific knockout mice, TRbeta knockout and TRalpha/TRbeta double-knockout mice both exhibited significantly less capillary density in LV compared with wild-type mice. In vitro, coronary ECs isolated from TRbeta knockout mice lacked the ability to form capillary networks. In addition, we identified that kinase insert domain protein receptor/fetal liver kinase-1 (vascular endothelial growth factor-2 receptor) was one of the angiogenic mediators controlled by T(3) administration in the AAC heart. These data suggest that TRbeta in the coronary ECs regulates capillary density during cardiac development, and down-regulation of TRbeta results in coronary microvascular rarefaction during pathological hypertrophy.

Figure 1

Sustained pressure overload reduces capillary density in LV myocardium and T₃ administration restores the level of capillary density. Pressure overload was mediated by AAC. A, Representative photographs showing capillary images in LV of control and AAC heart with T₃ administration (+ T₃) or without T₃ administration. Bar, 100 μm. B, Averaged data (mean ± se) showing the capillary density in control heart (cont; n = 5), control with T₃ administration (cont + T₃; n = 3), AAC heart (AAC; n = 4), and AAC with T₃ administration (AAC + T₃; n = 4). *, P < 0.05 vs. control; #, P < 0.05 vs. AAC.

Figure 2

Chronic T₃ administration in AAC mice restores TRβ protein expression level. Left panel, Western blots were obtained from HCECs transfected with TRβ-Adv or control Adv (SR-Adv) to show the molecular weight of TRβ (36 μg protein per each well). In coronary ECs, TRβ protein is very low expression, and to collect sufficient amount of protein for detection of TRβ expression level, at least four hearts as one sample are used to isolate mouse coronary ECs (MCECs). Right panel, Western blots demonstrate that TRβ protein level is markedly decreased by AAC and is markedly increased by T₃ administration (100 μg protein per each well).

Figure 3

TRβ plays a crucial role in angiogenesis during cardiac development. Representative photographs (left panels) showing capillary images in LV of ubiquitous TRα KO mouse, TRβ KO mouse, TRα/TRβ double KO mouse, and cardiac myocyte-specific TRβ KO mouse as well as their age-matched wild-type (Wt) mice. Bar, 100 μm. Averaged data (right panels) showing the capillary density in Wt (n = 6) and KO mice (n = 5) for TRα, Wt and KO mice for TRβ (each group, n = 3), Wt, and KO mice for TRα/TRβ (each group, n = 3), and Wt and KO mice for cardiac myocyte-specific TRβ (each group, n = 3). Data are mean ± se. *, P < 0.05 vs. Wt. In contrast to the ubiquitous TRβ KO with deletion in vascular ECs and cardiac myocytes, the cardiac myocyte-specific deletion of TRβ did not significantly decrease capillary density.

Figure 4

Treatment with 1 nmol/liter T₃ enhances rat vascular EC capillary network formation. A, Representative images showing capillary network formation in vehicle (V; 10 nmol/liter NaOH)-treated ECs and T₃-treated ECs. Bar, 100 μm. B, Summarized data showing total tube length (left panel) and network density (right panel) in vehicle (n = 6) and T₃-treated (n = 4) ECs.

Figure 5

Deletion of TRβ attenuates EC capillary network formation. Representative images (left panels) showing capillary network formation in coronary ECs isolated from wild-type (Wt), TRα KO, and TRβ KO mice. Bar, 100 μm. Right panels, Summarized data of total tube length and network density in coronary ECs from Wt (n = 6) and TRα KO (αKO, n = 6) and Wt (n = 4) and TRβ KO (βKO, n = 5) mice. *, P < 0.05 vs. ECs isolated from Wt. Data are mean ± se.

Figure 6

Protein expression level of KDR/Flk1, but not PECAM, is decreased in coronary ECs isolated from AAC heart and T₃ administration restored KDR/Flk1 protein level in AAC to the control level. Western blot (upper panel) showing KDR/Flk1, PECAM, and actin protein expression in coronary ECs from control (Cont) and AAC heart with T₃ administration (+ T₃) or without T₃ administration. Summarized data (lower panels) showing protein levels of KDR/Flk1 and PECAM (normalized by actin level) in ECs isolated from different groups of hearts. Data are mean ± se from five experiments in each group. *, P < 0.05 vs. control; #, P < 0.05 vs. AAC.

Figure 7

T₃ treatment during tube formation increased KDR/Flk1 protein expression in rat vascular ECs. Western blot (left panels) showing KDR/Flk1 and actin protein expression in ECs with 1 nmol/liter T₃ treatment or vehicle treatment (V; 10 nmol/liter NaOH). The summarized data (mean ± se) shown in the right panel are from four experiments in each group. *, P < 0.05 vs. vehicle-treated ECs.

Figure 8

T₃ administration in AAC mice significantly increases CVC. CVC was measured in Langendorff-perfused mouse hearts at a constant pressure of 60 mm Hg and paced at 400 beats/min. Groups included mice subjected to AAC for 8 wk to induce hypertrophy and treated with T₃ (3.5 ng/g) injections (+T₃, n = 9) or saline (−T₃, n = 11) for 3 wk. *, P < 0.05 vs. −T₃.