Thyroid Hormone Signaling in Male Mouse Skeletal Muscle Is Largely Independent of D2 in Myocytes

Abstract

The type 2 deiodinase (D2) activates the prohormone T4 to T3. D2 is expressed in skeletal muscle (SKM), and its global inactivation (GLOB-D2KO mice) reportedly leads to skeletal muscle hypothyroidism and impaired differentiation. Here floxed Dio2 mice were crossed with mice expressing Cre-recombinase under the myosin light chain 1f (cre-MLC) to disrupt D2 expression in the late developmental stages of skeletal myocytes (SKM-D2KO). This led to a loss of approximately 50% in D2 activity in neonatal and adult SKM-D2KO skeletal muscle and about 75% in isolated SKM-D2KO myocytes. To test the impact of Dio2 disruption, we measured soleus T3 content and found it to be normal. We also looked at the expression of T3-responsive genes in skeletal muscle, ie, myosin heavy chain I, α-actin, myosin light chain, tropomyosin, and serca 1 and 2, which was preserved in neonatal SKM-D2KO hindlimb muscles, at a time that coincides with a peak of D2 activity in control animals. In adult soleus the baseline level of D2 activity was about 6-fold lower, and in the SKM-D2KO soleus, the expression of only one of five T3-responsive genes was reduced. Despite this, adult SKM-D2KO animals performed indistinguishably from controls on a treadmill test, running for approximately 16 minutes and reached a speed of about 23 m/min; muscle strength was about 0.3 mN/m·g body weight in SKM-D2KO and control ankle muscles. In conclusion, there are multiple sources of D2 in the mouse SKM, and its role is limited in postnatal skeletal muscle fibers.

Figure 1.

Skeletal muscle and brown adipose tissue D2 activity in GLOB-D2KO and WT littermate mice. A, D2 activity in neonatal (hindlimb muscles; n = 3–6) and adult skeletal muscle (soleus; n = 3–5) of GLOB-D2KO and littermate WT mice. B, D2 activity in BAT (n = 3–4) of GLOB-D2KO and littermate control mice. C, D2 activity in slow-twitch red soleus (SOL), tibialis anterior (TA), and fast-twitch white gastrocnemius (WG) (n = 3–4) of WT mice. D, D2 activity in microsomes isolated and assayed as in (18) from BAT, SOL, and WG of WT mice (n = 3). Values are the mean ± SEM of the indicated number of animals. *, P < .05; **, P < .01; ***, P < .001 vs controls.

Figure 2.

Influence of systemic T₄ levels in D2 activity in soleus muscle and BAT. A, D2 activity in euthyroid, hypothyroid (Hypo), and hypothyroid treated with T₄ (Hypo + T4) adult soleus muscle of GLOB-D2KO and WT littermates. B, This is the same as in panel A except that BAT samples were used. Hypothyroidism was induced by feeding on a low-iodine diet containing 0.15% PTU and on water containing 0.05% MMI for 3 weeks. T₄ treatment consisted in one single injection (5 or 10 μg per 100 g of body weight, sc) 5 hours before the animals were killed. Values are the mean ± SEM of the indicated number of animals in parenthesis. **, P < .01, ***, P < .001 vs WT; ###, P < .001 vs euthyroid WT mice (fold induction is indicated in parentheses).

Figure 3.

Dio2 expression and D2 activity in skeletal muscle of SKM-D2KO. A, Serum TSH, T₃, and T₄ of SKM-D2KO (n = 5–7) and control mice (n = 5). B, D2 activity in BAT of SKM-D2KO (n = 5) and control mice (n = 11). C, This is the same as in panel B except that brain cortex samples were used. D, This is the same as in panel B except that pituitary samples were used. E, D3 activity in brain cortex of SKM-D2KO (n = 5) and control (n = 6) mice. F, D1 activity in liver of the same animals are as in panel A. G, relative Dio2 mRNA levels in neonatal (hindlimb; n = 3–10) and adult skeletal muscle (soleus; n = 4–5) as measured by quantitative RT-PCR and using cyclo-A or RNA polymerase II mRNA levels as internal control and H, D2 activity in neonatal and adult soleus of SKM-D2KO and control littermates. SKM-D2KO indicates Dio2 inactivation in skeletal muscle. Values are normalized by respective littermate controls. Values are the mean ± SEM of the indicated number of animals. *, P < .05, **, P < .01, ***, P < .001 vs control.

Figure 4.

D2 activity in skeletal muscle of WT, floxed D2 (Flox), and Cre-myosin light chain (Cre) expressing mice of SKM-D2KO strain and effects of hypothyroidism. A, D2 activity in neonatal (hindlimb) and adult (soleus) skeletal muscle of WT, Cre, and Flox mice (four to seven mice). B, D2 activity in euthyroid (n = 4) adult soleus of SKM-D2KO and control and hypothyroid mice (n = 4–6); hypothyroidism was induced by feeding on a low-iodine diet containing 0.15% PTU and on water containing 0.05% MMI for 6 weeks. Values are the mean ± SEM of the indicated number of animals. *, P < .05, ***, P < .001 vs respective control.

Figure 5.

Primary myocytes of SKM-D2KO mice. A, Dio2 mRNA levels in primary cultured myocytes or muscle fibroblasts (n = 3) of SKM-D2KO (black bars) and cre-MLC (white bars) hindlimb. Fibroblasts were obtained by plating muscle cultured cells for 30 minutes. B, Dio2 mRNA levels of primary myocytes treated with FSK (10 μM) or D2 activity after 6 hours of treatment with FSK and MG132 (a proteasome inhibitor, 1 μM). Numbers on top of black bars denote expression or activity relative to cre-MLC control. All mRNA results are normalized by myocytes of control mice in panel A. C, Illustration of the floxed Dio2 gene showing the loxP sites flanking the selenocysteine (Se) insertion site. Sense and antisense primers locations to determine DNA recombination are shown. Black bar denotes the PCR product size (∼2000 bp). CDS, coding sequence. D, Floxed Dio2 gene after cre-mediated recombination. Note the small PCR product (∼500b p) produced after DNA recombination. E, Evaluation of DNA recombination by PCR in primary cultured myocytes of Cre-MLC (lanes 2 and 4) and SKM-D2KO (lanes 3 and 5). In lane 5 the reaction was performed with no DNA. DNA ladder was added lane 1 (100–15 000 kb). Cre-MLC (F) and SKM-D2KO (G) myoblasts were differentiated for 3 days in 2% horse serum. Note the elongated myotubes in both groups. Images were taken at ×10 magnification. Values are the mean ± SEM of the indicated number of animals. ***, P < .001.

Figure 6.

Skeletal muscle T₃ content and mRNA levels in neonatal and adult SKM-D2KO mice. A, T₃ content in soleus muscle of SKM-D2KO and control mice. T₃ was determined by RIA after tissue extraction of iodothyronines. B, Relative skeletal muscle-related genes mRNA levels in neonatal hindlimb muscles measured by quantitative RT-PCR and using cyclo-A mRNA levels as internal control. C, This was the same as in panel B except that adult soleus muscle was used. Sample size is shown in parentheses for each group. Dashed bars represent systemic hypothyroid animals. Significance was set at P < .002 after Bonferroni's correction for multiple comparisons (two comparisons for each 11 genes). Hypothyroidism was induced by feeding on a low iodine diet containing 0.15% PTU and on water containing 0.05% MMI for 6 weeks. Values are the mean ± SEM of the indicated number of animals. ***, P < .002 vs controls.

Figure 7.

Treadmill maximal exercise capacity, muscle strength, and cross-sectional area of SKM-D2KO mice. A, Maximal exercise time and speed achieved during the treadmill test. The test starts at a speed of 10 m/min and progressively increased by 2 m/min every 2 minutes up to exhaustion (n = 7–9). B, Muscle tetanic force normalized by body weight. C, Soleus mean cross-sectional area assessed in 10-μm sections were stained with hematoxylin and eosin in SKM-D2KO mouse (n = 3). Representative soleus cross-section of control littermate (D) and SKM-D2KO mice (E) are shown. Bar scale, 50 μm. Values are the mean ± SEM of the indicated number of animals. ***, P < .001.